Suppression of microglial activation with innate lymphoid cells

Abstract

To suppress microglial activation with innate lymphoid cells.SOLUTION: The present invention provides a method for reducing microglial activation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of activated type II innate lymphoid cells (ILC2s). Compositions and methods of using ILC2 to reduce microglial activation or to reduce blood-brain barrier (BBB) permeability are described. Also described are methods and compositions that use an agent that increases the number of activated ILC2 to reduce microglial activation or BBB permeability.SELECTED DRAWING: Figure 8

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 62 / 698,545, filed July 16, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates generally to methods and compositions for inhibiting microglial activation, and more particularly to compositions and methods for inhibiting microglial activation or blood-brain barrier disruption by administering type II innate lymphoid cells or agents that increase the number or activity of type II innate lymphoid cells. [Background technology]

[0003] Innate lymphoid cells (ILCs), as their name suggests, exhibit features of both innate and adaptive immunity. Although ILCs arise from a common lymphoid precursor and differentiate into subsets similar to T helper cells, ILCs do not undergo somatic recombination, which underlies the receptor diversity that typifies adaptive immunity. Thus, unlike conventional T cells, ILC2s are not antigen-specific, nor do they lack the specific lineage markers that identify other lymphocytes, such as T cells, B cells, natural killer cells, and natural killer T cells. ILCs can be classified into three types (ILC1, ILC2, ILC3) based on the cytokines they produce, as well as the transcription factors that regulate their development and function (Spits et al., 2013, Nat Rev Immunol. 13(2):145-9).

[0004] ILC2s primarily produce type 2 cytokines, such as interleukin 4 (IL-4), IL-5, IL-9, and IL-13. As innate cells, they primarily respond to endogenous damage-associated molecular patterns (DAMPs), such as IL-25 and IL-33, alanine (Neill et al., 2010, Nature 464(7293):1367-1370). A subset of activated ILC2s can also produce the anti-inflammatory cytokine IL-10 (Seehus et al., 2017, Nat Commun. Dec 1;8(1):1900). This evidence suggests that ILC2s have diverse roles in immune responses. In mouse models of asthma, ILC2s have been shown to contribute to eosinophilic inflammation and hyperresponsiveness, and human studies have found increased numbers of ILC2s in patients with asthma, allergic rhinitis, eosinophilic esophagitis, and atopic dermatitis (Doherty et al., J Allergy Clin Immunol. 2017;139(5):1465-1467).

[0005] ILC2s are tissue resident and were initially shown to be abundant in mucosa, barrier, and adipose tissues. However, more recently, ILC2s have been unexpectedly detected within the environment of the peripheral and central nervous system (CNS, PNS) (Gadani et al., 2017, J Exp Med. 214(2):285-296). In the CNS, ILCs have only been examined pathologically, where ILC2s have been shown to respond after spinal cord injury, and ILC3s, normally resident in the meninges, have been found to exhibit disease-induced accumulation and activation in experimental autoimmune encephalomyelitis (EAE) (Hatfield and Brown, 2015, Cell Immunol. 297(2):69-79).

[0006] Inflammation in the CNS is thought to play a key role in pathways leading to neuronal cell death in many neurodegenerative diseases, including, for example, Parkinson's disease, Alzheimer's disease, prion diseases, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, and HIV dementia. The inflammatory response is mediated by activation of microglia, the resident immune cells of the CNS that normally respond to neuronal injury and remove damaged cells by phagocytosis.

[0007] Neuroinflammatory responses can be beneficial or detrimental to motor neuron survival. Activated microglia can release neurotoxic molecules such as inflammatory cytokines (e.g., tumor necrosis factor alpha, TNFα), nitric oxide, and superoxide (Chao et al., 1992, J Immunol. 149(8):2736-41). These neurotoxic molecules can damage or kill neurons and may precede or exacerbate certain neurological diseases. Many studies have found that activated microglia are a hallmark of brain pathology.

[0008] Many diseases and physiological stressors that affect the CNS also alter the functional integrity of the blood-brain barrier (BBB). They affect the barrier's ability to selectively restrict the passage of substances from the blood to the brain. BBB disruption can lead to disorders such as depression, anxiety, brain fog, and autoimmune brain diseases.

[0009] Inhibiting microglial activation reduces inflammation associated with neurodegenerative diseases and provides a promising therapeutic avenue for preventing or treating diseases associated with BBB disruption. Certain therapies utilizing compounds to modulate microglial activation have been proposed (US Patent Publication No. 2011021413, WO 9945950, US Patent Publication No. 20120237482), but the compounds are known to have off-target effects. In addition, stem cell therapies have been proposed as having the potential to alter microglial activation (Giunti et al., 2012, Stem Cells. 30(9):2044-53, WO 2011106476). The long-term effects of these therapies are unclear, as mesenchymal stem cells only survive for a few months in vivo (Volkman and Offen, 2017, Stem Cells, 35(8):1867-1880). Summary of the Invention [Problem to be solved by the invention]

[0010] Thus, there remains a need for effective therapies that can treat diseases such as neurodegenerative diseases associated with activated microglia. [Means for solving the problem]

[0011] The present invention fulfills this need by providing methods for inhibiting microglial activation in a subject in need thereof. These methods include increasing the number and / or activity of activated type II innate lymphoid cells (ILC2) in a subject.

[0012] The inventors unexpectedly found that increasing the number of meningeal ILC2s suppressed microglial activation. The inventors also unexpectedly found that meningeal ILC2s can be activated to produce IL-10, which mediates the suppression of microglial activation. In particular, the inventors found that ILC-deficient mice exhibited an inhibition of the microglial inflammatory response and increased blood-brain barrier (BBB) ​​permeability. Subsequent transcriptomics analysis unexpectedly revealed meningeal ILC2s as a source of IL-10. Thus, modulating the neuroinflammatory phenotype led to subsequent meningeal engraftment of adoptively transferred ILC2s, and to the suppression of IL-10 by IL-10 neutralizing antibodies or Il10. - / - These findings are accompanied by advantages that are abrogated by ILC2s. In addition, ILC2s derived from several mouse tissues exhibit IL-10 competence similar to human blood-derived ILC2s, suggesting a previously unrecognized immunomodulatory role for canonical ILC2s in addition to the recently described tissue-specific transcription-specific ILCREG subsets. Collectively, these findings indicate a role for meningeal ILC2s in suppressing neuroinflammation and BBB stability, implying that ILC2-based cell therapy could be used to treat neuroinflammatory pathologies.

[0013] In one general aspect, the invention relates to these methods in a subject in need of inhibiting microglial activation, or reducing BBB permeability, or increasing BBB stability.

[0014] In one embodiment, the present application provides a method for inhibiting microglial activation in a subject in need of inhibition of microglial activation, comprising administering a therapeutically effective amount of activated ILC2s to the subject.

[0015] In one embodiment, the present application provides a method for reducing BBB permeability or increasing BBB stability in a subject in need thereof, comprising administering a therapeutically effective amount of activated ILC2 to the subject.

[0016] In an embodiment of the present application, the activated ILC2s are activated by contacting the ILC2s with at least one cytokine selected from the group consisting of IL-33, IL-25, IL-2, and IL-7, or a combination thereof.

[0017] In other embodiments of the application, the cells are genetically modified, preferably the cells are genetically modified for increased IL-10 production compared to otherwise identical non-modified cells.

[0018] In an embodiment of the present application, a therapeutically effective amount of activated ILC2s is administered to a subject in need thereof by intravenous or intrathecal administration.

[0019] In another embodiment, the present application provides these methods in a subject in need of reducing microglial activation, reducing BBB permeability, or increasing BBB stability, comprising administering to the subject a therapeutically effective amount of an agent capable of increasing the number of activated ILC2s in the subject, preferably the activated ILC2s are meningeal ILC2s.

[0020] In yet another embodiment, the present application provides these methods in a subject in need of reducing microglial activation, reducing BBB permeability, or increasing BBB stability, comprising administering to the subject an effective amount of an agent capable of increasing production of IL-10 by activated type II innate lymphoid cells (ILC2s) in the subject.

[0021] In yet another embodiment, the present application provides these methods in a subject in need of reducing microglial activation, reducing BBB permeability, or increasing BBB stability, comprising administering to the subject an effective amount of an agent capable of increasing production of TImp1 by activated type II innate lymphoid cells (ILC2s) in the subject.

[0022] In certain embodiments of the present application, the subject is in need of treatment for a disease associated with microglial activation or BBB disruption, such as a neurodegenerative disease, an inflammatory disorder, meningitis, stroke, neuropsychological disorder, chronic pain, traumatic brain injury, spinal cord injury, optic neuritis, viral or bacterial infection.

[0023] In a preferred embodiment of the present application, the subject is in need of treatment for a neurodegenerative disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia, multiple sclerosis, prion diseases, amyotrophic lateral sclerosis, Huntington's disease, and aging.

[0024] In another preferred embodiment of the present application, the subject is in need of treatment for a neuropsychological disorder selected from the group consisting of depression, anxiety, bipolar depression, and schizophrenia.

[0025] In another general aspect, the application provides pharmaceutical compositions for reducing microglial activation in a subject in need thereof, and methods for preparing the same.

[0026] In one embodiment, the present application provides a pharmaceutical composition for reducing microglial activation in a subject in need thereof, the pharmaceutical composition comprising a therapeutically effective amount of isolated, activated ILC2s and a pharma- ceutical acceptable carrier. The present application also provides a method for preparing the pharmaceutical composition comprising combining a therapeutically effective amount of isolated, activated ILC2s with a pharma- ceutical acceptable carrier.

[0027] In another embodiment, the present application provides a pharmaceutical composition for reducing microglial activation in a subject in need thereof, comprising a therapeutically effective amount of an agent capable of increasing the number of activated type II innate lymphoid cells (ILC2) in a subject, and a pharma- ceutical acceptable carrier. The present application also provides a method for preparing the pharmaceutical composition, comprising combining a therapeutically effective amount of the agent with a pharma- ceutical acceptable carrier.

[0028] In yet another embodiment, the present application provides a pharmaceutical composition for reducing microglial activation in a subject in need thereof, comprising a therapeutically effective amount of an agent capable of increasing IL-10 or Timp1 production by activated type II innate lymphoid cells (ILC2) in a subject, and a pharma- ceutical acceptable carrier. The present application also provides a method for preparing the pharmaceutical composition, comprising combining a therapeutically effective amount of the agent with a pharma- ceutical acceptable carrier.

[0029] These and other aspects and advantages of the present invention will become apparent from the following detailed description and the accompanying drawings. It will be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. [Brief description of the drawings]

