Th9 cell and application of IL-9 secreted by Th9 cell

Abstract

The invention relates to application of Th9 cells and IL-9 secreted by the Th9 cells, the application refers to application of the Th9 cells and the IL-9 secreted by the Th9 cells as treatment targets of autoimmune diseases, the autoimmune diseases are autoimmune hepatitis, and the autoimmune hepatitis is immune-mediated inflammatory hepatitis. The compound is applied to treatment of autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to applications of Th9 cells and IL-9 secreted by them. Background Art

[0002] Autoimmune hepatitis (AIH) is a chronic autoimmune liver disease characterized by elevated transaminase levels, elevated autoantibodies, elevated γ-globulin or IgG levels, and interface hepatitis in liver sections. It can be acute or chronic, and can be accompanied by varying degrees of liver fibrosis. If not treated in time, it will eventually lead to cirrhosis and liver cancer.

[0005] T helper (Th) cells play a vital role in providing protection against invasive pathogens by secreting specific chemokines and cytokines. However, unrestrained T helper cell responses may lead to tissue damage and inflammatory diseases mediated by chronic inflammation. Evidence from human and animal studies has shown that CD4+Th cells play an important role in the pathogenesis of AIH, and it is known that the imbalance of Treg / Th17 is crucial for the development of AIH. At present, a new Th subset, Th9 cells, has attracted the attention of researchers, which mainly secrete the proinflammatory cytokine interleukin-9 (IL-9). Studies have found that Th9 has been shown to induce inflammation and aggravate inflammatory diseases, such as T cell-mediated allergic asthma, rheumatoid arthritis, colitis, and multiple sclerosis. This suggests that Th9 cells and IL-9 may become potential therapeutic targets.

[0006] It is well known that AIH is the result of the interaction of multiple genetic, immune and environmental factors. Environmental factors, especially those related to diet and hygiene habits, are considered to be modifiers of the disease. The relationship between dietary habits and the increased prevalence of inflammatory diseases of the digestive system is of particular interest. Previous studies have shown that non-alcoholic fatty liver disease (NAFLD) was induced in rats with a high-fat diet, and it was found that pre-existing NAFLD aggravated the severity of AIH. However, a characteristic factor in modern dietary habits that has been overlooked is its high salt (NaCl) content. Excessive salt intake affects multiple systems such as the cardiovascular and immune systems. At present, there are no reports on the effects of a high-salt diet on AIH.

[0007] In recent years, the incidence and prevalence of autoimmune diseases (AD) have increased. The pathogenesis of these diseases is multifactorial, involving a combination of genetic and environmental factors. Among them, dietary habits, especially excessive salt in food, are associated with the development of autoimmune diseases. Miranda et al. found that high levels of salt can regulate the composition and function of the intestinal microbiota, that is, reduce the relative abundance of lactobacilli and the level of butyrate, promote the pro-inflammatory state of the intestine, and thus aggravate inflammatory bowel disease (IBD). In addition to exacerbating colitis, Aguiar et al. found that eating HSD itself can also cause inflammation of the colonic mucosa of mice. The above changes induced by HSD are associated with increased frequencies of IL-23R+CD4+T cells and type 3 innate lymphoid cells (ILC3), and are dependent on IL-17, but not entirely dependent on T cells. In fact, high salt intake exacerbates experimental autoimmune encephalomyelitis (EAE) and may also affect the intestinal microbiota of mice, especially by depleting Lactobacillus and inducing Th17 activation.

[0008] A recent study found that HSD intake promoted immune activation of dendritic cells through the p38MAPK-STAT1 signaling pathway, thereby aggravating the symptoms of systemic lupus erythematosus (SLE). In addition, NaCl increased the differentiation of mouse and human Th17 cells in a dose-dependent manner. Compared with control collagen-induced arthritis (CIA) mice, CIA mice fed a high-salt diet had a higher proportion of Th17 cells in splenocytes and more severe clinical and histological arthritis. In summary, a high-salt diet has been shown to be associated with the development of a variety of autoimmune diseases, and its effects are mainly related to altering the intestinal microbiota and promoting the differentiation of helper T cells. These findings have certain significance for the treatment of patients with these autoimmune diseases because it is relatively easier to perform as a dietary intervention.

[0009] CD4+T helper cells are functionally differentiated into different subsets and participate in the occurrence and development of allergic diseases, inflammatory diseases, and autoimmune diseases. Th1 cells and Th2 cells are considered to be the two main typical prototypes differentiated from primitive CD4+T cells, and they have different cytokine secretion patterns. In recent years, several different Th cell subsets have also been discovered, including Th17, Th22, and regulatory T cells (Treg). These different subsets play complex roles in diseases through their respective expression of specific cytokines and transcription factors. Th9 cells are a new CD4+T cell subset differentiated from CD4+T cells induced by TGF-β and IL-4, and have the characteristic of secreting IL-9. Studies have shown that Th9 cells are associated with a series of autoimmune diseases such as allergic asthma, SLE, multiple sclerosis (MS), IBD, rheumatoid arthritis (RA), and psoriasis.

