Methods for treating lipid metabolism dysregulation

Abstract

Certain embodiments described herein provide a method of treating lipid metabolism dysregulation and / or inflammation in a mammal in need thereof, comprising administering to the mammal an effective amount of an agonist anti-TREM2 (triggering receptor expressed on myeloid cells 2) antibody.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent No. 62 / 771,456, filed November 26, 2018, U.S. Provisional Patent No. 62 / 817,955, filed March 13, 2019, and U.S. Provisional Patent No. 62 / 890,506, filed August 22, 2019. The entire contents of the above-referenced applications are incorporated herein by reference. [Background technology]

[0002] Lipids are a large and diverse class of biomolecules that perform multiple biochemical functions, such as storing energy, signaling, and serving as structural components of cell membranes and myelin sheaths. Lipid metabolism refers to the intracellular or extracellular synthesis and degradation of lipids, including the breakdown or storage of fats for energy. Lipid dysregulation in myeloid cells, including neutrophils, monocytes, macrophages, and microglia, has been shown to cause deleterious inflammatory responses as well as lipotoxicity in affected cells or tissues, mediating numerous disease processes. Although much research has been conducted to investigate lipid metabolism and its association with disease, additional work is needed to further clarify the molecular mechanisms underlying lipid processing, lipid-associated inflammation, and lipotoxicity, as well as to develop new therapies to cure lipid dysregulation and / or associated inflammatory responses in specific diseases and conditions. Summary of the Invention

[0003] Certain embodiments described herein provide a method for treating lipid metabolism dysregulation in a mammal in need of such treatment, comprising administering to the mammal an agonist anti-TREM2 (triggering receptor expressed on myeloid cells 2) antibody.

[0004] Certain embodiments described herein provide agonistic anti-TREM2 antibodies for use in treating dysregulated lipid metabolism in a mammal.

[0005] Certain embodiments described herein provide for the use of an agonist anti-TREM2 antibody for the preparation of a medicament for treating lipid metabolism dysregulation in a mammal.

[0006] Certain embodiments described herein provide methods of reducing intracellular accumulation of one or more lipids in a cell, comprising contacting the cell with an effective amount of an agonist anti-TREM2 antibody.

[0007] Certain embodiments described herein provide agonistic anti-TREM2 antibodies for use in reducing the intracellular accumulation of one or more lipids in a cell.

[0008] Certain embodiments described herein provide for the use of an agonist anti-TREM2 antibody for the preparation of a medicament for reducing intracellular accumulation of one or more lipids in a cell.

[0009] Certain embodiments described herein provide a method of treating Alzheimer's disease in a mammal in need thereof, the method comprising administering to the mammal an agonist anti-TREM2 antibody, wherein the mammal has, or has been determined to have, lipid metabolism dysregulation.

[0010] Certain embodiments described herein provide an agonistic anti-TREM2 antibody for use in treating Alzheimer's disease in a mammal, where the mammal has, or has been determined to have, lipid metabolism dysregulation.

[0011] Certain embodiments described herein provide for the use of an agonist anti-TREM2 antibody for the preparation of a medicament for treating Alzheimer's disease in a mammal, where the mammal has, or has been determined to have, dysregulated lipid metabolism.

[0012] Certain embodiments described herein provide a method of treating atherosclerosis in a mammal in need thereof, comprising administering to the mammal an effective amount of an agonist anti-TREM2 antibody.

[0013] Certain embodiments described herein provide agonistic anti-TREM2 antibodies for use in the treatment of atherosclerosis in a mammal.

[0014] Certain embodiments described herein provide for the use of an agonist anti-TREM2 antibody for the preparation of a medicament for treating atherosclerosis in a mammal.

[0015] Certain embodiments provide a method of treating inflammation in a mammal in need of such treatment, comprising administering to the mammal an effective amount of an agonist anti-TREM2 antibody.

[0016] Certain embodiments provide an agonistic anti-TREM2 antibody for use in treating inflammation in a mammal.

[0017] Certain embodiments provide the use of an agonist anti-TREM2 antibody for the preparation of a medicament for treating inflammation in a mammal. In certain embodiments, for example, the following are provided: (Item 1) An agonistic anti-TREM2 (triggering receptor expressed on myeloid cells 2) antibody for use in the treatment of lipid metabolism dysregulation in a mammal. (Item 2) A method for treating lipid metabolism dysregulation in a mammal in need of such treatment, comprising administering to said mammal an effective amount of an agonistic anti-TREM2 antibody. (Item 3) 3. The antibody or method of item 1 or 2, wherein cells expressing TREM2 in the mammal exhibit lipid metabolism dysregulation. (Item 4) 4. The antibody or method of item 3, wherein the cell is a microglial cell or a macrophage. (Item 5) 5. The antibody or method of any one of items 1 to 4, wherein the mammal has, or has been determined to have, reduced TREM2 activity, and optionally the mammal has, or has been determined to have, reduced apolipoprotein E (ApoE) activity. (Item 6) 6. The antibody or method according to any one of items 1 to 5, wherein the lipid metabolism dysregulation comprises increased accumulation of one or more lipids. (Item 7) 7. The antibody or method of item 6, wherein the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, bis(monoacylglycero)phosphate species (BMPs), diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, and combinations thereof. (Item 8) 8. The antibody or method of item 7, wherein the one or more lipids comprise a cholesteryl ester. (Item 9) 9. The antibody or method according to any one of items 1 to 8, wherein the mammal has or is susceptible to Alzheimer's disease, Nasu-Hakola disease (NHD), Lewy body disease, Parkinson's disease, retinal degeneration, Huntington's disease, frontotemporal lobar degeneration (FTD), amyotrophic lateral sclerosis (ALS), Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, multiple sclerosis or vanishing white matter disease. (Item 10) 9. The antibody or method according to any one of items 1 to 8, wherein the mammal has or is susceptible to obesity, type 2 diabetes, alcoholic or non-alcoholic steatohepatitis, alcoholic or non-alcoholic fatty liver disease, rheumatoid arthritis (RA) or atherosclerosis. (Item 11) 11. The antibody or method of any one of items 1 to 10, wherein the agonistic anti-TREM2 antibody is MAB17291 or 78.18. (Item 12) 12. The method of any one of items 2 to 11, wherein said administering reduces lipid accumulation and optionally reduces expression of at least one proinflammatory cytokine selected from the group consisting of G-CSF, INFy, IL-12(p40), IL-12(p70), LIX(CXCL5), MCP-1(CCL2), MIG(CXCL9), IL-1α, IL-1β, and IL-18. (Item 13) 13. The method of any one of items 2 to 12, further comprising administering a second therapeutic agent selected from the group consisting of an RXR agonist, an LXR agonist, and an acetyl-CoA acetyltransferase 1 (ACAT1) inhibitor. (Item 14) Use of an agonist anti-TREM2 antibody for the preparation of a medicament for treating lipid metabolism dysregulation in a mammal. (Item 15) An agonist anti-TREM2 antibody for use in reducing the intracellular accumulation of one or more lipids in a cell. (Item 16) A method for reducing intracellular accumulation of one or more lipids in a cell, the method comprising contacting the cell with an effective amount of an agonistic anti-TREM2 antibody. (Item 17) 17. The antibody or method of item 15 or 16, wherein the cell is a microglial cell or a macrophage. (Item 18) 18. The antibody or method according to any one of items 15 to 17, wherein the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, BMPs, diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, and combinations thereof. (Item 19) 19. The antibody or method of any one of items 15 to 18, wherein the cell has or has been determined to have reduced TREM2 activity, and optionally the cell has or has been determined to have reduced ApoE activity. (Item 20) 20. The antibody or method according to any one of items 15 to 19, wherein the cell is present in a mammal. (Item 21) Use of an agonist anti-TREM2 antibody for the preparation of a medicament for reducing the intracellular accumulation of one or more lipids in a cell. (Item 22) 13. An agonistic anti-TREM2 antibody for use in the treatment of Alzheimer's disease in a mammal, wherein the mammal has, or is determined to have, a lipid metabolism dysregulation. (Item 23) A method for treating Alzheimer's disease in a mammal in need of such treatment, comprising administering to the mammal an agonist anti-TREM2 antibody, wherein the mammal has, or has been determined to have, lipid metabolism dysregulation. (Item 24) 24. The antibody or method of item 22 or 23, wherein the mammal has or has been determined to have lipid metabolism dysregulation in TREM2 expressing cells. (Item 25) 25. The antibody or method of item 24, wherein the TREM2-expressing cells have, or have been determined to have, reduced TREM2 activity. (Item 26) 26. The antibody or method according to any one of items 22 to 25, wherein the lipid metabolism dysregulation comprises increased intracellular accumulation of one or more lipids selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, BMPs, diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, and combinations thereof. (Item 27) 13. Use of an agonist anti-TREM2 antibody for the preparation of a medicament for treating Alzheimer's disease in a mammal, wherein the mammal has, or has been determined to have, lipid metabolism dysregulation. (Item 28) An agonistic anti-TREM2 antibody for use in treating atherosclerosis in a mammal. (Item 29) 1. A method of treating atherosclerosis in a mammal in need of such treatment, comprising administering to the mammal an effective amount of an agonist anti-TREM2 antibody. (Item 30) 30. The antibody or method of item 28 or 29, wherein the mammal has or has been determined to have lipid metabolism dysregulation, and the lipid metabolism dysregulation comprises increased accumulation of one or more lipids. (Item 31) 31. The antibody or method of item 30, wherein the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, BMPs, diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, and combinations thereof. (Item 32) 32. The antibody or method according to any one of items 28 to 31, wherein macrophages in the mammal have, or have been determined to have, reduced TREM2 activity. (Item 33) Use of an agonist anti-TREM2 antibody for the preparation of a medicament for treating atherosclerosis in a mammal. (Item 34) An agonistic anti-TREM2 antibody for use in treating inflammation in a mammal. (Item 35) 13. A method of treating inflammation in a mammal in need of such treatment, comprising administering to the mammal an effective amount of an agonist anti-TREM2 antibody. (Item 36) 36. The method of claim 35, wherein the administration reduces expression of at least one proinflammatory cytokine selected from the group consisting of G-CSF, INFy, IL-12(p40), IL-12(p70), LIX(CXCL5), MCP-1(CCL2), MIG(CXCL9), IL-1α, IL-1β, and IL-18. (Item 37) 37. The antibody or method according to any one of items 34 to 36, wherein the mammal has or is susceptible to RA, gout, or inflammatory bowel disease (IBD). (Item 38) 37. The antibody or method according to any one of items 34 to 36, wherein the mammal has or is susceptible to Alzheimer's disease, Nasu-Hakola disease (NHD), Lewy body disease, Parkinson's disease, retinal degeneration, Huntington's disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, multiple sclerosis or vanishing white matter disease. (Item 39) 37. The antibody or method according to any one of items 34 to 36, wherein the mammal has or is susceptible to obesity, type 2 diabetes, alcoholic or non-alcoholic steatohepatitis, alcoholic or non-alcoholic fatty liver disease, or atherosclerosis. (Item 40) Use of an agonist anti-TREM2 antibody for the preparation of a medicament for treating inflammation in a mammal. (Item 41) 1. A method for selecting a population of CNS cells from a tissue sample, comprising: (a) contacting the tissue sample with a primary anti-CD45 antibody, a primary anti-CD11b antibody, and a primary anti-astrocyte surface antigen-2 (ACSA-2) antibody, each primary antibody being uniquely labeled, to provide a labeled tissue sample; (b) sorting the cells in the labeled tissue sample by flow cytometry; The method, wherein the method provides distinct cell populations of astrocytes and microglial cells. (Item 42) A population of CNS cells comprising two physically distinct cell populations, the first cell population expressing CD45 low / CD11b + / ACSA-2 - the second cell population comprising an enriched population of CD45 cells; - / CD11b - / ACSA-2 + The population of CNS cells comprising cells. [Brief description of the drawings]

