Methods and compositions for the assessment and treatment of fibrosis

Abstract

Staphylococcus nepalensis releases corisin, a peptide conserved among various staphylococci that induces apoptosis in lung epithelial cells. Therefore, methods and devices for detecting the presence of corisin in a patient's biological sample, as well as pharmaceutical compositions and methods, such as antibodies, for treating patients with or suspected of having fibrosis, are disclosed. For example, the present invention provides a method comprising detecting the presence of corisin in a patient's biological sample.

Description

[Technical Field]

[0001] cross reference This application claims priority to U.S. Patent Application No. 62 / 948,983, filed December 17, 2019, the contents of which are incorporated herein in their entirety.

[0002] Technical Field The present invention relates generally to a Staphylococcus pro-apoptotic peptide (referred to herein as "corisin") that has been found to induce acute exacerbations of pulmonary fibrosis, as well as methods, kits and devices for diagnosing or assessing fibrosis in patients, and methods and compositions for ameliorating or treating fibrosis, such as idiopathic pulmonary fibrosis. [Background technology]

[0003] Background technology Idiopathic pulmonary fibrosis (IPF) is a chronic, fatal disease with an unknown etiology, although alveolar epithelial cell apoptosis is known to play a role in disease progression. This intractable disease is associated with increased abundance of Staphylococcus and Streptococcus in the lungs, yet their role in disease pathogenesis remains elusive.

[0004] IPF is the most common form of idiopathic interstitial pneumonia, characterized by chronicity, progressive disease, and fatal clinical outcomes. See NPL1 and NPL2 (full citations for all non-patent literature identified herein by the designation "NPL" are provided at the end of this specification). The prognosis for IPF is worse than many other types of malignancies, with patients having an average life expectancy of only 2-3 years after diagnosis. See NPL3 and NPL4. Repeated trauma and / or apoptosis of pulmonary epithelial cells, excessive release of profibrotic factors, and enhanced recruitment of extracellular matrix-producing myofibroblasts to the lung play critical roles in the pathogenesis of the disease. See NPL2 and NPL5.

[0005] NPL6 suggests that the lung microbiome plays a causative role in IPF, with increased lung bacterial burden associated with acute disease exacerbations and higher mortality. As shown in NPL7, the relative abundance of the pulmonary microorganisms Staphylococcus and Streptococcus has also been associated with accelerated clinical progression of IPF. However, the role of these bacteria in the pathogenesis of pulmonary fibrosis remains unknown. Ideally, culturing bacteria associated with fibrotic tissue and characterizing their phenotype would be essential to unambiguously identify the organisms involved in the pathogenesis of IPF, but there are no previous reports of bacterial isolates associated with the pathogenesis of this disease.

[0006] NPL8 and NPL9 demonstrated that fibrotic lung tissue from IPF patients and from transforming growth factor (TGF) β1 transgenic (TG) mice with pulmonary fibrosis were characterized by an enrichment of halophilic bacteria, a finding also confirmed by NPL4. Summary of the Invention [Means for solving the problem]

[0007] Summary of the Invention The results in NPL8 and NPL9 led to the hypothesis that fibrotic tissue represents a salt-rich microenvironment and that the high salt conditions of pulmonary fibrotic tissue facilitate the growth of bacteria that release factors that play a role in the pathogenesis of IPF disease and its acute exacerbations.

[0008] In our study, which led to the new findings and insights described herein, we used a halophilic medium to enrich Staphylococcus strains from pulmonary fibrotic tissue samples from TGFβ1 TG mice. We found that the culture supernatant of one of the bacterial strains, S. nepalensis CNDG, contained a proapoptotic peptide that induced apoptosis in lung epithelial cells.

[0009] The inventors further found that this pro-apoptotic peptide, referred to herein as "corisin," is a component of a transglycosylase that is conserved in diverse members of the genus Stapylococcus, and that intratracheal instillation of either corisin or the corisin-encoding S. nepalensis CNDG strain into mice with established pulmonary fibrosis leads to an acute exacerbation of the disease.

[0010] Furthermore, by performing enhanced detection of colisin in human IPF patients with acute exacerbations and comparing the results with those of patients without disease exacerbations, we concluded that bacteria harboring and releasing proapoptotic peptides are involved in acute exacerbations of pulmonary fibrosis.

[0011] More specifically, we found that Staphylococcus nepalensis releases corisin, a peptide conserved among diverse staphylococci, to induce apoptosis of lung epithelial cells. Mice exhibit acute exacerbations after intrapulmonary instillation of corisin or pulmonary infection with corisin-containing S. nepalensis, compared with untreated mice or mice infected with bacteria lacking corisin. Correspondingly, human IPF patients with acute exacerbations have significantly elevated pulmonary corisin levels compared with patients without disease exacerbations. This leads to the conclusion that bacteria releasing corisin are involved in acute exacerbations of IPF, thereby providing insight into the molecular basis for the elevation of staphylococci in pulmonary fibrosis and the association between staphylococci and the worsening stage of pulmonary fibrosis.

[0012] Based on these new findings and insights, the inventors have developed the following aspects of the present teachings.

[0013] In one embodiment of the present teachings, methods, kits, and devices are disclosed that involve detecting the presence of corisin in a patient's biological sample, preferably in vitro. Corisin can have, for example, one of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13 disclosed herein. These methods, kits, and / or devices can be used in the evaluation and / or diagnosis of fibrosis in patients, such as idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and / or breast fibrosis. Preferably, these methods, kits, and / or devices are used in the detection and / or evaluation of idiopathic pulmonary fibrosis (IPF).

[0014] In such methods, kits, or devices, colisin can be detected by mass spectrometry, Western blotting, and / or enzyme-linked immunosorbent assay (ELISA), and involve binding of colisin to an antibody, preferably in vitro, such as an antibody capable of recognizing (binding to) one of the amino acid sequences set forth herein: SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0015] Another aspect of the present teachings is an antibody that binds to colisin. The antibody can recognize (bind to) one of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13 disclosed herein, and can be a polyclonal antibody.

[0016] The antibodies can be used as pharmaceuticals in the prevention, amelioration, and / or treatment of fibrosis in patient subjects having fibrosis or suspected of having or developing fibrosis. For example, the antibodies can be provided as a pharmaceutical composition for use as a pharmaceutical to be administered to a patient in need thereof.

[0017] Such pharmaceutical compositions may optionally contain one or more pharmaceutically acceptable additives, salts and / or excipients, such as preservatives, sugars, solubilizers, stabilizers, carriers, diluents, fillers, pH buffers, isotonicity agents, antibacterial agents, wetting agents, and / or emulsifiers, preferably in amounts of 0.005% to 99% by weight, e.g., 0.5% to 98% by weight (e.g., the combined amounts when two or more are present).

[0018] The antibody can be used in the prevention, amelioration, and / or treatment of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and / or breast fibrosis. For example, the antibody can be used in the prevention, amelioration, and / or treatment of idiopathic pulmonary fibrosis (IPF). The antibody can be a neutralizing antibody, e.g., an antibody that blocks or inhibits the negative effects of colisin in the lungs or other tissues of patients with fibrosis.

[0019] In a further aspect of the present teachings, a method for treating fibrosis in a patient in need thereof can include administering to the patient a therapeutically effective amount of any of the above-described antibodies. For example, the antibody can be administered to one or both lungs of the patient. Additionally or alternatively, the antibody can be administered intraperitoneally, by intratracheal instillation, or by inhalation. Preferably, administration of the antibody reduces at least the severity of fibrosis in the subject.

[0020] It is noted that all diagnostic and / or assessment methods are preferably performed in vitro on biological samples extracted, harvested, obtained, etc. from a patient having or suspected of having or developing fibrosis, such as any of the above or below mentioned types of fibrosis.

[0021] Other objects, aspects, embodiments and advantages of the present teachings will become apparent to those skilled in the art from the following detailed description when read in light of the drawings and appended claims. In certain embodiments, for example, the following items are provided: (Item 1) A method comprising detecting the presence of colisin in a biological sample from a patient. (Item 2) 2. The method of claim 1, wherein the colisin has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. (Item 3) 3. The method according to item 1 or 2, used to assess fibrosis in said patient. (Item 4) 4. The method of claim 3, wherein the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and mammary fibrosis. (Item 5) 4. The method of claim 3, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF). (Item 6) The method of any preceding item, wherein the colisin is detected by a method selected from the group consisting of mass spectrometry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). (Item 7) The method of any preceding item, wherein the colisin is detected by binding with an antibody. (Item 8) 8. The method of claim 7, wherein the antibody recognizes an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. (Item 9) Corisin-binding antibody. (Item 10) 10. The antibody of item 9, which recognizes an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. (Item 11) 11. The antibody of item 9 or 10, which is a polyclonal antibody. (Item 12) 12. The antibody of any one of items 9 to 11, for use in preventing, ameliorating and / or treating fibrosis in a patient subject having fibrosis or suspected of having or developing fibrosis. (Item 13) 13. The antibody of item 12, wherein the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and mammary fibrosis. (Item 14) 13. The antibody of item 12, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF). (Item 15) 15. The antibody of any one of items 9 to 14, which is a neutralizing antibody. (Item 16) 16. A method of treating fibrosis in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of the antibody of any one of items 9 to 15. (Item 17) 17. The method of claim 16, wherein the antibody is administered to one or both lungs of the patient. (Item 18) 18. The method of item 16 or 17, wherein the antibody is administered intraperitoneally or by intratracheal instillation or by inhalation. (Item 19) 19. The method of any one of items 16 to 18, wherein administering the antibody reduces the severity of the fibrosis in the subject. (Item 20) 1. A method for use in the assessment of a subject having, or suspected of having or developing, fibrosis, comprising: receiving an in vitro biological sample collected from the subject; and detecting the amount of colisin present in the in vitro biological sample. (Item 21) 21. The method of claim 20, further comprising the step of comparing the amount of colisin detected in the in vitro biological sample with one or more predetermined thresholds. (Item 22) 22. The method of claim 20 or 21, wherein the in vitro biological sample is obtained from one or both lungs of the subject. (Item 23) 23. The method of any one of items 20 to 22, wherein the in vitro biological sample is selected from the group consisting of sputum, bronchial secretions, pleural effusion, bronchoalveolar lavage fluid (BALF), and tissue taken from the bronchi or lungs. (Item 24) 24. The method of any one of items 20 to 23, wherein the in vitro biological sample is blood or bronchoalveolar lavage fluid (BALF). (Item 25) 25. The method of any one of items 20 to 24, wherein the colisin has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. (Item 26) 26. The method of any one of items 20 to 25, wherein the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and mammary fibrosis. (Item 27) 26. The method of any one of items 20 to 25, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF). (Item 28) 28. The method of any one of items 20 to 27, wherein the colisin is detected by a method selected from the group consisting of mass spectrometry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). (Item 29) 29. The method of any one of items 20 to 28, wherein corisin is detected by binding with an antibody. (Item 30) 30. The method of item 29, wherein the antibody recognizes an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. (Item 31) A pharmaceutical composition for use in treating fibrosis in a patient, comprising a corisin inhibitor that neutralizes at least a portion of corisin in the lungs of the patient and / or reduces the amount of corisin in the lungs of the patient. (Item 32) 32. The pharmaceutical composition of claim 31, wherein the corisin inhibitor is selected from the group consisting of a small molecule, an antagonist of corisin, or an antibody to corisin. (Item 33) 33. The pharmaceutical composition according to item 31 or 32, wherein the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and mammary fibrosis. (Item 34) 33. The pharmaceutical composition according to item 31 or 32, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF). (Item 35) 35. The pharmaceutical composition according to any one of items 31 to 34, further comprising at least one pharmaceutically acceptable additive, salt or excipient. (Item 36) A method for identifying corisin receptor proteins, comprising the step of searching for corisin-binding proteins present on the surface of epithelial cells. (Item 37) A method for identifying a corisin receptor protein, comprising the step of searching for an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13 in a binding protein present on the surface of an epithelial cell. (Item 38) 38. The method of any one of items 1 to 8, 20 to 30, or 37, wherein the colisin has the amino acid sequence of SEQ ID NO:1. (Item 39) 31. The method of any one of items 8, 16 or 30 or the antibody of any one of items 9 to 15, wherein the antibody recognizes the amino acid sequence of SEQ ID NO: 1. (Item 40) 38. A kit or device for use in carrying out the method according to any one of items 1 to 8, 20 to 30, 36 or 37. (Item 41) 1. A pharmaceutical composition for use in treating fibrosis in a patient, comprising: The antibody according to any one of items 9 to 15, at least one pharmaceutically acceptable additive, salt or excipient; A pharmaceutical composition comprising: (Item 42) 42. A method for treating fibrosis in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of the pharmaceutical composition of any one of items 31 to 35 or 41. (Item 43) 43. The method of claim 42, wherein the pharmaceutical composition is administered to one or both lungs of the patient. (Item 44) 44. The method according to item 42 or 43, wherein the pharmaceutical composition is administered intraperitoneally or by intratracheal instillation or by inhalation. (Item 45) 45. The method of any one of items 42 to 44, wherein administering the pharmaceutical composition reduces the severity of the fibrosis in the subject. [Brief explanation of the drawings]

[0022] [Figure 1A] Figure 1A shows chest computed tomography (CT) images of nine wild-type (WT) mice, six TGFβ1 TG mice without fibrosis, and six TGFβ1 TG mice with fibrosis; Figure 1B shows the CT scores for these mice; Figure 1C shows the salt content in the lung tissue of these mice, as measured by microwave analysis / inductively coupled plasma mass spectrometry. [Figure 1B-C] Figure 1A shows chest computed tomography (CT) images of nine wild-type (WT) mice, six TGFβ1 TG mice without fibrosis, and six TGFβ1 TG mice with fibrosis; Figure 1B shows the CT scores for these mice; Figure 1C shows the salt content in the lung tissue of these mice, as measured by microwave analysis / inductively coupled plasma mass spectrometry.

[0023] [Figure 2A-B] Figures 2A and 2B show CT images and CT fibrosis scoring of wild-type (WT) mice (n=3) and TGFβ1 transgenic (TG) mice (n=8), respectively.

[0024] [Figure 2C]Figure 2C shows fibrotic lung tissues excised under sterile conditions from wild-type (n = 3) and TGFβ1 transgenic (n = 8) mice after 48 hours of culture in high-salt culture medium. Analysis of bacterial colonies was performed by transmission electron microscopy. The scale bar indicates 100 nm.

[0025] [Figure 2D] Figure 2D shows flow cytometry analysis of A549 alveolar epithelial cells cultured for 48 h in DMEM medium containing a 1 / 10 dilution of spent culture supernatant of a mixture of Staphylococcus spp. (strain 6; n = 9), DMEM medium containing a 1 / 10 dilution of spent culture supernatant of Staphylococcus nepalensis strain CNDG (n = 9), or control medium (n = 9).

