Treatment of hepatic fibrosis with autologous macrophages

Abstract

The present disclosure provides a composition for treating or preventing fibrosis and / or inflammation in an organ or tissue in a subject. The present disclosure specifically provides a composition for treating or preventing fibrosis and / or inflammation in an organ or tissue in a subject, the composition containing macrophages derived from monocytes stimulated with IL-34. The composition can be produced by a method comprising: a step for preparing a sample comprising CD14-positive cells; an optional step for isolating the CD14-positive cells from the sample if required; and a step for bringing the sample or the isolated CD14-positive cells into contact with IL-34.

Description

Treatment of liver fibrosis with autologous macrophages

[0001] This application claims the benefit of priority from Japanese Patent Application No. 2024-90720 filed on June 4, 2024, and Japanese Patent Application No. 2024-188425 filed on October 25, 2024, the contents of which are incorporated herein by reference. The present invention relates to macrophages capable of treating or preventing fibrosis and / or inflammation in a target organ or tissue, compositions comprising such macrophages, methods for producing such macrophages, and methods of treatment using such macrophages.

[0002] In liver cirrhosis, abnormally accelerated fibrosis occurs within the liver. This fibrosis impairs normal liver tissue function, and if it becomes decompensated, a cure is impossible except through liver transplantation. Cell therapy using autologous or allogeneic cells has been proposed as a new treatment for liver cirrhosis. Various cell types, including hepatocytes and mesenchymal stem cells, have been considered. One of these is autologous macrophage therapy, a very simple method in which autologous macrophages obtained from the patient's peripheral blood are cultured and expanded and then administered to the patient. This therapy utilizes the tissue repair ability of macrophages, and is said to promote the degradation of fibrous tissue and the regeneration of liver tissue through the production of chemokines, immunosuppressive molecules, matrix metalloproteinases (MMPs), and Wnt3A. Several research groups have shown excellent results in animal experiments, and the results of a phase I human trial in the UK have recently been reported.

[0003] The cell preparation method for autologous macrophage therapy that has been reported to date involves collecting CD14-positive cells from peripheral blood and culturing them in the presence of CSF-1 (M-CSF) for approximately 7 days.

[0004] The present inventors have found that monocyte-derived macrophages stimulated with IL-34 are more effective in treating or preventing fibrosis or inflammation in organs or tissues than monocyte-derived macrophages stimulated with CSF-1. Historically, CSF-1 has been analyzed far more extensively, while IL-34 is a cytokine that was first reported in 2008. However, there has been no previous knowledge regarding IL-34-induced macrophages in hepatic fibrosis. Therefore, it was unexpected that monocyte-derived macrophages stimulated with IL-34 were more effective than monocyte-derived macrophages stimulated with CSF-1.

[0005] The present invention provides, for example, the following items: (Item 1) A composition for treating or preventing fibrosis and / or inflammation in a target organ or tissue, comprising macrophages derived from monocytes stimulated with IL-34. (Item 2) The composition described in the above items, wherein the macrophages are derived from monocytes further stimulated with a cytokine selected from the group consisting of IL-4, IL-6, and combinations thereof. (Item 3) The composition described in any one of the above items, wherein the fibrosis and / or inflammation is in an organ selected from the group consisting of the liver, lung, heart, and kidney. (Item 4) The composition described in any one of the above items, wherein the fibrosis and / or inflammation is in the liver. (Item 5) The composition described in any one of the above items, wherein the fibrosis and / or inflammation is cirrhosis or hepatitis. (Item 6) A method for producing a composition for treating or preventing fibrosis and / or inflammation in a target organ or tissue, the method comprising the steps of: preparing a sample containing CD14-positive cells; optionally isolating the CD14-positive cells from the sample; and contacting the sample or the isolated CD14-positive cells with IL-34. (Item 7) The method of any one of the above items, wherein the CD14-positive cells are further contacted with a cytokine selected from the group consisting of IL-4, IL-6, and combinations thereof. (Item 8) The method of any one of the above items, wherein the sample comprises peripheral blood mononuclear cells or bone marrow-derived cells. (Item 9) Macrophages derived from monocytes stimulated with (i) IL-34 and (ii) a cytokine selected from the group consisting of IL-4, IL-6, and combinations thereof. (Item 10) A method for producing macrophages with high IL-10 and / or CCL17 expression, the method comprising the steps of: preparing a sample containing CD14-positive cells; optionally isolating the CD14-positive cells from the sample; and culturing the sample or the isolated CD14-positive cells in the presence of (i) IL-34 and (ii) cytokines selected from the group consisting of IL-4, IL-6, and combinations thereof.(Item 11) A composition for treating or preventing fibrosis and / or inflammation in a target organ or tissue, comprising macrophages that express low levels of CXCL10 and / or iNOS. (Item 12) A composition for treating or preventing fibrosis and / or inflammation in a target organ or tissue, comprising macrophages that express high levels of MHC class II-related molecules, high levels of PD-L1, high levels of PD-L2, high levels of CD11c, or a combination thereof. (Item 13) The composition of any one of the above items, wherein the macrophages express low levels of CXCL10 and / or iNOS. (Item 1A) A method for treating or preventing fibrosis and / or inflammation in a target organ or tissue, comprising administering to the subject macrophages derived from monocytes stimulated with IL-34. (Item 2A) The method of the above items, wherein the macrophages are derived from monocytes further stimulated with a cytokine selected from the group consisting of IL-4, IL-6, and a combination thereof. (Item 3A) The method of any one of the above items, wherein the fibrosis and / or inflammation is in an organ selected from the group consisting of the liver, lung, heart, and kidney. (Item 4A) The method of any one of the above items, wherein the fibrosis and / or inflammation is in the liver. (Item 5A) The method of any one of the above items, wherein the fibrosis and / or inflammation is cirrhosis or hepatitis. (Item 6A) A method for treating or preventing fibrosis and / or inflammation in a subject's organ or tissue, comprising administering to the subject macrophages that express low levels of CXCL10 and / or iNOS. (Item 7A) A method for treating or preventing fibrosis and / or inflammation in a subject's organ or tissue, comprising administering to the subject macrophages that express high levels of MHC class II-related molecules, high levels of PD-L1, high levels of PD-L2, high levels of CD11c, or a combination thereof. (Item 8A) The method according to any one of the preceding items, wherein the macrophages are of the CXCL10 and / or iNOS low expression type.(Item 1B) Use of macrophages derived from monocytes stimulated with IL-34 in the manufacture of a medicament for treating or preventing fibrosis and / or inflammation in an organ or tissue of a target. (Item 2B) The use according to the above items, wherein the macrophages are derived from monocytes further stimulated with a cytokine selected from the group consisting of IL-4, IL-6, and combinations thereof. (Item 3B) The use according to any one of the above items, wherein the fibrosis and / or inflammation is in an organ selected from the group consisting of the liver, lung, heart, and kidney. (Item 4B) The use according to any one of the above items, wherein the fibrosis and / or inflammation is in the liver. (Item 5B) The use according to any one of the above items, wherein the fibrosis and / or inflammation is cirrhosis or hepatitis. (Item 6B) Use of macrophages with low CXCL10 and / or iNOS expression in the manufacture of a medicament for treating or preventing fibrosis and / or inflammation in an organ or tissue of a target. (Item 7B) Use of macrophages that express high MHC class II-related molecules, high PD-L1, high PD-L2, high CD11c, or a combination thereof, in the manufacture of a medicament for treating or preventing fibrosis and / or inflammation in a target organ or tissue. (Item 8B) The use according to any one of the above items, wherein the macrophages express low levels of CXCL10 and / or iNOS. (Item 1C) Macrophages derived from monocytes stimulated with IL-34 for treating or preventing fibrosis and / or inflammation in a target organ or tissue. (Item 2C) The macrophages according to any one of the above items, wherein the macrophages are derived from monocytes further stimulated with a cytokine selected from the group consisting of IL-4, IL-6, and a combination thereof. (Item 3C) The macrophages according to any one of the above items, wherein the fibrosis and / or inflammation is in an organ selected from the group consisting of the liver, lung, heart, and kidney. (Item 4C) The macrophage according to any one of the preceding items, wherein the fibrosis and / or inflammation is in the liver.(Item 5C) The macrophage according to any one of the preceding items, wherein the fibrosis and / or inflammation is cirrhosis or hepatitis. (Item 6C) A macrophage with low CXCL10 and / or iNOS expression for treating or preventing fibrosis and / or inflammation in a target organ or tissue. (Item 7C) A macrophage with high MHC class II-related molecule expression, high PD-L1 expression, high PD-L2 expression, high CD11c expression, or a combination thereof for treating or preventing fibrosis and / or inflammation in a target organ or tissue. (Item 8C) The macrophage according to any one of the preceding items, wherein the macrophage has low CXCL10 and / or iNOS expression.