[0030] The above Summary of the Invention and the following Detailed Description will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0031] The drawings are as follows: [Figure 1A] FACS plot of Rag2- / - meninges showing gating (left to right) of CD45+Lin-CD90+ and CD45+Lin-CD90+CD127+ cells expressing ILCs:Tbet (ILC1), GATA3 (ILC2) or Rorγt (ILC3), showing that meninges are enriched for ILC2s that display an IL-10 overt activation profile. [Figure 1B] Figure 2 shows that meninges are enriched for ILC2s that display an IL-10 overt activation profile. Relative frequency of ILC1, 2, and 3 found in meninges (n=4 mice x 2 experiments). [Figure 1C(i)]Figure 1. Meninges are enriched in ILC2s that display an IL-10 overt activation profile. Representative confocal images of Rag2- / - meninges in whole tissue preparations. Shown is the sagittal sinus labeled with antibodies against CD90 (green) and Lyve1 (red), showing CD90+ cells (ILCs) distributed within the sinus. [Figure 1C(ii)] Figure 2 shows that meninges are enriched in ILC2s that display an IL-10 overt activation profile. Representative confocal images of menILC2s in the Rag2- / - sagittal sinus labeled with antibodies against CD90 (green), Lyve1 (blue) and GATA3 (red). [Figure 1D] Figure 1 shows that meninges are enriched for ILC2s that display an IL-10 overt activation profile. Representative FACS plots showing cells isolated from post-mortem human meninges, CD45+ / live / single events shown, left plot shows Lin- (lineage cocktail, CD11c, Fcεr1α)IL7rα+ cells, right plot shows labeling for ILC2 markers CRTH2 and CD161, representative of six samples between two experiments. [Figure 1E] Figure 1 shows that meninges are enriched for ILC2s that display an IL-10 overt activation profile.Representative confocal images showing Rag2- / - sagittal sinus after 3 days of systemic IL-33 treatment.ILC2s labeled with CD90 (green) and GATA3 (red), one of three experiments shown. [Figure 1F] Volcano plot comparing transcripts expressed by ILC2s sorted from PBS and IL-33 treated mice showing that meninges are enriched for ILC2s displaying an IL-10 overt activation profile. [Figure 1G] Figure 1 shows that meninges are enriched for ILC2s that display an IL-10 overt activation profile. Heatmaps comparing meningeal ILC2s sorted from PBS and IL-33 treated mice for (i) transcription factors, (ii) cytokine factors, and (iii) extracellular markers. N=20 / 20 mice x 2 experiments. [Figure 1H]Figure 1 shows that meninges are enriched for ILC2s displaying an IL-10 overt activation profile. Representative FACS intracellular cytokine labeling of IL13, IL-5, and IL-10 in ILC2s sorted from Rag2- / - meninges cultured for 5 days with IL-2 / IL-7 and restimulated with IL-33. n=20 mice, 1 of 2 experiments shown. [Figure 1I] Figure 2 shows that meninges are enriched for ILC2s that display an IL-10 overt activation profile. Luminex measurements of IL-5, IL-13, and IL-10 from supernatants of mILC2s stimulated with IL-33. n=20 meninges, 4 wells. [Figure 1J] Figure 2 shows that meninges are enriched for ILC2s that display an IL-10 overt activation profile. Representative FACS plots of IL-10 and IL-13 intracellular cytokine labeling of meningeal CD90+ST2+ cells acutely isolated from mice following 3 days of systemic treatment with IL-33. n=3 mice, 1 out of 2 experiments shown. [Figure 1K] Showing that meninges are enriched in ILC2s that display an IL-10 overt activation profile. Still images from a two-photon time-lapse IL-10 antibody capture movie. Fluorescent antibodies against CD90 (green) and IL-10 (red) show increased IL-10 fluorescence at 12, 40, 60 and 120 minutes. A representative of three experiments is shown. [Figure 1L] Figure 1 shows that meninges are enriched for ILC2s displaying an IL-10 overt activation profile. Luminex measurements of IL-10 from unstimulated or IL-33 stimulated ILC2 supernatants isolated from blood of healthy human donors. Data shown are from 1 of 2 experiments, N=5 unique donors out of a total of 9. [Diagram 2] Quantification of meningeal ILC2s from mice after passive transfer as measured by FACS. [Figure 3A] Representative confocal images of Rag2- / - and Rag2- / -γc- / - brains (hippocampus). Iba1 (green) labels microglia. [Figure 3B]Figure 1 shows that ILC-deficient Rag2- / -γc- / - mice exhibit meningeal abnormalities at baseline. Number of Iba1+ cells / mm2 in untreated Rag2- / - and Rag2- / -γc- / - hippocampi. **, P<0.01, Student's T-test, n=3 / 3 mice. [Figure 3C] Figure 1 shows that ILC-deficient Rag2- / -γc- / - mice exhibit meningeal abnormalities at baseline. Representative FACS plots show side scatter (relative granularity) of acutely isolated microglia from untreated Rag2- / - and Rag2- / -γc- / - mice. n=3 / 3 (2 experiments). [Figure 3D] Representative histograms showing relative CD45 expression on microglia from Rag2- / - and Rag2- / -γc- / - mice showing that ILC-deficient Rag2- / -γc- / - mice exhibit meningeal abnormalities at baseline. [Figure 3E] Figure 1 shows that ILC-deficient Rag2- / -γc- / - mice exhibit meningeal abnormalities at baseline. Relative expression of a panel of markers associated with microglial activation in brains isolated from Rag2- / - and Rag2- / -γc- / - mice. Mean fluorescence intensity (MFI) is shown as a percentage of Rag2- / - controls. *, p<0.05; **, p<0.01; ***, p<0.001 ****, p<0.0001, Holm-Sidak multiple t-test, n=3 / 3 (2 experiments). [Figure 4A] Increased severity of experimental autoimmune encephalomyelitis (EAE) in ILC-deficient mice is consistent with T cell abnormalities and intrameningeal arrest. Mean clinical EAE scores in Rag2- / - and Rag2- / -γc- / - mice. n=10 / 10 (duplicate experiments). [Figure 4B] Increased severity of experimental autoimmune encephalomyelitis (EAE) in ILC-deficient mice is consistent with abnormalities in T cells and intrameningeal arrest of EAE incidence in Rag2- / - and Rag2- / -γc- / - mice following passive EAE induction. **, p<0.01, ANOVA, n=10 / 10 (duplicate experiments). [Figure 4C(i)]Figure 1 shows that the increased severity of experimental autoimmune encephalomyelitis (EAE) in ILC-deficient mice is consistent with abnormal T cell arrest within the meninges. Representative FACS plots of infiltrating T cells as a percentage of total CD45+ cells isolated from the brains of Rag2- / - and Rag2- / -γc- / - mice. [Figure 4C(ii)] Increased severity of experimental autoimmune encephalomyelitis (EAE) in ILC-deficient mice is consistent with T cell abnormalities and arrest within the meninges. Graph of all data in Figure 4C(i), n = 7 / 7 (2 experiments). [Figure 4D(i)] Figure 1 shows that the increased severity of experimental autoimmune encephalomyelitis (EAE) in ILC-deficient mice is consistent with T cell abnormalities and arrest within the meninges. Representative FACS plots of T cells as a percentage of total CD45+ cells isolated from the meninges of Rag2- / - and Rag2- / -γc- / - mice. [Figure 4D(ii)] Increased severity of experimental autoimmune encephalomyelitis (EAE) in ILC-deficient mice is consistent with T cell abnormalities and arrest within the meninges. Graph of all data in Fig. 4D(i), n = 7 / 7 (2 experiments). [Figure 5A] Figure 1 shows that ILC2-secreted factors suppress microglial inflammation and that neutralization of IL-10 abolishes the suppression. Microglia were cultured in the presence or absence of ILC2 supernatants and / or 1 μg / mL R848 (see matrix below each graph) and final supernatants were analyzed by Luminex for secreted factors. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ANOVA. 2 experiments x duplicate samples. [Figure 5B] Figure 1 shows that ILC2-secreted factors suppress microglial inflammation and that neutralization of IL-10 abolishes the suppression. Microglia were cultured in the presence or absence of ILC2 supernatants and / or 1 μg / mL R848 (see matrix below each graph) and final supernatants were analyzed by Luminex for secreted factors. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ANOVA. 2 experiments x duplicate samples. [Figure 5C]We show that ILC2-secreted factors suppress microglial inflammation and that neutralization of IL-10 abolishes the suppression.Microglia were cultured in the presence or absence of rIL-33 and supernatants were analyzed for Timp1 by Luminex. [Figure 5D] We show that ILC2-secreted factors suppress microglial inflammation and that neutralization of IL-10 abolishes the suppression.Microglia were cultured in the presence or absence of 1 μg / mL R848 and supernatants were analyzed by Luminex for Mmp2, 3, 8, 9 and 12. [Figure 5E] Figure 1 shows that ILC2-secreted factors suppress microglial inflammation and that neutralization of IL-10 abolishes the suppression. Microglia were cultured in the presence or absence of ILC2 supernatants and / or 1 μg / mL R848 (see matrix below each graph) and final supernatants were analyzed by Luminex for secreted factors. p<0.0001, ANOVA. A representative of two experiments is shown (2 experiments). [Figure 5F] Figure 1 shows that ILC2-secreted factors suppress microglial inflammation and that neutralization of IL-10 abolishes the suppression. Microglia were cultured as above but in the presence or absence of IL-10 and antibodies against IL-10rα and final supernatants were analyzed by Luminex for secreted factors. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ANOVA. Duplicate wells (2 experiments). [Figure 5G] Figure 1 shows that ILC2-secreted factors suppress microglial inflammation and that neutralization of IL-10 abolishes the suppression. Microglia were cultured as above but in the presence or absence of IL-10 and antibodies against IL-10rα and final supernatants were analyzed by Luminex for secreted factors. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ANOVA. Duplicate wells (2 experiments). [Figure 5H]ILC2-secreted factors suppress microglial inflammation and neutralization of IL-10 abolishes the suppression. Microglia were cultured as above but also in the presence of rmIL-10 and final supernatants were analyzed by Luminex for secreted factors. ****, p<0.0001, ANOVA. Duplicate wells (2 experiments). [Figure 5I] We show that ILC2-secreted factors suppress microglial inflammation and neutralization of IL-10 abolishes the suppression. Microglia were cultured with or without R848 and treated with simple medium containing IL-2, IL-7, and IL-33 with no ILC2s, supernatants from wild-type ILC2s, or supernatants from Il10- / - ILC2s, and final supernatants were analyzed by Luminex for Timp1. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ANOVA. [Figure 5J] Figure 1: ILC2-secreted factors suppress microglial inflammation and neutralization of IL-10 abolishes the suppression. Microglia were treated with ILC2 supernatants or medium, then stimulated with R848, and final supernatants were analyzed by Luminex for Mmp9 and Timp1. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ANOVA. [Figure 6A] How intrameningeal engraftment of passively transferred ILC2s suppresses neuroinflammation in an IL-10-dependent manner. Representative FACS plots showing intrameningeal Lin-CD90+ cells isolated from Rag2- / -γc- / - mice after iv passive transfer of ILC2s and GATA3 and ST2 labeling of these cells. [Figure 6B] Engraftment of passively transferred ILC2s in the meninges suppresses neuroinflammation in an IL-10-dependent manner. Percentage of ILC2s among CD45 cells derived from the meninges of engrafted mice, n=3 / 3 mice (2 experiments). [Figure 6C]How intrameningeal passively transferred ILC2 engraftment suppresses neuroinflammation in an IL-10-dependent manner. Representative FACS histograms showing comparative expression of lateral scatter in acutely isolated microglia from Rag2- / -γc- / - mice following iv transfer of ILC2 or PBS control. n=3 / 3 (2 experiments). [Figure 6D] How intrameningeal passively transferred ILC2 engraftment suppresses neuroinflammation in an IL-10-dependent manner. Comparative expression as measured by mean fluorescence intensity (MFI) of a panel of microglial activation markers in FACS of brains isolated from Rag2- / -γc- / - mice after iv transfer of ILC2s or PBS control. *, p<0.05; **, p<0.01; ***, p<0.001; multiple T-test, Holm-Sidak method, n=3 / 3 (2 experiments). [Figure 6E] Intrameningeal engraftment of passively transferred ILC2s suppresses neuroinflammation in an IL-10-dependent manner. Monocyte numbers sorted from the brains of Rag2- / -γc- / - mice after iv transfer of ILC2s or PBS control and 3 days of IMQ. Student's T-test, **, p<0.01, n=5 / 5, 3 / 3 (2 experiments). [Figure 6F] How intrameningeal passively transferred ILC2 engraftment suppresses neuroinflammation in an IL-10-dependent manner. Luminex measurements of factors secreted by microglia sorted from mouse brains in E. Student's T test, **, p<0.01; ****, p<0.0001, n=5 / 5 (1 of 2 experiments). [Figure 6G] Intrameningeal engraftment of passively transferred ILC2s suppresses neuroinflammation in an IL-10-dependent manner. Representative confocal images of Evans blue infiltration into the hippocampal parenchyma in slice brains from IMQ-treated Rag2- / -γc- / - mice following passive transfer of either wild-type or Il10- / - ILC2s or PBS. n=4 / 4 / 4 (2 experiments). [Figure 6H]Intrameningeal engraftment of passively transferred ILC2s suppresses neuroinflammation in an IL-10-dependent manner. Monocyte numbers sorted from the brains of Rag2- / -γc- / - mice after iv transfer of ILC2s or PBS control and 3 days of IMQ. Student's T-test, **, p<0.01, n=5 / 5, 3 / 3 (2 experiments). [Figure 6I] How intrameningeal engraftment of passively transferred ILC2s suppresses neuroinflammation in an IL-10-dependent manner. Luminex analysis of microglial secreted factors after 2 days of culture. **, p<0.01; ****, p<0.0001, ANOVA. n=6 / 5 / 5 (2 experiments) with similar results. [Figure 7A] ILC-deficient mice display a "segregated" skin-versus-brain response to IMQ challenge. Total clinical skin lesion scores of the dorsal hairy skin of wild-type, Rag2- / - and Rag2- / -γc- / - mice following IMQ treatment. **, p<0.01; ***, p<0.001; ANOVA, n=4 / 4 / 4. [Figure 7B] ILC-deficient mice show a "separate" skin vs. brain response to IMQ challenge. Representative H&E labeling of dorsal skin from three groups. "H", prominent hyperkeratosis, "M", prominent microabscesses, "A", prominent acanthosis, "I", prominent dermal infiltration, "DP", prominent dermal papillae, and "DC", dilated capillaries were prominent in wild-type dorsal hairy skin. "H", hyperkeratosis, "M", microabscesses, "A", acanthosis, and "I", dermal infiltration were prominent in Rag2- / -. Rag2- / -γc- / - showed no significant pathological changes. n=4 / 4 / 4. [Figure 7C] ILC-deficient mice display a "segregated" skin-versus-brain response to IMQ challenge. Representative H&E labeling of brain sections from the three groups. Minor microhemorrhages "H" were seen in wild-type and Rag2- / - and prominent in Rag2- / -γc- / - mice. n=4 / 4 / 4. [Figure 7D]ILC-deficient mice display a "separate" skin-versus-brain response to IMQ challenge. Representative FACS plots showing peripheral monocyte (Ly6cHiCD11bHi) content of total CD45+ cells isolated from wild-type, Rag2- / - and Rag2γc- / - brains following topical treatment with 0.8 mg / day of the TLR7 agonist IMQ for 3 days. [Figure 7E] ILC-deficient mice exhibit "segregated" skin-versus-brain responses to IMQ challenge. Quantitation of (D). p<0.01, ***, p<0.001, ANOVA. n=3 / 3 / 3 (duplicate experiments). [Figure 7F] ILC-deficient mice display a "segregated" skin-versus-brain response to IMQ challenge. Representative confocal images of brain slices (hippocampus) from IMQ-treated Rag2- / - and Rag2- / -γc- / - mice showing Evans blue infiltration of the brain parenchyma. n=3 / 3 (2 experiments). [Figure 7G] ILC-deficient mice display a "segregated" skin vs. brain response to IMQ challenge. Locomotor behavior of vehicle vs. IMQ-treated Rag2- / - and Rag2- / -γc- / - mice as measured by the open field test. No inhibition was measured in Rag2- / - mice, and the significant locomotor inhibition seen in Rag2γc- / - mice compared to Rag2- / - mice is consistent with the increased neuroinflammation also seen in Rag2- / -γc- / -. ****, p<0.0001, ANOVA. n=9 / 9 (duplicate experiments). [Figure 8] Figure 1 shows that blockade of IL-10 abolishes the suppressive effects associated with passive transfer of ILC2. Luminex detection of secreted factors in supernatants of microglial cells sorted from brains of mice transferred with ILC2 in the presence or absence of PBS (control) or neutralizing antibody to IL-10 and treated with 0.8 mg IMQ 3 days prior to harvest. Student's T-test, n=10 / 5 / 5 mice, results of two experiments combined are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Various publications, articles and patents are cited or described in the Background and throughout the specification. Each of these references is incorporated herein by reference in its entirety. Discussion of documents, operations, materials, devices, articles and the like which is included in the specification is for the purpose of providing a context for the invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention(s) disclosed or claimed.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, certain terms used herein have the meanings set forth herein. All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth herein in their entirety. It should be noted that the singular forms "a," "an," and "the" used in this specification and the appended claims include plural referents unless the context clearly dictates otherwise.

