Treatment of hepatic fibrosis by autologous macrophage

Abstract

To provide a composition for treating or preventing fibrosis and / or inflammation in an organ or a tissue of a subject.SOLUTION: Provided is a composition for treating or preventing fibrosis and / or inflammation in an organ or a tissue of a subject which contains a macrophage derived from a monocyte stimulated by IL-34. Such a composition is produced by a method including the steps of: preparing a sample including a CD14 positive cell; isolating the CD14 positive cell from the sample as needed; and bringing the sample or the isolated CD14 positive cell in contact with IL-34.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 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. [Background technology]

[0002] In liver cirrhosis, abnormally accelerated fibrosis occurs within the liver. This fibrosis impairs normal liver tissue function, and once decompensation occurs, liver transplantation is the only viable treatment option. Cell therapy using autologous or allogeneic cells has been proposed as a novel treatment for liver cirrhosis. Various cell types, including hepatocytes and mesenchymal stem cells, have been considered. One of these is autologous macrophage therapy, which involves culturing and expanding autologous macrophages obtained from the patient's peripheral blood and then administering them to the patient. This therapy utilizes the tissue repair capabilities of macrophages, which produce chemokines, immunosuppressive molecules, matrix metalloproteinases (MMPs), and Wnt3A, which are believed to promote the degradation of fibrous tissue and the regeneration of liver tissue. Several research groups have demonstrated promising results in animal studies, 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. Summary of the Invention [Means for solving the problem]

[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, whereas IL-34 was a cytokine first reported in 2008. However, there has been no previous knowledge regarding IL-34-induced macrophages in liver 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 preceding item, 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. (Item 3) The composition of any one of the preceding 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 of any one of the preceding items, wherein the fibrosis and / or inflammation is in the liver. (Item 5) The composition according to any one of the preceding items, wherein the fibrosis and / or inflammation is cirrhosis or hepatitis. (Item 6) 1. 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: providing a sample containing CD14-positive cells; optionally isolating CD14-positive cells from the sample; contacting the sample or the isolated CD14-positive cells with IL-34; A method comprising: (Item 7) The method according to any one of the preceding 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 preceding items, wherein the sample comprises peripheral blood mononuclear cells or bone marrow-derived cells. (Item 9) (i) macrophages derived from monocytes stimulated with 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: providing a sample containing CD14-positive cells; optionally isolating CD14-positive cells from the sample; Culturing the sample or the isolated CD14-positive cells in the presence of (i) IL-34 and (ii) a cytokine selected from the group consisting of IL-4, IL-6, and combinations thereof; A method comprising: (Item 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. (Item 12) A composition for treating or preventing fibrosis and / or inflammation in a target organ or tissue, comprising macrophages that have high expression of MHC class II-related molecules, high expression of PD-L1, high expression of PD-L2, high expression of CD11c, or a combination thereof. (Item 13) The composition according to any one of the preceding items, wherein the macrophages are low expressers of CXCL10 and / or iNOS.

[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. [Effects of the Invention]

[0007] The present invention uses monocyte-derived macrophages stimulated with IL-34 to more strongly suppress the expression of inflammatory chemokines and iNOS, a 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. [Brief explanation of the drawings]

