Use of CXCL11 for treating pulmonary fibrosis by inducing repolarization of m2 macrophages

Abstract

The present invention relates to use of CXCL11 for treating pulmonary fibrosis by inducing the repolarization of M2 macrophages. Specifically, a fibrosis treatment effect was confirmed through the injection of M0, M1 and M2 conditioned media (CM) by using a bleomycin-induced pulmonary fibrosis animal model, and a reduction in fibrosis markers such as collagen and a change in the M1 / M2 ratio close to a normal group were confirmed in the lungs of mice into which the M1 macrophage CM was injected. Also, the fibrosis treatment effect was confirmed through the intravenous injection of human recombinant CXCL11 by using a bleomycin-induced pulmonary fibrosis animal model, and a reduction in fibrosis markers such as collagen and a change in the M1 / M2 ratio close to a normal group were confirmed in the lungs of mice to which CXCL11 was administered. That is, the present invention aims to induce the repolarization of M2 macrophages through CXCL11 in the process of developing pulmonary fibrosis, and thus CXCL11 is presented as a potential target factor for the treatment of pulmonary fibrosis.

Description

Treatment of pulmonary fibrosis by inducing repolarization of M2 macrophages by CXCL11

[0001] The present invention relates to a treatment for pulmonary fibrosis by inducing repolarization of M2 macrophages by CXCL11.

[0002] Macrophages can be polarized into a classically activated M1 phenotype or an alternatively activated M2 phenotype depending on their microenvironment. Typically, M1 macrophages are responsible for wound healing after alveolar epithelial injury, while M2 macrophages play a role in resolving the wound healing process or terminating the inflammatory response in the lungs. Several studies have shown that activated M2 macrophages in fibrotic lesions produce large amounts of cytokines, such as IL-10 and TGFβ1, which enhance collagen synthesis and deposition. These proinflammatory and fibrotic cytokines indirectly suppress the production of anti-inflammatory cytokines through negative feedback, further promoting fibrosis.

[0003] Idiopathic pulmonary fibrosis (IPF) is a disease characterized by excessive accumulation of extracellular matrix (ECM) in the lungs, thickening of the alveolar walls and ultimately destruction of the alveolar structure, leading to respiratory failure. The five-year survival rate after diagnosis is less than 40%. While many new concepts have been developed to elucidate the pathogenesis, the exact cause of pulmonary fibrosis remains unknown, making it an incurable disease that is fundamentally difficult to treat. Currently, no medical technology exists to completely restore fibrotic tissue to its original state. Instead, drugs such as pirfenidone and nintendanib are used to slow the progression of fibrosis. While these drugs delay lung function decline, they do not halt the disease's progression. Furthermore, their severe side effects lead to a high rate of patient discontinuation. Many clinical studies are underway to develop treatments for idiopathic pulmonary fibrosis, but the drugs that have entered clinical trials and are effective against idiopathic pulmonary fibrosis are focused on improving symptoms by using mechanisms that suppress collagen production by reducing the action of proteins that affect collagen production or block inflammation and fibrosis using receptors involved in various physiological activities. However, there is still a high unmet medical need, so the development of new treatments is urgent.

[0004] The purpose of the present invention is to provide a pharmaceutical composition for treating pulmonary fibrosis containing CXCL11 as an active ingredient.

[0005] In addition, another object of the present invention is to provide a reagent composition for inducing repolarization of M2 macrophages into M1 macrophages in vitro, which comprises CXCL11 as an active ingredient, and a method for inducing repolarization of M2 macrophages into M1 macrophages using the same.

[0006] To achieve the above purpose, the present invention provides a pharmaceutical composition for treating pulmonary fibrosis containing CXCL11 as an active ingredient.

[0007] In addition, the present invention provides a reagent composition for inducing repolarization of M2 macrophages into M1 macrophages in vitro, comprising CXCL11 as an active ingredient.

[0008] In addition, the present invention provides a method for inducing repolarization of M2 macrophages into M1 macrophages, comprising a step of treating macrophages with CXCL11 in vitro.