[0010] It has been reported that peripheral blood Th9 cells and serum IL9 are elevated in patients with allergic asthma, and IL-9 neutralization can reverse airway remodeling with impaired lung function. In addition, children with allergic asthma have significantly elevated IL-9 levels and the presence of antigen-specific Th9 cells, suggesting that Th9 cells may be the main source of IL-9 in children with allergic asthma. IL-9 has recently been shown to be a key player in regulating autoimmune responses in experimental autoimmune encephalomyelitis. There is evidence that IL-9 may play a regulatory role in central nervous system autoimmunity and interact with pathogenic Th17 cells. On the other hand, in vitro differentiated Th9 cells can induce EAE and peripheral neuritis. SLE is a chronic autoimmune disease characterized by abnormal activation of T cells. Researchers have found that the proportion of Th9 cells in peripheral blood mononuclear cells of SLE patients is increased, and mononuclear cells and CD4+T cells of SLE patients show increased IL-9 expression. In studies of experimental mouse colitis models and patients, CD4+T cell-derived IL-9 is believed to play a role in IBD. The expression level of IL-9 mRNA in intestinal tissue of UC patients is correlated with histological disease activity, and higher IL-9 levels in serum are also related to the disease activity of UC. Summary of the invention

[0011] 1. Technical issues to be resolved

[0012] In view of the deficiencies of the prior art, the present invention provides an application of Th9 cells and IL-9 secreted by them, which can be applied in the treatment of autoimmune diseases.

[0013] (II) Technical solution

[0014] To achieve the above-mentioned purpose, the present invention provides the following technical solution: application of Th9 cells and IL-9 secreted by them, wherein the application is application of Th9 cells and IL-9 secreted by them as therapeutic targets for autoimmune diseases.

[0015] Preferably, the autoimmune disease is autoimmune hepatitis.

[0016] Preferably, the autoimmune hepatitis is due to immune-mediated inflammatory hepatitis.

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Protein peak of mouse liver homogenate after separation.

[0019] Figure 2 :HE staining of liver tissue (A) and pathological score (B) of AIH model mice (**P<0.01); changes in serum AST (C) and ALT (B) levels of AIH model mice (**P<0.01). Note: ▲ indicates inflammatory cell infiltration.

[0020] Figure 3 : Body weight changes (A) (**P<0.01) and survival status (B) of mice in each group during the experiment.

[0021] Figure 4 : Liver gross appearance (Figure A), liver index (Figure B), spleen gross appearance (Figure C), spleen index (Figure D) of mice in each group (*P<0.05, **P<0.01)

[0022] Figure 5 :HE staining of liver tissue (Figure A) and liver histopathological scores (Figure B) of mice in each group (*P<0.05, **P<0.01). Note: ▲ indicates inflammatory cell infiltration, scale bar is 50μm.

[0023] Figure 6 :Serum Na of mice in each group + , Cl - Levels of AST, ALT and ALB (*P<0.05.**P<0.01).

[0024] Figure 7: Immunofluorescence detection of CD4 and IL-9 in liver tissues of mice in each group (Figure A); Immunofluorescence detection of CD4 and IL-9 protein expression in liver tissues of mice in each group (scale bar, 50 μm); DAPI: blue; CD4: green; IL-9: red; Statistical analysis of the number of CD4 and IL-9 co-stained cells in the liver under 100X field of view (Figure B) (**P<0.01).

[0025] Figure 8 :Flow cytometry detection of spleen Th9 cells in each group of mice (Figure A) Relative percentage of spleen Th9 cells in each group of mice; Statistical analysis of relative percentage of spleen Th9 cells in each group of mice (Figure B) (**P<0.01).

[0026] Fig. 9 : Relative expression of IL-9 mRNA in liver tissue of mice in each group (*P<0.05, **P<0.01).

[0027] Fig.10 : IL-9 expression levels in liver homogenate of mice in each group (*P<0.05, **P<0.01).

[0028] Fig.11 : Expression of transcription factor IRF4 in liver tissue of mice in each group (Figure A); Western blot was used to detect the expression of IRF4 protein in liver tissue and semi-quantitative analysis of IRF4 protein level in liver tissue (Figure B) (*P<0.05, **P<0.01).

[0029] Fig.12 : CD4+CD62L+ cell sorting rate (Figure A); naive T cells were induced by TGF-β and IL-4 under normal or high salt conditions (Figure B) (*P<0.05); naive T cells were induced by only TGF-β under normal or high salt conditions (Figure C) (nsP>0.05); naive T cells were induced by only IL-4 under normal or high salt conditions (Figure D) (nsP>0.05).

