CN118813775A - Application of TNFSF12 in fibrotic diseases - Google Patents

Abstract

The application relates to the field of diagnosis and treatment of fibrotic diseases, in particular to an application of TNFSF12 in fibrotic diseases. The present application provides the use of TNFSF12 in the manufacture of a product for the diagnosis and/or treatment of fibrotic diseases. The present application provides the use of TNFSF12 selected from any one or more of the following: 1) As a target for diagnosis or treatment of fibrotic diseases; 2) As a marker for diagnosis or prognosis of fibrotic diseases; 3) Preparing a product for diagnosis or prognosis of a fibrotic disease; 4) As a target for screening drugs for treating fibrotic diseases. The application researches the role of TNFSF12/TNFRSF12A signal path in fibrosis, and aims to realize the regulation of the signal path in an adult, thereby achieving the purpose of treating the fibrosis.

Description

Application of TNFSF12 in fibrotic diseases

Technical Field

The present application relates to the field of diagnosis and treatment of fibrotic diseases, and in particular to the application of TNFSF12 in fibrotic diseases.

Background Art

TNFSF12 (TNF Superfamily Member 12), also known as TWEAK (Tumor necrosis factor (TNF)-like weak inducer of apoptosis), is a multifunctional cytokine that regulates a variety of cell activities including proliferation, migration, differentiation, apoptosis, angiogenesis and inflammatory response. TNFSF12 is a type II transmembrane protein composed of 249 amino acids. The extracellular region is located at the C-terminus, consisting of 206 amino acids, containing a typical TNF homology region; the transmembrane region is composed of 25 amino acids; the intracellular region is located at the N-terminus, consisting of 18 amino acids, and contains potential protein kinase C phosphorylation sites. Furin can be cut at positions 90-93 (RPRR) of TNFSF12 to convert it into a soluble protein of 156 amino acids. Therefore, TNFSF12 exists in two forms in the body. One is the full-length TNFSF12 that has not been cut, which is fixed on the cell membrane in a membrane-bound form (membrane-bound TNFSF12, mTNFSF12); the other is the soluble form after cutting (soluble TNFSF12, sTNFSF12), which is secreted outside the cell.

TNFRSF12A (TNF receptor superfamily member 12A), also known as Fn14 (Fibroblastgrowth factor-inducible 14), is the only known receptor for TNFSF12. TNFRSF12A is the smallest member of the TNF receptor superfamily and is a type I transmembrane protein composed of 129 amino acids. The extracellular segment of TNFRSF12A is located at the N-terminus and consists of 53 amino acids, including 1 to 4 cysteine-rich regions; the transmembrane region consists of 21 amino acids; the intracellular segment located at the C-terminus consists of 28 amino acids, which contains the binding site for TNF receptor-associated factor (TNFR-associated factor, TRAF). When TNFSF12 is synthesized, the C-terminal TNF homology domain is responsible for the trimerization of TWEAK and binding to the receptor. The trimerized TNFSF12 can bind to the extracellular domain of TNFRSF12A, and the C-terminal domain of TNFRSF12A bound to the ligand will aggregate together to form a trimerized TNFRSF12A, activating the TNFSF12-TNFRSF12A signaling pathway. Both trimerized mTNFSF12 and sTNFSF12 can bind to TNFRSF12A to activate downstream classical and non-classical NF-κB and MAPK signals, thereby exerting their biological functions.

Organ fibrosis is characterized by the formation of scar tissue and can occur in a variety of organs, including the heart, lungs, liver, and kidneys, affecting the physiological functions of the organs and ultimately leading to organ failure or even death. At present, there is an extreme lack of treatments for organ fibrosis, and only two drugs for idiopathic pulmonary fibrosis have been approved by the FDA. These two drugs, pirfenidone and nintedanib, can only slow down the progression of fibrosis but cannot cure the disease. The only way to cure fibrosis is still organ transplantation. However, organ transplant donors are limited, expensive, and cannot be widely used. Therefore, the treatment of fibrosis is still an unmet medical need. In-depth understanding of the pathogenesis of fibrosis, finding new targets for the treatment of fibrotic diseases, and developing anti-fibrotic drugs have important social value.