[0009] The present invention relates to a treatment for pulmonary fibrosis through induction of repolarization of M2 macrophages by CXCL11, and specifically, by using a bleomycin-induced pulmonary fibrosis animal model, the treatment effect of fibrosis was confirmed through injection of M0, M1, and M2 secretomes (conditioned medium, CM), and it was confirmed that in the lungs of mice injected with M1 macrophage secretomes, a decrease in fibrosis indicators such as collagen and a change in the M1 / M2 ratio close to the normal group were observed. In addition, by using a bleomycin-induced pulmonary fibrosis animal model, the treatment effect of fibrosis was confirmed through intravenous injection of human recombinant CXCL11, and it was confirmed that in the lungs of mice administered CXCL11, a decrease in fibrosis indicators such as collagen and a change in the M1 / M2 ratio close to the normal group were observed. That is, the present invention aims to induce repolarization of M2 macrophages through CXCL11 during the development of pulmonary fibrosis, and consequently, to suggest it as a possible target factor for the treatment of pulmonary fibrosis.

[0010] Figure 1 shows the results of confirming the reduction of fibrosis indicators by M1 macrophage secretions in an animal model of pulmonary fibrosis.

[0011] Figure 2 shows the results of confirming the regulation of macrophage polarity by M1 macrophage secretions in an animal model of pulmonary fibrosis.

[0012] Figure 3 shows the results of analysis using human cytokine array of M0, M1, and M2 macrophage secretomes (conditioned medium, CM).

[0013] Figure 4 shows the results of confirming the reduction of fibrosis indicators by CXCL11 in an animal model of pulmonary fibrosis.

[0014] Figure 5 shows the results of confirming the regulation of macrophage polarity by CXCL11 in an animal model of pulmonary fibrosis.

[0015] Tissue damage accompanied by cellular inflammation in the lung induces a fibrogenic response, playing a crucial role in the pathogenesis of fibrosis. Activated macrophages utilize a series of innate immune defense strategies, including the production of a series of antimicrobial mediators. However, a significant side effect of an efficient inflammatory response is tissue fibrosis, resulting from the accumulation of excessive extracellular matrix components due to the prolonged repair response for tissue regeneration. Activated M2 macrophages play a crucial role in wound healing and adopt a profibrotic phenotype. This phenotype is observed when the fibrogenic immune response peaks, suggesting that M2 macrophages are important inducers and regulators of fibrosis.

[0016] Research into the inhibition of pulmonary fibrosis through macrophage polarization is actively underway, and it has been reported that macrophage-derived extracellular vesicles, miRNA, and peptides are used to suppress the activity of M2 macrophages. CXCL11 has also been reported to inhibit pulmonary angiogenesis and restore collagen increase when administered to mice with pulmonary fibrosis. However, this study was conducted with simultaneous treatment of bleomycin, a fibrosis-inducing drug, and CXCL11, so the concept of prevention rather than treatment was pursued. Furthermore, the mechanism and therapeutic potential of fibrosis inhibition through the polarization control of M2 macrophages, which the present invention proposes, have not yet been elucidated. Since pulmonary fibrosis that has already progressed cannot be reversed, the development of a pulmonary fibrosis treatment drug is urgent. Therefore, the present invention can be utilized as a novel therapeutic strategy that maximizes treatment while minimizing side effects by selectively inhibiting M2 macrophages, immune cells that play a key role in pulmonary fibrosis.

[0017]

[0018] The present invention provides a pharmaceutical composition for treating pulmonary fibrosis, comprising CXCL11 as an active ingredient.

[0019] Preferably, the pulmonary fibrosis may be, but is not limited to, idiopathic pulmonary fibrosis (IPF).

[0020] Preferably, the pharmaceutical composition can induce a decrease in M2 macrophages and an increase in M1 macrophages.

[0021] Preferably, the pharmaceutical composition can induce repolarization of M2 macrophages into M1 macrophages.