[0026] [Figure 2E] Figure 2E shows flow cytometry analysis of normal human bronchial epithelial cells after 48 h of culture in DMEM medium containing a 1 / 10 dilution of spent culture supernatant of a mixture of Staphylococcus spp. (strain 6; n = 8), DMEM medium containing a 1 / 10 dilution of spent culture supernatant of Staphylococcus nepalensis strain CNDG (n = 8), or control medium (n = 4).

[0027] [Figure 2F-G] Figures 2F and 2G show TUNEL assays after culturing A549 alveolar epithelial cells in the presence of medium (n = 6) or supernatant of Staphylococcus nepalensis strain CNDG (n = 6). Scale bar indicates 20 μm.

[0028] [Figure 3A-B]Figure 3A shows the absorbance of fractions from the culture supernatant of a mixture of Staphylococcus spp. after gel filtration using a Sephadex G25 column; Figure 3B shows the cell viability after treatment of A549 alveolar epithelial cells with the culture supernatant of a mixture of Staphylococcus spp. (n = 3 for each fraction); Figure 3C shows cells in the sub-G1 phase after treatment of A549 cells with the culture supernatant of a mixture of Staphylococcus spp. (n = 3 for each fraction). [Figure 3C] Figure 3A shows the absorbance of fractions from the culture supernatant of a mixture of Staphylococcus spp. after gel filtration using a Sephadex G25 column; Figure 3B shows the cell viability after treatment of A549 alveolar epithelial cells with the culture supernatant of a mixture of Staphylococcus spp. (n = 3 for each fraction); Figure 3C shows cells in the sub-G1 phase after treatment of A549 cells with the culture supernatant of a mixture of Staphylococcus spp. (n = 3 for each fraction).

[0029] [Figure 3D] FIG. 3D shows a representative histogram of A549 cells in the sub-G1 phase after treatment with culture supernatants of a mixture of Staphylococcus spp.

[0030] [Figure 3E-F] Figure 3E shows the absorbance of fractions from the culture supernatant of Staphylococcus nepalensis strain CNDG after gel filtration; Figure 3F shows the cell viability after treating A549 cells with the culture supernatant of Staphylococcus nepalensis strain CNDG (n=3 for each fraction); Figure 3G shows cells in the sub-G1 phase after treating A549 cells with the culture supernatant of Staphylococcus nepalensis strain CNDG (n=3 for each fraction). [Figure 3G]Figure 3E shows the absorbance of fractions from the culture supernatant of Staphylococcus nepalensis strain CNDG after gel filtration; Figure 3F shows the cell viability after treating A549 cells with the culture supernatant of Staphylococcus nepalensis strain CNDG (n=3 for each fraction); Figure 3G shows cells in the sub-G1 phase after treating A549 cells with the culture supernatant of Staphylococcus nepalensis strain CNDG (n=3 for each fraction).

[0031] [Figure 3H] Figure 3H shows a representative histogram of A549 cells in sub-G1 phase after treatment with culture supernatant of Staphylococcus nepalensis strain CNDG. (1 mL of each sample was applied to a Sephadex G25 column. The eluted material was collected in 2 mL fractions, and the absorbance at 280 nm was then measured. Cell viability was assessed using a commercially available cell counting kit, and the percentage of cells in sub-G1 phase was assessed by flow cytometry.)

[0032] [Figure 3I] Figures 3I, 3J, and 3K show that bacteria were cultured in medium containing 2% or 8% salt. Culture supernatants of a mixture of Staphylococcus spp. (n = 9), Staphylococcus nepalensis CNDG strain (n = 9), or culture medium (n = 9) were then prepared by centrifugation and diluted 1 / 10 and added to the culture medium of A549 alveolar epithelial cells. Flow cytometry of A549 cells was performed after staining with propidium iodide and annexin V. [Figure 3J]Figures 3I, 3J, and 3K show that bacteria were cultured in medium containing 2% or 8% salt. Culture supernatants of a mixture of Staphylococcus spp. (n = 9), Staphylococcus nepalensis CNDG strain (n = 9), or culture medium (n = 9) were then prepared by centrifugation and diluted 1 / 10 and added to the culture medium of A549 alveolar epithelial cells. Flow cytometry of A549 cells was performed after staining with propidium iodide and annexin V. [Figure 3K] Figures 3I, 3J, and 3K show that bacteria were cultured in medium containing 2% or 8% salt. Culture supernatants of a mixture of Staphylococcus spp. (n = 9), Staphylococcus nepalensis CNDG strain (n = 9), or culture medium (n = 9) were then prepared by centrifugation and diluted 1 / 10 and added to the culture medium of A549 alveolar epithelial cells. Flow cytometry of A549 cells was performed after staining with propidium iodide and annexin V.

[0033] [Figure 4A] Figures 4A, 4B, and 4C show that bacterial culture supernatants were separated into <10 kDa and >10 kDa fractions by filtration, and each fraction was diluted 1 / 10 before being added to A549 alveolar epithelial cells, and apoptosis was determined by flow cytometry. [Figure 4B] Figures 4A, 4B, and 4C show that bacterial culture supernatants were separated into <10 kDa and >10 kDa fractions by filtration, and each fraction was diluted 1 / 10 before being added to A549 alveolar epithelial cells, and apoptosis was determined by flow cytometry. [Figure 4C] Figures 4A, 4B, and 4C show that bacterial culture supernatants were separated into <10 kDa and >10 kDa fractions by filtration, and each fraction was diluted 1 / 10 before being added to A549 alveolar epithelial cells, and apoptosis was determined by flow cytometry.

[0034] [Figure 5A-C]Figures 5A-5C show structural alignment analysis for corisin; Figures 5D and 5E show flow cytometry analysis of A549 alveolar epithelial cells after 48 hours of incubation in DMEM medium containing increasing concentrations of the pro-apoptotic peptide, demonstrating that synthetic corisin peptides dose-dependently inhibited the pro-apoptotic activity of supernatants from Staphylococcus aureus isolates; Figure 5F shows electron micrographs of A549 alveolar epithelial cells treated with saline or corisin, respectively. [Figure 5D] Figures 5A-5C show structural alignment analysis for corisin; Figures 5D and 5E show flow cytometry analysis of A549 alveolar epithelial cells after 48 hours of incubation in DMEM medium containing increasing concentrations of the pro-apoptotic peptide, demonstrating that synthetic corisin peptides dose-dependently inhibited the pro-apoptotic activity of supernatants from Staphylococcus aureus isolates; Figure 5F shows electron micrographs of A549 alveolar epithelial cells treated with saline or corisin, respectively. [Figure 5E-F] Figures 5A-5C show structural alignment analysis for corisin; Figures 5D and 5E show flow cytometry analysis of A549 alveolar epithelial cells after 48 hours of incubation in DMEM medium containing increasing concentrations of the pro-apoptotic peptide, demonstrating that synthetic corisin peptides dose-dependently inhibited the pro-apoptotic activity of supernatants from Staphylococcus aureus isolates; Figure 5F shows electron micrographs of A549 alveolar epithelial cells treated with saline or corisin, respectively.

[0035] [Figure 6A] FIG. 6A shows the schedule of treatment of mice with saline, scrambled peptide, or colisin.

[0036] [Figure 6B]Figure 6B shows bronchoalveolar lavage fluid cell counts for three saline-treated WT mice (WT / SAL), five saline-treated TGFβ1 TG mice (TGFβ1 TG / SAL), four scrambled peptide-treated TGFβ1 TG mice (TGFβ1 TG / Scrambled), and four colisin-treated TGFβ1 TG mice (TGFβ1 TG / corisin). Scale bar indicates 100 μm.

[0037] [Figure 6C-D] Figures 6C and 6D show quantification of collagen area by WinROOF software. Scale bar indicates 100 μm.

[0038] [Figure 6E] Figure 6E shows the concentrations of TGFβ1, monocyte chemotactic protein (MCP)-1, and type I collagen measured by enzyme immunoassay, where n = 3 for the WT / SAL group, n = 5 for the TGFβ1 TG / SAL and TGFβ1 TG / corisin groups, and n = 4 for the TGFβ1 TG / scrambled peptide group.

[0039] [Figure 6F-G] Figures 6F and 6G show DNA fragmentation assessed by terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) staining. Scale bars indicate 50 μm. n = 3 for the WT / SAL group, n = 5 for the TGFβ1 TG / SAL and TGFβ1 TG / corisin groups, and n = 4 for the TGFβ1 TG / scrambled peptide group.

[0040] [Figure 7A]Figures 7A and 7B show the number of cells in bronchoalveolar lavage fluid (BALF) counted 2 days after intratracheal instillation of saline or each bacterium and then stained with Giemsa. Scale bars indicate 100 μm. Figures 7A and 7B show DNA fragmentation assessed by staining with terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) and then quantified using Image WinROOF software. [Figure 7B-C] Figures 7A and 7B show the number of cells in bronchoalveolar lavage fluid (BALF) counted 2 days after intratracheal instillation of saline or each bacterium and then stained with Giemsa. Scale bars indicate 100 μm. Figures 7A and 7B show DNA fragmentation assessed by staining with terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) and then quantified using Image WinROOF software.

[0041] [Figure 7D] Figures 7A and 7B show the number of cells in bronchoalveolar lavage fluid (BALF) counted 2 days after intratracheal instillation of saline or each bacterium and then stained with Giemsa. Scale bars indicate 100 μm. Figures 7A and 7B show DNA fragmentation assessed by staining with terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) and then quantified using Image WinROOF software.

[0042] [Figure 8A-B] Figures 8A and 8B show photographs of Western blots of chorisin in lung tissue from four WT and four TGFβ1 TG mice, respectively, and the ratios of chorisin to β-actin. Quantitation was performed using ImageJ software.

[0043] [Figure 8C]FIG. 8C shows colisin levels measured using a competitive enzyme immunoassay for 8 healthy controls and 34 patients with stable idiopathic pulmonary fibrosis (IPF).

[0044] [Figure 8D] Figure 8D shows an analysis of bronchoalveolar lavage fluid levels of colisin before and after acute exacerbations in 14 IPF patients.

[0045] [Figure 9A] 9A and 9B show the criteria for scoring pulmonary radiological findings and the correlation of CT scores with Ashcroft fibrosis score and with lung hydroxyproline content. [Figure 9B] 9A and 9B show the criteria for scoring pulmonary radiological findings and the correlation of CT scores with Ashcroft fibrosis score and with lung hydroxyproline content.

[0046] [Figure 10] 10A-10D show the abnormal immune response in fibrotic lung tissue, showing the percentages of monocytes / macrophages, CD4Cd25 cells, T cells, and B cells in fibrotic lung tissue from mice treated in three different ways, respectively.

[0047] [Figure 11] FIG. 11 shows that sodium levels correlate with the number of immune cells in fibrotic lung tissue, as well as with the expression of fibrotic markers and sodium channels.

[0048] [Figure 12A] 12A-12D show that the pro-apoptotic factors in bacterial culture supernatants are heat stable. [Figure 12B] 12A-12D show that the pro-apoptotic factors in bacterial culture supernatants are heat stable. [Figure 12C]12A-12D show that the pro-apoptotic factors in bacterial culture supernatants are heat stable. [Figure 12D] 12A-12D show that the pro-apoptotic factors in bacterial culture supernatants are heat stable.

[0049] [Figure 13] FIG. 13 is a schematic diagram describing the sample fractionation steps and the biological activity of each fraction.

[0050] [Figure 14] FIG. 14 shows the pro-apoptotic activity of each of the fractions obtained by fractionation of bacterial supernatant from Staphylococcus nepalensis on A549 alveolar epithelial cells.

[0051] [Figure 15] FIG. 15 shows that apoptosis of lung epithelial cells was induced by the ethanol fraction, methanol fraction, and acetonitrile fraction of the culture supernatant of Staphylococcus nepalensis CNDG strain.

[0052] [Figure 16A] 16A, 16B and 16C show that the pro-apoptotic activity of fractions obtained from the supernatant of cultured Staphylococcus nepalensis strain CNDG is sensitive to proteinase K treatment. [Figure 16B] 16A, 16B and 16C show that the pro-apoptotic activity of fractions obtained from the supernatant of cultured Staphylococcus nepalensis strain CNDG is sensitive to proteinase K treatment. [Figure 16C] 16A, 16B and 16C show that the pro-apoptotic activity of fractions obtained from the supernatant of cultured Staphylococcus nepalensis strain CNDG is sensitive to proteinase K treatment.

[0053] [Figure 17] FIG. 17 is a photograph of silver staining of fractions that exhibited pro-apoptotic activity.

[0054] [Figure 18A] Figures 18A-18E show that synthetic corisin peptides prepared by different manufacturers induced dose-dependent apoptosis of alveolar epithelial cells, and the apoptotic activity of corisin was significantly more potent than that of an equivalent concentration of supernatant protein. [Figure 18B] Figures 18A-18E show that synthetic corisin peptides prepared by different manufacturers induced dose-dependent apoptosis of alveolar epithelial cells, and the apoptotic activity of corisin was significantly more potent than that of an equivalent concentration of supernatant protein. [Figure 18C] Figures 18A-18E show that synthetic corisin peptides prepared by different manufacturers induced dose-dependent apoptosis of alveolar epithelial cells, and the apoptotic activity of corisin was significantly more potent than that of an equivalent concentration of supernatant protein. [Figure 18D] Figures 18A-18E show that synthetic corisin peptides prepared by different manufacturers induced dose-dependent apoptosis of alveolar epithelial cells, and the apoptotic activity of corisin was significantly more potent than that of an equivalent concentration of supernatant protein. [Figure 18E] Figures 18A-18E show that synthetic corisin peptides prepared by different manufacturers induced dose-dependent apoptosis of alveolar epithelial cells, and the apoptotic activity of corisin was significantly more potent than that of an equivalent concentration of supernatant protein.

[0055] [Figure 19A] Figures 19A to 19E show that the pro-apoptotic peptide (corisin) induced apoptosis in normal human bronchial epithelial cells, but the scrambled sequence of the pro-apoptotic peptide (corisin) did not induce apoptosis in normal human bronchial epithelial cells. [Figure 19B-C]Figures 19A to 19E show that the pro-apoptotic peptide (corisin) induced apoptosis in normal human bronchial epithelial cells, but the scrambled sequence of the pro-apoptotic peptide (corisin) did not induce apoptosis in normal human bronchial epithelial cells. [Figure 19D-E] Figures 19A to 19E show that the pro-apoptotic peptide (corisin) induced apoptosis in normal human bronchial epithelial cells, but the scrambled sequence of the pro-apoptotic peptide (corisin) did not induce apoptosis in normal human bronchial epithelial cells.