[0006] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0007] The present invention uses monocyte-derived macrophages stimulated with IL-34 to more strongly suppress the expression of inflammatory chemokines and iNOS, which is characteristic of M1 macrophages, and to highly express MHC class II. Furthermore, monocyte-derived macrophages stimulated with IL-34 can reduce fibrosis in a mouse liver fibrosis model. Therefore, monocyte-derived macrophages stimulated with IL-34 can be used to treat or prevent fibrosis or inflammation in organs or tissues.

[0008] Figure 1 shows a schematic diagram of how CSF-1 and IL-34 bind to the CSF-1 receptor (CSF-1R) on monocytes and induce macrophage survival, proliferation, and differentiation (Science, 320:807, 2008). Figure 2 shows a graph demonstrating that IL-34 strongly induces the immunosuppressive molecule IL-10 in an M1 environment and induces CCL-17, which promotes wound healing, in an M2 macrophage environment (Sci Rep 8:256, 2018). Figure 3 shows the results of flow cytometry analysis of various surface antigens in macrophages stimulated with CSF-1, IL-34, IL-34 + CSF2, IL-34 + IL-6, or IL-34 + IL-4. Figure 4 shows the results of liver observations of wild-type mice (BALB / c) and T cell-deficient nude mice (BALB / c-nu / nu) harvested on day 14 of bile duct ligation (BDL) to induce liver fibrosis. The vertical axis of the graph indicates the area of ​​fibrosis (%) (Student's t-test, p**<0.01). Figure 5 shows the results of analyzing the role of CD8+ T cells in liver fibrosis in C57BL / 6J mice administered a CD8 T cell-depleting antibody 1 and 6 days before bile duct ligation (BDL). The vertical axis of the graph indicates the area of ​​fibrosis (%) (Student's t-test, p**<0.05, N=4). Figure 6 shows the results of administering various macrophages (in the figure, macrophages are referred to as MΦ or Mf) on day 10 after bile duct ligation (BDL), and measuring the fibrosis rate after removing the liver on day 14. The vertical axis of the graph represents the area of ​​fibrosis (%) (Tukey-Kramer, p*<0.05, p***<0.001). Figure 7 shows the results of administering macrophages induced from bone marrow cells with IL-34 or M-CSF in a mouse model of liver cirrhosis induced with carbon tetrachloride, and measuring hepatic enzymes (AST, ALT) as indicators of hepatocellular damage (blood test) and pathological examination. 8 shows the results of measuring the effect of culturing macrophages on cell proliferation in the presence of IL-34 or CSF1 with additional cytokines (IL-6, IL-4, CSF2, TNFα, IL-2, IL-18), saline, DMSO, or PGE2. The left bar in each group represents IL-34, and the right bar represents CSF1.Figure 9 shows the results of measuring the fibrosis rate in a mouse model in which liver fibrosis is induced by bile duct ligation (BDL). Various macrophages were administered on day 10 after BDL, and the liver was removed on day 14. Image analysis of Sirius Red staining was performed using ImageJ. The vertical axis of the graph indicates the area of ​​fibrosis (%) (Tukey's multiple comparisons test, p*<0.05). Figure 10 shows a graph of the change in body weight over time in the group administered with various macrophages. The vertical axis of the graph indicates the percentage change, with day 0 set as 100% (Tukey's multiple comparisons test, p*<0.05). Figure 11 shows Kaplan-Meier survival curves in the group administered with various macrophages. Figure 12A shows the results of CCl. ４Figure 12 shows Sirius red staining of liver sections from C57BL / 6J mice in which liver fibrosis was induced by IFN-γ. (A) Saline or macrophages were intravenously injected at week 8, and liver samples were collected at week 12 (n=6 per group). The scale bar represents 200 μm. The bar graph represents the quantified Sirius red-positive area ratio (mean ± SEM). *P<0.05, ***P<0.001. Similar results were obtained from two independent experiments. Figure 12B shows the appearance of macrophages. The scale bar represents 100 μm. Figure 12C shows a comparison of CSF-1 and IL-34 macrophage mRNA expression. Figure 13A shows a comparison of macrophage mRNA expression levels. The vertical axis represents fragments per million mapped reads (FPKM) per kilobase of exon. ND indicates not detected. Figure 13B shows the number of viable macrophage cells detected by MTT assay. ***P<0.001. Figure 14A shows Sirius red staining of liver sections from mice with BDL-induced liver fibrosis (n>6 per group). Saline or macrophages were intravenously injected on day 10, and liver samples were collected on day 14 (n=6 per group). The scale bar represents 200 μm. Figure 14B shows Sirius red staining of liver sections from mice with TAA-induced liver fibrosis (n>6 per group). Saline or macrophages were intravenously injected on week 6, and liver samples were collected on week 10. The scale bar represents 200 μm. The bar graph shows the quantification of the Sirius red-positive area ratio (mean ± SEM). Similar results were obtained from two independent experiments. Figures 14C and D show immunohistochemistry of liver sections for COL1A (C) and α-SMA (D) expression. Bar graphs show quantification of immunostaining-positive area ratios (mean ± SEM, n = 6 per group). *P < 0.05. Figure 15A shows the gating strategy for flow cytometry analysis. Figure 15B shows histograms showing CFSE fluorescence intensity of CD4 and CD8, or cells gated on CD3 (left, green) for T cell proliferation without co-culture. CFSE-stained T cells selected from splenocytes with magnetic beads were co-cultured with IL-34 + IL-4 macrophages and stimulated with anti-CD3 and anti-CD28 antibodies for 4 days.Culture conditions included control IgG (red), anti-PD-L1 and anti-PD-L2 antibodies (Abs, blue), and T cells cultured without growth stimulation (unstimulated, gray). Bar graphs show the proliferation rates (non-gray area / live gate) of cells in the Abs and control IgG groups (mean ± SEM, n = 3 per group). ***P<0.001. Figure 15C shows immunohistochemistry of CD8-positive cells in liver sections. Bar graphs show quantification of the number of immunostained cells (mean ± SEM, n = 3 per group). ***P<0.001. Figure 16A shows flow cytometry analysis of human monocyte-derived macrophages stimulated with IL-34 and IL-4 for various surface antigens. Green histograms show cells expressing the indicated surface proteins. Gray histograms show isotype control staining. Figure 16B shows histograms showing CFSE fluorescence intensity of CD4- and CD8-gated cells. Human IL-34 + IL-4 macrophages were co-cultured with CFSE-stained T cells stimulated with anti-CD3 and anti-CD28 antibodies for 4 days. Figure 16C shows Sirius red staining of liver sections from NSG mice (n=3 per group) injected with human PBMCs. Saline or IL-34 + IL-4 macrophages were intravenously injected at 4 weeks, and liver samples were collected at 8 weeks. The scale bar represents 200 μm. The scale bar represents 200 μm. The bar graph shows quantification of the Sirius red-positive area ratio (mean ± SEM). *P<0.05.

[0009] It should be understood that the terms used in this specification are used in the meanings commonly used in the relevant field unless otherwise specified.Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.In the event of any discrepancy, this specification (including definitions) shall prevail.

[0010] <Definitions> First, terms and general techniques used in this disclosure will be explained.

[0011] As used herein, "about" means ±10% of the indicated value.

