KR102635831B1 - Use for treating pulmonary fibrosis by inducing repolarization of M2 macrophage by CXCL11 - Google Patents

Abstract

The present invention relates to the use of pulmonary fibrosis treatment by inducing repolarization of M2 macrophages by CXCL11, and specifically, injection of M0, M1, and M2 secretions (conditioned medium, CM) using a bleomycin-induced pulmonary fibrosis animal model. The effectiveness of fibrosis treatment was confirmed, and a decrease in fibrosis indicators such as collagen and a change in the M1/M2 ratio close to the normal group were confirmed in the lungs of mice injected with M1 macrophage secretory body. In addition, using a bleomycin-induced pulmonary fibrosis animal model, the fibrosis treatment effect was confirmed through intravenous injection of human recombinant CXCL11. In the lungs of mice administered CXCL11, fibrosis indicators such as collagen were reduced and the M1/M2 ratio was close to that of the normal group. It was confirmed that has changed. In other words, the present invention seeks to induce repolarization of M2 macrophages through CXCL11 during the development of pulmonary fibrosis and consequently present it as a possible target factor for the treatment of pulmonary fibrosis.

Description

Use for treating pulmonary fibrosis by inducing repolarization of M2 macrophage by CXCL11}

The present invention relates to the use for treating pulmonary fibrosis through inducing repolarization of M2 macrophages by CXCL11.

Macrophages can be polarized into either a classically activated M1 phenotype or an alternatively activated M2 phenotype depending on their microenvironment. Generally, M1 macrophages are responsible for wound healing after alveolar epithelial injury, while M2 macrophages are responsible for resolving the wound healing process or terminating the inflammatory response in the lung. Several studies have shown that activated M2 macrophages in fibrotic lesions produce large amounts of cytokines such as IL-10 and TGFβ1 that enhance collagen synthesis and deposition, and that these pro-inflammatory and pro-fibrotic cytokines exert anti-inflammatory properties through negative feedback. It is known to further promote fibrosis by indirectly suppressing the production of cytokines.

Idiopathic pulmonary fibrosis (IPF) is a disease in which excessive extracellular matrix (ECM) accumulates in the lungs, thickens the alveolar walls, and ultimately destroys the alveolar structure, leading to respiratory failure. The 5-year survival rate after diagnosis is 40%. It is only less than %. To date, many new concepts have been established to reveal the etiology, but unfortunately, the exact cause of pulmonary fibrosis is not known, making it an incurable disease that is difficult to fundamentally treat. Currently, there is no technology to completely return fibrotic tissue to its original state in medical technology, and drugs such as pirfenidone and nintendanib are used to suppress the progression of fibrosis. Although these drugs delay the decline in lung function, they still do not stop the disease progression, and the side effects are so severe that the rate of patients giving up taking the drug is high. Many clinical studies are in progress to develop treatments for idiopathic pulmonary fibrosis, but drugs that are currently in clinical trials and are shown to be effective for idiopathic pulmonary fibrosis inhibit collagen production by reducing the action of proteins that affect collagen production, or suppress collagen production in various ways. The focus is on improving symptoms by using a mechanism to prevent inflammation and fibrosis using receptors involved in physiological activity, but the development of new treatments is urgently needed due to the high unmet medical demand.

US published patent US 2021-0330734 A1 (published on October 28, 2021)

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

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

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating pulmonary fibrosis containing CXCL11 as an active ingredient.

Additionally, the present invention provides a reagent composition for inducing repolarization of M2 macrophages to M1 macrophages in vitro, comprising CXCL11 as an active ingredient.

Additionally, the present invention provides a method of inducing repolarization of M2 macrophages into M1 macrophages, comprising treating macrophages with CXCL11 in vitro.