[0030] Fig.13a : Th9 cell RNA quality test results (Figure A); Th9 cell RNA bands (Figure B); Obtain high-quality effective sequence statistics (Figure C); Effective sequencing data alignment to the reference genome statistics (Figure D); Gene expression level statistics (Figure E); Control group VS NaCl 40mM group sample clustering diagram (Figure F); Control group VS NaCl 40mM group differential gene expression volcano map (Figure G); Control group VS NaCl 40mM group differential gene heat map (Figure H) Control group VS NaCl 40mM group differential gene GO functional classification items (Figure I);

[0031] Fig.13b : GO enrichment analysis of biological processes of differentially down-regulated genes in the Control group VS NaCl 40mM group (Figure J); KEGG enrichment analysis of differentially up-regulated genes in the Control group VS NaCl 40mM group (Figure K); KEGG enrichment analysis of differentially down-regulated genes in the Control group VS NaCl 40mM group (Figure L).

[0032] Fig.14 : Detection of splicing sites in the sequencing data of each sample (Figure A); Proportion of detected alternative splicing events (Figure B); Statistics of differential alternative splicing events between the Control group and the NaCl 40mM group (Figure C); Five-dimensional Venn diagram analysis of the top five differentially alternatively spliced ​​genes (Figure D); GO functional classification items of differentially alternatively spliced ​​genes between the Control group and the NaCl 40mM group (Figure E); GO enrichment analysis of biological processes of differentially alternatively spliced ​​genes between the Control group and the NaCl 40mM group (Figure F); KEGG enrichment analysis of differentially alternatively spliced ​​genes between the Control group and the NaCl 40mM group (Figure G).

[0033] (III) Beneficial effects

[0034] A use of Th9 cells and the IL-9 secreted by them, specifically the use of Th9 cells and the IL-9 secreted by them as therapeutic targets for autoimmune diseases, which proposes the therapeutic application of Th9 cells and the IL-9 secreted by them in autoimmune diseases. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The present invention provides an application of Th9 cells and IL-9 secreted therefrom, specifically, an application of Th9 cells and IL-9 secreted therefrom as a target for treating autoimmune diseases, wherein the autoimmune disease is autoimmune hepatitis, which is an inflammatory hepatitis mediated by immunity.

[0037] The present invention provides a new use of Th9 and the IL-9 secreted by it, and determines that the expression level thereof is associated with autoimmune diseases, including autoimmune hepatitis, and can be used as a new target for preventing and treating autoimmune hepatitis.

[0038] In one embodiment of the present invention, a liver-specific S100 antigen was used to induce C57BL / 6 mice to construct an autoimmune hepatitis male mouse model.

[0039] The present invention is described in detail below in conjunction with specific embodiments and drawings.

[0040] Embodiment 1:

[0041] Construction of an autoimmune hepatitis mouse model

[0042] The mice used in the embodiment are male SPF-grade C57BL / 6 mice C (16.7g ± 1.0g, 6 weeks old) obtained from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (certificate number: 0001766), and are raised in the Experimental Animal Center of Lanzhou University under constant temperature and humidity conditions. In order to adapt the mice to the new environment, the experiment started 1 week after arriving at the central animal facility, and diet and drinking water were not restricted during the period. This experimental process complies with the "Regulations on the Administration of Experimental Animals of the People's Republic of China" and ethical requirements, and is approved by the Ethics Committee of the First Hospital of Lanzhou University, and the ethics approval number is: LDYYLL2022-347.

[0043] 1. Experimental Animal Grouping

[0044] Thirty-six male C57BL / 6 mice (16.7 g ± 1.0 g, 6 weeks old) were randomly divided into 4 groups (n = 9) according to the random number table method, including: ① control group; ② high salt diet (HSD) group; ③ autoimmune hepatitis model (AIH) group and ④ autoimmune hepatitis model + high salt diet (AIH + HSD) group. Figure 2 As shown in A, mice in the HSD group and AIH+HSD group received a high-salt diet containing 4% NaCI and tap water containing 1% NaCI at will, and the other two groups received a normal diet (ND) and tap water. After the four groups of mice received the corresponding diets for 3 weeks, the AIH group and AIH+HSD group mice began to establish the AIH model by intraperitoneal injection of freshly prepared mouse liver S100 antigen.

[0045] 2. Preparation and Isolation of Liver-Specific S100 Antigen

[0046] (1) Four healthy male C57BL / 6 mice were selected and disinfected after anesthesia. The mice were dissected under sterile conditions to expose the abdominal organs and heart. A small cut was made in the right atrial appendage with ophthalmic scissors to allow pre-cooled sterile saline to be injected from the left ventricle to flow through the entire systemic circulation and flush out the blood in the liver. The perfusion was stopped when the liver turned grayish white.

[0047] (2) The mouse liver was completely separated and placed in a culture dish containing 1x PBS. It was completely minced on ice and then transferred to a glass homogenizer and ground thoroughly to destroy the cell structure.

[0048] (3) To further disrupt the cells and fully release the protein, the homogenate was transferred into a 15 mL centrifuge tube and repeatedly frozen and thawed three times at -80°C.

[0049] (4) Centrifugation: Centrifuge the liver tissue homogenate at 150Xg and 40℃ for 15 min and collect the supernatant.

[0050] (5) Ultracentrifugation: Transfer the supernatant into a centrifuge bottle and centrifuge at 100,000 × g, 40°C for 1 h. Collect the supernatant, which is the liver S100 antigen.