[0034] Unless otherwise specified, any numerical values, such as concentrations or concentration ranges, described herein should be understood in all cases as being modified by the word "about". Thus, numerical values ​​typically include ±10% of the described value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible subranges, including integers and fractions of values ​​within the range, and all individual numerical values ​​within the range, unless the context clearly indicates otherwise.

[0035] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are understood to imply the inclusion of a specified integer or group of integers, but not the exclusion of any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or not inherently present in such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0036] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the present invention.

[0037] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to meet the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also understood to be included in the meaning and thus meets the requirements of the term "and / or".

[0038] As used herein, the term "consists of" or variations such as "consist of" or "consisting of," as used throughout the specification and claims, indicates that any recited integer or group of integers is inclusive, but that no additional integer or group of integers is added to the specified method, structure, or composition.

[0039] As used herein, the term "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of" as used throughout the specification and claims indicates the inclusion of any recited integer or group of integers, optionally including any recited integer or group of integers that do not materially change the basic or novel characteristics of the specified method, structure, or composition. See MPEP §2111.03.

[0040] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human, that is to be treated or has been treated by a method according to an embodiment of the present invention. As used herein, the term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

[0041] In certain embodiments, the subject is in need of treatment for a disorder associated with microglial activation, examples of which include, but are not limited to, neurodegenerative diseases, inflammatory disorders, neuropsychological disorders, chronic pain, spinal cord injury, optic neuritis, viral or bacterial infections.

[0042] In certain embodiments, the subject is in need of treatment for a neurodegenerative disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia, multiple sclerosis, prion diseases, amyotrophic lateral sclerosis, Huntington's disease, and aging.

[0043] In certain preferred embodiments, the subject is in need of treatment for a neuropsychological disorder selected from the group consisting of depression, anxiety, bipolar depression, and schizophrenia.

[0044] It should also be understood that the terms "about," "approximately," "generally," "substantially," and the like, used herein when referring to dimensions or features of preferred inventive components, indicate that the described dimensions / features are not precise boundaries or parameters, and do not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters will include variations that do not change the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement, or other systematic errors, manufacturing tolerances, and the like).

[0045] As used herein, the term "combined" refers to the use of multiple therapeutic agents in the context of administering two or more therapeutic agents to a subject. The use of the term "combined" does not limit the order in which therapeutic agents are administered to a subject. For example, a first therapeutic agent (e.g., a composition described herein) can be administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent to a subject.

[0046] As used herein, the term "microglial activation" refers to processes associated with the innate or adaptive activation of microglial cells. Such activation can include morphological changes in microglial cells, including shortening of cellular processes and enlargement of their cell bodies, as well as release of inflammatory cytokines and chemokines, reactive oxygen and / or nitrogen intermediates, proteases and complement proteins, and upregulation of cell surface activation antigens.

[0047] We demonstrate for the first time that meningeal ILC2s exert a potent regulatory role at the brain border by secreting IL-10. ILC-deficient mice showed increased microglial reactivity at baseline and exacerbated neuroinflammatory responses to either adaptive or innate immune challenge in models of EAE and IMQ-mediated systemic inflammation, respectively. In both of these models, ILC-deficient mice showed increased brain infiltration of peripheral cells and impaired BBB integrity, as indicated by leakage into the parenchyma of molecules normally excluded from the brain.

[0048] Characterization of meningeal ILC subsets in mice showed that they were predominantly ILC2 and located in the sagittal sinus, with potential access to both CSF and blood, and later examination of human meningeal samples suggested a similar population of ILC2. RNAseq analysis of meningeal ILC2s sorted from control and IL-33-stimulated mice revealed an activation profile that mapped closely to canonical ILC2s by transcription factors and extracellular markers, but with an unexpected predominance of the Il10 signature.

[0049] Protein levels of IL-10 production and release by meningeal ILC2s stimulated with IL-33, as well as several other factors associated with resolution of inflammation and tissue protection, were subsequently identified, including Timp1, a factor shown to be protective at the BBB by inhibiting Mmps, extracellular matrix and tight junction degrading peptidases. Thus, it was shown that supernatants from activated ILC2s potently inhibited cytokine, chemokine, and Mmp release from microglia in vitro, and the protective effect was largely abolished when IL-10 signaling was blocked. In vivo, passively transferred ILC2s inhibited Rag2 - / - γc - / - Mice demonstrated engraftment of meningeal ILC2s. Compared to PBS-treated controls, engrafted Rag2 - / - γc - / - Mice showed modulation of the hyperactivated microglial phenotype at baseline and a marked amelioration of neuroinflammation in the IMQ model. Critically, neuroprotection was achieved by neutralizing IL-10 or by inhibiting IL-10. - / - It disappeared after ILC2 transfer, indicating an important role for ILC2-derived IL-10.

[0050] Since their initial discovery, the diversity of ILC subtypes has increased substantially, and several functionally similar to T cell types have been well characterized (Artis et al., Nature, 2015, 517:293-301). Nevertheless, ILCs, i.e., regulatory innate lymphoid cells, resulting from regulatory T cells, have remained conspicuously absent. Recently, however, gut-resident lineage-negative cells, lacking canonical ILC2 markers and bearing a transcription factor signature distinguished by Id3 versus the predicted Id2 common to ILCs, have been designated as a source of protective IL-10 in an anti-CD40-induced colitis model (Wang et al., Cell, 2017, 171(1):201-216). Thus, this cell has emerged as a novel and potentially useful alternative to the long-standing enigma of “ILCs” ( REG It was suggested that the ILCs were the first to produce IL-10 and IL-13. Furthermore, it was noted that the ILCs exhibited extremely high tissue specificity. Subsequent discoveries revealed a second, specialized ILC in the lung that selectively produces IL-10 and refrains from producing IL-13, and they were named "ILC2 10 " (Seehus et al., Nat Commun. 2017, 8(1):1900). Thus, tissue specificity leaves comprehensive immune regulatory functions yet to be addressed by the ILC family.

[0051] Transcriptional and functional analysis of meningeal ILC2s described in this application confirmed IL-10 to be highly upregulated after IL-33 activation. The further detection of IL-10 production in canonical ILC2s from other mouse tissues and subsequently from human blood as described herein suggests that IL-10 production by ILC2s may be the rule rather than the exception. It is noted that Gata3, Irf4 and Nfil3 highlighted in the RNAseq dataset described herein are truly responsible for IL-10 production in other cell types, thus eliminating the search for further new factors. Thus, ILC2s may unexpectedly represent a major innate lymphoid cell population with extensive regulatory functions. This previously unrecognized classical ILC2 suggests ILC2 cell therapy as a new therapeutic approach. Moreover, given the demonstrated importance of IL-33 for IL-10 production, the understanding of alarmins may need to be reconsidered to some extent to include repair signals depending on the temporal or molecular context.

[0052] The observations described herein suggest that meningeal ILC2s are multi-competent factors involved in neuroimmune homeostasis, brain-directed chemotaxis of peripheral immune cells through regulation of chemokines such as CCL3 and CCL4, and further play an unexpected role in BBB integrity via both IL-10 and Timp1, and possibly other mechanisms. In view of the present disclosure, additional factors / agents useful for regulating neuroinflammation and barrier integrity can be identified. Such factors / agents may ultimately be useful for either cell or small molecule therapy in the amelioration of CNS inflammatory pathologies such as BBB disruption in multiple sclerosis, meningitis or stroke, or psychiatric disorders complicated by chronic inflammation (Bhattacharya, et al., 2016, Psychopharmacology (Berl). 233:1623-36).

[0053] In a general aspect, the invention relates to methods of reducing or inhibiting microglial activation or reducing BBB breakdown in a patient in need of these methods.

[0054] According to an embodiment of the invention, the method comprises administering to the subject a therapeutically effective amount of type II innate lymphoid cells (ILC2s), preferably activated ILC2s.

[0055] As used herein, the terms "type II innate lymphoid cells," "ILC2," and "ILC2 cells" each refer to a lymphoid-class population that has lymphoid morphology and lacks T cell receptors, but can express type 2 helper T (Th2) cell-associated cytokines upon activation. Examples of Th2 cytokines include interleukin 4 (IL-4), IL-5, IL-9, IL-10, and IL-13. ILC2s are considered to be innate helper cells.

[0056] ILC2s can be identified by expression of one or more markers using methods known in the art in view of the present disclosure. In some embodiments, ILC2 cells test positive for expression of lymphoid marker CD90, positive for expression of lymphoid marker ICOS, and negative for expression of a lineage marker associated with immune cell fate commitment and maturation (hereinafter referred to as Lin). In some embodiments, ILC2 cells test positive for expression of lymphoid markers CD90 and ICOS, negative for expression of Lin, and further test positive for expression of one or more of the following additional markers: IL7Ra (CD127), CD161, ST2, stem cell antigen 1 (Sca1), IL2Ra (CD25), and CRTH2. For example, human ILC2 cells are found in the gastrointestinal tract and lungs and can be identified by CD161 and the chemoattractant receptor homolog molecule expressed on Th2 cells (CRTH2) (Mjosberg et al., Nat Immunol. 2011;12(11):1055-1062). ILC2s can be obtained according to known methods in view of the present disclosure. For example, cells can be isolated from bone marrow and enriched for ILC2s using commercially available kits and fluorescence activated cell sorting (FACS).

[0057] As used herein, the term "activated ILC2" refers to ILC2 that have been contacted with an agent that induces production of Th2 cytokines by the ILC2. In some embodiments, the ILC2 are activated by cytokines such as IL-33, IL-25, IL-7, IL-2, and / or combinations thereof.