[0018] [Figure 1] Attenuated expression of genes involved in lipid metabolism in Trem2 mutant mice with chronic demyelination induced by cuprizone diet. (A) Venn diagram of the number of differentially expressed genes in bulk microglia isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - mouse brains without treatment. (B) Venn diagram of the number of differentially expressed genes in bulk microglia isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - mouse brains with chronic demyelination (12 weeks cuprizone treatment). (C) Log2 fold expression change in individual genes related to lipid metabolism in Trem2+ / +, Trem2+ / -, and Trem2- / - bulk microglia with control diet (left inset) versus 5 or 12 weeks cuprizone treatment (right inset, top or bottom, respectively) (left inset). [Figure 2A] Attenuated expression of genes involved in lipid metabolism in Trem2 knockout mice with chronic demyelination. Microglial clusters of single-cell RNA sequencing data from individually isolated Trem2+ / + control diet microglia (Trem2+ / + Ctrl) compared to microglia isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - mice with 12 weeks cuprizone treatment (Trem2+ / + CPZ, Trem2+ / - CPZ, Trem2- / - CPZ). Left: % of all aggregated samples represented in each cluster; Right: heatmap of the percentage of each individual sample represented in the cluster. [Figure 2B] Attenuated expression of genes involved in lipid metabolism in Trem2 knockout mice with chronic demyelination. Heatmap clustering showing the proportion of top differentially expressed genes up- and downregulated in clusters knn5, 8, and 10. [Figure 3A]Increased abundance of cholesteryl esters and myelin lipids in Trem2 knockout forebrains during chronic demyelination. Unaltered forebrain total free cholesterol levels in Trem2+ / +, Trem2+ / -, and Trem2- / - mice on a control or cuprizone diet. Seen in forebrains from Trem2- / - mice on a 12-week cuprizone diet compared with Trem2+ / +, Trem2+ / -, and Trem2- / - mice on a control diet or cuprizone for 5 weeks, and Trem2+ / + and Trem2+ / - mice on cuprizone for 12 weeks. [Figure 3B] Increased abundance of cholesteryl esters and myelin lipids in Trem2 knockout forebrains during chronic demyelination. Increased cholesteryl esters. [Figure 3C] Increased abundance of cholesteryl esters and myelin lipids in Trem2 knockout forebrains during chronic demyelination. Oxidized cholesteryl esters. [Figure 3D] Increased abundance of cholesteryl esters and myelin lipids in Trem2 knockout forebrains during chronic demyelination. Bis(monoacylglycero)phosphate (BMP). [Figure 3E] Increased abundance of cholesteryl esters and myelin lipids in Trem2 knockout forebrains during chronic demyelination. Increased triacylglycerides. *p<0.05, **p<0.01, ***p<0.001, 2-way ANOVA, Tukey's test, ***: + / + compared to control, +++: comparison between genotypes with CPZ for 12 weeks. Lipids were quantified by LC / MS. [Figure 3F] Increased abundance of cholesteryl esters and myelin lipids in Trem2 knockout forebrains during chronic demyelination. Increased ganglioside levels. For each ganglioside species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5 weeks CPZ; + / + 12 weeks CPZ; + / - control; + / - 5 weeks CPZ; + / - 12 weeks CPZ; - / - control; - / - 5 weeks CPZ; and - / - 12 weeks CPZ. [Figure 4A]Increased abundance of cholesteryl esters and myelin-derived lipids in Trem2 knockout isolated microglia during chronic demyelination. Increased cholesteryl esters were detected in microglia isolated from Trem2- / - brains (12-week cuprizone diet) compared with Trem2+ / +, Trem2+ / -, and Trem2- / - microglia on control diet or 5-week cuprizone, and Trem2+ / + and Trem2+ / - microglia on 12-week cuprizone. [Figure 4B] Increased abundance of cholesteryl esters and myelin-derived lipids in Trem2 knockout isolated microglia during chronic demyelination. Increased BMPs detected in microglia isolated from Trem2- / - brains (12-week cuprizone diet) compared with Trem2+ / +, Trem2+ / -, and Trem2- / - microglia on control diet or 5-week cuprizone, and Trem2+ / + and Trem2+ / - microglia on 12-week cuprizone. [Figure 4C] Increased abundance of cholesteryl esters and myelin-derived lipids in Trem2 knockout isolated microglia during chronic demyelination. Increased hexosylceramide was detected in microglia isolated from Trem2- / - brains (12-week cuprizone diet) compared with Trem2+ / +, Trem2+ / -, and Trem2- / - microglia on control diet or 5-week cuprizone, and Trem2+ / + and Trem2+ / - microglia on 12-week cuprizone. [Figure 4D] Increased abundance of cholesteryl esters and myelin-derived lipids in Trem2 knockout isolated microglia during chronic demyelination. Increased galactosylceramide levels were detected in microglia isolated from Trem2- / - brains (12-week cuprizone diet) compared with Trem2+ / +, Trem2+ / -, and Trem2- / - microglia on control diet or 5-week cuprizone, and Trem2+ / + and Trem2+ / - microglia on 12-week cuprizone. [Figure 4E]There was no change in cholesteryl ester lipid levels in astrocyte-enriched cell populations isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - brains on either the control or cuprizone diets. [Figure 4F] There is no change in BMP lipid levels in astrocyte-enriched cell populations isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - brains on either the control or cuprizone diets. [Figure 4G] There was no change in hexosylceramide lipid levels in astrocyte-enriched cell populations isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - brains on either the control or cuprizone diets. [Figure 4H] There was no change in galactosylceramide lipid levels in astrocyte-enriched cell populations isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - brains on either the control or cuprizone diets. [Figure 4I] Increased ceramide detected in microglia isolated from Trem2- / - brains on the 12-week cuprizone diet compared with Trem2+ / +, Trem2+ / -, and Trem2- / - microglia on control diet or 5-week cuprizone, and Trem2+ / + and Trem2+ / - microglia on 12-week cuprizone. [Figure 4J] Increased GM3 detected in microglia isolated from Trem2- / - brains on the 12-week cuprizone diet compared with Trem2+ / +, Trem2+ / -, and Trem2- / - microglia on control diet or 5-week cuprizone, and Trem2+ / + and Trem2+ / - microglia on 12-week cuprizone. [Figure 4K] Increased phosphatidylglycerol detected in microglia isolated from Trem2- / - brains on the 12-week cuprizone diet compared with Trem2+ / +, Trem2+ / -, and Trem2- / - microglia on control diet or 5-week cuprizone, and Trem2+ / + and Trem2+ / - microglia on 12-week cuprizone. [Figure 4L] Increased sulfatide levels detected in microglia isolated from Trem2- / - brains on the 12-week cuprizone diet compared with Trem2+ / +, Trem2+ / -, and Trem2- / - microglia on control diet or 5-week cuprizone, and Trem2+ / + and Trem2+ / - microglia on 12-week cuprizone. [Figure 4M] No changes in ceramide lipid levels were detected in astrocyte-enriched cell populations isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - brains on either the control or cuprizone diets. Lipids were quantified by LC / MS. [Figure 4N] No changes in GM3 lipid levels were detected in astrocyte-enriched cell populations isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - brains on either the control or cuprizone diets. Lipids were quantified by LC / MS. [Figure 4O] No changes in phosphatidylglycerol lipid levels were detected in astrocyte-enriched cell populations isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - brains on either the control or cuprizone diets. Lipids were quantified by LC / MS. [Figure 4P] No changes in sulfatide lipid levels were detected in astrocyte-enriched cell populations isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - brains on either the control or cuprizone diets. Lipids were quantified by LC / MS. [Diagram 5]TREM2 KO BMDMs show increased neutral lipid staining upon treatment with oxidized low density lipoprotein (oxLDL). (A) Nile red staining (63x resolution) of cultured TREM2 WT or KO bone marrow derived macrophages (BMDMs) treated for 48 hours with either oxidized LDL (oxLDL, 50ug / mL) or vehicle. (B). Quantification of total spot area shows accumulation of neutral lipids in TREM2 KO, both to a lesser extent under vehicle conditions as well as to a greater extent under lipid challenge (oxLDL) conditions. Data are shown as the mean and standard deviation of three technical replicates. [Figure 6A] TREM2 KO BMDMs show increased lipid accumulation upon treatment with oxidized LDL. Increased levels of cholesteryl esters were detected in cultured TREM2 KO BMDMs compared to WT BMDMs when administered 50ug / mL oxLDL. [Figure 6B] TREM2 KO BMDMs show increased lipid accumulation upon treatment with oxidized LDL. Increased levels of ganglioside GM3 are detected in cultured TREM2 KO BMDMs compared to WT BMDMs when administered 50ug / mL oxLDL. [Figure 6C] TREM2 KO BMDMs show increased lipid accumulation upon treatment with oxidized LDL. Increased levels of triacylglycerides are detected in cultured TREM2 KO BMDMs compared to WT BMDMs when administered 50ug / mL oxLDL. [Figure 6D] TREM2 KO BMDMs show increased lipid accumulation upon treatment with oxidized LDL. Increased levels of hexosylceramide are detected in cultured TREM2 KO BMDMs compared to WT BMDMs when administered 50ug / mL oxLDL. [Figure 6E]TREM2 KO BMDMs show increased lipid accumulation upon treatment with oxidized LDL. No changes in phosphatidylcholine were observed in TREM2 KO compared to WT when administered 50ug / mL oxLDL. Data are presented as the mean and standard deviation of three technical replicates, and all data are normalized to the average cell number per well. Lipids were measured by LC / MS. [Figure 7A] TREM2 KO BMDMs show increased lipid accumulation upon treatment with myelin. Cultured Trem2 KO BMDMs showed increased accumulation of cholesteryl esters, cholesteryl esters, and lipids when treated with purified mouse brain myelin (25ug / mL). [Figure 7B] TREM2 KO BMDMs show increased lipid accumulation upon treatment with myelin. Cultured Trem2 KO BMDMs show greater accumulation of oxidized cholesteryl esters when treated with purified mouse brain myelin (25ug / mL). Data are presented as mean and SEM of three technical replicates, and all data are normalized to the average cell number per well. Lipids were measured by LC / MS. [Figure 7C] TREM2 KO BMDMs show increased lipid accumulation upon treatment with myelin. Cultured Trem2 KO BMDMs show greater accumulation of diacylglycerides when treated with purified mouse brain myelin (25ug / mL). Data are shown as the mean and SEM of three technical replicates, and all data are normalized to the average cell number per well. Lipids were measured by LC / MS. [Figure 7D] TREM2 KO BMDMs show increased lipid accumulation upon treatment with myelin. Cultured Trem2 KO BMDMs show greater accumulation of triacylglycerides when treated with purified mouse brain myelin (25ug / mL). Data are shown as the mean and SEM of three technical replicates, and all data are normalized to the average cell number per well. Lipids were measured by LC / MS. [Figure 7E]TREM2 KO BMDMs show increased lipid accumulation upon treatment with myelin. Cultured Trem2 KO BMDMs show greater accumulation of hexosylceramide when treated with purified mouse brain myelin (25ug / mL). Data are shown as the mean and SEM of three technical replicates, and all data are normalized to the average cell number per well. Lipids were measured by LC / MS. [Figure 7F] TREM2 KO BMDMs show increased lipid accumulation upon treatment with myelin. Cultured Trem2 KO BMDMs show greater accumulation of lactosylceramide when treated with purified mouse brain myelin (25ug / mL). Data are shown as the mean and SEM of three technical replicates, and all data are normalized to the average cell number per well. Lipids were measured by LC / MS. [Figure 7G] TREM2 KO BMDMs show increased lipid accumulation upon treatment with myelin. Cultured Trem2 KO BMDMs show greater accumulation of gangliosides when treated with purified mouse brain myelin (25ug / mL). Data are presented as mean and SEM of three technical replicates, and all data are normalized to the average cell number per well. Lipids were measured by LC / MS. [Figure 8A] TREM2 KO induced pluripotent stem cell (iPSC) derived human microglia show increased lipid accumulation upon treatment with myelin. TREM2 KO human iPSC derived microglia show greater accumulation of free cholesterol when treated with myelin (25ug / mL). Data are shown as the mean and standard deviation of three technical replicates, and all data are normalized to the average cell number per well. Lipids were analyzed by LC / MS. [Figure 8B]TREM2 KO induced pluripotent stem cell (iPSC) derived human microglia show increased lipid accumulation upon treatment with myelin. TREM2 KO human iPSC derived microglia show greater accumulation of phosphatidylserine 38:4 when treated with myelin (25ug / mL). Data are shown as the mean and standard deviation of three technical replicates, and all data are normalized to the average cell number per well. Lipids were analyzed by LC / MS. [Figure 8C] TREM2 KO induced pluripotent stem cell (iPSC) derived human microglia show increased lipid accumulation upon treatment with myelin. TREM2 KO human iPSC derived microglia show greater accumulation of BMP44:12 when treated with myelin (25ug / mL). Data are presented as the mean and standard deviation of three technical replicates, and all data are normalized to the average cell number per well. Lipids were analyzed by LC / MS. [Figure 8D] TREM2 KO induced pluripotent stem cell (iPSC) derived human microglia show increased lipid accumulation upon treatment with myelin. TREM2 KO human iPSC derived microglia show greater accumulation of lysophosphatidylcholine 16:0 when treated with myelin (25ug / mL). Data are shown as the mean and standard deviation of three technical replicates, and all data are normalized to the average cell number per well. Lipids were analyzed by LC / MS. [Figure 8E] TREM2 KO induced pluripotent stem cell (iPSC) derived human microglia show increased lipid accumulation upon treatment with myelin. TREM2 KO human iPSC derived microglia show greater accumulation of platelet activating factor when treated with myelin (25ug / mL). Data are presented as the mean and standard deviation of three technical replicates, and all data are normalized to the average cell number per well. Lipids were analyzed by LC / MS. [Figure 8F]TREM2 KO induced pluripotent stem cell (iPSC) derived human microglia show increased lipid accumulation upon treatment with myelin. TREM2 KO human iPSC derived microglia show greater accumulation of cholesterol sulfate when treated with myelin (25ug / mL). Data are presented as the mean and standard deviation of three technical replicates, and all data are normalized to the average cell number per well. Lipids were analyzed by LC / MS. [Figure 8G] TREM2 KO induced pluripotent stem cell (iPSC) derived human microglia show increased lipid accumulation upon treatment with myelin. TREM2 KO human iPSC derived microglia show greater accumulation of lysophosphatidylethanolamine when treated with myelin (25ug / mL). Data are presented as the mean and standard deviation of three technical replicates, and all data are normalized to the average cell number per well. Lipids were analyzed by LC / MS. [Figure 8H] TREM2 KO induced pluripotent stem cell (iPSC) derived human microglia show increased lipid accumulation upon treatment with myelin. TREM2 KO human iPSC derived microglia show greater accumulation of the plasmalogen phosphatidylethanolamine (PEp) when treated with myelin (25ug / mL). Data are presented as the mean and standard deviation of three technical replicates, and all data are normalized to the average cell number per well. Lipids were analyzed by LC / MS. [Figure 9] Myelin-derived cholesterol is converted to cholesteryl esters via ACAT1 in BMDM. Free cholesterol and cholesteryl ester (CE) levels in BMDM from wild-type mice treated with or without myelin for 2 h were then extracted immediately after myelin uptake (T0) or after myelin washout and a 2-h (T2) or 4-h (T4) chase. ACAT1 inhibitor was added during myelin uptake and maintained through the 4-h washout (T4+ACAT1 inhibitor). Lipids were measured by LC / MS. [Figure 10]Recombinant human APOE3 ameliorates neutral lipid accumulation in Trem2 KO BMDMs upon myelin treatment. Trem2 KO BMDMs accumulate more neutral lipids than WT BMDMs when treated with myelin debris (25ug / mL) for 24 hours, as quantified by Nile Red staining. This accumulation is ameliorated by the addition of recombinant human APOE3 (10ug / mL) to the culture medium. [Figure 11A] ACAT1 inhibition abolishes the increase in cholesteryl esters in iPSC-derived human microglia upon myelin treatment. ACAT inhibition prevents the accumulation of all cholesteryl ester species measured in both WT and Trem2 KO iPSC-derived human microglia treated with purified myelin. [Figure 11B] ACAT1 inhibition abolishes the increase in cholesteryl esters in iPSC-derived human microglia upon myelin treatment. Specific example of cholesteryl ester 22:6 is shown. [Figure 11C] ACAT1 inhibition abolishes the increase in cholesteryl esters in iPSC-derived human microglia upon myelin treatment. As a control, cholesterol levels are shown to be unaffected by the presence of ACAT1 inhibitors. Data are presented as the mean and standard deviation of three technical replicates, and all data are normalized to the average number of cells per well. Lipids were measured by LC / MS. [Figure 12A] Agonistic anti-TREM2 antibodies reduce neutral lipid accumulation in iPSC-derived human microglia upon myelin treatment. Nile red images of iPSC-derived human microglia treated with either vehicle or myelin (50ug / mL) and then treated 24 hours later with either RSV control or agonistic anti-TREM2 antibodies. [Figure 12B]Agonistic anti-TREM2 antibodies reduce neutral lipid accumulation in iPSC-derived human microglia upon myelin treatment. Spot quantification of triacylglycerides shows reduced lipid accumulation upon treatment with TREM2 antibodies compared to isotype control (RSV). Lipidomic data are normalized to the average cell number per well. Triglycerides were measured by LC / MS. [Figure 12C] Agonistic anti-TREM2 antibodies reduce neutral lipid accumulation in iPSC-derived human microglia upon myelin treatment. Lipidology shows reduced lipid accumulation upon treatment with TREM2 antibodies compared to isotype control (RSV). Lipidomic data are normalized to the average number of cells per well. Triglycerides were measured by LC / MS. [Figure 12D] Agonistic anti-TREM2 antibodies reduce neutral lipid accumulation in iPSC-derived human microglia upon myelin treatment. Includes bar graphs showing quantified levels of triacylglyceride lipid species (in iPSC-microglia treated with myelin followed by incubation with an exemplary anti-TREM2 antibody). [Figure 12E] Agonistic anti-TREM2 antibodies reduce neutral lipid accumulation in iPSC-derived human microglia upon myelin treatment. Includes bar graphs showing quantified levels of triacylglyceride lipid species (in iPSC-microglia treated with myelin followed by incubation with an exemplary anti-TREM2 antibody). Data is presented for iPSC-microglia in which a myelin washout step was included prior to incubation with an exemplary anti-TREM2 antibody. [Figure 13A] Effect of bexarotene on myelin storage in TREM2 KO BMDMs. Trem2 KO BMDMs accumulate more neutral lipids than WT BMDMs when treated with myelin debris (25ug / mL) for 48 hours, as quantified by Nile Red staining. This accumulation is reduced by co-treatment with bexarotene (10uM). [Figure 13B]ACAT1 inhibitors and LXR agonists reduce the levels of cholesteryl esters (CE) in human iPSC-derived TREM2 KO microglia. Human iPSC-derived microglia were treated with myelin (25ug / mL) in C+++ medium for 48 hours with no drug, ACAT1 inhibitor K604 (500nM) and LXR agonist GW3965 (10uM). CE levels were rescued by the drugs in both WT and TREM2 KO cells, but the increased cholesterol seen in TREM2 KO cells was not rescued by the drugs. CE and cholesterol levels were measured in lysates by LC / MS. N=3 biological replicates. [Figure 14A] TREM2 deficiency prevents DAM conversion during chronic demyelination. Log2 fold expression changes in individual genes related to lysosomal function in Trem2+ / +, Trem2+ / -, and Trem2- / - bulk microglia following a control diet (left inset) versus cuprizone treatment for 5 or 12 weeks (right inset, top or bottom, respectively). [Figure 14B] TREM2 deficiency prevents DAM conversion during chronic demyelination. Log2 fold expression changes in individual genes related to lipid metabolism in Trem2+ / +, Trem2+ / -, and Trem2- / - bulk microglia following a control diet (left inset) versus cuprizone treatment for 5 or 12 weeks (right inset, top or bottom, respectively). [Figure 14C] TREM2 deficiency prevents DAM conversion during chronic demyelination. Heatmap of bulk microglial expression changes (log 2 fold change) from 5 and 12 week control vs. CPZ-treated Trem2+ / +, Trem2+ / -, and Trem2- / - in the top 69 DAM genes downregulated (up) or upregulated (down) in 5XFAD compared to wild-type microglia (Keren-Shaul, et al. (2017). Cell 169,1276-1290 e1217). Camera, p<1x10-41. [Figure 14D]TREM2 deficiency prevents DAM conversion during chronic demyelination. Log2 fold expression change in individual homeostatic DAM genes in Trem2+ / +, Trem2+ / -, and Trem2- / - bulk microglia with control (left inset) or CPZ treatment (right inset) for 5 weeks (top) or 12 weeks (bottom). [Figure 14E] TREM2 deficiency prevents DAM conversion during chronic demyelination. Log2 fold expression change in first stage DAM genes in Trem2+ / +, Trem2+ / -, and Trem2- / - bulk microglia with control (left inset) or CPZ treatment (right inset) for 5 weeks (top) or 12 weeks (bottom). [Figure 14F] TREM2 deficiency prevents DAM conversion during chronic demyelination. Log2 fold expression change in stage 2 DAM genes in control (left inset) or CPZ-treated (right inset) Trem2+ / +, Trem2+ / -, and Trem2- / - bulk microglia for 5 weeks (top) or 12 weeks (bottom). [Figure 15A] TREM2 deficiency prevents DAM conversion during chronic demyelination. Log2 fold change for genes upregulated in DAMs vs. homeostatic microglia from 5XFAD mice in Trem2+ / + 12 week control vs. CPZ treated mice (top) and Trem2- / - 12 week control vs. CPZ treated mice (bottom) (see Keren-Shaul, et al. (2017) Cell 169, 1276-1290 e1217). [Figure 15B]TREM2 deficiency prevents DAM conversion during chronic demyelination. Microglia isolated from aged wild-type brains expressed injury-associated microglial features. Comparison of t-statistics calculated from RNA-seq profiles of microglia sorted from aged vs. young Trem2+ / + mice (x-axis) and aged vs. young Trem2- / - mice (y-axis); n=7 mice per group. Genes are colored by membership in gene sets of interest. Homeostatic, DAM1, and DAM2 gene sets are defined in (Keren-Shaul, et al. (2017). Cell 169,1276-1290 e1217). Cholesterol metabolism-related gene sets identified from a subset of differentially expressed genes from 12-week CPZ vs. control treated mice previously known to be related to cholesterol metabolism. [Figure 15C] TREM2 deficiency prevents DAM conversion during chronic demyelination. Microglia isolated from aged wild-type brains expressed injury-associated microglial features. Heatmap of differentially expressed genes from Trem2 cuprizone and aged cohorts that are upregulated in DAM2 microglia as defined in (Keren-Shaul,et al.(2017).Cell 169,1276-1290 e1217) or in the cholesterol metabolism related gene set as defined in (15B). Gene expression values ​​are plotted as zero mean and unit variance. [Figure 16A] ScRNA-seq confirms that Trem2- / - microglia exhibit an attenuated transition to an injury-associated microglial state upon demyelination. Expression profile of selected marker genes across the dataset plotted as normalized counts per cell. Legend on the left indicates upregulated (up arrow) vs downregulated (down arrow) marker genes in the indicated clusters. [Figure 16B]ScRNA-seq confirms that Trem2- / - microglia exhibit an attenuated transition to an injury-associated microglial state upon demyelination. Normalized counts of unique molecular identifier (UMI) reads for individual marker genes defining the clusters were compared across expression in all clusters. Legend on the left indicates upregulated (up arrow) vs downregulated (down arrow) marker genes in the indicated clusters. [Figure 16C-1] ScRNA-seq confirms that Trem2- / - microglia exhibit an attenuated transition to an injury-associated microglial state upon demyelination.Normalized expression scores of upregulated marker genes from cluster 8 compared across all other clusters. Dots represent individual cells shaded by cluster. [Figure 16C-2] ScRNA-seq confirms that Trem2- / - microglia exhibit an attenuated transition to an injury-associated microglial state upon demyelination.Normalized expression scores of upregulated marker genes from cluster 8 compared across all other clusters. Dots represent individual cells shaded by cluster. [Figure 17A] TREM2 deficiency leads to cholesteryl ester accumulation in the brain. Neurotropic light chain (Nf-L) levels in plasma isolated from control, Trem2+ / +, Trem2+ / -, and Trem2- / - mice fed a cuprizone (CPZ) diet for 5 or 12 weeks. ***p<0.001, interaction p=0.06, two-way ANOVA, Tukey's test, n=7-16 mice / condition. [Figure 17B] TREM2 deficiency leads to cholesteryl ester accumulation in the brain. Neurotropic light chain (Nf-L) levels in plasma isolated from Trem2+ / + and Trem2- / - mice at 2 and 17 months (2-way ANOVA, FDR<0.05, interaction age genotype p<0.05). [Figure 17C] TREM2 deficiency leads to cholesteryl ester accumulation in the brain. Heatmap comparison of significantly altered lipids in control vs. 5-week CPZ treatment. N=3 mice per condition, 2-way ANOVA, FDR<0.05. [Figure 17D-1] TREM2 deficiency leads to cholesteryl ester accumulation in the brain. Heatmap of lipids significantly altered by genotype and / or 12-week CPZ treatment in Trem2+ / +, Trem2+ / -, and Trem2- / - mouse forebrains. Two-way ANOVA, FDR<0.05; columns represent data from individual mice, n=3–7 mice per condition. [Figure 17D-2] TREM2 deficiency leads to cholesteryl ester accumulation in the brain. Heatmap of lipids significantly altered by genotype and / or 12-week CPZ treatment in Trem2+ / +, Trem2+ / -, and Trem2- / - mouse forebrains. Two-way ANOVA, FDR<0.05; columns represent data from individual mice, n=3–7 mice per condition. [Figure 17E] TREM2 deficiency causes cholesteryl ester accumulation in the brain. Concentrations of cholesteryl ester (CE) species extracted from Trem2+ / +, Trem2+ / -, and Trem2- / - mouse forebrains on control, 5-week or 12-week CPZ diet. Data represent mean ± SEM and are presented on a log10 scale. Two-way ANOVA, FDR<0.05; genotype-treatment interaction shown for the indicated lipid species from Trem2- / - on the 12-week CPZ diet as indicated by asterisks. *p<0.05, **p<0.01, ***p<0.001. For each CE species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5-week CPZ; + / + 12-week CPZ; + / - control; + / - 5-week CPZ; + / - 12-week CPZ; - / - control; - / - 5-week CPZ; and - / - 12-week CPZ. [Figure 17F] TREM2 deficiency leads to cholesteryl ester accumulation in the brain. Quantification of APP-positive puncta, APP-positive areas, and APP intensity in the hippocampus of Trem2+ / + and Trem2- / - mice on a control diet or after 5 or 12 weeks of CPZ. [Figure 18A]TREM2 deficiency causes cholesteryl ester accumulation in isolated microglia. (A) Heatmap of lipids significantly altered by treatment and / or genotype in microglia isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - mouse brains upon control, 5- or 12-week cuprizone (CPZ) treatment. Two-way ANOVA, FDR<0.05; columns represent data from individual mice, n=6 mice per condition. (B) Heatmap comparison of lipids detected in cerebrospinal fluid (CSF) isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - mice upon control, 5- or 12-week CPZ treatment. Columns represent data from individual mice, n=5-6 mice per condition. As shown in A-B, certain lipidomic changes are present in Trem2 knockout isolated microglia (A) but absent in CSF (B) upon chronic demyelination. [Figure 18B] TREM2 deficiency causes cholesteryl ester accumulation in isolated microglia. (A) Heatmap of lipids significantly altered by treatment and / or genotype in microglia isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - mouse brains upon control, 5- or 12-week cuprizone (CPZ) treatment. Two-way ANOVA, FDR<0.05; columns represent data from individual mice, n=6 mice per condition. (B) Heatmap comparison of lipids detected in cerebrospinal fluid (CSF) isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - mice upon control, 5- or 12-week CPZ treatment. Columns represent data from individual mice, n=5-6 mice per condition. As shown in A-B, certain lipidomic changes are present in Trem2 knockout isolated microglia (A) but absent in CSF (B) upon chronic demyelination. [Figure 18C]TREM2 deficiency leads to cholesteryl ester accumulation in isolated microglia. Heatmap comparison of lipids detected in astrocytes isolated from Trem2+ / +, Trem2+ / -, and Trem2- / - mice upon control, 5 weeks, or 12 weeks of CPZ treatment. Columns represent data from individual mice, n=5-6 mice per condition. [Figure 18D] TREM2 deficiency causes cholesteryl ester accumulation in isolated microglia. Increased (D) cholesteryl ester levels were detected in microglia isolated from Trem2- / - brains on the 12-week cuprizone diet compared to Trem2+ / +, Trem2+ / -, and Trem2- / - microglia on a control diet or 5 weeks cuprizone, and Trem2+ / + and Trem2+ / - microglia on a 12-week cuprizone diet. In general, no changes in lipid levels of cholesteryl esters were detected in astrocyte-enriched cell populations (E) or CSF (F) isolated from control or cuprizone diet Trem2+ / +, Trem2+ / -, and Trem2- / - brains. Lipids were quantified by LC / MS. *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA, Tukey's test, ***: + / + comparison with control, +++: comparison between genotypes with 12 weeks CPZ. (D-F) For each sterol species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5 weeks CPZ; + / + 12 weeks CPZ; + / - control; + / - 5 weeks CPZ; + / - 12 weeks CPZ; - / - control; - / - 5 weeks CPZ; and - / - 12 weeks CPZ. [Figure 18E]TREM2 deficiency causes cholesteryl ester accumulation in isolated microglia. Increased (D) cholesteryl ester levels were detected in microglia isolated from Trem2- / - brains on the 12-week cuprizone diet compared to Trem2+ / +, Trem2+ / -, and Trem2- / - microglia on a control diet or 5 weeks cuprizone, and Trem2+ / + and Trem2+ / - microglia on a 12-week cuprizone diet. In general, no changes in lipid levels of cholesteryl esters were detected in astrocyte-enriched cell populations (E) or CSF (F) isolated from control or cuprizone diet Trem2+ / +, Trem2+ / -, and Trem2- / - brains. Lipids were quantified by LC / MS. *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA, Tukey's test, ***: + / + comparison with control, +++: comparison between genotypes with 12 weeks CPZ. (D-F) For each sterol species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5 weeks CPZ; + / + 12 weeks CPZ; + / - control; + / - 5 weeks CPZ; + / - 12 weeks CPZ; - / - control; - / - 5 weeks CPZ; and - / - 12 weeks CPZ. [Figure 18F]TREM2 deficiency causes cholesteryl ester accumulation in isolated microglia. Increased (D) cholesteryl ester levels were detected in microglia isolated from Trem2- / - brains on the 12-week cuprizone diet compared to Trem2+ / +, Trem2+ / -, and Trem2- / - microglia on a control diet or 5 weeks cuprizone, and Trem2+ / + and Trem2+ / - microglia on a 12-week cuprizone diet. In general, no changes in lipid levels of cholesteryl esters were detected in astrocyte-enriched cell populations (E) or CSF (F) isolated from control or cuprizone diet Trem2+ / +, Trem2+ / -, and Trem2- / - brains. Lipids were quantified by LC / MS. *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA, Tukey's test, ***: + / + comparison with control, +++: comparison between genotypes with 12 weeks CPZ. (D-F) For each sterol species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5 weeks CPZ; + / + 12 weeks CPZ; + / - control; + / - 5 weeks CPZ; + / - 12 weeks CPZ; - / - control; - / - 5 weeks CPZ; and - / - 12 weeks CPZ. [Figure 18G] TREM2 deficiency leads to cholesteryl ester accumulation in isolated microglia. Concentrations of lipid species, sorted astrocytes and CSF from control, Trem2+ / +, Trem2+ / - and Trem2- / - mouse brains on a CPZ diet for 5 or 12 weeks: microglial BMP. Data represent mean ± SEM and are presented on a log10 scale. Two-way ANOVA; genotype-treatment interaction shown for the indicated lipid species from Trem2- / - on a CPZ diet for 12 weeks, as indicated by asterisks, ***p<0.0001. For each species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5 weeks CPZ; + / + 12 weeks CPZ; + / - control; + / - 5 weeks CPZ; + / - 12 weeks CPZ; - / - control; - / - 5 weeks CPZ; and - / - 12 weeks CPZ. [Figure 18H]TREM2 deficiency leads to cholesteryl ester accumulation in isolated microglia. Concentrations of lipid species, sorted astrocytes and CSF from control, Trem2+ / +, Trem2+ / - and Trem2- / - mouse brains on a CPZ diet for 5 or 12 weeks: Astrocyte BMP. Data represent mean ± SEM and are presented on a log10 scale. Two-way ANOVA; genotype-treatment interaction shown for the indicated lipid species from Trem2- / - on a CPZ diet for 12 weeks, as indicated by asterisks, ***p<0.0001. For each species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5 weeks CPZ; + / + 12 weeks CPZ; + / - control; + / - 5 weeks CPZ; + / - 12 weeks CPZ; - / - control; - / - 5 weeks CPZ; and - / - 12 weeks CPZ. [Figure 18I] TREM2 deficiency leads to cholesteryl ester accumulation in isolated microglia. Concentrations of lipid species, sorted astrocytes and CSF from control, Trem2+ / +, Trem2+ / - and Trem2- / - mouse brains on a CPZ diet for 5 or 12 weeks: CSF sterols. Data represent mean ± SEM and are presented on a log10 scale. Two-way ANOVA; genotype-treatment interaction shown for the indicated lipid species from Trem2- / - on a CPZ diet for 12 weeks, as indicated by asterisks, ***p<0.0001. For each species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5 weeks CPZ; + / + 12 weeks CPZ; + / - control; + / - 5 weeks CPZ; + / - 12 weeks CPZ; - / - control; - / - 5 weeks CPZ; and - / - 12 weeks CPZ. [Figure 18J]TREM2 deficiency causes cholesteryl ester accumulation in isolated microglia. Concentrations of lipid species, sorted astrocytes and CSF from control, Trem2+ / +, Trem2+ / - and Trem2- / - mouse brains on a CPZ diet for 5 or 12 weeks:CSF sulfatides. Data represent mean±SEM and are presented on a log10 scale. Two-way ANOVA; genotype-treatment interaction shown for the indicated lipid species from Trem2- / - on a CPZ diet for 12 weeks as indicated by asterisks, ***p<0.0001. For each species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5 weeks CPZ; + / + 12 weeks CPZ; + / - control; + / - 5 weeks CPZ; + / - 12 weeks CPZ; - / - control; - / - 5 weeks CPZ; and - / - 12 weeks CPZ. [Figure 18K] TREM2 deficiency leads to cholesteryl ester accumulation in isolated microglia. Concentrations of lipid species, sorted astrocytes and CSF from control, Trem2+ / +, Trem2+ / - and Trem2- / - mouse brains on a CPZ diet for 5 or 12 weeks: CSF BMP. Data represent mean ± SEM and are presented on a log10 scale. Two-way ANOVA; genotype-treatment interaction shown for the indicated lipid species from Trem2- / - on a CPZ diet for 12 weeks as indicated by asterisks, ***p<0.0001. For each species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5 weeks CPZ; + / + 12 weeks CPZ; + / - control; + / - 5 weeks CPZ; + / - 12 weeks CPZ; - / - control; - / - 5 weeks CPZ; and - / - 12 weeks CPZ. [Figure 18L]TREM2 deficiency causes cholesteryl ester accumulation in isolated microglia. Concentrations of lipid species from sorted microglia, sorted astrocytes and CSF from control, Trem2+ / +, Trem2+ / - and Trem2- / - mouse brains on a CPZ diet for 5 or 12 weeks: CSF hexosylceramide. Data represent mean ± SEM and are presented on a log10 scale. Two-way ANOVA; genotype-treatment interaction shown for the indicated lipid species from Trem2- / - on a CPZ diet for 12 weeks as indicated by asterisks, ***p<0.0001. For each species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5 weeks CPZ; + / + 12 weeks CPZ; + / - control; + / - 5 weeks CPZ; + / - 12 weeks CPZ; - / - control; - / - 5 weeks CPZ; and - / - 12 weeks CPZ. [Figure 18M] TREM2 deficiency causes cholesteryl ester accumulation in isolated microglia. Concentrations of lipid species, sorted astrocytes and CSF from control, Trem2+ / +, Trem2+ / - and Trem2- / - mouse brains on a CPZ diet for 5 or 12 weeks: CSF ceramide. Data represent mean ± SEM and are presented on a log10 scale. Two-way ANOVA; genotype-treatment interaction shown for the indicated lipid species from Trem2- / - on a CPZ diet for 12 weeks as indicated by asterisks, ***p<0.0001. For each species, data corresponding to the following conditions are shown from left to right: + / + control; + / + 5 weeks CPZ; + / + 12 weeks CPZ; + / - control; + / - 5 weeks CPZ; + / - 12 weeks CPZ; - / - control; - / - 5 weeks CPZ; and - / - 12 weeks CPZ. [Figure 19A]Myelin sulfatides bind TREM2 and promote downstream signaling. Phospho-SYK (pSYK) fold changes of TREM2 / DAP12-expressing stable HEK293 cells stimulated with liposomes consisting of 30% of the indicated test lipid and 70% phosphatidylcholine (PC) were normalized to buffer control (dotted line) and compared to TREM2 agonist antibody and isotype control. N>2 experimental replicates from ≥2 average technical replicates; SM: sphingomyelin, PE: phosphatidylethanolamine, PS: phosphatidylserine, PI: phosphatidylinositol, GalCer: galactosylceramide. *p<0.05, **p<0.01, ***p<0.001, 2-way ANOVA, Sidak test. For each species, Trem2 / DAP12 is shown on the left and DAP12 is shown on the right. [Figure 19B] Myelin sulfatide binds to TREM2 and promotes downstream signaling. Liposome titration curves of pSYK fold change in human macrophage cells from 2-4 donors upon stimulation with the indicated test lipids, normalized to buffer control and compared to TREM2 agonist antibody and isotype control. [Figure 19C] Myelin sulfatide binds to TREM2 and promotes downstream signaling. Liposome stimulation of the indicated test lipids in human macrophage cells from 4-5 donors with liposomes alone (stimulated) or liposomes with 3 μM recombinant TREM2 or TREM1 extracellular domain (ECD) protein normalized to buffer control (dotted line). *p<0.05, two-way ANOVA, Tukey's test. For each species, from left to right, stimulated, Trem2-ECD, and TREM1-ECD conditions are shown. [Figure 19D] Myelin sulfatide binds to TREM2 and promotes downstream signaling. Surface plasmon resonance binding response to increasing concentrations of wild-type (dark shading) and mutant (light shading) R47H hTREM2 protein up to 30% sulfatide / 70% PC. [Figure 19E]Myelin sulfatide binds to TREM2 and promotes downstream signaling. Surface plasmon resonance binding response of wild-type (dark shading) and mutant (light shading) R47H hTREM2 proteins to 30% PS / 70% PC 100 nm liposomes. [Figure 19F] Myelin sulfatide binds to TREM2 and promotes downstream signaling. Intensity of vehicle or pHrodo-myelin (5 μg / mL) phagocytosis in Trem2+ / + and Trem2- / - BMDMs with 5 ng / mL M-CSF. Data represent the mean of three biological replicates ± SEM from three technical replicates, *p<0.05 comparing Trem2+ / + and Trem2- / - areas under the curve under two-tailed tests. [Figure 19G] Myelin sulfatide binds to TREM2 and promotes downstream signaling. Intensity of vehicle or pHrodo-myelin (5 μg / mL) phagocytosis in Trem2+ / + and Trem2- / - BMDMs with 50 ng / mL M-CSF. Data represent three biological replicates ± SEM from three averaged technical replicates. [Figure 19H] Myelin sulfatides bind to TREM2 and promote downstream signaling. Surface plasmon resonance kinetic analysis of (H) hTREM2 vs. (I) hTREM2 R47H mutant protein and (J-K) hTREM2 vs. (K) hTREM2 R47H mutant protein on phosphatidylserine (PS) liposomes (D-E) sulfatide liposomes. ka: association rate constant, kd: dissociation rate constant, KD: dissociation constant, s: seconds, M: moles. [Figure 19I] Myelin sulfatides bind to TREM2 and promote downstream signaling. Surface plasmon resonance kinetic analysis of (H) hTREM2 vs. (I) hTREM2 R47H mutant protein and (J-K) hTREM2 vs. (K) hTREM2 R47H mutant protein on phosphatidylserine (PS) liposomes (D-E) sulfatide liposomes. ka: association rate constant, kd: dissociation rate constant, KD: dissociation constant, s: seconds, M: moles. [Figure 19J]Myelin sulfatides bind to TREM2 and promote downstream signaling. Surface plasmon resonance kinetic analysis of (H) hTREM2 vs. (I) hTREM2 R47H mutant protein and (J-K) hTREM2 vs. (K) hTREM2 R47H mutant protein on phosphatidylserine (PS) liposomes (D-E) sulfatide liposomes. ka: association rate constant, kd: dissociation rate constant, KD: dissociation constant, s: seconds, M: moles. [Figure 19K] Myelin sulfatides bind to TREM2 and promote downstream signaling. Surface plasmon resonance kinetic analysis of (H) hTREM2 vs. (I) hTREM2 R47H mutant protein and (J-K) hTREM2 vs. (K) hTREM2 R47H mutant protein on phosphatidylserine (PS) liposomes (D-E) sulfatide liposomes. ka: association rate constant, kd: dissociation rate constant, KD: dissociation constant, s: seconds, M: moles. [Figure 20A] TREM2 KO BMDMs show sterol accumulation upon myelin treatment. Increased neutral lipid accumulation in Trem2 KO BMDMs (25ug / ml) treated with myelin compared to WT BMDMs as shown by Nile red staining (left). Cells were imaged and Nile red was quantified as total spot area (right). Data represent 5 biological replicates out of 3 averaged technical replicates ± SEM, *p<0.05, one-tailed t-test for comparison of Trem2+ / + with myelin vs. Trem2- / - with myelin. [Figure 20B]TREM2 KO BMDMs show sterol accumulation upon myelin treatment. Cholesteryl esters do not accumulate in the presence of ACAT inhibitor in both WT and TREM2 KO BMDMs administered myelin, indicating that cholesteryl ester accumulation is ACAT dependent. Cholesterol is shown as a control, and cholesterol is slightly elevated in Trem2 KO BMDMs with myelin treatment and ACAT inhibition (C). (B) For each CE species, from left to right, the following conditions are indicated: + / +, - / -, + / + myelin, - / - myelin, + / + myelin / K604, - / - myelin / K604. (C) Bars from left to right indicate the following conditions: + / +, - / -, + / + myelin, - / - myelin, + / + myelin / K604, - / - myelin / K604. [Figure 20C] TREM2 KO BMDMs show sterol accumulation upon myelin treatment. Cholesteryl esters do not accumulate in the presence of ACAT inhibitor in both WT and TREM2 KO BMDMs administered myelin, indicating that cholesteryl ester accumulation is ACAT dependent. Cholesterol is shown as a control, and cholesterol is slightly elevated in Trem2 KO BMDMs with myelin treatment and ACAT inhibition (C). (B) For each CE species, from left to right, the following conditions are indicated: + / +, - / -, + / + myelin, - / - myelin, + / + myelin / K604, - / - myelin / K604. (C) Bars from left to right indicate the following conditions: + / +, - / -, + / + myelin, - / - myelin, + / + myelin / K604, - / - myelin / K604. [Figure 21A-1]TREM2 deficiency-associated cholesteryl ester accumulation is rescued in vitro by ACAT1 inhibitors and LXR agonists. (A-B) Quantification of cholesteryl esters (CE), free cholesterol, triacylglycerol (TG), diacylglycerol (DG), and hexosylceramide (HexCer) (B) from cultured Trem2+ / + and Trem2- / - BMDMs, and sterols from cultured iPSC-derived microglia (iMGs) treated for 48 h with the ACAT1 inhibitor K604 (500 nM) or the LXR agonist GW (10 μM). Differential abundance between experimental groups was identified by fitting the following analysis of variance model for each lipid: log10(abundance)~treatment+genotype+challenge:genotype+batch. Each plot shows the batch-adjusted mean and its 95% confidence interval for each group (N=3 biological replicates per group). Significant baseline differences (main effects) between genotypes are indicated as #p<0.05, ##p<0.01, and ###p<0.001. Significant drug treatment effects were identified within each genotype by paired t-tests on log10 transformed abundance comparing myelin / myelin+inhibitor groups (N=3 biological replicates per condition) and are indicated as *p<0.05 and **p<0.01. (A) For each species, from left to right, vehicle, myelin, and myelin+K604 conditions are shown. (B) For each species, from left to right, vehicle, myelin, myelin+K604, and myelin+GW conditions are shown. [Figure 21A-2]TREM2 deficiency-associated cholesteryl ester accumulation is rescued in vitro by ACAT1 inhibitors and LXR agonists. (A-B) Quantification of cholesteryl esters (CE), free cholesterol, triacylglycerol (TG), diacylglycerol (DG), and hexosylceramide (HexCer) (B) from cultured Trem2+ / + and Trem2- / - BMDMs, and sterols from cultured iPSC-derived microglia (iMGs) treated for 48 h with the ACAT1 inhibitor K604 (500 nM) or the LXR agonist GW (10 μM). Differential abundance between experimental groups was identified by fitting the following analysis of variance model for each lipid: log10(abundance)~treatment+genotype+challenge:genotype+batch. Each plot shows the batch-adjusted mean and its 95% confidence interval for each group (N=3 biological replicates per group). Significant baseline differences (main effects) between genotypes are indicated as #p<0.05, ##p<0.01, and ###p<0.001. Significant drug treatment effects were identified within each genotype by paired t-tests on log10 transformed abundance comparing myelin / myelin+inhibitor groups (N=3 biological replicates per condition) and are indicated as *p<0.05 and **p<0.01. (A) For each species, from left to right, vehicle, myelin, and myelin+K604 conditions are shown. (B) For each species, from left to right, vehicle, myelin, myelin+K604, and myelin+GW conditions are shown. [Figure 21B]TREM2 deficiency-associated cholesteryl ester accumulation is rescued in vitro by ACAT1 inhibitors and LXR agonists. (A-B) Quantification of cholesteryl esters (CE), free cholesterol, triacylglycerol (TG), diacylglycerol (DG), and hexosylceramide (HexCer) (B) from cultured Trem2+ / + and Trem2- / - BMDMs, and sterols from cultured iPSC-derived microglia (iMGs) treated for 48 h with the ACAT1 inhibitor K604 (500 nM) or the LXR agonist GW (10 μM). Differential abundance between experimental groups was identified by fitting the following analysis of variance model for each lipid: log10(abundance)~treatment+genotype+challenge:genotype+batch. Each plot shows the batch-adjusted mean and its 95% confidence interval for each group (N=3 biological replicates per group). Significant baseline differences (main effects) between genotypes are indicated as #p<0.05, ##p<0.01, and ###p<0.001. Significant drug treatment effects were identified within each genotype by paired t-tests on log10 transformed abundance comparing myelin / myelin+inhibitor groups (N=3 biological replicates per condition) and are indicated as *p<0.05 and **p<0.01. (A) For each species, from left to right, vehicle, myelin, and myelin+K604 conditions are shown. (B) For each species, from left to right, vehicle, myelin, myelin+K604, and myelin+GW conditions are shown. [Figure 22A] TREM2 deficiency causes ACAT1-dependent cholesteryl ester accumulation in vitro. Phospho-SYK (pSYK) fold change in TREM2 / DAP12-expressing stable HEK293 cells stimulated with LDL or oxidized LDL (oxLDL) normalized to buffer control (dotted line) and compared to TREM2 agonist antibody and isotype control. ≥2 averaged technical replicates from ≥2 experimental replicates; ***p<0.001 (by two-way ANOVA, Sidak test). For each condition, Trem2 / DAP12 is shown on the left and DAP12 on the right. [Figure 22B]TREM2 deficiency causes ACAT1-dependent cholesteryl ester accumulation in vitro. Liposome titration curves of pSYK fold change in human macrophage cells from 2–4 donors upon oxLDL stimulation, normalized to buffer control (dotted line) and compared to TREM2 agonist antibody and isotype control. [Figure 22C] TREM2 deficiency causes ACAT1-dependent cholesteryl ester accumulation in vitro. oxLDL stimulation of the indicated test lipids in human macrophage cells from 4-5 donors with oxLDL alone (stimulated) or oxLDL with 3 μM or 9 μM recombinant TREM2 or TREM1 extracellular domain (ECD) protein normalized to buffer control (dotted line). For each condition, shown from left to right are stimulated, Trem2-ECD, and Trem1-ECD. [Figure 22D] TREM2 deficiency causes ACAT1-dependent cholesteryl ester accumulation in vitro. Phospho-SYK (pSYK) fold change in Trem2+ / + (left) or Trem2- / - (right) BMDM cells stimulated with oxLDL, normalized to buffer control (dotted line) and compared to TREM2 agonist antibody and isotype control. [Figure 22E] TREM2 deficiency causes ACAT1-dependent cholesteryl ester accumulation in vitro. Quantification of total spot area of ​​Nile Red staining in vehicle or 25ug / ml oxLDL-treated Trem2+ / + and Trem2- / - BMDMs. Data represent seven biological replicates from three averaged technical replicates ± SEM, *p<0.05, one-tailed t-test for comparison of Trem2+ / + with oxLDL vs. Trem2- / - with oxLDL. [Figure 22F] TREM2 deficiency causes ACAT1-dependent cholesteryl ester accumulation in vitro. Mean fluorescence intensity per cell in cultured Trem2+ / + and Trem2- / - BMDMs treated with vehicle or 50 ng / mL oxLDL over a 200 min time course. Data represent three biological replicates ± SEM from three averaged technical replicates. [Figure 22G] TREM2 deficiency causes ACAT1-dependent cholesteryl ester accumulation in vitro. Quantification of cholesteryl esters (CE), free cholesterol, triacylglycerol (TG), and hexosylceramide (HexCer) from cultured Trem2+ / + and Trem2- / - BMDMs treated with vehicle, oxLDL, or oxLDL for 48 h with the ACAT1 inhibitor K604 (500 nM). Differential abundance between experimental groups was identified by fitting the following analysis of variance model for each lipid: log10(abundance)~treatment+genotype+challenge:genotype+batch. Each plot shows the batch-adjusted mean and its 95% confidence interval for each group (N=3 biological replicates per group). Significant baseline differences (main effects) between genotypes are indicated as #p<0.05, ##p<0.01, and ###p<0.001. Significant drug treatment effects were identified within each genotype by paired t-tests on log10 transformed abundance comparing oxLDL / oxLDL+inhibitor groups (N=3 biological replicates per condition) and are indicated as *p<0.05 and **p<0.01. For each species, from left to right, vehicle, oxLDL, and oxLDL+K604 conditions are shown. [Figure 23A] TREM2 KO BMDMs show filipin staining accumulation upon myelin treatment, and anti-TREM2 antibodies reduce filipin staining in human iPSC-derived microglia. Figure 1. Increased endolysosomal free cholesterol accumulation in Trem2 KO BMDMs treated with myelin compared to WT BMDMs, as shown by filipin staining. [Figure 23B] TREM2 KO BMDMs show filipin staining accumulation upon myelin treatment, and anti-TREM2 antibodies reduce filipin staining in human iPSC-derived microglia. Quantification of filipin fluorescence as total spot area is shown. Data are presented as the mean and standard deviation of three technical replicates. [Figure 23C]TREM2 KO BMDMs show filipin staining accumulation with myelin treatment and anti-TREM2 antibody reduces filipin staining in human iPSC-derived microglia. Shown is iPSC-microglia have reduced filipin staining when treated with TREM2 antibody compared to RSV control. Positive control cells were treated with NPC1 inhibitor U18666A at 3ug / mL. Representative image of endolysosomal free cholesterol stained by filipin. [Figure 23D] TREM2 KO BMDMs show filipin staining accumulation upon myelin treatment, and anti-TREM2 antibody reduces filipin staining in human iPSC-derived microglia. Quantification of filipin fluorescence is shown. Data are presented as the mean and standard deviation of one to three technical replicates. [Figure 24] ApoE KO forebrains show cholesteryl ester (CE) accumulation in the presence or absence of chronic demyelination. CE accumulation occurs in wild-type forebrains receiving a 4-week cuprizone diet compared to a normal diet. CE accumulation is exacerbated in ApoE knockout mice on cuprizone compared to a normal diet. Acylphosphatidylserine (acyl-PS) abundance is increased by cuprizone treatment in both wild-type and ApoE knockout mice. *p<0.05, ***p<0.001, two-way ANOVA, Dunnett's post-hoc test, corrected for multiple comparisons. Lipids were quantified by liquid chromatography mass spectrometry (LCMS). Animals were 6 months old with N=8 animals (3 males, 5 females) per group. [Diagram 25] ApoE KO forebrains show accumulation of multiple cholesteryl ester (CE) species in the presence or absence of chronic demyelination. CE species accumulation occurs in wild-type forebrains receiving a 4-week cuprizone diet compared to a normal diet. CE species accumulation is exacerbated in ApoE knockout mice on cuprizone compared to a normal diet. *p<0.05, ***p<0.001, 2-way ANOVA, Dunnett's post-hoc test, corrected for multiple comparisons. Lipids were quantified by LCMS. Animals were 6 months old, N=8 animals per group (3 males, 5 females). [Figure 26] A-C, Increased levels of cholesteryl esters (CE) in microglia, astrocytes, and neurons isolated from ApoE KO mice during chronic demyelination. Most CE species levels are higher in microglia isolated from APOE Ko mice (A) compared to wild-type (WT) brains, and the 12-week cuprizone diet increases levels in both groups compared to the control diet. Astrocytes from ApoE KO mice on the 12-week cuprizone diet (B) show exacerbated CE accumulation compared to ApoE KO mice on the control diet, as well as WT mice on either cuprizone or control diets. Several species of CE, including CE (20:4) and CE (22:6), are increased in neurons sorted from ApoE KO mice on either diet (C) compared to WT mice on either diet. Lipids were quantified by LCMS. Animals were 6 months old with N=6 animals (3 males, 3 females) per group. [Figure 27A] APOE deficiency causes cholesteryl ester accumulation in brain, sorted microglia and astrocytes, and CSF. Heatmap of the top 50 lipids altered by genotype and / or 12-week CPZ treatment in Apoe+ / + and Apoe- / - mouse forebrains, ranked by p-value (one-way ANOVA). [Figure 27B]APOE deficiency causes cholesteryl ester accumulation in brain, sorted microglia and astrocytes, and CSF. Concentrations of free cholesterol, cholesteryl ester (CE), BMP, and triacylglyceride species from forebrain extracts of Apoe+ / + and Apoe- / - mice fed a control or 12-week CPZ diet. For B, data represent mean ± SEM (n=6) and are presented on a log10 scale. Two-way ANOVA, FDR<0.05; genotype effects are indicated by hashtags and genotype-treatment interactions by asterisks. *p<0.05, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001. For each species, from left to right: Apoe+ / + control; Apoe+ / + 12-week CPZ; Apoe- / - control; Apoe- / - 12-week CPZ. [Figure 27C] APOE deficiency causes cholesteryl ester accumulation in brain, sorted microglia and astrocytes, and CSF. Heatmap of the top 50 lipids altered by genotype and / or 12-week CPZ treatment in Apoe+ / + and Apoe- / - sorted microglia, ranked by p-value (one-way ANOVA). [Figure 27D] APOE deficiency causes cholesteryl ester accumulation in brain, sorted microglia and astrocytes, and CSF. Concentrations of free cholesterol, CE, hexosylceramide, ceramide, sulfatide, and ganglioside species from control or Apoe+ / + and Apoe- / - microglia sorted on a 12-week CPZ diet. For D, data represent mean ± SEM (n=6) and are presented on a log10 scale. Two-way ANOVA, FDR<0.05; genotype effects are indicated by hashtags and genotype-treatment interactions by asterisks. *p<0.05, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001. For each species, from left to right: Apoe+ / + control; Apoe+ / + 12 week CPZ; Apoe- / - control; Apoe- / - 12 week CPZ. [Figure 27E]APOE deficiency causes cholesteryl ester accumulation in brain, sorted microglia and astrocytes, and CSF. Heatmap of the top 50 lipids altered by genotype and / or 12-week CPZ treatment in Apoe+ / + and Apoe- / - sorted astrocytes, ranked by p-value (one-way ANOVA). [Figure 27F] APOE deficiency causes cholesteryl ester accumulation in brain, sorted microglia and astrocytes, and CSF. Concentrations of free cholesterol, CE, hexosylceramide, ceramide, sulfatide, and ganglioside species from control or Apoe+ / + and Apoe- / - sorted astrocytes fed a 12-week CPZ diet. For F, data represent mean ± SEM (n=6) and are presented on a log10 scale. Two-way ANOVA, FDR<0.05; genotype effects are indicated by hashtags and genotype-treatment interactions by asterisks. *p<0.05, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001. For each species, from left to right: Apoe+ / + control; Apoe+ / + 12 week CPZ; Apoe- / - control; Apoe- / - 12 week CPZ. [Figure 27G] APOE deficiency causes cholesteryl ester accumulation in brain, sorted microglia and astrocytes, and CSF. Heatmap of the top 50 lipids altered by genotype and / or 12-week CPZ treatment from Apoe+ / + and Apoe- / - CSF, ranked by p-value (one-way ANOVA). [Fig. 27H]APOE deficiency causes cholesteryl ester accumulation in brain, sorted microglia and astrocytes, and CSF. Concentrations of CE, sulfatides, gangliosides, and phosphatidic acid species from Apoe+ / + and Apoe- / - CSF with control or 12-week CPZ diet. For H, data represent mean ± SEM (n=6) and are presented on a log10 scale. Two-way ANOVA, FDR<0.05; genotype effects are indicated by hashtags and genotype-treatment interactions by asterisks. *p<0.05, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001. For each species, from left to right: Apoe+ / + control; Apoe+ / + 12-week CPZ; Apoe- / - control; Apoe- / - 12-week CPZ. [Figure 28A] Increased abundance of cholesteryl esters (CE) in microglia and astrocytes derived from the brains of 5XFAD mice. CE levels are higher in microglia isolated from 5XFAD mice compared to wild type (WT) mice. Animals were 14 months old. N=4 animals / group. [Figure 28B] Increased abundance of cholesteryl ester (CE) in microglia and astrocytes from the brains of 5XFAD mice. CE levels are higher in astrocytes compared to wild-type (WT) mice. Animals were 14 months old. N=4 animals / group. [Figure 29A] Increased inflammatory cytokine production in mouse TREM2 KO BMDMs upon LPS stimulation and myelin treatment. WT and TREM2 KO BMDMs were seeded at 100,000 cells / well in 50ng / mL mCSF before treatment with vehicle or 25ug / mL purified mouse myelin for 48 hours. For the last 16 hours of myelin treatment, cells were stimulated with either 0 or 10ng / mL LPS. Cell culture media was collected and levels of (A) were measured by quantitative immunoassay (Eve Technologies) G-CSF. Data represent mean ± SEM, n=2 technical replicates. [Figure 29B]Increased inflammatory cytokine production in mouse TREM2 KO BMDMs upon LPS stimulation and myelin treatment. WT and TREM2 KO BMDMs were seeded at 100,000 cells / well in 50 ng / mL mCSF before treatment with vehicle or 25 ug / mL purified mouse myelin for 48 hours. For the last 16 hours of myelin treatment, cells were stimulated with either 0 or 10 ng / mL LPS. Cell culture media was collected and (B) levels of INFy were measured by quantitative immunoassay (Eve Technologies). Data represent mean ± SEM, n = 2 technical replicates. [Figure 29C] Increased inflammatory cytokine production in mouse TREM2 KO BMDMs upon LPS stimulation and myelin treatment. WT and TREM2 KO BMDMs were seeded at 100,000 cells / well in 50ng / mL mCSF before treatment with vehicle or 25ug / mL purified mouse myelin for 48 hours. For the final 16 hours of myelin treatment, cells were stimulated with either 0 or 10ng / mL LPS. Cell culture media was collected and levels of IL-12(p40) were measured by quantitative immunoassay (Eve Technologies). Data represent mean ± SEM, n=2 technical replicates. [Figure 29D] Increased inflammatory cytokine production in mouse TREM2 KO BMDMs upon LPS stimulation and myelin treatment. WT and TREM2 KO BMDMs were seeded at 100,000 cells / well in 50ng / mL mCSF before treatment with vehicle or 25ug / mL purified mouse myelin for 48 hours. For the final 16 hours of myelin treatment, cells were stimulated with either 0 or 10ng / mL LPS. Cell culture media was collected and levels of IL-12(p70) were measured by quantitative immunoassay (Eve Technologies). Data represent mean ± SEM, n=2 technical replicates. [Figure 29E]Increased inflammatory cytokine production in mouse TREM2 KO BMDMs upon LPS stimulation and myelin treatment. WT and TREM2 KO BMDMs were seeded at 100,000 cells / well in 50ng / mL mCSF before treatment with vehicle or 25ug / mL purified mouse myelin for 48 hours. For the final 16 hours of myelin treatment, cells were stimulated with either 0 or 10ng / mL LPS. Cell culture media was collected and levels of LIX (CXCL5) were measured by quantitative immunoassay (Eve Technologies). Data represent mean ± SEM, n=2 technical replicates. [Figure 29F] Increased inflammatory cytokine production in mouse TREM2 KO BMDMs upon LPS stimulation and myelin treatment. WT and TREM2 KO BMDMs were seeded at 100,000 cells / well in 50ng / mL mCSF before treatment with vehicle or 25ug / mL purified mouse myelin for 48 hours. For the final 16 hours of myelin treatment, cells were stimulated with either 0 or 10ng / mL LPS. Cell culture media was collected and levels of MCP-1 (CCL2) were measured by quantitative immunoassay (Eve Technologies). Data represent mean ± SEM, n=2 technical replicates. [Figure 29G] Increased inflammatory cytokine production in mouse TREM2 KO BMDMs upon LPS stimulation and myelin treatment. WT and TREM2 KO BMDMs were seeded at 100,000 cells / well in 50ng / mL mCSF before treatment with vehicle or 25ug / mL purified mouse myelin for 48 hours. For the final 16 hours of myelin treatment, cells were stimulated with either 0 or 10ng / mL LPS. Cell culture media was collected and levels of MIG (CXCL9) were measured by quantitative immunoassay (Eve Technologies). Data represent mean ± SEM, n=2 technical replicates. [Figure 29H]Increased inflammatory cytokine production in mouse TREM2 KO BMDMs upon LPS stimulation and myelin treatment. WT and TREM2 KO BMDMs were seeded at 100,000 cells / well in 50ng / mL mCSF before treatment with vehicle or 25ug / mL purified mouse myelin for 48 hours. For the last 16 hours of myelin treatment, cells were stimulated with either 0 or 10ng / mL LPS. Cell culture media was collected and levels of IL-1a were measured by quantitative immunoassay (Eve Technologies). Data represent mean ± SEM, n=2 technical replicates. [Figure 29I] Increased inflammatory cytokine production in mouse TREM2 KO BMDMs upon LPS stimulation and myelin treatment. WT and TREM2 KO BMDMs were seeded at 100,000 cells / well in 50ng / mL mCSF before treatment with vehicle or 25ug / mL purified mouse myelin for 48 hours. For the final 16 hours of myelin treatment, cells were stimulated with either 0 or 10ng / mL LPS. Cell culture media was collected and levels of IL-1b were measured by quantitative immunoassay (Eve Technologies). Data represent mean ± SEM, n=2 technical replicates. [Figure 30A]Increased IL-1β cytokine response in human iPSC-derived TREM2 KO microglia and attenuated IL-1β mRNA response by anti-TREM2 antibody. (A) WT and TREM2 KO iPSC-derived microglia were treated with LPS / ATP (1ug / ml and 5mM, respectively) for 4 hours to stimulate NLRP3 inflammasome activation, after which cell culture medium was collected and levels of IL-1β were measured by quantitative immunoassay (Eve Technologies). **p<0.01; two-way ANOVA with Tukey post-hoc test, corrected for multiple comparisons. Data represent mean ± SEM, N=4 biological replicates. The following conditions within each group are shown: control on the left, LPS+ATP in the middle, and LPS+ATP+Caspase-1 inhibitor VX-765 (InvivoGen) on the right. (B) iPSC-microglia were treated with 25ug / mL myelin for 24 hours, then treated with control antibody (anti-RSV) or anti-TREM2 antibody for 48 hours. IL-1β mRNA levels were measured by QPCR and normalized to GAPDH. N=2 biological replicates. n=2 technical replicates. [Figure 30B]Increased IL-1β cytokine response in human iPSC-derived TREM2 KO microglia and attenuated IL-1β mRNA response by anti-TREM2 antibody. (A) WT and TREM2 KO iPSC-derived microglia were treated with LPS / ATP (1ug / ml and 5mM, respectively) for 4 hours to stimulate NLRP3 inflammasome activation, after which cell culture medium was collected and levels of IL-1β were measured by quantitative immunoassay (Eve Technologies). **p<0.01; two-way ANOVA with Tukey post-hoc test, corrected for multiple comparisons. Data represent mean ± SEM, N=4 biological replicates. The following conditions within each group are shown: control on the left, LPS+ATP in the middle, and LPS+ATP+Caspase-1 inhibitor VX-765 (InvivoGen) on the right. (B) iPSC-microglia were treated with 25ug / mL myelin for 24 hours, then treated with control antibody (anti-RSV) or anti-TREM2 antibody for 48 hours. IL-1β mRNA levels were measured by QPCR and normalized to GAPDH. N=2 biological replicates. [Figure 31A] TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have higher levels of ABCA1 mRNA compared to TREM2 WT iMG both at baseline (vehicle treatment) and upon treatment with 25ug / mL purified myelin for 24 hours. [Figure 31B] TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have higher levels of ABCA7 mRNA compared to TREM2 WT iMG both at baseline (vehicle treatment) and upon treatment with 25ug / mL purified myelin for 24 hours. [Figure 31C] TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have higher levels of ABCG1 mRNA compared to TREM2 WT iMG both at baseline (vehicle treatment) and upon treatment with 25ug / mL purified myelin for 24 hours. [Figure 31D] TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have lower levels of APOC1 mRNA compared to TREM2 WT iMG. N=4 biological replicates. Data are presented as mean and SEM. [Figure 31E] TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have lower levels of APOE mRNA compared to TREM2 WT iMG. N=4 biological replicates. Data are presented as mean and SEM. [Fig. 31F] TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have lower levels of CH25H mRNA compared to TREM2 WT iMG. N=4 biological replicates. Data are presented as mean and SEM. [Figure 31G]TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have lower levels of FABP3 mRNA compared to TREM2 WT iMG. N=4 biological replicates. Data are presented as mean and SEM. [Fig. 31H] TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have lower levels of FABP5 mRNA compared to TREM2 WT iMG. N=4 biological replicates. Data are presented as mean and SEM. [Fig. 31I] TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have lower levels of LPL mRNA compared to TREM2 WT iMG. N=4 biological replicates. Data are presented as mean and SEM. [Figure 31J] TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have lower levels of OLR1 mRNA compared to TREM2 WT iMG. N=4 biological replicates. Data are presented as mean and SEM. [Figure 31K]TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have higher levels of LDLR mRNA compared to TREM2 WT iMG both at baseline (vehicle treatment) and upon treatment with 25ug / mL purified myelin for 24 hours. [Figure 31L] TREM2 KO human iPSC-derived microglia (iMG) show differential regulation of lipid metabolism genes at baseline and upon treatment with myelin compared to TREM2 WT iMG. TREM2 KO iMG have lower levels of LIPA mRNA compared to TREM2 WT iMG. N=4 biological replicates. Data are presented as mean and SEM. [Diagram 32] A-B, Treatment with 25ug / mL purified myelin for 48 hours increases secreted APOE (A) and APOC1 (B) protein in both TREM2 KO and TREM2 WT human iMGs derived microglia (iMGs). Secreted APOE (A) and APOC1 (B) levels are decreased in TREM2 KO iMGs under both vehicle and myelin treatment conditions compared to TREM2 WT iMGs. N=3 technical replicates. Data are presented as median and interquartile range. For each genotype group, "vehicle" is shown on the left and "myelin" is shown on the right. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] As described herein, it has been determined that a reduction in the functional level of TREM2 results in dysregulation of lipid metabolism. When the equivalent dysregulation is induced in cells expressing TREM2 upon lipid challenge, it can be improved with agonistic anti-TREM2 antibodies. Similarly, lipid dysregulation can be improved in cells with reduced TREM2 activity by treating with ACAT1 inhibitors, apolipoprotein E (ApoE), RXR agonists, LXR agonists, or combinations thereof (see Examples). RXR and LXR agonists as well as ACAT1 inhibitors have been used to improve lipid clearance, but their mechanisms of action target various cell types and may result in undesirable side effects. In contrast, TREM2 expression is restricted to cells of myeloid lineage (e.g., microglia, dendritic cells, and macrophages). Thus, agonistic anti-TREM2 antibodies can be used as a more targeted approach to promote lipid clearance in various conditions. For example, a wide range of diseases and disorders, including certain neurodegenerative disorders (e.g., Alzheimer's disease), atherosclerosis, diseases associated with metabolic syndrome, and certain lysosomal storage diseases (e.g., Niemann-Pick disease type C (NPC)), are associated with dysregulated lipid metabolism in myeloid lineages. Thus, as described herein, agonistic anti-TREM2 antibodies may be used to treat dysregulated lipid metabolism in mammals having such conditions.