[0051] (6) Place the sterilized 90 cm glass chromatography column vertically on an iron stand, dilute the agarose gel CL-6B with sterile water and mix thoroughly on a magnetic stirrer, slowly and evenly pour it into the chromatography column along the tube wall, open the water stop clamp at the lower end, and use eluent (a mixed solution of 0.05 mmol / L Na3PO4·12Ha0 and 0.15 mmol / L NaCI, PH=7.0) to circulate and rinse the gel to allow it to settle freely. When the upper end of the gel is 8 cm away from the top of the column, close the water stop clamp. Do not allow the gel to dry during the entire process.

[0052] (7) Open the water stop clamp of the chromatography column. When the liquid level is close to the gel surface, use a micropipette to slowly and evenly add the S100 protein to the top of the gel. After all the S100 protein has entered the gel, continue to add the pre-cooled eluent at a uniform speed.

[0053] (8) Observe the migration of the light brown S100 protein. When it descends to the middle of the column, start collecting the sample with 1.5 mL of EP, 1 mL per tube. At the same time, detect the OD (A280) value on an ultra-micro nucleic acid protein detector. Stop the chromatography when the OD value is continuously 0.

[0054] (9) Draw an OD value curve and retain the S100 protein corresponding to the first peak as shown in the curve. Calculate the protein concentration (mg / mL) based on the A280 value. This peak is the non-toxic small molecular weight part.

[0055] (10) For better immunity, the first peak S100 protein with a concentration of 0.5-2g / L and an equal volume of complete Freund's adjuvant (CFA) were respectively loaded into a 5 ml syringe, connected to a three-way tube, and the two were thoroughly mixed on ice until they did not disperse after being left for a period of time. The mixture was the liver S100 antigen required for modeling. Figure 1As shown, gel chromatography can effectively separate the molecular peaks of S100. After separation by a 90 cm column, three protein peaks were present. The last non-toxic first peak (small molecule portion) was selected to induce the mouse AIH model. This peak was present in approximately the 58th to 78th tube.

[0056] 3. Establishing AIH model

[0057] After the four groups of mice received the corresponding diet for 3 weeks, the mice in the AIH group and the AIH+HSD group were intraperitoneally immunized with 0.5 mL of liver S100 antigen on the 1st and 7th day, and the other two groups were injected with an equal amount of normal saline. The mice were fixed in a head-down position, and a 1 mL insulin syringe was used to pierce the abdominal cavity from one side of the lower abdomen. The antigen could be injected when no blood or urine was aspirated. On the 28th day, the mice were anesthetized and killed for research. The corresponding diet continued to be maintained during the modeling period, and the weight, food and water intake, activity and mortality of the mice were monitored throughout the animal experiment. Figure 2 As shown, in order to test whether the mouse AIH model was successfully established in this study, the liver tissue of the mice in the model group was stained with HE to examine its pathological changes, and the changes in AST and ALT levels in the serum of the mice in the model group were detected. Figure 2 As shown in Figure 2A, HE staining results showed that compared with the mice in the Control group, a large number of inflammatory cells infiltrated the portal vein and central vein of the liver tissue of the mice in the AIH group, showing typical manifestations of interface hepatitis. The histopathological scores of the mice in the AIH group were increased ( Figure 2 -B, P<0.01). Compared with the mice in the Control group, the serum AST level of mice in the AIH group was significantly increased ( Figure 2 -C, P<0.01, serum ALT level was also significantly increased ( Figure 2 -DP<0.01).

[0058] Example 2:

[0059] High-salt diet aggravates S100-induced autoimmune hepatitis in mice

[0060] 1. General changes

[0061] During the entire experimental period, after the HSD group and AIH+HSD group were given a 4% high-salt diet and 1% high-salt drinking water, their water intake and urine output increased rapidly, their mental state became irritable, and their activity level increased compared with the normal diet group. Figure 3) There was no significant difference in the initial grouping (P = 0.92). Within one week after the first intraperitoneal injection of S100 liver antigen, the AIH group and the AIH + HSD group showed depression, decreased activity, unformed stool, easy to adhere to the perianal area, dry and dull hair, and weight loss. Among them, the condition of 3 mice in the AIH + HSD group continued to deteriorate, and their weight gradually decreased, and they died one after another within the 5th week. At this mild concentration of S100 liver antigen, no mice in the AIH group died. One week after the first administration of S100 liver antigen, the mental state of the remaining mice in the AIH group and the AIH + HSD group gradually improved, and their weight increased. At the end of the experiment, the AIH+HSD group had the lowest body weight (22.13g±1.51g), and the AIH group had the highest body weight (26.52g±2.90g). The difference between the two groups was statistically significant (P<0.01). The differences between the two groups and the Control group (24.42g±1.13g) were statistically significant (P<0.05). The AIH group mice were dissected and found to have different amounts of ascites in the abdominal cavity.

[0062] 2. Changes in the gross morphology of the liver and spleen and the liver and spleen index.

