An application of cGAS

Abstract

The present invention provides an application of cGAS, namely cyclic GMP-AMP synthase. The application includes the use of cGAS or a cGAS analog in the preparation of a product for diagnosing, preventing or treating overweight or obesity. The present invention for the first time clearly demonstrates that the lack of cGAS in intestinal CD4+ T cells exacerbates diet-induced obesity and metabolic dysfunction, and exacerbates obesity by reducing thermogenesis and energy consumption. At the same time, knocking out cGAS in intestinal CD4+ T cells significantly affects the generation of the strain Alistipes dispar, and specifically leads to a decrease in the content of the strain Alistipes dispar in the intestine. And the strain Alistipes dispar is closely related to overweight and obesity in the population. Oral administration of this strain helps to enhance thermogenic metabolism, reduce weight gain, and achieve the effect of weight loss.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to an application of a cyclic synthetase. Background Art

[0002] cGAS, the full name is cyclic GMP-AMP synthase, was discovered by Professor Zhijian Chen, who won the 2019 Breakthrough Prize in Science for this. The full name of cGAS is "cyclic GMP-AMP synthase", which is located in the cytoplasm. It can recognize DNA that should not appear in the cytoplasm, and catalyze GTP and ATP to synthesize "cyclic GMP-AMP" (cGAMP). cGAMP binds to and activates the endoplasmic reticulum protein STING, and then transduces the signal into the cell nucleus through the STING pathway, regulates gene transcription, and turns on the immune response. This means that cGAS can recognize exogenous DNA in cells to turn on immune responses, and is an "accelerator" of the immune system. Strengthen the body's "attack power" against tumors and pathogenic microorganisms.

[0003] According to current research, because cGAS is the "accelerator" of the immune system, its abnormal expression in the body will cause a series of immune disorders, leading to an overly strong immune response in the body, and then causing the occurrence of autoimmune diseases. Systemic lupus erythematosus is a common autoimmune disease. Chen Zhijian's research group has discovered the correlation between cGAS and systemic lupus erythematosus. Therefore, the use of drugs that inhibit cGAS may be used to treat systemic lupus erythematosus. In addition, PD-1 is the brake of the immune system, and cGAS is the accelerator of the immune system. If PD-1 inhibitors and cGAS analogs can be used at the same time, the immune system can release the brakes and press the accelerator at the same time, strengthening the body's immunity and producing a stronger killing effect on tumor cells. The success of PD-1 inhibitors has shown us the important potential of regulating the immune system in treating diseases. If more immune regulation-related targets can be found and applied to clinical practice, humans will be one step closer to defeating tumors.

[0004] That is to say, the application of cGAS is currently mainly concentrated in the fields of systemic lupus erythematosus and tumor treatment, specifically using "drugs that inhibit cGAS" to treat systemic lupus erythematosus, and using "cGAS analogs" to kill more tumor cells. Therefore, it is necessary to actively study and expand the application of cGAS in other fields. Summary of the invention

[0005] Therefore, the present invention provides an application of cGAS, wherein the cGAS is cyclic GMP-AMP synthase, and the application includes the use of cGAS or a cGAS analog in the preparation of a product for diagnosing, preventing or treating overweight or obesity.

[0006] In a specific embodiment, the application includes the application of cGAS in intestinal CD4+ T cells.

[0007] In a specific embodiment, the application includes the application of cGAS in CD4+ T cells in the lamina propria of the small intestine and large intestine.

[0008] In a specific embodiment, the application includes the use of cGAS in the preparation of drugs for preventing or treating overweight or obesity.

[0009] In a specific embodiment, the application includes the use in promoting the production of the bacterial species Alistipes dispair in the intestine.

[0010] The present invention also provides an application of cGAS, and the application includes the use of cGAS in the preparation of products for diagnosing, preventing or treating metabolic dysfunction in humans or animals.

[0011] The present invention also provides an application of the bacterial species Alistipes dispair, and the application includes the use of the bacterial species Alistipes dispair in the preparation of products for diagnosing, preventing or treating overweight or obesity.

[0012] In a specific embodiment, the bacterial species Alistipes dispair is prepared as an oral preparation.

[0013] The present invention first clarifies that intestinal immune balance is the key to regulating fat thermogenesis; among them, cGAS in CD4+ T cells is a key factor regulating intestinal immunity. The present invention shows through experiments that under a high-fat diet, knocking out cGAS in CD4+ T cells will significantly affect intestinal microbiota dysbiosis, leading to intestinal immune dysregulation, and further leading to changes in intestinal microbiota and metabolites, inhibiting fat thermogenesis and promoting obesity. At the same time, the regulatory role of cGAS in CD4+ T cells first clarifies that cGAS can promote the differentiation of Tregs and the production of its secreted factor TGFβ in intestinal CD4+ T cells. At the same time, knocking out cGAS in intestinal CD4+ T cells will significantly affect the production of the bacterial species Alistipes dispair. The present invention reveals the key role of intestinal immunity in fat thermogenesis, and at the same time clarifies the key target role of cGAS in CD4+ T cells therein.