[0022] The pharmaceutical composition of the present invention can be manufactured using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and the adjuvants may include solubilizers such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents. The pharmaceutical composition of the present invention can be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and may include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.

[0023] The pharmaceutical composition of the present invention may be in the form of a pharmaceutical formulation, such as granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of the active compound. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Therefore, it can be adjusted according to various factors, including the type of disease, the severity of the disease, the types and contents of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, body weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs.

[0024]

[0025] In addition, the present invention provides a reagent composition for inducing repolarization of M2 macrophages into M1 macrophages in vitro, comprising CXCL11 as an active ingredient.

[0026] In addition, the present invention provides a method for inducing repolarization of M2 macrophages into M1 macrophages, comprising a step of treating macrophages with CXCL11 in vitro.

[0027] Below, the present invention is described in detail using examples that do not limit the scope of the invention. The following examples are intended to concretize the invention and do not limit or restrict the scope of the invention. Therefore, anything that a specialist in the technical field can easily infer from the detailed description and examples of the invention is interpreted as falling within the scope of the invention.

[0028]

[0029] <Experimental Example>

[0030] The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

[0031]

[0032] 1. Laboratory animals

[0033] Eight-week-old male C57BL / 6 mice were purchased from Dooyeol Biotech (Seoul, Korea), and the Animal Care and Use Committee approved the KW-211006-1 animal experiment. Lung fibrosis was induced in the mice by intratracheal instillation of 1 mg / kg bleomycin for 9 days after treatment with zoletil 50 (Virbac Korea, Korea) and Rumpun muscle relaxant (Bayer Healthcare, 20 μl / kg, IP injection). Macrophage secretion products (50 μg / mouse) were intravenously administered to mice with pulmonary fibrosis on days 7, 9, and 11 after bleomycin injection. Mice were sacrificed 14 days after bleomycin injection, and lung tissues were removed for efficacy evaluation.

[0034]

[0035] 2. Trichrome stain (abcam, ab150686)

[0036] 1) The deparaffinized slide was immersed in Bouin's solution at 50-60℃, reacted for 60 minutes, and cooled for 10 minutes.

[0037] 2) After washing three times with water, soak in Weigert's Iron Hematoxylin for 5 minutes.

[0038] 3) After washing three times with water, soak in Biebrich Scarlet / Acid Fuchsin solution for 15 minutes.

[0039] 4) After washing three times with water, it was reacted in a phosphomolybdic / phosphotungstic acid solution for 10-15 minutes.

[0040] 5) Immerse the slides in Aniline Blue solution for 5-10 minutes.

[0041] 6) After washing with water three times, soak in acetic acid solution for 3-5 minutes.

[0042] 7) After removing moisture, mount.

[0043]

[0044] 3. sirius stain (abcam,ab150681)

[0045] 1) The deparaffinized slides were reacted with picro-sirius red solution for 60 minutes.

[0046] 2) After washing in acetic acid solution, it was washed again in absolute alcohol.

[0047] 3) After removing moisture, mount.

[0048]

[0049] 4. Fluorescent staining

[0050] 1) Deparaffinization: Slides were immersed in antigen retrieval solution at 121°C for 1 minute. After washing in distilled water for 10 minutes, they were immersed in PBS for 3 minutes and washed in PBS-T (0.1% tween 20) for 3 minutes.

[0051] 2) After completing the hydrogen peroxide process, the slides were reacted with peroxidase-blocking solution for 20 minutes and then washed with PBS-T. After blocking with 10% Normal Goat Serum, they were reacted with primary antibodies against collagen (abcam, ab34710, 1:400), α-SMA (Santa Cruz, sc-53015, 1:500), iNOS (NB300-605, NOVUS, 1:400), CD206 (abcam, ab64693, 1:800), and F4 / 80 (Biorad, MCA497, 1:400) at 4℃ for one day.