[0063] like Figure 4 -A, compared with the Control group, there was no significant difference in the gross appearance of the liver in the HSD group, while the gross appearance of the liver in the AIH group was significantly changed, including changes in overall shape, increased hardness, tissue edema, increased volume, thickened edges, and dull color. The diaphragmatic surface of the liver was covered with some white proliferative connective tissue, which was difficult to separate from the diaphragm and surrounding organs during dissection. Compared with the AIH group, the above-mentioned gross morphological changes in the liver were more obvious in the AIH+HSD group, with more connective tissue covering and a larger liver index (Figure B) (0.079±0.020vs 0.066±0.009, P<0.05). As shown in Figure C, compared with the Control group, there was no significant change in the gross morphology of the spleen in the HSD group, while the spleen in the AIH group was significantly larger, darker in color, and more tightly adhered to the surrounding tissues during dissection. The spleen volume of mice in the AIH+HSD group was longer and wider than that of mice in the AIH group. Similarly, the spleen index (Figure D) was also larger (0.011±0.005 vs 0.007±0.002, P<0.01).

[0064] 3. Pathological changes of liver tissue

[0065] HE staining of mouse liver tissue ( Figure 5-A) showed that compared with the mice in the Control group, the AIH group mice had a large number of inflammatory cell infiltrations in the portal vein area and near the central vein of the liver tissue, with typical manifestations of interface hepatitis, and the histopathological score (Figure B) was also higher (1.50±0.76 vs 0.00±0.00, P<0.01). There was basically no inflammatory cell infiltration in the liver tissue of the HSD group mice, and the liver histopathological score of the AIH+HSD group mice was also higher than that of the HSD group mice (2.67±0.52 vs 0.13±0.35, P<0.01). Compared with the AIH group mice, the liver tissue sections of the AIH+HSD group mice had more inflammatory cell infiltration, and cell fatty degeneration and dissolution necrosis were observed, and the tissue score was also higher (2.67±0.52 vs 1.50±0.76, P<0.05).

[0066] 4. Changes in serum biochemical indicators.

[0067] like Figure 6As shown in the figure, serum biochemical tests showed that compared with the mice in the Control group, the serum Na+ concentration of mice in the HSD group (Figure A) was increased to a certain extent (159.33mmol / L±3.93mmol / L vs 153.33mmol / L±1.03mmol / L, P<0.01). Compared with the mice in the AIH group, the serum Na+ concentration of mice in the AIH+HSD group was also increased to a certain extent (156.80mmol / L±2.77mmol / L vs 152.83mmol / L±1.33mmol / L, P<0.05). However, there was no statistically significant difference in serum Na+ concentration between the mice in the Control group and the AIH group, and between the mice in the HSD group and the AIH+HSD group. There was no statistically significant difference in serum Cl- concentration (Figure B) among the four groups of mice (P=0.81). As shown in Figure C, compared with the mice in the Control group, the serum AST level of mice in the AIH group was increased (232.53U / L±62.04U / L vs 99.25U / L±8.96U / L, P<0.01). Compared with the mice in the HSD group, the serum AST level of mice in the AIH+HSD group was increased (319.183U / L±104.42U / L vs 113.70U / L±7.64U / L, P<0.01). The serum AST level of mice in the AIH+HSD group was higher than that in the A group (319.183U / L±104.42U / L vs 232.53U / L±62.04U / L, P<0.05). As shown in Figure D, compared with the mice in the Control group, the serum ALT level of mice in the AIH group was increased (124.68U / L±50.72U / L vs 36.82U / L±9.00U / L, P<0.01). Compared with the mice in the HSD group, the serum ALT level of mice in the AIH+HSD group was increased (183.90U / L±72.42U / L vs 37.61U / L±6.10U / L, P<0.01). The serum ALT level of mice in the AIH+HSD group was higher than that in the AIH group (183.90U / L±72.42U / L vs 124.68U / L±50.72U / L, P<0.05). There was no significant difference in serum ALB (Figure E) among the four groups of mice (P=0.43).

[0068] Example 3:

[0069] High-salt diet promotes Th9 cell differentiation in AIH mice

[0070] 1. Infiltration of Th9 cells in liver tissue

[0071] CD4 and IL-9 immunofluorescence co-staining method was used to detect the level of Th9 cells in liver tissue. Figure 7-A, green fluorescence represents CD4 expression, red fluorescence represents IL-9 expression, and the number of CD4+IL-9+ cells under the 100X field of view is counted. The results are shown in Figure B. Compared with the mice in the Control group, the mice in the AIH group had more CD4+IL-9+ cells in the liver tissue (17.00±3.39vs0.00±0.00, P<0.01). Similarly, compared with the mice in the HSD group, the mice in the AIH+HSD group also had more CD4+IL-9+ cells in the liver tissue (33.20±5.81vs 0.60±0.89, P<0.01. Compared with the mice in the AIH group, the number of CD4+IL-9+ cells in the liver tissue of the mice in the AIH+HSD group increased significantly (33.20±5.81vs 17.00±3.39, P<0.01).