[0008] [Figure 1] 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 the survival, proliferation, and differentiation of macrophages (Science, 320:807, 2008). [Figure 2] 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] Figure 3 shows the results of flow cytometry of macrophages stimulated with CSF1, IL-34, IL-34+CSF2, IL-34+IL-6, or IL-34+IL-4 for various surface antigens. [Figure 4]Figure 4 shows the results of liver fibrosis induction in wild-type mice (BALB / c) and T cell-deficient nude mice (BALB / c-nu / nu) by bile duct ligation (BDL), followed by observation of the livers harvested on day 14 after BDL. The vertical axis of the graph shows the fibrotic area (%) (Student's t-test, p**<0.01). [Figure 5] 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 shows the fibrotic area (%) (Student's t-test, p**<0.05, N=4). [Figure 6] Figure 6 shows the results of administering various macrophages (referred to as MΦ or Mf in the figure) 10 days after bile duct ligation (BDL), and then measuring the fibrosis rate after removing the liver on day 14. The vertical axis of the graph shows the fibrotic area (%) (Tukey-Kramer, p*<0.05, p***<0.001). [Figure 7] Figure 7 shows the results of administering macrophages induced from bone marrow cells with IL-34 or M-CSF to a mouse model of liver cirrhosis induced with carbon tetrachloride, and measuring (blood test) liver enzymes (AST, ALT), which are indicators of liver cell damage, and pathological examination. [Figure 8] Figure 8 shows the results of measuring the cell proliferation effect of macrophages when cultured 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] Figure 9 shows the results of administering various macrophages 10 days after bile duct ligation (BDL) to a mouse model of liver fibrosis. The liver was removed on day 14 and the fibrosis rate was measured by image analysis using ImageJ with Sirius Red staining. The vertical axis of the graph shows the area of ​​fibrosis (%) (Tukey's multiple comparisons test, p<0.05). [Figure 10] Figure 10 shows a graph of changes in body weight over time in the groups administered with various macrophages. The vertical axis of the graph shows the percentage change, with day 0 taken as 100% (Tukey's multiple comparisons test, p*<0.05). [Figure 11] FIG. 11 shows Kaplan-Meier survival curves for the groups administered with various macrophages. [Figure 12] Figure 12A shows Sirius Red staining of liver sections from C57BL / 6J mice with CCl4-induced liver fibrosis. (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 mRNA expression in macrophages. [Figure 13] Figure 13A shows a comparison of mRNA expression levels in macrophages. The vertical axis is expressed as fragments per kilobase of exon per million mapped reads (FPKM). ND indicates not detected. Figure 13B shows the viable cell counts of macrophages detected by MTT assay. ***P<0.001. [Figure 14]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 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. The bar graph shows quantification of the immunostaining-positive area ratio (mean ± SEM, n = 6 per group). *P<0.05. [Figure 15] Figure 15A shows the gating strategy for flow cytometry analysis. Figure 15B shows histograms showing CD3-gated cells (left, green) or CFSE fluorescence intensity of CD4 and CD8 for T cell proliferation without coculture. CFSE-stained T cells selected with magnetic beads from splenocytes were cocultured 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 proliferation 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 immunostained cell numbers (mean ± SEM, n = 3 per group). ***P < 0.001. [Figure 16]Figure 16A shows flow cytometry analysis of human monocyte-derived macrophages stimulated with IL-34+IL-4 for various surface antigens. Green histograms indicate cells expressing the indicated surface proteins. Gray histograms indicate isotype control staining. Figure 16B shows histograms showing CFSE fluorescence intensity of CD4- and CD8-gated cells. Human IL-34+IL-4 macrophages were cocultured 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 harvested at 8 weeks. Scale bar indicates 200 μm. Scale bar indicates 200 μm. Bar graphs show quantification of Sirius Red-positive area ratio (mean ± SEM). *P<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0009] It should be understood that the terms used in this specification are used in the meanings generally used in the relevant field unless otherwise specified.Therefore, unless otherwise defined, all technical terms 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] <Definition> First, we explain the terms and general techniques used in this disclosure.

[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 degrades 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" refer to macrophages obtained by contacting monocytes with IL-34 during the process of monocyte differentiation into macrophages. Macrophages derived from monocytes stimulated with IL-34 are further characterized by CD14 positivity and higher 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 monocyte differentiation into macrophages, or by contacting macrophages differentiated from monocytes with CSF-1.

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

[0015] As used herein, "high expression of MHC class II-related molecules" refers to a state in which the expression level of MHC class II-related molecules is higher than that of 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 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 of PD-L1 expressed 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 in macrophages derived from monocytes stimulated with CSF-1.