[0056] [Figure 20A] 20A-20E show that a synthetic pro-apoptotic peptide (corisin) is heat stable. [Figure 20B] 20A-20E show that a synthetic pro-apoptotic peptide (corisin) is heat stable. [Figure 20C] 20A-20E show that a synthetic pro-apoptotic peptide (corisin) is heat stable. [Figure 20D] 20A-20E show that a synthetic pro-apoptotic peptide (corisin) is heat stable. [Figure 20E] 20A-20E show that a synthetic pro-apoptotic peptide (corisin) is heat stable.

[0057] [Figure 21A] 21A to 21F show that the apoptotic peptide (corisin) does not induce apoptosis in fibroblasts, vascular endothelial cells, or T cells. [Figure 21B] 21A to 21F show that the apoptotic peptide (corisin) does not induce apoptosis in fibroblasts, vascular endothelial cells, or T cells. [Figure 21C]21A to 21F show that the apoptotic peptide (corisin) does not induce apoptosis in fibroblasts, vascular endothelial cells, or T cells. [Figure 21D] 21A to 21F show that the apoptotic peptide (corisin) does not induce apoptosis in fibroblasts, vascular endothelial cells, or T cells. [Figure 21E] 21A to 21F show that the apoptotic peptide (corisin) does not induce apoptosis in fibroblasts, vascular endothelial cells, or T cells. [Figure 21F] 21A to 21F show that the apoptotic peptide (corisin) does not induce apoptosis in fibroblasts, vascular endothelial cells, or T cells.

[0058] [Figure 22] 22A and 22B show bands at the molecular weight corresponding to that of colisin observed in Western blotting of mouse lung tissue samples and culture supernatants of Staphylococcus nepalensis, respectively, using colisin antibodies.

[0059] [Figure 23A] 23A-23D show that antibodies against colisin inhibit the pro-apoptotic activity of both colisin and the supernatant of Staphylococcus nepalensis strain CNDG. [Figure 23B] 23A-23D show that antibodies against colisin inhibit the pro-apoptotic activity of both colisin and the supernatant of Staphylococcus nepalensis strain CNDG. [Figure 23C] 23A-23D show that antibodies against colisin inhibit the pro-apoptotic activity of both colisin and the supernatant of Staphylococcus nepalensis strain CNDG. [Figure 23D] 23A-23D show that antibodies against colisin inhibit the pro-apoptotic activity of both colisin and the supernatant of Staphylococcus nepalensis strain CNDG.

[0060] [Figure 24A] 24A-24E show that the full-length transglycosylase 351 containing the colisin sequence does not have apoptotic activity. [Figure 24B-C] 24A-24E show that the full-length transglycosylase 351 containing the colisin sequence does not have apoptotic activity. [Figure 24D] 24A-24E show that the full-length transglycosylase 351 containing the colisin sequence does not have apoptotic activity. [Figure 24E] 24A-24E show that the full-length transglycosylase 351 containing the colisin sequence does not have apoptotic activity.

[0061] [Figure 25A] Figures 25A and 25B show CT images and findings, respectively, for mice used for intratracheal instillation of colisin, scrambled peptide, or saline. [Figure 25B] Figures 25A and 25B show CT images and findings, respectively, for mice used for intratracheal instillation of colisin, scrambled peptide, or saline.

[0062] [Figure 26A] Figures 26A and 26B show CT images and findings, respectively, for mice used for intratracheal instillation of Staphylococcus nepalensis, Staphylococcus epidermidis, or saline. [Figure 26B]Figures 26A and 26B show CT images and findings, respectively, for mice used for intratracheal instillation of Staphylococcus nepalensis, Staphylococcus epidermidis, or saline.

[0063] [Figure 27A] Figures 27A and 27B show that a synthetic peptide (corisin) containing the sequence of the transglycosylase segment derived from Staphylococcus nepalensis CNDG strain induced apoptosis of alveolar epithelial cells, but neither a scrambled peptide of corisin nor a synthetic peptide containing the sequence of the transglycosylase segment derived from Staphylococcus epidermidis induced apoptosis of alveolar epithelial cells. [Figure 27B] Figures 27A and 27B show that a synthetic peptide (corisin) containing the sequence of the transglycosylase segment derived from Staphylococcus nepalensis CNDG strain induced apoptosis of alveolar epithelial cells, but neither a scrambled peptide of corisin nor a synthetic peptide containing the sequence of the transglycosylase segment derived from Staphylococcus epidermidis induced apoptosis of alveolar epithelial cells.

[0064] [Figure 28A] Figures 28A and 28B show worsening radiological findings after intratracheal instillation of Staphylococcus nepalensis in germ-free TGFβ1 TG mice. [Figure 28B] Figures 28A and 28B show worsening radiological findings after intratracheal instillation of Staphylococcus nepalensis in germ-free TGFβ1 TG mice.

[0065] [Figure 29A]Figures 29A-29D show phylogenetic analysis of Staphylococcus nepalensis strain CNDG transglycosylases and their relatives within the genus Staphylococcus. [Figure 29B] Figures 29A-29D show phylogenetic analysis of Staphylococcus nepalensis strain CNDG transglycosylases and their relatives within the genus Staphylococcus. [Figure 29C] Figures 29A-29D show phylogenetic analysis of Staphylococcus nepalensis strain CNDG transglycosylases and their relatives within the genus Staphylococcus. [Figure 29D] Figures 29A-29D show phylogenetic analysis of Staphylococcus nepalensis strain CNDG transglycosylases and their relatives within the genus Staphylococcus.

[0066] [Figure 30A] Figures 30A, 30B, and 30C show multiple sequence alignments of conserved sequences of pro-apoptotic segments of transglycosylases in several species of Staphylococcus and Streptococcus. Corisins shown in Figures 30A-30C include, for example: [ka] and can be used in one or more embodiments of the present teachings. [Figure 30B] Same as above. [Figure 30C] Same as above.

[0067] [Figure 31A]Figures 31A-31F show the genomic context and multiple sequence alignments for the conserved sequences of the pro-apoptotic segments of transglycosylases in several species of Staphylococcus and Streptococcus. More specifically, Figure 31A shows the genomic context of transglycosylases containing the peptide IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1) or its derivatives in Staphylococcus nepalensis strain SNUC 4025 and Staphylococcus cohnii subsp. cohnii. Figure 31B shows that Streptococcus pneumoniae contains transglycosylases (COE35810 and COE67256) with peptide sequences nearly identical to colisin. Figure 31C shows the query and subject sequences from S. pneumoniae strain N and S. warneri, respectively, in an alignment (complementary nucleotide sequence encoding COE67256 and highly identical proteins in Staphylococcus warneri strains SWO, SGI, NCTC 11044, NCTC 7291, and 22.1). Figure 31D shows the genomic context of transglycosylases containing the colisin sequence or its derivatives in Streptococcus pneumoniae strain N and Staphylococcus warneri. Figure 31E shows that the genome of a strain of the emerging pathogen Mycobacterium abscessus has a transglycosylase (SKT99287) that is nearly identical to the transglycosylase of Staphylococcus hominis (WP_049379270). FIG. 31F shows the genomic context of transglycosylases containing colisin sequences or derivatives thereof in Mycobacterium [Mycobacteroides] abscessus and Staphylococcus hominis. [Figure 31B] Same as above. [Figure 31C] Same as above. [Figure 31D] Same as above. [Figure 31E] Same as above. [Figure 31F] Same as above.

[0068] [Figure 32A] Figures 32A and 32B show that synthetic peptides derived from Streptococcus pneumoniae strain N transglycosylase have pro-apoptotic activity. [Figure 32B] Figures 32A and 32B show that synthetic peptides derived from Streptococcus pneumoniae strain N transglycosylase have pro-apoptotic activity.

[0069] [Figure 33] FIG. 33 is a model of fibrotic tissue developed based on the work disclosed herein, and in particular the contribution of colisin to the pathogenesis of idiopathic pulmonary fibrosis (IPF).

[0070] [Figure 34A] Figures 34A-34C show the flow cytometry gating strategy used for the experiments shown in Figure 12A (Figure 34A), Figure 19A (Figure 34B), and Figure 20A (Figure 34C), where SSC means side scatter and FSC means forward scatter. [Figure 34B] Figures 34A-34C show the flow cytometry gating strategy used for the experiments shown in Figure 12A (Figure 34A), Figure 19A (Figure 34B), and Figure 20A (Figure 34C), where SSC means side scatter and FSC means forward scatter. [Figure 34C] Figures 34A-34C show the flow cytometry gating strategy used for the experiments shown in Figure 12A (Figure 34A), Figure 19A (Figure 34B), and Figure 20A (Figure 34C), where SSC means side scatter and FSC means forward scatter. DETAILED DESCRIPTION OF THE INVENTION

[0071] Detailed Description of the Invention In another aspect of the present teachings, a method for evaluating or diagnosing a subject having fibrosis or suspected of having or developing fibrosis can include receiving an in vitro biological sample, e.g., collected, recovered, or obtained from the subject; and detecting the amount of chorisin present in the biological sample. Such a method can further include comparing the amount of chorisin detected in the biological sample with one or more predetermined thresholds. The predetermined thresholds can be set, for example, based on levels of chorisin typically (usually) present in healthy individuals.

[0072] The biological sample may be taken from one or both lungs of the subject.

[0073] The biological sample can be, for example, sputum, bronchial secretions, pleural effusion, bronchoalveolar lavage fluid (BALF), and tissue taken from the bronchi or lungs.

[0074] The biological sample can be blood or bronchoalveolar lavage fluid (BALF).

[0075] In any of these methods, detection of one of the amino acid sequences SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13 preferably serves as detection of colisin.

[0076] In any of these methods, the patient may have, or be suspected of having or developing, idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and / or breast fibrosis. In particular, the method is advantageous for use in patients with idiopathic pulmonary fibrosis (IPF).

[0077] Corisin can be detected by mass spectrometry, Western blotting, or enzyme-linked immunosorbent assay (ELISA, e.g., by detecting corisin bound to an antibody that recognizes, e.g., one of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13, e.g., by binding a labeled antibody to corisin bound to an antibody bound to a substrate, e.g., a substrate.) Kits for carrying out such methods can include such antibodies and one or more reagents for effecting detection of corisin in a biological sample.

[0078] In another aspect of the present teachings, a pharmaceutical composition for use in treating fibrosis in a patient is disclosed, the pharmaceutical composition preferably comprising a corisin inhibitor capable of neutralizing and / or reducing the amount of corisin in the patient's lungs.

[0079] Corisin inhibitors can be, for example, small molecules, antagonists of corisin, or antibodies to corisin. Corisin inhibitors can act, for example, by binding to corisin, by degrading corisin, or by blocking or inhibiting the production of corisin.

[0080] Corisin inhibitors can be used to treat patients who have or are suspected of having or developing idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and / or breast fibrosis, particularly idiopathic pulmonary fibrosis (IPF).

[0081] In another aspect of the present teachings, a method for identifying a chorisin receptor protein can include searching for chorisin-binding proteins present on the surface of epithelial cells.

[0082] In another aspect of the present teachings, a method for identifying a corisin receptor protein may include searching for one of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13 in binding proteins present on the surface of epithelial cells.

[0083] The results of this research that led to the present teachings, as well as a discussion of the particular methods used in this research, are now presented below. result Fibrotic lung tissue has a salt-rich microenvironment

[0084] TGFβ1 (transforming growth factor-1) is thought to be the most important mediator of IPF. Therefore, in the experiments described in more detail below, we used transgenic (TG) mice with pulmonary fibrosis induced by pulmonary overexpression of human TGFβ1, as previously reported in NPL8, NPL10, NPL11, and NPL12. Similar to IPF disease in humans, these TGFβ1 TG mice spontaneously develop pulmonary fibrosis characterized by a predominant and progressive scarring process, fatal outcome, and typical lung histopathological findings (diffuse collagen deposition, honeycomb cysts, and fibroblastic foci). See NPL8 and NPL11. As a control, we used a TGFβ1 TG mouse strain that expresses a human transgene but not the protein, and does not have fibrosis. See NPL8 and NPL13.

[0085] To investigate the hypothesis that pulmonary fibrosis is a salt-rich microenvironment, we assigned TGFβ1 TG mice and wild-type (WT) mice to groups according to computed tomography-based fibrosis scores (see Figures 9A and 9B). We then analyzed the Na+ levels in pulmonary fibrotic tissue from TGFβ1 TG mice with pulmonary fibrosis. + The content was measured (see NPL8).

[0086] More specifically, Figure 9A shows computed tomography (CT) images obtained according to the following method. The criteria for scoring CT findings were as follows: score 1: normal findings; score 2: intermediate; score 3: mild fibrosis; score 4: intermediate; score 5: moderate fibrosis; score 6: intermediate; and score 7: severe fibrosis. The CT score for each mouse was calculated by averaging the scores from six pulmonologists.

[0087] Figure 9B shows the Ashcroft fibrosis score and hydroxyproline content measured according to the following methods. Ten-week-old male mice weighing 20–25 g were used in the experiment. N = 23 mice. CT score significantly correlated with Ashcroft score (r = 0.78; p < 0.0001) and lung hydroxyproline content (r = 0.84; p < 0.0001). Statistical analysis was performed according to Pearson's product-moment correlation.

[0088] As a result of these experiments, we found significantly higher concentrations of Na in the lung tissue of TGFβ1 TG mice with pulmonary fibrosis compared with TG mice without pulmonary fibrosis and WT mice. + These findings demonstrated that pulmonary fibrosis is a microenvironment rich in salt. Abnormal immune responses in pulmonary fibrosis

[0089] We isolated lung immune cells from WT mice without fibrosis, TGFβ1 TG mice without pulmonary fibrosis, and TGFβ1 TG mice with fibrosis, and compared the percentages of cells between groups. We found that monocytes / macrophages and regulatory cells (CD4) were significantly higher in TGFβ1 TG mice with pulmonary fibrosis than in WT mice and TGFβ1 TG mice without pulmonary fibrosis. + CD25 +We found that the percentage of T cells was significantly increased in TGFβ1 TG mice with pulmonary fibrosis compared with WT mice and TGFβ1 TG mice without pulmonary fibrosis (see Figures 10A and 10B and Table 1 below). The percentage of total T cells did not differ between groups, but the percentage of B cells was significantly decreased in TGFβ1 TG mice with pulmonary fibrosis compared with WT mice and TGFβ1 TG mice without pulmonary fibrosis (see Figures 10C and 10D). These findings provide evidence that the immune response is impaired in fibrotic lung tissue.