[0072] 2. Changes in the proportion of Th9 cells in spleen mononuclear cells.

[0073] Percp / Cyanine 5.5anti-mouse CD4 and PE anti-mouse IL-9 were used to label spleen mononuclear cells, and flow cytometry was used to detect the proportion of CD4+IL-9+ cells in all spleen mononuclear cells. Figure 8 -A shows that compared with the mice in the Control group, the proportion of CD4+IL-9+ cells in the spleen mononuclear cells of the mice in the AIH group was higher (1.54%±0.25% vs 0.33%±0.08%, P<0.01). Similarly, compared with the mice in the HSD group, the proportion of CD4+IL-9+ cells in the spleen mononuclear cells of the mice in the AIH+HSD group was higher (2.46%±0.87% vs 0.38%±0.08%, P<0.01). Compared with the mice in the AIH group, the proportion of CD4+IL-9+ cells in the spleen mononuclear cells of the mice in the AIH+HSD group was significantly increased (Figure B) (C2.46%±0.87% vs 1.54%±0.25%, P<0.01).

[0074] Example 4:

[0075] High-salt diet promotes IL-9 mRNA expression in liver tissue of AIH mice.

[0076] like Fig. 9As shown, Realtime PCR was used to detect the expression of IL-9 mRNA in mouse liver tissue. Compared with the mice in the Control group, the expression of IL-9 mRNA in the liver tissue of the mice in the AIH group was significantly increased (0.000015±0.0000093 vs 0.0000035±0.0000023, P<0.01). Compared with the mice in the HSD group, the expression of IL-9 mRNA in the liver tissue of the mice in the AIH+HSD group was significantly increased (0.000021±0.000010 vs 0.0000065±0.0000059, P<0.01). Similarly, compared with the mice in the AIH group, the expression of IL-9 mRNA in the liver tissue of the mice in the AIH+HSD group was significantly increased (0.000021±0.000010 vs 0.000015±0.0000093, P<0.05).

[0077] Example 5:

[0078] High-salt diet promotes IL-9 secretion in liver tissue of AIH mice

[0079] like Fig.10 As shown, ELISA kit was used to detect the expression of IL-9 protein in liver tissue homogenate of mice. Compared with the mice in the Control group, the expression of IL-9 protein in liver tissue homogenate of mice in the AIH group was significantly increased (16.75ng / mL±8.93ng / mL vs 6.08ng / mL±3.17ng / mL, P<0.01). Compared with the mice in the HSD group, the expression of IL-9 protein in liver tissue homogenate of mice in the AIH+HSD group was significantly increased (24.92ng / mL±3.86ng / mL vs 8.79ng / mL±3.24ng / mL, P<0.01). Similarly, compared with the mice in the AIH group, the expression of IL-9 protein in liver tissue homogenate of mice in the AIH+HSD group was also significantly increased (24.92ng / mL±3.86ng / mL vs 16.75ng / mL±8.93ng / mL, P<0.05).

[0080] Example 6:

[0081] High-salt diet promotes the expression of IRF4 mRNA and protein in liver tissue of AIH mice

[0082] like Fig.11As shown, Realtime PCR was used to detect the expression of IRF4 mRNA in mouse liver tissue. Compared with the mice in the Control group, the expression of IRF4 mRNA in the liver tissue of the mice in the AIH group was significantly increased (0.00065±0.00026 vs 0.00027±0.000053, P<0.01). Compared with the mice in the HSD group, the expression of IRF4 mRNA in the liver tissue of the mice in the AIH+HSD group was significantly increased (0.00087±0.00010 vs 0.00021±0.000088, P<0.01). Similarly, compared with the mice in the AIH group, the expression of IRF4 mRNA in the liver tissue of the mice in the AIH+HSD group was significantly increased (0.00087±0.00010 vs 0.00065±0.00026, P<0.05). Western blot was used to detect the expression of IRF4 protein in mouse liver tissue. Compared with the mice in the Control group, the expression of IRF4 protein in the liver tissue of the mice in the AIH group was significantly increased (0.59±0.07 vs 0.15±0.01, P<0.01). Compared with the mice in the HSD group, the expression of IRF4 protein in the liver tissue of the mice in the AIH+HSD group was significantly increased (0.80±0.07 vs 0.25±0.11, P<0.01). Similarly, compared with the mice in the AIH group, the expression of IRF4 protein in the liver tissue of the mice in the AIH+HSD group was significantly increased (0.80±0.07 vs 0.59±0.07, P<0.01).

[0083] Example 7:

[0084] High salt promotes the differentiation of Th9 cells in vitro.

[0085] 1. Identification of the purity of CD4+Naive T cells sorting.

[0086] like Fig.12 As shown in A, CD4+Naive T cells were obtained from spleen mononuclear cells by two-step sorting using MACS magnetic cell sorter, and cells were labeled with Percp / Cyanine 5.5anti-mouse CD4 and APC anti-mouse CD62L, and the sorting rate of CD4+CD62L+ cells was identified by flow cytometry. The results showed that the purity of CD4+Naive T cells sorted from spleen exceeded 96%, which could meet the requirements of subsequent experiments.