[0052] 3) After washing with PBS-T, the secondary antibody Goat-anti-rabbit 488, 594 was reacted at room temperature for 1 hour.

[0053] 4) After washing with PBS-T, the cells were mounted using Fluoreshield Mounting Medium with DAPI (Abcam, #ab104139).

[0054]

[0055] 5. Protein analysis (SDS-PAGE)

[0056] Protein lysis buffer was prepared with 10 ml of protein extraction solution (RIPA) (ELPIS BIOTECH, EBA-1149), 100 μl of 100X protease inhibitor cocktail (Thermo scientific, 1860932), and 100 μl of 100X EDTA solution (Thermo scientific, 1860851), and 300 μl was used to isolate lung tissue proteins. For protein quantification, standards were prepared with BSA 1000 μg / ml, 500 μg / ml, 250 μg / ml, 125 μg / ml, 62.25 μg / ml, 31.125 μg / ml, and 0 μg / ml (DW), and 20 μl each of sample and standard were dispensed into 96 wells. Pierce TMBCA protein assay reagent A (Thermo scientific, 23228) and B (Thermo scientific, 23224) were added to the standard well and sample well, respectively, at 196 μl and 4 μl, and incubated at 36°C for 20 minutes. Afterwards, the protein was quantified by measuring the absorbance corresponding to 562 nm using a microplate spectrophotometer. The quantified protein was made into a concentration of 20 μg / 10 μl using protein 5X sample buffer (ELPIS BIOTECH, EBA-1052) and protein lysis buffer, and then heated at 100°C for 10 minutes and cooled on ice for 10 minutes. A 10% running gel and staking gel were prepared, and 10 μg of sample was loaded. The protein levels of Col1a1 (Santa cruz, sc-293182, 1:500), α-SMA (Santa cruz, sc-53015, 1:500), and actin (Santa cruz, sc-47778, 1:5000) were confirmed.

[0057]

[0058] 6. Human XL cytokine array (R&D systems, ARY022B)

[0059] The Proteome Profiler Human XL Cytokine Array is a membrane-based immunoassay. Capture antibodies, detected on a dual nitrocellulose membrane, bind to specific target proteins present in the sample. The membrane-bound proteins are detected with a biotinylated detection antibody and then visualized using a chemiluminescent detection reagent. The luminescent signal is proportional to the amount of bound analyte. A total of 105 human cytokines and chemokines can be detected.

[0060] 1) Each membrane was placed in a separate well, the array buffer was filled into the well, and the reaction was performed on a rocking platform shaker for 1 hour.

[0061] 2) After blocking, the array buffer was suctioned and the sample was loaded into the well. The reaction was carried out on a rocking platform shaker at 4℃ for one day.

[0062] 3) The membrane was transferred to an individual plastic container and washed with 1X Wash Buffer for 10 minutes. This was repeated twice.

[0063] 4) The antibody to detect the protein was added and reacted on a rocking platform shaker for 1 hour.

[0064] 5) After washing with 1X Wash Buffer for 10 minutes, 1X Streptavidin-HRP was added and reacted on a rocking platform shaker for 30 minutes.

[0065] 6) After washing with 1X Wash Buffer for 10 minutes, the Chemi Reagent Mix was evenly spread over the membrane and reacted for 1 minute.

[0066] 7) The membrane was placed on autoradiography film and exposed for 1-10 minutes.

[0067]

[0068] 7. Bone marrow-derived macrophage (BMDM) differentiation and M2 polarity changes

[0069] 1) Bone marrow from mice was collected through PBS flush.

[0070] 2) The isolated bone marrow cells were cultured in DMEM + 10% FBS + 1% penicillin / streptomycin + 50 ng / ml GM-CSF medium and differentiated into macrophages.

[0071] 3) Differentiated macrophages were differentiated into M2 macrophages by exposing them to 20 ng / ml of IL-4 for 12 hours.

[0072] 4) The experimental groups are as follows.

[0073] - Naive: negative control

[0074] - IL4: M2 macrophages

[0075] - I+L: Simultaneous exposure to IL4 and LPS induced repolarization into M1 macrophages.