[0090] More specifically, Figures 10A-10D show the percentages of monocytes / macrophages, CD4+ / CD25+ cells, T cells, and B cells in pulmonary fibrotic tissue from wild-type (WT) mice (n = 4) and TGFβ1 transgenic (TG) mice with and without fibrosis (n = 4), respectively, as enumerated by flow cytometry using specific antibodies, as further described in the methods below. Bars indicate mean ± SD. Statistical analysis was performed using ANOVA with Tukey's test. * p<0.05, ** p<0.01.

[0091] [Table 1] Sodium, immune cells, fibrotic markers, and sodium channels

[0092] The relative mRNA expression in lung tissue of fibrotic markers (connective tissue growth factor, fibronectin 1, type I collagen) and profibrotic cytokines (TGFβ1, tumor necrosis factor α, interferon γ), chemokines (monocyte chemoattractant protein-1), vascular endothelial growth factor, or inducible nitric oxide synthase was significantly increased in TGFβ1 TG mice with pulmonary fibrosis compared with WT mice and TGFβ1 TG mice without fibrosis (see Table 2 below).

[0093] However, the relative mRNA expression of chloride channels (cystic fibrosis transmembrane conductance regulator) and sodium channels (Scnnγ, Scnnβ) in lung tissue was significantly reduced in TGFβ1 TG mice with pulmonary fibrosis compared with WT mice and TGFβ1 TG mice without pulmonary fibrosis (see Table 2 below). Therefore, we evaluated the correlations between variables in all WT mice and all TGFβ1 TG mice with and without fibrosis.

[0094] [Table 2]

[0095] As a result, we found that tissue sodium levels were significantly inversely correlated with chloride channel and sodium channel mRNA expression and with the number of B cells. In contrast, tissue sodium levels were significantly proportionally correlated with fibrotic markers, profibrotic cytokines, and the number of monocytes / macrophages and regulatory T cells (see Figure 11).

[0096] More specifically, the concentrations of sodium, expression of fibrotic factors, profibrotic cytokines, chemokines, angiogenic factors, and percentage of immune cells in lung tissue were evaluated in lung tissue from wild-type mice (n=4) and TGFβ1 TG mice with and without pulmonary fibrosis (n=4). Spearman correlation r values ​​are shown in Figure 11. Ctfr, cystic fibrosis transmembrane conductance regulator; Scnn1α, sodium channel epithelial 1 α subunit; Scnn1β, sodium channel epithelial 1 β subunit; Scnn1γ, sodium channel epithelial 1 γ subunit; TNFα, tumor necrosis factor α; IFNγ, interferon gamma; Ctgf, connective tissue growth factor; mTGFβ1, mouse transforming growth factor β1; Vegf, vascular endothelial growth factor; iNOS, inducible nitric oxide synthase; Mcp-1, monocyte chemoattractant protein-1; αSMA, α-smooth muscle actin; Fn1, fibronectin 1; Col1α1, collagen 1 α1. Statistical analysis was performed by Spearman correlation. * p<0.05.

[0097] These findings provide evidence for an unfavorable role of a salt-rich microenvironment in the process of tissue fibrosis and for the intimate involvement of tissue sodium levels in regulating immune responses. See also NPL14. Growth of bacteria from fibrotic lung tissue

[0098] After confirming that fibrotic tissues are salt-rich microenvironments, we hypothesized that a high-salt culture medium would best mimic the in vivo fibrotic tissue condition and therefore favor the growth of microorganisms implicated in disease pathogenesis.

[0099] Therefore, we incubated lung fibrosis tissue specimens from TGFβ1 TG mice and WT mice in medium containing 8% NaCl for 48 hours (see Figures 2A and 2B). Bacterial growth was detected in the medium inoculated with lung fibrosis specimens from TGFβ1 TG mice, but not in the medium inoculated with lung fibrosis specimens from WT mice. We then performed streaking to isolate bacterial colonies and observed bacterial morphology compatible with Staphylococcus spp. using phase-contrast microscopy (see Figure 2C). The identity of the bacterial strains was confirmed by sequencing their 16S rRNA genes amplified by polymerase chain reaction.

[0100] However, whole genome sequencing revealed that one of the colonies (strain 8) corresponded to a strain of Staphylococcus nepalensis, while another colony (strain 6) was a mixture of Staphylococcus spp. The whole genome sequences of the cultures designated strain 6 and strain 8 have been deposited in the Genbank database under accession number PRJNA544423.

[0101] To further confirm the identity of strain 8, we compared its whole genome sequence with that of other Staphylococcus nepalensis strains in the GenBank database and found identities of 99.52%, 99.61%, 99.60%, 99.53%, and 99.50% to strains JS9, SNUC4337, DSM15150, JS11, and JS1, respectively. Therefore, based on the purity of strain 8 and its very high genome homology with other Staphylococcus nepalensis strains, we assigned strain 8 to Staphylococcus nepalensis, with the strain name CNDG.

[0102] Apoptosis of lung cells induced by culture supernatant

[0103] To assess the potential relevance of these bacterial isolates from fibrotic tissues in disease pathogenesis, we cultured normal human bronchial epithelial (NHBE) cells and A549 alveolar epithelial cells in the presence of bacterial culture supernatants and assessed cell survival. Cells cultured in the presence of supernatants from Staphylococcus nepalensis CNDG and mixed bacteria showed significant levels of apoptosis, caspase-3 activation, and DNA fragmentation compared with cells cultured in control medium (Figures 2D-2G). Culture supernatant with the highest apoptotic activity

[0104] Culture supernatants of mixed Staphylococcus spp. (strain 6; see Figures 3A-3D) and Staphylococcus nepalensis CNDG (strain 8; see Figures 3E-3H) were separated into several fractions using a Sephadex column, and the peaks of protein concentration corresponded well with the lowest values ​​of cell viability in the MTT assay and with the sub-G1 fraction peaks in the cell cycle analysis. Apoptosis depends on the salt concentration of the bacterial medium

[0105] Staphylococcus nepalensis CNDG and mixed Staphylococcus spp. were cultured in media containing 0%, 2%, or 8% NaCl, and apoptosis was assessed by flow cytometry using culture supernatants. We found that apoptotic activity significantly depended on the salt concentration of the media used to culture both isolates in vitro (see Figures 3I, 3J, and 3K). Apoptotic factors are heat-stable, low-molecular-weight peptides

[0106] The bacterial culture supernatants were incubated at 85°C for 15 min and then diluted 1 / 10 to assess their proapoptotic activity against A549 alveolar epithelial cells. The apoptotic activity of both the culture supernatants of Staphylococcus nepalensis CNDG and the culture supernatants of mixed Staphylococcus spp. remained stable after heating, and the activity was significantly stronger than that of the unheated culture supernatants (see Figures 12A–12D). To gain insight into the identity of the proapoptotic factors, we fractionated the bacterial supernatant proteins into low-molecular-weight proteins (<10 kDa) and high-molecular-weight proteins (>10 kDa) and repeated this experiment. We found that the fractions containing low-molecular-weight proteins had stronger and significantly stronger apoptotic activity than the fractions containing high-molecular-weight proteins (Figures 4A, 4B, 4C, 12A, and 12B).

[0107] More specifically, Figures 12A and 12B show flow cytometry of A549 cells after staining with propidium iodide and Annexin V. n = 3 per group. Bars indicate mean ± SD. Statistical analysis was performed by ANOVA with Newman-Keuls test. * p<0.001 vs. medium; †p<0.05 vs. unheated supernatant from Staphylococcus nepalensis (strain CNDG) or strain 6.

[0108] Furthermore, Figures 12C and 12D show the activation of caspase-3 by culture supernatants after stimulation of A549 alveolar epithelial cells in the presence of medium or a mixture of Staphylococcus spp. or the supernatant of Staphylococcus nepalensis CNDG strain, as assessed by Western blotting. n = 3 per group. Bars indicate mean ± SD. Statistical analysis was performed by ANOVA with the Newman-Keuls test. * p<0.05 vs. medium.

[0109] These findings provide evidence that the apoptosis-inducing factor is a low-molecular-weight protein and that this soluble factor, released by bacteria enriched in fibrotic tissue, contributes to the mechanism of pulmonary fibrosis by determining the fate of lung epithelial cells. Identification of pro-apoptotic peptides

[0110] Next, we proceeded to purify the soluble pro-apoptotic factor from the culture supernatant of Staphylococcus nepalensis strain CNDG. Sequential extraction of the supernatant proteins into n-hexane, water, ethyl acetate, and ethanol was performed, followed by fractionation using octadecyl-silane gel flash column chromatography and Sep-Pak followed by high-performance liquid chromatography (HPLC) (Figure 13), to isolate the biologically active protein (see Figures 14 and 15). After treatment of the sample with proteinase K, biological activity was significantly reduced (see Figures 16A, 16B, and 16C). Silver staining of the sample after gel electrophoresis revealed a protein / peptide with an apparent molecular weight of 2 kDa (see Figure 17).

[0111] More specifically, fractionation of the culture supernatant was performed as described in the method below. The pro-apoptotic activity of the fractions on A549 alveolar epithelial cells was evaluated by flow cytometry and shown in Figure 13 as bioactivity (+) or no bioactivity (-). Figure 14 shows the pro-apoptotic activity of each fraction on A549 alveolar epithelial cells. Figure 15 shows the pro-apoptotic activity of each fraction on A549 alveolar epithelial cells cultured in the presence of each fraction for 48 hours. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, where DAPI is the abbreviation for 4',6-diamidino-2-phenylindole. Representative photomicrographs from two experiments are shown. The scale bar indicates 100 μm.

[0112] Staphylococcus nepalensis culture supernatants and their ethanol, methanol, or acetonitrile fractions were then incubated at 37°C in the presence of 200 μg / ml proteinase K (PK) and then diluted 1 / 10 and added to the culture medium of A549 alveolar epithelial cells. Each group consisted of three cells. Figures 16A, 16B, and 16C show the results of flow cytometry of A549 alveolar epithelial cells after staining with propidium iodide and annexin V. Bars indicate the mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test. * p<0.01. PK is the abbreviation for proteinase K.

[0113] Five micrograms of the biologically active HPLC fraction (fraction 3) was then loaded onto a 15% sodium dodecyl sulfate polyacrylamide gel and silver stained using a commercially available kit. A representative photomicrograph from three experiments with similar results is shown in Figure 17.

[0114] The peptides were then analyzed by mass spectrometry, and the raw data were compared against a custom database of Staphylococcus nepalensis CNDG strain protein sequences based on its unpublished genome sequence data (GenBank accession number PRJNA544423). Consistent with the purified biological activity in the culture supernatant, mass spectrometry analysis identified a 19-amino acid residue peptide (IVMPESSGNPNAVNPAGYR-SEQ ID NO: 1) corresponding to a molecular mass of 1.94 kDa. The inventors named this newly discovered peptide "corisin." A homology search revealed that the colisin sequence corresponds to a segment of transglycosylase IsaA (MW: 25.6 kDa) from Staphylococcus nepalensis CNDG strain. Structural prediction and apoptotic activity of colisin

[0115] Structural alignment using the homology modeling server (swissmodel.expasy.org) showed that corisin shares 46.88% identity with a segment of endo-membrane-bound lytic murein transglycosylase A (see Figures 5A-5C). Therefore, we commissioned two different commercial manufacturers (Peptide Institute, Inc., Osaka, Japan, and ThermoFisher Scientific, Waltham, MA, USA) to prepare synthetic corisin peptides (i.e., with the predicted amino acid sequence) for us. We then used each of these synthetic corisin peptides to treat A549 alveolar epithelial cells.

[0116] Both synthetic corisin peptides reproduced the proapoptotic effects of staphylococcal isolate supernatants in A549 lung epithelial cells in a dose-dependent manner (see Figures 5D, 5E, 18A, and 18B). The apoptotic activity of synthetic corisin was significantly stronger than that of supernatants from Staphylococcus nepalensis strain CNDG and mixed Staphylococcus spp. (strain 6) at equivalent protein concentrations (see Figures 18C–18E).

[0117] More specifically, Figures 18A and 18B show flow cytometry analysis of A549 alveolar epithelial cells after 48 hours of culture in DMEM medium containing various concentrations of colisin. n = 3 per group. Bars indicate mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test. ** p<0.001 vs. control (0 μg / ml); †p<0.001 vs. 0.5 μg / ml colisin.

[0118] Figures 18C-18E show flow cytometry analysis of A549 alveolar epithelial cells after 48 hours of culture in DMEM medium containing various concentrations of colisin (5 μg / ml or 10 μg / ml), supernatant proteins from mixed Staphylococcus spp. or strain 6 (10 μg / ml or 100 μg / ml), or supernatant proteins from Staphylococcus nepalensis CNDG strain or strain 8 (10 μg / ml or 100 μg / ml). n = 3 for each group. Overlapping bars indicate mean ± SD. Statistical analysis was performed by ANOVA and Tukey's test. ‡ p < 0.05 vs. saline or scrambled peptide; § p < 0.001 vs. supernatant proteins from mixed Staphylococcus spp. or Staphylococcus nepalensis (10 μg / ml and 100 μg / ml).

[0119] Normal human bronchial epithelial cells also showed significantly enhanced apoptosis in the presence of colisin, but not in the presence of a synthetic peptide composed of a scrambled amino acid sequence (see Figures 19A-19B), which was associated with increased cleavage of caspase-3 and reduced Akt activation (see Figures 19C-19E).

[0120] More specifically, Figures 19A-19B show flow cytometry analysis of normal human bronchial epithelial (NHBE) cells after 48 hours of culture in DMEM medium containing 10 μM colisin or its scrambled sequence. n=4 for each treatment group. Bars indicate mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test. * p<0.001.

[0121] Figure 19C shows Western blotting of lysates from NHBE cells treated with colisin or scrambled peptide. Each treatment group had n=4. A representative blot from each treatment group is shown.

[0122] Figures 19D and 19E show the intensity of Western blot membrane bands quantified by densitometry using ImageJ software. n = 4 per treatment group. Bars indicate mean ± SD. Statistical analysis was performed by one-tailed Mann-Whitney U test. * p<0.05.

[0123] In additional experiments using A549 alveolar epithelial cells, the proapoptotic activity of synthetic corisin was found to be heat-resistant, as observed in the culture supernatant (see Figures 20A-20B), and transmission electron micrographs confirmed the apoptotic properties of corisin (see Figure 5F). However, corisin did not exhibit apoptotic activity against lung fibroblasts, vascular endothelial cells, or lymphocyte cell lines (see Figures 21A-21F).

[0124] More specifically, synthetic corisin (5 μM; Peptide Institute, Inc.) or scrambled peptide (5 μM; Peptide Institute, Inc.) was incubated at 85°C for 15 minutes and then added to the culture medium of A549 alveolar epithelial cells for 48 hours. Figures 20A-20B show flow cytometry analysis of A549 alveolar epithelial cells after staining with propidium iodide and annexin V. Each treatment group contained three samples. Bars indicate the mean ± SD. Statistical analysis was performed by ANOVA with the Newman-Keuls test. * p<0.001 versus unheated or heated scrambled peptide.