The present invention relates to the use of pulmonary fibrosis treatment by inducing repolarization of M2 macrophages by CXCL11, and specifically, injection of M0, M1, and M2 secretions (conditioned medium, CM) using a bleomycin-induced pulmonary fibrosis animal model. The effectiveness of fibrosis treatment was confirmed, and a decrease in fibrosis indicators such as collagen and a change in the M1/M2 ratio close to the normal group were confirmed in the lungs of mice injected with M1 macrophage secretory body. In addition, using a bleomycin-induced pulmonary fibrosis animal model, the fibrosis treatment effect was confirmed through intravenous injection of human recombinant CXCL11. In the lungs of mice administered CXCL11, fibrosis indicators such as collagen were reduced and the M1/M2 ratio was close to that of the normal group. It was confirmed that has changed. In other words, the present invention seeks to induce repolarization of M2 macrophages through CXCL11 during the development of pulmonary fibrosis and consequently present it as a possible target factor for the treatment of pulmonary fibrosis.

Figure 1 shows the results of confirming the reduction of fibrosis indicators by M1 macrophage secretome in a pulmonary fibrosis animal model.
Figure 2 shows the results of confirming the regulation of macrophage polarity by the M1 macrophage secretome in an animal model of pulmonary fibrosis.
Figure 3 shows the results of analysis of M0, M1, and M2 macrophage secretome (conditioned medium, CM) using human cytokine array.
Figure 4 shows the results confirming the decrease in fibrosis indices by CXCL11 in a pulmonary fibrosis animal model.
Figure 5 shows the results of confirming macrophage polarity regulation by CXCL11 in a pulmonary fibrosis animal model.

Tissue damage accompanied by cellular inflammation in the lung induces a fibrotic response and plays an important role in the pathogenesis of fibrosis. Activated macrophages employ a series of innate immune defense strategies, such as the production of a series of antibacterial mediators. An important side effect of an efficient inflammatory response is tissue damage due to the accumulation of excessive extracellular matrix components followed by the continuation of the repair response for tissue regeneration. It is fibrosis. In general, activated M2 macrophages play an important role in wound healing and acquire a pro-fibrotic phenotype. Since this phenotype is observed when the fibrotic immune response is at its peak, M2 macrophages are being studied as important inducers and regulators of fibrosis.

Research on suppressing pulmonary fibrosis through macrophage polarization is actively underway, and it has been reported that the activity of M2 macrophages is suppressed using macrophage-derived extracellular vesicles, miRNA, and peptides. It has been reported that CXCL11 also inhibited angiogenic activity in the lung and restored the increase in collagen when administered to mice inducing pulmonary fibrosis. However, this study showed that simultaneous treatment of bleomycin, a fibrosis-inducing drug, and CXCL11 was carried out, so that it was not a treatment concept, but rather a prevention concept. A similar study was conducted. In addition, the mechanism and therapeutic potential of suppressing fibrosis through controlling the polarity of M2 macrophages proposed in the present invention have not yet been revealed. Since pulmonary fibrosis that is already in progress cannot be returned to normal, the development of a treatment drug for pulmonary fibrosis is urgent. The present invention provides treatment while minimizing side effects by selectively inhibiting M2 macrophages, immune cells that play a key role in pulmonary fibrosis. It can be used as a new treatment strategy to maximize treatment.

The present invention provides a pharmaceutical composition for treating pulmonary fibrosis containing CXCL11 as an active ingredient.

Preferably, the pulmonary fibrosis may be idiopathic pulmonary fibrosis (IPF), but is not limited thereto.

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

Preferably, the pharmaceutical composition is capable of inducing repolarization of M2 macrophages into M1 macrophages.

The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable auxiliaries in addition to the active ingredients, and the auxiliaries include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, and glidants. Alternatively, solubilizers such as flavoring agents may be used. For administration, the pharmaceutical composition of the present invention can be preferably formulated as a pharmaceutical composition containing one or more pharmaceutically acceptable carriers in addition to the active ingredient. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The pharmaceutical preparation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release preparations of the active compound. You can. The pharmaceutical composition of the present invention can be administered in any conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. It can be administered. The effective amount of the active ingredient of the pharmaceutical composition of the present invention means the amount required for the prevention or treatment of disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form and the patient's age, weight, general health condition, gender and diet, administration time, administration route and composition. It can be adjusted depending on a variety of factors, including secretion rate, duration of treatment, and concurrent medications.