[0020] Furthermore, it has been shown that a decrease in the functional level of TREM2 is proinflammatory (e.g., leading to upregulation of proinflammatory cytokines, including IL-1β, a cytokine in the inflammatory pathway). Conversely, stimulating TREM2 with an antibody attenuates such inflammation (e.g., reduces inflammasome responses). Thus, various diseases and disorders associated with inflammation and inflammasome responses can also be treated with agonistic anti-TREM2 antibodies.

[0021] The TREM2 gene encodes a single-pass transmembrane protein that is a member of the immunoglobulin (Ig) receptor superfamily. TREM2 was originally cloned as a cDNA encoding a TREM1 homolog (Bouchon, A et al., J Exp Med, 2001.194(8):p.1111-22). The receptor is a glycoprotein of approximately 40 kDa that is reduced to 26 kDa after N-deglycosylation. The TREM2 gene encodes a 230 amino acid long protein that includes an extracellular domain, a transmembrane region, and a short cytoplasmic tail (UniProtKB Q9NZC2; NCBI Reference Sequence: NP_061838.1). The extracellular region encoded by exon 2 is composed of a single V-type Ig-SF domain that contains three potential N-glycosylation sites. The putative transmembrane region contains a charged lysine residue. The cytoplasmic tail of TREM2 lacks signaling motifs and is thought to signal through the signaling adaptor molecules DAP12 / TYROBP and through DAP10. TREM2 is found on the surface of osteoclasts, immature dendritic cells, and macrophages. In the central nervous system, TREM2 is expressed exclusively in microglia.

[0022] Accordingly, certain embodiments disclosed herein provide a method for treating dysregulation of lipid metabolism and / or inflammation in a mammal (e.g., a human) in need of such treatment, comprising administering to the mammal an effective amount of an agonist anti-TREM2 antibody.

[0023] In certain embodiments, such methods may be used to treat lipid metabolism dysregulation. In certain embodiments, such methods may be used to treat inflammation (e.g., inflammation associated with lipid metabolism dysregulation). In certain embodiments, such methods may be used to treat both lipid metabolism dysregulation and inflammation.

[0024] In certain embodiments, cells expressing TREM2 in a mammal exhibit lipid metabolism dysregulation. In certain embodiments, the cells are microglial cells. In certain embodiments, the cells are macrophages.

[0025] As used herein, the term "dysregulated lipid metabolism" refers to altered lipid metabolism in a cell / mammal compared to a control (e.g., compared to a healthy control mammal or control animal that does not have dysregulated lipid metabolism, reduced TREM2 activity, reduced ApoE activity, or APOE ε4 allele). For example, dysregulated lipid metabolism can include altered levels (e.g., through increased formation or decreased degradation) or altered localization / storage of one or more classes or species of lipid. In certain embodiments, dysregulated lipid metabolism includes increased accumulation (e.g., intracellular or extracellular accumulation) of one or more classes or species of lipid compared to a control.

[0026] In certain embodiments, lipid metabolism dysregulation comprises the increase in intracellular accumulation of one or more lipids.In certain embodiments, one or more lipids accumulate intracellularly in microglial cells.In certain embodiments, one or more lipids accumulate intracellularly in macrophages.In certain embodiments, one or more lipids do not accumulate intracellularly in astrocytes.

[0027] In certain embodiments, lipid metabolism dysregulation comprises increased extracellular accumulation of one or more lipids.

[0028] In certain embodiments, the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, bis(monoacylglycero)phosphate species (BMPs), diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, sphingomyelin (e.g., SMd18:1 / 18:0), phosphatidylglycerol (e.g., PG d16:0 / 18:1), phosphatidylethanolamine (e.g., PE38:6), and combinations thereof. In certain embodiments, the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, bis(monoacylglycero)phosphate species (BMPs), diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, and combinations thereof. In certain embodiments, the one or more lipids include those lipids described herein, such as in the figures or examples.

[0029] In certain embodiments, the one or more lipids comprise a cholesteryl ester.

[0030] In certain embodiments, the one or more lipids comprise an oxidative metabolite of a cholesteryl ester (e.g., CE oxoODE, CEHODE, CE HpODE, CE oxoHETE, or CE HETE).

[0031] In certain embodiments, agonistic anti-TREM2 antibodies reduce lipid accumulation (e.g., intracellular or extracellular accumulation). In certain embodiments, agonistic anti-TREM2 antibodies reduce cholesteryl ester accumulation.

[0032] In certain embodiments, lipid accumulation is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% compared to a control. Lipid accumulation levels in cells can be established by assessing a sample (e.g., a sample comprising one or more cells) using an assay described herein or known in the art.

[0033] In certain embodiments, the cells expressing TREM2 in mammals exhibit inflammatory or proinflammatory responses, such as upregulation of proinflammatory cytokines.In certain embodiments, inflammasomes are upregulated in the cells expressing TREM2.In certain embodiments, the cells are microglial cells.In certain embodiments, the cells are macrophages.

[0034] In certain embodiments, the expression of at least one proinflammatory cytokine is upregulated in a mammal (e.g., in a TREM2-expressing cell, such as a macrophage or microglial cell). In certain embodiments, the at least one cytokine is selected from the group consisting of G-CSF, INFy, IL-12(p40), IL-12(p70), LIX(CXCL5), MCP-1(CCL2), MIG(CXCL9), IL-1α, IL-1β, and IL-18. In certain embodiments, the at least one cytokine is associated with the inflammasome pathway (e.g., IL-1β or IL-18). In certain embodiments, the at least one cytokine is IL-1β.

[0035] In certain embodiments, the agonistic anti-TREM2 antibody reduces a proinflammatory response (e.g., an inflammasome response) in a mammal. For example, in certain embodiments, the expression of at least one proinflammatory cytokine is reduced (e.g., compared to a control, such as a corresponding mammal that was not administered the agonistic anti-TREM2 antibody). In certain embodiments, the at least one cytokine is selected from the group consisting of G-CSF, INFy, IL-12(p40), IL-12(p70), LIX(CXCL5), MCP-1(CCL2), MIG(CXCL9), IL-1α, IL-1β, and IL-18. In certain embodiments, the at least one cytokine is associated with the inflammasome pathway (e.g., IL-1β or IL-18). In certain embodiments, the at least one cytokine is IL-1β. In certain embodiments, the expression of at least one cytokine is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% compared to a control. Cytokine expression levels in a cell / mammal can be established by evaluating a sample (e.g., a sample comprising one or more cells) using an assay described herein or known in the art. For example, the assay can evaluate RNA (e.g., mRNA) or protein expression levels (e.g., compared to a control).

[0036] Certain embodiments described herein also include a method of treating lipid metabolism dysregulation in a patient in need thereof, comprising: 1) obtaining or having obtained a biological specimen from a patient; and 2) analyzing the biological sample or analyzing the sample to detect the presence of lipid metabolism dysregulation, thereby diagnosing the patient as having lipid metabolism dysregulation; 3) administering to the diagnosed patient an effective amount of an agonist anti-TREM2 antibody.

[0037] Certain embodiments described herein also provide a method of treating a patient with an agonist anti-TREM2 antibody, the method comprising: 1) obtaining or having obtained a biological specimen from a patient; and 2) analyzing the biological sample or analyzing the sample to detect the presence of lipid metabolism dysregulation, thereby diagnosing the patient as having lipid metabolism dysregulation; 3) administering to the diagnosed patient an effective amount of an agonist anti-TREM2 antibody.

[0038] Decreased TREM2 activity, decreased ApoE activity and ApoE4 expression In certain embodiments, the mammal treated using the methods described herein has, or has been determined to have, normal TREM2 activity (eg, compared to a healthy control subject).

[0039] In certain other embodiments, the mammal treated using the methods described herein has or has been determined to have reduced TREM2 activity. As used herein, the term "reduced TREM2 activity" refers to a cell or a mammal containing a cell that has reduced TREM2 function compared to a control cell / mammal (e.g., a corresponding cell from a healthy subject). In certain embodiments, the reduced level of functional protein can be due to reduced expression of TREM2 (e.g., inhibition of transcription, inhibition of RNA maturation, inhibition of RNA translation, altered post-translational modification, or increased degradation of RNA or protein) or reduced cell surface levels of TREM2 protein. In certain embodiments, the reduced level of functional TREM2 is caused by loss or partial loss of function gene mutations in the TREM2 gene (e.g., R47H, R62H, H157Y, Q33X, T66M, or Y38C). In certain embodiments, the reduced level of functional TREM2 is caused by reduced TREM2 protein levels. In certain embodiments, reduced levels of functional TREM2 are caused by increased cleavage of the receptor by disintegrin and metalloproteinase (ADAM) proteases (e.g., ADAM10 and ADAM17), which releases soluble TREM2 (sTREM2) into the extracellular environment. In certain embodiments, reduced TREM2 activity includes reduced signal transduction.

[0040] The presence of reduced TREM2 activity in a cell / mammal can be established by evaluating a sample (e.g., a sample containing one or more cells) using an assay described herein or known in the art. For example, the assay may evaluate RNA or protein expression levels, cell surface TREM2 protein levels, or may examine TREM2 activity (e.g., signal transduction) (e.g., compared to a control). In other embodiments, the assay may measure levels of sTREM2 (e.g., compared to a control). Other functional measures of TREM2 activity, such as reduced pSyk activity or class I PI 3 kinase activity, compared to control cells, may be used to identify cells or mammals with reduced TREM2 activity.

[0041] In certain embodiments, the level of functional TREM2 in the sample is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% compared to a control. In certain embodiments, the cell / mammal does not express functional TREM2.

[0042] In certain embodiments, the mammal has altered expression of one or more additional genes, such as genes associated with lysosomal function or lipid metabolism (eg, genes described herein).

[0043] ApoE is the major cholesterol carrier that supports lipid transport and injury repair in the brain. In peripheral tissues, ApoE is mainly produced by the liver and macrophages and mediates cholesterol metabolism in an isoform-dependent manner. The human APOE gene exists as three polymorphic alleles (ε2, ε3, and ε4) that code for ApoE2, ApoE3, and ApoE4 (genomic coordinates (GRCh38): 19: 44,905,748-44,909,394; see UniProtKB P02649). ApoE consists of 299 amino acids and has a molecular weight of approximately 34 kDa. The differences between the three ApoE isoforms are restricted to amino acids 112 and 158, where either a cysteine ​​or an arginine is present: ApoE2 (Cys112, Cys158), ApoE3 (Cys112, Arg158) and ApoE4 (Arg112, Arg158). The single amino acid difference at these two positions affects the structure of the ApoE isoforms and influences their ability to bind lipids, receptors and Aβ.

[0044] In certain embodiments, the mammal treated using the methods described herein has, or has been determined to have, normal ApoE activity (eg, compared to a healthy control subject).

[0045] In certain embodiments, the mammal treated using the methods described herein has or has been determined to have reduced ApoE activity. As used herein, the term "reduced ApoE activity" refers to a cell or a mammal containing a cell that has reduced ApoE function compared to a control cell / mammal (e.g., a corresponding cell from a healthy subject). In certain embodiments, the reduced level of functional protein may be due to reduced expression of ApoE (e.g., inhibition of transcription, inhibition of RNA maturation, inhibition of RNA translation, altered post-translational modification, or increased RNA or protein degradation). In certain embodiments, the reduced level of functional ApoE is caused by loss or partial loss of function gene mutation or coding variant in the APOE gene. In certain embodiments, the reduced level of functional ApoE is caused by reduced ApoE protein level. In certain embodiments, the reduced level of functional ApoE is caused by reduced ApoE secretion. In certain embodiments, the reduced level of functional ApoE is caused by reduced intracellular or extracellular ApoE transport. In certain embodiments, the reduction in the level of functional ApoE is caused by abnormal cellular transport, including reduced recycling to plasma membrane, reduced retrograde transport from endolysosome to Golgi complex, and reduced transport along biosynthetic pathways.In certain embodiments, the reduction in the level of functional ApoE is caused by reduced transport of ApoE cargo, such as lipids.In certain embodiments, the reduction in the level of functional ApoE is caused by reduced efflux of cellular lipids.In certain embodiments, the reduction in the level of functional ApoE is caused by reduced antioxidant properties.

[0046] The presence of reduced ApoE activity in a cell / mammal can be established by evaluating a sample (e.g., a sample containing one or more cells) using an assay described herein or known in the art. For example, the assay may evaluate RNA or protein expression levels, or may examine ApoE activity (e.g., compared to a control).

[0047] In certain embodiments, the level of functional ApoE in the sample is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% compared to the control. In certain embodiments, the cell / mammal does not express functional ApoE.

[0048] In certain embodiments, the mammal treated using the methods described herein does not have, or has been determined to not have, the APOE ε4 allele.

[0049] In certain embodiments, the mammal treated using the methods described herein has or has been determined to have the APOE ε4 allele. In certain embodiments, the mammal is heterozygous for the APOE ε4 allele. In certain embodiments, the mammal is homozygous for the APOE ε4 allele. The APOE ε4 allele can be detected in a sample (i.e., a sample comprising one or more cells from a mammal) using an assay described herein or known in the art. In certain embodiments, the assay is a genotyping assay, such as a sequencing assay.

[0050] As described herein, mammals expressing ApoE4 may have lipid metabolism dysregulation and / or inflammation that may be treated with agonistic anti-TREM2 antibodies. Thus, in certain embodiments, mammals having the APOE ε4 allele may be treated using the methods described herein.

[0051] Therefore, certain embodiments disclosed herein also provide a method for treating lipid metabolism dysregulation in a mammal in need thereof, comprising administering to the mammal an effective amount of an agonist anti-TREM2 antibody, wherein the mammal has or is determined to have reduced TREM2 activity, reduced ApoE activity, and / or an APOE ε4 allele. In certain embodiments, the mammal has or is determined to have reduced TREM2 activity. In certain embodiments, the mammal has or is determined to have reduced ApoE activity. In certain embodiments, the mammal has or is determined to have an APOE ε4 allele.

[0052] Certain embodiments also provide a method for treating inflammation in a mammal in need of such treatment, comprising administering to the mammal an effective amount of an agonist anti-TREM2 antibody, wherein the mammal has, or has been determined to have, reduced TREM2 activity.

[0053] Certain embodiments disclosed herein include a method of treating lipid metabolism dysregulation in a patient in need thereof, comprising: 1) obtaining or having obtained a biological specimen from a patient; and 2) detecting or having detected reduced TREM2 activity, reduced ApoE activity, or the APOE ε4 allele in the sample; 3) diagnosing the patient as having lipid metabolism dysregulation when reduced TREM2 activity, reduced ApoE activity, or the APOE ε4 allele is detected; 4) administering to the diagnosed patient an effective amount of an agonistic anti-TREM2 antibody.