[0058] Activated ILC2s can be obtained using methods known in the art in light of the present disclosure. ILC2 cells can be isolated from a variety of suitable sources of ILC2 cells, including, but not limited to, lung tissue, gastrointestinal (GI) tract, CNS tissue, mammalian blood and / or blood products. In some embodiments, ILC2 cells can be derived from human umbilical cord blood cells. Such human umbilical cord blood cells may be derived from the donor subject's and / or the patient's own umbilical cord blood. During isolation, cells may be filtered through a Dacron mesh of dimensions corresponding to the cells of interest and then washed twice for 1 min each at 50×g. Cell viability can be determined by trypan blue dye exclusion. Cells with >90% viability can be used for transplantation. Ex vivo cells can be adult somatic cells, adult progenitor cells, adult stem cells, embryonic progenitor cells, or embryonic stem cells. Such cell sources are well known to those skilled in the art. After isolation, cells can be activated and optionally modified ex vivo.

[0059] ILC2 cells can be activated by co-culturing with other cells and / or by culturing with one or more stimulatory or activating molecules, such as various cytokines. For example, similar to mouse ILC2s, human ILC2s can be expanded and activated in vitro or ex vivo to produce significant amounts of IL-5 and IL-13 in response to IL-2, IL-7, and IL-33 or IL-25. In some embodiments, activated ILC2 cells can be generated using an in vitro method that includes collecting human cord blood from a subject, isolating c-Kit positive cells from the cord blood, and culturing the c-Kit positive cells in the presence of IL-33 cytokines to generate IL-33-activated ILC2 cells. In some embodiments, ILC2 cells are activated by exposing the c-Kit positive cells in culture to one or more of the cytokines IL-33, IL-25, IL-2, and IL-7. In some embodiments, the in vitro method further comprises analyzing the generated ILC2 cells by measuring expression of one or more markers for ILC2, such as expression of ST2, killer cell lectin-like receptor subfamily G member 1 (KLRG1), SCA-1 or CD127 in the ILC2 cells. In some embodiments, the activated ILC2 cells are substantially free of multipotent progenitor 2 (MPP2), which are IL-25 responsive but distinct from ILC2s.

[0060] IL-33 is a cytokine belonging to the IL-1 superfamily. It is a dual-function protein that acts as a nuclear transcription factor and a proinflammatory cytokine. Nuclear localization and association with heterochromatin is mediated by the N-terminal domain, allowing IL-33 to function as a novel transcriptional regulator of the p65 subunit of the NF-κB complex. The C-terminal domain is sufficient to bind to the ST2 receptor and activate the production of type 2 cytokines (e.g., IL-5 and IL-13) from polarized Th2 and ILC2 cells. Thus, in some embodiments, activated ILC2 cells can be generated by contacting ILC2 cells with IL-33 or its C-terminal domain.

[0061] The length of activation can be determined experimentally and may depend on factors such as the source of ILC2, the cytokine, and / or the conditions used for activation. In certain embodiments, ILC2 are contacted with an agent such as IL-33, IL-25, IL-2, IL-7, thymic stromal lymphopoietin (TSLP), etc. for at least 30 minutes (e.g., at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours). In a preferred embodiment, ILC2 are contacted with a cytokine for at least 4 hours for activation before being administered to a subject.

[0062] In some embodiments, activated ILCs useful in the present invention express CD90, SCA-1, iCOS and / or ST2, respond to IL-25 and / or IL-33, and produce IL-10, IL-5, and IL-13.

[0063] In certain embodiments of the present application, the ILC2 administered is genetically modified.Those skilled in the art will recognize that, in view of the present disclosure, genetic modification can be introduced into cells using methods known in the art.For example, one or more viral vectors or a viral vector, as well as other gene delivery and editing tools, including the use of mRNA, siRNA, miRNA, or other genetic modification, can be used to manipulate the gene expression of any given relevant factor.

[0064] In certain embodiments, ILC2s may be engineered to express immune-modulating molecules, including but not limited to cytokines, preferably ILC2s are engineered to express IL-10. In certain embodiments, ILC2s may be genetically engineered to express an agent that results in increased ILC2 numbers and / or increased IL-10 production, preferably ILC2s are engineered to express the IL-33 receptor. In certain embodiments, ILC2 cells may be genetically engineered to express one or more other products of interest, such as one or more proteins known to be effective in reducing microglial activation, e.g., TGF-β.

[0065] As used herein, "autologous" refers to biological material or cells derived from the tissues or cells of a subject or host. The activated ILC2s administered to a subject may be autologous.

[0066] As used herein, "xenogeneic" refers to biological material or cells derived from a different species than the subject or host, or from a different individual tissue or cell of the same species (e.g., allogeneic or xenogenic). Activated ILC2s administered to a subject may be xenogeneic.

[0067] In certain embodiments of the present application, a method for microglial activation or reducing BBB permeability in a subject in need thereof comprises administering to the subject a therapeutically effective amount of an agent capable of increasing the number of activated ILC2s in the subject. Examples of agents capable of increasing the number of activated ILC2s include, but are not limited to, agents capable of activating ILCs, such as IL-33, IL-25, thymic stromal lymphopoietin (TSLP), IL-2, and IL-7.

[0068] Increasing the number of activated ILC2s in a subject can mean increasing the number of total activated ILC2s in the subject, or increasing the number of specific tissue-resident activated ILC2s. ILC2s are present in mammalian skin, lung, liver, intestine, fat, and brain. In a preferred embodiment, the subject has an increased number of activated meningeal ILC2s as a result of one or more treatments of the invention.

[0069] In addition to agents known to be capable of activating ILC2s and increasing the number of activated ILCs, other agents useful for reducing microglial activation or BBB disruption in a subject in need thereof include: (1) contacting ILC2 with a test compound under conditions suitable for proliferation and activation of ILC2; (2) measuring the number of activated ILC2s.

[0070] Suitable conditions for the proliferation and activation of ILC2 are known to those skilled in the art. Suitable growth media for the ex vivo proliferation of cells are well known in the art and are disclosed, for example, in "Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications" RIFreshney, 2010, Wiley-Blackwell. Optimal media for various cells can be obtained from specialized cell suppliers (e.g., ATCC-LGC (MI, Italy); CDC (Atlanta, Ga., USA)). Activated ILC2 can be identified and quantified by the expression of one or more ILC2 markers, such as CD90 and ICOS, IL7Ra (CD127), CD161, ST2, stem cell antigen 1 (Sca1), IL2Ra (CD25), and CRTH2, and negative Lin expression.

[0071] In certain other embodiments of the present application, a method of reducing microglial activation in a subject in need thereof comprises administering to the subject a therapeutically effective amount of an agent capable of increasing the production of IL-10 by ILC2 in the subject. Examples of agents capable of increasing the production of IL-10 by ILC2 include, but are not limited to, IL-25, IL-33, IL-2, IL-4, or combinations thereof. Not all activated ILC2s increase the production of IL-10. The tissue location of the ILC2 appears to play a role. For example, ILC2 in the lungs produce very little IL-10 when present after treatment with IL-33 alone. However, when ILC2 in the lungs are stimulated by IL-33 and IL-2 and / or IL-4, they produce high levels of IL-10. In certain embodiments, a combination of cytokines can be used to obtain a synergistic response in increasing the production of IL-10 by ILC2. For example, a combination of IL-25 and IL-33 can result in much higher production of IL-10 than each separately. In certain other embodiments, a combination of cytokines can be used to obtain a synergistic response in increasing the production of Timp1 by ILC2s.

[0072] Increased production of IL-10 or Timp1 by activated ILC2 in a subject can mean increased production of IL-10 or Timp1 by all activated ILC2 in the subject, or increased production of IL-10 or Timp1 by specific tissue-resident activated ILC2. In a preferred embodiment, the subject has increased production of IL-10 or Timp1 by activated meningeal ILC2 as a result of one or more treatments of the invention.

[0073] In addition to agents known to be capable of increasing IL-10 or Timp1 production by ILC2s, other agents useful for reducing microglial activation in subjects in need thereof include: (1) contacting ILC2s with a test compound under conditions suitable for the production of IL-10 by the ILC2s; (2) measuring the levels of IL-10 or Timp1 produced by ILC2s.

[0074] Conditions suitable for the production of IL-10 by ILC2s are known to those of skill in the art. IL-10 or Timp1 produced by ILC2s can be identified and quantified using methods known in the art in light of the present disclosure, for example, with antibodies specific for IL-10 or Timp1.

[0075] In a particular aspect, the present invention relates to a pharmaceutical composition for the reduction of microglial activation in a patient in need thereof.

[0076] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of activated ILC2 and a pharma- ceutical acceptable carrier.

[0077] In another embodiment, a pharmaceutical composition comprises a therapeutically effective amount of an agent capable of increasing the number of activated type II innate lymphoid cells (ILC2) in a subject, and a pharma- ceutical acceptable carrier.

[0078] In yet another embodiment, the pharmaceutical composition comprises a therapeutically effective amount of an agent capable of increasing the production of IL-10 or Timp1 by ILC2s in a subject, and a pharma- ceutically acceptable carrier. The production of Timp1 can be increased directly by activated ILC2s, or indirectly by another factor (e.g., IL-10) whose production and / or activity is increased by activated ILC2s.

[0079] The pharma- ceutically acceptable carrier should be non-toxic and should not interfere with the effectiveness of the active ingredient. The pharma- ceutically acceptable carrier may include, but is not limited to, one or more of water, glycols, sugars, oils, amino acids, alcohols, preservatives, emollients, stabilizers, coloring agents, and the like. Any suitable pharma- ceutical acceptable carrier may be used with the activated ILC2, the agent that increases the number of ILC2, or the agent that increases the production of IL-10 or Timp1 for administration to a subject. For example, suitable formulations may include aqueous and non-aqueous sterile injections that may include antioxidants, buffers, bacteriostatic agents, bactericidal antibiotics, and solutes that render the formulation isotonic with the body fluids of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. The formulations may be in single-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Some exemplary components are SDS, mannitol or another sugar, and phosphate buffered saline (PBS).

[0080] It will be understood that in addition to the ingredients particularly mentioned above, the formulations of the presently disclosed subject matter may include other agents conventional in the art having relevance to the type of formulation in question. For example, sterile pyrogen-free aqueous and non-aqueous solutions can be used.

[0081] The therapeutic regimens and compositions of the presently disclosed subject matter can be used in conjunction with additional drugs or biological response modifiers, including, but not limited to, cytokines.

[0082] The administration of the compositions of the presently disclosed subject matter may be by any method known to those skilled in the art, including, but not limited to, intravenous administration, intrasynovial administration, transdermal administration, intramuscular administration, subcutaneous administration, topical administration, rectal administration, intravaginal administration, intratumoral administration, oral administration, buccal administration, nasal administration, parenteral administration, inhalation, and insufflation. In some embodiments, suitable methods for administration of the compositions of the presently disclosed subject matter include, but are not limited to, intravenous injection. Alternatively, the compositions may be deposited at the site requiring treatment in any other manner. The particular manner of administration of the compositions of the presently disclosed subject matter depends on a variety of factors, including the distribution and abundance of the cells to be treated, additional tissue or cell targeting properties of the composition, and mechanisms for metabolism or removal of the composition from the site of administration.

[0083] According to embodiments of the present invention, administration of the isolated ILC2 or pharmaceutical composition thereof can be systemic or local. In certain embodiments, administration is parenteral. In preferred embodiments, administration of the isolated ILC2 or pharmaceutical composition thereof to a subject is by injection, infusion, intravenous (IV), intrathecal, or intrafemoral administration. In yet further preferred embodiments, administration of the isolated ILC2 or pharmaceutical composition thereof to a subject is by intravenous or intrathecal administration.

[0084] The administration of the agent, such as a cytokine that increases the number of activated ILC2 or the production of IL-10 by ILC2, may be intramuscular, subcutaneous, or intravenous. However, other administration methods, such as cutaneous, intradermal, or intranasal, may be envisaged as well. Intramuscular administration of the agent can be achieved by using a needle to inject a suspension of the pharmaceutical composition. Alternatively, the use of a needleless injection device (e.g., using Biojector™) to administer the composition or the use of a lyophilized powder of the pharmaceutical composition.

[0085] In certain embodiments, ILC2s can be harvested from a patient, optionally genetically modified and activated by ex vivo processing, and then administered back to the patient to reduce microglial activation.

[0086] For intravenous, cutaneous or subcutaneous injection, the pharmaceutical composition may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability.Those skilled in the art can prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactate Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as necessary.Slow-release preparations may also be used.