Additionally, the present invention provides a reagent composition for inducing repolarization of M2 macrophages to M1 macrophages in vitro, comprising CXCL11 as an active ingredient.

Additionally, the present invention provides a method of inducing repolarization of M2 macrophages into M1 macrophages, comprising treating macrophages with CXCL11 in vitro.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

< Experiment example >

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

1. Experimental animals

C57BL/strain 6 male 8-week-old mice were purchased from Duyeol Biotech (Seoul, Korea), and the Animal Experiment Ethics Committee approved KW-211006-1 animal testing. In mice, pulmonary fibrosis was induced for 9 days by intratracheal injection of 1mg/kg bleomycin after zoletil 50 (Vervac Korea, Korea) and Rumpun muscle relaxation (Bayer Healthcare, 20μl/kg, IP injection). Macrophage secretory 50ug/mouse was intravenously administered to mice induced with pulmonary fibrosis 7, 9, and 11 days after bleomycin injection. Mice were sacrificed 14 days after bleomycin injection, lung tissue was removed, and efficacy was evaluated.

2. Trichrome stain ( abcam , ab150686 )

1) Deparaffinized slides were immersed in Bouin's solution at 50-60°C, reacted for 60 minutes, and cooled for 10 minutes.

2) After washing with water three times, it was soaked in Weigert's Iron Hematoxylin for 5 minutes.

3) After washing with water three times, it was soaked in Biebrich Scarlet / Acid Fuchsin solution for 15 minutes.

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

5) The slide was soaked in Aniline Blue solution for 5-10.

6) After washing with water three times, it was soaked in acetic acid solution for 3-5 minutes.

7) After removing the moisture, it was mounted.

3. sirius stain ( abcam,ab150681 )

1) Deparaffinized slides were reacted in picro-sirius red solution for 60 minutes.

2) After washing with acetic acid solution, it was washed again with absolute alcohol.

3) After removing the moisture, it was mounted.

4. Fluorescent staining

1) Deparaffinization Slides were immersed in Antigen retrieval solution and carried out at 121°C for 1 minute. After washing in distilled water for 10 minutes, it was soaked in PBS for 3 minutes and washed in PBS-T (0.1% tween 20) for 3 minutes.

2) Slides that have completed the hydrogen peroxide process are After reacting in peroxidase-blocking solution for 20 minutes, it was washed with PBS-T. After blocking using 10% Normal Goat Serum, collagen (abcam, ab34710, 1:400), α-SMA (Santa cruz, sc-53015,1:500), iNOS (NB300-605,NOVUS,1:400), It was reacted with CD206 (abcam, ab64693, 1:800) and F4/80 (Biorad, MCA497, 1:400) primary antibodies at 4°C for one day.

3) After washing with PBS-T, Goat-anti-rabbit 488 and 594 secondary antibodies were reacted at room temperature for 1 hour.

4) After washing with PBS-T, it was mounted using Fluoreshield Mounting Medium with DAPI (Abcam, #ab104139).

5. Protein analysis (SDS-PAGE)

Protein lysis buffer was prepared with 10ml of protein extraction solution (RIPA) (ELPIS BIOTECH, EBA-1149), 100μl of 100X protease inhibitor cocktail (Thermo scientific, 1860932), and 100μl of 100X EDTA solution (Thermo scientific, 1860851). Tissue proteins were isolated. For protein quantification, standards were made with BSA 1000μg/ml, 500μg/ml, 250μg/ml, 125μg/ml, 62.25μg/ml, 31.125μg/ml, 0μg/ml (DW), and 20μl each of sample and standard were 96 It was busy in the well. Pierce ^TM BCA protein assay reagent A (Thermo scientific, 23228) and B (Thermo scientific, 23224) were added to standard wells and sample wells (196 μl and 4 μl, respectively) and reacted at 36°C for 20 minutes. Afterwards, the protein was quantified by measuring the absorbance at 562 nm using a micro plate spectrophotometer. The quantified protein was adjusted to a concentration of 20 μg/10 μl using protein 5X sample buffer (ELPIS BIOTECH, EBA-1052) and protein lysis buffer, then heated at 100°C for 10 minutes and cooled on ice for 10 minutes. A 10% running gel and staking gel were created, and 10 μg of sample was loaded. 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.