[0076] - I+FA: Simultaneous exposure to IL4 and CXCL11 induced CXCL11-induced repolarization in M2 macrophages.

[0077] - L: M1 macrophages were induced by treating with LPS, an M1 macrophage inducer.

[0078]

[0079] After setting up the experimental group as above and treating it for 12 hours, M1 markers (iNOS, Socs3) and M2 markers (Arg1, Mrc3) were confirmed at the mRNA level.

[0080]

[0081] <Example 1> Confirmation of reduction in fibrosis indicators by M1 macrophage secretory body in pulmonary fibrosis animal model

[0082] Using a bleomycin-induced pulmonary fibrosis animal model, we investigated the anti-fibrotic effects of injections of M0, M1, and M2 conditioned medium (CM). Expression of fibrotic markers col1a1 and αSMA in mouse lung tissues was confirmed. A decrease in fibrotic markers was observed in mice injected with M1 CM compared to mice injected with M0 and M2 CM (Fig. 1A).

[0083] As a result of quantifying the level of protein expression and displaying it graphically, a decrease in the fibrosis index was confirmed in mice injected with the M1 secretome compared to mice injected with the M0 and M2 secretomes (Fig. 1B).

[0084] The degree of collagen fiber accumulation was confirmed through trichrome and sirius staining in paraffin-slides of mouse lung tissue, and the expression of collagen and αSMA was confirmed through fluorescent staining in the same tissue. A decrease in collagen fibers was confirmed in mice injected with the M1 secretome compared to mice injected with the M0 and M2 secretomes. In addition, the expression of collagen and αSMA confirmed by fluorescent staining was also significantly reduced in mice injected with the M1 secretome (Fig. 1C).

[0085] A decrease in fibrosis indices was confirmed in mice injected with M1 secretion compared to mice injected with M0 and M2 secretion, and when the degree of expression was quantified as intensity and displayed as a graph, a decrease in the expression of collagen and αSMA was confirmed in mice injected with M1 secretion (Fig. 1D).

[0086]

[0087] <Example 2> Confirmation of macrophage polarity regulation by M1 macrophage secretory body in pulmonary fibrosis animal model

[0088] Using a bleomycin-induced pulmonary fibrosis animal model, the polarity of M1 and M2 macrophages was confirmed through injection of M0, M1, and M2 secretomes (conditioned medium, CM). When F4 / 80+iNOS (M1) and F4 / 80+CD206 (M2) in the lung tissues of mice were confirmed by fluorescent staining, F4 / 80+CD206 (M2), which was increased in the bleomycin model, showed a decrease in F4 / 80+CD206 (M2) in mice injected with the M1 secretome compared to mice injected with the M0 and M2 secretomes. On the other hand, F4 / 80+iNOS (M1), which was decreased in the bleomycin model, showed an increase in mice injected with the M1 secretome. This confirmed that the M1 secretome induces a decrease in M2 macrophages and an increase in M1 macrophages (Fig. 2A).

[0089] When the expression of M1 and M2 markers was counted per field and analyzed as a ratio, it was confirmed that the M1 / M2 ratio changed to near normal in mice injected with M1 macrophage secretome. This was quantified and presented graphically. This confirmed that the M1 secretome induced changes in the M1 / M2 ratio similar to the normal group (Fig. 2B).

[0090]

[0091] <Example 3> Analysis of M0, M1, and M2 macrophage secretomes (conditioned medium, CM) using human cytokine array

[0092] Experiments were conducted to compare the relative levels of cytokines and chemokines in the secretory body to identify factors affecting anti-fibrosis and macrophage repolarization (Fig. 3A).

[0093] We confirmed that the expression of CXCL9, CXCL11, and Pentraxin3 was increased in M1 macrophage secretomes compared to M0 and M2 macrophage secretomes. The visualized detection amount of expression was expressed as intensity (Fig. 3B).