[0087] An effective dose of the composition of the present disclosure is administered to a subject in need thereof. As used herein, "effective amount" refers to an amount of a composition that, when administered to a subject in need thereof, provides the subject with a desired local or systemic effect. In some embodiments, an effective amount is an amount sufficient to achieve a beneficial or desired clinical result of reducing microglial activation in a subject. An effective amount can be provided all at once in one administration, or in an amount that provides an effective amount in several administrations.

[0088] The actual dosage level of the active ingredient in the composition of the subject of the present disclosure can be varied to administer an effective amount of the active compound to achieve the desired therapeutic response for a particular subject.The selected dosage level may depend on the activity of the therapeutic composition, the route of administration, the combination with other drugs or treatments, the severity of the condition being treated, and each subject's own factors, such as body weight, age, injury and / or disease state, and medical history, and the time since the onset of the disease or the onset of the disease.However, in view of the present disclosure, it is within the skill of the art to start administering the composition at a level lower than the level required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0089] For example, after reviewing the disclosure of the subject matter of the present disclosure presented herein, one of ordinary skill in the art can adjust the dosage for an individual patient, taking into account the particular formulation, the administration method used with the composition, and the severity of the condition. Further calculation of the dosage may take into account the patient's height and weight, the severity and stage of symptoms, and the presence of additional adverse health conditions. Such adjustments or changes, as well as the evaluation of the timing and manner of making such adjustments or changes, are well known to those of ordinary skill in the medical arts.

[0090] The contents of all cited references (including article references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

[0091] Embodiment The present invention also provides the following non-limiting embodiments.

[0092] Embodiment 1 is a method of reducing or inhibiting microglial activation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of activated type II innate lymphoid cells (ILC2s).

[0093] Embodiment 1a is a method of reducing the permeability of the blood-brain barrier (BBB) ​​in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of activated type II innate lymphoid cells (ILC2s).

[0094] Embodiment 2 is the method of embodiment 1 or 1a, wherein the ILC2s are activated by contacting the ILC2s with at least one cytokine selected from the group consisting of IL-33, IL-25, IL-2, IL-7, or a combination thereof.

[0095] Embodiment 2a is the method of embodiment 2, wherein the ILC2s are activated by contacting the ILC2s with two cytokines selected from the group consisting of IL-33, IL-25, IL-2, and IL-7.

[0096] Embodiment 2b is the method of embodiment 2, wherein the ILC2s are activated by contacting the ILC2s with three cytokines selected from the group consisting of IL-33, IL-25, IL-2, and IL-7.

[0097] Embodiment 2c is the method of embodiment 2, wherein the ILC2s are activated by contacting the ILC2s with a cytokine that is IL-33, IL-25, IL-2, IL-7.

[0098] Embodiment 2d is the method of embodiment 2, wherein the ILC2s are activated by contacting the ILC2s with IL-33 and IL-25.

[0099] Embodiment 2e is a method according to any one of embodiments 2 to 2d, wherein the ILC2s are activated by contacting the ILC2s with at least one cytokine for at least 30 minutes, preferably at least 2 hours, more preferably at least 4 hours.

[0100] Embodiment 3 is the method according to any one of embodiments 1 to 2d, wherein the administered activated ILC2 cells are autologous.

[0101] Embodiment 3(a) is the method of any one of embodiments 1 to 2d, wherein the administered activated ILC2 cells are allogeneic.

[0102] Embodiment 3(b) is the method of any one of embodiments 1 to 2d, wherein the administered activated ILC2 cells are syngeneic.

[0103] Embodiment 4 is the method of any one of embodiments 1 to 3(b), wherein the ILC2s are genetically modified.

[0104] Embodiment 4a is the method of embodiment 4, wherein the cells are genetically modified for increased IL-10 production compared to otherwise identical unmodified cells.

[0105] Embodiment 4b is the method of embodiment 4, wherein the cells are genetically modified for an increase in the number of activated ILC2s compared to otherwise identical unmodified cells.

[0106] Embodiment 4c is the method of embodiment 4, wherein the cells are genetically modified for increased Timp1 production compared to otherwise identical unmodified cells.

[0107] Embodiment 5 is the method of any one of Embodiments 1 to 4c, wherein a therapeutically effective amount of activated ILC2s is administered intravenously or intrathecally.

[0108] Embodiment 6 is a method of reducing microglial activation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent capable of increasing the number of activated type II innate lymphoid cells (ILC2s) in the subject.

[0109] Embodiment 6a is a method of reducing the permeability of the blood-brain barrier (BBB) ​​in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent capable of increasing the number of activated type II innate lymphoid cells (ILC2s) in the subject.

[0110] Embodiment 7 is the method of embodiment 6 or 6a, comprising administering to the subject at least one cytokine selected from the group consisting of IL-33, IL-25, IL-2, IL-7, or a combination thereof.

[0111] Embodiment 7a is the method of embodiment 7, comprising administering to the subject two cytokines selected from the group consisting of IL-33, IL-25, IL-2, and IL-7.

[0112] Embodiment 7b is the method of embodiment 7, comprising administering to the subject three cytokines selected from the group consisting of IL-33, IL-25, IL-2, and IL-7.

[0113] Embodiment 7c is the method of embodiment 7, comprising administering IL-33, IL-25, IL-2, IL-7 to the subject.

[0114] Embodiment 8 is a method of reducing microglial activation in a subject in need thereof, comprising administering to the subject an effective amount of an agent capable of increasing production of IL-10 by activated type II innate lymphoid cells (ILC2s) in the subject.

[0115] Embodiment 8a is a method for reducing microglial activation in a subject in need thereof, comprising administering to the subject an effective amount of an agent capable of increasing production of Timp1 by activated type II innate lymphoid cells (ILC2s) in the subject.

[0116] Embodiment 8b is a method of reducing the permeability of the blood-brain barrier (BBB) ​​in a subject in need thereof, comprising administering to the subject an effective amount of an agent capable of increasing the production of IL-10 by activated type II innate lymphoid cells (ILC2s) in the subject.

[0117] Embodiment 8c is a method of reducing the permeability of the blood-brain barrier (BBB) ​​in a subject in need thereof, comprising administering to the subject an effective amount of an agent capable of increasing the production of Timp1 by activated type II innate lymphoid cells (ILC2s) in the subject.

[0118] Embodiment 9 is a method according to any one of embodiments 8-8c, comprising administering to the subject a therapeutically effective amount of IL-33, IL-25, IL-2, IL-4, or a combination thereof.

[0119] Embodiment 10 is the method of any one of embodiments 1 to 9, wherein the subject is in need of treatment for a disorder associated with microglial activation.

[0120] Embodiment 11 is the method of embodiment 10, wherein the subject is in need of treatment for a neurodegenerative disease, an inflammatory disorder, a neuropsychological disorder, chronic pain, a traumatic brain injury, a spinal cord injury, optic neuritis, a viral or bacterial infection.

[0121] Embodiment 12 is the method of embodiment 10, wherein the subject is in need of treatment for a neurodegenerative disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia, multiple sclerosis, prion diseases, amyotrophic lateral sclerosis, Huntington's disease, aging, meningitis, and stroke.

[0122] Embodiment 13 is the method of embodiment 10, wherein the subject is in need of treatment for a neuropsychological disorder selected from the group consisting of depression, anxiety, bipolar depression, and schizophrenia.

[0123] Embodiment 14 is a pharmaceutical composition for reducing microglial activation in a subject in need thereof, comprising a therapeutically effective amount of isolated activated type II innate lymphoid cells (ILC2s) and a pharma- ceutical acceptable carrier.

[0124] Embodiment 14a is a pharmaceutical composition according to embodiment 14, further comprising at least one agent capable of activating ILC2 or maintaining ILC2 in an activated state in the composition.

[0125] Embodiment 14b is a pharmaceutical composition according to embodiment 14a, wherein at least one agent is selected from the group consisting of IL-33, IL-25, IL-2, and IL-7.

[0126] Embodiment 14c is a pharmaceutical composition according to embodiment 14a, wherein the at least one agent comprises two agents selected from the group consisting of IL-33, IL-25, IL-2, and IL-7.

[0127] Embodiment 14d is a pharmaceutical composition of embodiment 14a, wherein the at least one agent comprises three agents selected from the group consisting of IL-33, IL-25, IL-2, and IL-7.

[0128] Embodiment 14e is a pharmaceutical composition of embodiment 14a, wherein at least one agent comprises IL-33, IL-25, IL-2, and IL-7.

[0129] Embodiment 14f is a pharmaceutical composition according to embodiment 14a, wherein at least one agent comprises IL-33 and IL-25.

[0130] Embodiment 14g is a pharmaceutical composition according to any one of embodiments 14 to 14f, wherein the ILC2s are meningeal ILC2s.

[0131] Embodiment 15 is a pharmaceutical composition for reducing microglial activation in a subject in need thereof, comprising a therapeutically effective amount of an agent capable of increasing the number of activated type II innate lymphoid cells (ILC2) in a subject, and a pharma- ceutical acceptable carrier.

[0132] Embodiment 15a is the pharmaceutical composition of embodiment 15, wherein the pharmaceutical composition comprises at least one agent selected from the group consisting of IL-33, IL-25, thymic stromal lymphopoietin (TSLP), IL-2, and IL-7.

[0133] Embodiment 15b is a pharmaceutical composition according to embodiment 15, wherein the pharmaceutical composition comprises two agents selected from the group consisting of IL-33, IL-25, thymic stromal lymphopoietin (TSLP), IL-2, and IL-7.

[0134] Embodiment 15c is a pharmaceutical composition according to embodiment 15, wherein the pharmaceutical composition comprises three agents selected from the group consisting of IL-33, IL-25, thymic stromal lymphopoietin (TSLP), IL-2, and IL-7.

[0135] Embodiment 15d is a pharmaceutical composition according to embodiment 15, wherein the pharmaceutical composition comprises four agents selected from the group consisting of IL-33, IL-25, thymic stromal lymphopoietin (TSLP), IL-2, and IL-7.

[0136] Embodiment 15e is the pharmaceutical composition of embodiment 15, wherein the pharmaceutical composition comprises IL-33, IL-25, thymic stromal lymphopoietin (TSLP), IL-2, and IL-7.

[0137] Embodiment 15f is the pharmaceutical composition of embodiment 15, wherein the pharmaceutical composition comprises IL-33 and IL-25.

[0138] Embodiment 15g is a pharmaceutical composition according to any one of embodiments 15 to 15f, wherein the ILC2s are meningeal ILC2s.

[0139] Embodiment 16 is a pharmaceutical composition for reducing microglial activation in a subject in need thereof, comprising a therapeutically effective amount of an agent capable of increasing production of IL-10 by activated type II innate lymphoid cells (ILC2) in a subject, and a pharma- ceutical acceptable carrier.

[0140] Embodiment 16a is the pharmaceutical composition of embodiment 16, wherein the pharmaceutical composition comprises at least one agent selected from the group consisting of IL-33, IL-25, thymic stromal lymphopoietin (TSLP), IL-2, and IL-7.

[0141] Embodiment 16b is the pharmaceutical composition of embodiment 16, wherein the pharmaceutical composition comprises two agents selected from the group consisting of IL-33, IL-25, thymic stromal lymphopoietin (TSLP), IL-2, and IL-7.

[0142] Embodiment 16c is a pharmaceutical composition according to embodiment 16, wherein the pharmaceutical composition comprises three agents selected from the group consisting of IL-33, IL-25, thymic stromal lymphopoietin (TSLP), IL-2, and IL-7.

[0143] Embodiment 16d is a pharmaceutical composition according to embodiment 16, wherein the pharmaceutical composition comprises four agents selected from the group consisting of IL-33, IL-25, thymic stromal lymphopoietin (TSLP), IL-2, and IL-7.

[0144] Embodiment 16e is the pharmaceutical composition of embodiment 16, wherein the pharmaceutical composition comprises IL-33, IL-25, thymic stromal lymphopoietin (TSLP), IL-2, and IL-7.

[0145] Embodiment 16f is the pharmaceutical composition of embodiment 16, wherein the pharmaceutical composition comprises IL-33 and IL-25.

[0146] Embodiment 16g is a pharmaceutical composition according to any one of embodiments 16 to 16f, wherein the ILC2s are meningeal ILC2s.

[0147] Embodiment 17 is a method for preparing a pharmaceutical composition according to any one of embodiments 14 to 14g, comprising mixing a therapeutically effective amount of isolated activated type II innate lymphoid cells (ILC2s) with a pharma- ceutical acceptable carrier.

[0148] Embodiment 18 is a method for preparing a pharmaceutical composition according to any one of embodiments 15 to 15g, comprising mixing a therapeutically effective amount of an agent capable of increasing the number of ILC2 in a subject with a pharma- ceutical acceptable carrier.