[0125] Figure 20C shows separate experiments in which synthetic chorisin (5 μM) or scrambled peptide was incubated at 85°C for 15 minutes, then added to the culture medium of A549 alveolar epithelial cells for 48 hours. Cells were harvested and prepared for Western blotting of cleaved caspase-3, β-actin, total Akt, and phosphorylated Akt (p-Akt). n=3 per treatment group. Representative blots from each treatment group are shown.

[0126] Figures 20D and 20E show the intensity of Western blot membrane bands quantified by densitometry using ImageJ software. n = 3 for each treatment group. Bars indicate mean ± SD. Statistical analysis was performed by ANOVA with Newman-Keuls test. * p<0.01 vs. saline.

[0127] 21A and 21B show flow cytometry analysis of HFL1 lung fibroblasts after culturing them in DMEM medium containing 10 μg / ml of colisin for 48 hours (n=4 for each).

[0128] Figures 21C and 21D show flow cytometry analysis of human umbilical vein endothelial cells after culturing them in DMEM medium containing 10 μg / ml of colisin for 48 hours (n=4 for each group).

[0129] Figures 21E and 21F show flow cytometry analysis of human Jurkat T cells after 48 hours of culture in DMEM medium containing 10 μg / ml of colisin. n = 4 for each treatment group. Bars indicate mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test. Anti-corisin antibody inhibits corisin-induced apoptosis

[0130] We then developed a polyclonal antibody against colisin using the methods described further below, which was able to detect colisin in mouse lung tissue and in culture supernatants of Staphylococcus nepalensis (see Figures 22A-22B).

[0131] More specifically, 5 micrograms of lung tissue homogenate prepared from WT and TGFβ1 TG mice (Figure 22A) and several volumes of Staphylococcus nepalensis culture supernatant concentrated by precipitation with trichloroacetic acid (Figure 22B) were loaded onto a 5-15% gradient sodium dodecyl sulfate polyacrylamide gel, followed by Western blotting using an anti-corisin antibody. Representative micrographs from two experiments with similar results are shown in Figures 22A and 22B. Synthetic corisin was used as a control. MW is the abbreviation for molecular weight in kDa. The arrow indicates the corisin band.

[0132] We then stimulated A549 alveolar epithelial cells with colisin or culture supernatant of Staphylococcus nepalensis CNDG strain in the presence of saline, control rabbit IgG, or rabbit anti-corisin IgG, and assessed apoptotic cells by flow cytometry. We found that the presence of polyclonal anti-corisin antibody significantly inhibited synthetic colisin-induced lung epithelial cell apoptosis (Figures 23A-23B) and Staphylococcus nepalensis culture supernatant-induced apoptosis (Figures 23C-23D) compared with control IgG.

[0133] More specifically, A549 alveolar epithelial cells (2 × 10 cells) 5Cells (cells / well) were cultured in 12-well plates and stimulated with 5 μM colisin for 48 hours in the presence of saline (saline / corisin), 10 μg / ml control rabbit IgG (control IgG / corisin), or 10 μg / ml rabbit anti-corisin IgG (anti-corisin IgG / corisin). Cells cultured in the presence of saline and treated with saline (saline / saline), control rabbit IgG (control IgG / saline), or rabbit anti-corisin IgG (anti-corisin IgG / saline) served as controls. n = 3 for each treatment group (triplicates). Results are shown in Figures 23A and 23B. Bars indicate mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test (by by). * p<0.001.

[0134] Furthermore, A549 alveolar epithelial cells cultured in 12-well plates were stimulated with a 1 / 10 dilution of Staphylococcus nepalensis CNDG culture supernatant for 48 hours in the presence of saline (saline / Staphylococcus nepalensis CNDG culture supernatant), 10 μg / ml control rabbit IgG (control IgG / Staphylococcus nepalensis CNDG culture supernatant), or 10 μg / ml rabbit anti-corisin IgG (anti-corisin IgG / Staphylococcus nepalensis CNDG culture supernatant). Cells cultured in medium and treated with saline (saline / medium), control rabbit IgG (control IgG / medium), or rabbit anti-corisin IgG (anti-corisin IgG / medium) served as controls. Each treatment group consisted of three samples. Flow cytometry of A549 cells was performed after staining with propidium iodide and Annexin V. The results are shown in Figures 23C and 23D. Again, bars indicate the mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test. * p<0.001. Full-length transglycosylase does not have apoptotic activity

[0135] We prepared recombinant full-length transglycosylase 351 with a 6-histidine tag (His tag) or without the tag (His tag truncated), expressed it in E. coli cells, and evaluated its apoptotic activity against A549 cells. Unheated or heated recombinant His-tagged transglycosylase 351 (see Figures 24A-24B) and untagged recombinant transglycosylase 351 (Figures 24C-24E) were unable to induce apoptosis in lung epithelial cells, providing evidence that polypeptide processing and colisin release are required for biological activity.

[0136] More specifically, Figures 24A and 24B show flow cytometry analysis of A549 alveolar epithelial cells after 48 hours of culture in DMEM medium containing 10 μg / ml of colisin, unheated, or heated His-tagged recombinant transglycosylase. n=3 for each treatment group. Bars indicate mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test. * p<0.001.

[0137] Figure 24C shows the results of gel electrophoresis and silver staining of His-tagged recombinant transglycosylase 351 from Staphylococcus nepalensis strain CNDG, treated with or without thrombin, using a sodium dodecyl sulfate polyacrylamide gel (10-20%). A representative photomicrograph from two experiments with similar results is shown.

[0138] Figures 24D and 24E show flow cytometry analysis of A549 alveolar epithelial cells after culturing them in DMEM medium containing 10 μg / ml of colisin, His-tagged recombinant transglycosylase, or untagged recombinant transglycosylase for 48 hours. n = 3 for each treatment group. Bars indicate mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test. * p<0.001. Corisin exacerbates pulmonary fibrosis in hTGF-β1 TG mice

[0139] To investigate whether colisin can exacerbate pulmonary fibrotic disease in vivo, we divided TGFβ1 TG mice into three groups matched for the level of pulmonary fibrosis (see Figures 25A and 25B) and treated them with saline, scrambled peptide, or colisin by the intratracheal route once daily for 2 days, after which they were euthanized on day 3 (see Figure 6A).

[0140] TGFβ1 TG mice receiving corisin showed significantly increased infiltration of macrophages, lymphocytes, and neutrophils, increased collagen deposition and levels of inflammatory cytokines and chemokines, and enhanced epithelial cell apoptosis in the lungs compared with control mice (see Figures 6B–6G), thereby demonstrating the detrimental effects of the proapoptotic activity of corisin in vivo.

[0141] More specifically, Figures 25A and 25B show computed tomography (CT) images and CT fibrosis scoring of WT (n=3) and TGFβ1 TG mice before treatment with saline (n=5), scrambled peptide (n=4), or corisin (n=5), respectively, performed as described in the Methods section below. Bars indicate mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test. * p<0.05. There was no statistical difference among the TGFβ1 TG / SAL group, the TGFβ1 TG / scrambled peptide group, and the TGFβ1 TG / corisin group (p=0.9). Intravenous instillation of S. nepalensis exacerbates pulmonary fibrosis

[0142] We evaluated whether bacteria expressing colisin-containing transglycosylases also exacerbate pulmonary fibrosis in vivo. To this end, we intratracheally administered the colisin-containing Staphylococcus nepalensis CNDG strain or Staphylococcus epidermidis [ATCC14990] as a negative control to germ-free TGFβ1 TG mice, divided into three groups and matched for pulmonary fibrosis CT score (Figures 26A and 26B).

[0143] Prior to this in vivo experiment, we confirmed in vitro that a synthetic peptide (IIARESNGQLHARNASGAA—SEQ ID NO: 2) corresponding to the peptide sequence of the “corisin site” of transglycosylase from Staphylococcus epidermidis exerted no proapoptotic effect on lung epithelial cells (not shown) (see Figures 27A and 27B). TGFβ1 mice instilled with Staphylococcus nepalensis CNDG strain exhibited significantly worse pulmonary radiological findings (see Figures 28A and 28B), significantly increased neutrophil infiltration, and enhanced alveolar epithelial cell apoptosis compared with mice receiving Staphylococcus epidermidis (see Figures 7A–7D), further confirming the role of proapoptotic peptides in the acute exacerbation of pulmonary fibrosis.

[0144] More specifically, Figures 26A and 26B show computed tomography (CT) images and CT fibrosis scoring of TGFβ1 TG mice before intratracheal instillation with Staphylococcus nepalensis (n=6), Staphylococcus epidermidis (n=6), or saline (n=4), respectively, as further described in the methods below. Bars in Figure 26B represent mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test. There was no statistical difference between the mouse groups (p=0.5).

[0145] Figures 27A and 27B show flow cytometry analysis of A549 alveolar epithelial cells cultured for 24 hours in DMEM medium containing 10 μM of a synthetic peptide (corisin) containing the sequence of the transglycosylase segment derived from Staphylococcus nepalensis CNDG strain (IVMPESSGNPNAVNPAGYR - SEQ ID NO: 1), its scrambled peptide (NRVYNGPAASPVSEGMPIN - SEQ ID NO: 3), or a synthetic peptide of the transglycosylase segment derived from Staphylococcus epidermidis (ATCC14990) (IIARESNGQLHARNASGAA - SEQ ID NO: 2). Each treatment group consisted of three groups (triplicates). Bars in Figure 27B represent the mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test. * p<0.001.

[0146] Figures 28A and 28B show computed tomography (CT) images and CT fibrosis scoring of TGFβ1 TG mice, respectively, performed before and after intratracheal instillation of saline (n = 4), Staphylococcus epidermidis (n = 6), or Staphylococcus nepalensis (n = 6) into germ-free TGFβ1 TG mice, as further described in the Methods below. Bars in Figure 28A represent the mean ± SD. Statistical analysis was performed by two-tailed Mann-Whitney U test. * p<0.05. Detection of colisin in the lungs of mice and human patients

[0147] We investigated the presence of chorisin in WT mice without fibrosis, as well as in TGFβ1 TG mice with and without fibrosis. We found that chorisin levels in TGFβ1 TG mice with pulmonary fibrosis were significantly enhanced compared with those in WT mice and TGFβ1 TG mice without fibrosis (see Figures 8A and 8B).

[0148] To clarify the clinical relevance of this finding, we also evaluated colisin in human IPF patients. To this end, we collected bronchoalveolar lavage fluid from 34 IPF patients and 8 male healthy controls. The characteristics of the IPF patients are listed in Table 3 below.

[0149] [Table 3]

[0150] IPF patients with stable disease or acute exacerbations had significantly increased levels of corisin in bronchoalveolar lavage fluid (BALF) compared with healthy controls (see Figures 8C and 8D). BALF corisin levels were also significantly elevated in IPF patients with acute exacerbations compared with patients with stable disease (again, see Figures 8C and 8D). The difference in corisin levels between men (50.6 ± 4.9 pg / ml) and women (58.8 ± 10.7 pg / ml) was not statistically significant (p = 0.07). There was also no significant correlation between corisin levels and patient age (r = 0.1, p = 0.5). These results provide evidence of the clinical relevance of corisin in IPF.

[0151] A dramatic increase in apoptotic epithelial cells occurs in the lungs of IPF patients with acute exacerbations. See NPL15 and NPL16. Our results provide evidence that excessive release of bacterially derived pro-apoptotic colisin contributes to this fatal disease complication. Phylogenetic analysis reveals conservation of colisin

[0152] To clarify the evolutionary relationships of transglycosylases expressed by different bacteria, we constructed a phylogenetic tree based on the amino acid sequences of six transglycosylases identified in the genomes of Staphylococcus nepalensis strain CNDG and its homologs in a publicly available database (www.ncbi.nlm.nih.gov / pubmed), as further described below.

[0153] Phylogenetic tree topology indicates that transglycosylase derivatives close to the ancestral sequence are separated into two IsaA clusters (IsaA-1 and IsaA-2), and that proteins designated as SceD members (SceD-1, SceD-2, SceD-3, and SceD-4) likely evolved from IsaA-1-related sequences (see Figures 29A-29D). Multiple alignments of IsaA and SceD amino acid sequences reveal that amino acid residues representing proapoptotic chorisins are generally conserved, thus highlighting their functional significance (see Figures 30A, 30B, and 30C).

[0154] The amino acid sequence identity of the chorisin-homologous transglycosylases from Staphylococcus xylosus, Staphylococcus cohnii, and Staphylococcus nepalensis was 100%. Furthermore, these staphylococci shared over 98% identity with the corresponding chorisin regions of transglycosylases from other members of the IsaA-1 and IsaA-2 clusters and 60% identity with the corresponding regions in members of the SceD cluster (see Figures 30A, 30B, and 30C). The genomic context (synteny) of gene clustering around the transglycosylases tended to be conserved in Staphylococcus cohnii and Staphylococcus nepalensis (see Figure 31A).

[0155] In particular, Figures 30A-30C show, for example, the following amino acid sequences that are considered to be "corisin" in the context of the present teachings or that fall within the scope of the term "corisin," namely: [ka] Shows. Horizontal gene transfer of the corisin-encoding gene

[0156] Sequence alignment and comparative genomic analysis revealed that a pathogenic strain of Streptococcus associated with respiratory tract disease, Streptococcus pneumoniae strain N, contains a transglycosylase (COE35810) with a peptide sequence nearly identical to that of colisin (a single amino acid change).

[0157] Further investigation of this bacterial genome revealed a second homologue (COE67256) of the colisin-containing polypeptide (Figures 30A, 30B and 30C).

[0158] Because the corisin polypeptide sequence is highly conserved among diverse Staphylococcus spp., to understand how Streptococcus pneumoniae strain N was able to acquire the gene encoding corisin, we conducted a search in the GenBank database and found that the polypeptide (COE35810) shares 98–100% identity with transglycosylases from different strains of Staphylococcus warneri (WP_002467055, WP_050969398, WP_126403073, and WP107532308) (see Figures 31B and 31C). Despite one or two amino acid changes in the N-terminal region of the polypeptide, the corisin peptide sequence in these transglycosylases is invariant.

[0159] We further investigated the genomic context of these genes in Streptococcus pneumoniae N strains compared to Staphylococcus warneri strains and found clear conservation of synteny despite some differences in annotation (see Figure 31D).