[0012] As used herein, "monocyte" refers to a mononuclear leukocyte present in the blood that can differentiate into macrophages upon migration to tissues. "Macrophage" refers to a phagocyte that differentiates from monocytes and ingests and decomposes foreign substances such as dead cells, their debris, and pathogens. Monocytes and macrophages are characterized by CD14 positivity. CD14 positivity can be measured using methods known in the art, such as flow cytometry using an anti-CD14 antibody.

[0013] As used herein, "macrophages derived from monocytes stimulated with IL-34" refers to macrophages obtained by contacting monocytes with IL-34 during the process of differentiation of monocytes into macrophages. Macrophages derived from monocytes stimulated with IL-34 are further characterized by being CD14-positive and by high expression of IL-10 and / or CCL17 compared to macrophages derived from monocytes stimulated with CSF-1. Here, "macrophages derived from monocytes stimulated with CSF-1" refers to macrophages obtained by contacting monocytes or macrophages with CSF-1 during the process of differentiation of monocytes into macrophages, or by contacting macrophages differentiated from monocytes with CSF-1.

[0014] As used herein, the term "IL-10 and / or CCL17 highly expressing macrophages" refers to macrophages that express higher levels of IL-10 and / or CCL17 than the levels of IL-10 and / or CCL17 expressed in macrophages derived from monocytes stimulated with CSF-1.

[0015] As used herein, the term "high expression of MHC class II-related molecules" refers to the expression of an MHC class II-related molecule at a level higher than that of MHC class II-related molecules in macrophages derived from monocytes stimulated with CSF-1. "MHC class II-related molecules" refers to molecules such as H2-Aa, H2-DMB2, H2-Ab1, H2-DMa, H2-Eb1, H2-DMb1, CD74, and CIITA in the case of mice, and to molecules such as HLA-DP, HLA-DQ, and HLA-DR in the case of humans.

[0016] As used herein, "high PD-L1 expression" refers to the expression of PD-L1 at a level higher than that in macrophages derived from monocytes stimulated with CSF-1.

[0017] As used herein, "high PD-L2 expression" refers to the expression of PD-L2 at a level higher than that of PD-L2 expressed in macrophages derived from monocytes stimulated with CSF-1.

[0018] As used herein, the term "high CD11c expression type" refers to the expression of CD11c at a level higher than that of macrophages derived from monocytes stimulated with CSF-1.

[0019] As used herein, "CXCL10 and / or iNOS low expressor" refers to macrophages that express lower amounts of CXCL10 and / or iNOS than the amounts of CXCL10 and / or iNOS expressed in macrophages derived from monocytes stimulated with CSF-1.

[0020] As used herein, the term "subject" refers to a subject to which the cells, compositions, or methods of the present invention are administered, and examples of subjects include mammals, such as humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, and monkeys.

[0021] As used herein, the term "tissue" refers to a cell aggregate that performs a specific function and constitutes an organ or part of the body. Examples of tissues include, but are not limited to, epithelial tissue, connective tissue, muscle tissue, nerve tissue, vascular tissue, bone tissue, cartilage tissue, and adipose tissue. An "organ" refers to a group of tissues that is composed of multiple tissues and performs one or more functions. Examples of organs include, but are not limited to, the liver, lungs, heart, kidneys, ovaries, pancreas, uterus, small intestine, large intestine, bladder, brain, breast, esophagus, fallopian tubes, gallbladder, ovaries, prostate, placenta, spinal cord, spleen, stomach, testes, thymus, thyroid gland, trachea, and umbilical cord.

[0022] As used herein, "fibrosis" refers to the hardening of organs or tissues caused by excessive deposition of extracellular matrix, such as collagen, by fibroblasts in response to chronic inflammation. "Inflammation" refers to the body's defense response to tissue irritation or injury. Inflammation is characterized by fenestrations in the capillary system, leakage of blood elements into the interstitial space, and migration of leukocytes into inflamed tissue. Redness, heat, swelling, and pain are the four signs of inflammation. Inflammation is distinguished from chronic inflammation. "Acute inflammation" refers to short-term inflammation lasting a few days, referring to transient inflammation. "Chronic inflammation" refers to long-term inflammation lasting several weeks to several years, referring to inflammation with an unclear or indefinite end date. Liver fibrosis refers to fibrosis in the liver caused by persistent inflammation, such as hepatitis B, hepatitis C, fatty liver, or alcoholic hepatitis, or by liver damage due to cholestasis or hepatic congestion. Prolonged inflammation or damage can progress to cirrhosis.

[0023] As used herein, the term "sample" refers to any substance obtained from a subject, and includes whole blood, serum, plasma, saliva, urine, tears, cerebrospinal fluid, bone marrow, etc. Blood samples include whole blood, serum, plasma, and blood cells.

[0024] As used herein, "treatment" refers to preventing the worsening of, maintaining the current state, alleviating, or partially or completely eliminating a certain disease, disorder, or symptom. As used herein, "prevention" refers to preventing a certain disease, disorder, or symptom from occurring before it occurs.

[0025] Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0026] <Monocyte-derived macrophages stimulated with IL-34> IL-34 was discovered in 2008 as a novel cytokine other than CSF-1 that binds to the CSF-1 receptor (CSF-1R), which is important for macrophage survival, proliferation, and differentiation (Figure 1). IL-34 and CSF-1 bind to different sites on the CSF-1R and transmit similar but distinct signals (Cell Death Differ 17:1917, 2010). Furthermore, there is another receptor (Syndecan-1, PTP-ζ) that only IL-34 binds (J Leukoc Biol 104:931, 2018). Therefore, although CSF-1 and IL-34 appear to have similar effects on macrophage proliferation and differentiation, detailed analysis reveals differences in molecular expression and other aspects (Sci Rep 8:256, 2018). For example, IL-34 strongly induces the immunosuppressive molecule IL-10 in an M1 environment, and induces CCL-17, which promotes wound healing, in an M2 environment (Fig. 2).The present inventors found that when macrophages activated with IFN-γ were exposed to CSF-1 or IL-34, the IL-34-treated macrophages more strongly suppressed the expression of inflammatory chemokines and iNOS, which is characteristic of M1 macrophages.

[0027] Macrophages derived from monocytes stimulated with IL-34 of the present invention can be obtained by contacting IL-34 with monocytes or macrophages during the process of monocyte differentiation into macrophages, or by contacting IL-34 with macrophages differentiated from monocytes. Macrophages derived from monocytes stimulated with IL-34 can be characterized by CD14 positivity and high expression of IL-10 and / or CCL17. Thus, IL-34 strongly induces the immunosuppressive molecule IL-10 and induces CCL-17, which promotes wound healing. Macrophages derived from monocytes stimulated with IL-34 of the present invention can be further characterized by low expression of CXCL10 and / or iNOS when activated with IFNγ. IFNγ strongly induces the expression of the proinflammatory cytokine CXCL10 and iNOS, a characteristic of M1 macrophages, in induced macrophages. However, unexpectedly, the addition of IL-34 to IFNγ strongly suppressed the expression of the proinflammatory cytokine CXCL10 and iNOS, a characteristic of M1 macrophages. Therefore, without intending to be bound by theory, IL-34 can induce both inflammatory M1 macrophages and anti-inflammatory M2 macrophages responsible for tissue repair, but it appears to more strongly induce M2 macrophages than M1 macrophages. Induction with IL-4 in combination with IL-34 demonstrated a stronger antifibrotic effect than induction with IL-34 alone (Figures 9 and 14). Furthermore, macrophages induced with IL-34 and IL-4 exhibited a stronger tendency toward the M2 macrophage phenotype than those induced with IL-34 alone (Figure 13).

[0028] Macrophages induced with IL-4 in combination with IL-34 can be characterized by high expression of MHC class II-associated molecules, high expression of PD-L1, high expression of PD-L2, high expression of CD11c, or a combination thereof. MHC class II-associated molecules include, but are not limited to, I-A, I-E, H2-Aa, H2-DMB2, H2-Ab1, H2-DMa, H2-Eb1, H2-DMb1, CD74, and CIITA in mice, and HLA-DP, HLA-DQ, and HLA-DR in humans.