[0149] Embodiment 19 is a method for preparing a pharmaceutical composition according to any one of embodiments 16 to 16g, comprising mixing a therapeutically effective amount of an agent capable of increasing the production of IL-10 by activated type II innate lymphoid cells (ILC2) in a subject with a pharma- ceutical acceptable carrier.

[0150] Embodiment 20 is a method for identifying an agent useful for reducing microglial activation in a subject in need thereof, comprising: (1) contacting innate lymphoid cells (ILC2) with an agent under conditions favorable for proliferation of the ILC2; (2) measuring the number of ILC2; The method, wherein an increase in the number of ILC2 compared to control levels indicates the agent is useful for reducing microglial activation in a subject in need thereof.

[0151] Embodiment 21 is a method for identifying an agent useful for reducing microglial activation in a subject in need thereof, comprising: (1) contacting innate lymphoid cells (ILC2) with a drug; (2) measuring the level of IL-10 produced by the ILC2; The method further comprises: an increase in the amount of IL-10 produced by ILC2s compared to control levels indicating that the ILC2s are a useful agent for reducing microglial activation in a subject in need thereof.

[0152] Embodiment 21a is a method of identifying an agent useful for reducing microglial activation in a subject in need thereof, comprising: (1) contacting innate lymphoid cells (ILC2) with a drug; (2) measuring the level of Timp1 produced by the ILC2s; The method further comprises: an increase in the amount of Timp1 produced by ILC2s compared to control levels indicating that the agent is useful for reducing microglial activation in a subject in need thereof.

[0153] Embodiment 21b is a method for identifying an agent useful for reducing BBB permeability in a subject in need thereof, comprising: (1) contacting innate lymphoid cells (ILC2) with a drug; (2) measuring the level of IL-10 produced by the ILC2; The method further comprises: an increase in the amount of IL-10 produced by ILC2s compared to control levels indicating that the ILC2 is a useful agent for reducing BBB permeability in a subject in need thereof.

[0154] Embodiment 21c is a method of identifying an agent useful for reducing BBB permeability in a subject in need thereof, comprising: (1) contacting innate lymphoid cells (ILC2) with a drug; (2) measuring the level of Timp1 produced by the ILC2s; The method further comprises: an increase in the amount of Timp1 produced by ILC2s compared to control levels indicating that the ILC2 is a useful agent for reducing BBB permeability in a subject in need thereof.

[0155] Embodiment 22 is a method according to any one of embodiments 1 to 13, a pharmaceutical composition according to any one of embodiments 14 to 16, or a method according to any one of embodiments 17 to 21c, wherein ILC2 express one or more of CD90, ICOS, IL7Ra (CD127), CD161, ST2, stem cell antigen 1 (Sca1), IL2Ra (CD25), and CRTH2, and are negative for expression of Lin.

[0156] Embodiment 22a is the method of embodiment 22, wherein the ILC2 express two of CD90, ICOS, IL7Ra (CD127), CD161, ST2, stem cell antigen 1 (Sca1), IL2Ra (CD25), and CRTH2, and are negative for expression of Lin.

[0157] Embodiment 22b is the method of embodiment 22, wherein the ILC2 express three of CD90, ICOS, IL7Ra (CD127), CD161, ST2, stem cell antigen 1 (Sca1), IL2Ra (CD25), and CRTH2, and are negative for expression of Lin.

[0158] Embodiment 22c is the method of embodiment 22, wherein the ILC2 express four of CD90, ICOS, IL7Ra (CD127), CD161, ST2, stem cell antigen 1 (Sca1), IL2Ra (CD25), and CRTH2, and are negative for expression of Lin.

[0159] Embodiment 22d is the method of embodiment 22, wherein the ILC2 express five of CD90, ICOS, IL7Ra (CD127), CD161, ST2, stem cell antigen 1 (Sca1), IL2Ra (CD25), and CRTH2, and are negative for expression of Lin.

[0160] Embodiment 22e is the method of embodiment 22, wherein the ILC2 express six of the following: CD90, ICOS, IL7Ra (CD127), CD161, ST2, stem cell antigen 1 (Sca1), IL2Ra (CD25), and CRTH2, and are negative for expression of Lin.

[0161] Embodiment 22f is the method of embodiment 22, wherein the ILC2 express seven of CD90, ICOS, IL7Ra (CD127), CD161, ST2, stem cell antigen 1 (Sca1), IL2Ra (CD25), and CRTH2, and are negative for expression of Lin.

[0162] Embodiment 22g is the method of embodiment 22, wherein the ILC2 express eight of the following: CD90, ICOS, IL7Ra (CD127), CD161, ST2, stem cell antigen 1 (Sca1), IL2Ra (CD25), and CRTH2, and are negative for expression of Lin.

[0163] Embodiment 22h is the method of any one of embodiments 22 to 22g, wherein the activated ILC2s produce IL-10.

[0164] Embodiment 22i is a method according to any one of embodiments 22 to 22h, wherein the activated ILC2s further produce at least one of IL-4, IL-5, IL-9, and IL-13.

[0165] Embodiment 22j is the method of embodiment 22i, wherein the activated ILC2s further produce two of IL-4, IL-5, IL-9, and IL-13.

[0166] Embodiment 22k is the method of embodiment 22j, wherein the activated ILC2s further produce three of IL-4, IL-5, IL-9, and IL-13.

[0167] Embodiment 22l is the method of embodiment 22j, wherein the activated ILC2s further produce IL-4, IL-5, IL-9, and IL-13.

[0168] Embodiment 22m is a method according to any one of embodiments 22 to 22l, wherein the activated ILC2s produce Timp1.

[0169] Embodiment 22n is the method of any one of embodiments 1 to 22m, wherein the activated ILC2s are meningeal ILC2s. EXAMPLES

[0170] The following examples are included to further illustrate various embodiments of the subject matter of the present disclosure. However, those skilled in the art will understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the subject matter of the present disclosure. The examples do not limit the present invention in any manner. They merely serve to clarify the present invention.

[0171] Example 1. ILC2s are abundant in the medullary cavity Transcription factor labeling of CD45+Lin-FCεr1a-DX5-CD90+IL7rα+ cells (Figure 1A) was used to separate ILC1 (Tbet+), ILC2 (Gata3Hi) and ILC3 (RORγt+Gata3- / lo) subsets. First, meningeal innate lymphoid cells (mILCs) were profiled to determine the most abundant type. Untreated mouse brains were carefully dissected and immediately placed in 4% paraformaldehyde for fixation. After 48 hours (hr) of fixation at 4°C, brains were incubated in 30% sucrose in H2O for 24 hours and subsequently stored at -80°C. Calvaria were placed in 20 mL fluorescence-activated cell sorting (FACS) buffer (DMEM / F-12 + 2% FBS) in a Petri dish and the meninges were carefully peeled off using forceps. The meninges were placed into a 70 μm cell strainer and gently dissociated using a back-swirling motion (minimum 100 times) of the syringe plunger. The cells were then washed through the filter three times to further release the cells from the dissociated tissue. The tubes were centrifuged at 1500 RPM for 7 minutes (min) at 4°C and then carefully decanted. The pellet was resuspended in 200 μL of FACS buffer and the cells were placed on ice. The cells were extracellularly labeled with antibodies against lineage markers FCεr1α, DX5, CD45, CD90, IL7rα, fixed, permeabilized, and then nuclear labeled for TBET, GATA3, and RoRγt using the TrueNuclear® labeling kit (Biolegend) according to the manufacturer's protocol. Transcription factor FACS revealed that ILC2s (ILC2s) were the predominant population in mouse dural lymphatics (Figure 1B).

[0172] To explore possible anatomical routes by which ILC2s may affect the CNS, we performed immunolabeling and high-resolution confocal imaging of intact meninges with CD90, which labels ILCs and Lyve1, to represent lymphoid versus non-lymphoid tissues (Louveau et al., 2015, Nature 523, 337-341). CD90+ ILCs were visualized primarily within the venous sinuses, but not restricted to the Lyve1+ lymphatic system itself (Figure 1C(i)), and the addition of in situ Gata3 immunolabeling confirmed the presence of ILC2s in the venous sinuses (Figure 1C(ii)). As conduits for both cerebrospinal fluid (CSF) and CNS blood flow, the venous sinuses suggested secreted factors as a possible mechanistic link allowing meningeal ILCs to interact with other cells in the CNS.

[0173] ILCs have not previously been characterized (or even identified) in human meninges. Their presence in fresh postmortem meninges was investigated in this study. Briefly, upon receipt, sagittal sinus, pia mater and choroid plexus tissues 18-24 hours postmortem were dissected, carefully washed with PBS, dissociated and labeled for analysis by flow cytometry. CD45+Lin-CD11c-FcεR1-CD127+CRTH2+CD161+ cells were successfully isolated from all samples (Figure 1D). Initial results from studies with fresh postmortem meninges suggest that human meninges also harbor an ILC2 population similar to that found in the mouse meningeal cavity.

[0174] Example 2. IL-33 and IL-25 activate meningeal ILC2 To examine the relative functional responses of IL-25 and IL-33, Rag2 - / -Mice were treated with each cytokine by intraperitoneal (ip) injection, with mice receiving 0.033 mg / kg daily every 24 hours for 3 days. Brains and meninges were removed as described in Example 1. Cells were isolated and labeled with antibodies against lineage markers FCεr1α, DX5, CD45, CD90, IL7rα, KLRG1, ST2, GATA3, KI-67, and analyzed by FACS. Increases in CD45+ mILC2 percentage, number, and proliferation as measured by KI-67 labeling were seen with injection of either factor, but were most robust with IL-33.

[0175] Example 3. ILC2-deficient mice exhibit microglial activity Microglia, the primary immune system within the CNS, are engaged in constant surveillance and readily respond to protect the surrounding delicate neural tissue. To investigate the role of mILC2-derived factors in CNS immune regulation, we transformed microglia into ILC2-deficient Rag2 cells. - / - γ□ - / - Rag2 control isolated from mouse brain using FACS - / - compared with mice.

[0176] Immunolabeling with Iba-1 indeed suggested a visible difference in microglial density in the brains of ILC-deficient mice (Figure 3A), indicating an increased number of microglia per unit area (Figure 3B). Subsequent analysis by flow cytometry of acutely isolated brain cell suspensions revealed elevated side scatter (a general indicator of cell granularity, and therefore intracellular activity) by microglia from ILC-deficient mice (Figure 3C), and increased expression of CD45 (Figure 3D) and other surface activation markers, such as FcRII / III and MARCO (Figure 3E). These observations suggest that indeed the baseline status of microglia was altered in association with ILC deficiency. Activation markers associated with both type I and type II responses were similarly elevated, suggesting a general activation rather than M1 or M2 skewing.

[0177] To investigate the functional role of mILC2s, control and ILC2-deficient mice were injected with rIL-33 at 0.033 mg / kg daily, every 24 hours for 3 days to activate ILC2s. Meningeal and brain cells were then isolated and placed together in culture at 37°C in RPMI containing 10% FBS, 1:100 P / S, 1:100 L-glutamine, 1:100 NEAA, 1:100 Na-pyruvate, 1:1000 β-mercaptoethanol (all Gibco). IL-33 treatment magnified the difference between ILC2-sufficient and -deficient mice, and the secretion profile showed increased levels of several cytokines and chemokines in ILC2-deficient mice when analyzed by Luminex bead assay.

[0178] This "hyperactivated" microglial state in ILC2-deficient mice could be explained by a loss of immune suppressive action by ILCs, which could enhance microglial de-inhibition in a feasible way. Alternatively, however, a leaky blood-brain barrier and / or blood-cerebrospinal fluid barrier (BBB, BCB) could also lead to increased parenchymal levels of immune stimulatory factors normally restricted to the periphery, resulting in "hyperactivation" of microglia. The first scenario is consistent with a direct immunomodulatory action of ILCs, while the second is consistent with another possibility that meningeal ILCs, similar to intestinal ILCs, are involved in regulating barrier function. To comprehensively explore these possibilities, we characterized responses to both adaptive and innate immune exposure. We chose experimental autoimmune encephalomyelitis (EAE), with direct autoimmune attacks of the CNS, as an adaptive model, and local administration of imiquimod (IMQ), an immunostimulant shown to induce both skin and brain inflammation, to investigate the interplay between innate effectors at the brain interface, including ILCs and microglia.