[0160] Therefore, we hypothesized that the transglycosylase gene and other associated genes in Streptococcus pneumoniae strain N were acquired from Staphylococcus warneri or related species. Importantly, other pathogenic bacterial strains are known to inhabit the human lung. For example, Mycobacterium abscessus contains a transglycosylase variant (SKT99287). Based on the same analysis of Streptococcus pneumoniae strain N described above, we hypothesized that transmission was from Staphylococcus hominis or related species (see Figures 31E and 31F). We then conducted experiments to confirm that synthetic corisin derived from Streptococcus pneumoniae transglycosylase (containing a single amino acid change from the Staphylococcus nepalensis derivative) also induced apoptosis in A549 alveolar epithelial cells (see Figures 30A-30C, 32A, and 32B).

[0161] More specifically, Figures 32A and 32B show flow cytometry analysis of A549 alveolar cells after 48 hours of culture in DMEM medium containing 5 μM of synthetic colisin (IVMPESSGNPNAVNPAGYR) derived from Staphylococcus nepalensis (CNDG strain) transglycosylase 351, its scrambled peptide (NRVYNGPAASPVSEGMPIN), or synthetic peptide (IVMPESGGNPNAVNPAGYR) derived from Streptococcus pneumoniae N strain transglycosylases (COE35810 and COE6725). Each group contained 3 peptides. The bars in Figure 32B represent the mean ± SD. Statistical analysis was performed by ANOVA with Tukey's test. * p<0.001.

[0162] These findings led to the conclusion that a non-Staphylococcus organism carrying a gene encoding a transglycosylase with very high homology to Staphylococcus nepalensis transglycosylase 351 is associated with the lung, thereby providing evidence for a case of horizontal gene transfer from a Staphylococcus strain inhabiting the lung. Consideration

[0163] Transforming growth factor-1 (TGF-β1) is a pleiotropic cytokine with a central role in the pathogenesis of pulmonary fibrosis due to its potent stimulatory activity on extracellular matrix synthesis, myofibroblast activation, differentiation, and migration, epithelial-to-mesenchymal transition, production of profibrotic factors, and apoptosis of alveolar epithelial cells (see NPL17 and NPL18). The development of pulmonary fibrosis in TG mice overexpressing TGF-β1 is proof-of-concept for the pivotal role of this cytokine in tissue fibrosis (see NPL11). Furthermore, TGF-β1 may promote the progression of pulmonary fibrosis by directly suppressing both the innate and adaptive immune systems, thereby increasing the host's susceptibility to infection (see NPL19, NPL20, and NPL21).

[0164] NPL22, NPL23, and NPL24 have shown that high salt concentrations impair host defense mechanisms by suppressing the activity of antimicrobial peptides or by altering immune cell populations. Thus, TGFβ1 may also indirectly affect host immune responses by favoring salt accumulation in the extracellular space. See NPL25 and NPL26. Abnormal extracellular salt storage may result in TGFβ1-mediated negative regulation of the surface expression of epithelial sodium and chloride channels, thereby inhibiting Na+. + ions and Cl - This results in a decrease in the transport of ions from the alveolar airspace across the epithelium. See also NPL27-NPL29.

[0165] Consistent with these findings, as shown in this disclosure, the inventors found that sodium levels in lung tissue were significantly increased in TGFβ1 TG mice with pulmonary fibrosis compared to WT mice, that sodium levels were significantly positively correlated with fibrotic markers and profibrotic cytokines, and that sodium levels were significantly negatively correlated with lymphocyte counts and sodium and chloride channels.

[0166] Recent single-cell RNA sequencing studies have demonstrated that the expression of several plasma membrane sodium and chloride transporters is significantly altered in alveolar epithelial cells from IPF patients, suggesting that ion transmembrane transport is disrupted in pulmonary fibrosis, favoring salt accumulation in this fibrotic disease (see NPL30). Since no difference in lung sodium levels was found between TGFβ1 TG and WT mice without fibrosis, sodium storage appears to require the presence of a fibrotic matrix. In this context, previous studies have shown that sodium is stored in the extracellular space in an osmotically inactive form by binding to negatively charged glycosaminoglycans, which are abundant in the extracellular matrix of fibrotic tissues (see NPL31–NPL35).

[0167] Overall, these findings suggest that fibrotic tissues represent a salt-rich microenvironment accompanied by abnormal immune and healing responses (see model in Figure 33). More specifically, transforming growth factor (TGF)-β1 can increase extracellular salt concentration by downregulating the cell surface expression of ion transporters, and the salt-rich microenvironment stimulates the growth of Staphylococcus spp., which release colisin and induce apoptosis of alveolar epithelial cells. Excessive apoptosis and / or activation of epithelial cells contributes to acute exacerbations of pulmonary fibrosis. Previous studies have identified halophilic bacteria in the lungs of IPF patients, supporting these findings. See NPL8 and NPL9.

[0168] Acute exacerbations are a devastating complication of IPF. See NPL 36. Nearly 50% of patients who die from IPF have a history of a previous acute exacerbation, and patients with a previous acute exacerbation have a life expectancy of only 3-4 months. See NPL 37-NPL 41.

[0169] Currently, there is no optimal treatment for acute exacerbations of IPF. See NPL36. In 2016, an international research group proposed classifying this complication as provoked acute exacerbation (confirmed events: postoperative, drug toxicity, infection, aspiration) or idiopathic acute exacerbation (unconfirmed inciting event). Ibid. Recent data link acute exacerbations to the lung microbiome and the state of host immunosuppression, and retrospective studies demonstrating the preventative effect of antibiotic treatment suggest a role for infection in the pathogenesis of acute exacerbations and the progression of pulmonary fibrosis. See NPL7 and NPL42–NPL45. Further support for the role of bacterial pathogenicity in pulmonary fibrosis comes from a double-blind, randomized, placebo-controlled trial showing improvement in symptoms and exercise capacity in patients with advanced IPF treated with co-trimoxazole, as well as a subsequent double-blind follow-up and multicenter trial showing significantly reduced mortality with better quality of life and fewer respiratory tract infections in IPF patients treated with co-trimoxazole. See NPL46 and NPL47.

[0170] In NPL7, Staphylococcus and Streptococcus bacteria were shown to worsen clinical outcomes in patients with IPF, suggesting their close relationship with disease progression and pathogenesis. Multiple studies demonstrating a significant correlation between the relative abundance of Staphylococcus or Streptococcus in fibrotic lungs and the host immune response in IPF patients further support the contribution of these bacterial genera to the pathogenesis of pulmonary fibrosis. (See NPL6, NPL42, and NPL48–NPL52.) However, the exact mechanism remains unclear.

[0171] In the research that led to this disclosure, the inventors hypothesized that high-salt culture medium mimics the salt-rich fibrotic tissue in vivo and thus favors the growth of bacteria involved in the pathogenesis of pulmonary fibrosis. The inventors detected the growth of Staphylococcus bacteria in high-salt medium inoculated with fibrotic tissue from hTGFβ1 TG mice with advanced fibrosis. Whole-genome sequencing of a pure bacterial culture revealed that it corresponded to Staphylococcus nepalensis, which the inventors categorized as a "CNDG strain." The culture supernatant of this bacteria induced apoptosis in alveolar epithelial cells. Subsequent chromatography, mass spectrometry, and gene sequencing analyses demonstrated that apoptosis was induced by a peptide, designated "corisin," corresponding to a segment of transglycosylase 351 from Staphylococcus nepalensis CNDG strain. The increased apoptotic activity of supernatants from bacteria cultured under high salt conditions may be due to salt-dependent stimulation of bacterial growth or to increased bacterial expression of a related protein, colisin-containing transglycosylase, whose expression has been reported to be enhanced under similar conditions in Staphylococcus aureus. See NPL53.

[0172] In additional experiments, we detected the peptide in lungs from hTGFβ1 TG mice with progressive pulmonary fibrosis and lungs from patients with IPF and found that intratracheal instillation of synthetic corisin or Staphylococcus nepalensis CNDG strain induced acute exacerbations of pulmonary fibrosis associated with widespread apoptosis of alveolar epithelial cells (see model in Figure 33). Accelerated apoptosis of alveolar epithelial cells plays a central role in the pathogenesis of acute exacerbations in pulmonary fibrosis. See NPL16 and NPL54. Therefore, based on these findings, corisin appears as a likely candidate microbial factor that may induce acute exacerbations in patients with idiopathic pulmonary fibrosis.

[0173] The present inventors have found that the sequence of colisin shares high homology with a region of membrane-bound lytic transglycosylases. Lytic transglycosylases are bacterial enzymes that have been reported to cleave the peptidoglycan component of bacterial cell walls (see NPL55) and also perform other essential cellular functions, such as cell wall synthesis, remodeling, antibiotic resistance, secretion system insertion, flagellar assembly, virulence factor release, sporulation, and germination (ibid.). Transglycosylases are ubiquitous in bacteria, and individual species can produce multiple transglycosylases with functional redundancy to compensate if any member is lost or inactivated. See NPL56 and NPL57.

[0174] In the results described herein, the complete genome sequence showed that Staphylococcus nepalensis strain CNDG produces six transglycosylases, of which transglycosylase 351, a member of the IsaA-1 cluster, contains a colisin sequence. Full-length transglycosylase 351 did not induce apoptosis in lung epithelial cells, providing evidence that the colisin peptide is active only when released from the full-length protein. Although the mechanism for releasing this peptide is unknown, the genome context of Staphylococcus nepalensis strain CNDG, showing the presence of peptidases surrounding transglycosylase 351, provides evidence that these peptidases may be involved in the release of the lethal peptide.

[0175] We found that sequences similar to colisin were highly conserved in several transglycosylases from some members of the microbial communities inhabiting normal and fibrotic lungs, including Staphylococcus nepalensis CNDG strain, other Staphylococcus species, and strains of Streptococcus pneumoniae and Mycobacterium abscessus (see NPL51 and NPL58-60). This observation provides evidence that a wide range of bacteria may be a source of colisin in pulmonary fibrosis.

[0176] While this disclosure is believed to be the first report of the virulence of peptides derived from IsaA homologs in Staphylococcus strains, we note that homologous proteins in Staphylococcus aureus (i.e., IsaA and SceD) have been reported to be involved in virulence. See NPL53. Staphylococcus aureus IsaA in NPL53 corresponds to YP_501340 in the alignment shown in Figures 30A, 30B, and 30C, while SceD in the same report has a variant of colisin similar to that in the SceD-1 through SceD-4 polypeptides (ibid.). Thus, the characterized transglycosylase in Staphylococcus aureus is related but distinct from the Staphylococcus nepalensis transglycosylase characterized in previous studies. However, we note that Staphylococcus aureus possesses an uncharacterized IsaA transglycosylase with a highly conserved colisin sequence (Figures 29A-29D, IsaA-2, SUK04795.1), which may suggest that a similar mechanism for colisin processing as described in this disclosure exists in Staphylococcus aureus.

[0177] Streptococcus pneumoniae and Staphylococcus species also frequently cause severe pulmonary infections with high in-hospital mortality in patients with IPF. See NPL20, NPL58, and NPL61. Given the growing evidence that alveolar cell apoptosis plays a central role in the pathogenesis and exacerbation of IPF (see NPL62), it is reasonable to hypothesize that the release of lethal peptides constitutes an important contributor to the loss of functional alveolar cells and poor clinical outcome in patients with comorbid microbial infections.

[0178] Another mechanism that may further contribute to bacterial virulence and invasiveness is horizontal bacterial gene transfer. See NPL63. Here, we found that strains of Streptococcus pneumoniae, Mycobacterium [Mycobacteroides] abscessus, and several Staphylococcus species share highly similar genomic contexts (synteny) and sequence homology of transglycosylases containing colisin sequences, thereby providing evidence for the involvement of horizontal gene transfer in the acquisition of this virulence factor. The genera Staphylococcus and Streptococcus are common members of the human microbiota. See NPL64. Therefore, if the colisin-related peptides identified in this study are determined to have similar apoptotic effects on human cells from other sites or organs, such as the kidney and liver, our understanding of infections by these bacteria will need to be reevaluated.

[0179] Given the growing evidence implicating the lung microbial community in the pathogenesis of IPF, the identification of colisin as a disease-promoting factor validates the role of apoptosis in fibrotic disease, provides novel diagnostic markers and therapeutic targets for IPF, and opens new avenues for investigating the role of the microbiome in organ fibrosis. method reagent

[0180] Human lung epithelial cell line A549 and high-salt medium (ATCC medium 1097, 2168) were obtained from the American Type Culture Collection (Manassas, VA), Dulbecco's modified Eagle's medium (DMEM) was obtained from Sigma-Aldrich (Saint Louis, MO), and fetal bovine serum (FBS) was obtained from BioWhittaker (Walkersville, MD). L-glutamine, penicillin, and streptomycin were obtained from Invitrogen (Carlsbad, CA). Normal human bronchial epithelial (NHBE) cells were obtained from Clonetics (Walkersville, MD). Synthetic peptides were prepared and provided by Peptide Institute Co., Ltd. (Osaka, Japan) and ThermoFisher Scientific (Waltham, MA, USA). subject

[0181] The study described herein included 34 Japanese patients with stable idiopathic pulmonary fibrosis (IPF; mean age: 71.7-6.6 years, 29 men, 5 women) and 8 healthy Japanese male volunteers (38.3 ± 6.1 years). Patient characteristics are listed in Table 3 above. The diagnosis of idiopathic pulmonary fibrosis was made according to accepted international criteria according to NPL65 and NPL66. Bronchoscopy was performed according to the American Thoracic Society guidelines, and bronchoalveolar lavage fluid (BALF) samples were collected from all 34 IPF patients and 8 healthy volunteers. See NPL65. BALF samples during acute exacerbations of the disease were available from 14 of the 34 IPF participants. Aliquots of unprocessed BALF collected in sterile tubes were stored at -80°C until analysis. animal

[0182] We used transgenic (TG) mice on a C57BL / 6J background with lung-specific overexpression of a previously characterized latent form of human TGFβ1 (see NPL8 and NPL11). These TGFβ1 TG mice spontaneously developed pulmonary fibrosis from 10 weeks of age, showing similarities to the disease in humans (ibid.). C57BL / 6J wild-type (WT) mice were used as controls. In some experiments, TGFβ1 TG mice without pulmonary fibrosis were used as controls; however, the number of mice born positive for the human TGFβ1 transgene but without the phenotype (pulmonary fibrosis) was very low or rare, making it very difficult to include them in all experiments. All mice were maintained in a specific pathogen-free environment with a 12-hour light / dark cycle in the Mie University Laboratory Animal Facility. Genotyping of TG mice was performed as described in NPL11 using standard PCR analysis, DNA isolated from mouse tails, and primer pairs (Supplementary Table 5). Computed tomography (CT)