[0087] 2. Effect of high salt on the differentiation of Th9 cells

[0088] like Fig.12As shown in BD, the co-stimulation of cytokines TGF-β and IL-4 is a necessary condition for inducing CD4+Naive T cells to differentiate into Th9 cells. In this experiment, the effect of high salt (40mMNaCI) on Th9 cell differentiation was observed under the conditions of adding or not adding TGF-β and IL-4. When the culture conditions contained both TGF-β (5ng / mL) and IL-4 (50ng / mL), the proportion of Th9 cells in the TGF-β+IL-4 group was 14.69%±9.12%, and the proportion of Th9 cells in the TGF-β+IL-4+NaCl group was 29.90%±4.85%, and the difference was statistically significant (P<0.05) (Figure B). When the culture conditions only contained TGF-β (5ng / mL), the proportion of Th9 cells in the TGF-β group was 2.90%±0.80%, and the proportion of Th9 cells in the TGF-β+NaCI group was 3.50%±1.26%, and the difference was not statistically significant (P=0.53) (Figure C). When the culture conditions only contained IL-4 (50 ng / mL), the proportion of Th9 cells in the IL-4 group was 2.37%±1.80%. The proportion of Th9 cells in the IL-4+NaCl group was 2.31%±1.52%, with no significant difference (P=0.97) (Figure D).

[0089] Example 8:

[0090] Transcriptome analysis of Th9 cells induced by high salt

[0091] 1. RNA quality inspection results of Th9 cells.

[0092] The RNA quality test results of two samples of Th9 cells in each group of the Control group and the NaCI40 mM group are shown in Fig.13a As shown in A, the RNA extracted from all samples meets the quality control standards, the total amount is qualified, the purity is qualified, and the bands are clear, that is, the samples meet the requirements for library construction. Fig.13a B.

[0093] 2. Transcription sequencing data analysis.

[0094] Transcriptome sequencing was performed on all samples. Fig.13aAs shown in Figure C, after the transcription sequencing data was quality controlled, the analysis data results showed that the sample data had good uniformity, the Q20 of each sample was above 97.89%, the Q30 was above 93.89%, the GC was between 48%-50%, and the cleanreads of the sample ranged from 96.85% to 98.46%. The quality-controlled data was sequenced with the reference genome, and the sequence alignment Totalmapped indicated the sequence located on the genome. As shown in Figure D, the proportion of sequence reads with alignment positions in all Th9 cell samples exceeded 92% of all reads; the proportion of reads with only one alignment position (Uniquely mapped) accounted for more than 89% of all reads; the proportion of sequence reads with multiple alignment positions (Multiple mapped) accounted for less than 10.29% of all reads, and finally each sample obtained a total of 72.4M±2.0M high-quality sequences.

[0095] 3. Gene expression level.

[0096] The gene expression abundance is characterized by calculating FPKM (the number of reads per kilobase length of a gene per million reads). The gene expression level statistics obtained are as follows: Fig.13a The FPKM values ​​of all 55,360 genes shown in E were used to calculate the correlation matrix based on the Pearson correlation coefficient. The diagonal of the heat map shows the Pearson correlation between high-salt-induced Th9 cells and control cells, where the correlation matrix is ​​symmetrical, indicating that the two biological replicates are highly correlated ( Fig.13a F).

[0097] 4. Analysis of differentially expressed genes.

[0098] Based on the above RNA-seq data obtained from high-salt-induced Th9 cells and control cells, genes that may be affected by high salt were explored at the transcriptional level. Volcano plot ( Fig.13a G) Analysis showed that there were 58 genes that changed significantly in the Control group compared with the NaCI 40mM group, of which 29 genes were significantly upregulated and 29 genes were significantly downregulated. In order to explore whether the gene expression change trends in the biological replicate groups were similar, all significantly differentially expressed genes were taken as a union, and a heat map was made based on the expression levels. As shown in Figure H, the clustering results of the 58 differentially expressed genes showed that under the same conditions, the gene expression change trends in the two biological replicate samples were similar and could be clustered into one category. The Control group was clustered into two clusters, high expression and low expression, respectively, which was significantly different from the NaCI 40mM group.

[0099] 5. Gene Ontology enrichment analysis of differentially expressed genes.

[0100] GO functional classification is divided into three categories: biological process (BP), molecular function (MF) and cellular component (CC). The GO functional classification items of differentially expressed genes in Th9 cells between the control group and the NaCI40 mM group and their enrichment in MF, BP and CC are shown in the figure. Fig.13a As shown in I. In terms of biological process analysis, the differentially downregulated genes in high salt-induced Th9 cells were mainly enriched in immune system processes and adaptive immune responses ( Fig.13b J), while the differentially up-regulated genes had no significantly enriched GO terms.

[0101] 6. KEGG Pathway enrichment analysis of differentially expressed genes.