[0019] As used herein, "low CXCL10 and / or iNOS expressor" refers to macrophages that express lower levels of CXCL10 and / or iNOS than those 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 defensive response to tissue irritation or injury. Inflammation is characterized by fenestration of 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, and refers to transient inflammation. "Chronic inflammation" refers to long-term inflammation lasting weeks to years, and refers 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 and 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] <Macrophages derived from monocytes stimulated with IL-34> In 2008, IL-34 was discovered as a novel cytokine other than CSF-1 that binds to the CSF-1 receptor (CSF-1R), which is important for the survival, proliferation, and differentiation of macrophages (Figure 1). IL-34 and CSF-1 bind to different sites on the CSF-1R and transmit similar but different signals (Cell Death Differ 17:1917, 2010). In addition, there are other receptors (Syndecan-1, PTP-ζ) that bind only IL-34 (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 the like (Sci Rep 8:256, 2018). For example, IL-34 strongly induces the immunosuppressive molecule IL-10 in the M1 environment and induces CCL-17, which promotes wound healing, in the M2 environment (Figure 2). The inventors found that when CSF-1 or IL-34 was applied to macrophages activated with IFN-γ, the macrophages treated with IL-34 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 these cytokines. Therefore, without intending to be bound by theory, IL-34 can induce both proinflammatory M1 macrophages and anti-inflammatory M2 macrophages, responsible for tissue repair. It appears that IL-34 induces M2 macrophages more strongly 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 by IL-4 in combination with IL-34 can be characterized by high expression of MHC class II-related molecules, high expression of PD-L1, high expression of PD-L2, high expression of CD11c, or combinations thereof. MHC class II-related molecules refer to molecules such as I-A, I-E, H2-Aa, H2-DMB2, H2-Ab1, H2-DMa, H2-Eb1, H2-DMb1, CD74, CIITA, etc. in mice, and molecules such as HLA-DP, HLA-DQ, HLA-DR, etc. in humans, but are not limited thereto.

[0029] In one embodiment, macrophages derived from IL-34-stimulated monocytes of the present invention can be characterized by (i) CD14 positive 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 can be a macrophage 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. 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 can further enhance the proliferative activity and therapeutic effect compared to macrophages stimulated with IL- alone, which is advantageous.

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

[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 the 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 appropriately adjusted 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 x 10 3 ~Approx. 1×10 8 pieces, about 5×10 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 approximately 1 × 10 8There 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 x 10 3 ~Approx. 1×10 8 pieces, about 5×10 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 within the range 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 approximately 1 × 10 8 There can be one.

[0036] Monocytes are produced in the bone marrow, enter the bloodstream, and differentiate into macrophages after migrating to tissues. Monocytes are characterized by CD14 positivity. Therefore, the cells that can be used in this method include CD14-positive cells. To produce macrophages more efficiently, CD14-positive cells may be isolated from the sample, but isolation is not necessary. CD14-positive cells can be isolated by isolation methods known in the art, such as immunomagnetic beads.

[0037] In one embodiment, contacting CD14-positive cells with IL-34 may involve 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, for example, 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 concentration within the range defined 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 have advantageously high proliferative activity and therapeutic effects.

[0039] IL-4 may be contacted with CD14-positive cells to a final concentration of at least about 1 ng / ml, for example, 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 the 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, or about 19 ng / ml. IL-6 may be at 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, 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. In certain embodiments, the final concentration of IL-6 can 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 110 ng / ml, about 120 ng / ml, about 130 ng / ml, 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™ 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 to induce M2 macrophages more strongly than M1 macrophages. M2 macrophages are involved in suppressing inflammatory responses and tissue repair. Surprisingly, the macrophages disclosed herein exhibit superior therapeutic effects against fibrosis and / or inflammation in a target organ or tissue compared with conventional macrophages, i.e., monocyte-derived macrophages stimulated with CSF-1, which are known to be effective in degrading fibrotic tissue and regenerating 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 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 can 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 can 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 x 10 5 pieces~1×10 9 pieces, about 1×10 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, about 5×10 5 pieces, about 1×106 pieces, about 5×10 6 pieces, about 1×10 7 pieces, about 5×10 7 pieces, about 1×10 8 pieces, about 5×10 8 pieces, about 1×10 9 pieces, or approximately 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. [Example]

[0046] (mouse) 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) 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 maintained in a humidified incubator at 37°C with 5% CO2.

[0048] Example 1: Expression of MHC class II (IA / IE), PD-L1, PD-L2, and CD11c 5×10 6Mouse 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 IL-34 or CSF-1, 10 ng / mL IL-6, or 10 ng / mL IL-4 were added. After 6 days, 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 the expression of 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 acting as inhibitors of 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: Suppression of inflammation and fibrosis by IL-34-stimulated monocyte-derived macrophages (Macrophage proliferation assay) 5×10 4 Mouse 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 IL-34 or Csf-1 was added. After 6 days, 100 ng / ml IL-6, 10 ng / ml IL-4, 20 ng / ml Csf2, 20 ng / ml TNFα, 10 ng / ml IL-2, 10 ng / ml IL-18, or 2 μM 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 an MTT assay. The MTT assay was performed 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). Similar experiments were performed with human CD14+ macrophages.