[0014] The present invention has the following beneficial effects: The present invention first clarifies intestinal CD4 +The lack of cGAS in T cells exacerbates diet-induced obesity and metabolic dysfunction by reducing thermogenesis and energy consumption, thus exacerbating obesity. At the same time, knocking out cGAS in intestinal CD4+ T cells significantly affects the production of the bacterium Alistipes dispar, specifically leading to a decrease in the content of Alistipes dispar in the intestine. And the bacterium Alistipes dispar is closely related to overweight and obesity in the population. Oral administration of Alistipes dispar helps to enhance thermogenic metabolism, reduce weight gain, and achieve the effect of weight loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Study result diagram of the effect of high-fat diet on intestinal CD4 + T cells and cGAS expression in Example 1 of the present invention, wherein,

[0016] A: The number of CD4 + T cells in the small intestine of mice fed a normal diet (ND) and a high-fat diet (HFD);

[0017] B: The number of CD4 + T cells in the colon of mice fed ND and HFD;

[0018] C: The number of CD4 + T cells in the spleen of mice fed ND and HFD;

[0019] D: The number of CD4 + T cells in the thymus of mice fed ND and HFD;

[0020] E: Flow cytometry detection of the number change results of CD4 + T cells and CD8 + T cells in the small intestine of mice fed HFD;

[0021] F: Flow cytometry detection of the number change results of CD4 + T cells and CD8 + T cells in the colon of mice fed HFD;

[0022] G: Western blot detection of the expression results of cGAS in different tissues of 3-month-old male wild-type C57 mice;

[0023] H: Flow chart for isolating CD4 + T cells from lamina propria lymphocytes;

[0024] I: Expression of cGAS protein in CD4 + or CD4-T cells in different tissues of C57 mice, such as spleen, thymus, intestine, lamina propria of colon, and gut-associated lymphoid tissue (PP);

[0025] J: Expression of cGAS protein in CD4 + T cells in the lamina propria of the small intestine under ND and HFD feeding conditions;

[0026] K: Expression of cGAS protein in CD4 + T cells in the lamina propria of the colon under ND and HFD feeding conditions;

[0027] L: Expression of cGAS protein in CD4 + T cells in the spleen under ND and HFD feeding conditions;

[0028] M: Expression of cGAS protein in CD4 + T cells in the thymus under ND and HFD feeding conditions;

[0029] N: Expression of cGAS protein in the intestinal epithelium and lamina propria of C57 mice;

[0030] O: Results of immunofluorescence experiments detecting the expression of cGAS protein in the intestine of people with different BMIs.

[0031] Figure 2 Panel A in [reference] shows the Western blot verification results of cGAS knockout mice (cGASCD4-KO) in Example 2 of the present invention; Figure 2 Panels B-I in [reference] show some results of the study on the effects of CD4 + T cell-specific cGAS deficiency on obesity and metabolic syndrome in Example 3 of the present invention, where

[0032] A: Expression of cGAS protein in CD4 + T cells of cGASCD4-KO mice verified by Western blot in Example 2 of the present invention;

[0033] B: Weight gain of Flox control mice (n = 3) and cGASCD4-KO mice (n = 3) under HFD feeding;

[0034] C: Fat content and lean body mass of Flox control mice (n = 3) and cGASCD4-KO mice (n = 3) after 12 weeks of HFD feeding;

[0035] D: Glucose tolerance of Flox control mice (n = 3) and cGASCD4-KO mice (n = 3) under HFD feeding;

[0036] E: Insulin tolerance in Flox control mice (n = 3) and cGAS CD4-KO mice (n = 3) under HFD feeding;

[0037] F: Images of fat pads and liver sizes and relative tissue weights of cGAS CD4-KO mice and Flox control mice fed with HFD for 17 weeks;

[0038] G: Adipocyte sizes and liver fibrosis conditions of cGAS CD4-KO mice and Flox control mice fed with HFD for 17 weeks. The scale bar in the figure is 50 μm;

[0039] H: Fasting insulin levels of cGAS CD4-KO mice and Flox control mice fed with HFD for 17 weeks;

[0040] I: Fasting blood glucose levels of cGAS CD4-KO mice and Flox control mice fed with HFD for 17 weeks.