[0054] In certain embodiments, the method comprises diagnosing the patient as having dysregulated lipid metabolism when decreased TREM2 activity is detected. In certain embodiments, the method comprises diagnosing the patient as having dysregulated lipid metabolism when decreased ApoE activity is detected. In certain embodiments, the method comprises diagnosing the patient as having dysregulated lipid metabolism when APOE ε4 allele is detected.

[0055] Certain embodiments disclosed herein provide a method of treating a patient with an agonist anti-TREM2 antibody, the method comprising: 1) obtaining or having obtained a biological specimen from a patient; and 2) analyzing the biological sample or having the sample analyzed to detect reduced TREM2 activity, reduced ApoE activity, or the APOE ε4 allele, thereby diagnosing the patient as having a lipid metabolism dysregulation; 4) administering to the diagnosed patient an effective amount of an agonist anti-TREM2 antibody.

[0056] In certain embodiments, the method comprises analyzing biological sample or analyzing sample to detect reduced TREM2 activity.In certain embodiments, the method comprises analyzing biological sample or analyzing sample to detect reduced ApoE activity.In certain embodiments, the method comprises analyzing biological sample or analyzing sample to detect APOE ε4 allele.

[0057] TREM2-expressing cells As described herein, reduced TREM2 activity has been specifically shown to cause dysregulation of lipid metabolism in certain cell types (e.g., microglial cells and macrophages) but not in certain other cell types (e.g., astrocytes) (see Examples). Reduction of functional TREM2 has also been shown to cause an increase in proinflammatory responses.

[0058] Thus, certain embodiments disclosed herein provide a method for reducing intracellular accumulation of one or more lipids in a cell, comprising contacting the cell with an effective amount of an agonist anti-TREM2 antibody. Certain embodiments also provide a method for reducing expression of at least one proinflammatory cytokine in a cell, comprising contacting the cell with an effective amount of an agonist anti-TREM2 antibody.

[0059] In certain embodiments, the cell expresses TREM2. In certain embodiments, the cell is a microglial cell. In certain embodiments, the cell is a macrophage.

[0060] In certain embodiments, the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, bis(monoacylglycero)phosphate species (BMPs), diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, sphingomyelin (e.g., SMd18:1 / 18:0), phosphatidylglycerol (e.g., PG d16:0 / 18:1), phosphatidylethanolamine (e.g., PE38:6), and combinations thereof. In certain embodiments, the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, BMPs, diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, and combinations thereof. In certain embodiments, the one or more lipids comprise cholesteryl esters. In certain embodiments, the one or more lipids comprise lipids described herein.

[0061] In certain embodiments, lipid accumulation is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% compared to a control.

[0062] In certain embodiments, the at least one cytokine is selected from the group consisting of G-CSF, INFy, IL-12(p40), IL-12(p70), LIX(CXCL5), MCP-1(CCL2), MIG(CXCL9), IL-1α, IL-1β, and IL-18. In certain embodiments, the at least one cytokine is associated with the inflammasome pathway (e.g., IL-1β or IL-18). In certain embodiments, the at least one cytokine is IL-1β.

[0063] In certain embodiments, expression of at least one cytokine is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% compared to a control (e.g., corresponding control cells that were not administered the agonistic anti-TREM2 antibody).

[0064] In certain embodiments, the cells have, or have been determined to have, reduced TREM2 activity, hi other embodiments, the cells have normal TREM2 activity.

[0065] In certain embodiments, the cells have or have been determined to have reduced ApoE activity (e.g., the cells have an APOE loss or partial loss of function mutation or coding variant). In other embodiments, the cells have normal ApoE activity.

[0066] In certain embodiments, the cells express or have been determined to express ApoE4. In certain other embodiments, the cells do not express or have been determined to not express ApoE4.

[0067] In certain embodiments, the cells are contacted with an agonistic anti-TREM2 antibody described herein (eg, an agonistic anti-TREM2 antibody described herein, such as MAB17291 or 78.18).

[0068] In certain embodiments, the cells are contacted with the agonist anti-TREM2 antibody in vitro, in vivo, or ex vivo. In certain embodiments, the cells are contacted with the agonist anti-TREM2 antibody in vitro. In certain embodiments, the cells are contacted with the agonist anti-TREM2 antibody in vivo. In certain embodiments, the cells are contacted with the agonist anti-TREM2 antibody ex vivo.

[0069] In certain embodiments, the cells are present in a mammal and are contacted with the agonist anti-TREM2 antibody in vivo. In such embodiments, the cells may be contacted through administration of the antibody. In certain embodiments, the administration is systemic.

[0070] In certain embodiments, the mammal has inflammation associated with intracellular lipid accumulation. In certain embodiments, the agonist anti-TREM2 antibody reduces expression of at least one proinflammatory cytokine (e.g., a proinflammatory cytokine described herein, such as G-CSF, INFy, IL-12(p40), IL-12(p70), LIX(CXCL5), MCP-1(CCL2), MIG(CXCL9), IL-1alpha, IL-1beta, or IL-18). In certain embodiments, the at least one cytokine is IL-1β.

[0071] In certain embodiments, the mammal has or is susceptible to developing a disease or condition described herein.

[0072] Treating a specific disease or condition As described in the Examples, loss of TREM2 function in macrophages and microglia results in an inability to process and metabolize lipids (e.g., cholesterol, cholesteryl esters (CE), triglycerides, and sphingolipids). Furthermore, accumulation of these lipids has been shown to lead to proinflammatory responses (e.g., upregulation of proinflammatory cytokines, including IL-1β, a cytokine in the inflammasome pathway). Conversely, stimulating TREM2 with antibodies reduces lipid load and attenuates inflammation. Thus, as described herein, agonistic anti-TREM2 antibodies may be used to correct lipid dysregulation and inflammatory responses in macrophages, microglia, or other cell types expressing TREM2, and treat associated diseases and disorders.

[0073] For example, increased lipid loading and associated inflammation in microglial cells are key features of various neurodegenerative diseases, including Alzheimer's disease (AD). CE is known to accumulate in the brains of AD patients and AD mouse models (Astarita, et al. (2011). PLoS One 6, e24777; Chan, et al. (2012). J Biol Chem 287, 2678-2688; Morel, et al. (2013). Nat Commun 4, 2250; Shibuya, et al. (2015). Future Med Chem 7, 2451-2467) and LOAD-binding TREM2 variants result in partial loss of function (Ulland, TK, and Colonna, M. (2018). Nat Rev Neurol 14, 667-675). Thus, in light of the results described herein, enhancing TREM2 function may be beneficial in AD, in part by promoting lipid clearance in microglia. Similarly, agonistic anti-TREM2 antibodies may also be useful to reduce lipid burden and inflammation in other neurodegenerative disorders characterized by these pathologies. Such diseases include, but are not limited to, Nasu-Hakola disease (NHD), Lewy body disease, Parkinson's disease, retinal degeneration (e.g., macular degeneration), Huntington's disease, frontotemporal lobar degeneration (FTD), and amyotrophic lateral sclerosis (ALS).

[0074] As described in the Examples, TREM2 also plays a role in regulating lysosomal cholesterol, and agonistic anti-TREM2 antibodies were shown to reduce the accumulation of endolysosomal free cholesterol in cells with reduced TREM2 expression. Thus, agonizing TREM2 may be useful in treating certain lysosomal storage disorders associated with cholesterol accumulation, such as Niemann-Pick disease (types A, B, or C).

[0075] In addition, TREM2 has been shown to be involved in the control of microglial gene expression and cholesterol transport during chronic myelin phagocytosis, a program that, if not properly executed, leads to widespread neuronal damage in the brain (see Examples). These results indicate that increasing TREM2 activity may be neuroprotective (e.g., for aging) and may stimulate remyelination in certain neurodegenerative diseases, such as multiple sclerosis and vanishing white matter disease.

[0076] TREM2 is also expressed on a subset of macrophages outside the CNS (e.g., in adipose tissue, liver, skeletal muscle, and atherosclerotic lesions in arteries). TREM2 modulation can be used to alter lipid metabolism and inflammatory responses in these tissues to treat various related diseases. For example, metabolic syndrome, which includes a range of conditions such as obesity, type 2 diabetes, atherosclerosis, alcoholic and nonalcoholic fatty liver disease, and alcoholic and nonalcoholic steatohepatitis, is typically associated with low-grade chronic (i.e., unresolved) inflammation in various tissues, including adipose tissue, liver, and skeletal muscle. Myeloid cells, such as monocytes and macrophages, are the primary mediators of these inflammatory responses, which can ultimately lead to insulin resistance, glucose intolerance, and atherosclerosis. Central to the inflammatory response in these tissues is the interaction of myeloid cells with other fat-containing cells, such as adipocytes or hepatocytes, as well as the degree of lipid dysregulation in the myeloid cells themselves. Therefore, regulating lipid metabolism / inflammation with TREM2 agonists may be useful for treating metabolic syndrome and conditions related to metabolic syndrome. Furthermore, rheumatoid arthritis (RA) is an autoimmune disease that causes chronic inflammation of the joints and is also associated with lipid dysregulation. These lipid abnormalities increase the risk of developing various cardiovascular diseases. Therefore, regulating lipid metabolism / inflammation with TREM2 agonists may be useful for treating RA.

[0077] The reduction of functional TREM2 also results in the upregulation of various proinflammatory cytokines, including IL-1β inflammasome pathway-related cytokines. As described in the Examples, this inflammasome response is reduced by agonistic anti-TREM2 antibodies, demonstrating their anti-inflammatory effects and their usefulness in treating diseases and conditions associated with inflammation (e.g., inflammasome-related diseases and disorders). For example, administration of anti-TREM2 antibodies can be useful in treating diseases such as RA, gout, and certain intestinal conditions (e.g., inflammatory bowel disease (IBD)).

[0078] Thus, in certain embodiments, the methods disclosed herein may be used to treat a mammal having or susceptible to Alzheimer's disease, NHD, Lewy body disease, Parkinson's disease, retinal degeneration (e.g., macular degeneration), FTD, ALS, or Huntington's disease. The methods disclosed herein may be used to treat obesity, type 2 diabetes, alcoholic and nonalcoholic steatohepatitis, alcoholic and nonalcoholic fatty liver disease, atherosclerosis, and other diseases associated with metabolic syndrome. In certain embodiments, the methods described herein are not used to treat nonalcoholic steatohepatitis. In certain embodiments, the methods described herein may be used to treat lysosomal storage disorders, such as Niemann-Pick disease types A, B, or C. In certain other embodiments, the methods described herein may be used to treat diseases associated with demyelination (e.g., multiple sclerosis or vanishing white matter disease). The methods disclosed herein may be used to treat a mammal having or susceptible to inflammation or inflammation-related diseases or disorders, such as inflammasome-related diseases and disorders. In certain embodiments, the methods described herein may be used to treat rheumatoid arthritis (RA), gout, and certain bowel diseases (e.g., inflammatory bowel disease (IBD)). The methods disclosed herein may also be used to treat aging or the effects associated with aging. In certain embodiments, such methods reduce cellular senescence and / or improve cellular function / activity. In certain embodiments, such methods extend the lifespan of a cell.

[0079] Treating lipid dysregulation and / or inflammation in a mammal having one or more of these conditions can alter the natural history of the disease (e.g., by preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, ameliorating or palliating the disease state, or improving remission or prognosis).

[0080] As used herein, the term "predisposed" refers to a mammal that is at increased risk of developing a particular disease or condition (e.g., due to genetic risk factors, such as expressing an ApoE4 isoform or TREM2 mutation, due to lifestyle choices, such as consuming a diet high in fat, or due to a condition resulting from a combination of genetic and lifestyle factors, such as metabolic syndrome).

[0081] In certain embodiments, the mammal treated using the methods described herein has NHD. In certain embodiments, the mammal is susceptible to developing NHD.

[0082] In certain embodiments, the mammal treated using the methods described herein has Niemann-Pick disease type C. In certain embodiments, the mammal is predisposed to developing Niemann-Pick disease type C.

[0083] In certain embodiments, the mammal treated using the methods described herein has Alzheimer's disease. In certain embodiments, the mammal is susceptible to developing Alzheimer's disease. Thus, certain embodiments described herein provide a method of treating Alzheimer's disease in a mammal in need thereof, the method comprising administering an agonist anti-TREM2 antibody to the mammal, and the mammal has or has been determined to have lipid metabolism dysregulation. In certain embodiments, the mammal has or has been determined to have lipid metabolism dysregulation in a TREM2-expressing cell (e.g., a microglial cell). In certain embodiments, the TREM2-expressing cell has or has been determined to have reduced TREM2 activity. In certain embodiments, the lipid metabolism dysregulation comprises increased intracellular accumulation of one or more lipids (e.g., cholesteryl esters) described herein. In certain embodiments, the mammal has inflammation associated with lipid metabolism dysregulation (e.g., at least one proinflammatory cytokine, such as a cytokine described herein (e.g., IL-1β), is upregulated).

[0084] In certain embodiments, the mammal treated using the methods described herein has atherosclerosis. In certain embodiments, the mammal is susceptible to developing atherosclerosis. Thus, certain embodiments disclosed herein also provide a method of treating atherosclerosis in a mammal in need of treatment, comprising administering to the mammal an effective amount of an agonist anti-TREM2 antibody (e.g., MAB17291 or 78.18) described herein. In certain embodiments, the mammal has or has been determined to have a lipid metabolism dysregulation. In certain embodiments, the lipid metabolism dysregulation comprises increased accumulation (e.g., intracellular or extracellular accumulation) of one or more lipids described herein. In certain embodiments, the one or more lipids are accumulated intracellularly in macrophages (e.g., macrophages having or determined to have reduced TREM2 activity). In certain embodiments, the mammal has inflammation associated with lipid metabolism dysregulation (e.g., at least one proinflammatory cytokine, such as a cytokine described herein (e.g., IL-1β), is upregulated). In certain embodiments, the method further comprises administering a second therapeutic agent (for example, the therapeutic agent described herein).In certain embodiments, the second therapeutic agent is a drug useful for treating atherosclerosis.In certain embodiments, the second therapeutic agent is the LXR agonist or RXR agonist described herein, or the ACAT1 inhibitor described herein.

[0085] Agonist anti-TREM2 antibodies As described herein, in certain embodiments, an effective amount of an agonist anti-TREM2 antibody is administered to a mammal to treat dysregulation of lipid metabolism or a disease or condition associated with dysregulation of lipid metabolism, such as Alzheimer's disease or atherosclerosis. In certain other embodiments, an effective amount of an agonist anti-TREM2 antibody is administered to a mammal to treat inflammation or a disease or condition associated with inflammation.

[0086] As used herein, the term "TREM2 protein" refers to the triggering receptor 2 expressed on myeloid cells protein encoded by the gene Trem2. As used herein, "TREM2 protein" refers to the native (i.e., wild-type) TREM2 protein of any vertebrate, including, but not limited to, human, non-human primate (e.g., cynomolgus monkey), rodent (e.g., mouse, rat), and other mammals. In some embodiments, the TREM2 protein is a human TREM2 protein having the sequence identified in UniprotKB Accession No. Q9NZC2.

[0087] As used herein, the term "TREM2" also includes protein variants and recombinant TREM2 or fragments thereof.

[0088] As used herein, the term "anti-TREM2 antibody" refers to an antibody that specifically binds to a TREM2 protein (eg, human TREM2).

[0089] As used herein, the term "agonist anti-TREM2 antibody" refers to an antibody that is capable of binding to and activating TREM2 or increasing at least one biological activity of TREM2.

[0090] Certain anti-TREM2 antibodies (e.g., agonist antibodies) and fragments thereof are known in the art. For example, anti-TREM2 antibodies include, but are not limited to, MAB17291 (clone number 237920, R&D Systems) and 78.18 (catalog number MCA4772; Bio-Rad). Antibodies against TREM2 are also described, for example, in patent / publication numbers WO2016 / 023019, WO2017 / 062672, WO2017 / 058866, WO2018 / 195506, WO2019 / 118513, and WO2019 / 028292.

[0091] In certain embodiments, the agonist anti-TREM2 antibody or fragment thereof is MAB17291 or 78.18.

[0092] In one embodiment, the agonist anti-TREM2 antibody or fragment thereof specifically binds to TREM2 and increases its activity. In certain embodiments, the agonist anti-TREM2 antibody is a full-length antibody (e.g., an IgG1 or IgG4 antibody). In certain embodiments, a fragment of an agonist anti-TREM2 antibody is used in the methods disclosed herein and comprises only an antigen-binding portion (e.g., a Fab, F(ab')2 or scFv fragment). In certain embodiments, the agonist anti-TREM2 antibody or fragment thereof is modified to affect functionality, for example, to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933). Mutations that can eliminate effectors include "LALA" mutations (L234A / L235A mutations, numbered according to the EU numbering scheme).

[0093] In certain embodiments, the agonist anti-TREM2 antibody is a monoclonal antibody, or a fragment thereof. In certain embodiments, the agonist anti-TREM2 antibody is an isolated recombinant monoclonal antibody, or a fragment thereof, that specifically binds to TREM2. In certain embodiments, the agonist anti-TREM2 antibody, or a fragment thereof, is a human antibody, or a fragment thereof. In certain embodiments, the antibody is fully human.

[0094] In certain embodiments, the antibody or antigen-binding fragment is bispecific, comprising a first binding specificity for TREM2 and a second binding specificity for a second target epitope, which may be another epitope on TREM2 or on a different protein.

[0095] As used herein, the term "Fc receptor" refers to a surface receptor protein found on immune cells, including B lymphocytes, natural killer cells, macrophages, basophils, neutrophils, and mast cells, that has binding specificity for the Fc region of an antibody. The term "Fc receptor" includes, but is not limited to, Fcy receptors (e.g., FcyRI (CD64), FcyRI IA (CD32), FcyRII B (CD32), FcyRI IIA (CD16a), and FcyRIII B (CD16b), Fca receptors (e.g., FcaRI or CD89), and Fcs receptors (e.g., FcsRI and FcsRII (CD23)).

[0096] As used herein, the term "antibody" refers to a protein having an immunoglobulin fold that specifically binds to an antigen through its variable region. The term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and multispecific antibodies such as human antibodies. As used herein, the term "antibody" also includes antibody fragments that retain binding specificity, including, but not limited to, Fab, F(ab')2, Fv, scFv, and bivalent scFv. Antibodies may contain light chains classified as either kappa or lambda. Antibodies may contain heavy chains classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0097] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer to these light and heavy chains, respectively.

[0098] The term "variable region" or "variable domain" refers to the domain in an antibody heavy or light chain that is derived from the germline variable (V), diversity (D), or joining (J) genes (and not from constant (Cμ and Cδ) gene segments) and that confers to the antibody its specificity for binding to antigen. Typically, antibody variable regions contain four conserved "framework" regions interspersed with three hypervariable "complementarity determining regions".

[0099] The term "complementarity determining region" or "CDR" refers to the three hypervariable regions of each chain that interrupt the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for antibody binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and typically identified by the chain in which the particular CDR is located. Thus, the VH CDR3 or CDR-H3 is located in the variable region of the heavy chain of the antibody in which it is found, while the VL CDR1 or CDR-L1 is the CDR1 from the variable region of the light chain of the antibody in which it is found.

[0100] The "framework regions" or "FRs" of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serve to position and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBASE2" germline variable gene sequence database of human and mouse sequences.

[0101] The amino acid sequences of the CDRs and framework regions can be determined using a variety of definitions well known in the art, such as Kabat, Chothia, the international ImMunoGeneTics database (IMGT), AbM, and observed antigen contact ("contact"). In some embodiments, the CDRs are determined according to the contact definition. See MacCallum et al., J. Mol. Biol., 262:732-745 (1996). In some embodiments, the CDRs are determined by a combination of the Kabat, Chothia, and / or contact CDR definitions.

[0102] The terms "antigen-binding portion" and "antigen-binding fragment" are used interchangeably herein and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a TREM2 protein) via its variable region. Examples of antigen-binding fragments include, but are not limited to, a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains), a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), a single chain Fv (scFv), a disulfide-linked Fv (dsFv), a complementarity determining region (CDR), a VL (light chain variable region), and a VH (heavy chain variable region).

[0103] The term "epitope" refers to the area or region of an antigen to which the CDR of an antibody specifically binds, and may include several amino acids or portions of several amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or portions thereof. For example, if the target is a protein, the epitope may be composed of contiguous amino acids (e.g., linear epitopes), or amino acids from different parts of the protein that are brought into close proximity by protein folding (e.g., discontinuous or conformational epitopes). In some embodiments, the epitope is phosphorylated at one amino acid (e.g., at a serine or threonine residue).

[0104] As used herein, the phrase "recognizes an epitope," when used in reference to an anti-TREM2 antibody, means that the antibody CDR interacts with or specifically binds to an antigen (i.e., a TREM2 protein) at that epitope or a portion of the antigen that contains that epitope.

[0105] As used herein, the term "multispecific antibody" refers to an antibody that comprises two or more different antigen-binding portions, each of which comprises a different variable region that recognizes a different antigen, or a fragment or portion of an antibody that binds to two or more different antigens through its variable regions. As used herein, the term "bispecific antibody" refers to an antibody that comprises two different antigen-binding portions, each of which comprises a different variable region that recognizes a different antigen, or a fragment or portion of an antibody that binds to two different antigens through its variable regions.

[0106] "Monoclonal antibody" refers to an antibody produced by a single clone of cells or a single cell line and consisting of, or consisting essentially of, antibody molecules which are identical in their primary amino acid sequence.

[0107] A "polyclonal antibody" refers to an antibody obtained from a heterogeneous population of antibodies, where different antibodies within the population bind to different epitopes of an antigen.

[0108] "Chimeric antibody" refers to an antibody molecule in which the constant region or a portion thereof is altered, replaced, or exchanged such that the antigen binding site (i.e., variable region, CDR, or a portion thereof) is linked to a constant region of a different or altered class, effector function, and / or species, or the variable region or a portion thereof is altered, replaced, or exchanged with a variable region (e.g., CDR and framework region from a different species) having a different or altered antigen specificity. In some embodiments, a chimeric antibody is a monoclonal antibody that comprises a variable region from one source or species (e.g., mouse) and a constant region from a second source or species (e.g., human). Methods for producing chimeric antibodies have been described in the art.

[0109] A "humanized antibody" is a chimeric antibody derived from a non-human source (e.g., mouse) that contains minimal sequence derived from a non-human immunoglobulin outside of the CDRs. Generally, a humanized antibody contains at least one (e.g., two) antigen-binding variable domain(s), in which the CDR regions correspond substantially to those of a non-human immunoglobulin and the framework regions correspond substantially to those of a human immunoglobulin sequence. In some instances, certain framework region residues of a human immunoglobulin may be substituted with corresponding residues from a non-human species to improve, for example, specificity, affinity, and / or serum half-life. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin sequence. Methods for antibody humanization are known in the art.

[0110] A "human antibody" or "fully human antibody" is an antibody that has human heavy and light chain sequences, typically derived from human germline genes. In some embodiments, the antibodies are produced by human cells, by non-human animals that utilize the human antibody repertoire (e.g., transgenic mice genetically engineered to express human antibody sequences), or by phage display platforms.

[0111] Terms such as "specifically bind" or "specifically binds to" mean that a binding molecule (e.g., an antibody or antigen-binding fragment thereof) can bind to a target (e.g., an antigen such as TREM2) without substantially recognizing and binding to other unrelated molecules present in a sample, e.g., a biological sample. In some cases, specific binding can be greater than about 10 -6 , 10 -7 , or 10 -8 M or less (smaller K Dindicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., BIACORE™), and the like. Furthermore, multispecific antibodies that bind to one domain of TREM2, and one or more additional antigens or bispecific antibodies that bind to two different regions of TREM2, are still considered to be "specifically binding" antibodies as used herein.

[0112] The term "high affinity" antibody refers to an antibody that has a K D Binding affinity for TREM2 of at least 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, or 10 -11 M.

[0113] The agonist anti-TREM2 antibody, or fragment thereof, may be conjugated to a moiety such as a ligand or therapeutic moiety (an "immunoconjugate"), such as a second agonist anti-TREM2 antibody, or an antibody against another antigen.

[0114] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody that specifically binds TREM2, or a fragment thereof, is substantially free of Abs that specifically bind antigens other than TREM2.

[0115] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows analysis of real-time biomolecular interactions by detection of changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0116] As used herein, "K D The term "antibody-antigen interaction" is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0117] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 80% sequence identity, 90% sequence identity, or at least 95%, 98%, or 99% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT, using a predefined gap weight. Residue positions that are not identical may differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions are not expected to substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:SOT-SSI, incorporated herein by reference. Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45, which is incorporated herein by reference. A "reasonably conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0118] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the best overlap region between the query sequence and the search sequence and the percentage of sequence identity (Pearson (2000) supra). Another algorithm for comparing the sequences disclosed herein to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0119] The antibodies and antigen-binding fragments that may be used as disclosed herein specifically bind to TREM2. Agonist anti-TREM2 antibodies may bind to TREM2 with high or low affinity. They may be used alone or as adjunctive therapy with other therapeutic moieties or modalities (i.e., at least one additional therapeutic agent) known in the art to treat dysregulation of lipid metabolism and / or inflammation.

[0120] Certain agonist anti-TREM2 antibodies are capable of binding to TREM2 and increasing the activity of TREM2 as determined by in vitro or in vivo assays. The ability of an antibody to bind to TREM2 and increase the activity of TREM2 can be measured using any standard method known to those of skill in the art, including binding or activity assays.

[0121] Antibodies specific for TREM2 may contain no additional label or moiety, or may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label may be a radionuclide or a fluorescent dye. In certain embodiments, such labeled antibodies may be used in diagnostic assays.

[0122] Preparation of anti-human TREM2 antibody Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used as disclosed herein to generate human antibodies that specifically bind to TREM2.

[0123] To generate antibodies against TREM2 or a fragment thereof, an immunogen comprising any one of the following can be used. For example, the primary immunogen may be full-length TREM2 (see UniProtKB Q9NZC2), or a recombinant form of TREM2 or a modified human TREM2 fragment or a modified cynomolgus TREM2 fragment. In certain embodiments, the primary immunogen may be followed by immunization with a secondary immunogen, or with an immunogenic active fragment of TREM2. Alternatively, TREM2 or a fragment thereof may be generated and modified using standard biochemical techniques and used as an immunogen. The immunogen may be a biologically active and / or immunogenic fragment of TREM2, or DNA encoding the active fragment.

[0124] In certain embodiments, the immunogen may be a peptide from the N-terminus or C-terminus of TREM2. In one embodiment, the immunogen is a specific domain of TREM2. In some embodiments, the immunogen may be a recombinant TREM2 peptide expressed in E. coli or in any other eukaryotic or mammalian cell, such as Chinese Hamster Ovary (CHO) cells. The peptide may be modified to include the addition or substitution of certain residues for tagging or for the purpose of conjugation to a carrier molecule, such as KLH. For example, a cysteine ​​may be added to either the N-terminus or C-terminus of the peptide, or a linker sequence may be added to prepare the peptide for conjugation to KLH, for example, for immunization.

[0125] Agonistic anti-TREM2 antibodies, including Fc variants An agonist anti-TREM2 antibody may comprise an Fc domain that contains one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, an agonist anti-TREM2 antibody may comprise an Fc domain that contains one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C H The antibody may contain mutations in the 3 region, where the mutation(s) increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., within an endosome at a pH range of about 5.5 to about 6.0). Such mutations may result in an increase in the serum half-life of the antibody when administered to an animal.

[0126] Biological properties of agonist anti-TREM2 antibodies or fragments thereof Generally, antibodies useful as disclosed herein function by binding to TREM2, and include agonist anti-TREM2 antibodies and antigen-binding fragments thereof that, for example, bind to the TREM2 molecule with high affinity, e.g., a K of less than about 50 nM as measured by surface plasmon resonance. DAntibodies and antigen-binding fragments of antibodies that bind to TREM2 at 25° C. (e.g., at 25° C. or 37° C.) may be used as disclosed herein. In certain embodiments, the antibodies or antigen-binding fragments thereof have a K of less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, or less than about 1 nM as measured by surface plasmon resonance or a substantially similar assay. D The antibodies or antigen-binding fragments thereof disclosed herein also bind to TREM2 with a K of less than about 35 nM as measured by surface plasmon resonance. D In certain embodiments, the antibody or antigen-binding fragment thereof may have a K of less than about 30 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, or less than about 5 nM as measured by surface plasmon resonance or a substantially similar assay. D Binds to cynomolgus monkey TREM2.

[0127] The methods and uses described herein also include the use of antibodies and antigen-binding fragments thereof that bind to TREM2 with a dissociation half-life (t1 / 2) of greater than about 1.1 minutes as measured by surface plasmon resonance at 25° C. or 37° C., or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments bind to TREM2 with a t1 / 2 of greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1200 minutes as measured by surface plasmon resonance (e.g., mAb capture or antigen capture formats) at 25° C. or 37° C., or a substantially similar assay.

[0128] In some embodiments, the antibodies may bind to a specific domain or fragment of a domain of TREM2. In some embodiments, the antibodies for use disclosed herein may bind to more than one domain (cross-reactive antibodies).

[0129] In certain embodiments, the antibody for use disclosed herein may be a bispecific antibody. A bispecific antibody may bind to one epitope in one domain and also to a second epitope in a different domain of TREM2. In certain embodiments, a bispecific antibody may bind to two different epitopes in the same domain.

[0130] In one embodiment, there is provided the use of an isolated fully human monoclonal antibody or antigen-binding fragment thereof that binds to TREM2.

[0131] Species selectivity and species cross-reactivity According to certain embodiments, the agonist anti-TREM2 antibody may bind to human TREM2 but not to TREM2 from other species. Alternatively, the agonist anti-TREM2 antibody may bind to human TREM2 and to TREM2 from one or more non-human species. For example, the agonist anti-TREM2 antibody may or may not bind to human TREM2, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey or chimpanzee TREM2. In certain embodiments, the agonist anti-TREM2 antibody may bind to human and cynomolgus monkey TREM2 with the same or different affinity, but not to rat and mouse TREM2.

[0132] Additional medications In certain embodiments, the methods described herein further comprise administering one or more additional therapeutic agents (eg, a second therapeutic agent).

[0133] The one or more additional therapeutic agents may be administered simultaneously or sequentially with the agonist anti-TREM2 antibody. In certain embodiments, the one or more additional therapeutic agents are administered simultaneously with the agonist anti-TREM2 antibody. In certain embodiments, a pharmaceutical composition comprising an agonist anti-TREM2 antibody and one or more additional therapeutic agents is administered. In certain embodiments, the agonist anti-TREM2 antibody and one or more additional therapeutic agents are administered sequentially. In certain embodiments, the agonist anti-TREM2 antibody is administered before the one or more additional therapeutic agents. In certain embodiments, the one or more additional therapeutic agents are administered before the agonist anti-TREM2 antibody.

[0134] In certain embodiments, the additional therapeutic agent is an agent useful in the treatment of Alzheimer's disease or atherosclerosis.

[0135] In certain embodiments, the additional therapeutic agent is an agent useful for treating inflammation.

[0136] In certain embodiments, the additional therapeutic agent is an LXR agonist. As described herein, an effective amount of an LXR agonist may be administered to a mammal to treat dysregulation of lipid metabolism or a disease or condition associated with such dysregulation. LXR is part of the superfamily of ligand-dependent nuclear receptor transcription factors. Oxidized derivatives of cholesterol (oxysterols) are natural ligands of LXR and have the ability to both stimulate and antagonize LXR activation. LXRα (encoded by NR1H3) is highly expressed in the liver, macrophages, and other highly metabolic tissues, while LXRβ (NR1H2) is ubiquitously expressed. Upon ligand activation, LXR forms a heterodimer with RXR and plays a role in regulating lipid metabolism and inflammatory signaling.