[0094] A schematic diagram of the experiment confirming the regulation of macrophage polarity by CXCL11 in bone-marrow derived macrophage (BMDM) cells extracted from mice is shown in Figure 3C.

[0095] After differentiating mouse BMDM into M2 macrophages through exposure to IL-4, we examined the regulation of macrophage polarization at the mRNA level by treating them with LPS (an M1 polarization inducer) and CXCL11. Repolarization of M2 macrophages was confirmed through M1 markers (iNOS, Socs3) and M2 markers (Arg1, Mrc1). When CXCL11 was treated in macrophages differentiated into M2 macrophages, a decrease in M2 markers and an increase in M1 markers were observed. This indicates that CXCL11 induces repolarization of M2 macrophages into M1 macrophages (Fig. 3D and Fig. 3E).

[0096]

[0097] <Example 4> Confirmation of reduction in fibrosis markers by CXCL11 in an animal model of pulmonary fibrosis

[0098] Using a bleomycin-induced pulmonary fibrosis animal model, we confirmed the antifibrotic effect of intravenous CXCL11 injection. Expression of the fibrotic markers col1a1 and αSMA in mouse lung tissue was confirmed, and a decrease in fibrotic markers was observed in mice injected with CXCL11 (Fig. 4A).

[0099] The level of protein expression was quantified and presented graphically in Figure 4B.

[0100] The degree of collagen fiber accumulation was confirmed in paraffin-slide lung tissues of mice using trichrome and Sirius staining, and the expression of collagen and αSMA was confirmed in the same tissues using fluorescent staining. In mice injected with CXCL11, a decrease in collagen fibers and fluorescent expression of collagen and αSMA were also confirmed (Fig. 4C).

[0101] When the degree of fluorescence expression of collagen and αSMA was quantified as intensity and displayed in a graph, the anti-fibrotic effect by CXCL11 was confirmed (Fig. 4D).

[0102]

[0103] <Example 5> Confirmation of macrophage polarity regulation by CXCL11 in an animal model of pulmonary fibrosis

[0104] Using a bleomycin-induced pulmonary fibrosis animal model, we confirmed the polarity of M1 and M2 macrophages through CXCL11 injection. When F4 / 80+iNOS (M1) and F4 / 80+CD206 (M2) in mouse lung tissues were confirmed by fluorescent staining, F4 / 80+CD206 (M2), which increased in the bleomycin model, showed a decrease in F4 / 80+CD206 (M2) in mice injected with CXCL11, and F4 / 80+iNOS (M1), which decreased in the bleomycin model, showed an increase in mice injected with CXCL11. This confirmed that CXCL11 induces a decrease in M2 macrophages and an increase in M1 macrophages (Fig. 5A).

[0105] When the expression of M1 and M2 markers was counted per field and analyzed as a ratio, the M1 / M2 ratio in CXCL11-injected mice was confirmed to change to near normal. This was quantified and presented graphically. This confirmed that CXCL11 induces changes in the M1 / M2 ratio similar to those in the normal group (Fig. 5B).

[0106]

[0107] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for treating pulmonary fibrosis containing CXCL11 as an active ingredient.

2. A pharmaceutical composition for treating pulmonary fibrosis, characterized in that the pulmonary fibrosis in paragraph 1 is idiopathic pulmonary fibrosis (IPF).

3. A pharmaceutical composition for treating pulmonary fibrosis, characterized in that the pharmaceutical composition induces a decrease in M2 macrophages and an increase in M1 macrophages in the first paragraph.

4. A pharmaceutical composition for treating pulmonary fibrosis, characterized in that the pharmaceutical composition induces repolarization of M2 macrophages into M1 macrophages in the first paragraph.

5. A reagent composition for inducing repolarization of M2 macrophages into M1 macrophages in vitro, comprising CXCL11 as an active ingredient.

6. A method for inducing repolarization of M2 macrophages into M1 macrophages, comprising a step of treating macrophages with CXCL11 in vitro.

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