[0041] Figure 3 Shows the results of the study on the effect of CD4 + T cell-specific cGAS deletion on the number of immune cells in Example 3 of the present invention. Among them,

[0042] A: Effect of CD4 + T cell-specific cGAS knockout mice (7 weeks old) on the number of thymocytes;

[0043] B: CD4 + and CD8 + T cell populations and cell numbers (n = 4 / group) in the spleen and thymus of cGAS CD4-KO mice and Flox control mice;

[0044] C: Quantitative analysis of CD44 and CD62 staining for the frequencies and total numbers of naive and effector T cell subsets of CD4 + T cells and CD8 + T cells;

[0045] D: Representative FACS plots of Tregs in the spleen and thymus (left panel) and frequencies and total numbers of CD4 + T cells (right panel) (n = 4 / group);

[0046] E: Numbers of naive and effector CD4 + T cells in the spleen and the small intestinal lamina propria (siLP);

[0047] F: Levels of naive CD4 + T and effector CD4 + T cell numbers in the siLP of cGAS CD4-KO mice and Flox control mice;

[0048] G: Naive CD4 and effector CD4 T cell numbers in siLEL of cGAS CD4-KO mice and Flox control mice + T + cell number levels.

[0049] Figure 4 Shows partial results of the study on the effect of CD4 T cell-specific cGAS deletion on obesity and metabolic syndrome in Example 3 of the present invention, where + A: Body weights of cGAS CD4-KO mice and Flox control mice under ND feeding conditions (n = 6 / group);

[0050] A: Body weights of cGAS CD4-KO mice and Flox control mice under ND feeding conditions (n = 6 / group);

[0051] B: Fat mass and lean mass of cGAS CD4-KO mice and Flox control mice under ND feeding conditions (n = 6 / group);

[0052] C: Fasting blood glucose levels of cGAS CD4-KO mice and Flox control mice under ND feeding conditions (n = 6 / group);

[0053] D: Fasting insulin levels of cGAS CD4-KO mice and Flox control mice under ND feeding conditions (n = 6 / group);

[0054] E: Food intake of cGAS CD4-KO mice and Flox control mice under ND feeding conditions (n = 6 / group).

[0055] Figure 5 Shows partial results of the study on the effect of CD4+ T cell-specific cGAS deletion on energy metabolism in Example 4 of the present invention, where

[0056] A: Oxygen consumption of cGAS CD4-KO mice and Flox control mice at 5 weeks of HFD feeding (n = 6 / group);

[0057] B: Average oxygen consumption of cGAS CD4-KO mice and Flox control mice under HFD feeding (n = 6 / group);

[0058] C: Energy expenditure (RER) of cGAS CD4-KO mice and Flox control mice under HFD feeding (n = 6 / group);

[0059] D: mRNA levels of thermogenesis-related genes in BAT of cGAS CD4-KO mice and Flox control mice under cold exposure conditions (n = 8 / group);

[0060] E: Western blot analysis of UCP1 in BAT and sWAT of cGAS CD4-KO mice and Flox control mice under cold exposure conditions (n = 5 / group);

[0061] F: Surface temperature and rectal temperature of cGAS CD4-KO mice and Flox control mice at room temperature and under cold exposure conditions (n = 9 / group).

[0062] Figure 6 Shows CD4 of Example 4 of the present invention + Partial results of the study on the effect of CD4 T cell-specific cGAS deletion on energy consumption and BAT thermogenesis, where

[0063] A: mRNA levels of thermogenesis-related genes in BAT of cGAS CD4-KO mice and Flox control mice at room temperature and under cold exposure conditions (n = 4 / group);

[0064] B: mRNA levels of thermogenesis-related genes in sWAT of cGAS CD4-KO mice and Flox control mice under cold exposure conditions (n = 8 / group);

[0065] C: Intestinal length of cGAS CD4-KO mice and Flox control mice fed with HFD for 5 weeks;

[0066] D: Total fecal calories of cGAS CD4-KO mice and Flox control mice during HFD feeding (n = 4 / group);

[0067] E: Results of H&E staining of intestinal villi of cGAS CD4-KO mice and Flox control mice fed with HFD for 5 weeks. The scale bar in the figure is 100 μm;

[0068] F: Serum concentration of FITC-dextran orally administered to cGAS CD4-KO mice and Flox control mice fed with HFD for 5 weeks;

[0069] G: Serum total cholesterol (TC) levels of cGAS CD4-KO mice and Flox control mice fed with HFD for 5 weeks;

[0070] H: Serum triglyceride (TG) levels of cGAS CD4-KO mice and Flox control mice fed with HFD for 5 weeks.

[0071] Figure 7 Is the gating strategy for GC-Tfh cells and GC-B cells in Example 5 of the present invention.