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

Proteome Profiler Human XL Cytokine Array is a membrane-based immunoassay. Capture antibodies found double on the nitrocellulose membrane bound to specific target proteins present in the sample. Proteins bound to the membrane were detected with a biotinylated detection antibody and then visualized using a chemiluminescent detection reagent. The signal emitted is proportional to the amount of bound analyte. A total of 105 human cytokines and chemokines can be detected.

1) Each membrane was placed in a separate well, filled with array buffer, and reacted on a rocking platform shaker for 1 hour.

2) After blocking, the array buffer was suctioned and the sample was loaded into the well. The reaction was performed for one day on a rocking platform shaker where the temperature was maintained at 4°C.

3) The membrane was transferred to an individual plastic container and washed with 1X Wash Buffer for 10 minutes. Repeated twice.

4) Antibodies to detect proteins were added and reacted on a rocking platform shaker for 1 hour.

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.

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

7) The membrane was placed in autoradiography film and exposed for 1-10 minutes.

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

1) The bone marrow of the mouse was collected through a PBS flush.

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

3) Differentiated macrophages were exposed to 20ng/ml of IL-4 for 12 hours to differentiate into M2 macrophages.

4) The experimental group is as follows.

- Naive: negative control

- IL4: M2 macrophages

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

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

- L: M1 macrophages were induced by treating LPS as an M1 macrophage inducer.

The experimental group as above was set up and treated for 12 hours, and M1 markers (iNOS, Socs3) and M2 markers (Arg1, Mrc3) were confirmed at the mRNA level.

< Example 1> A decrease in fibrosis indicators due to M1 macrophage secretion was confirmed in a pulmonary fibrosis animal model.

Using a bleomycin-induced pulmonary fibrosis animal model, the fibrosis treatment effect was confirmed through injection of M0, M1, and M2 secretions (conditioned medium, CM). As a result of confirming the expression of fibrosis indicators col1a1 and αSMA in the lung tissue of mice, a decrease in fibrosis indicators was confirmed in mice injected with M1 secretion compared to mice injected with M0 and M2 secretion (Figure 1A).

As a result of quantifying the protein expression level and graphing it, a decrease in fibrosis index was confirmed in mice injected with M1 secretion compared to mice injected with M0 and M2 secretion (Figure 1B).

The degree of accumulation of collagen fibers was confirmed through trichrome and sirius staining in paraffin-slides of mouse lung tissue, and the expression of collagen and αSMA in the same tissue was confirmed through fluorescence staining. A decrease in collagen fibers was confirmed in mice injected with M1 secretion compared to mice injected with M0 and M2 secretion, and the expression of collagen and αSMA, confirmed by fluorescent staining, was also confirmed to be significantly reduced in mice injected with M1 secretion ( Figure 1C).

A decrease in fibrosis indicators was confirmed in mice injected with M1 secretion compared to mice injected with M0 and M2 secretion, and when the expression level was quantified by intensity and displayed in a graph, in mice injected with M1 secretion, collagen, A decrease in the expression of αSMA was confirmed (Figure 1D).