[0179] The increased severity of EAE in ILC-deficient mice was found to be arrested within the meninges, consistent with T cell abnormalities. 35~55 CD4+ T cells were sorted from peptide-immunized wild-type mice and were then subjected to Rag2 - / - and Rag2 - / -γc - / - Mice were transferred i.v. with EAE. Passive induction of EAE ensured equal numbers and virulence of T cells between groups. Of note, the initial experiment required early termination in all cases, except for one case in which Rag2 - / - γc - / - Mice rapidly progress to complete paralysis and / or moribundity within 24-36 hours of first symptom detection, whereas Rag2 - / - Mice exhibited the expected progression. Thus, adjusting CD4 counts by 50% slowed the disease course, where ILC-deficient mice still showed early symptoms slightly earlier than controls and subsequently showed reduced mortality, although disease severity increased (Figure 4A). Along with severity, the incidence of EAE was significantly increased in the Rag2 - / - γc - / - In parallel with the higher clinical scores, ILC-deficient mice showed more T cell infiltration into the parenchyma (Fig. 4Ci-ii). Interestingly, however, Rag2 - / - The meninges of - / - γc - / - The meninges contained significantly more T cells than the brain (Fig. 4D-ii), the opposite of what was observed in the brain and spinal cord. Quantification of serum factors revealed that Rag2 - / - No differences or trends towards increased proinflammatory cytokines in mice were noted, arguing against the hypothesis that circulating T cells reduce the pathogenicity of peripheral ILCs when present in lymph nodes or spleen.

[0180] Rag2 - / - and Rag2 - / - γc - / - The inverse correlation between the parenchyma and meninges in T cell accumulation in the CNS of mice suggested that ILCs may be involved in arresting encephalitogenic T cells from crossing the BBB.

[0181] In addition, ILC-deficient mice showed a dissociated skin-versus-brain response to local IMQ. IMQ, a TLR7 / 8 agonist, has been widely used topically to induce psoriatic skin inflammation in rodent models and has also been shown to promote vigorous microglial responses and immune infiltration of the brain. - / - Mice maintain a moderate psoriatic response to IMQ, whereas Rag2 - / - γc - / - Together with the finding that the inflammatory response of the peripheral inflammatory cells is not maintained, this suggests that this is the only model to simultaneously examine microglia, ILCs and barrier integrity. It is noted that peripheral inflammatory models may have relevance to CNS pathologies, given the well-documented clinical comorbidity of psychiatric disorders with peripheral inflammatory diseases, e.g., mood disorders with psoriasis.

[0182] Wild type, Rag2 - / - and Rag2 - / - γc - / - We compared the skin and brain responses to IMQ in mice. As expected, the clinical scores (Figure 7A) and histology (Figure 7B) of the skin were significantly higher in the wild-type, Rag2 - / - and Rag2 - / - γc - / - Mice showed severe, moderate, and mild to no pathology, respectively (Figure 7B). Surprisingly, however, hematoxylin and eosin staining (H&E) of brains from the same groups revealed different patterns of microhemorrhages, i.e., wild-type and Rag2 - / - Surprisingly, the disease was milder in both the Rag2 and Rag3 mice. - / - γc - / - The morbidity and mortality of ILC-deficient mice was severe (Figure 7C). Flow cytometry analysis of dissociated brains showed a similar pattern of monocyte infiltration (Figure 7D), with the brains of ILC-deficient mice exhibiting significantly more monocytes (Figure 7E). Thus, surprisingly, ILCs, but not T cells, appeared to be crucial for the observed phenotype of BBB disruption.

[0183] Although parenchymal microhemorrhages and immune infiltration can be considered valid measures of integrity at the macroscopic level, changes in BBB permeability to intracellular particles were also assessed. IMQ treatment was monitored by transcardial injection of Evans Blue dye, which is normally excluded from the brain by the intact blood-brain and blood-CSF barriers (BCB). Consistent with measurements of cellular infiltration, brains of ILC-deficient mice showed increased levels of parenchymal Evans Blue dye compared to Rag2- / - controls (Figure 7F).

[0184] Example 4. ILC2 transfer suppresses microglial activation To examine the ILC2-specific effects on microglia, Rag2 - / - γc - / - Or Rag2 - / - Control mice were subjected to intravenous passive transfer of ILC2s or PBS (control).

[0185] Isolation of ILC2s Rag2 - / - Mice were injected intraperitoneally (ip) with 0.033 mg / kg recombinant IL-33 daily for 3 days. Mice were euthanized and tibias and femurs were isolated in a sterile hood. Muscles were removed from the bones with forceps and then kept on ice in sterile PBS. Bones were gently crushed 4 at a time in sterile PBS using a mortar and pestle. The released bone marrow cells and all bone material were transferred using a pipetman into a 50 mL conical tube and filtered through a 70 μm cell strainer to remove large particles. The cells were then passed through a 40 μm cell strainer to further remove any cellular debris. The tubes were centrifuged at 1500 RPM for 10 minutes at 4° C. and then decanted. The pellet was resuspended in red blood cell lysis buffer and incubated at room temperature for 2 minutes. Ice-cold PBS was added to dilute and stop cell lysis. The tubes were centrifuged at 1500 RPM for 10 minutes at 4°C and then decanted. The pellet was resuspended in 2% BSA and viable cell counted to obtain a cell concentration of 1 x 10 8 The cells were adjusted to 1 / mL and placed on ice. ILC2 cells were enriched using the EasyStep™ Mouse ILC2 Enrichment Kit according to the manufacturer's instructions.

[0186] Sorting of ILC2s The enriched cells were incubated with 2μL / 10 of the following antibody cocktail: 6 Use at a cell concentration of 1 μL / 10 6 Cells were labeled: Zombie Aqua Live / Dead, lineage cocktail, +FCεr1a, +CD49b, CD45, CD90.2, IL7rα, ST2. Live, single, CD45+lineage-CD49b-FCεr1a-CD90.2+IL7rα+ST2+ events were sorted into ILC2 medium.

[0187] ILC2 expansion Sorted cells were plated at 1 × 10 in 100 μL of ILC2 medium supplemented with 50 units / mL recombinant IL-2 and 50 ng / mL recombinant IL-7 in 96-well TC-coated round-bottom plates. 6 Cells were grown in these plates for 2 days and then transferred to 48-well TC-coated flat-bottom plates for an additional 3 days. On day 5, 100 ng / mL rIL-33 (final concentration) was added. On day 6, cells were harvested, washed twice with sterile PBS, and plated at 5 × 10 cells / mL in sterile saline. 5 / mL.

[0188] Passive transfer of ILC2s Female Rag2 - / - γc - / - Inject 100 μL of ILC2 cell suspension (0.5 × 10 6 Mice were injected with 100% ethanol (100% ethanol), 100% ethanol (100% ethanol), or an equal volume of saline (control). On day 41, mice were euthanized by CO2 inhalation. Euthanized mice were immediately perfused transcardially with perfusion buffer using a variable speed pump (Fisher Scientific, Cat #13-876-1) at speed 4.5 "fast" for 3 minutes to remove all blood. Bone marrow was isolated as above. Single meningeal cells were isolated as described in Example 1. Cells were then analyzed using FACS.

[0189] Five weeks after transfer, all animals showed ILC2 engraftment in the meninges (Figure 2), lungs, and femoral bone marrow, with the engrafted cells having distinct marker signatures corresponding to the adoptive tissue. Microglia from mice with ILC2 meningeal engraftment showed reduced activation markers relative to PBS controls (see, e.g., Figures 6A-6I). Microglial density was reduced in the hippocampus of mice transferred with ILC2, likely as a result of reduced activation.

[0190] Example 5. IL-10 is produced by ILC2s following alarmin activation ILC2s in peripheral tissues have been shown to be activated by alarmins (Vannella et al., 2016, Sci Transl Med., 8(337):337ra65). IL-33 has previously been shown to activate meningeal ILC2s (Gadani et al., J Exp Med. 2017;214(2):285-296), and has been shown to increase the absolute numbers, Ki-67 labeling, cytokine labeling (data not shown), and Gata3 expression in the venous sinuses of IL-33-treated mice. + This was confirmed by whole-tissue labeling of a dense population of ILCs ( Fig. 1E ).

[0191] To investigate the whole transcriptome of mILC2s, mice (n=20 / group, two independent groups) were injected with PBS, rIL-25 or rIL-33 (both at 0.033mg / kg). mILC2s were then sorted, RNA extracted and RNAseq analysis was performed. Hierarchical clustering showed clear differences in treated vs. PBS groups. As expected from the purity test, hierarchical clustering reflected the presence of canonical ILC2-associated factors (Figure 1F, 1G). i~iii ). Surprisingly, however, IL-10 was expressed even in these and was upregulated by >11 log2-fold (Fig. 1G ii IL-10 production by meningeal ILC2s was unexpected. REG " and "ILC2" in the lungs. 10IL-10 production by a tissue-specific, non-canonical, novel innate subset of ILCs has been reported (Seehus et al., 2017; Wang et al., 2017). However, ILCs REG In contrast to the Id3 dependency described for , the results described here showed Id2 dominance and a lack of Id3 (Figure 1G i ). Similarly, robust Il13 transcript levels (Figure 1F, 1G ii ) are lung-specific ILC2s that have been reported to lack Il13 expression. 10 In general, meningeal ILC2s are clearly mapped by both transcriptomic and phenotypic markers as identical to canonical ILC2s rather than a novel lineage.

[0192] To confirm the transcriptional data, we investigated IL-10 protein production using several methods. First, meningeal ILC2s were sorted from IL-33-treated mice, maintained in cultures containing IL-2 and IL-7, and then restimulated with IL-33. FACS analysis showed that these cells expressed ST2 and were also strongly positive for IL-5, IL-13, and IL-10 (Figure 1H), and Luminex analysis of culture supernatants confirmed IL-10 release (Figure 1I). To exclude possible cell culture artifacts, cells were then acutely isolated from IL-33-treated mice, and intracellular labeling revealed a significant portion of meningeal ILC2s as IL-10 positive (Figure 1J). Finally, an IL-10 antibody capture reagent was used to identify IL-33-treated Rag2s. - / - Two-photon time-lapse microscopy was performed on acute ex vivo preparations of mouse meninges. + Cells accumulated anti-IL-10 antibody fluorescence over time in living tissue ( Fig. 1K ), consistent with direct IL-10 capture after release.

[0193] In the above experiments, ILC2s were also acutely isolated from non-meningeal tissues for comparison. Surprisingly, ILC2s from calvaria, tibia, and lung also showed positive labeling for IL-10 (data not shown), suggesting that IL-10 production by ILCs may not be tissue specific as previously suggested. Based on these observations, we tested human ILC2s for their ability to respond to IL-10. To do this, we used CD45 + Lin - CD11c - FcεR1 - CD127 + CRTH2 + CD161 + The cells were selected from the blood of healthy donors and cultured under the same conditions as mouse ILC2s. Subsequent analysis of the supernatants showed IL-10 release after IL-33 stimulation (Figures 1K and 1L), indicating that human ILC2s are indeed capable of IL-10 production. Overall, these results provide some evidence that ILC2s generate IL-10 after IL-33 stimulation. Furthermore, these data suggest that IL-10 production may be a previously unrecognized property common to most ILC2s.

[0194] Example 6. ILC2-derived factors suppress microglial LPS responses Next, to determine whether IL-10 production by ILC2s suppresses microglial activation, mILC2s and microglia were co-cultured together. First, ILC2s were sorted from IL-33-treated mice, and microglia were sorted from untreated mice. Microglia were then cultured alone, with ILC2s, or with ILC2 supernatants, followed by LPS stimulation. Soluble ST2 was included in all conditions at a concentration of 300 ng / mL to bind IL-33 and block any direct effects still present in microglia that also express the receptor for IL-33.

[0195] As measured by Luminex, protein levels of IL-5, IL-13, and IL-10 were higher in wells containing either ILC2s or ILC2 supernatants compared to wells containing only microglia with or without LPS. Levels of both IL-10 and IL-13 were reduced in microglia treated with supernatant and LPS compared to supernatant alone, suggesting active consumption and receptor binding of IL-10 and IL-13 by activated microglia. This interpretation was strengthened by the observation that no difference was seen in IL-5 levels, for which microglia do not express a receptor. Several other cytokines and chemokines were also analyzed, with some showing near complete suppression by ILC2s or ILC2 supernatants (e.g. IL-6), others partial suppression (TNF-a) or no suppression (CXCL10), suggesting specificity rather than simple toxic factors for microglia. To specifically determine the IL-10-mediated effects, the experiment was repeated with the addition of both IL-10 (300 ng / mL) and IL10ra (300 ng / mL) neutralizing antibody for 24 h. Neutralization of IL-10 abolished the suppression of microglial responses as evidenced by the reappearance of elevated cytokine and chemokine levels. This result strongly suggested that ILC2-derived IL-10 was primarily responsible for the observed microglial suppression.