Among them, pulmonary fibrosis is a progressive respiratory disease characterized by the proliferation of fibroblasts and the accumulation of extracellular matrix to form scar tissue, leading to the loss of gas exchange function. The main type of pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF). Once the disease is diagnosed, the median survival period is only 2-3 years, and it is called "cancer that is not cancer."

It is currently believed that IPF originates from abnormal repair of alveolar epithelial cells after repeated damage, leading to continuous activation of (myo) fibroblasts. After epithelial cell damage, cytokines and chemokines are released, causing an inflammatory response, recruiting fibroblasts to the site of damage, fibroblasts proliferate and become activated, and secrete extracellular matrix to help epithelial repair; under the multiple effects of known or unknown genetic/environmental factors and even aging, repeated damage leads to abnormal or insufficient regeneration and repair of epithelial cells, continuous activation of (myo) fibroblasts, and then excessive extracellular matrix is produced and accumulated in large quantities in the lung interstitium, leading to fibrosis and decreased lung function.

Inflammatory monocytes and resident tissue macrophages are key regulators of tissue repair, regeneration, and fibrosis. Macrophages have many functions, such as promoting inflammatory responses, phagocytizing apoptotic cells, supporting cell proliferation after injury, and promoting fibrosis, etc., and they play an important role in maintaining the balance in the body. Macrophages in the lungs play two roles. On the one hand, macrophages polarize into the M2 phenotype in the presence of IL4 and IL13, thereby accelerating the resolution of inflammation and promoting the proliferation of type II alveolar epithelial cells (Alveolar epithelial type II cells, AEC2s). After bleomycin injury in mice, alveolar macrophages can produce Wnt ligands to promote the proliferation of AEC2s; bone marrow-derived monocytes can be recruited to the lungs through CCL2/CCR2, polarized into M2 macrophages in the lung microenvironment, and promote the proliferation of AEC2 cells. On the other hand, a large number of aggregated macrophages may also secrete excessive TGFβ and other profibrotic factors, promote the proliferation of lung fibroblasts, and aggravate the progression of pulmonary fibrosis. In addition, monocyte-derived macrophages may play a more important role in the repair of lung injury and the development of fibrosis. When resident macrophages were removed by liposomal clodronate and then bleomycin injury was performed, the level of fibrosis did not change; however, when monocyte-derived macrophages were removed, fibrosis could be improved. This suggests that different macrophage subsets play different roles in the process of fibrosis. The recruitment of monocytes/macrophages and their role in pulmonary fibrosis need to be further studied.

Under steady state, the expression of TNFSF12 and TNFRSF12A is relatively broad. TNFSF12 is expressed in a variety of tissues and organs, mainly in macrophages, natural killer cells and monocytes; TNFRSF12A is also expressed in a variety of tissues and organs, mainly in epithelial cells and fibroblasts. The relationship between TNFSF12/TNFRSF12A signaling and fibrotic diseases is still unclear.

Summary of the invention

In view of the shortcomings of the prior art described above, in order to solve the technical problem that the relationship between TNFSF12/TNFRSF12A signaling and fibrotic diseases in the prior art is still unclear, the purpose of this application is to provide the application of TNFSF12 in fibrotic diseases. This application studies the role of the TNFSF12/TNFRSF12A signaling pathway in fibrosis, aiming to achieve the purpose of regulating the signaling pathway in adults, thereby achieving the purpose of treating fibrosis.

To achieve the above objectives and other related objectives, the first aspect of the present application provides the use of TNFSF12, selected from any one or more of the following:

1) As a target for diagnosis or treatment of fibrotic diseases;

2) As a marker for the diagnosis or prognosis of fibrotic diseases;

3) Preparation of products for diagnosis or prognosis of fibrotic diseases;

4) As a target for screening drugs for treating fibrotic diseases.