[0102] The results of KEGG enrichment analysis of differentially expressed genes showed that the 10 signaling pathways with the highest risk factors of up-regulated genes in the main enriched signaling pathways between the Control group and the NaCI40 mM group included malaria pathway, inflammatory bowel disease pathway, graft-versus-host disease pathway, pertussis pathway, rheumatoid arthritis pathway, Th17 cell differentiation pathway, herpes simplex virus type 1 infection pathway, C-type lectin receptor signaling pathway, Jak-STAT signaling pathway, and tuberculosis pathway ( Fig.13b K). The 10 signaling pathways with the highest risk of down-regulated genes included the B cell receptor signaling pathway, the hematopoietic cell lineage pathway, the primary immunodeficiency pathway, the intestinal immune network pathway for IgA production, the Epstein-Barr virus infection pathway, the Staphylococcus aureus infection pathway, the asthma pathway, the renin-angiotensin system pathway, the cell attachment molecule pathway, and the tuberculosis pathway ( Fig.13b L).

[0103] Example 9:

[0104] Analysis of alternative splicing events in Th9 cells induced by high salt

[0105] 1. Splicing site detection and statistics of alternative splicing events.

[0106] The splice junction (SJ) of each sample in the Control group and the NaCl40 mM group was analyzed using HISAT2 software. The results are as follows Fig.14As shown in A, Allsplice junction represents the number of splicing sites detected, Knownsplice junction represents the number of known splicing sites detected, and Novelsplice junction represents the number of new splicing sites detected. The detected SJ results were subjected to overall analysis and classification of splicing modes, and various alternative splicing events (ASE) were counted. The involved ASEs were divided into 9 categories in total: exon skipping (ES), alternative 5'splice site (ASSS), alternative 3'splice site (A3SS), mutually exclusive exons (MXE), variable 5'UTRs (mutually exclusive 5'UTRs, SpMXE), variable 3'UTRs (mutually exclusive 3'UTRs, 3pMXE), variable 3'splice site and exon skipping occurring simultaneously (A3SS&ES), variable 5'splice site and exon skipping occurring simultaneously (ASSS&ES), and cassette exon. Statistics of splicing events of each splicing type between the control group and the NaCI 40mM group, a total of 39340 ASEs were identified, including 18285 known ASEs and 21055 new ASEs. Fig.14 As shown in B.

[0107] 2. Analysis of differences in alternative splicing events between samples.

[0108] like Fig.14 As shown in Figure C, a total of 341 differential alternative splicing events (DASE) were screened, of which 171 were DASEs (up DASEs) with a higher proportion in the NaCI 40mM group than in the Control group, and 170 were DASEs (down DASEs) with a lower proportion in the NaCI 40mM group than in the Control group. A3SS and ASSS were the main DASEs, accounting for 28.7% each. These data indicate that high salt globally regulates ASE in Th9 cell differentiation. Next, based on the top five DASE types, a five-dimensional Venn diagram was drawn for the genes that underwent DASE. The results are shown in Figure 2. Fig.14 As shown in D, the same gene can undergo different types of ASE, but a single alternative splicing type is predominant.

[0109] 2. Overlap analysis of alternative splicing regulation and differential expression. A total of 307 differential alternative splicing genes (DASGs) were screened. DASGs and differentially expressed genes (DEGs) in each sample were integrated and analyzed. The Venn diagram ( Fig.14 E) The number of genes showing significant differences in both expression level and alternative splicing level is 0.

[0110] 4. Gene Ontology enrichment analysis of differentially alternatively spliced ​​genes.

[0111] GO functional classification items of differentially alternatively spliced ​​genes in Th9 cells between the control group and the NaCI 40mM group and their enrichment in MF, BP and CC are shown in Figure 2 Figure 3 As shown. In terms of biological process analysis, high salt-induced DASG in Th9 cells is mainly related to the regulation of chromatin organization, cell response to DNA damage stimuli, and histone acetylation ( Fig.14 F).

[0112] 5. KEGG Pathway enrichment analysis of differentially alternatively spliced ​​genes

[0113] The results of KEGG enrichment analysis of differentially spliced ​​genes showed that the main enriched signaling pathways of the differential DASG between the Control group and the NaCI 40mM group included homologous recombination pathway, Fanconi anemia pathway, Yersinia infection pathway, endocrine resistance pathway, cell senescence pathway, chlorophyll metabolism pathway, TNF signaling pathway, lysosomal pathway, Notch signaling pathway, and ubiquitin-mediated proteolysis pathway ( Fig.14 G).

[0114] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of Th9 cells and IL-9 secreted therefrom, characterized in that: The application is the application of Th9 cells and the IL-9 secreted by them as therapeutic targets for autoimmune diseases.

2. The use of Th9 cells and IL-9 secreted therefrom according to claim 1, characterized in that: The autoimmune disease is autoimmune hepatitis.

3. The use of Th9 cells and IL-9 secreted therefrom according to claim 2, characterized in that: The autoimmune hepatitis is due to immune-mediated inflammatory hepatitis.

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