< Example 2> Control of macrophage polarity by M1 macrophage secretome was confirmed in an animal model of pulmonary fibrosis

Using a bleomycin-induced pulmonary fibrosis animal model, the polarity of M1 and M2 macrophages was confirmed through injection of M0, M1, and M2 secretome (conditioned medium, CM). When F4/80+iNOS(M1) and F4/80+CD206(M2) in mouse lung tissue were confirmed by fluorescent staining, F4/80+CD206(M2) increased in the bleomycin model in mice injected with M0 and M2 secretions. In contrast, mice injected with M1 secretome showed a decrease in F4/80+CD206(M2), and F4/80+iNOS(M1), which was decreased in the bleomycin model, showed an increase in mice injected with M1 secretion. It was confirmed that this led to a decrease in M2 macrophages and an increase in M1 macrophages in the M1 secretome (Figure 2A).

When the expression of M1 and M2 markers was analyzed as a ratio by counting the number per field, it was confirmed that the M1/M2 ratio changed close to normal in mice injected with M1 macrophage secretion. This was quantified and displayed in a graph. This confirmed that the M1 secretome induces a change in the M1/M2 ratio similar to that in the normal group (Figure 2B).

< Example 3> M0, M1 and M2 macrophages secretory body human in conditioned medium (CM) cytokine Analysis through array

An experiment was conducted to find factors that affect anti-fibrosis and macrophage repolarization by comparing the relative levels of cytokines and chemokines in the secretome (Figure 3A).

It was confirmed that the expression of CXCL9, CXCL11, and Pentraxin3 was increased in the M1 macrophage secretome compared to the M0 and M2 macrophage secretome. The visualized detection amount of the expression level was expressed as intensity (Figure 3B).

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

Mouse BMDM were differentiated into M2 macrophages through exposure to IL-4, and then treated with LPS (M1 polarity inducer) and CXCL11 to confirm macrophage polarity regulation at the mRNA level. As a result of confirming the repolarization of M2 macrophages through M1 markers (iNOS, Socs3) and M2 markers (Arg1, Mrc1), when CXCL11 was treated with macrophages differentiated into M2 macrophages, there was a decrease in M2 markers and an increase in M1 markers. Confirmed. This indicates that CXCL11 induces repolarization of M2 macrophages into M1 macrophages (Figures 3D and 3E).

< Example 4> CXCL11 Reduction of fibrosis index by pulmonary fibrosis Confirmed in animal model

Using a bleomycin-induced pulmonary fibrosis animal model, the anti-fibrotic effect was confirmed through intravenous injection of CXCL11. As a result of confirming the expression of fibrosis indicators col1a1 and αSMA in mouse lung tissue, a decrease in fibrosis indicators was confirmed in mice injected with CXCL11 (Figure 4A).

The protein expression level was quantified and shown graphically in Figure 4B.

The degree of accumulation of collagen fibers was confirmed through trichrome and sirius staining in paraffin-slides of mouse lung tissue, and the expression of collagen and αSMA in the same tissue was confirmed through fluorescence staining. It was confirmed that in mice injected with CXCL11, collagen fibers decreased and the fluorescence expression of collagen and αSMA also decreased (Figure 4C).

When the level of fluorescence expression of collagen and αSMA was quantified by intensity and displayed in a graph, the anti-fibrotic effect of CXCL11 was confirmed (Figure 4D).

< Example 5> CXCL11 Control of macrophage polarity by pulmonary fibrosis Confirmed in animal model

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

When the expression of M1 and M2 markers was analyzed by ratio by counting the number per field, it was confirmed that the M1/M2 ratio changed close to normal in mice injected with CXCL11. This was quantified and displayed in a graph. This confirmed that CXCL11 induced changes in the M1/M2 ratio similar to the normal group (Figure 5B).

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

delete delete A reagent composition for inducing repolarization of M2 macrophages into M1 macrophages in a pulmonary fibrosis mouse model, comprising CXCL11 as an active ingredient. A method for inducing repolarization of M2 macrophages into M1 macrophages, comprising treating a pulmonary fibrosis mouse model with CXCL11. A reagent composition for inducing repolarization of M2 macrophages into M1 macrophages in vitro, comprising CXCL11 as an active ingredient. A method of inducing repolarization of M2 macrophages into M1 macrophages, comprising treating macrophages with CXCL11 in vitro.