[0137] The term "LXR agonist" refers to an agent that can activate, enhance, increase, or otherwise stimulate one or more functions of a target LXR. An agonist of an LXR can induce any LXR activity, such as LXR-mediated signal transduction, either directly or indirectly. As used herein, an LXR agonist may, but may not, be required to bind to an LXR and may or may not directly interact with an LXR. An LXR agonist may specifically stimulate LXRα, LXRβ, or both. An LXR agonist may affect other receptors / pathways in addition to stimulating LXR.

[0138] LXR agonists include natural oxysterols, synthetic oxysterols, synthetic nonoxysterols, and natural nonoxysterols. Exemplary natural oxysterols include 20(S) hydroxycholesterol, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25(S) epoxycholesterol, and 27-hydroxycholesterol. Exemplary synthetic oxysterols include N,N-dimethyl-3.beta.-hydroxycholenamide (DMHCA). Exemplary synthetic nonoxysterols include N-(2,2,2-trifluoroethyl)-N-{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl-yl)ethyl]phenyl}benzenesulfonamide (TO901317, Tularik 0901317), [3-(3-(2-chloro-trifluoromethylbenzyl-2,2-diphenylethylamino)propoxy)-phenylacetic acid] (GW3965), N-methyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-1-ethyl)-phenyl-1]-benzenesulfonamide (TO314407), 4,5-dihydro-1-(3-(3-trifluoromethyl-7-propyl-benzisoxazol-6-yloxy)propyl)-2,6-pyrimidinedione, 3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenylacetic acid (F.sub.3-MethylAA), and acetylpodocarpic acid dimer. Exemplary natural nonoxysterols include paxillin, desmosterol, and stigmasterol.

[0139] Other useful LXR agonists are described in, e.g., U.S. Patent Application Publication Nos. 2006 / 0030612, 2005 / 0131014, 2005 / 0036992, 2005 / 0080111, 2003 / 0181420, 2003 / 0086923, 2003 / 0207898, 2004 / 0110947, and 2005 / 0080111. Nos. 2004 / 0087632, 2005 / 0009837, 2004 / 0048920, and 2005 / 0123580, U.S. Patent Nos. 6,316,503, 6,828,446, 6,822,120, and 6,900,244, WO2008036239, WO2001 / 41704, Menke JG et al., Endocrinology 143:2548-58(2002), Joseph SB et al., Proc.Natl.Acad.Sci.USA 99:7604-09(2002), Fu X et al., J.Biol.Chem.276:38378-87(2001), Schultz JR et al., Genes Dev.14:2831-38(2000), Sparrow CP et al.,J.Biol.Chem.277:10021-27(2002), Yang C et al.,J.Biol.Chem.,Manuscript M603781200(Jul.20,2006), Bramlett KS et al. al., J. Pharmacol. Exp. Ther. 307:291-96(2003); Ondeyka JG et al., J. Antibiot (Tokyo):58:559-65(2005).

[0140] In certain embodiments, the LXR agonist is hypocholamide, T0901317, GW3965, IMB-808, or N,N-dimethyl-3β-hydroxycholenamide (DMHCA). In certain embodiments, the LXR agonist is GW3965.

[0141] In certain other embodiments, the additional therapeutic agent is an RXR agonist. As described herein, an effective amount of an RXR agonist may also be administered to a mammal to treat dysregulation of lipid metabolism or a disease or condition associated with such dysregulation. RXR is a type of nuclear receptor that is activated by 9-cis-retinoic acid and 9-cis-13,14-dihydro-retinoic acid. There are three RXR forms: RXR-α, RXR-β, and RXR-γ, which are encoded by the RXRA, RXRB, and RXRG genes, respectively. RXR heterodimerizes with subfamily 1 nuclear receptors, including CAR, FXR, LXR, PPAR, PXR, RAR, TR, and VDR. As with other type II nuclear receptors, RXR heterodimers in the absence of ligand bind to hormone response elements complexed with corepressor proteins. Binding of an agonist ligand to RXR results in the dissociation of the corepressor and recruitment of coactivator proteins, which in turn promote the transcription of downstream target genes into mRNA and ultimately into protein.

[0142] The term "RXR agonist" refers to an agent that can activate, enhance, increase, or otherwise stimulate one or more functions of a target RXR (e.g., enhance the transcriptional regulatory activity of RXR homo- and heterodimers). An agonist of RXR can induce any RXR activity, e.g., RXR-mediated signal transduction, either directly or indirectly. As used herein, an RXR agonist may, but may not, be required to bind to RXR, and may or may not directly interact with RXR. An RXR agonist may specifically stimulate RXRα, RXRβ, or RXRγ, or a combination thereof. An RXR agonist may affect other receptors / pathways in addition to stimulating RXR.

[0143] Certain RXR agonists and methods for synthesizing such agonists are known. For example, RXR agonists include, but are not limited to, those described in Boehm et al. J. Med. Chem. 38:3146 (1994), Boehm et al. J. Med. Chem. 37:2930 (1994), Antras et al., J. Biol. Chem. 266:1157-61 (1991), Salazar-Olivo et al., Biochem. Biophys. Res. Commun. 204:10 257-263 (1994), Safanova, Mol. Cell. Endocrin. 104:201 (1994), MLDawson and WHOkamura, Chemistry and Biology of Synthetic Retinoids, Chapters 3, 8, 14 and 16, CRC Press, Inc., Florida (1990), MLDawson and PDHobbs,The Retinoids,Biology,Chemistry and Medicine,MBSporn et al.,Eds.(2nd ed.),Raven Press,New York,NY,pp.5-178(1994),Liu et al.,Tetrahedron,40:1931(1984),Cancer Res.,43:5268(1983), Eur.J.Med.Chem.15:9(1980), Allegretto et al.,J.Bio.Chem.,270:23906(1995), Bissonette et al.,Mol.Cell.Bio.,15:5576(1995), Beard et al. al., J. Med. Chem., 38:2820 (1995), Koch et al. al., J.Med.Chem.,39:3229(1996), US4,326,055, US4,578,498, US5,399,586, US5,466,86 1, US5,721,103, US5,780,676, US5,801,253, US5,830,959, US6,083,977, US6,131,050, US20160324874, US20180185342, US20180263939, US20180318241, WO93 / 11755, WO93 / 21146, WO94 / 15902, W094 / 23068, WO95 / 04036, WO96 / 20913, WO20100105728, WO2013056232, and WO2013090616.

[0144] In certain embodiments, the RXR agonist is CD3254, docosahexaenoic acid, fluorobexarotene, bexarotene (LGD1069), IRX4204, HX630, PA024, isotretinoin, retinoic acid, SR 11237, LG101506, LGD100268 or LGD100324. In certain embodiments, the RXR agonist is bexarotene.

[0145] In certain other embodiments, the additional therapeutic agent is an ACAT1 inhibitor.As described herein, an effective amount of an ACAT1 inhibitor may also be administered to a mammal to treat dysregulation of lipid metabolism or a disease or condition associated with such dysregulation.

[0146] The ACAT1 gene encodes mitochondrial acetyl-CoA acetyltransferase (UniProtKB P24752), a short-chain-length specific thiolase. As used herein, "ACAT1 inhibitor" includes any compound or treatment that can inhibit the expression and / or function of ACAT1, either directly or indirectly (e.g., inhibit transcription, RNA maturation, RNA translation, post-translational modification, or enzymatic activity). As used herein, an ACAT1 inhibitor may, but may not, be required to bind to ACAT1 and may or may not directly interact with the enzyme. In certain embodiments, the inhibitor detectably inhibits the expression level or biological activity of ACAT1, e.g., as measured using an assay described herein or known in the art. In certain embodiments, the inhibitor inhibits the expression level or biological activity of ACAT1 by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0147] The inhibitor can be of natural or synthetic origin. For example, it can be a nucleic acid, a polypeptide, a protein, a peptide, or an organic compound. In one embodiment, the inhibitor is an siRNA, an shRNA, a small molecule, or an antibody.

[0148] In certain embodiments, the inhibitor is an antisense nucleic acid (e.g., siRNA or shRNA) that can inhibit the transcription of ACAT1 or the translation of the corresponding messenger RNA.Those skilled in the art can use commercially available software and the gene sequence of ACAT1 to design antisense nucleic acid.

[0149] In certain embodiments, the inhibitor is a polypeptide, such as an antibody against ACAT1, or a fragment or derivative thereof, such as a Fab fragment, a CDR region, or a single chain antibody.

[0150] The term "small molecule" includes organic molecules with a molecular weight of less than about 1000 amu. In one embodiment, a small molecule may have a molecular weight of less than about 800 amu. In another embodiment, a small molecule may have a molecular weight of less than about 500 amu.

[0151] Certain ACAT1 inhibitors are known in the art.For example, in certain embodiments, the ACAT1 inhibitor is the ACAT1 inhibitor described in US2004 / 0038987, US20140044757, US20170292128, or WO2015 / 038585. In certain embodiments, the ACAT1 inhibitor is selected from the group consisting of avasimibe (CI-1011), pactimibe, perpactin, manassantin A, diphenylpyridazine derivatives, grisoprenin A, CP113, 818, K604, beauveriolide I, beauveriolide III, U18666A, TMP-153, YM750, GERI-BP002-A, Sandoz Sah 58-035, VULM 1457, lovastatin, CI976, CL-283, 546, CI-999, E5324, YM17E, FR182980, ATR-101 (PD132301 or PD132301-2), F-1394, HL-004, F-12511 (eflucimibe), cinnamic acid derivatives, cinnamic derivatives, Dup 128, RP-73163, pyripyropene C, FO-1289, AS-183, SPC-15549, FO-6979, Angekica, carrot, decursin, terpendol C, beauvericin, spiridon, pentacecilides, CL-283, 546, betulinic acid, shikonin derivatives, esculeogenin A, Wu-V-23, pyripyropene derivatives A, B and D, glisoprenin BD, saucerneol B, sespendol, diethyl pyrocarboxylate, beauveriolide analogs, acaterin, DL-melinamide, PD 138142-15, CL277, 082, EAB-309, Enniatin antibiotics, epi-cochlioquinone A, FCE-27677, FR186485, FR190809, NTE-122, Ovobatol, Panaxadiol, Protopanaxadiol, Polyacetylene, SaH 57-118, AS-186, BW-447A, 447C88, T-2591, TEI-6522, TEI-6620, XP 767, XR 920, GERI-BP001, Gomisin N, Gypsetin, Helminthosporol, TS-962, A 922500 (CAS959122-11-3), N-[3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-L-phenylalanine, methyl ester (CAS 615264-52-3), 3,4-dihydroxyhydrocinnamic acid (L-aspartic acid dibenzyl ester) amide (CAS 615264-62-5), CI-976, FR14523 (Fujisawa Pharmaceutical Co. Ltd.), F1394 (Fujirebio Inc.), isochromophilones, kudingosides, lateritin, naringenin, and combinations thereof. In certain embodiments, the ACAT1 inhibitor is CP-113, 818, CI-1011, or K-604. In certain embodiments, the ACAT1 inhibitor is K-604.

[0152] In certain embodiments, the additional therapeutic agent is an agent described herein.

[0153] Administration The therapeutic agent (e.g., an agonist anti-TREM2 antibody or an additional therapeutic agent) can be formulated as a pharmaceutical composition and administered to a mammalian host, such as a human patient, in a variety of forms adapted for the selected route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

[0154] Thus, the therapeutic agents may be administered systemically, for example orally (e.g., added to drinking water), in combination with a pharma- ceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of the patient's diet. For oral therapeutic administration, the therapeutic agents may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations generally contain at least 0.1% of the agent. The percentage of the compositions and preparations may, of course, be varied and may conveniently be from about 2 to about 60% of the weight of a given unit dosage form. The amount of agent in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0155] Tablets, troches, pills, capsules, etc. may also contain: binders, such as gum tragacanth, acacia, corn starch, or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, fructose, lactose, or aspartame; or flavorings, such as peppermint, wintergreen oil, or cherry flavoring, may be added. When the unit dosage form is a capsule, in addition to the above types of materials, it may contain a liquid carrier, such as vegetable oil or polyethylene glycol. Various other materials may be present as coatings or otherwise to modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, and the like. A syrup or elixir may contain the therapeutic agent, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and a flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharma- ceutically acceptable and substantially non-toxic in the amounts used. In addition, the therapeutic agent may be incorporated into sustained-release preparations and devices.

[0156] The therapeutic agent may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the therapeutic agent or its salts may be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof, and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0157] Pharmaceutical forms suitable for injection or injection may include sterile aqueous solutions, or dispersions or sterile powders containing the active ingredient adapted for the extemporaneous preparation of sterile injectable or insoluble solutions or dispersions, optionally encapsulated in liposomes. In any case, the final dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or solvent may be, for example, a solvent or liquid dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Prevention of microbial action can be brought about by various antibacterial and antifungal agents, such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0158] Sterile injectable solutions are prepared by incorporating the therapeutic agent in the required amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Sterile powders for preparing sterile injectable solutions can be prepared by vacuum drying and freeze-drying techniques to yield a powder of the active ingredient and any additional desired ingredient present in a previously sterile-filtered solution.

[0159] For topical administration, the therapeutic agents may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which may be a solid or liquid.

[0160] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Useful liquid carriers include water, alcohol or glycol, or water-alcohol / glycol blends in which the therapeutic agent can be dissolved or dispersed at effective levels, optionally with non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resulting liquid composition can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0161] Thickening agents such as synthetic polymers, fatty acids, fatty acids and esters, fatty alcohols, modified celluloses, or modified mineral materials may also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for application directly to the user's skin.

[0162] Examples of useful dermatological compositions that can be used to deliver therapeutic agents to the skin are known in the art, for example, Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157), and Wortzman (U.S. Pat. No. 4,820,508).

[0163] Useful dosages of Therapeutic Agents can be determined by comparing their in vitro and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; see, for example, U.S. Patent No. 4,938,949.

[0164] The amount of Therapeutic Agent, or an active salt or derivative thereof, required for use in therapy will vary with the particular salt selected, as well as the route of administration, the nature of the condition being treated, and the age and condition of the patient, and is ultimately at the discretion of the attending physician or clinician.

[0165] The therapeutic agent may be conveniently formulated in a unit dosage form, in one embodiment, a composition comprising the therapeutic agent formulated in such a unit dosage form may be used.

[0166] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, e.g., two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into several discrete loosely spaced administrations, e.g., by multiple inhalations from an insufflator, or by applying a plurality of drops to the eye.

[0167] In certain embodiments, an agonist anti-TREM2 antibody is administered to the mammal. In certain embodiments, an additional therapeutic agent is further administered to the mammal (e.g., an RXR agonist, an LXR agonist, an ACAT1 inhibitor, or other agent useful for treating lipid dysregulation / inflammation). When a combination of two or more of these agents is administered, they may be administered simultaneously or sequentially. In certain embodiments, two or more agents are administered sequentially. In certain embodiments, two or more agents are administered simultaneously. In certain embodiments, a pharmaceutical composition comprising a combination of two or more agents is administered. For example, in one embodiment, a composition comprising an agonist anti-TREM2 antibody, at least one other therapeutic agent, and a pharma- ceutically acceptable diluent or carrier is provided for use in treating lipid metabolism dysregulation and / or inflammation.

[0168] Certain embodiments also provide kits comprising an agonist anti-TREM2 antibody, packaging materials, and instructions for administering the agonist anti-TREM2 antibody to an animal to treat lipid metabolism dysregulation and / or inflammation, hi certain embodiments, the kit further comprises at least one other therapeutic agent.

[0169] Methods for isolating enriched CNS cell populations Provided herein are methods for isolating enriched populations of CNS cell types from brain tissue (eg, enriched populations of astrocytes or microglial cells).

[0170] Accordingly, certain embodiments provide a method for selecting a population of CNS cells from a tissue sample, comprising: (a) contacting a tissue sample with a primary anti-CD45 antibody, a primary anti-CD11b antibody, and a primary anti-astrocyte surface antigen-2 (ACSA-2) antibody, each primary antibody being uniquely labeled, to provide a labeled tissue sample; (b) sorting the cells in the labeled tissue sample by flow cytometry; A method is provided, which provides distinct cell populations of astrocytes and microglial cells.

[0171] As used herein, the term "distinct cell population" refers to a physically distinct population of cells enriched for a particular CNS cell type (eg, neurons or astrocytes).

[0172] Certain embodiments also provide compositions comprising a sorted distinct cell population isolated using the methods described herein (e.g., a sorted microglial cell population or a sorted astrocyte cell population).

[0173] In certain embodiments, the microglial cell population is selected based on the following marker profile: CD45 low / CD11b + / ACSA-2 - .

[0174] In certain embodiments, the astrocyte cell population is sorted based on the following marker profile: CD45 - / CD11b - / ACSA-2 + .

[0175] Certain embodiments include a population of CNS cells comprising two physically distinct cell populations, the first cell population expressing CD45low / CD11b + / ACSA-2 - the second cell population comprising an enriched population of CD45 cells; - / CD11b - / ACSA-2 + The present invention provides a population of CNS cells, comprising the cells.

[0176] As described herein, a combination of anti-CD45 primary and anti-CD11b antibodies can be used to isolate enriched microglial cell populations from other CNS cell types. CD45, also known as leukocyte common antigen (LCA) and protein tyrosine phosphatase receptor type C (PTPRC), is a cell surface antigen expressed at various levels by most hematopoietic cells, except for erythrocytes and platelets. CD45 is expressed at low levels in microglial cells, not by astrocytes, and at high levels in certain non-microglial non-astroglial cells, such as macrophages. Thus, anti-CD45 primary antibodies can be used to detect the CD45 cell surface marker (CD45 + ) and flow cytometry can be used to identify labeled cells that express low levels of CD45 (e.g., CD45 low Microglial cells) can be isolated. low Identify cells and identify CD45 high For example, the reference value may be a control cell or a control cell population, such as a known microglial cell(s) or a known CD45 high It can be the amount of CD45 expression in cells.In some embodiments, the reference value is, for example, a range of values ​​when the reference value is obtained from multiple samples.Furthermore, the reference value can be presented as a single value (e.g., measured abundance value, mean value, or median value) or a range of values, with or without standard deviation or error criteria.

[0177] CD11b is an integrin family member that pairs with CD18 to form the CR3 heterodimer. CD11b is expressed on a variety of cell types, including macrophages and microglial cells, but is not expressed by astrocytes. Thus, anti-CD11b primary antibodies may be used to label cells expressing the CD11b cell surface marker, and labeled cells (e.g., CD11b + Flow cytometry may be used to isolate cells (microglial cells).

[0178] The anti-ACSA-2 antibody recognizes a glycosylated surface molecule expressed by astrocytes. In contrast, this surface molecule is expressed by neurons, oligodendrocytes, and NG2 cells. + It is not expressed by non-astroglial cells in the CNS, such as cells, microglia, endothelial cells, leukocytes, or erythrocytes (Kantzer et al., 2017, Glia, 65:990-1004). Therefore, anti-ACSA-2 primary antibodies may be used to label cells that express the ACSA-2 cell surface marker, and labeled cells (e.g., ACSA-2 + Flow cytometry may be used to isolate cells (astrocytic cells).

[0179] In certain embodiments, the primary antibody is included in the composition and the tissue sample is contacted with the composition (e.g., a composition comprising an anti-ACSA-2 antibody, an anti-CD11b antibody, and an anti-CD45 antibody). In certain embodiments, each primary antibody is uniquely labeled (i.e., each antibody in the composition comprises a different label) with a label (e.g., a fluorescent label) suitable for sorting by flow cytometry. In certain embodiments, the composition further comprises a viability dye, which can be used to distinguish viable and non-viable cells by flow cytometry (e.g., Fixable Viability Stain BV510). In certain other embodiments, a viability dye is not included with the composition and the tissue sample is contacted with the viability dye simultaneously or sequentially with the composition.

[0180] In certain embodiments, cells present within the tissue sample are dissociated prior to contacting with the viability dye and / or composition.

[0181] In certain embodiments, the tissue sample is contacted with the composition under conditions suitable for the antibody to bind to its corresponding marker and label the cells. In certain embodiments, the labeled tissue sample prior to sorting by flow cytometry is contacted with the composition under conditions suitable for the antibody to bind to its corresponding marker and label the cells. + Cells, labeled CD45 + Cells and labeled CD11b + In certain embodiments, the cells are further labeled with a viability dye.

[0182] In certain embodiments, cells present within a tissue sample are sorted by flow cytometry (eg, using a viability dye) into a population of non-viable cells and a population of viable cells.

[0183] In certain embodiments, cells present within a tissue sample are identified by flow cytometry as CD45 + Cell populations and CD45 - In certain embodiments, the population of cells is selected to contain CD45 + Populations of cells were identified as CD45 low Cell populations and CD45 high A population of cells is selected.

[0184] In certain embodiments, cells present in the tissue sample are identified by flow cytometry as CD11b + Cell populations and CD11b - A population of cells is selected.

[0185] In certain embodiments, cells present in the tissue sample are identified by flow cytometry as ACSA-2 + Cell populations and ACSA-2 - The cells are sorted into populations.

[0186] Labeled cells are CD45 low / CD11b + / ACSA-2 - Isolated populations of microglial cells and CD45 - / CD11b - / ACSA-2 + Any gating combination that results in an isolated population of astrocytes may be used to sort by flow cytometry. For example, in certain embodiments, cells present within a tissue sample are sorted by flow cytometry into a population of non-viable cells and a population of viable cells (e.g., using a viability dye). In certain embodiments, the population of viable cells is sorted by flow cytometry into a population of CD11b + Cell populations and CD11b - In certain embodiments, the population of cells is selected to include CD11b + A population of cells expressing CD45 + Cell populations and CD45 - In certain embodiments, the population of cells is further selected to be CD11b + / CD45 + The cell population was identified by flow cytometry as CD11b + / CD45 low Cell populations and CD11b + / CD45 high In certain embodiments, the population of cells is selected to include CD11b - The cell population was analyzed by flow cytometry to identify ACSA-2 + Cell populations and ACSA-2 - Such selection is performed by selecting a population of cells that express CD45 low / CD11b + / ACSA-2 - Microglial cell populations and CD45 - / CD11b - / ACSA-2 + This results in a population of astrocytes.

[0187] In certain embodiments, a sorted population of enriched astrocytes (e.g., viable, CD45 - , CD11b - , ACSA-2+ cells) contain less than about 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% non-astroglial cells. In certain embodiments, the sorted population of enriched astrocytes contains no non-astroglial cells.

[0188] In certain embodiments, a sorted population of enriched astrocytes (e.g., viable, CD45 low , CD11b + , ACSA-2 - cells) contain less than about 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% non-astroglial cells. In certain embodiments, the sorted population of enriched microglial cells does not contain (non-microglial cells.

[0189] In certain embodiments, one or more enriched cell populations are analyzed for quantification of metabolic species (e.g., lipid species) or nucleic acid species. In certain embodiments, an enriched astrocyte cell population is analyzed for quantification of metabolic or nucleic acid species. In certain embodiments, an enriched microglial cell population is analyzed for quantification of metabolic or nucleic acid species. In certain embodiments, enriched astrocyte cell populations and microglial cell populations are analyzed for quantification of metabolic or nucleic acid species. In certain embodiments, one or more enriched cell populations are analyzed for quantification of metabolic species. In certain embodiments, one or more enriched cell populations are analyzed for quantification of two or more metabolic species (e.g., 2, 3, 4, 5, 10, 25, 50 or more). In certain embodiments, one or more enriched cell populations are analyzed for quantification of nucleic acid species. In certain embodiments, one or more enriched cell populations are analyzed for quantification of two or more nucleic acid species (e.g., 2, 3, 4, 5, 10, 25, 50 or more). In certain embodiments, one or more enriched cell populations are analyzed for quantification of metabolic and nucleic acid species. In certain embodiments, one or more enriched cell populations are analyzed for quantification of two or more metabolic species and two or more nucleic acid species.

[0190] As used herein, the term metabolic species includes macromolecules such as lipid species. For example, in certain embodiments, metabolic species is lipid species such as lipid species (e.g., cholesteryl ester species) described herein. In certain embodiments, a combination of metabolic species is quantified, such as a combination of lipids described herein. Metabolic species can be quantified using methods known in the art. For example, metabolic species can be quantified using liquid chromatography mass spectrometry (LCMS) assay (see, for example, Examples).

[0191] The nucleic acid may be RNA or DNA, such as, for example, genomic DNA, RNA transcribed from genomic DNA, or cDNA generated from RNA. In certain embodiments, the nucleic acid species is RNA. In certain embodiments, the nucleic acid species is DNA. In certain embodiments, the nucleic acid species is genomic DNA. Methods for quantifying nucleic acid species are known in the art. For example, such methods include, but are not limited to, polymerase chain reaction (PCR), including quantitative PCR (qPCR) and real-time quantitative reverse transcription PCR (qRT-PCR), RNA-seq, Northern blot analysis, expression microarray analysis, next generation sequencing (NGS), and fluorescent in situ hybridization (FISH). In certain embodiments, the nucleic acid species is quantified using the assays described herein.

[0192] In certain embodiments, one or more enriched cell populations are analyzed for quantification of administered therapeutic agents. In certain embodiments, enriched astrocyte cell populations are analyzed for quantification of administered therapeutic agents. In certain embodiments, enriched microglial cell populations are analyzed for quantification of administered therapeutic agents. In certain embodiments, enriched astrocyte cell populations and microglial cell populations are analyzed for quantification of administered therapeutic agents. Methods for quantifying therapeutic agents are known in the art and described herein.

[0193] Specific Technology As used herein, the phrase "physiological sample" is meant to refer to a biological sample obtained from a subject that contains proteins, lipids, and / or nucleic acids. Thus, the sample may be evaluated at the nucleic acid, lipid, or protein level. In certain embodiments, the physiological sample comprises tissue, cerebrospinal fluid (CSF), urine, blood, serum, or plasma. In certain embodiments, the sample comprises tissue (e.g., containing microglia). In certain embodiments, the sample comprises CSF. In certain embodiments, the sample comprises blood and / or plasma.

[0194] Biological samples can be obtained using methods known to those skilled in the art. Biological samples can be obtained from vertebrates, particularly mammals. DNA, RNA or protein diversity (e.g., mutation, expression or localization) can be detected from the sample.

[0195] The nucleic acid may be, for example, genomic DNA, RNA transcribed from genomic DNA, or cDNA generated from RNA. The nucleic acid or protein may be derived from a vertebrate, for example, a mammal. A nucleic acid or protein is said to be "derived from" a particular source if it is obtained directly from that source or is a copy of a nucleic acid found in that source.

[0196] In certain embodiments, genomic DNA can be isolated from biological samples and analyzed in detection assays. In certain embodiments, mRNA can be isolated from biological samples and analyzed in detection assays. In certain embodiments, mRNA isolated from biological samples can be reverse transcribed to generate cDNA.

[0197] Nucleic acid and amino acid sequence variations can be detected by certain methods known to those of skill in the art. Similarly, nucleic acid expression (e.g., mRNA expression) can be detected using methods known in the art. Such methods include polymerase chain reaction (PCR), including quantitative PCR (qPCR) and real-time quantitative reverse transcription PCR (qRT-PCR), Northern blot analysis, expression microarray analysis, next generation sequencing (NGS), fluorescent in situ hybridization (FISH), DNA sequencing, primer extension assays, including allele-specific nucleotide incorporation assays and allele-specific primer extension assays (e.g., allele-specific PCR, allele-specific ligation chain reaction (LCR), and gap-LCR), allele-specific oligonucleotide hybridization assays (e.g., oligonucleotide ligation assays), cleavage protection assays that use protection from cleaving agents to detect mismatched bases in a nucleic acid duplex, analysis of MutS protein binding, electrophoretic analysis to compare the mobility of variant and wild-type nucleic acid molecules, denaturing gradient gel electrophoresis (DGGE, see, e.g., Myers et al. (1985) Nature 103:131-135), and methods for detecting variants and wild-type nucleic acid molecules. 313:495), analysis of RNase cleavage at mismatched base pairs, analysis of chemical or enzymatic cleavage of heteroduplex DNA, mass spectrometry (e.g., MALDI-TOF), genetic bit analysis (GBA), 5' nuclease assays (e.g., TaqMan®, and assays using molecular beacons.

[0198] In certain embodiments, protein expression may also be detected. Assays for detecting and measuring protein expression are known in the art and include, for example, Western blot analysis, immunofluorescence, immunohistochemistry (e.g., of tissue arrays), and the like.

[0199] In certain embodiments, macrophages are evaluated using assays known in the art or described herein.In certain embodiments, iPSCs are evaluated using assays known in the art or described herein.In certain embodiments, microglial cells are evaluated using assays known in the art or described herein.In certain embodiments, microglial cells differentiated from iPSCs are evaluated using assays known in the art or described herein.

[0200] Specific Definitions The term "control" or "control sample" refers to any sample appropriate for the detection technique used. A control sample may contain the product of the detection technique used or the material being tested. Additionally, a control may be a positive control or a negative control.

[0201] The term "gene" is used broadly to refer to any fragment of nucleic acid associated with a biological function. Genes include coding sequences and / or regulatory sequences necessary for their expression. For example, genes refer to nucleic acid fragments that express mRNA, functional RNA, or specific proteins, including their regulatory sequences. Genes also include non-expressed DNA fragments, for example, that form recognition sequences for other proteins. Genes can include sequences obtained from various sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and designed to have desired parameters. In addition, "gene" or "recombinant gene" refers to a nucleic acid molecule that includes an open reading frame and includes at least one exon and (optionally) intron sequences. The term "intron" refers to DNA sequences present within a given gene that are not translated into protein and are generally found between exons.

[0202] "Mutated gene" or "mutation" or "functional mutation" refers to an allelic form of a gene that can alter the phenotype of a subject carrying the mutant gene compared to a subject not carrying the mutant gene.

[0203] A "variant" of a molecule is a sequence that is substantially similar to the sequence of a natural molecule. For nucleotide sequences, variants include sequences that, due to the degeneracy of the genetic code, code for the same amino acid sequence of a natural protein. Naturally occurring allelic variants such as these can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated by using site-directed mutagenesis to code for natural proteins, as well as those that code for polypeptides with amino acid substitutions. Generally, the nucleotide sequence variants disclosed herein, in at least one embodiment, have 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81% to 84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence.

[0204] As used herein, the terms "specifically hybridize" or "specifically detect" with respect to a nucleic acid refer to the ability of a nucleic acid molecule to hybridize to at least about 6 consecutive nucleotides of a sample nucleic acid.

[0205] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein.

[0206] "Naturally occurring," "native," or "wild type" are used to describe objects that can be found in nature as distinct from those that are artificially produced. For example, a nucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified in a laboratory occurs naturally. Additionally, "wild type" refers to a normal gene or organism found in nature without any known mutations.

[0207] The following terms are used to describe the sequence relationships between two or more nucleic acids, polynucleotides, or polypeptides: (a) "reference sequence," (b) "comparison window," (c) "sequence identity," (d) "percentage of sequence identity," and (e) "substantial identity." (a) As used herein, a "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a particular sequence, for example, as a segment of a full-length cDNA or gene sequence, or as a complete cDNA or gene sequence. (b) As used herein, "comparison window" refers to a specific contiguous segment of a polynucleotide sequence, and the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (not containing additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides long, and can be optionally 30, 40, 50, 100 or longer. Those skilled in the art will understand that in order to avoid high similarity with the reference sequence due to the inclusion of gaps in the polynucleotide sequence, gap penalties are typically introduced and subtracted from the number of matches.

[0208] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm.

[0209] For comparison of sequences to determine sequence identity, computer implementations of these mathematical algorithms can be used. Such implementations include, but are not limited to, the CLUSTAL of the PC / gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (version 2.0) of the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA), and GAP, BESTFIT, BLAST, FASTA, and TFASTA. Alignments using these programs can be performed using default parameters.

[0210] Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (see the World Wide Web at ncbi.nlm.nih.gov). The algorithm identifies high-scoring sequence pairs (HSPs) by first identifying short words of length W in the query sequence that match or meet a positive threshold score T when aligned with words of the same length in a database sequence. T is called the neighborhood word score threshold. These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by a quantity X from its maximum achieved value, when the accumulation of one or more negatively scoring residue alignments causes the cumulative score to fall below zero, or when the end of either sequence is reached.