[0196] Example 7. Human ILC2 cells produce IL-10 To confirm that human ILC2 cells could produce IL-10 as seen in mouse ILC2, a similar experiment was performed with human cells. ILC2 (Lin-IL7ra+CRTH2+CD161+) were sorted from six separate normal human blood donors and cultured in conditions similar to mouse ILC2 with or without IL-33 stimulation, and supernatants were analyzed for cytokine content using Luminex bead assays. ILC2 from five of the six donors had increased levels of IL-10 (and IL-13 as a positive control) after IL-33 stimulation, suggesting that human ILC2 were capable of IL-10 production. To examine whether ILC2 were unique in their ability to produce IL-10, further samples were obtained, but this time sorted for ILC1, 2, and 3. Cells were incubated in medium containing factors appropriate for recruitment of ILC1, 2, or 3 subtypes (IL-12+IL-15, IL-33, and IL-1b+IL-6+IL-23, respectively). Cytokine levels in the supernatants of all three cell types were measured as described above. Wells containing ILC2s showed significantly more IL-10 and IL-13 than wells containing either ILC1 or 3. ILC3 supernatants from one donor showed IL-10, suggesting that ILC3s may also produce IL-10. These results provided further evidence that human ILCs, particularly ILC2s, can produce IL-10.

[0197] Example 8. ILC2-secreted factors suppress microglial inflammation and neutralization of IL-10 abolishes the suppression ILC2s from IL-33-treated mice and microglia from untreated mice were isolated to test the ability of ILC2s to suppress microglial inflammatory factors. Microglia were cultured alone, directly with ILC2s, or with ILC2-derived supernatants, and then changed to medium containing the TLR7 / 8 agonist R848 (similar to IMQ, well suited for cell culture) or control medium without agonist. Soluble ST2 was included to block any possible direct effects on ST2+ microglia by ILC2-supernatant-derived IL-33. IL-5, IL-13, and IL-10 were all measured from wells containing either ILC2s (Extended Data) or ILC2 supernatants, but were not detectable (IL-5, IL-13) or were minimal (IL-10) in the supernatants from wells containing only microglia (Figure 5A), indicating that microglia are not a significant source of these factors. Several other cytokines and chemokines were also measured, with a heterogeneous inhibitory pattern suggesting specificity (Figure 5B). As both ILC2s and supernatants showed similar effects, we concluded that soluble factors are probably preferred over direct cell contact. In addition to proinflammatory cytokines, microglia produce matrix metalloproteinases (Mmps) by enzymatic degradation of extracellular matrix components, in particular Mmp9, which has an important role in neurotoxicity, and BBB25-27. Mmps are in turn counterregulated by tissue inhibitory members of the metalloproteinase (Timp) family. Timp1 message was highly (6.7 Log2-fold, adjusted p<0.02) upregulated in activated ILC2s by RNAseq, and confirmed by protein analysis of supernatants from IL-33-activated ILC2s (Figure 5C). As expected, microglia showed a strongly increased production of Mmp9 after R848 loading ( Fig. 5D ), which was potently suppressed by supernatants from activated ILC2s ( Fig. 5E ).

[0198] To examine the importance of IL-10 in the suppression of these factors by ILC2 supernatants, neutralizing antibodies against IL-10 and IL-10rα were then included in the assay. The almost complete disappearance of the IL-10 signal in wells treated with antibody alone indicated that antibody blockade was effective (Figure 5F). Thus, neutralization of IL-10 abolished the suppression, as evidenced by the reappearance of elevated cytokine and chemokine (Figure 5G) levels. The relationship between IL2, microglia, and the regulation of Mmp9 was somewhat complex with respect to IL-10 and Timp1, as expected. It was shown that Mmp9 production is directly negatively regulated by IL-10, but also by Timp1, which itself can be induced by IL-1031. Timp1 was also shown to act in an autocrine as well as paracrine manner. Thus, it is expected that the combination of IL-10 and Timp1 will show superior Mmp9 suppression to IL-10 in the absence of Timp1. In support of this, rmIL-10 did indeed suppress Mmp9, but to a lesser extent than ILC2 supernatants, which were intact (Figure 5H). In a further test of this possibility, we compared the effects of supernatants of ILC2s from Il10- / - mice and wild-type mice on unstimulated and R848-stimulated microglia in terms of total Timp1 protein secretion, and found that the presence of IL-10 increased Timp1 levels in either condition (Figure 5I). A synergistic effect between IL-10 and Timp1 was suggested by the Mmp9 / Timp1 ratio, and blockade of IL-10 and IL-10ra was sufficient to strongly reverse the suppression mediated by ILC2 supernatants (Figure 5J).

[0199] Overall, these data suggest that meningeal ILC2s likely function in multiple ways to suppress not only inflammatory cytokine and chemokine production but also BBB degradation by Mmps such as Mmp9. IL-10 is further suggested to play a dual role, directly as a suppressor of Mmp9 and indirectly via Timp1. Other factors highlighted by the RNAseq data that are upregulated in activated meningeal ILC2s, e.g., amphiregulin, arginase, also play roles in BBB protection and suppression of neuroinflammation and could be amenable to further investigation.

[0200] Passively transferred ILC2s engraft within the meninges and display IL-10-dependent suppression of neuroinflammation To investigate whether ILC2s alone could affect microglial phenotype and suppress neuroinflammation in vivo, we also investigated Rag2 - / - and Rag2 - / - γc - / - To exclude the possibility that the differences observed between the adult and mouse models were due to either developmental differences or a defect in the common γ-chain of the microglial IL-2 receptor, we - / - γc - / - We performed iv adoptive transfer of ILC2s into mice. Robust meningeal engraftment of CD45+Lin-CD90+ST2+GATA3+ cells was confirmed (Figure 6A), and the functionality of engrafted meningeal ILC2s was confirmed by the Rag2 ILC2-transferred - / - γc - / - This was established as cultured meninges supernatants from mice were positive for detection of IL-5, whereas controls were not (Figure 6B). Subsequent examination of acutely isolated brains showed that side scatter was significantly reduced (Figure 6C-D) and expression of microglial activation markers was markedly attenuated compared to PBS-treated ILC-deficient controls (Figure 6D). These data suggested that ILC2s alone were sufficient to modulate baseline microglial phenotype, prompting us to test whether engrafted ILC2s could suppress neuroinflammation, especially considering the effect on possible ILC2-based therapeutic applications.

[0201] To examine this, we also performed adoptive transfer of ILC2s prior to IMQ loading. When whole brain samples were sorted for myeloid cells, the number of microglia (CD11b+CD45Mid) did not differ between groups (data not shown), but brains of mice engrafted with ILC2s contained significantly fewer monocytes (CD11b+CD45HiLy6cHi) than brains of ILC-deficient controls, suggesting that supplementation with ILC2s was sufficient to blunt the infiltration (Figure 6E). We also sorted equivalent numbers of microglia per mouse from brains, placed them in culture, and analyzed the supernatants for secreted factors, revealing that microglia from the brains of mice engrafted with ILC2s displayed a suppressed profile, primarily in terms of chemokine levels (Figure 6F).

[0202] To further determine whether IL-10 blockade affects the ability of ILC2 transfer to inhibit cell infiltration and microglial inflammation, we repeated the above experiment, but this time injecting a neutralizing antibody against IL-10 on days 2 and 3 of IMQ treatment. Similar to the in vitro experiments, IL-10 blockade abolished the inhibitory effect of ILC2 transfer on microglial release of proinflammatory factors (Figure 8). Finally, mice were treated with either wild-type or Il-10 - / - We investigated the importance of cell-specific ILC2-derived IL-10 by adoptively transferring either wild-type or Il10 ILC2s. Again, as suggested by the in vitro results, wild-type but not Il10 - / - Transfer of ILC2s, but not ILC2s, blocked both Evans blue staining (Figure 6G) and parenchymal infiltration by monocytes (Figure 6H). Consistent with these observations, microglial supernatants demonstrated suppression of immune factors in mice transferred with wild-type ILC2s, but not Il10s. - / - Microglia isolated from mice transferred with ILC2s showed little evidence of suppression ( Fig. 6I ), suggesting the importance of ILC2-derived IL-10.

[0203] Altogether, these results suggested that ILC2-secreted factors could play one of several possible mechanistic roles in strengthening the barrier protecting delicate CNS tissues and suppressing associated microglial inflammation. Indeed, such activity was consistent with ILC-mediated barrier regulation shown in peripheral mucosal tissues such as the intestine, where IL-10 also plays a prominent role.

[0204] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined by this description.

[0205] [Embodiment] (1) A method for reducing microglial activation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of activated type II innate lymphoid cells (ILC2s). (2) The method of embodiment 1, wherein the activated ILC2 is activated by contacting the ILC2 with at least one cytokine selected from the group consisting of IL-33, IL-25, IL-2, and IL-7, or a combination thereof. (3) The method of embodiment 1, wherein the ILC2s are genetically modified, preferably wherein the ILC2s are genetically modified for increased IL-10 production compared to otherwise identical non-modified cells. (4) The method of embodiment 1, wherein the therapeutically effective amount of activated ILC2 is administered intravenously or intrathecally to the subject. (5) The method of embodiment 1, wherein the ILC2 is a meningeal ILC2.

[0206] (6) A method for reducing microglial activation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent capable of increasing the number of activated type II innate lymphoid cells (ILC2) in the subject. (7) The method of embodiment 6, wherein the agent increases the number of meningeal ILC2 in the subject. (8) The method of embodiment 6 or 7, wherein the agent is IL-33, IL-25, thymic stromal lymphopoietin (TSLP), or a combination thereof. (9) A method for reducing microglial activation in a subject in need thereof, comprising administering to the subject an effective amount of an agent capable of increasing production of IL-10 by activated type II innate lymphoid cells (ILC2) in the subject. (10) The method of embodiment 9, wherein the agent increases production of IL-10 by activated meningeal ILC2s in the subject.

[0207] (11) The method according to embodiment 9 or 10, wherein the agent is IL-33, IL-25, IL-2, IL-4, or a combination thereof. (12) The method according to any one of embodiments 1 to 11, wherein the subject is in need of treatment for a disorder associated with microglial activation or increased permeability of the BBB. (13) The method of embodiment 12, wherein the subject is in need of treatment for a neurodegenerative disease, an inflammatory disorder, a neuropsychological disorder, chronic pain, a traumatic brain injury, a spinal cord injury, optic neuritis, or a viral or bacterial infection. (14) The method of embodiment 13, wherein the subject is in need of treatment for a neurodegenerative disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia, multiple sclerosis, prion diseases, amyotrophic lateral sclerosis, Huntington's disease, aging, meningitis, and stroke. (15) The method of embodiment 13, wherein the subject is in need of treatment for a neuropsychological disorder selected from the group consisting of depression, anxiety, bipolar depression, and schizophrenia.

[0208] (16) A method for reducing microglial activation in a subject in need thereof, comprising administering to the subject an effective amount of an agent capable of increasing production of Timp1 by activated type II innate lymphoid cells (ILC2) in the subject. (17) A method for reducing the permeability of the blood-brain barrier (BBB) ​​in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of activated type II innate lymphoid cells (ILC2s), an agent capable of increasing the number of activated type II innate lymphoid cells (ILC2s) in the subject, or an agent capable of increasing the production of IL-10 or Timp1 by activated type II innate lymphoid cells (ILC2s) in the subject. (18) A pharmaceutical composition for reducing microglial activation in a subject in need thereof, comprising a therapeutically effective amount of isolated activated type II innate lymphoid cells (ILC2s) or genetically modified activated ILC2s and a pharma- ceutical acceptable carrier. (19) A pharmaceutical composition for reducing microglial activation in a subject in need thereof, comprising a therapeutically effective amount of an agent capable of increasing the number of activated type II innate lymphoid cells (ILC2) in the subject, and a pharma- ceutical acceptable carrier. (20) A pharmaceutical composition for reducing microglial activation in a subject in need thereof, comprising a therapeutically effective amount of an agent capable of increasing IL-10 production by activated type II innate lymphoid cells (ILC2) in the subject, and a pharma- ceutical acceptable carrier.

[0209] (21) A pharmaceutical composition for reducing microglial activation in a subject in need thereof, comprising a therapeutically effective amount of an agent capable of increasing production of Timp1 by activated type II innate lymphoid cells (ILC2) in the subject, and a pharma- ceutical acceptable carrier.

Claims

[Claim 1] 1. A method for reducing microglial activation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of activated type II innate lymphoid cells (ILC2).

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