[0183] We performed radiological evaluation of the mouse chest using a micro-CT (Latheta LCT-200, Hitachi Aloka Medical Co., Ltd., Tokyo, Japan). Mice were anesthetized with isoflurane and placed in a prone position for data acquisition according to NPL67. Six respiratory disease specialists blinded to the treatment groups scored the chest CT findings based on the following criteria: score 1, normal lung findings; 2, intermediate findings; 3, slight lung fibrosis; 4, intermediate findings; 5, moderate lung fibrosis; 6, intermediate findings; and 7, advanced lung fibrosis (Figure 9A). See NPL67. We validated the CT findings using the Ashcroft pulmonary fibrosis score and lung hydroxyproline content (Figure 9B). Assessment of pulmonary fibrosis in mice

[0184] Under deep anesthesia, we collected bronchoalveolar lavage fluid for biochemical analysis and cell counting. Briefly, bronchoalveolar lavage was performed according to NPL68 by cannulating the trachea using a 20-gauge needle and injecting saline solution into the lungs. Samples were centrifuged, and the supernatant was stored at -80°C until analysis. The cell pellet was resuspended in physiological saline solution and counted. A ChemoMetec (Allerod, Denmark) nucleocounter was used for cell counting, and cells were stained with May-Gruenwald-Giemsa (Merck, Darmstadt, Germany) to count differential cells. Mice were sacrificed by anesthesia overdose, and the lungs were removed, fixed in formalin, embedded in paraffin, and prepared for hematoxylin-eosin staining. The severity of pulmonary fibrosis was quantified based on the Ashcroft criteria. See NPL 67. TGFβ1 levels were measured using a commercially available enzyme immunoassay kit from BD Biosciences Pharmingen (San Diego, Calif.). Ethics Statement

[0185] All subjects participating in the clinical study provided written informed consent, and the study protocol was approved by the Mie University Clinical Research Ethics Committee (approval number: H2019064, approval date: 25 / 04 / 2019), Matsusaka City Hospital (approval date: 11 / 06 / 2014), and Chuo Medical Center (approval number 2014-6, approval date: 02 / 09 / 2014), and was conducted in accordance with the principles of the Declaration of Helsinki. The experimental protocol was approved by the Recombinant DNA Experiment Safety Committee (Approval number: I-614 (henkol); Approval date: 2013 / 15 / 12; Approval number: I-708, Approval date: 13 / 02 / 2019) and the Mie University Animal Experimentation Committee (Approval number: 25-20-hen1-sai1, Approval date: 23 / 07 / 2015; Approval number: 29-23, Approval date: 15 / -01 / 2019), and all procedures were performed in accordance with the internationally accepted principles for laboratory animal care published by the National Institutes of Health. Lung sampling for in vitro culture

[0186] Under sterile conditions, the inventors euthanized the mice by intraperitoneal injection of an overdose of pentobarbital, then removed the left and right lungs. The tissues were placed in sterile tubes and immediately stored at −80°C until use. Na in lung tissue + Measurement of

[0187] Lungs were removed from TGFβ1 mice and WT mice with or without pulmonary fibrosis. Samples were sent to Shimadzu Techno Research Corporation (Kyoto, Japan) for measurement of tissue sodium content using microwave analysis / inductively coupled plasma mass spectrometry (ICP-MS) with a microwave ashing system ETHOS-TC (Milestone General) and an ICP-MS system 7700× (Agilent Technologies, Santa Clara, CA). See NPL69 and NPL70. The results are shown in Figure 1C. Assessment of lung tissue immune cells

[0188] To isolate lung immune cells, mice were sacrificed by anesthesia overdose and then dissected. We minced the lung tissue into 2-3 mm pieces with scissors, incubated it in 0.5 mg / ml collagenase solution at 37°C for 30 minutes, and then filtered it through a stainless steel mesh. Lung cells were isolated and purified using an isotonic 33% Percoll (Sigma-Aldrich, St. Louis, MO) solution. We then detected lung immune cells by flow cytometry using the antibodies listed in Table 4 below.

[0189] [Table 4] Evaluation of the effects of pro-apoptotic colisin in mice

[0190] Three groups of TGFβ1 TG mice (n = 5 or n = 4, respectively) matched for the grade (level) of pulmonary fibrosis assessed by CT score were intratracheally instilled with colisin or scrambled peptide or 0.9% NaCl solution on days 1 and 2 and sacrificed on day 3 to evaluate changes in lung inflammation and fibrosis. WT mice (n = 3) without pulmonary fibrosis treated with 0.9% NaCl solution served as controls. Intratracheal instillation of Staphylococcus nepalensis

[0191] We administered 200 μl of a solution containing a cocktail of antibiotics, including vancomycin (0.5 mg / ml), neomycin (1 mg / ml), ampicillin (1 mg / ml), metronidazole (1 mg / ml), and gentamicin (1 mg / ml), by oral gavage once daily for 4 days to three groups of TGFβ1 TG mice. The grade of pulmonary fibrosis, as assessed by CT score, was matched across all mice. On day 5, one group of mice received 1 × 10 8Colony-forming units (75 μl) of Staphylococcus nepalensis CNDG strain or Staphylococcus epidermidis ATCC14990 were instilled intratracheally and sacrificed 2 days later. Germ-free TGFβ1 TG mice treated with 0.9% NaCl solution served as controls. Bacterial isolation, culture, and spent media preparation

[0192] Lungs from TGFβ1 TG mice with pulmonary fibrosis and lungs from WT mice were used for in vitro microbial culture. Lung tissue specimens were washed with PBS and inoculated into ATCC medium 1097 (8% NaCl) and incubated at 37°C with shaking at 220 rpm until growth was visible. Bacterial colonies were isolated by plating the liquid-cultured organisms onto ATCC medium 1097 agar plates. Individual single colonies were inoculated into liquid ATCC medium 1097 (8% NaCl) and incubated at 37°C and 220 rpm for 24 hours. The cultures were centrifuged at 4,000 rpm for 5 minutes at 4°C to pellet the cells, and the resulting supernatant was filtered through a MILLEXGP filtration unit (0.22 μm, Millipore) to remove any remaining cells and used as spent bacterial culture medium. Phase contrast microscopy

[0193] Following NPL71, we harvested bacterial cells from single colonies growing in the exponential phase, immersed them in fixative overnight at 4°C, and collected micrographs using phase-contrast microscopy (Frederick Seitz Materials Research Lab, UIUC). Genomic DNA sequencing and genome annotation

[0194] Genome sequencing was performed using a combination of Oxford Nanopore Sequencing and Illumina Miseq nano sequencing, resulting in 6.3 Gb and 1.6 million (2 × 250) nucleotides with a perfect Qscore. Briefly, genomic DNA (400 ng) from the bacterial strain was converted into a Nanopore library using the Rapid Barcoding library kit SQK-RAD004. Library sequencing was performed over 48 hours on a GridION sequencer with a SpotON R9.4.1 FLO-MINI 06 flow cell. Base calling was performed using Guppy 1.4.3, and demultiplexing was performed using Porechops 0.2.3. The majority of reads were between 6 kb and 30 kb in length, but reads as long as 94 kb were also obtained. Illumina Miseq sequencing was performed by preparing a shotgun genomic library using the Hyper Library construction kit from Kapa Biosystems (Roche). Libraries were quantified by qPCR and sequenced from each end of the fragments over 251 cycles on one MiSeq Nano flow cell using the MiSeq 500-cycle sequencing kit version 2. Fastq files were generated and demultiplexed using bcl2fastq v2.20 Conversion Software (Illumina).

[0195] Four assemblies were performed using various assembly strategies, primarily to assess quality and identify the best overall assembly. A workflow was developed as follows: An initial assembly of Oxford Nanopore data was performed using Canu (NPL72), followed by refinement using Nanopolish (NPL73) and Pilon (utilizing Illumina MiSeq reads—NPL74), and finally, the genome was reoriented using Circlator (NPL75). Another hybrid genome assembly was performed using SPAdes (NPL76), followed by genome reorientation using Circlator. A hybrid genome assembly was also performed using Unicycler (NPL77). The final hybrid genome assembly was generated using Unicycler, using the above Canu assembly as the scaffold.

[0196] Quality assessment of all assemblies was performed using BUSCO (NPL78) and QUAST (NPL79) and compared to relevant reference genomes using MUMmer (see NPL80). Assembly was then followed by an annotation run using the Prokka tool (NPL81). After assessment, the best overall BUSCO score was used in combination with global assembly metrics to determine the best overall assembly. Evaluation of the molecular weight of apoptotic factors

[0197] Bacterial culture supernatants were prepared from cultures grown with shaking at 37°C in Halomonas medium (8% NaCl, 0.75% casamino acids, 0.5% proteose peptone, 0.1% yeast extract, 0.3% sodium citrate, 2% magnesium sulfate heptahydrate, 0.05% dipotassium phosphate, and 0.05% ammonium iron(II) sulfate hexahydrate). Bacterial cells were removed by centrifugation (17,000 × g, 10 min at 4°C) and filtration through a 0.2 μm filter (Corning). The supernatant was size-fractionated into high-molecular-weight (HMW) and low-molecular-weight (LMW) fractions by ultrafiltration using Ultracel-10K filters (Amicon), aliquoted, and frozen at -20°C. In some experiments, bacterial culture supernatants were heat-treated (85°C, 15 min) before size fractionation. Equal volumes of supernatant were separated by 17.5% tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver stained using the Daiichi 2-D Silver Staining Kit (Daiichi, Tokyo, Japan). cell culture

[0198] A549 and NHBE cells were cultured in DMEM supplemented with 10% fetal bovine serum, 0.03% (w / v) L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin in a humidified, 5% CO atmosphere at 37°C. We used the A549 cell line for the majority of experiments because it has greater growth potential than primary NHBE cells and mimics the phenotype of alveolar type II cells (NPL82, NPL83). Furthermore, these primary cells typically undergo phenotypic change or begin to senesce after short-term culture.

[0199] The bacterial culture supernatant (2 L) was partitioned sequentially between n-hexane and water, followed by ethyl acetate and water (2 L each, twice) (Figure 13). The concentrated protein was further concentrated under reduced pressure and then extracted with ethanol (2 L each, twice). The ethanol-soluble portion (7.96 g) was fractionated by octadecylsilane gel flash column chromatography (5%; 10%, 20%, 50% methanol and methanol, 0.5 L each) to obtain 42 fractions (fractions 1–42). Fraction 42 (185.3 mg protein) was further separated by Sep-Pak (80% acetonitrile, methanol, and chloroform). Fraction 42–80% acetonitrile (75.6 mg protein) was separated by reverse-phase HPLC (C8, 80% methanol) to yield 22 fractions (fractions 42–80% acetonitrile-1–22).

[0200] mass spectrometry

[0201] The dried sample was suspended in 0.1% formic acid (FA) in 5% acetonitrile (ACN), and 2 μg of peptide was injected into a Thermo UltiMate 3000 UHPLC system. Reversed-phase separation of the sample peptides was achieved using a 15 cm Acclaim PepMap 100 C18 column with a mobile phase of 0.1% FA in water (A) and 0.1% FA in ACN (B). Peptides were eluted using a gradient of 2% B to 35% B over 60 min, followed by a gradient of 35% to 50% B over 5 min at a flow rate of 300 μl per min. The UHPLC system was online coupled to a Thermo Orbitrap Q-Exactive HFX (Biopharma Option) mass spectrometer operated in data-dependent mode. A precursor scan from 300 m / z to 1,500 m / z (120,000 resolution) was followed by collision-induced dissociation (CID) of the most abundant precursor over a maximum cycle time of 3 s (3e4 AGC, 35% NCE, 1.6 m / z isolation window, 60 s dynamic exclusion window).

[0202] The raw data were analyzed using Mascot 1.6 against a custom database containing a protein library of Staphylococcus nepalensis CNDG genomic DNA and polypeptides encoded by large and small plasmids (a total of 3,541 protein sequences). No enzymes were identified. Peptide mass tolerance and fragment mass tolerance were set at 10 ppm and 0.1 Da, respectively. Variable modifications included oxidation of methionine residues (see mass spectrometry data in the Supplementary Information). Apoptosis assay

[0203] A549 cells and NBHE cells (4 × 10 cells) 5 Cells (cells / well) were seeded into 12-well plates, grown to subconfluence, washed, and then cultured in serum-free medium containing 10% of each bacterial supernatant for 48 hours. Uninoculated high-salt medium was used as a control. Cells were stained with fluorescein-labeled annexin V and propidium iodide (FITC Annexin V Apoptosis Detection Kit with PI, Biolegend, San Diego, CA) and then analyzed for apoptosis by flow cytometry (FACScan, BD Biosciences, Oxford, UK). The flow cytometry gating strategy used in this experiment is described in Figures 34A-34C. Under physiological conditions, phosphatidylcholine is exposed externally, while phosphatidylserine (PS) is located on the inner surface of the lipid bilayer of the cell membrane. See NPL84. During apoptosis, PS translocates from the cytoplasmic face of the plasma membrane to the cell surface. Ibid. Annexin V interacts with Ca upon binding to phosphatidylserine. 2+ It exhibits strong affinity in apoptosis-dependent manner and is therefore commonly used as a probe to detect apoptosis (see NPL85). Western blotting

[0204] For Western blot analysis, cells were washed twice with ice-cold phosphate-buffered saline and then lysed in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease / phosphatase inhibitors (1 mM orthovanadate, 50 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 5 μg / mL leupeptin, 2 μg / mL aprotinin, 5 mM sodium fluoride). The suspension was centrifuged (17,000 × g for 10 min at 4°C), and protein content was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Incorporation, Waltham, MA). Equal amounts of cell lysate protein were mixed with Laemmli sample buffer and separated by SDS-PAGE. Proteins were then electrophoretically transferred from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose membranes, followed by Western blotting using anti-phospho-Akt, anti-Akt, anti-cleaved caspase-3, or anti-β-actin antibodies (Cell Signaling, Danvers, MA). See NPL67. Band intensity was quantified by densitometry using the public domain NIH imageJ program (Wayne Rasband, NIH, Research Service Branch). Immunohistochemistry

[0205] Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) staining was performed at Biopathology Research Institute Co., Ltd. (Kunisaki, Oita, Japan) using Alexa Fluor 594 goat anti-rabbit IgG and slow-fade gold antifade reagent with 4',6-diamidino-2-phenylindole (DAPI) or ApopTag terminal deoxynucleotidyl transferase (Merck Millipore, Burlington, MA), anti-digoxigenin-peroxidase, and 3,3'-diaminobenzidine. Quantification of apoptotic areas was performed using WinROOF software (Mitani Corporation, Tokyo, Japan), and values ​​for each individual mouse were averaged. Gene expression assessment

[0206] We extracted total RNA from cells or lung tissues using Sepasol RNA-I Super G reagent (Nacalai Tesque, Kyoto, Japan), synthesized cDNA from 2 μg of total RNA using oligo-dT primers and ReverTra Ace Reverse Transcriptase (Toyobo Life Science Department, Osaka, Japan), and then performed standard PCR using the primers listed in Table 5 below.