[0211] In addition to calculating the percentage of sequence identity, BLAST algorithm also performs statistical analysis of the similarity between two sequences.One measure of similarity provided by BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that the match between two nucleotides or the match between amino acid sequences occurs by chance.For example, if the minimum total probability in the comparison between the test nucleic acid sequence and the reference nucleic acid sequence is less than about 0.1, less than about 0.01, or even less than about 0.001, the test nucleic acid sequence is considered to be similar to the reference sequence.

[0212] To obtain gapped alignments for comparison purposes, Gapped BLAST (BLAST 2.0) can be utilized. Alternatively, PSI-BLAST (BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. When using BLAST, Gapped BLAST, or PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See the World Wide Web at ncbi.n1m.nih.gov. Alignments can also be performed manually by visual inspection.

[0213] Comparison of nucleotide sequences to determine the percentage of sequence identity with the promoter sequences disclosed herein can be performed using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program. By "equivalent program" is intended any sequence comparison program that produces an alignment having identical nucleotide or amino acid residue matches and the same percentage of sequence identity for any two sequences of interest when compared to the corresponding alignment produced by the BLAST program.

[0214] (c) As used herein, "sequence identity" or "identity" in reference to two nucleic acid or polypeptide sequences refers to a certain percentage of residues in the two sequences that are the same when aligned for maximum matching over a specified comparison window, as measured by a sequence comparison algorithm or by visual inspection. When percentages of sequence identity are used in reference to proteins, it is recognized that non-identical residue portions often differ by conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue that has similar chemical properties (e.g., charge or hydrophobicity), thus not changing the functional properties of the molecule. When sequences differ in terms of conservative substitutions, the percentage of sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those skilled in the art. Usually, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of zero, conservative substitutions are given a score between zero and 1. Scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0215] (d) As used herein, "percentage of sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0216] (e)(i) The term "substantial identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes usually means at least 70%, or at least 80%, 90%, or even at least 95% sequence identity.

[0217] Another indication that nucleotide sequences are substantially identical is when two molecules hybridize to each other under stringent conditions (see below). Generally, stringent conditions are determined by the thermal melting point (T m) is selected to be about 5°C lower than the desired temperature. However, stringent conditions encompass temperatures ranging from about 1°C to about 20°C, depending on the degree of stringency desired, as otherwise limited herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This can occur, for example, when copies of nucleic acids are made using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid.

[0218] (e)(ii) The term "substantial identity" in the context of a peptide indicates that the peptide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity with the reference sequence over a specified comparison window. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with an antibody raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, when the two peptides differ only by conservative substitutions.

[0219] For sequence comparison, one sequence usually serves as a reference sequence to which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence(s) to reference sequence based on the designated program parameters.

[0220] The term "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. If the RNA transcript is a perfect complementary copy of the DNA sequence, it is called a primary transcript, or it may be an RNA sequence derived from post-transcriptional processing of the primary transcript, and is called a mature RNA. "Messenger RNA" (mRNA) refers to RNA that does not contain introns and can be translated into protein by the cell. "cDNA" refers to single- or double-stranded DNA that is complementary to and derived from mRNA.

[0221] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention to alter the natural course of the individual being treated, and may be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improvement or palliative of the disease state, and remission or improved prognosis.

[0222] The phrase "effective amount" refers to an amount of a compound described herein that (i) treats or prevents a particular disease, condition or disorder, (ii) reduces, ameliorate or eliminates one or more symptoms of a particular disease, condition or disorder, or (iii) prevents or delays one or more symptoms of a particular disease, condition or disorder described herein.

[0223] The "therapeutically effective amount" of a substance / molecule disclosed herein may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the substance / molecule that induces the desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or adverse effects of the substance / molecule are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective at the dosages and for the period of time necessary to achieve the desired prophylactic result. Typically, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount, since a prophylactic dose is used in subjects before or at an early stage of disease.

[0224] The term "mammal" refers to any mammalian species, such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, farm animals, etc.

[0225] "Obtaining a sample from a patient," "obtained from a patient," and similar phrases are used to refer to obtaining a sample directly from a patient, as well as obtaining a sample indirectly from a patient through an intermediate entity (e.g., obtaining a sample from a courier who obtains the sample from a nurse who obtains the sample from the patient).

[0226] The term "enriched population" in the context of a cell composition means that the population contains a substantially greater proportion of a particular cell type than is found in the tissue from which the cells are derived. The specified type of cells may be enriched by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 500%, or 1000% compared to the native tissue. Alternatively, the cell population may contain at least 20%, 50%, 70%, 80%, 90%, or 95% of the specified cell type.

[0227] Specific Embodiments Particular embodiments described herein are included below.

[0228] Embodiment 1. A method for treating lipid metabolism dysregulation in a mammal in need of such treatment, comprising administering to said mammal an effective amount of an agonist anti-TREM2 (triggering receptor expressed on myeloid cells 2) antibody.

[0229] Embodiment 2. The method of embodiment 1, wherein cells expressing TREM2 in the mammal exhibit lipid metabolism dysregulation.

[0230] Embodiment 3. The method of embodiment 2, wherein the cell is a microglial cell or a macrophage.

[0231] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the mammal has, or has been determined to have, reduced TREM2 activity.

[0232] Embodiment 5. The method of embodiment 4, wherein said decreased TREM2 activity is caused by decreased TREM2 protein levels.

[0233] Embodiment 6 The method of embodiment 4, wherein said decreased TREM2 activity is caused by decreased cell surface protein levels.

[0234] Embodiment 7. The method of embodiment 4, wherein said reduced TREM2 activity is caused by a TREM2 loss-of-function or partial loss-of-function mutation.

[0235] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the mammal has, or has been determined to have, reduced apolipoprotein E (ApoE) activity.

[0236] Embodiment 9. The method of embodiment 8, wherein the mammal has, or has been determined to have, an APOE loss-of-function or partial loss-of-function mutation or coding variant.

[0237] Embodiment 10. The method of any one of embodiments 1 to 7, wherein the mammal has, or has been determined to have, an APOE ε4 allele.

[0238] Embodiment 11. The method of any one of embodiments 1 to 7, wherein the mammal does not have, or has been determined to not have, the APOE ε4 allele.

[0239] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the lipid metabolism dysregulation comprises increased accumulation of one or more lipids.

[0240] Embodiment 13 The method of embodiment 12, wherein said increased accumulation of said one or more lipids is intracellular.

[0241] Embodiment 14 The method of embodiment 13, wherein the one or more lipids accumulate intracellularly in microglial cells or macrophages.

[0242] Embodiment 15 The method of embodiment 12, wherein said increased accumulation of said one or more lipids is extracellular.

[0243] Embodiment 16. The method of any one of embodiments 12-15, wherein the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, bis(monoacylglycero)phosphate species (BMPs), diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, and combinations thereof.

[0244] Embodiment 17 The method of embodiment 16, wherein the one or more lipids comprise a cholesteryl ester.

[0245] Embodiment 18. The method of any one of embodiments 1 to 17, wherein the mammal has inflammation associated with said lipid metabolism dysregulation.

[0246] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the mammal has or is susceptible to developing Alzheimer's disease, Nasu-Hakola disease (NHD), Lewy body disease, Parkinson's disease, retinal degeneration (e.g., macular degeneration), Huntington's disease, frontotemporal lobar degeneration (FTD), amyotrophic lateral sclerosis (ALS), Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, obesity, type 2 diabetes, alcoholic or non-alcoholic steatohepatitis, alcoholic or non-alcoholic fatty liver disease, multiple sclerosis, vanishing white matter disease, rheumatoid arthritis (RA) or atherosclerosis.

[0247] Embodiment 20. The method of any one of embodiments 1 to 18, wherein the mammal has or is susceptible to developing Alzheimer's disease, Nasu-Hakola disease (NHD), Lewy body disease, Parkinson's disease, retinal degeneration, Huntington's disease, frontotemporal lobar degeneration (FTD), amyotrophic lateral sclerosis (ALS), Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, multiple sclerosis or vanishing white matter disease.

[0248] Embodiment 21. The method of any one of embodiments 1-18, wherein the mammal has or is susceptible to obesity, type 2 diabetes, alcoholic or non-alcoholic steatohepatitis, alcoholic or non-alcoholic fatty liver disease, rheumatoid arthritis (RA), or atherosclerosis.

[0249] Embodiment 22 The method of embodiment 19, wherein the mammal has or is susceptible to developing Alzheimer's disease.

[0250] Embodiment 23 The method of embodiment 19, wherein the mammal has or is susceptible to developing NHD.

[0251] Embodiment 24 The method of embodiment 19, wherein the mammal has or is susceptible to developing atherosclerosis.

[0252] Embodiment 25. The method of embodiment 19, wherein the mammal has or is susceptible to developing Niemann-Pick disease type C.

[0253] Embodiment 26. The method of any one of embodiments 1 to 25, wherein the agonistic anti-TREM2 antibody is MAB17291 or 78.18.

[0254] Embodiment 27. The method of any one of embodiments 1 to 26, wherein the agonistic anti-TREM2 antibody reduces lipid accumulation.

[0255] Embodiment 28 The method of embodiment 27, wherein the agonist anti-TREM2 antibody reduces accumulation of cholesteryl esters.

[0256] Embodiment 29 The method of embodiment 27 or 28, wherein the agonistic anti-TREM2 antibody reduces intracellular lipid accumulation.

[0257] Embodiment 30 The method of embodiment 27 or 28, wherein said agonistic anti-TREM2 antibody reduces extracellular lipid accumulation.

[0258] Embodiment 31 The method of any one of embodiments 1 to 30, wherein said administering reduces expression of at least one proinflammatory cytokine.

[0259] Embodiment 32. The method of embodiment 31, wherein the at least one cytokine is selected from the group consisting of G-CSF, INFy, IL-12(p40), IL-12(p70), LIX(CXCL5), MCP-1(CCL2), MIG(CXCL9), IL-1α, IL-1β, and IL-18.

[0260] Embodiment 33 The method of embodiment 32, wherein the at least one cytokine is IL-1β.

[0261] Embodiment 34. The method of any one of embodiments 1 to 33, further comprising administering a second therapeutic agent.

[0262] Embodiment 35. The method of embodiment 34, wherein the second therapeutic agent is selected from the group consisting of an RXR agonist, an LXR agonist, and an acetyl-CoA acetyltransferase 1 (ACAT1) inhibitor.

[0263] Embodiment 36 The method of embodiment 35, wherein the second therapeutic agent is an RXR agonist.

[0264] Embodiment 37 The method of embodiment 36, wherein the RXR agonist is bexarotene.

[0265] Embodiment 38. The method of embodiment 35, wherein the second therapeutic agent is an LXR agonist.

[0266] Embodiment 39. The method of embodiment 38, wherein the LXR agonist is GW3965.

[0267] Embodiment 40 The method of embodiment 35, wherein the second therapeutic agent is an acetyl-CoA acetyltransferase 1 (ACAT1) inhibitor.

[0268] Embodiment 41. The method of embodiment 40, wherein the ACAT1 inhibitor is CP-113, 818, CI-1011, or K-604.

[0269] Embodiment 42 The method of embodiment 41, wherein the ACAT1 inhibitor is K-604.

[0270] Embodiment 43 The method of embodiment 34, wherein the second therapeutic agent is an agent useful for treating Alzheimer's disease or atherosclerosis.

[0271] Embodiment 44. A method for treating lipid metabolism dysregulation in a patient in need thereof, comprising: 1) obtaining or having obtained a biological sample from said patient; 2) detecting or having detected reduced TREM2 activity, reduced ApoE activity, or the APOE ε4 allele in said sample; 3) diagnosing the patient as having lipid metabolism dysregulation when reduced TREM2 activity, reduced ApoE activity, or the APOE ε4 allele is detected; 4) administering to the diagnosed patient an effective amount of an agonistic anti-TREM2 antibody.

[0272] Embodiment 45. A method of treating a patient with an agonist anti-TREM2 antibody, the method comprising: 1) obtaining or having obtained a biological sample from said patient; 2) analyzing the biological sample or analyzing the sample to detect reduced TREM2 activity, reduced ApoE activity, or the presence of the APOE ε4 allele, thereby diagnosing the patient as having a lipid metabolism dysregulation; 3) administering to the diagnosed patient an effective amount of an agonistic anti-TREM2 antibody.

[0273] Embodiment 46. An agonistic anti-TREM2 antibody for use in treating lipid metabolism dysregulation in said mammal.

[0274] Embodiment 47. The antibody for use according to embodiment 46, wherein the mammal has, or has been determined to have, reduced TREM2 activity.

[0275] Embodiment 48. The antibody for use according to embodiment 46 or 47, wherein the mammal has, or has been determined to have, reduced ApoE activity.

[0276] Embodiment 49. The antibody for use according to embodiment 46 or 47, wherein the mammal has, or has been determined to have, an APOE loss-of-function or partial loss-of-function mutation or coding variant.

[0277] Embodiment 50. The antibody for use according to embodiment 46 or 47, wherein the mammal has, or is determined to have, the APOE ε4 allele.

[0278] Embodiment 51. The antibody for use according to embodiment 46 or 47, wherein the mammal does not have, or has been determined to not have, the APOE ε4 allele.

[0279] Embodiment 52. Use of an agonistic anti-TREM2 antibody for the preparation of a medicament for treating lipid metabolism dysregulation in a mammal.

[0280] Embodiment 53. The use of embodiment 52, wherein the mammal has, or has been determined to have, reduced TREM2 activity.

[0281] Embodiment 54. The use according to embodiment 52 or 53, wherein the mammal has reduced or has been determined to have reduced ApoE activity.

[0282] Embodiment 55. The use of embodiment 52 or 53, wherein the mammal has, or has been determined to have, an APOE loss-of-function or partial loss-of-function mutation or coding variant.

[0283] Embodiment 56. The use of embodiment 52 or 53, wherein the mammal has, or is determined to have, the APOE ε4 allele.

[0284] Embodiment 57. The use of embodiment 52 or 53, wherein the mammal does not have, or has been determined to not have, the APOE ε4 allele.

[0285] Embodiment 58. A method for reducing intracellular accumulation of one or more lipids in a cell, comprising contacting the cell with an effective amount of an agonistic anti-TREM2 antibody.

[0286] Embodiment 59. The method of embodiment 58, wherein the cells are microglial cells.

[0287] Embodiment 60 The method of embodiment 58, wherein the cell is a macrophage.

[0288] Embodiment 61. The method of any one of embodiments 58-60, wherein the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, BMPs, diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, and combinations thereof.

[0289] Embodiment 62. The method of embodiment 61, wherein the one or more lipids comprise a cholesteryl ester.

[0290] Embodiment 63 The method of any one of embodiments 58 to 62, wherein the cell has, or has been determined to have, reduced TREM2 activity.

[0291] Embodiment 64. The method of any one of embodiments 58 to 63, wherein the cells have, or have been determined to have, reduced ApoE activity.

[0292] Embodiment 65. The method of any one of embodiments 58 to 63, wherein the cell has, or has been determined to have, an APOE loss-of-function or partial loss-of-function mutation or coding variant.

[0293] Embodiment 66 The method of any one of embodiments 58 to 63, wherein the cells express, or have been determined to express, ApoE4.

[0294] Embodiment 67. The method of any one of embodiments 58 to 63, wherein the cells do not express, or have been determined to not express, ApoE4.

[0295] Embodiment 68. The method of any one of embodiments 58 to 67, wherein the agonistic anti-TREM2 antibody is MAB17291 or 78.18.

[0296] Embodiment 69. The method of any one of embodiments 58 to 68, wherein the cells are contacted with an agonistic anti-TREM2 antibody in vitro, in vivo, or ex vivo.

[0297] Embodiment 70 The method of any one of embodiments 58 to 68, wherein the cell is present in a mammal.

[0298] Embodiment 71. The method of any one of embodiments 58 to 68, wherein the cell is present in a mammal and is contacted with the agonistic anti-TREM2 antibody in vivo.

[0299] Embodiment 72 The method of embodiment 70 or 71, wherein the mammal has inflammation associated with intracellular lipid accumulation.

[0300] Embodiment 73. The method of embodiment 72, wherein said agonistic anti-TREM2 antibody reduces expression of at least one proinflammatory cytokine.

[0301] Embodiment 74. The method of embodiment 73, wherein the at least one cytokine is selected from the group consisting of G-CSF, INFy, IL-12(p40), IL-12(p70), LIX(CXCL5), MCP-1(CCL2), MIG(CXCL9), IL-1α, IL-1β, and IL-18.

[0302] Embodiment 75. The method of embodiment 74, wherein the at least one cytokine is IL-1β.

[0303] Embodiment 76. The method of any one of embodiments 70 to 75, wherein the mammal has or is susceptible to developing Alzheimer's disease, NHD, Lewy body disease, Parkinson's disease, retinal degeneration (e.g., macular degeneration), Huntington's disease, FTD, ALS, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, obesity, type 2 diabetes, alcoholic or non-alcoholic steatohepatitis, alcoholic or non-alcoholic fatty liver disease, multiple sclerosis, vanishing white matter disease, RA or atherosclerosis.

[0304] Embodiment 77. The method of embodiment 76, wherein the mammal has or is susceptible to developing NHD.

[0305] Embodiment 78. The method of embodiment 76, wherein the mammal has or is susceptible to developing atherosclerosis.

[0306] Embodiment 79. The method of embodiment 76, wherein the mammal has or is susceptible to developing Niemann-Pick disease type C.

[0307] Embodiment 80 The method of any one of embodiments 58-79, further comprising contacting the cells with a second therapeutic agent.

[0308] Embodiment 81 The method of embodiment 80, wherein the second therapeutic agent is an RXR agonist.

[0309] Embodiment 82. The method of embodiment 81, wherein the RXR agonist is bexarotene.

[0310] Embodiment 83. The method of embodiment 80, wherein the second therapeutic agent is an LXR agonist.

[0311] Embodiment 84. The method of embodiment 83, wherein the LXR agonist is GW3965.

[0312] Embodiment 85 The method of embodiment 80, wherein the second therapeutic agent is an ACAT1 inhibitor.

[0313] Embodiment 86. The method of embodiment 85, wherein the ACAT1 inhibitor is CP-113, 818, CI-1011, or K-604.

[0314] Embodiment 87. The method of embodiment 86, wherein the ACAT1 inhibitor is K-604.

[0315] Embodiment 88. An agonistic anti-TREM2 antibody for use in reducing the intracellular accumulation of one or more lipids in a cell.

[0316] Embodiment 89. Use of an agonistic anti-TREM2 antibody for the preparation of a medicament for reducing the intracellular accumulation of one or more lipids in a cell.

[0317] Embodiment 90. A method for treating Alzheimer's disease in a mammal in need of such treatment, comprising administering to the mammal an agonist anti-TREM2 antibody, wherein the mammal has, or has been determined to have, lipid metabolism dysregulation.

[0318] Embodiment 91 The method of embodiment 90, wherein the mammal has, or has been determined to have, lipid metabolism dysregulation in TREM2-expressing cells.

[0319] Embodiment 92. A method for treating Alzheimer's disease in a mammal in need of such treatment, comprising administering to the mammal an agonistic anti-TREM2 antibody, wherein the mammal has, or has been determined to have, lipid metabolism dysregulation in TREM2-expressing cells.

[0320] Embodiment 93 The method of embodiment 91 or 92, wherein the TREM2-expressing cells are microglial cells.

[0321] Embodiment 94. The method of any one of embodiments 91 to 93, wherein the TREM2-expressing cells have, or have been determined to have, reduced TREM2 activity.

[0322] Embodiment 95. The method of any one of embodiments 90 to 94, wherein the lipid metabolism dysregulation comprises increased intracellular accumulation of one or more lipids.

[0323] Embodiment 96. The method of embodiment 95, wherein the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, BMPs, diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, and combinations thereof.

[0324] Embodiment 97. The method of embodiment 96, wherein the one or more lipids comprise a cholesteryl ester.

[0325] Embodiment 98. The method of any one of embodiments 90 to 97, wherein the mammal has inflammation associated with said lipid metabolism dysregulation.

[0326] Embodiment 99. The method of embodiment 98, wherein said administering reduces expression of at least one proinflammatory cytokine.

[0327] Embodiment 100. The method of embodiment 99, wherein the at least one cytokine is selected from the group consisting of G-CSF, INFy, IL-12(p40), IL-12(p70), LIX(CXCL5), MCP-1(CCL2), MIG(CXCL9), IL-1α, IL-1β, and IL-18.

[0328] Embodiment 101. The method of embodiment 100, wherein the at least one cytokine is IL-1β.

[0329] Embodiment 102. The method of any one of embodiments 90 to 101, wherein the agonistic anti-TREM2 antibody is MAB17291 or 78.18.

[0330] Embodiment 103. The method of any one of embodiments 90 to 102, further comprising administering to the mammal a second therapeutic agent.

[0331] Embodiment 104. The method of embodiment 103, wherein the second therapeutic agent is an RXR agonist.

[0332] Embodiment 105. The method of embodiment 104, wherein the RXR agonist is bexarotene.

[0333] Embodiment 106. The method of embodiment 103, wherein the second therapeutic agent is an LXR agonist.

[0334] Embodiment 107. The method of embodiment 106, wherein the LXR agonist is GW3965.

[0335] Embodiment 108. The method of embodiment 103, wherein the second therapeutic agent is an ACAT1 inhibitor.

[0336] Embodiment 109. The method of embodiment 108, wherein the ACAT1 inhibitor is CP-113, 818, CI-1011, or K-604.

[0337] Embodiment 110. The method of embodiment 109, wherein the ACAT1 inhibitor is K-604.

[0338] Embodiment 111. An agonistic anti-TREM2 antibody for use in treating Alzheimer's disease in a mammal, wherein the mammal has, or is determined to have, a lipid metabolism dysregulation.

[0339] Embodiment 112. An agonistic anti-TREM2 antibody for use in treating Alzheimer's disease in a mammal, wherein the mammal has, or has been determined to have, lipid metabolism dysregulation in TREM2-expressing cells.

[0340] Embodiment 113. Use of an agonistic anti-TREM2 antibody for the preparation of a medicament for treating Alzheimer's disease in a mammal, wherein the mammal has, or is determined to have, a lipid metabolism dysregulation.

[0341] Embodiment 114. Use of an agonistic anti-TREM2 antibody for the preparation of a medicament for treating Alzheimer's disease in a mammal, wherein the mammal has, or has been determined to have, lipid metabolism dysregulation in TREM2-expressing cells.

[0342] Embodiment 115. A method of treating atherosclerosis in a mammal in need of such treatment, comprising administering to the mammal an effective amount of an agonistic anti-TREM2 antibody.

[0343] Embodiment 116 The method of embodiment 115, wherein the mammal has, or has been determined to have, a lipid metabolism dysregulation.

[0344] Embodiment 117. The method of embodiment 115 or 116, wherein the lipid metabolism dysregulation comprises increased accumulation of one or more lipids.

[0345] Embodiment 118. The method of embodiment 117, wherein the increased accumulation of the one or more lipids is intracellular.

[0346] Embodiment 119. The method of embodiment 117, wherein the one or more lipids accumulate intracellularly in macrophages.

[0347] Embodiment 120. The method of embodiment 119, wherein the macrophages have, or have been determined to have, reduced TREM2 activity.

[0348] Embodiment 121 The method of embodiment 117, wherein the increased accumulation of the one or more lipids is extracellular.

[0349] Embodiment 122. The method of any one of embodiments 117 to 121, wherein the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, BMPs, diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, and combinations thereof.

[0350] Embodiment 123. The method of embodiment 122, wherein the one or more lipids comprise a cholesteryl ester.

[0351] Embodiment 124. The method of any one of embodiments 116 to 123, wherein the mammal has inflammation associated with said lipid metabolism dysregulation.

[0352] Embodiment 125. The method of embodiment 124, wherein said administering reduces expression of at least one proinflammatory cytokine.

[0353] Embodiment 126. The method of embodiment 125, wherein the at least one cytokine is selected from the group consisting of G-CSF, INFy, IL-12(p40), IL-12(p70), LIX(CXCL5), MCP-1(CCL2), MIG(CXCL9), IL-1α, IL-1β, and IL-18.

[0354] Embodiment 127. The method of embodiment 126, wherein said at least one cytokine is IL-1β.

[0355] Embodiment 128. The method of any one of embodiments 115 to 127, wherein the agonistic anti-TREM2 antibody is MAB17291 or 78.18.

[0356] Embodiment 129. The method of any one of embodiments 115 to 128, further comprising administering a second therapeutic agent.

[0357] Embodiment 130. The method of embodiment 129, wherein the second therapeutic agent is an agent useful for treating atherosclerosis.

[0358] Embodiment 131. The method of embodiment 129, wherein the second therapeutic agent is an RXR agonist, an LXR agonist, or an ACAT1 inhibitor.

[0359] Embodiment 132. An agonistic anti-TREM2 antibody for use in treating atherosclerosis in a mammal.

[0360] Embodiment 133. Use of an agonistic anti-TREM2 antibody for the preparation of a medicament for treating atherosclerosis in a mammal.

[0361] Embodiment 134. A method of treating inflammation in a mammal in need of such treatment, comprising administering to the mammal an effective amount of an agonistic anti-TREM2 antibody.

[0362] Embodiment 135. The method of embodiment 134, wherein said administering reduces expression of at least one proinflammatory cytokine.

[0363] Embodiment 136 The method of embodiment 135, wherein the at least one cytokine is associated with an inflammasome response.

[0364] Embodiment 137. The method of embodiment 135 or 136, wherein the at least one cytokine is selected from the group consisting of G-CSF, INFy, IL-12(p40), IL-12(p70), LIX(CXCL5), MCP-1(CCL2), MIG(CXCL9), IL-1α, IL-1β, and IL-18.

[0365] Embodiment 138. The method of embodiment 137, wherein the at least one cytokine is IL-1β.

[0366] Embodiment 139. The method of any one of embodiments 134 to 138, wherein the mammal has or is susceptible to an inflammasome-associated disease or disorder.

[0367] Embodiment 140. The method of any one of embodiments 134-138, wherein the mammal has or is susceptible to developing rheumatoid arthritis, gout, or inflammatory bowel disease (IBD).

[0368] Embodiment 141. The method of any one of embodiments 134 to 138, wherein the inflammation is associated with lipid metabolism dysregulation.

[0369] Embodiment 142 The method of embodiment 141, wherein administration of the agonistic anti-TREM2 antibody reduces lipid accumulation.

[0370] Embodiment 143. The method of embodiment 141 or 142, wherein the mammal has or is susceptible to developing Alzheimer's disease, Nasu-Hakola disease (NHD), Lewy body disease, Parkinson's disease, retinal degeneration (e.g., macular degeneration), Huntington's disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, obesity, type 2 diabetes, alcoholic or non-alcoholic steatohepatitis, alcoholic or non-alcoholic fatty liver disease, multiple sclerosis, vanishing white matter disease, or atherosclerosis.

[0371] Embodiment 144. The method of embodiment 141 or 142, wherein the mammal has or is susceptible to developing Alzheimer's disease, Nasu-Hakola disease (NHD), Lewy body disease, Parkinson's disease, retinal degeneration, Huntington's disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, multiple sclerosis or vanishing white matter disease.

[0372] Embodiment 145. The method of embodiment 141 or 142, wherein the mammal has or is susceptible to obesity, type 2 diabetes, alcoholic or non-alcoholic steatohepatitis, alcoholic or non-alcoholic fatty liver disease, or atherosclerosis.

[0373] Embodiment 146. The method of any one of embodiments 134 to 145, wherein the agonistic anti-TREM2 antibody is MAB17291 or 78.18.

[0374] Embodiment 147. The method of any one of embodiments 134 to 146, further comprising administering a second therapeutic agent.

[0375] Embodiment 148. The method of embodiment 147, wherein the second therapeutic agent is an RXR agonist, an LXR agonist, or an ACAT1 inhibitor.

[0376] Embodiment 149. An agonistic anti-TREM2 antibody for use in treating inflammation in a mammal.

[0377] Embodiment 150. Use of an agonistic anti-TREM2 antibody for the preparation of a medicament for treating inflammation in a mammal.

[0378] Embodiment 151. The antibody of embodiment 149 or the use of embodiment 150, wherein the inflammation is associated with lipid metabolism dysregulation.

[0379] Embodiment 152. A method for selecting a population of CNS cells from a tissue sample, comprising: (a) contacting the tissue sample with a primary anti-CD45 antibody, a primary anti-CD11b antibody, and a primary anti-astrocyte surface antigen-2 (ACSA-2) antibody, each primary antibody being uniquely labeled, to provide a labeled tissue sample; (b) sorting the cells in the labeled tissue sample by flow cytometry; The method, wherein the method provides separate cell populations of astrocytes and microglial cells.

[0380] Embodiment 153. The method of claim 152, wherein the primary anti-CD45 antibody, the primary anti-CD11b antibody, and the primary anti-ACSA-2 antibody are present in a composition.

[0381] Embodiment 154. A method for selecting a population of CNS cells from a tissue sample, comprising: (a) contacting the tissue sample with a composition comprising a primary anti-CD45 antibody, a primary anti-CD11b antibody, and a primary anti-astrocyte surface antigen-2 (ACSA-2) antibody, each primary antibody being uniquely labeled, to provide a labeled tissue sample; (b) sorting the cells in the labeled tissue sample by flow cytometry; The method, wherein the method provides separate cell populations of astrocytes and microglial cells.

[0382] Embodiment 155. The method of embodiment 153 or 154, wherein the composition further comprises a viability dye.

[0383] Embodiment 156. The method of any one of embodiments 152 to 154, further comprising contacting the tissue sample with a viability dye.

[0384] Embodiment 157. The method of any one of embodiments 152 to 156, providing a distinct population of microglial cells comprising less than about 20% non-microglial cells.

[0385] Embodiment 158. The method of any one of embodiments 152 to 156, providing a distinct population of distinct astrocytes comprising less than about 20% non-astroglial cells.

[0386]

[0081] Embodiment 159. The microglial cell population has the following marker profile: CD45 low / CD11b + / ACSA-2 - The method of any one of embodiments 152 to 158, wherein the method is selected based on:

[0387]

[0023] Embodiment 160. The astrocyte population has the following marker profile: CD45 - / CD11b - / ACSA-2 + The method of any one of embodiments 152 to 159, wherein the method is selected based on:

[0388] Embodiment 161. The method of any one of embodiments 152 to 160, wherein the distinct cell populations are analyzed for quantification of metabolic or nucleic acid species.

[0389] Embodiment 162. The method of embodiment 161, wherein the metabolic species is a lipid species.

[0390] Embodiment 163. The method of embodiment 161, wherein the nucleic acid species is selected from RNA, DNA, and genomic DNA.

[0391] Embodiment 164. The method of any one of embodiments 152 to 160, wherein the distinct cell populations are analyzed for quantification of the therapeutic agent administered.

[0392] Embodiment 165. A composition comprising a distinct cell population isolated by the method of any one of embodiments 152 to 160.

[0393] Embodiment 166 The composition of embodiment 165, wherein the distinct cell population is a microglial cell population.

[0394] Embodiment 167. The composition of embodiment 165, wherein the distinct cell population is an astrocyte cell population.

[0395] Embodiment 168. A population of CNS cells comprising two physically distinct cell populations, wherein the first cell population is CD45 low / CD11b + / ACSA-2 - the second cell population comprising an enriched population of CD45 cells; - / CD11b - / ACSA-2 + The population of CNS cells comprising cells.

[0396] The following examples are intended to be non-limiting. EXAMPLES

[0397] Example 1: Attenuated expression of genes involved in lipid metabolism in Trem2 knockout mice with chronic demyelination In this example, mouse microglial gene expression analysis is described.