[0029] In one embodiment, the macrophages derived from monocytes stimulated with IL-34 of the present invention can be characterized by (i) CD14 positivity, and (ii) at least one of high expression of IL-10, high expression of CCL17, low expression of CXCL10, and low expression of iNOS.

[0030] In another aspect, the present invention may provide macrophages derived from monocytes stimulated with (i) IL-34 and (ii) a cytokine selected from the group consisting of IL-4, IL-6, and a combination thereof. Macrophages derived from monocytes stimulated with (i) IL-34 and (ii) a cytokine selected from the group consisting of IL-4, IL-6, and a combination thereof are advantageous in that they can further enhance proliferative activity and therapeutic effects compared to macrophages stimulated with IL-34 alone. In yet another aspect, macrophages obtained by stimulating monocytes with (i) IL-34 and (iii) a cytokine or compound selected from the group consisting of IL-13, IL-10, prostaglandin E2 (PGE2), vitamin D3 (VitD3), lipoprotein(a) (LPA), glucocorticoids, and a combination thereof may also be used.

[0031] In one embodiment, the macrophages derived from monocytes stimulated with IL-34 and IL-4 of the present invention can be characterized by (i) CD14 positivity, and (ii) high expression of one, two, three, or four of MHC class II-associated molecules, high expression of PD-L1, high expression of PD-L2, and high expression of CD11c.

[0032] <Method for producing IL-34-stimulated macrophages> In one aspect, the present invention provides a method for producing a composition for treating or preventing fibrosis and / or inflammation in an organ or tissue of a subject, the method comprising the steps of: preparing a sample containing CD14-positive cells; optionally isolating CD14-positive cells from the sample; and contacting the sample or the isolated CD14-positive cells with IL-34.

[0033] In another aspect, the present invention provides a method for producing macrophages that highly express IL-10 and / or CCL17, the method comprising the steps of: preparing a sample containing CD14-positive cells; optionally isolating CD14-positive cells from the sample; and contacting the sample or the isolated CD14-positive cells with (i) IL-34 and (ii) cytokines selected from the group consisting of IL-4, IL-6, and combinations thereof. Macrophages further stimulated with cytokines such as IL-4 and IL-6 have advantageously high proliferative activity and therapeutic effects.

[0034] In one embodiment, the sample contains peripheral blood mononuclear cells and bone marrow-derived cells. The number of peripheral blood mononuclear cells and bone marrow-derived cells contained in the sample may be any number depending on the number of cells to be administered, and can be adjusted appropriately by those skilled in the art. The number of peripheral blood mononuclear cells and bone marrow-derived cells is preferably at least about 1 × 10 ３ pieces, for example, about 1 × 10 ３ ~Approx. 1×10 ８ pieces, approximately 5 x 10 ３ ~Approx. 1×10 ５ In certain embodiments, the sample contains about 1 x 10 peripheral blood mononuclear cells, bone marrow-derived cells, or any number of cells within a range of about 1 x 10 peripheral blood mononuclear cells, bone marrow-derived cells, or any number of cells described herein. ３ , about 5×10 ３ , about 1×10 ４ , about 5×10 ４ , about 1×10 ５ , about 5×10 ５ , about 1×10 ６ , about 5×10 ６ , about 1×10 ７ , about 5×10 ７ , or about 1 × 10８ There can be one.

[0035] In one embodiment, the number of CD14-positive cells contained in the sample may be any number depending on the number of cells to be administered, and can be adjusted appropriately by those skilled in the art. ３ pieces, for example, about 1 × 10 ３ ~Approx. 1×10 ８ pieces, approximately 5 x 10 ３ ~Approx. 1×10 ５ In certain embodiments, the sample contains about 1 x 10 CD14-positive cells or a range of about 1 x 10 CD14-positive cells or any number of cells described herein. ３ , about 5×10 ３ , about 1×10 ４ , about 5×10 ４ , about 1×10 ５ , about 5×10 ５ , about 1×10 ６ , about 5×10 ６ , about 1×10 ７ , about 5×10 ７ , or about 1 × 10 ８ There can be one.

[0036] Monocytes are produced in the bone marrow, enter the blood, and differentiate into macrophages upon migration to tissues. Monocytes are characterized by CD14 positivity. Therefore, cells that can be used in the present method include CD14-positive cells. To produce macrophages more efficiently, CD14-positive cells may be isolated from a sample, but isolation is not necessarily required. Isolation of CD14-positive cells can be performed by isolation means known in the art, such as immunomagnetic beads.

[0037] In one embodiment, contacting CD14-positive cells with IL-34 may include culturing a sample containing CD14-positive cells or isolated CD14-positive cells in the presence of IL-34. IL-34 may be contacted with CD14-positive cells to a final concentration of at least about 1 ng / ml, e.g., about 1 ng / ml to about 1000 ng / ml, about 10 ng / ml to about 200 ng / ml, about 10 ng / ml to about 100 ng / ml, or any of the concentrations described herein. In certain embodiments, the final concentration of IL-34 may be about 1 ng / ml, about 5 ng / ml, about 10 ng / ml, about 20 ng / ml, 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, about 500 ng / ml, about 600 ng / ml, about 700 ng / ml, about 800 ng / ml, about 900 ng / ml, or about 1000 ng / ml.

[0038] In one embodiment, the cells may be further contacted with a cytokine selected from the group consisting of IL-4, IL-6, and a combination thereof, in addition to IL-34. Macrophages further stimulated with cytokines such as IL-4 and IL-6 are advantageous in that they have high proliferative activity and therapeutic effects. In another embodiment, the cells may be further contacted with a cytokine or compound selected from the group consisting of IL-13, IL-10, prostaglandin E2 (PGE2), vitamin D3 (VitD3), lipoprotein(a) (LPA), glucocorticoids, and a combination thereof, in addition to IL-34.

[0039] IL-4 may be contacted with CD14-positive cells to a final concentration of at least about 1 ng / ml, e.g., about 1 ng / ml to about 1000 ng / ml, about 1 ng / ml to about 100 ng / ml, about 5 ng / ml to about 50 ng / ml, or any of the concentrations described herein as lower and upper limits. In certain embodiments, the final concentration of IL-4 is about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, or any of the concentrations described herein as lower and upper limits. 1, about 20 ng / ml, 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, about 500 ng / ml, about 600 ng / ml, about 700 ng / ml, about 800 ng / ml, about 900 ng / ml, or about 1000 ng / ml. IL-6 may be contacted with CD14-positive cells to a final concentration of at least about 1 ng / ml, e.g., about 1 ng / ml to about 1000 ng / ml, about 10 ng / ml to about 200 ng / ml, about 50 ng / ml to about 150 ng / ml, or any of the concentrations described herein as lower and upper limits. In certain embodiments, the final concentration of IL-6 is about 1 ng / ml, about 5 ng / ml, about 10 ng / ml, about 20 ng / ml, 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 110 ng / ml, about 120 ng / ml, about 130 ng / ml, It can be about 140 ng / ml, about 150 ng / ml, about 160 ng / ml, about 170 ng / ml, about 180 ng / ml, about 190 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, about 500 ng / ml, about 600 ng / ml, about 700 ng / ml, about 800 ng / ml, about 900 ng / ml, or about 1000 ng / ml.

[0040] Typically, blood samples are collected using tubes containing ethylenediaminetetraacetic acid to isolate mononuclear cells, followed by cell enrichment. Positive selection involving antibodies targeting CD14 is performed on the monocyte concentrate. Purified monocytes are then cultured in GlutaMAX 1000 ng / ml medium supplemented with 10% (vol / vol) fetal bovine serum (FBS). ＴＭ Monocytes are grown in RPMI 1640 medium containing IL-1 (Life Technologies, 61870044). Monocytes are seeded into tissue culture treated flasks or wells and induced to differentiate into macrophages by adding IL-34 to the medium.