On the other hand, the present application provides the use of TNFSF12 or a promoter thereof in the preparation of a product, wherein the product has at least one of the following effects:

1) Inhibit the activation of fibroblasts;

2) inhibit the expression of extracellular matrix genes;

3) inhibit the expression of fibroblast markers;

4) inhibiting the expression of extracellular matrix genes and fibroblast markers induced by TGFβ1;

5) Induce the migration of bone marrow-derived macrophages;

6) Promote the repair of alveolar damage;

7) Inhibit organ fibrosis.

On the other hand, the present application provides a drug for treating fibrotic diseases, comprising an effective dose of TNFSF12 or a promoter thereof in the aforementioned use.

Another aspect of the present application provides a method for treating fibrotic diseases, comprising administering the TNFSF12 or a promoter thereof in the aforementioned use to a subject.

Compared with the prior art, the beneficial effects of this application are:

1. The present study shows that TNFSF12/TNFRSF12A can play a protective role in fibrotic diseases.

2. The present invention found that in IPF patient tissues and mouse pulmonary fibrosis models, despite the increase in receptor levels, TNFSF12 showed a downward regulation trend.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a single cell transcriptome data analysis. Figures 1A and 1B are CellChat analysis of published scRNAseq data of IPF lung (GSE135893). Figure 1C shows the expression of TNFSF12 and TNFRSF12A in different cell populations of normal lung and IPF lung. Figure 1D shows the cell-to-cell communication involved in the TNFSF12/TNFRSF12A signaling pathway in IPF lung.

Figure 2 shows the distribution and expression of TNFSF12/TNFRSF12A in fibrotic tissues. Figure 2A and Figure 2B show the mRNA and protein levels of TNFSF12 in IPF lungs, respectively. Figure 2C shows the mRNA level of Tnfsf12 in mouse lungs. Figure 2D and Figure 2E show the immunofluorescence staining levels of TNFRSF12A in IPF patients and fibrotic mouse lungs, respectively.

Figure 3 shows the effect of TNFSF12 on fibroblasts. Figures 3A and 3B show all upregulated and downregulated genes (log2 FC absolute value >= 1, p-value ≤ 0.05) after 100 ng/mL TNFSF12 stimulated mouse fibroblasts for 48 hours. Figures 3C and 3E show the expression of fibrosis-related genes and chemokines after TNFSF12 stimulated fibroblasts for 48 hours, respectively. Figure 3D shows the effect of TNFSF12 stimulation on fibroblast proliferation, with 10% FBS as a positive control. Figure 3F shows a schematic diagram of a macrophage migration experiment. Figure 3G shows macrophage migration caused by the conditioned medium of fibroblasts stimulated by TNFSF12. Figure 3H shows macrophage migration caused by the conditioned medium of fibroblasts with knockdown of Ccl2 or Ccl5 under TNFSF12 stimulation.

Figure 4 shows the effect of TNFSF12 on alveolar epithelial cells. Figures 4A and 4B show the clone formation efficiency of mouse AEC2s after 8 days of stimulation with 30ng/ml TNFSF12. Figure 4C shows the area of the organoid. Figures 4D and 4E show the proliferation of AEC2 organoids characterized by immunofluorescence staining. RAGE: characterizes AEC1; SFTPC: characterizes AEC2; Phospho-Histone H3: characterizes proliferation signals.

Figure 5 is a pulmonary fibrosis model of Tnfrsf12a knockout mice. Figure 5A is a survival curve of mice after 17 days of bleomycin treatment. Figure 5B is the change in Collagen1 mRNA level after 14 days of bleomycin treatment. Figure 5C is immunofluorescence staining of myofibroblast marker ACTA2, AEC1 marker RAGE, AEC2 marker SFTPC and profibrotic macrophage marker CD163 after 14 days of bleomycin treatment.