[0398] Dietary cuprizone induces demyelination in mice. Trem2 - / - Mice were purchased from Jackson Laboratory (Stock #:027197) and backcrossed to C57BL / 6J mice to express Trem2 + / - We generated mice. Trem2 + / - The mice were further crossed to generate littermates of three genotypes for this study: Trem2 + / + , Trem2 + / - and Trem2 - / - ) were generated. Mice around 9-11 months of age were used. Each genotype of mice was divided into two groups (6-11 mice / group) with either a treatment paradigm of normal diet (Envigo TD.160766) or cuprizone diet (0.2% cuprizone, Envigo TD.160765) for 5 or 12 weeks. The weight of each animal was recorded weekly to monitor the effect of cuprizone.

[0399] Fluorescence-activated cell sorting (FACS) of microglia, astrocytes, and other cells from mouse brain To prepare single cell suspensions for sorting CNS cells, mice were perfused with PBS, brains were dissected, and processed into single cell suspensions using an Adult Brain Dissociation Kit (Miltenyi Biotec 130-107-677) according to the manufacturer's protocol. Cells were Fc blocked and stained for flow cytometry analysis with Fixable Viability Stain BV510 to exclude dead cells (BD Biosciences 564406), CD11b-BV421 (BD Biosciences 562605), CD45-APC (BD Biosciences 559864), and ACSA-2-PE (Milteni Biotec 130-102-365). Cells were washed twice with Hibernate A (BrainBits LLC) and strained through a 100 μm filter before being sorted on a FACS Aria III (BD Biosciences) with a 100 μm nozzle for CD11b. + Microglia and ACSA-2 + Astrocytes were sorted and sorted cells were processed for downstream analyses including RNAseq, scRNAseq or lipidomics.

[0400] FACS-RNAseq analysis of microglial gene expression Live cells were stained with CD11b against all other unstained cells (100,000–200,000 cells). +Microglia (100,000–120,000 cells) were sorted and collected directly into RLT+ buffer (Qiagen) containing 1:100 β-mercaptoethanol. RNA was extracted using the RNeasy Plus Micro Kit (Qiagen, 74034) and resuspended in 14 μl nuclease-free water. RNA quantity and quality were assessed with an RNA 6000 Pico chip (Agilent 5067-1513) on a 2100 Bioanalyzer (Agilent). RNA was processed using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) according to the manufacturer-defined "low input" protocol. Barcoded samples were quantified using the NEBNext Library Quant Kit for Illumina (NEB, E7630S). All samples were pooled in equimolar ratios into one sequencing library, which was quantified on a Bioanalyzer with a High Sensitivity DNA chip (Agilent, 5067-4626). Single-end reads of 50 bp were generated on an Illumina HiSeq 4000 lane at the UCSF Center for Advanced Technology.

[0401] Reads were aligned to the mouse genome version GRCm 38_p6. A STAR index (Dobin, A et al., Bioinformatics, 2013.29(1):p.15-21; version 2.5.3a) was constructed using the --sjdbOverhang=50 argument. Splice junctions from Gencode gene models (release M17) were provided via the --sjdbGTFfile argument. STAR alignments were generated using the following parameters: --outFilterType BySJout, --quantMode TranscriptomeSAM, --outFilterIntronMotifs RemoveNoncanonicalUnannotated, --outSAMstrandField intronMotif, --outSAMattributes NH HI AS nM MD XS, and --outSAMunmapped Within. Alignments were obtained using the following parameters:--readFilesCommand zcat--outFilterType BySJout--outFilterMultimapNmax 20--alignSJoverhangMin 8--alignSJDBoverhangMin 1--outFilterMismatchNmax 999--outFilterMismatchNoverLmax 0.6--alignIntronMin 20--alignIntronMax 1000000--alignMatesGapMax 1000000--quantMode GeneCounts--outSAMunmapped Within--outSAMattributes NH HI AS nM MD XS-outSAMstrandField intronMotif-outSAMtype BAM SortedByCoordinate-outBAMcompression 6. Gene-level counts were obtained using featureCounts from the subread package (Liao, Y et al., Nucleic Acids Res, 2013.41(10):e108; version 1.6.2).Gene symbols and Entrez gene identifiers were mapped using Ensembl (version 91) with R (version 3.4.3) via the biomaRt R package (Durinck, S et al., Nat Protoc, 2009.4(8):p.1184-91; version 2.34.2).

[0402] To identify differentially expressed genes, linear models were fitted using the limma Bioconductor package (Liu, R et al., Nucleic Acids Res, 2015.43:p.e97). Statistical analysis included only genes with sufficiently large counts, as determined by the "filterByExpr" function in edgeR. TMM scaling coefficients for each sample were calculated with the "calcNormFactors" function (Robinson, MD et al., Genome Biol, 2010.11(3):p.R25). We estimated the mean-variance relationship of log2 transformation counts and derived observation-level weights with the "voom" function from the limma Bioconductor package (Liu, R et al., Nucleic Acids Res 2015.43:p.e97). Linear models were fitted with the "lmFit" and "eBayes" functions. Results were plotted using the ggplot2 R package (Wickham, H et al., ggplot2: Elegant Graphics for Data Analysis, 2016). Competitive gene set testing was performed using the "camera" algorithm from the limma R package (Wu, D et al., Nucleic Acids Res, 2012.40(17):p.e133).

[0403] Single-cell RNA-seq library preparation and analysis (2) Control diet Trem2 + / + Hemibrain, and (2) Trem2 + / + (2) Trem2 + / - , and (2) Trem2 - / -Dissociated cells from 12-week cuprizone-treated hemi-brains were processed and stained as described above. 30,000 live CD11b+ / CD45lo microglia were sorted from each hemi-brain and (2) hemi-brains per condition were pooled in PBS+0.5% BSA to generate a total of four sequencing groups. Microglia were counted, diluted to 500,000 cells / ml in 70 μl, and confirmed to be >70% viable. Single-cell libraries were barcoded and prepared at the Stanford Functional Genomics Facility using the Chromium Single Cell 3'Library Kit with v2 chemistry (10X Genomics, product # 120267) with a Chromium Controller (10X Genomics). ScRNASeq libraries were sequenced using a NovaSeq S4 at the Center for Advanced Technology at UCSF.

[0404] Four single-cell datasets were generated. See DropletUtils (Lun, ATL et al., biorXiv, 2018. doi:10.1101 / 234872), Scater (McCarthy DJ et al., Bioinformatics, 2017.33(8):p.1179-1186), and scran (Lun, ATL et al., Genome Biology, 2016.17:p.75) Bioconductor (v3.8) packages. Each experiment was independently quality controlled and filtered in the following manner: (i) droplets containing only ambient RNA (FDR<0.01) were identified and removed using the "empty droplets" function, and (ii) cells with low read counts, low gene counts, or high mitochondrial load were identified and removed with the "isOutlier" function. The remaining cells from each experiment were then compiled into a universal atlas of cells for downstream analysis. The "computeSumFactors" function was used to account for differences in sequencing depth per cell, and normalized log counts for each gene were calculated with the "normalize" function. Initial PCA and subsequent tSNE analysis further identified cell groups from each sample where high mitochondrial loading was the only real distinguishing characteristic. This cell group was considered to be a technical artifact and was removed to compose the final high-quality cell atlas.

[0405] Ten clusters of microglial cells were identified in the atlas by first building a shared nearest neighbor graph using the "buildSNNGraph" function (k=6), followed by community detection using the Louvian method. The largest cluster contains 595 cells (knn_07) and the smallest cluster contains 147 cells (knn_10). Clusters were first characterized by identifying the fraction of cells across the atlas that belonged to each cluster (Figure 2A, left), followed by the fraction of cells per sample that belonged to each cluster (Figure 2A, right). Marker gene and gene set enrichment analysis was performed for each cluster under two scenarios: universal - each cluster was tested against the entire dataset; or restricted - only the three interesting cell clusters (knn_05, knn_05, and knn_10) were tested against each other. Marker genes per cluster were identified by Wilcoxon Test using the "pairwiseWilcox" function and filtered using an FDR threshold of 0.001. Fold changes per gene were reported by taking the average of the log2 fold changes of genes in a given cluster and the average of the log2 fold changes of each remaining gene (calculated by the “pairwiseTTests” function). Table 1: Genes altered in microglial clusters knn 5, 8, and 10. [Table 1]

[0406] As shown in Figures 1A-C, 2A-B, and Table 1, genes involved in lipid metabolism were upregulated in Trem2 during acute and chronic demyelination. + / + and Trem2 + / - Trem2 is upregulated in mouse microglia (5 and 12 weeks of cuprizone treatment, respectively) with chronic and acute demyelination - / - Figure 2 shows reduced upregulation in mouse microglia. Bulk isolated Trem2 in mice on a control diet. + / + , Trem2 + / - , and Trem2- / - There is little genotypic variation among mouse microglia (Figure 1A). Hundreds of genes are involved in the bulk Trem2 expression in mice with chronic demyelination. + / + and Trem2 + / - Upregulated in mouse microglia, but Trem2 - / - Only a few genes were upregulated in microglia (Figure 1B). + / + and Trem2 + / - Among the genes that were upregulated in Trem2- / - but not in Trem2- / - microglia, many were involved in lipid metabolism (Figure 1C). + / + , Trem2 + / - , and Trem2 - / - The log2 fold change in gene expression of individual genes related to lipid metabolism in bulk microglia is shown compared to 5- or 12-week cuprizone treatment (right inserts, top or bottom, respectively). + / + , Trem2 + / - , and Trem2 - / - Mouse (Trem2 + / + CPZ, Trem2 + / - CPZ, Trem2 - / - Compared with microglia isolated from the CPZ, individually isolated Trem2 + / + Control diet microglia (Trem2 + / + Identifying microglial clusters in single-cell RNA sequencing data from 14C-T10 ... + / + CPZ and Trem2 + / - Upregulated in chronic demyelination in CPZ mice, but not in controls or Trem2 - / - Identifying a population of microglia with genes that are not upregulated in CPZ mice. Cluster knn_10 identifies Trem2 - / - Trem2, up- or down-regulated in CPZ mice + / + CPZ, Trem2 + / -Identifying microglial populations with genes that are not up- or down-regulated in CPZ or control mice. Table 1 lists the genes identified in clusters knn 5, 8, and 10, the log fold change and direction of change, and the false discovery rate (FDR).

[0407] Example 2. Increased cholesteryl esters and myelin lipids in Trem2 knockout forebrains and isolated microglia during chronic demyelination In this example, we describe the lipidology of mouse forebrains and isolated microglia and astroglial cell populations.

[0408] Forebrain lipid extraction After PBS perfusion, sagittal mouse hemibrains were flash frozen in liquid nitrogen and coronally frozen at -20°C in alternating 100 μm (lipidology) or 20 μm (histology) widths using a Leica CM 1950 cryostat. Two 100 μm sections from matched forebrain regions containing the corpus callosum were placed in 1.5 mL LoBind tubes (Eppendorf) containing 3 mm stainless steel beads (Qiagen) with 200 μl of LC-MS grade methanol containing internal standards. Tubes were lysed 2x:1 min, 25 Hz, 4°C using a TissueLyser (Qiagen). 20 μl samples were removed for protein concentration measurement using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). Lysates were spun for 20 min at 18,000xg, 4°C. Supernatants were transferred to glass LC-MS vials (Waters).

[0409] FACS lipid extraction Dissociated cells were stained according to the FACS protocol described above, except that all staining buffers contained PBS + 1% fatty acid free BSA (Sigma). 400 μl of LC-MS grade internal standard containing methanol was added to a 2 mL lo-bind tube (Eppendorf). After sorting, the total volume was adjusted to 800 μl with deionized water (Milli-Q). Samples were vortexed at 2500 rpm for 5 min at room temperature. 800 μl of methyl tertiary butyl ether (MTBE) was added and samples were vortexed at 2500 rpm for 5 min at room temperature, followed by spinning at 21000xg for 10 min at 4°C. 600 μl of MTBE supernatant was transferred to a glass LC-MS vial and dried under nitrogen gas. Samples were resuspended in 100 μl of LC-MS grade methanol.

[0410] Mass spectrometry of lipids Lipid analysis was performed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) coupled to electrospray mass spectrometry (QTRAP 6500+, Sciex, Framingham, MA, USA). For each analysis, 5 μL of sample was injected onto a BEH C18 1.7 μm, 2.1×100 mm column (Waters Corporation, Milford, Massachusetts, USA) using a flow rate of 0.25 mL / min at 55° C. For positive ionization mode, mobile phase A consisted of 60:40 acetonitrile / water (v / v) with 10 mM ammonium formate+0.1% formic acid, and mobile phase B consisted of 90:10 isopropyl alcohol / acetonitrile (v / v) with 10 mM ammonium formate+0.1% formic acid. For negative ionization mode, mobile phase A consisted of 60:40 acetonitrile / water (v / v) with 10 mM ammonium acetate, and mobile phase B consisted of 90:10 isopropyl alcohol / acetonitrile (v / v) with 10 mM ammonium acetate. The gradient was programmed as follows: 0.0–8.0 min at 45% B to 99% B, 8.0–9.0 min at 99% B, 9.0–9.1 min to 45% B, and 9.1–10.0 min at 45% B. Electrospray ionization was performed in either positive or negative ion mode applying the following settings: curtain gas was set at 30, collision gas was set at medium, ion spray voltage was 5500 (positive mode) or 4500 (negative mode), temperature was 250 °C (positive mode) or 600 °C (negative mode), ion source gas 1 was 50, and ion source gas 2 was 60.Data acquisition was performed using Analyst 1.6.3 (Sciex) in multiple reaction monitoring mode (MRM) with the following parameters: residence time (msec) and collision energy (CE) for each species reported in Table 2 (positive mode) or Table 3 (negative mode), declustering potential (DP) of 80, entrance potential (EP) of 10 (positive mode) or -10 (negative mode), and collision cell exit potential (CXP) of 12.5 (positive mode) or -12.5 (negative mode). Lipids were quantified using a mixture of non-endogenous internal standards as reported in Tables 2 and 3. Lipids were identified based on their retention time and MRM characteristics of commercial reference standards (Avanti Polar Lipids, Birmingham, AL, USA). Quantification was performed using MultiQuant 3.02 (Sciex). Metabolites were normalized to either total protein or cell number. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 3-1] [Table 3-2] [Table 3-3]

[0411] Mass spectrometry of GlcCer and GalCer Glucosylceramide (GlcCer), GalCer, Glucosylsphingosine and Galatosylsphingosine analyses were performed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) coupled to electrospray mass spectrometry (QTRAP 6500+, Sciex, Framingham, MA, USA). For each analysis, 10 μL of sample was injected onto a HALO HILIC 2.0 μm, 3.0 × 150 mm column (Advanced Materials Technology) using a flow rate of 0.45 mL / min at 45 °C. Mobile phase A consisted of 92.5 / 5 / 2.5 ACN / IPA / H2O with 5 mM ammonium formate and 0.5% formic acid. Mobile phase B consisted of 92.5 / 5 / 2.5 HO / IPA / ACN with 5 mM ammonium formate and 0.5% formic acid. The gradient was programmed as follows: 100% B for 0.0-3.1 min, 95% B for 3.2 min, 85% B for 5.7 min, held at 85% B for 7.1 min, dropped to 0% B at 7.25 min, held for 8.75 min, returned to 100% at 10.65 min, and held for 11 min. Electrospray ionization was performed in positive ion mode applying the following settings: curtain gas was set to 25, collision gas was set to medium, ion spray voltage was 5500, temperature was 350 °C, ion source gas 1 was set to 55, and ion source gas 2 was set to 60. Data acquisition was performed using Analyst 1.6 (Sciex) in multiple reaction monitoring mode (MRM) with the following parameters: various residence times (msec) and collision energies (CE) reported in Table 4, declustering potential (DP) of 45, entrance potential (EP) of 10, and collision cell exit potential (CXP) of 12.5. Lipids were quantified using a mixture of internal standards reported in Table 4. Glucosylceramide and galactosylceramide were identified based on their retention times and MRM characteristics of commercial reference standards (Avanti Polar Lipids, Birmingham, AL, USA). Quantification was performed using MultiQuant 3.02 (Sciex). Metabolites were normalized to cell number. [Table 4]

[0412] Figures 3A-F and 4A-P show Trem2 with chronic demyelination. - / - We highlight the elevation of cholesteryl esters and myelin-enriched lipids in isolated microglia, but not in astrocytes, from the forebrain and mice. + / +、 Trem2 + / -、 and Trem2 - / - Forebrain total cholesterol levels were unchanged in mice (Figure 3A). Cholesteryl esters (Figure 3B), oxidized cholesteryl esters (Figure 3C), BMP (Figure 3D), and triacylglycerides (Figure 3E) levels were significantly increased after either a control diet or cuprizone-induced Trem2 upregulation for 5 weeks. + / + , Trem2 + / - , and Trem2 - / - Mice and Trem2 inhibition by cuprizone for 12 weeks + / + , and Trem2 + / - Compared with 12-week cuprizone treatment, Trem2 - / - The levels of GM3 d38:1 and GM3 d40:1 (FIG. 3F) were increased in the forebrain from mice fed a control diet or with cuprizone for 5 weeks, indicating that Trem2 + / + , Trem2 + / - , and Trem2 - / - Mice and Trem2 inhibition by cuprizone for 12 weeks + / + , and Trem2 + / - Compared with mice fed a diet containing cuprizone for 12 weeks, Trem2 - / - In Figure 4, Trem2 was increased in the forebrain of mice fed cuprizone for 12 weeks. - / - Microglia isolated from the brain were fed a control diet or cuprizone-induced Trem2 upregulation for 5 weeks. + / + , Trem2 + / - , and Trem2 - / - Microglia and Trem2 by 12-week cuprizone + / + and Trem2 + / -Increases in cholesteryl esters (FIG. 4A), BMP (FIG. 4B), hexosylceramide (FIG. 4C), and galactosylceramide levels (FIG. 4D) are shown in mice treated with either the control or cuprizone diet compared to control or cuprizone diet. + / + , Trem2 + / - , and Trem2 - / - No changes in lipid levels of cholesteryl esters (FIG. 4E), BMPs (FIG. 4F), hexosylceramide (FIG. 4G), and galactosylceramide (FIG. 4H) were detected in astrocyte-enriched cell populations isolated from the brain.

[0413] Example 3. Increased lipid storage in vitro in BMDMs cultured from Trem2 KO mice and iPSC microglia In this example, we describe by both immunocytochemistry and mass spectrometry the lipid storage phenotype observed in Trem2 KO BMDMs cultured in vitro and treated with oxidized low-density lipoprotein (oxLDL) or myelin.

[0414] Harvesting and culture of mouse BMDMs Mouse femurs and tibiae were dissected and briefly sterilized with 70% ethanol. Bones were washed twice with HBSS and then crushed with a mortar and pestle in 10 mL of HBSS. The cell suspension was filtered through a 70 μm cell strainer, spun at 300xg for 5 min, and the supernatant was discarded. The cell pellet was resuspended in ACK lysis buffer (Thermo Fisher A1049201) for 4 min at room temperature. ACK lysis was stopped by adding 10 mL of RPMI-1640 (Thermo Fisher) + 10% Hyclone FBS (GE Healthcare) + Penicillin Streptomycin (Thermo Fisher), then spun at 300xg for 5 min, and the supernatant was discarded. Cells were resuspended in RPMI culture medium containing 50 ng / mL mouse M-CSF (Life Technologies, PMC2044), counted, and collected at 1 × 10 6The cells were diluted to 1 × 10 cells / mL and then plated onto non-tissue culture treated Petri dishes. Three days after seeding, fresh mouse M-CSF (50 ng / mL) was added. Five days after seeding, cell culture medium was aspirated and cells were washed once with PBS. Cells were resuspended in RPMI / FBS / Pen-Strep and harvested with a cell scraper. Cells were spun at 300xg for 5 min, the supernatant discarded, and 1 × 10 cells were plated for direct culture onto tissue culture treated plates. 6 Either diluted to cells / mL or frozen in RPMI / FBS / Pen-Strep+10% DMSO for later use.

[0415] iPSC-Microglia Method Generation of human iPSC-derived microglia Method Overview: Hematopoietic progenitor cells are generated from wild-type and knockout iPS cells (generation of knockout lines protocol below) using a commercially available kit (StemCell Technologies catalog number 05310) following the manufacturer's instructions. At day 12 of hematopoietic stem cell differentiation, cells are positive for HSC markers CD34, CD43, and CD45, at which point floating and adherent cells are transferred to 6-well plates containing primary human astrocytes. Re-seeded cells are co-cultured with astrocytes in medium C (Source: Pandya, H et al., Nat Neurosci, 2017.20(5):p.753-759) (IMDM, 10% Hyclone FBS, PenStrep, 20ng / ml Il3, 20ng / ml GM-CSF, 20ng / ml M-CSF) for 14-21 days, during which time progenitor cells are progressively removed and floating cells are predominantly (>80%) mature microglia. Mature microglia are transferred to homeostatic culture conditions (Source: Muffat, J et al., Nat Med, 2016.(11):p.1358-1367) (MGdM medium) for 3-7 days prior to assay.

[0416] Generation of stable knockout cell lines A CRISPR-based approach was used with an RNP-based protocol using reagents from IDT (Alt-R system: https: / / www.idtdna.com / pages / products / crispr-genome-editing / alt-r-crispr-cas9-system) and NEB (Cas9 cat #M0646M) introduced via nucleofection using Lonza cat #V4XP-3032.

[0417] Myelin purification Myelin was purified from wild-type C57Bl / 6 mouse brains (Jackson Laboratories) using the method described in Safaiyan, S et al. Nat Neurosci, 2016.19(8):p.995-8. After purification, myelin was resuspended in PBS and adjusted to a protein concentration of 1 mg / mL using the DC Protein Assay Kit 2 (BioRad, 5000112).

[0418] BMDMs were seeded in RPMI / 10% FBS / Pen-Strep at a density of 100,000 cells per well in tissue culture-treated 96-well plates (CellCarrier, PerkinElmer) supplemented with 5 ng / mL mouse M-CSF. The ACAT inhibitor K604 was prepared according to a published protocol (US2004 / 0038987A1).

[0419] Nile Red staining, filipin staining or in vitro lipid storage assay for lipidology Either WT or TREM2 KO iPSC-microglia (30,000 / well) or BMDM (100,000 / well) were seeded onto PDL-coated 96-well plates in their respective full serum medium containing 20 ng / mL mCSF. After 24 h at 37 °C, purified myelin (isolated from mouse brain as described above, 5 μg / mL (2 h uptake) or 25 μg / mL or 50 μg / mL (48–72 h uptake) final concentration) or oxLDL (Thermo Fisher L34357, 50 μg / mL final concentration) was added to the wells. For experiments with oxLDL, a second addition of the same amount of oxLDL was added to the wells 24 h after the first addition. For ACAT inhibitor experiments, 500 nM of the ACAT inhibitor K604 or vehicle control was added together with the first lipid dose. Cells were harvested or imaged 2 h after lipid treatment (Figure 9) or after 48–72 h at 37 °C. For myelin washout experiments, myelin was removed after a 24 h incubation period and subsequently replaced with antibody-containing medium for 24–48 h of incubation.

[0420] For LC-MS, cells were extracted according to the following protocol: For Nile Red imaging, the supernatant was removed and cells were incubated for 30 min at 37 °C in live cell imaging buffer (Life Technologies, A14291DJ) containing 1 μM Nile Red (Thermo Fisher N1142) and 1 drop / mL Nucblue (Thermo Fisher R37605). After incubation, the staining solution was removed and cells were fixed in 4% paraformaldehyde. Cells were then imaged using 568 and DAPI illumination settings on an Opera Phoenix high content confocal imager. For filipin staining, the supernatant was removed and cells were fixed using 4% paraformaldehyde. Cells were washed three times for 5 min each with cholesterol detection wash buffer (Abcam ab133116). Flipin III was diluted 1:100 in cholesterol detection assay buffer (Abcam ab133116) and added to cells for 30 min. Cells were washed two more times with wash buffer and imaged using the DAPI illumination setting. Lipid and filipin spots were analyzed using the spot survey algorithm on the Harmony software provided with the instrument. Figure 5A-B shows increased lipid accumulation in Trem2 KO BMDMs treated with oxLDL (50 µg / mL) for 48 h compared to WT BMDMs, as shown by Nile red staining (Figure 5A). Cells were imaged at 63x resolution and Nile red was quantified as total spot area using the spot survey algorithm on the Harmony software (Figure 5B).

[0421] Figure 9 shows cholesterol and cholesteryl ester (CE) levels in bone marrow from differentiated macrophages from wild-type mice administered 5 μg / mL myelin for 2 hours, followed by extraction immediately after myelin uptake (T0) or after myelin washout and a 2-hour (T2) or 4-hour (T4) chase. ACAT inhibitor was added during myelin uptake and maintained through the 4-hour washout (T4+ACAT inhibitor).

[0422] Figure 11A-C show that cholesteryl esters do not accumulate in the presence of ACAT inhibitor in both myelin-treated WT and TREM2 KO iPSC microglia, indicating that cholesteryl ester accumulation is ACAT-dependent. Cholesterol is shown as a control and is not affected by ACAT inhibition.

[0423] Lipid extraction protocol for in vitro samples The cells treated with either oxLDL or myelin as above were then washed with PBS while kept on ice. 70 μl of 9:1 methanol:water solution containing internal standard was added to the cells in the 96-well plate. The plate was agitated on a shaker at 4C and 1200 rpm for 20 minutes, then spun down at 300xg for 5 minutes. 50 μl of the supernatant was transferred to an LC-MS vial and kept at -80C until run on the instrument. For mass spectrometry protocol, see Example 2.

[0424] Mass spectrometry was performed on WT and Trem2 KO BMDMs treated with oxLDL for 48 hours to characterize the lipid species accumulated in the cells (Figure 6A-E). OxLDL treatment increases lipid abundance in both WT and Trem2 KO BMDMs, but the increase in cholesteryl esters (Figure 6A), gangliosides (Figure 6B), triacylglycerides (Figure 6C), and hexosylceramide (Figure 6D) is exacerbated in Trem2 KO BMDMs.

[0425] Mass spectrometry was performed on WT and Trem2 KO BMDMs treated with myelin for 48 h to characterize the lipid species that accumulate within the cells. Trem2 KO BMDMs show greater accumulation of cholesteryl esters (Figure 7A), oxidized cholesteryl esters (Figure 7B), diacylglycerides (Figure 7C), triacylglycerides (Figure 7D), hexosylceramide (Figure 7E), lactosylceramide (Figure 7F), and gangliosides (Figure 7G) compared to WT BMDMs.

[0426] In Figure 8A-H, mass spectrometry was performed on WT and TREM2 KO iPSC microglia treated with myelin (25ug / mL) for 72 hours to characterize the lipid species that accumulate within the cells. TREM2 KO iPSCs show greater accumulation of cholesterol (Figure 8A), phosphatidylserine 38:4 (Figure 8B), bis(monoacylglycero)phosphate 44:12 (Figure 8C), lysophosphatidylcholine 16:0 (Figure 8D), platelet activating factor (Figure 8E), cholesterol sulfate (Figure 8F), and lysophosphatidylethanolamine (Figure 8G).

[0427] Figures 23A and 23B show that Trem2 KO BMDMs accumulate more free cholesterol in the endolysosomal system than Trem2 WT BMDMs after treatment with myelin (25ug / mL) and staining for free cholesterol with filipin.

[0428] Figures 23C and 23D show that anti-TREM2 antibodies reduce free cholesterol levels in human iPSC-derived microglia compared to a control antibody (anti-RSV). Figure 23C shows staining of free cholesterol with filipin under various conditions, and Figure 23D shows quantification of filipin puncta.

[0429] Example 4. Amelioration of lipid accumulation in iPSC-derived microglia by TREM2 antibody or exogenous APOE iPSCs were generated and BMDMs were harvested / cultured using methods similar to those in Example 3.

[0430] The protocol from Example 3 above for in vitro lipid storage assay and Nile Red staining or lipidology was modified as follows: After 24 hours of lipid treatment at 37°C, TREM2 antibody or RSV control was added to the wells to a final concentration of 100 nM. Cells were incubated for an additional 48 hours at 37°C before harvesting or imaging. For experiments with APOE3, cells were treated with myelin (25 μg / mL) for 24 hours, and then the medium was replaced with medium containing 10 μg / mL APOE3. After 24 hours, cells were harvested for analysis. Lipidomics or Nile Red staining was performed according to the protocol above.

[0431] Figure 10 shows that Trem2 KO BMDMs accumulate more lipids than WT BMDMs when fed myelin (24 hours), as quantified by total spot area of ​​Nile Red staining. This accumulation is ameliorated by the addition of exogenous human APOE3, which has been shown to mediate lipid efflux (PMID:9541497, PMID:10693931, PMID:15485881).

[0432] 12A-12C show that TREM2 antibodies can reduce the amount of myelin-induced lipid accumulation in human iPSC-derived WT microglia, as shown by both Nile Red staining and measuring triacylglyceride levels on LC-MS.

[0433] FIG. 12D shows the levels of triacylglyceride (TAG) lipid species detected by mass spectrometry in cell lysates of iPSC-microglial cells treated with several different anti-TREM2 antibodies at 72 hours after 24 hours of myelin treatment. Anti-TREM2 antibodies A and B bind to the stalk region of TREM2, whereas anti-TREM2 antibodies C, D, and E bind to the IgV region of TREM2. FIG. 12E shows the levels of TAG lipid species detected by mass spectrometry in cell lysates of iPSC-microglial cells subjected to myelin washout experiments with anti-TREM2 antibodies. The LC / MS data generated in FIG. 12D and 12E were normalized to myelin plus isotype control for each lipid species.

[0434] Lipid accumulation in iPSC-microglia is induced by myelin treatment as reflected by an increase in neutral lipid staining (Nile Red) and by LC / MS for detection of specific lipid species in cell lysates. The data illustrated in Figures 12A-12E collectively show that treatment of iPSC-microglia cells after myelin challenge with multiple anti-TREM2 antibodies reduced lipid species accumulation, as shown by a decrease in TAG lipid species levels measured by LC / MS. A decrease in lipid levels as a result of antibody treatment was observed at different time points ranging from 24 h to 72 h. To exclude the possibility that the decrease in lipid levels was caused by a blockade of lipid uptake, a myelin washout experiment was performed in which myelin was removed prior to anti-TREM2 antibody addition. Figure 12E shows that anti-TREM2 antibodies also reduced lipid levels in iPSC-microglia with myelin washout prior to antibody treatment compared to isotype control.

[0435] Example 5. Effects of ACAT1 inhibitors, bexarotene, and GW3965 on myelin or oxLDL storage in TREM2 KO cells Either WT or TREM2 KO iPSC-microglia (30,000 / well) or BMDM (100,000 / well) were seeded onto PDL-coated 96-well plates in their respective full serum medium containing 20 ng / mL mCSF. After 24 h at 37 °C, purified myelin (isolated from mouse brain as described above, 5 μg / mL (2 h uptake) or 25 μg / mL or 50 μg / mL (48-72 h uptake) final concentration) or oxLDL (Thermo Fisher L34357, 50 μg / mL final concentration) was added to the wells. For experiments with oxLDL, a second addition of the same amount of oxLDL was added to the wells 24 h after the first addition. For experiments with ACAT inhibitors, 500 nM of the ACAT inhibitor K604 or vehicle control was added together with the first lipid dose. In experiments with bexarotene, 10 uM bexarotene or vehicle control was added along with purified myelin. In experiments with GW3965, 10 uM GW3965 or vehicle control was added along with purified myelin. Cells were harvested or imaged after 2 hours of lipid treatment (Figure 9) or after 48-72 hours at 37 °C.

[0436] Figure 13A shows that Trem2 KO BMDMs accumulate more neutral lipids than WT BMDMs when treated with myelin debris (25ug / mL) for 48 hours, as quantified by Nile Red staining, and this accumulation is reduced by co-treatment with bexarotene (10uM).

[0437] FIG. 13B shows that human iPSC-derived TREM2 KO microglia accumulate various cholesteryl ester (CE) species and that this accumulation is reduced by co-treatment with the ACAT inhibitor K604 (500 nM) and the LXR agonist GW3965 (10 μM).