[0052] (Induction of liver fibrosis and treatment with macrophages) For the induction of liver fibrosis using thioacetamide, mice were given 0.03% thioacetamide in sterile water ad libitum, and the concentration of thioacetamide in the drinking water was adjusted to 0.015% for the first week.

[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 locations 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+ 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 biochemical analysis) Biochemical 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 over 10 fields of view. The percentage of fibrotic area within the captured fields of view was calculated.

[0059] (Flow cytometry) 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 used were anti-mouse CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), B220 (RA3-6B2), CD11b (M1 / 70), CD11c (N418), F4 / 80 (BM8), and Ly6g (1A8). 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). 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 instructions provided with the kit. 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 acquired 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 the mean ± SEM. Statistical significance was determined using Student's t-test (two-tailed, unpaired) or Tukey's honest significant difference (HSD) test. Mouse survival was analyzed by Kaplan-Meier survival curve analysis using the log-rank test. When three or more experimental groups were performed, the analysis results were adjusted by Bonferroni correction.

[0062] (result) (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 on day 14 after BDL. As shown in Figure 4, wild-type mice showed more severe fibrosis than T cell-deficient nude mice, demonstrating the importance of T cells in liver fibrosis. Furthermore, C57BL / 6J mice were administered a CD8 T cell-depleting antibody 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 T cells reduced the rate of fibrosis.

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

[0064] (Therapeutic effect in carbon tetrachloride (CCl4) model) We investigated acute and chronic hepatitis models in mice with carbon tetrachloride-induced liver cirrhosis. Macrophages induced from bone marrow cells with IL-34 or M-CSF were administered for treatment, and hepatic enzymes (AST, ALT), which are indicators of hepatocellular damage, were measured (blood tests) and pathological examinations were 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 reduces 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 it 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: Effects of additional cytokines To identify IL-34 macrophages with a more therapeutic effect, we cultured them in the presence of IL-34 and additional cytokines. First, we selected those with high proliferative activity, because high proliferative activity allows for the generation of a large number of therapeutic cells 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 further analysis was performed to determine the therapeutic effects of these macrophages.

[0069] In a mouse model in which liver fibrosis was induced by bile duct ligation (BDL), various macrophages were administered on day 10 after BDL, and the liver was removed on day 14. Fibrosis rates were measured by Sirius Red staining and image analysis using ImageJ. Administration of IL-34+IL-4 macrophages or IL-34+IL-6 macrophages showed a significantly higher anti-fibrotic effect (Figure 9). Administration of IL-34+CSF2 macrophages did not result in any anti-fibrotic effect.

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

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

[0072] (material and method) (mouse) Six-week-old male C57BL / 6 mice were purchased from Japan SLC Co., Ltd. (Shizuoka Prefecture, Japan). 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 Care and Use 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 chow were available ad libitum. (macrophage culture) Mouse macrophage culture medium was 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). 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 maintained in a humidified incubator at 37°C and 5% CO2. The viable macrophage cell count 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). 1 × 10 6 The sorted cells were cultured in 2 ml of TexMACS TM The cells were cultured in a 3.5 cm dish 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] (Macrophage-mediated induction and treatment of liver fibrosis) Two experimental models were used to induce chronic liver fibrosis. First, mice were given 0.1% sucralose in sterile water containing 0.03% thioacetamide (TAA) ad libitum. Specifically, for the first week, the concentration of thioacetamide in the drinking water was adjusted to 0.015%. Second, 20 μl of carbon tetrachloride (CCl4) dissolved in 180 μl of corn oil was administered intraperitoneally to the mice twice weekly.

[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 turned over to expose the bile duct and separate it from adjacent vessels. The bile duct was ligated in three locations 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] For human macrophage experiments, NSG mice were inoculated with 1 × 10 7 We administered human PBMCs to NSG mice to reconstitute their human immune status. In preliminary experiments, we also administered TAA to NSG mice administered human PBMCs. However, under these conditions, all mice exhibited severe wasting syndrome and died, so we decided not to administer any further hepatotoxic substances. On the other hand, in NSG mice administered only 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 fibrosis 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. The fibrotic area was 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) Flow cytometry was performed using 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 included anti-mouse F4 / 80 (clone; BM8), CD206 (C068C2), IA / IE (M5 / 114.15.2), and H-2 D. b / K bThe antibodies 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 the lack of DAPI, propidium iodide, or 7-AAD uptake. All antibodies were used at a 1:200 dilution. 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, gated on CD4 and CD8.