[0207] [Table 5]

[0208] PCR was performed using 26-35 cycles depending on the gene: denaturation at 94°C for 30 seconds, annealing at 65°C for 30 seconds, elongation at 72°C for 1 minute, followed by a further extension at 72°C for 5 minutes. See NPL67. mRNA expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. Transmission electron microscopy of apoptotic cells

[0209] A549 cells (cells 10 x 104 Cells (cells / ml) were plated onto collagen-coated 8-well chamber slides (BD Bioscience, San Jose, CA) and cultured until semiconfluent. Cells were serum-starved for 6 hours and stimulated with a proapoptotic peptide (5 μM) for 16 hours. Cells were fixed with 2% fresh formaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 hours at room temperature. After washing with 0.1 M cacodylate buffer (pH 7.4), cells were post-fixed with 1% OsO₄ in the same buffer for 2 hours at 4°C. Samples were rinsed with distilled water, stained with 1% aqueous uranyl acetate for 2 hours or overnight at room temperature, dehydrated with ethanol and propylene oxide, and embedded in Epon (Epon 812 resin, Nakarai). After the cells were removed from the glass, they were cut into ultrathin sections (94 nm), stained with uranyl acetate and Reynolds lead citrate, and observed under a transmission electron microscope (JEM-1010, JEOL, Tokyo, Japan). Cell cycle analysis and cell viability assays

[0210] We cultured cells with or without the bacterial supernatant fraction for 48 hours, and then performed DNA content / cell cycle analysis by flow cytometry. Cells were treated with propidium iodide and then cell cycle distribution was assessed. Cell viability was measured using a commercially available cell counting kit (Dojindo Laboratories, Tokyo, Japan). Samples used in the assay were fractionated after gel filtration using a Sephadex G25 column. Expression of S. nepalensis IsaA transglycosylase

[0211] The genes encoding Staphylococcus nepalensis strain CNDG transglycosylase 351 and transglycosylase 531 were synthesized using E. coli-optimized codons, amplified to add terminal A's, and cloned into the TA-cloning vector pGEM-T Easy (Promega, Madison, WI). The genes were then excised, cloned into a modified pET28a vector, and transformed into E. coli BL21 DE3 cells, where they were expressed and purified as hexa-histidine-tagged (His-tag) proteins. See NPL86. Preparation of antibodies against pro-apoptotic peptides

[0212] A protein A-purified rabbit polyclonal antibody against a pro-apoptotic peptide (corisin) was developed by Eurofins Genomics Inc. (Tokyo, Japan) using the sequence NH2-C+IVMPESSGNPNAVNPAGYR-COOH (SEQ ID NO: 1).

[0213] A band of the molecular weight corresponding to the target peptide can be observed in Western blotting of mouse lung tissue samples and culture supernatants of Staphylococcus nepalensis strain CNDG (FIGS. 22A and 22B). Detection and measurement of colisin in tissues and body fluids

[0214] Purified anti-corisin IgG antibody was used for Western blotting of lung tissue at a 1 / 1000 dilution. We measured the concentration of corisin in body fluids using a competitive enzyme immunoassay. Briefly, purified corisin from transglycosylase 351 was coated onto 96-well plates at a final concentration of 2 μg / ml in phosphate-buffered saline overnight at 4°C. After blocking and appropriate washing, standards, samples, and 5 ng / ml anti-corisin were added to the wells and incubated overnight at 4°C. After washing the wells, horseradish peroxidase-conjugated goat anti-rabbit IgG (R&D System) in phosphate-buffered saline containing 5 μg / ml human IgG was added as the secondary antibody. After appropriate washing and incubation, a substrate solution was added for color development and absorbance reading at 450 nm. Values ​​were estimated from a standard curve prepared using several concentrations of the peptide. Phylogenetic analysis

[0215] To search for homologous proteins, the GenBank protein database (ncbi.nlm.nih.gov / protein / ) was searched using five transglycosylase polypeptides (CNDG_8p_00351, CNDG_8p_00513, CNDG_8p_00157, CNDG_8p_00159, and CNDG_8p_00845). Protein sequences were aligned using the MUltiple Sequence Comparison with Log-Expectation (MUSCLE) program, and the alignment was used to construct a phylogenetic tree based on the neighbor-joining method with a bootstrap value of 1,000 replicates. All of these programs are available in Geneious Prime 2016 version (www.geneious.com).

[0216] More specifically, the phylogenetic tree shown in Figure 29 was constructed by the neighbor-joining method. Bootstrapping was performed with 1,000 replicates. GenBank accession numbers for this tree are as follows: CLUSTER IsaA-1 [WP_112369066.1 (transglycosylase, S. arlettae), WP_061853755.1 (hypothetical protein, S. kloosii), WP_107393111.1 (transglycosylase, S. auricularis), WP_049409534.1 (hypothetical protein, S. pettenkoferi), WP_103371985.1 (transglycosylase, S. argensis), WP_046466985.1 (transglycosylase, S. pasteuri), COE35810.1 (transglycosylase, Streptococcus pneumoniae), WP_002467055.1 (hypothetical protein, S. warneri), WP_050969684.1 (transglycosylase, Streptococcus pneumoniae type N), WP_002449188.1 (hypothetical protein, S. hominis), WP_103166037.1 (transglycosylase, S. devriesei), WP_053024542.1 (transglycosylase, S. haemolyticus), WP_103328722.1 (transglycosylase, S. petrasii), WP_126565453.1 (transglycosylase, S. carnosus), WP_107511677.1 (transglycosylase, S. gallinarum), WP_069823097.1 (transglycosylase) transglycosylase, S. succinus), WP_069833173.1 (transglycosylase, S. equorum), WP_057513458.1 (hypothetical protein, S. sp. NAM3COL9), WP_002506616.1 (hypothetical protein, S. sp. OJ82), WP_107552346.1 (transglycosylase, S. xylosus), WP_069827045.1 (transglycosylase, S. saprophyticus), WP_099091381.1 (transglycosylase, S. edaphicus), WP_073344326.1.1 (transglycosylase, S. cohnii), WP_119487699.1 (transglycosylase, S. nepalensis), CNDG_8p_00351 (putative transglycosylase IsaA-1, S. nepalensis)] CLUSTER IsaA-2 ■ [SUK04795.1 SceA (S. aureus), WP_105995336.1 (hypothetical protein, S. agnetis), WP_105986821.1 (hypothetical protein, S. chromogenes), WP_009384111.1 (hypothetical protein, S. massiliensis), WP_126510217.1 (transglycosylase, S. epidermidis), WP_049407882.1 (hypothetical protein, S. pettenkoferi), WP_103371892.1 (hypothetical protein, S. argensis), WP_061853631.1 (hypothetical protein, S. kloosii), WP_107376802.1 (hypothetical protein, S. arlettae), WP_022791177.1 LysM peptidoglycan-binding domain-containing protein (Weissella halotolerans), WP_105993143.1 (hypothetical protein, S. simulans), WP_114602723.1 (hypothetical protein, S. sp. EZ-P03), WP_095089569.1 (hypothetical protein, S. stepanovicii), WP_017000663.1 (hypothetical protein, S. lentus), WP_119634381.1 (hypothetical protein, S. fleurettii), WP_126476519.1 (hypothetical protein, S. schleiferi), WP_107573021 ...lentus) Protein, S. sciuri), WP_069822945.1 (Hypothetical protein, S. succinus), WP_119484130.1 (Hypothetical protein, S. gallinarum), WP_099090334.1 (Hypothetical protein, S. edaphicus), WP_107558872.1 (Hypothetical protein, S. xylosus), WP_069995535.1 (Hypothetical protein, S. saprophyticus), WP_057513315.1 (Hypothetical protein, S. sp. NAM3COL9), WP_069817445.1 (hypothetical protein, S. equorum), WP_107384366.1 (hypothetical protein, S. cohnii), CNDG_8p_00513 (putative transglycosylase IsaA-2, S. nepalensis), WP_096808504.1 (hypothetical protein, S. nepalensis)] CLUSTER SceD-1 ■ [WP_101118359.1 (transglycosylase, S. succinus), WP_107530874.1 (transglycosylase, S. xylosus), WP_011302117.1 transglycosylase SceD 1 (S. saprophyticus), WP_105873943.1 (transglycosylase, S. cohnii), WP_107644182.1 (transglycosylase, S. nepalensis), CNDG_8p_00157 (putative transglycosylase SceD-1, S. nepalensis), WP_071564462.1 (transglycosylase, S. equorum)]CLUSTER SceD-2 [WP_070812670.1 (transglycosylase, S. sp. HMSC034G07), WP_119486153.1 (transglycosylase, S. gallinarum), WP_047504891.1 (transglycosylase, S. sp. ZWU0021), WP_057513650.1 (transglycosylase, S. sp. NAM3COL9), WP_096808177.1 (transglycosylase, S. nepalensis), CNDG_8p_00159 (putative transglycosylase SceD-2, S. nepalensis)] CLUSTER SceD-3 [WP_107564333.1 (transglycosylase, S. succinus), WP_115347167.1 (transglycosylase, S. saprophyticus), WP_107557548.1 (transglycosylase, S. xylosus), WP_099091190.1 (transglycosylase, S. edaphicus), WP_064263215.1 (transglycosylase, S. cohnii), CNDG_8p_00161 (putative transglycosylase SceD-3, S. nepalensis), WP_107644349.1 (transglycosylase, S. nepalensis)] CLUSTER SceD-4 [WP_119569949.1 (transglycosylase, S. succinus), WP_107385877.1 (transglycosylase, S. cohnii), CNDG_8p_00845 (putative transglycosylase SceD-4, S. nepalensis), WP_096808795.1 (transglycosylase, S. nepalensis)].WP_050969685.1 (transglycosylase, Streptococcus pneumoniae type N), YP_501340.1 (transglycosylase, S. aureus subsp. aureus NCTC 8325), WP_046206716.1 (transglycosylase, S. cohnii) subs.cohnii). statistical analysis

[0217] Unless otherwise specified, data are presented as mean ± standard deviation (SD). Statistical differences between two variables were assessed by the Mann-Whitney U test, and differences between three or more variables were assessed by analysis of variance using Tukey's test for post-hoc analysis. A P value of <0.05 was considered statistically significant. We performed statistical analyses using GraphPad Prism vs 7 (GraphPad Software, Inc., San Diego, CA).

[0218] Further embodiments of the present disclosure include, but are not limited to:

[0219] 1. A method for assessing fibrosis, comprising the step of detecting colisin as a target substance.

[0220] 2. A method according to embodiment 1 above, which detects a sequence of 19 amino acids within colisin (IVMPESSGNPNAVNPAGYR - SEQ ID NO: 1).

[0221] 3. The method according to embodiment 1 or 2 above, wherein said fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and mammary fibrosis.

[0222] 4. Antibodies that bind to colisin and prevent and / or treat fibrosis.

[0223] 5. The antibody according to embodiment 5 above, which recognizes the sequence of 19 amino acids (IVMPESSGNPNAVNPAGYR - SEQ ID NO: 1).

[0224] 6. The antibody according to embodiment 4 or 5 above, which is a polyclonal antibody.

[0225] 7. A method for identifying a corisin receptor protein, comprising the step of searching for corisin-binding proteins present on the surface of epithelial cells.

[0226] 8. A method for identifying colisin receptor proteins, comprising the step of searching for the 19 amino acid sequence (IVMPESSGNPNAVNPAGYR - SEQ ID NO: 1) of a binding protein present on the surface of epithelial cells.

[0227] Non-Patent Literature ("NPL") References Mentioned in the Description Above

[0228] [ka] [ka] [ka] [ka] [ka] [ka]

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Claims

1. 1. A method for detecting the amount of corisin present in an in vitro biological sample obtained from a subject as an indication of having fibrosis or being suspected of having or developing fibrosis, comprising: detecting the amount of corisin present in the in vitro biological sample obtained from the subject; and optionally comparing the amount of corisin detected in said in vitro biological sample to one or more predetermined thresholds; The method, wherein the corisin has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:

106.

2. A method comprising detecting the presence of corisin in an in vitro biological sample from a subject, The method, wherein the corisin has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:

106.

3. The method described in claim 2, wherein the presence is used in assessing fibrosis in the subject.

4. The method described in claim 1 or 3, wherein the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and breast fibrosis.

5. 5. The method of any one of claims 1 to 4, wherein the in vitro biological sample is obtained from one or both lungs of the subject and / or is selected from the group consisting of sputum, bronchial secretions, pleural effusion, bronchoalveolar lavage fluid (BALF), blood, and tissue obtained from the bronchi or lungs.

6. 6. The method of claim 1, wherein the corisin is detected by a method selected from the group consisting of mass spectrometry, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

7. A method described in any one of claims 1 to 6, wherein the corisin is detected by antibody binding.

8. The method described in claim 7, wherein the antibody recognizes the amino acid sequence of SEQ ID NO: 1 or 4.

9. 1. A method for identifying a corisin receptor protein, comprising the step of searching for a corisin-binding protein present on the surface of an epithelial cell, The method, wherein the corisin has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:

106.

10. 1. A pharmaceutical composition for use in treating fibrosis in a subject, comprising a corisin inhibitor that neutralizes at least a portion of corisin in the lungs of said subject and / or reduces the amount of corisin in the lungs of said subject; the corisin has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:106; A pharmaceutical composition optionally further comprising at least one pharmaceutically acceptable additive, salt or excipient.

11. 11. The pharmaceutical composition of claim 10, wherein the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and breast fibrosis.

12. The pharmaceutical composition described in claim 11, wherein the corisin inhibitor is selected from the group consisting of a small molecule, a corisin antagonist, or an antibody against corisin.

13. The pharmaceutical composition described in claim 12, wherein the antibody recognizes an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO:

106.

14. The pharmaceutical composition described in claim 13, wherein the antibody recognizes the amino acid sequence of SEQ ID NO: 1 or 4.

15. An antibody that binds to corisin, An antibody wherein the corisin has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:

106.

16. An antibody described in claim 15, which recognizes the amino acid sequence of SEQ ID NO: 1 or 4.

17. An antibody described in claim 15 or 16, which is a polyclonal antibody and / or a neutralizing antibody.

18. 1. A pharmaceutical composition for use in treating fibrosis in a subject, comprising: An antibody according to any one of claims 15 to 17; at least one pharmaceutically acceptable additive, salt or excipient; A pharmaceutical composition comprising:

19. 19. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition is administered to one or both lungs of the subject and / or is administered intraperitoneally or by intratracheal instillation or by inhalation.

20. 20. The pharmaceutical composition of claim 18 or 19, wherein administration of the pharmaceutical composition reduces the severity of the fibrosis in the subject.

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