DETAILED DESCRIPTION

In order to make the invention purpose, technical scheme and beneficial effect of the present application clearer, the present application is further described below in conjunction with the examples. It should be understood that the examples are only used to explain the present application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified, and those familiar with the technology can easily understand other advantages and effects of the present application from the contents disclosed in this description.

After extensive exploration and research, the present application discovered the application of TNFSF12 in fibrotic diseases, and completed the present application on this basis.

In one aspect, the present application provides a use of TNFSF12, selected from any one or more of the following:

1) As a target for diagnosis or treatment of fibrotic diseases;

2) As a marker for the diagnosis or prognosis of fibrotic diseases;

3) Preparation of products for diagnosis or prognosis of fibrotic diseases;

4) As a target for screening drugs for treating fibrotic diseases.

In the use provided in the present application, the fibrotic disease is selected from pulmonary fibrosis, renal fibrosis, liver fibrosis, or cardiac fibrosis.

On the other hand, the present application provides the use of TNFSF12 or a promoter thereof in the preparation of a product, wherein the product has at least one of the following effects:

1) Inhibit the activation of fibroblasts;

2) inhibit the expression of extracellular matrix genes;

3) inhibit the expression of fibroblast markers;

4) inhibiting the expression of extracellular matrix genes and fibroblast markers induced by TGFβ1;

5) Induce the migration of bone marrow-derived macrophages;

6) Promote the repair of alveolar damage;

7) Inhibit organ fibrosis.

A promoter refers to a substance that can increase the activity and/or content of the TNFSF12 gene or its protein in vivo or in vitro. The substance can be a synthetic or natural compound, protein, nucleotide, etc.

In the use provided by the present application, the extracellular matrix gene includes Col1a1 and/or Col1a2. The fibroblast marker is selected from Acta2. In a specific embodiment of the present application, the fibroblast is a lung fibroblast, the extracellular matrix gene is a lung extracellular matrix gene, and the organ fibrosis is selected from pulmonary fibrosis, renal fibrosis, liver fibrosis or cardiac fibrosis.

On the other hand, the present application provides a drug for treating fibrotic diseases, comprising an effective dose of TNFSF12 or a promoter thereof in the aforementioned use.

The medicine provided in the present application also includes a pharmaceutically acceptable carrier or excipient.

Pharmaceutically acceptable carriers should be compatible with the gene editing system, that is, they can be blended with it without significantly reducing the effect of the pharmaceutical composition. Substances that can be used as carriers include sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tablets, stabilizers, antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffers, etc. These substances can be used to help the stability of the formulation, improve activity or produce an acceptable taste and smell in the case of oral administration as needed.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and can be made into any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops. The choice of pharmaceutical dosage form should match the mode of administration.

The administration method may be a conventional method, such as intravenous injection, intravenous drip, arterial infusion, gastrointestinal administration, parenteral administration, etc.

Among the drugs provided in the present application, the drug for treating fibrotic diseases is selected from the group consisting of a drug for treating pulmonary fibrosis, a drug for treating renal fibrosis, a drug for treating liver fibrosis or a drug for treating cardiac fibrosis.

On the other hand, the present application provides a method for treating fibrotic diseases, which is to administer the aforementioned TNFSF12 or its promoter to a subject. The subject is a mammal. Mammals are, for example, rodents, even-toed ungulates, odd-toed ungulates, lagomorphs, primates, etc. Primates are, for example, monkeys, apes or Homo sapiens. The treatment of fibrotic diseases is selected from the treatment of pulmonary fibrosis, the treatment of renal fibrosis, the treatment of liver fibrosis or the treatment of cardiac fibrosis.

The present application is further described below by way of examples, but the scope of the present application is not limited thereby.