[0041] <Composition for Treatment or Prevention> In one aspect, the present invention provides a composition for treating or preventing fibrosis and / or inflammation in a target organ or tissue, comprising macrophages derived from monocytes stimulated with IL-34. IL-34 induces immunosuppressive molecules (e.g., IL-10), induces cytokines that induce tissue repair (e.g., CCL17), suppresses inflammatory cytokines (e.g., CXCL10), and suppresses M1 macrophage markers (e.g., iNOS) ( Figure 2 ). Therefore, it is believed that IL-34 induces M2 macrophages more strongly than M1 macrophages. M2 macrophages are involved in the suppression of inflammatory responses and tissue repair. Surprisingly, the macrophages of the present disclosure exhibit superior therapeutic effects against fibrosis and / or inflammation in a target organ or tissue compared to conventional macrophages, i.e., monocyte-derived macrophages stimulated with CSF-1, which are known to be effective in the degradation of fibrous tissue and the regeneration of liver tissue.

[0042] Because M2 macrophages are universally involved in the suppression of inflammatory responses and tissue repair, not limited to specific organs or tissues, it is understood that the macrophages of the present disclosure, derived from monocytes stimulated with IL-34, can treat or prevent inflammation in any organ or tissue. Furthermore, because fibrosis is caused by excessive deposition of extracellular matrix, such as collagen, by fibroblasts in response to inflammation, the macrophages of the present disclosure can prevent fibrosis by suppressing the inflammation that causes fibrosis. Furthermore, the macrophages of the present disclosure can treat fibrosis in any organ or tissue by utilizing the tissue repair function of M2 macrophages. In one embodiment, the fibrosis and / or inflammation described herein can occur in an organ selected from the group consisting of the liver, lungs, heart, and kidneys. In certain embodiments, the fibrosis and / or inflammation can occur in the liver. In certain embodiments, the fibrosis and / or inflammation can be caused by cirrhosis or hepatitis.

[0043] In another aspect, the present invention provides a composition for treating or preventing fibrosis and / or inflammation in a target organ or tissue, comprising macrophages that highly express IL-10 and / or CCL17. In one embodiment, the macrophages may highly express MHC class II-related molecules, highly express PD-L1, highly express PD-L2, highly express CD11c, or a combination thereof. In one embodiment, the macrophages may lowly express CXCL10 and / or iNOS.

[0044] The number of macrophage cells to be administered can be determined appropriately by those skilled in the art, but is preferably at least about 1 × 10 ５ pieces, for example, about 1 × 10 ５ pieces ~ 1×10 ９ pieces, approximately 1 x 10 ６ pieces ~ approx. 1×10 ８ or any cell number described herein. In certain embodiments, the number of macrophages administered is about 1 x 10 ５ pieces, approximately 5 x 10 ５ pieces, approximately 1 x 10 ６ pieces, approximately 5 x 10６ pieces, approximately 1 x 10 ７ pieces, approximately 5 x 10 ７ pieces, approximately 1 x 10 ８ pieces, approximately 5 x 10 ８ pieces, approximately 1 x 10 ９ pieces, or about 5 x 10 ９ There can be one.

[0045] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0046] (Mice) C57BL / 6 mice, BALB / c, and BALB / c ｎｕ／ｎｕ Mice (nude mice) were purchased from Japan SLC (Shizuoka, Japan). B6-Ly5.1 mice were purchased from Sankyo Labo Service (Tokyo, Japan). NOD / ShiJic-scid mice (NOD / SCID mice) were purchased from CLEA Japan (Tokyo, Japan). All animal procedures were approved by the Hokkaido University Animal Care and Use Committee (approval number: 20-0086).

[0047] Macrophage culture: Macrophages were maintained in RPMI-1640 (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum (Sigma), 1% penicillin / streptomycin (Nacalai Tesque), and 1% non-essential amino acids (Nacalai Tesque). Depending on the experiment, cytokines such as IL-34 or Csf1 were added. All cells were cultured in a 5% CO atmosphere. ２ The cells were maintained in a humidified incubator at 37°C.

[0048] (Example 1: Expression of MHC class II (IA / IE), PD-L1, PD-L2, and CD11c) 5 x 10 ６ Mouse bone marrow cells were suspended in 10 mL of macrophage maintenance medium (RPMI-1640 containing 10% FBS and GlutaMAX-I) and cultured in a 10 cm dish. During this process, 50 ng / mL of IL-34 or CSF-1, 10 ng / mL of IL-6, or 10 ng / mL of IL-4 was added and the cells were cultured. After 6 days, the cells were harvested, stained with various antibodies, and analyzed by flow cytometry.

[0049] The results are shown in Figure 3. Expression of four surface antigens, MHC class II (IA / IE), PD-L1, PD-L2, and CD11c, was increased in monocyte-derived macrophages stimulated with IL-34 and IL-4 compared with macrophages derived from monocytes stimulated with CSF-1. Little difference was observed in these four surface antigens between monocyte-derived macrophages stimulated with CSF-1 and those stimulated with IL-34. Monocyte-derived macrophages stimulated with CSF-1 and IL-4 had a similar phenotype to those stimulated with IL-34 and IL-4, but monocyte-derived macrophages stimulated with IL-34 and IL-4 showed higher expression of MHC class II.

[0050] Macrophage markers, such as F4 / 80 and CD206 (M2 markers), were shown to be expressed in all macrophages. As previously mentioned, MHC class II expression was enhanced in IL-34+IL-4 macrophages compared to other macrophages. PD-L1 and PD-L2 are known to negatively regulate T cells by binding to PD-1 and suppressing MHC-mediated TCR signaling. PD-L1 expression was unchanged in IL-34+IL-4 macrophages, but PD-L2 expression was clearly upregulated. These results suggest that IL-34+IL-4 macrophages may negatively regulate T cells via PD-1 inhibitory signaling.

[0051] (Example 2: Inhibition of inflammation and fibrosis by IL-34-stimulated monocyte-derived macrophages) (Macrophage proliferation assay) 5 x 10 ４Mouse bone marrow cells were suspended in 100 μl of macrophage maintenance medium and cultured in a 96-well plate. During this process, 50 ng / ml of IL-34 or Csf-1 was added. After 6 days, 100 ng / ml of IL-6, 10 ng / ml of IL-4, 20 ng / ml of Csf2, 20 ng / ml of Tnfα, 10 ng / ml of IL-2, 10 ng / ml of IL-18, or 2 μM of PGE2 was added, and the cells were further cultured for another 2 days. Subsequently, a cell proliferation assay was performed. Macrophage proliferation was then analyzed using the MTT assay. The MTT assay was performed using the MTT Cell Count Kit (Nacalai Tesque). The absorbance was measured at a test wavelength of 570 nm and a reference wavelength of 650 nm using a Multiskan FC (Thermo Fisher Scientific). Similar experiments were performed on human CD14+ macrophages.

[0052] Induction of liver fibrosis and macrophage therapy: For the induction of liver fibrosis using thioacetamide, mice were given sterilized water containing 0.03% thioacetamide ad libitum to induce liver fibrosis. During the first week, the concentration of thioacetamide in the drinking water was adjusted to 0.015%.

[0053] For induction of liver fibrosis by carbon tetrachloride, 20 μl of carbon tetrachloride was dissolved in 180 μl of corn oil and administered intraperitoneally to mice twice a week.

[0054] Induction of liver fibrosis by bile duct ligation (BDL) was performed according to the method outlined by Tag et al. J Vis Exp. 2015; (96): 52438. Briefly, mice were anesthetized to reduce pain and provide sedation, and a midline abdominal incision was made. The liver was carefully inverted to expose the bile duct, which was then separated from adjacent blood vessels. The bile duct was ligated in three places using surgical sutures. The liver was then repositioned, and the peritoneum and skin were sutured closed. Mice were kept warm until they regained consciousness.

[0055] To induce mouse macrophages, mouse bone marrow cells containing CD14-positive cells were cultured for 6 days with mouse IL-34 (50 ng / ml) or Csf1 (50 ng / ml) alone or in combination with 10 ng / ml each of IL-4, IL-6, and Csf2 (all cytokines were purchased from BioLegend). ６ The macrophages were suspended in saline and carefully administered via the tail vein.

[0056] (Blood Biochemistry Analysis) Biochemistry analysis of mouse serum, including AST and ALT levels, was performed by SRL (Tokyo, Japan).