[0079] (Immunohistochemical analysis) Liver samples were fixed in 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.) for 24 hours at 4°C and then embedded in paraffin. The embedded samples were sliced ​​into 5-μm-thick sections. The sections were deparaffinized and endogenous peroxidase was blocked with 0.3% H2O2 in distilled water for 20 minutes. 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 overnight at room temperature with anti-COL1A1 (E8F4L, CST), α-SMA antibody (1A4, BioLegend), or CD8 (4SM15, Invitrogen) in PBS. After washing, sections were incubated with horseradish peroxidase-conjugated secondary antibodies (BioLegend) for 1 hour at room temperature, followed by incubation with 3,3'-diaminobenzidine tetrahydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) in Tris-HCl containing HO for 5–20 minutes and counterstained with hematoxylin. Sections were mounted with Marinol (Muto Chemical Co., Ltd.) and observed under a BX53F (Olympus) light microscope. Immunostained areas or cells were quantified using Image J software (NIH) for at least 10 fields per liver sample.

[0080] (RNA sequence 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 sequencing 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 tested using Student's t-test (unpaired, two-tailed) or Tukey's honest significant difference (HSD) test.

[0082] (result) In both acute and chronic liver injury experiments, interleukin-34-induced macrophages significantly ameliorated liver fibrosis. Adding interleukin-4 to the differentiation process resulted in a higher number of macrophages, which were more inclined toward alternatively activated macrophages (the so-called M2 phenotype). These alternatively activated macrophages (M2 type) exhibited reproducible therapeutic effects on liver fibrosis while suppressing parameters of stellate cell and T cell activation. Similar macrophages expressed 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 administered to mice with CCl4-induced liver fibrosis. 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 a bile duct ligation (BDL)-induced acute liver injury model, 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 were similar in appearance, both round, some spindle-shaped, and some attached 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 MHC class II protein expression 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 and adhered more strongly to plastic surfaces than those induced with IL-34 alone (Figure 12B).

[0086] We performed RNA sequencing to examine the mRNA expression of induced macrophages (Figure 13A). Expression of MHC-related genes was upregulated 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 significantly 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 immunosuppression, 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 more cells using a similar culture protocol is considered to be one of the advantages in developing autologous macrophage therapy.

[0088] IL-34+IL-4 macrophages protect against both acute and chronic liver fibrosis. Next, we investigated whether treatment with selectively activated macrophages induced by IL-34+IL-4 was also effective in liver fibrosis models. First, we confirmed the therapeutic effect in an acute liver injury model using BDL. In this experiment, IL-34+IL-4 macrophages were the most effective in treating liver fibrosis among the macrophages tested (Figure 14A).

[0089] In mice with thioacetamide (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 (HSC) activation. Expression of type I collagen alpha 1 (COL1A1), a major component of liver fibrosis produced by activated HSCs, was confirmed by immunohistochemistry. COL1A1 expression was significantly reduced by IL-34+IL-4 macrophage treatment (Figure 14C). 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 suppressed HSC activation and reduced fibrosis.

[0090] (IL-34+IL-4 macrophages suppress T cell proliferation) Because 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, we investigated whether IL-34+IL-4 macrophages could 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). However, the addition of inhibitory antibodies against PD-L1 and PD-L2 to the culture shifted the attenuated CFSE peak toward the mitogenic side (Abs). These results indicate that IL-34+IL-4 macrophages inhibit the proliferation of CD4 and CD8 T cells via 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 via 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. Therefore, human IL-34+IL-4 macrophages were effective against liver fibrosis, similar to that in mice. [Industrial Applicability]

[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. 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. 2. 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. 1. 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: providing a sample containing CD14-positive cells; Optionally, isolating CD14-positive cells from the sample; contacting the sample or the isolated CD14-positive cells with IL-34; A method comprising:

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

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

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 of claim 12, wherein the macrophages are low expressers of CXCL10 and / or iNOS.

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