Unless otherwise indicated, the conventional techniques of molecular biology, microbiology, cell biology, biochemistry, immunology and the like employed in the practice of the present invention are within the understanding and knowledge of the skilled person. These techniques are widely used and can be found in the following literature, such as: "Molecular cloning: A Laboratory Manual, Fourth edition" (M.R. Green, et al. 2014); "Oligonucleotide Synthesis" (M.J. Gait, et al. 1984); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting" (M.E. Babar, et al. 2011); "Short Protocols in Molecular Biology, Fifth edition" (F.M. Ausubel, et al. 2002); "Methods in Molecular Biology" (Humana Press); "Gene Transfer Vectors for Mammalian Cells" (J.H. Miller and M.P. Calos. 1987); "Culture of Animal Cell" (R.I. Freshney, et al.

2010); "Methods in Enzymology" (Academic Press, Inc); "Using Antibodies: A Laboratory Manual" (E. Harlow and D. Lane. 1999); "Handbook of Experimental Immunology" (L. A. Herzenberg, et al. 1997); " Current Protocols in Immunology" (J.E. Coligan, et al. 2002).

Example 1

This application analyzes the published IPF single cell transcriptome data and finds that there is cell communication between macrophages and fibroblasts in IPF. The secretome of macrophages and the cell surface receptors expressed by fibroblasts were analyzed, and the TNFSF12/TNFRSF12A signal was enriched (Figure 1). TNFSF12 is mainly expressed in immune cells, and its receptor TNFRSF12A is highly expressed in fibroblasts and epithelial cells (Figures 1, 2). This suggests that TNFSF12/TNFRSF12A may regulate the development of fibrosis by regulating intercellular communication.

Example 2

The present application stimulated fibroblasts with 100ng/mL TNFSF12 recombinant protein for 48 hours, resulting in the inhibition of extracellular matrix genes (Figure 3A), immune cell recruitment and upregulation of cell chemotaxis-related genes (Figure 3B). TNFSF12 recombinant protein stimulation not only inhibits the expression of background levels of extracellular matrix genes such as type I collagen Col1a1, Col1a2 and myofibroblast marker Acta2, but also inhibits the expression of these genes induced by TGFβ1 (Figure 3C). TGFβ1 is a classic profibrotic factor. In addition, TNFSF12 does not affect the proliferation of fibroblasts (Figure 3D). In general, TNFSF12 can not only inhibit the activation of fibroblasts, but also inhibit the activation of fibroblasts induced by TGFβ1.

Mouse lung fibroblasts were cultured in vitro and stimulated with 30 ng/mL recombinant TNFSF12 protein for 5 days. The conditioned medium was collected to induce the migration of bone marrow-derived macrophages (Figure 3F, G). Using siRNA to knock down the chemokines Ccl2 or Ccl5, the migration of mononuclear macrophages induced by the conditioned medium stimulated by TNFSF12 was inhibited (Figure 3H). These results indicate that TNFSF12 acts on fibroblasts, induces fibroblasts to produce chemokines, and recruits bone marrow-derived macrophages.

Example 3

Alveoli are important places for gas exchange, but they are easily damaged due to close contact with the external environment. Alveoli have two types of epithelial cells, namely type I alveolar epithelial cells (AlveolarepithelialtypeIcells, AEC1s) and type II alveolar epithelial cells (Alveolar epithelial typeII cells, AEC2s). The former complete the gas exchange process, while the latter reduce the surface tension of the alveoli by secreting surfactants. Under normal conditions, the lungs are highly quiescent tissues. Studies have shown that type II alveolar epithelial cells in the alveoli are alveolar stem cells, which can respond quickly after lung injury, proliferate and differentiate to repair alveolar damage.