[0438] Example 6. Altered expression of genes involved in lipid metabolism and lysosomal function in Trem2 knockout mice with chronic demyelination In this example, we describe mouse microglial gene expression analyses. In particular, these analyses demonstrate that 1) TREM2 deficiency prevents DAM conversion during chronic demyelination, 2) TREM2 deficiency blocks age-dependent conversion to an injury-associated microglial state, and 3) Trem 2 - / - We demonstrate that microglia exhibit an attenuated transition to an injury-associated microglial state upon demyelination, as shown by single-cell RNAseq.

[0439] Generally, using methods similar to those described in Example 1, single-cell RNA-seq clustering and expression analysis was performed as shown below.

[0440] Single-cell RNA-seq cluster and expression analysis A PCA was performed on the Log2 normalized gene expression matrix and the top 21 principal components were retained. A shared nearest neighbor graph (Xu, C., and Su, Z. (2015). Bioinformatics 31, 1974-1980) was constructed over the data in PC space, after which community detection was performed using the Louvain method (Blondel, et al., (2008) Journal of Statistical Mechanics: Theory and Experiment 2008) to assign cells to one of the eight clusters. Marker genes per cluster were identified by exhaustive pairwise Wilcoxon tests, as implemented in the scran package. Briefly, the expression level of each gene in a cluster was tested individually against each of the other seven clusters. The seven p-values ​​obtained were combined using the Sims method (Simes, RJ (1986). Biometrika 73, 751-754) to provide a final p-value for the differential expression status of genes per cluster, and then adjusted to correct for multiple testing using the Benjamini-Hochberg method (Benjamini, Y., and Hochberg, Y. (1995) Journal of the Royal Statistical Society 57, 289-300). The effective size for each comparison is calculated as the "overlap fraction", i.e., the probability that a randomly selected cell in the source cluster has a higher expression of gene X than a random cell in the query cluster. The overlap fraction (Wilcoxon effect size) is averaged across all pairwise comparisons to provide the final effect size of genes in the cluster. Finally, marker genes per cluster were extracted by identifying genes with FDR<0.05 and an average overlap fraction less than 0.4 or greater than 0.6.

[0441] TREM2 deficiency prevents DAM conversion during chronic demyelination To characterize the effects of acute and chronic demyelination on Trem2-dependent gene expression in microglia, we used Trem2 + / + , Trem2+ / - , and Trem2 - / - Mice were fed a diet containing 0.2% CPZ for either 5 or 12 weeks. + Microglia were isolated from hemibrains by FACS and transcriptome analysis was performed using RNAseq. + The majority of cells are CD45 low In fact, CD45 high Macrophage infiltration in the Trem2+ / Trem2+ cells represented less than 0.2% of total live cells in the absence of CPZ and less than 0.5% in the presence of the CPZ diet for all three genotypes, suggesting a minimal infiltration of macrophages in this model. Principal component analysis (PCA) showed that CPZ treatment reduced the Trem2+ / Trem2+ cells. + / + and Trem2 + / - CPZ challenge induced transcriptional changes in microglial samples from animals, but not Trem2 - / - Microglia showed clustering with those of untreated animals. Gene expression variability analysis failed to reveal significant genotype-dependent differences under normal dietary conditions (see Example 1, Figures 1A-B, absolute log2 fold change >0.5, FDR <0.2). In contrast, applying the same threshold to the effect of CPZ identified hundreds of significantly up- or down-regulated genes with changes after 5 weeks and stronger effects after 12 weeks (see Example 1, Figures 1A-1B). Genotype-specific comparison between CPZ vs. control diet for 5 and 12 weeks revealed that these gene expression changes were almost entirely attributable to Trem2. + / + and Trem2 + / - While restricted to animal-derived microglia, Trem2 - / - It was determined that mouse-derived microglia were unable to respond to a myelin challenge.

[0442] Using gene set analysis from the Reactome database (Fabregat, et al. (2018). Nucleic Acids Res 46, D649-D655), significant Trem2-dependent upregulation of genes involved in lysosomal and phagosomal function, AD, oxidative phosphorylation, and cholesterol metabolism was detected. For example, as shown in Figures 14A-14B, genes involved in lysosomal function and lipid metabolism were upregulated by Trem2 during acute and chronic demyelination. + / + and Trem2 + / - Trem2 is upregulated in mouse microglia (5 and 12 weeks of cuprizone treatment, respectively) with chronic and acute demyelination - / - Figure 14A shows reduced upregulation of Trem2 in mouse microglia. + / + , Trem2 + / - , and Trem2 - / - The log2 fold change in gene expression of individual genes related to lysosomal function in bulk microglia is shown compared to 5- or 12-week cuprizone treatment (right inserts, top or bottom, respectively). + / + , Trem2 + / - , and Trem2 - / - The log2 fold change in gene expression for individual genes related to lipid metabolism in bulk microglia is shown compared to 5 or 12 weeks of cuprizone treatment (right insert, top or bottom, respectively). For example, key genes in the lysosomal degradation pathway such as Ctse and Ctsl are downregulated by Trem2. + / + and Trem2 + / - Trem2 was upregulated two-fold (FDR<0.05) during chronic demyelination in microglia - / - No change was noted in microglia (Figure 14A, interaction p-value ≤ 0.05). Several Trem2 genes appear to regulate multiple aspects of cholesterol transport and metabolism, including Ch25h, Lipa, Nceh1, Npc2, and Soat1 in addition to Apoe. Gene set enrichment also revealed that previously described DAM genes (Keren-Shaul, et al. (2017). Cell 169, 1276-1290 e1217) were significantly increased in Trem2 at both time points. + / +and Trem2 + / - The DAM-like response was significantly upregulated in response to CPZ treatment in microglia from both animals (Fig. S14C). - / - This was attenuated in samples from animals (Figures 14C and 15C), suggesting that chronic CPZ demyelination induces microglial expression changes that mirror those observed in 5XFAD and SOD1 microglia (Keren-Shaul, et al. (2017). Cell 169, 1276-1290 e1217).

[0443] Microglial transition from homeostasis to DAM has been described as a two-step process, with a TREM2-independent transition to an intermediate state (DAM stage 1), followed by a second TREM2-dependent change (DAM stage 2) (Keren-Shaul et al. (2017) Cell 169, 1276-1290 e1217). Consistent with previous models, the homeostatic genes P2ry12 and Tmem119 downregulate Trem2 in response to CPZ. + / + and Trem2 + / - (FDR<0.01), but Trem2 - / - It was not downregulated in microglia (FIG. 14D, genotype-diet interaction p-value <0.05). Trem2 after CPZ treatment + / + and Trem2 + / - Compared to microglia, Trem2 - / - We also observed a reduced induction of stage 1 DAM genes such as Apoe (interaction p-value < 0.001), Fth1 (interaction p-value < 0.005), and Tyrobp (interaction p-value < 0.1) in mice (Figure 14E). For example, a reduced induction of Trem2 after 12 weeks was observed, similar to that observed for stage 2 DAM genes such as Axl (interaction p-value < 0.05), Cd9 (interaction p-value < 0.001), or Csf1 (interaction p-value < 0.1). + / + and Trem2 + / - CPZ treatment of animals upregulated Apoe expression by more than eightfold, a response that was mediated by upregulation of Trem2. - / -In mice, TREM2 was attenuated in mice (Figure 14F). In summary, these data confirm TREM2 as a regulator of phagocytic clearance of myelin debris and point to a role in endolysosomal processing and lipid metabolism, with cholesterol transport and metabolism clearly indicated. Furthermore, they show that CPZ chronic demyelination induces an injury-associated microglial state, which is related to Trem2 - / - This suggests that it is not initiated in microglia.

[0444] TREM2 deficiency blocks the age-dependent transformation of microglia into an injury-associated state. Gene expression studies show that aged microglia acquire a DAM transcriptional state, suggesting that microglia may respond to age-induced parenchymal damage (Keren-Shaul, et al. (2017). Cell 169, 1276-1290 e1217). Compared to wild-type microglia, aged Trem2 - / - To test whether microglia have a reduced ability to transition to an injury-associated microglial state, we analyzed young (2-month-old) and aged (15-17-month-old) wild-type and Trem2 - / - Gene expression analysis was performed on sorted microglia derived from mice.

[0445] It was confirmed that microglia isolated from aged wild-type brains expressed injury-associated microglial characteristics based on the downregulation of various constitutive genes and the upregulation of the DAM1 and DAM2 genes (Figures 15B and 15C). This response was consistent with the CPZ-treated Trem2 - / - As observed in microglia, Trem2 - / - The DAM2 gene set was largely attenuated in microglia. - / -The DAM1 gene set was more affected, as exemplified by the more pronounced upregulation of Lpl and Spp1 in aged wild-type microglia compared to control microglia (Figures 15B and 15C). This is consistent with the Trem2 dependency of the DAM2 profile shown in 5XFAD microglia (Keren-Shaul, et al. (2017). Cell 169, 1276-1290 e1217). However, the cholesterol metabolism-related gene module was more significantly upregulated in CPZ-treated Trem2 compared to control microglia. - / - As with microglia, aging Trem2 - / - Microglia were not significantly affected (Figures 15B and 15C). Collectively, these data suggest that CPZ-challenged Trem2 - / - Maladaptive functions observed in mouse-derived microglia are related to aging Trem2 - / - This suggests that it is also present in mouse-derived microglia, although gene expression in the latter is not as dramatically altered when compared to CPZ challenge.

[0446] Trem2 - / - Microglia exhibit an attenuated transition to an injury-associated microglial state upon demyelination as shown by single-cell RNA-seq. The following experiments were designed to determine 1) whether the population-based CPZ-induced transcriptional changes observed in bulk RNAseq profiles are universal to all microglia or 2) whether heterogeneous transcriptional responses occur at the single-cell level. To address this question, we used scRNAseq to characterize Trem2 microglia with chronic demyelination. + / + , Trem2 + / - , and Trem2 - / - Compared with microglia from mice, Trem2 + / + CD11b isolated from control brain + / CD45 low This was done with microglia.

[0447] These experiments generated data for 3,023 individual cells. To identify transcriptionally distinct microglial subpopulations within these data, we performed unsupervised graph-based clustering across single-cell expression profiles using a shared nearest neighbor approach (Xu, C., and Su, Z. (2015). Bioinformatics 31, 1974-1980). This identified eight subpopulations of microglia, each accounting for 2%-19% of all analyzed cells. Quantification of cluster membership across groups was performed using Trem2. + / + We identified two clusters (4 and 8) that were essentially absent in controls (<3%) but showed strong treatment- and genotype-specific expansion and collapse. The expression profile of cells in cluster 4 contained at least one functional copy of Trem2 (Trem2 + / + Approximately 1% of control, Trem2 + / + CPZ and Trem2 + / - 16%-19% of CPZ, Trem2 - / - The expression profile of microglia in cluster 8 was also significantly higher than that of Trem2. + / + It was almost absent in the control (less than 2.3%), and Trem2 + / + CPZ mice and Trem2 + / - Their relative abundance was mildly increased (approximately 10%) in CPZ mice, and Trem2 - / - It was most abundant in CPZ mice (approximately 20%).

[0448] To further characterize the expression profile within each cluster, marker gene analysis was performed to identify genes within a given cluster that showed cluster-specific over- and under-expression with respect to the remaining seven clusters at FDR < 0.05. The relative expression of the top 15 up- and down-regulated genes per cluster provides confirmation that these clusters are transcriptionally distinct, although it is rare to find genes that are exclusive in expression between clusters. Consistent with findings from bulk microglial data, Trem2 + / +CPZ and Trem2 + / - The top upregulated marker genes in CPZ-enriched cluster 4 were composed of lysosomal genes such as Ctsb, Ctsd, and Ctsz, and genes involved in lipid metabolism such as Apoe and Lpl (Figure 16A). The downregulated marker genes in cluster 4 included microglial homeostasis genes such as P2ry12 and Tmem119, suggesting that microglia in this cluster are in a more reactive state (Figures 16A and 16B). The top upregulated marker genes in cluster 8 are similar to those in upregulated cluster 4, but to a lesser extent (Figures 16A and 16C). Thus, the upregulated marker genes in cluster 8 were similarly composed of lysosomal and lipid metabolism-related genes such as Ctsb, Ctsd, and Apoe (Figure 16A). This data supports the conclusion that the downregulated marker genes in cluster 4 include microglial homeostasis genes such as P2ry12 and Tmem119, suggesting that microglia in this cluster are in a more reactive state (Figures 16A and 16B). - / - We reinforce the inability of CPZ microglia to fully upregulate the transcription of lysosomal and lipid metabolism-related genes to allow for an appropriate transformation to a reactive state in the presence of demyelination. However, Trem2 + / + Mouse and Trem2 + / - Not all microglia in the mouse brain change gene expression upon chronic demyelination: rather, approximately 20% of the total microglial population upregulates the above genes.

[0449] Consistent with findings from bulk microglial data, Trem2 + / + and Trem2 + / -The transcriptional program found in CPZ microglial cluster 4 was also composed largely of upregulated marker genes previously characterized with DAM2 expression (Keren-Shaul, et al. (2017). Cell 169, 1276-1290 e1217), including Lgals3, Cd63, Spp1, Cst7, Cd68, Capg, and Fth1 (Figure 16A). To characterize the extent to which clusters 4 and 8 are associated with DAM stage 1 and 2 genes, a set of previously reported marker genes for each DAM state was used to provide an aggregated DAM score for each cell. These scores were then averaged by cluster to summarize the extent to which each cluster resembled a given DAM state. Cluster 4 showed the highest enrichment for DAM2 gene expression, followed by cluster 8, followed by Trem2 + / + CPZ and Trem2 + / - CPZ microglia express Trem2 - / - Our results suggest that CPZ microglia display a DAM2-like transition in response to chronic demyelination, which was significantly attenuated in CPZ microglia. Furthermore, the TREM2-independent DAM1 gene expression was upregulated by Trem2 + / + CPZ, Trem2 + / - CPZ and Trem2 - / - Upregulated in CPZ microglia, but Trem2 + / + CPZ and Trem2 + / - CPZ microglia express Trem2 - / - Compared with CPZ microglia, they showed higher DAM1 gene expression. Thus, during chronic demyelination, Trem2 - / - A subset of microglia shows attenuated expression of specific DAM genes. These data support the notion that Trem2 - / - We demonstrate that CPZ microglia fail to fully upregulate lysosomal, lipid metabolism, and DAM gene transcription, as do microglia with at least one functional copy of Trem2, to allow for an appropriate conversion to a reactive state during chronic demyelination.

[0450] Example 7. TREM2 deficiency causes neuronal damage during chronic demyelination This example describes the effect of TREM2 deficiency on neuronal damage during chronic demyelination.

[0451] Dietary cuprizone induces demyelination in mice. A method similar to that described in Example 1 was used for the demyelination protocol.

[0452] reagent Primary antibodies: 1:100 rabbit anti-β-APP (Life Technologies, 512700); 1:1000 mouse anti-SMI-32 (Millipore 559844). Secondary antibodies: goat anti-mouse-555, goat anti-mouse-647, goat anti-rabbit-555, and goat anti-rabbit-647 (Invitrogen).

[0453] Immunofluorescence After PBS perfusion, sagittal mouse hemibrains were flash frozen in liquid nitrogen and coronally frozen at -200C in alternating 100 μm (lipidology) or 20 μm (histology) widths using a Leica CM 1950 cryostat. For each hemibrain, a set of 10 consecutive histological slides representing the rostral to caudal brain region were collected and frozen at -800C. Prior to staining, slides were thawed at room temperature until dry and then fixed in 4% paraformaldehyde (Electron Microscopy Sciences 15710) for 10 min. Slides were washed twice in PBS for 5 min and then blocked with PBS+0.3% TritonX-100+5% Normal Goat Serum (Vector Labs S-1000) for 1 h at room temperature. Primary antibodies were diluted in blocking buffer and added to the slides overnight at 40°C. Slides were washed 3x15 min in PBS and incubated with secondary antibodies diluted 1:1000 in blocking buffer for 2 h at room temperature. Slides were washed 3x15 min in PBS, dried at room temperature, and mounted with Fluoromount G (Southern Biotech 0100-01). Images of immunostained brain sections were captured on a Zeiss AxioScan automated slide imager using a 20x objective and a Zeiss Cell Observer SD, then cropped to focus on the hippocampal region.

[0454] Neurofilament optical detection Mouse blood was collected into EDTA tubes (Sarstedt 201341102) using capillary tubes (Sarstedt 201278100), spun at 15,000xg for 7 min at 40 C, and the upper plasma layer was transferred to 1.5 mL tubes and stored at -800 C. Frozen plasma samples were thawed on ice, diluted 10-fold, and run on an SR-X (Quanterix) using the Simoa NF-light advantage kit (Quanterix 103186) according to the manufacturer's protocol.

[0455] The accumulation of dystrophic APP-positive puncta was significantly greater in Trem2 mice after 5 or 12 weeks of CPZ compared with all other groups. - / - APP puncta were identified in the hippocampus and corpus callosum of mice (Figure 17F). Although APP puncta were the size of cell nuclei, they did not colocalize with DAPI. Instead, puncta were identified in SMI32 + They were surrounded or contiguous with nonphosphorylated neurofilament staining, suggesting dystrophic neuritis. Quantification of APP-positive dystrophic neuritic puncta, performed semi-automated or manually, showed a strong genotype effect, as well as an interaction between genotype and CPZ treatment, for 5 and / or 12 weeks of treatment, depending on the parameter quantified (puncta number, intensity, or area) (Figure S17F).

[0456] To confirm that chronic demyelination causes neuronal damage in the absence of TREM2, we also measured plasma neurofilament-light chain (Nf-L) levels. Consistent with APP staining, Nf-L levels were significantly higher in control or CPZ Trem2 mice. + / + and Trem2 + / - They were not altered in plasma, but were downregulated by Trem2 during chronic demyelination. - / - In plasma, Trem2 was increased (Figure 17A). - / - Consistent with the increased neuronal damage observed in the CNS, aging Trem2 - / - Mice showed increased levels of Nf-L in their plasma (Figure 17B, 2-way ANOVA, FDR<0.05, interaction age genotype p<0.05). These data support the notion that TREM2 plays a neuroprotective role.

[0457] Example 8. Lipidological changes in Trem2 knockout mice with chronic demyelination In this example, we describe the lipidology of forebrains and isolated microglia, astrocytes and CSF from Trem2 knockout mice with chronic demyelination.

[0458] Dietary cuprizone induces demyelination in mice. A method similar to that described in Example 1 was used for the demyelination protocol.

[0459] CSF separation For CSF isolation, mice were anesthetized using 2.5% avertin / tert-amyl alcohol. After sedation, a sagittal incision was made behind the animal's skull to expose the cisterna magna, and a needle attached to a glass capillary tube was used to puncture the cisterna magna to collect CSF. CSF was transferred to a 0.5 mL lo-bind tube (Eppendorf) and spun at 12,000 rpm for 10 min at 4 °C. Prior to LCMS analysis, 2 μL of supernatant was transferred to a glass LCMS vial and 50 μL of methanol containing an internal standard was added.

[0460] FACS of microglia, astrocytes, and other cells from mouse brain Methods similar to those described in Example 1 were used for brain dissociation and FACS protocols.

[0461] FACS lipid extraction and mass spectrometry Lipid extraction and mass spectrometry of forebrain, microglia, astrocytes, and CSF were performed using methods similar to those described in Example 2.

[0462] TREM2 deficiency leads to accumulation of cholesteryl esters in the brain. As described in Example 6, the expression of genes involved in lipid metabolism was increased in wild-type microglia but not in Trem2, including six genes encoding proteins directly involved in cholesterol metabolism, mediating extracellular transport (Apoe), hydrolysis of cholesteryl esters in lysosomes (Lipa), release of non-esterified cholesterol from lysosomes (Npc2), synthesis and storage of cholesteryl esters in lipid droplets (Soat1), hydrolysis of cholesteryl esters in lipid droplets (Nceh1), and 25-hydroxylation (Ch25h). - / -It is strongly induced during chronic demyelination (see, e.g., Figures 14B and 16A), but not in microglia (Figures 14B and 16A). Thus, Trem2 after CPZ challenge - / - To test whether intra- and extracellular cholesterol transport is defective in microglia, we performed LCMS analysis of lipid extracts from coronal forebrain sections containing the corpus callosum.

[0463] Control Trem2 under control conditions + / + , Trem2 + / - , and Trem2 - / - No differences were observed in the brain lipid profile (Figure 17D). Minimal changes were detected upon acute demyelination (5 weeks CPZ), with enhanced levels of cholesteryl ester (CE) and oxidized forms of CE (oxCE) in all three genotypes (Figure 17C). Chronic demyelination (12 weeks CPZ) significantly elevated CE and oxCE lipid species (Figures 17D-17E; 2-way ANOVA, FDR<0.05, interaction p<0.01, 12 weeks CPZ; see also Example 2, Figures 3A-3C). Further comparisons showed that Trem2 + / + and Trem2 + / - Compared with 12-week CPZ brains, TREM2-deficient brains with chronic demyelination exhibited a significant accumulation of CE species containing polyunsaturated fatty acids, such as CE22:6 (docosahexaenoic acid, DHA) and to a lesser extent CE20:4 (arachidonic acid) (Figure 17E, interaction p<0.0001, two-way ANOVA). CE22:6 is a key regulator of Trem2 expression in chronic demyelination. - / - The most significant increase was in the brain, with Trem2 - / - 38-fold higher than control brain, Trem2 + / + 2.5-fold higher in the 12-week CPZ brain compared to the 12-week CPZ brain (Figure 17E). Similarly, for oxCE species previously reported only in atherosclerotic lesions (see Choi, et al. (2017). Biochim Biophys Acta Mol Cell Biol Lipids 1862, 393-397; Hutchins, et al. (2011). J Lipid Res 52, 2070-2083), Trem2 - / -In the brain, Trem2 + / + Although significantly upregulated with chronic demyelination compared to control brains, they were found at much lower levels than CE (see Example 2, Figure 3C). Despite the significant accumulation of CE, cholesterol levels remained unchanged in the brain (see Example 2, Figure 3A), consistent with the fact that it is present in greater amounts than CE (Martin, et al. (2014). EMBO Rep 15, 1036-1052). Ganglioside GM3 levels were significantly upregulated in Trem2 with chronic demyelination compared to control brains. - / - It was also increased in the brain (see Example 2, FIG. 3F), reminiscent of the endolysosomal defects seen in the lysosomal storage disease Niemann-Pick disease type C (Bissig, C., and Gruenberg, J. (2013). Cold Spring Harb Perspect Biol 5, a016816; Zervas, et al. (2001). J Neuropathol Exp Neurol 60, 49-64). Other neutral lipids such as TG were also upregulated in Trem2 upon CPZ treatment compared to controls. - / - was elevated in the brain (see Example 2, FIG. 3E).

[0464] Overall, these data indicate that chronic demyelination causes profound alterations in cholesterol metabolism, as well as specific lipid changes in TREM2-deficient brains.

[0465] TREM2 deficiency causes cholesteryl ester accumulation in microglia isolated from CPZ-treated mice To investigate whether CE accumulation was primarily intracellular or extracellular, a FACS-based lipidomic approach was developed to measure lipid species in a cell-type-specific manner.CSF was also collected and analyzed to assess circulating levels of lipids in the CNS.

[0466] As observed in whole tissue analysis from the forebrain, chronic demyelination was more severe in Trem2 mice compared to untreated genotype controls. + / + , Trem2 + / - , and Trem2 - / -Increased levels of certain lipid species in microglia. Trem2 - / - Microglia upregulate Trem2 upon exposure to chronic demyelination + / + and Trem2 + / - Compared with microglia, astrocytes showed a dramatic increase in the abundance of certain lipid species during chronic demyelination, but there was no significant genotype-dependent effect in control microglia without CPZ treatment (Figure 18A, 2-way ANOVA, FDR<0.05). Surprisingly, the changes in the lipid profile during chronic demyelination were specific to microglia, as astrocyte-enriched populations or CSF did not show any significant genotype- or CPZ treatment-specific changes (Figures 18B and 18C; 2-way ANOVA, FDR<0.05).

[0467] Increased cholesteryl ester levels (Figure 18D) were measured using Trem2-treated control diet or cuprizone for 5 weeks. + / + , Trem2 + / - , and Trem2 - / - Microglia and Trem2 by 12-week cuprizone + / + and Trem2 + / - Compared to microglia, Trem2 - / - In general, Trem2 upregulation by the control or cuprizone diet was not observed in isolated microglia. + / + , Trem2 + / - , and Trem2 - / - No changes in cholesteryl ester lipid levels were detected in astrocyte-enriched cell populations isolated from the brain (Figure 18E) or in CSF (Figure 18F). - / - Lipids specifically increased in microglia versus astrocytes included some of the same species identified in the forebrain, such as CE18:1, CE20:4, and CE22:6 (Figures 18D and 18E; interaction p<0.01, 12 weeks CPZ, two-way ANOVA), which were associated with increased levels of Trem2 upon 12 weeks CPZ treatment compared to controls. - / - It increased 8-43 fold in microglia. Trem2 during demyelination - / -We further confirmed the accumulation of myelin debris in microglia, myelin-enriched lipids or their metabolites, and found that ganglioside GM3 upregulates Trem2 during chronic demyelination. - / - It was enriched exclusively in microglia, but not in astrocytes (see Example 2, FIG. 4). This data supports the idea that myelin lipids regulate Trem2 - / - These results suggest that although it can be phagocytosed by microglia, it is not properly metabolized and accumulates over time in microglia. In addition, certain BMPs, such as BMP 36:2, upregulated Trem2 expression during 12 weeks of CPZ treatment compared to controls. - / - Trem2 was elevated in microglia (Figures 18G and 18H), indicating possible lysosomal stress or dysfunction (Bissig, C., and Gruenberg, J. (2013). Cold Spring Harb Perspect Biol 5, a016816; Miranda, et al. (2018). Nat Commun 9, 291). - / - LCMS analysis of CSF from mice showed that Trem2 was upregulated in mice with or without CPZ treatment. + / + , Trem2 + / - , or Trem2 - / - No significant lipidomic differences from the CSF were evident (Figures 18I-18M), suggesting that the lipid accumulation observed in bulk forebrain tissue does not reflect extracellular accumulation, although alterations in interstitial fluids were not excluded.

[0468] These data indicate that TREM2-deficient microglia are able to phagocytose myelin debris during demyelination, but are unable to properly metabolize or mediate the excretion of myelin lipids.

[0469] Example 9. Myelin sulfatide binds to TREM2 and promotes downstream signaling In this example, Trem2 - / -We describe the use of an in vitro system to more precisely delineate the mechanisms underlying increased lipid accumulation in microglia. In particular, we characterized specific myelin lipids that may bind to and signal through TREM2, and in turn, regulate phagocytic clearance of myelin.

[0470] Liposome preparation 70 mol% DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipid) and 30 mol% of one test lipid were combined in chloroform in a glass vial and dried under a stream of N2 gas for 1-2 h or until completely dry. Test lipids included sulfatides (Avanti), POPS (Avanti), SM (Avanti), PI (Avanti), GalCer (Avanti), PE (Echelon Biosciences), and free cholesterol (Echelon Biosciences). The lipid mixture was resuspended in HBSS (final lipid concentration of 1-2 mg / mL) and vortexed for 2-3 min. The lipid suspension was then bath sonicated for 10 min. For surface plasmon resonance experiments, liposomes were extruded 10 times using an Avanti mini-extruder constructed with a membrane with one pore size of 100 nm to form small unilamellar vesicles.

[0471] TREM2 / DAP12 and DAP12 HEK293 stable cell lines HEK293 cells were transfected with pBudCE4.1 mammalian expression vector (ThermoFisher) expressing wild-type human TREM2 and DAP12, as well as DAP12 alone. Stable expressing clones were selected and cell surface TREM2 expression was assessed by flow cytometry using APC-conjugated rat-anti-human / mouse-TREM2 monoclonal (R&D MAB17291). The highest wild-type TREM2 expressing clone was selected for expansion. Clones stably expressing DAP12 were analyzed by Western blot.

[0472] pSYK AlphaLISA Activation of TREM2-dependent pSYK signaling was measured using a commercially available AlphaLISA assay (PerkinElmer). HEK293 cells: Two days prior to the experiment, HEK293 cells stably overexpressing TREM2 and DAP12 were seeded at 40,000 cells / well onto 96-well poly-D-lysine coated plates. Differentiated human macrophages and BMDMs were seeded at 100,000 cells / well onto tissue culture treated 96-well plates. Cells were washed once with HBSS before adding 50 μL of liposome mixture per well. For competition experiments, hTREM2-ECD or TREM1-his (Novoprotein Scientific) was i...

Claims

1. A composition for use in a method for treating lipid metabolism dysregulation in a mammal in need of such treatment, the composition comprising an agonist anti-TREM2 (triggering receptor 2 expressed on myeloid cells) antibody, the method comprising administering the composition to the mammal, and the lipid metabolism dysregulation comprises increased accumulation of one or more lipids.

2. The composition of claim 1 , wherein cells expressing TREM2 in the mammal exhibit dysregulated lipid metabolism.

3. The composition of claim 2 , wherein the cell is a microglial cell or a macrophage.

4. The composition of any one of claims 1 to 3, wherein the mammal has, or has been determined to have, reduced TREM2 activity.

5. The composition of any one of claims 1 to 4, wherein the mammal has, or has been determined to have, reduced apolipoprotein E (ApoE) activity.

6. The composition according to any one of claims 1 to 5, wherein the lipid metabolism dysregulation comprises increased intracellular accumulation of one or more lipids.

7. 7. The composition of any one of claims 1 to 6, wherein the one or more lipids are selected from the group consisting of cholesteryl esters, oxidized cholesteryl esters, bis(monoacylglycero)phosphate species (BMPs), diacylglycerides, triacylglycerides, hexosylceramides, galactosylceramides, lactosylceramides, sulfatides, gangliosides, phosphatidylserine 38:4, bis(monoacylglycero)phosphate 44:12, lysophosphatidylcholine 16:0, platelet activating factor, cholesterol sulfate, lysophosphatidylethanolamine, and combinations thereof.

8. The composition of claim 7 , wherein the one or more lipids comprise a cholesteryl ester.

9. The mammal is a patient suffering from Alzheimer's disease, Nasu-Hakola disease (NHD), Lewy body disease, Parkinson's disease, retinal degeneration, Huntington's disease, frontotemporal lobar degeneration (FTD), amyotrophic lateral sclerosis (ALS), or amyloidosis. The composition of any one of claims 1 to 8, wherein the patient has ALS, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, multiple sclerosis or vanishing white matter disease.

10. 9. The composition of any one of claims 1 to 8, wherein the mammal has obesity, type 2 diabetes, alcoholic or non-alcoholic steatohepatitis, alcoholic or non-alcoholic fatty liver disease, rheumatoid arthritis (RA), or atherosclerosis.

11. The composition according to any one of claims 1 to 8, wherein the mammal has inflammation.

12. The composition of claim 11, wherein the mammal has rheumatoid arthritis (RA), gout, or inflammatory bowel disease (IBD).

13. The composition of any one of claims 1 to 12, wherein the agonist anti-TREM2 antibody is MAB17291 or 78.

18.

14. The composition of any one of claims 1 to 13, wherein said administration reduces lipid accumulation.

15. 15. The composition of any one of claims 1-14, wherein said administration reduces expression of at least one proinflammatory cytokine selected from the group consisting of G-CSF, IFNy, IL-12(p40), IL-12(p70), LIX (CXCL5), MCP-1 (CCL2), MIG (CXCL9), IL-1α, IL-1β, and IL-18.

16. 16. The composition of any one of claims 1 to 15, wherein the method further comprises administering a second therapeutic agent selected from the group consisting of an RXR agonist, an LXR agonist, and an acetyl-CoA acetyltransferase 1 (ACAT1) inhibitor.

17. 13. Use of an agonist anti-TREM2 antibody for the preparation of a medicament for treating lipid metabolism dysregulation in a mammal, said lipid metabolism dysregulation comprising increased accumulation of one or more lipids.

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