[0057] Sirius Red Staining and Measurement of Fibrosis Area Sirius Red solution was purchased from Muto Chemical Co., Ltd. (Tokyo, Japan). Sirius Red staining was performed according to the method described by Junqueira et al. Histochem J. 1979 Jul;11(4):447-55.

[0058] After Sirius Red staining, specimen images were taken using a microscope (Keyence). Fibrotic areas were quantified using Image J software (NIH) for more than 10 fields. The percentage of fibrotic area within the captured fields was calculated.

[0059] Flow cytometry was performed using an FC500 or BD FACSCelesta™ (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Antibodies against mouse CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), B220 (RA3-6B2), CD11b (M1 / 70), CD11c (N418), F4 / 80 (BM8), and Ly6g (1A8) were used. Corresponding isotype controls were purchased from BioLegend. For analysis, live cells were gated based on forward and side scatter and the lack of DAPI or propidium iodide uptake. All antibodies were used at a 1:100 dilution.

[0060] (RNA Sequencing Analysis) To induce mouse macrophages, mouse bone marrow cells were cultured for 6 days with mouse IL-34 (50 ng / ml) or Csf1 (50 ng / ml) alone or in combination with 10 ng / ml each of IL-4, IL-6, and Csf2 (all cytokines were purchased from BioLegend). For macrophage administration, 2 × 10 ６ Macrophages were suspended in saline and carefully administered via the tail vein. RNA was extracted from macrophages (Mφ) using the NucleoSpin RNA Kit (TakaraBio). Subsequently, cDNA was synthesized from the RNA using the SureSelect Strand-Specific RNA Library Prep Kit (Agilent Technologies) according to the kit's instructions. Samples were prepared for sequencing analysis using the NextSeq 500 / 550 High Output Kit v2.5 (75 cycles) (Illumina). Sequencing was performed using the NextSeq 500 platform (Illumina). Fastq files were generated from the obtained reads using the bcl2fastq program (Illumina).

[0061] Statistics: Statistical analysis was performed using JMP software (JMP version 16.0.0, SAS Institute Inc.). Data represent mean ± SEM. Statistical significance was determined using Student's t-test (unpaired, two-tailed) or Tukey's honest significant difference (HSD) test. Mouse survival was analyzed by Kaplan-Meier survival curve analysis using the log-rank test. When more than two experimental groups were performed, the results were adjusted by Bonferroni correction.

[0062] (Results) (Effect of T Cells on Fibrosis) Liver fibrosis was induced in wild-type mice (BALB / c) and T cell-deficient nude mice (BALB / c-nu / nu) by bile duct ligation (BDL). Livers were harvested 14 days after BDL. As shown in Figure 4, stronger fibrosis was observed in wild-type mice compared to T cell-deficient nude mice, indicating the importance of T cells in liver fibrosis. Furthermore, CD8 T cell-depleting antibodies were administered to C57BL / 6J mice 1 and 6 days before BDL to analyze the role of CD8 T cells in liver fibrosis. As shown in Figure 5, depletion of CD8 cells reduced the rate of fibrosis.

[0063] (Anti-fibrotic effect) Various macrophages were administered on day 10 after bile duct ligation (BDL), and the liver was excised on day 14 to measure the fibrosis rate. As shown in Figure 6, administration of macrophages derived from monocytes stimulated with IL-34 (IL-34-BMMs) showed a higher anti-fibrotic effect than administration of saline or CSF-1 macrophages.

[0064] (Carbon tetrachloride (CCl ４ ) Therapeutic effect in models) Mouse models of liver cirrhosis induced with carbon tetrachloride were examined in acute and chronic hepatitis models. Macrophages induced from bone marrow cells with IL-34 or M-CSF were administered for treatment, and hepatic enzymes (AST, ALT), which serve as indicators of hepatocellular damage, were measured (blood test) and pathological examination was performed. The results are shown in Figure 7.

[0065] (1) Acute model (4-week continuous administration of carbon tetrachloride) Treatment with IL-34-induced macrophages significantly reduced blood AST (which becomes high in the presence of liver damage) and ALT also tended to decrease, suggesting that this may reduce liver damage associated with liver fibrosis.

[0066] (2) Chronic model (12-week continuous administration of carbon tetrachloride): Treatment with IL-34-induced macrophages significantly reduced blood ALT (which, like AST, becomes elevated in the presence of liver damage), and AST also tended to decrease, suggesting that this may be reducing liver damage associated with liver fibrosis. Pathological analysis (Sirius Red staining: fibrotic areas stain red) showed a tendency for fibrosis to be suppressed compared to the livers of untreated mice.

[0067] (Example 3: Effect of additional cytokines) To search for IL-34 macrophages with a higher therapeutic effect, we cultured them in the presence of IL-34 and additional cytokines. First, we selected those with high proliferation activity. This is because high proliferation activity allows a large number of therapeutic cells to be produced from a small number of original cells.

[0068] As shown in Figure 8, a high cell proliferation effect was observed in both mice and humans when cultured in the presence of IL-34 (or CSF1) and IL-6, IL-4, or CSF2, and the therapeutic effects of these macrophages were further analyzed.

[0069] In a mouse model in which liver fibrosis was induced by bile duct ligation (BDL), various macrophages were administered 10 days after BDL, and the liver was excised on day 14. The fibrosis rate was measured by image analysis using ImageJ with Sirius Red staining. Administration of IL-34+IL-4 macrophages or IL-34+IL-6 macrophages produced a significantly higher antifibrotic effect (Figure 9). Administration of IL-34+CSF2 macrophages did not produce any antifibrotic effect.

[0070] Furthermore, the groups administered IL-34+IL-4 or IL-34+IL-6 showed a tendency toward recovery of body weight (Figure 10), and analysis using the Kaplan-Meier survival curve showed a high survival rate (Figure 11).

[0071] Example 4: Improvement of Liver Fibrosis by Autologous Macrophages Induced by IL-34-Based Conditions In this example, acute (bile duct ligation (BDL)) and chronic (administration of carbon tetrachloride or thioacetamide) liver fibrosis models were employed. These models were used to evaluate the therapeutic potential of macrophages induced by IL-34-based conditions. In most experiments, IL-4 was also added to the differentiation process to induce alternative activated macrophages. For mechanistic analysis, the status of stellate cells and the immunosuppressive potential of macrophages were investigated. Human macrophages were transfected with CD14 ＋ They were differentiated from monocytes and analyzed.

[0072] (Materials and Methods) (Mice) Six-week-old male C57BL / 6 mice were purchased from Japan SLC Co., Ltd. (Shizuoka Prefecture). Five-week-old male NOD.Cg-Prkdc mice were used. ｓｃｉｄ Il2rg ｔｍ１Ｗｊｌ / SzJ (NSG mice) were purchased from Jackson Laboratory Japan. All animal experiments were approved by the Hokkaido University Animal Experiment Committee (approval number: 20-0086). All mice were housed at 25°C under a 12-hour light-dark cycle with darkness from 9 PM to 9 AM. Water and standard feed were available ad libitum. (Macrophage Culture) Mouse macrophage culture medium was RPMI-1640 (Fujifilm Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum (Sigma), 1% penicillin / streptomycin (Nacalai Tesque), and 1% non-essential amino acids (Nacalai Tesque). To induce mouse macrophages, 5 × 10 ６ Mouse bone marrow cells were suspended in 10 ml of macrophage medium and cultured in a 10 cm dish for 6 days in the presence of 50 ng / ml IL-34 or CSF-1 (BioLegend). In some experiments, 10 ng / ml IL-4 (BioLegend) was added from day 0. All cells were incubated at 37°C in 5% CO. ２The cells were maintained in a humidified incubator at 4°C. The viable cell count of macrophages was analyzed using an MTT Cell Count Kit (Nacalai Tesque). The absorbance was measured at a test wavelength of 570 nm and a reference wavelength of 650 nm using a Multiskan FC (Thermo Fisher Scientific). To induce human macrophages, CD14 ＋ Monocytes were magnetically sorted from peripheral blood mononuclear cells (PBMCs) using CD14 microbeads (Miltenyi Biotec). ６ The sorted cells were placed in 2 ml of TexMACS ＴＭ The cells were cultured in 3.5-cm dishes in the presence of 100 ng / ml IL-34 (BioLegend) and 10 ng / ml IL-4 (Miltenyi Biotec) for 6 days. The human study was approved by the Hokkaido University Hospital Committee (approval number: 22-0020).