The present application uses flow cytometry to sort out mouse AEC2s (Live/CD45-/CD31-/Epcam+/Integrinβ4-) for in vitro organoid culture. 30ng/ml TNFSF12 was added on the second day of culture, and cells were collected on the tenth day. From the results of bright field microscopy imaging and clone formation efficiency, TNFSF12 stimulation significantly promoted the clone formation efficiency and clone size of AEC2 (Figures 4A-4C). The organoids were embedded, sliced, and stained, and it was found that TNFSF12 stimulation significantly promoted the proliferation of AEC2 (Figures 4D, 4E). This shows that TNFSF12 has the ability to promote the rapid proliferation of AEC2, and therefore may promote the repair of alveolar damage.

Example 4

In order to reveal the function of TNFSF12/TNFRSF12A signal in mice, the present application established TNFRSF12A knockout mice (Tnfrsf12a-/- mice). TNFRSF12A gene knockout does not affect the survival and reproduction of mice themselves, but in the bleomycin-induced fibrosis model, the degree of fibrosis is significantly increased. After 17 days of bleomycin injury, the mortality rate of Tnfrsf12a-/- mice was much higher than that of wild mice (Figure 5A). Extracellular matrix gene expression was upregulated (Figure 5B), and more activated fibroblasts and pro-fibrotic CD163+ macrophages appeared in the alveolar region (Figure 5C). In addition, it can be seen that in Tnfrsf12a-/- mice, AEC2 was greatly reduced after bleomycin injury (Figure 5C). The above results are consistent with the results of in vitro experiments. This shows that the TNFSF12/TNFRSF12A signaling pathway plays a protective role in the fibrosis model.

Claims (10)

1. The purpose of TNFSF12 is selected from any one or more of the following: 1) As a target for diagnosis or treatment of fibrotic diseases; 2) As a marker for the diagnosis or prognosis of fibrotic diseases; 3) Preparation of products for diagnosis or prognosis of fibrotic diseases; 4) As a target for screening drugs for treating fibrotic diseases. 2. The use according to claim 1, characterized in that the fibrotic disease is selected from pulmonary fibrosis, renal fibrosis, liver fibrosis or cardiac fibrosis. 3. Use of TNFSF12 or its promoter in the preparation of a product, wherein the product has at least one of the following effects: 1) Inhibit the activation of fibroblasts; 2) inhibit the expression of extracellular matrix genes; 3) inhibit the expression of fibroblast markers; 4) inhibiting the expression of extracellular matrix genes and fibroblast markers induced by TGFβ1; 5) Induce the migration of bone marrow-derived macrophages; 6) Promote the repair of alveolar damage; 7) Inhibit organ fibrosis. 4. The use according to claim 3, wherein the extracellular matrix gene comprises Col1a1 and/or Col1a2; and/or, the fibroblast marker is selected from Acta2; And/or, the fibroblasts in 1) are lung fibroblasts; And/or, the extracellular matrix gene in 2) is a lung extracellular matrix gene; And/or, the organ fibrosis in 7) is selected from pulmonary fibrosis, renal fibrosis, liver fibrosis or cardiac fibrosis. 5. The use according to claim 3, wherein the promoter is a substance that can increase the activity and/or content of the TNFSF12 gene or its protein in vivo or in vitro; the substance is selected from a compound, a protein, or a nucleic acid. 6. A drug for treating fibrotic diseases, comprising an effective dose of TNFSF12 or a promoter thereof for use according to any one of claims 3 to 5. 7. The medicine according to claim 6, further comprising a pharmaceutically acceptable carrier or excipient. 8. The drug according to claim 6, characterized in that the drug for treating fibrotic diseases is selected from the group consisting of drugs for treating pulmonary fibrosis, drugs for treating renal fibrosis, drugs for treating liver fibrosis or drugs for treating cardiac fibrosis. 9. A method for treating fibrotic diseases, characterized by administering the TNFSF12 or a promoter thereof according to any one of claims 3 to 5 to a subject. 10. The method of claim 9, wherein the subject is a mammal; And/or, the treatment of fibrotic diseases is selected from the group consisting of treating pulmonary fibrotic diseases, treating renal fibrotic diseases, treating liver fibrotic diseases or treating cardiac fibrotic diseases.