[0073] (Induction and Treatment of Liver Fibrosis by Macrophages) Two experimental models were used to induce chronic liver fibrosis. First, mice were given sterilized water containing 0.03% thioacetamide (TAA) containing 0.1% sucralose ad libitum. In particular, the concentration of thioacetamide in the drinking water was adjusted to 0.015% for the first week. Next, mice were given 20 μl of carbon tetrachloride (CCl ４ ) was dissolved in 180 μl of corn oil and administered intraperitoneally to mice twice a week.

[0074] To induce acute liver injury and fibrosis, bile duct ligation (BDL) was performed according to the methodology outlined by Tag et al. (J Vis Exp. 2015;96:52438). Briefly, mice were anesthetized for analgesia and sedation, and a midline abdominal incision was made. The liver was carefully inverted to expose the bile duct and separate it from adjacent vessels. The bile duct was ligated in three places using surgical sutures. The liver was then replaced, and the peritoneum and skin were sutured closed. Mice were kept warm until they regained consciousness.

[0075] In the human macrophage experiment, 1 × 10 ７Human PBMCs were administered to NSG mice to reconstitute the human immune system. In preliminary experiments, TAA was also administered to NSG mice administered with human PBMCs. However, under these conditions, all mice exhibited severe wasting syndrome and died, so it was decided not to administer any further hepatotoxic substances. On the other hand, in NSG mice administered only with human PBMCs, a graft-versus-host disease-like xenogeneic response was induced, and liver fibrosis was detected after 8 weeks (Figure 16C).

[0076] For therapeutic macrophage administration, 2 × 10 ６ Macrophages were suspended in saline and carefully administered via the tail vein at the indicated time points.

[0077] (Sirius Red Staining and Measurement of Fibrotic Area) Sirius Red solution was purchased from Muto Chemical Co., Ltd. (Tokyo, Japan). Sirius Red staining was performed according to the method of Junqueira et al. (Histochem J. 1979;11:447). Images of specimens were taken using a microscope (Keyence) after Sirius Red staining. Fibrotic areas were quantified using Image J software (NIH) for at least 10 fields per liver sample. The percentage of fibrotic area within the photographed fields was calculated.

[0078] Flow cytometry was performed using a FACSCelesta™ (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA) or Kaluza (Beckman Coulter). Unless otherwise noted, fluorescently labeled monoclonal antibodies and corresponding isotype controls were purchased from BioLegend. Antibodies used were anti-mouse F4 / 80 (clone; BM8), CD206 (C068C2), I-A / I-E (M5 / 114.15.2), and H-2 D. ｂ / K ｂThe antibodies used were anti-human CD206 (19.2, BD Bioscience), HLA-DR (Tu39), HLA-ABC (W6 / 32, Invitrogen), PD-L1 (29E.2A3), PD-L2 (24F.10C12), CD4 (OKT4, eBioscience), and CD8 (SK1). For analysis, live cells were gated based on forward and side scatter and lack of DAPI, propidium iodide, or 7-AAD uptake. All antibodies were used at a dilution of 1:200. For T cell and macrophage coculture experiments, T cells were magnetically isolated from mouse spleens or human PBMCs using mouse CD90.2 microbeads or a human pan T cell isolation kit (Miltenyi Biotec), respectively. T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated for 4 days with anti-CD3 and anti-CD28 antibodies (BioLegend) for mice or Dynabeads Human T-Activator CD3 / CD28 (Gibco) for humans. From the start of the culture, macrophages were cocultured with CFSE-stained T cells. After coculture, CFSE fluorescence intensity and cell counts were analyzed by flow cytometry for cells gated on CD4 and CD8.

[0079] (Immunohistochemical analysis) Liver samples were fixed in 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.) at 4°C for 24 hours and then embedded in paraffin. The embedded samples were sliced ​​to prepare 5 μm-thick sections. The sections were deparaffinized and then immobilized in 0.3% H in distilled water. ２ O ２Endogenous peroxidase was blocked for 20 minutes with PBS. The sections were then incubated with BlockAce (DS Pharma Biomedical) in PBS for 1 hour to block nonspecific reactions. After protein blocking, the sections were incubated with anti-COL1A1 (E8F4L, CST), α-SMA antibody (1A4, BioLegend), or CD8 (4SM15, Invitrogen) in PBS overnight at room temperature. After washing, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (BioLegend) for 1 hour at room temperature, followed by H ２ O ２ The sections were incubated with 3,3'-diaminobenzidine tetrahydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) in Tris-HCl containing HCl for 5 to 20 minutes and counterstained with hematoxylin. The sections were mounted with Marinol (Muto Chemical Co., Ltd.) and observed under a BX53F light microscope (Olympus). The immunostained areas or cells were quantified using Image J software (NIH) for at least 10 fields per liver sample.

[0080] (RNA Seq Analysis) RNA was extracted using the NucleoSpin RNA Kit (Takara Bio). Subsequently, cDNA was synthesized from the RNA using the SureSelect Strand-Specific RNA Library Prep Kit (Agilent Technologies). Samples were prepared for sequence analysis using the NextSeq 500 / 550 High Output Kit v2.5 (75 cycles) (Illumina). Sequencing was performed using the NextSeq 500 platform (Illumina). Fastq files were generated from the acquired reads using the bcl2fastq program (Illumina).

[0081] Statistics: Statistical analysis was performed using JMP software (JMP version 16.0.0, SAS Institute Inc.) and R (version 4.2.3). Data represent the mean ± SEM. Statistical significance was determined using Student's t-test (unpaired, two-tailed) or Tukey's honest significant difference (HSD) test.

[0082] Results: In both acute and chronic liver injury experiments, interleukin-34-induced macrophages significantly ameliorated liver fibrosis. Addition of interleukin-4 to the differentiation process resulted in a higher number of macrophages, more inclined toward alternatively activated macrophages (the so-called M2 phenotype). Alternatively activated macrophages (M2 type) showed reproducible therapeutic effects on liver fibrosis while suppressing parameters of stellate cell and T cell activation. Similar macrophages were also able to express human CD14 in the presence of interleukin-34 and interleukin-4. ＋ Interleukin-34-induced macrophages, which could be differentiated from monocytes, further ameliorated liver fibrosis, especially when further stimulated with interleukin-4.

[0083] (Macrophages induced by IL-34 from bone marrow cells suppress liver fibrosis) Macrophages were induced from mouse bone marrow cells using IL-34, and CCl ４ IL-34 macrophages were administered to mice with induced liver fibrosis. The results showed that 4 weeks after macrophage administration, fibrosis deposition was inhibited to the same extent as with CSF-1 macrophages (Figure 12A). As demonstrated in Example 2, in an acute liver injury model induced by bile duct ligation (BDL), CSF-1 macrophages showed minimal effect, whereas IL-34 macrophages inhibited fibrosis (Figure 6). Thus, administration of IL-34 macrophages is effective in treating liver fibrosis.

[0084] We compared the appearance and gene expression of CSF-1 and IL-34 macrophages. The macrophages had similar appearances: both were round, some spindle-shaped, and some adhered to the plastic plate (Figure 12B). Next, we analyzed mRNA expression by RNA sequencing. As shown in Figure 12C, the scatter plots were consistent. A closer look revealed that IL-34 macrophages exhibited enhanced expression of MHC class II-related genes, such as H2-A, H2-E, Cd74, and Ciita. The expression of these genes may be related to the enhanced expression of MHC class II proteins when IL-4 was added to IL-34 macrophage differentiation cultures (Figure 3).

[0085] IL-34+IL-4-induced macrophages exhibit M2-biased immunosuppressive properties. IL-34 macrophages were alternatively activated by adding IL-4 to differentiated cultures from bone marrow cells. IL-34+IL-4 macrophages appeared more spindle-shaped than those induced with IL-34 alone and adhered more strongly to plastic surfaces (Figure 12B).

[0086] We performed RNA sequencing to examine the mRNA expression of induced macrophages (Figure 13A). Expression of MHC-related genes was elevated in IL-34+IL-4 macrophages. Expression of M1 macrophage markers Il1b, Il16, and Cxcl16 was reduced by approximately 40%, whereas expression of M2 macrophage markers Arg1, Retnla, and Chi3l3 was elevated. These results suggest that IL-34+IL-4 macrophages are polarized toward the M2 type. On the other hand, expression of Tgfb1, an M2-specific gene that activates hepatic stellate cells and promotes fibrosis, was actually reduced. Some matrix metalloproteinases (MMPs), such as MMP12, 13, and 19, were significantly upregulated. Cd274 (PD-L1) and Pdcd1Ig2 (PD-L2), which are associated with immune suppression, were also upregulated in IL-34+IL-4 macrophages. Several hepatic growth factors, such as Osm (oncostatin M) and Hgf (hepatocyte growth factor), were also upregulated in IL-34+IL-4 macrophages.

[0087] Furthermore, when cell proliferation was examined using the MTT assay, the IL-34 + IL-4 macrophages showed significantly higher cell proliferation than the CSF-1 and IL-34 group (Figure 13B). The ability to obtain a larger number of cells using a similar culture protocol is considered to be one of the advantages in the development of autologous macrophage therapy.

[0088] (IL-34 + IL-4 macrophages prevent both acute and chronic liver fibrosis models) Next, we investigated whether treatment with selectively activated macrophages induced by IL-34 + IL-4 is also effective in liver fibrosis models. First, we confirmed the therapeutic effect in an acute liver injury model using BDL. In this experiment, of the macrophages tested, IL-34 + IL-4 macrophages had the greatest therapeutic effect on liver fibrosis (Figure 14A).

[0089] In mice with TAA-induced liver fibrosis, IL-34 + IL-4 macrophages significantly suppressed liver fibrosis (Figure 14B). Next, we examined the effect of IL-34 + IL-4 macrophages on hepatic stellate cell activation. The expression of type I collagen α1 (COL1A1), a major component of liver fibrosis produced by activated hepatic stellate cells, was confirmed by immunohistochemistry. COL1A1 expression was significantly reduced by IL-34 + IL-4 macrophage treatment (Figure 14C). The expression of α-SMA, a marker of stellate cell activation, was also significantly reduced by IL-34 + IL-4 macrophage treatment (Figure 14D). Without wishing to be bound by theory, these findings suggest that IL-34 + IL-4 macrophages suppress hepatic stellate cell activation and reduce fibrosis.

[0090] (IL-34 + IL-4 macrophages suppress T cell proliferation.) T cells, particularly CD8+ T cells, are known to activate hepatic stellate cells and secrete proinflammatory cytokines (TNF-α and IFN-γ) to promote the production of fibrotic proteins. Therefore, we investigated whether IL-34 + IL-4 macrophages have the ability to suppress T cell activation. Spleen-derived T cells were first labeled with CFSE and stimulated with anti-CD3 and anti-CD28 antibodies. Next, T cells were cocultured with IL-34 + IL-4 macrophages in the presence or absence of inhibitory antibodies against PD-L1 and PD-L2. After 4 days, T cell proliferation was analyzed by flow cytometry. The FACS gating strategy is shown in Figure 15A. As shown in Figure 15B, the peaks indicating CD4 and CD8 T cell proliferation were attenuated by coculture with IL-34 + IL-4 macrophages (control). On the other hand, when inhibitory antibodies against PD-L1 and PD-L2 were added to the culture, the attenuated CFSE peak shifted toward the mitogenic side (Abs). These results indicate that IL-34+IL-4 macrophages inhibit CD4 and CD8 T cell proliferation through PD-1 negative signaling. Next, we examined CD8 T cells in the liver in vivo using immunohistochemistry. Consistent with the in vitro results, the number of CD8 T cells in the liver was significantly reduced in the IL-34+IL-4 macrophage-treated group (Figure 15C). Whether this effect is due to inhibition of CD8 T cell migration to the liver or inhibition of proliferation in the liver is currently unknown. However, without being bound by theory, it appears that IL-34+IL-4 macrophages exert their therapeutic effect through CD8 T cell suppression.

[0091] (Human IL-34 + IL-4 macrophages exhibit similar phenotypes and functions to mouse macrophages.) We attempted to differentiate human macrophages. ＋Human IL-34+IL-4 macrophages were cultured in the presence of IL-34 and IL-4. Flow cytometry analysis revealed that human IL-34+IL-4 macrophages highly expressed CD206, an M2 marker (Figure 16A). Similar to mouse macrophages (Figure 16B), they also highly expressed HLA class I and II, PD-L1, and PD-L2 (Figure 16A). When cocultured with human T cells stimulated with anti-CD3 and anti-CD28 antibodies, human IL-34+IL-4 macrophages significantly inhibited the mitogenic shift of the CFSE peak, effectively suppressing T cell activation (Figure 16B). Finally, we examined the therapeutic effect of human IL-34+IL-4 macrophages in a liver fibrosis model using immunodeficient NSG mice administered with human PBMCs. Eight weeks after PBMC injection, clear fibrosis was observed in the control group (Figure 16C). In the treatment group, IL-34 + IL-4 macrophages were administered at week 4, and liver samples were examined after another 4 weeks. As shown in Figure 16C, IL-34 + IL-4 macrophage treatment significantly suppressed liver fibrosis. Thus, human IL-34 + IL-4 macrophages were effective against liver fibrosis, similar to that in mice.

[0092] The present invention provides macrophages that have excellent therapeutic effects against fibrosis. The present invention can be used in the pharmaceutical industry.

Claims

1. A composition for treating or preventing fibrosis and / or inflammation in an organ or tissue of a subject, comprising macrophages derived from monocytes stimulated with IL-34.

2. The composition of claim 1, wherein the macrophages are derived from monocytes that have been further stimulated with a cytokine selected from the group consisting of IL-4, IL-6, and combinations thereof.

3. The composition of claim 1, wherein the fibrosis and / or inflammation is in an organ selected from the group consisting of the liver, lung, heart, and kidney.

4. The composition of claim 1, wherein the fibrosis and / or inflammation is in the liver.

5. The composition of claim 1, wherein the fibrosis and / or inflammation is cirrhosis or hepatitis.

6. A method for producing a composition for treating or preventing fibrosis and / or inflammation in a target organ or tissue, the method comprising the steps of: preparing a sample containing CD14-positive cells; optionally isolating the CD14-positive cells from the sample; and contacting the sample or the isolated CD14-positive cells with IL-34.

7. The method of claim 6, wherein the CD14 positive cells are further contacted with a cytokine selected from the group consisting of IL-4, IL-6, and combinations thereof.

8. The method of claim 6, wherein the sample comprises peripheral blood mononuclear cells or bone marrow-derived cells.

9. Macrophages derived from monocytes stimulated with (i) IL-34 and (ii) a cytokine selected from the group consisting of IL-4, IL-6 and combinations thereof.

10. A method for producing macrophages with high expression of IL-10 and / or CCL17, the method comprising the steps of: preparing a sample containing CD14-positive cells; optionally isolating the CD14-positive cells from the sample; and culturing the sample or the isolated CD14-positive cells in the presence of (i) IL-34 and (ii) cytokines selected from the group consisting of IL-4, IL-6, and combinations thereof.

11. A composition for treating or preventing fibrosis and / or inflammation in a target organ or tissue, comprising macrophages with low CXCL10 and / or iNOS expression.

12. A composition for treating or preventing fibrosis and / or inflammation in a target organ or tissue, comprising macrophages that highly express MHC class II-related molecules, highly express PD-L1, highly express PD-L2, highly express CD11c, or a combination thereof.

13. The composition according to claim 12, wherein the macrophages are of the CXCL10 and / or iNOS low expressing type.

Images