[0072] Figure 8 Is CD4 of Example 5 of the present invention +Results of the study on the effect of T cell-specific cGAS deficiency on intestinal immune homeostasis, where

[0073] A: Relative levels of PPs and mLN cells in GC-Tfh cells of cGAS CD4-KO mice and Flox control mice;

[0074] B: Relative levels of PPs and mLN cells in GC-B cells of cGAS CD4-KO mice and Flox control mice;

[0075] C: Levels of Treg, Th1, and Th17 cells in the intestinal lamina propria (siLP) of cGAS CD4-KO mice and Flox control mice;

[0076] D: Levels of Treg, Th1, and Th17 cells in the intestinal Paneth nodules of cGAS CD4-KO mice and Flox control mice;

[0077] E: Levels of Treg, Th1, and Th17 cells in the colonic lamina propria (cLP) of cGAS CD4-KO mice and Flox control mice;

[0078] F: Frequencies of Treg, Th1, and Th17 cells in siLP of cGAS CD4-KO mice and Flox control mice;

[0079] G: Frequencies of Treg, Th1, and Th17 cells in cLP of cGAS CD4-KO mice and Flox control mice;

[0080] H: Levels of Treg-related cytokines in siLP of cGAS CD4-KO mice and Flox control mice;

[0081] I: Levels of Treg-related cytokines in cLP of cGAS CD4-KO mice and Flox control mice;

[0082] J: In vitro Treg culture results of naive CD4 + T cells isolated from the spleens of cGAS CD4-KO mice and control mice.

[0083] Figure 9 For the CD4 of Example 6 of the present invention + Results of the exploration of the cause of obesity caused by T cell-specific cGAS deficiency, where

[0084] A: Weight gain in grams of HFD co-housed Flox control mice (n = 7) and cGAS CD4-KO (n = 8) mice during feeding;

[0085] B: Fasting blood glucose levels of HFD co-housed Flox control mice (n = 3) and cGAS CD4-KO (n = 4) mice after 12 weeks of feeding;

[0086] C: Fasting insulin levels of HFD co-housed Flox control mice (n = 3) and cGAS CD4-KO (n = 4) mice after 12 weeks of feeding;

[0087] D: Glucose tolerance of HFD co-housed Flox control mice (n = 3) and cGAS CD4-KO (n = 4) mice after 12 weeks of feeding;

[0088] E: Insulin tolerance of HFD co-housed Flox control mice (n = 3) and cGAS CD4-KO (n = 4) mice after 12 weeks of feeding;

[0089] F: Fat pad and liver size of mice after 12 weeks of feeding;

[0090] G: Fat pad and liver weights of HFD co-housed Flox control mice (n = 3) and cGAS CD4-KO (n = 4) mice after 12 weeks of feeding;

[0091] H: Weight gain of HFD co-housed Flox control mice and cGAS CD4-KO fed with or without broad-spectrum antibiotics (ABX);

[0092] I: Fat content of HFD co-housed Flox control mice and cGAS CD4-KO fed with or without broad-spectrum antibiotics;

[0093] J: Lean body mass content of HFD co-housed Flox control mice and cGAS CD4-KO fed with or without broad-spectrum antibiotics;

[0094] K: Fat size and liver fibrosis of ABX-treated cGAS CD4-KO mice and Flox control mice. Scale bar in the figure is 50 μm;

[0095] L: Weight gain of mice after fecal microbiota transplantation (FMT);

[0096] M: Glucose tolerance of mice after fecal microbiota transplantation (FMT);

[0097] N: Insulin tolerance of mice after fecal microbiota transplantation (FMT);

[0098] O: Core temperature changes of mice at room temperature and under cold exposure after fecal microbiota transplantation (FMT).

[0099] Figure 10 CD4 of Example 7 of the present invention is shown +Partial results of the study on the effect of T cell-specific cGAS deletion on the intestinal flora, where

[0100] A: Microbial diversity of the fecal microbiota of cGAS CD4-KO mice and Flox control mice;

[0101] B: Principal coordinate analysis (PCoA) plot to analyze the differences in microbial diversity of the fecal microbiota between cGAS CD4-KO mice and Flox control mice;

[0102] C: Microbial community analysis of cGAS CD4-KO mice and Flox control mice;

[0103] D: Top 10 bacterial genera affecting weight gain and the random forest linearized standard error of the intestinal microbiota;

[0104] E: Top 10 bacterial genera affecting weight gain and the random forest linearized standard error of the intestinal microbiota.

[0105] Figure 11 Shows the CD4 of Example 7 of the present invention + Partial results of the study on the effect of T cell-specific cGAS deletion on the intestinal flora, where

[0106] A: Microbial communities between cohoused and non-cohoused cGAS CD4-KO mice and Flox mice;

[0107] B: PCoA plot to analyze the differences in fecal flora between cGAS CD4-KO mice and Flox control mice fed a high-fat diet (HFD) for 12 weeks;

[0108] C: PCoA plot to analyze the differences in fecal flora between cohoused Flox mice and non-cohoused Flox control mice fed a HFD for 12 weeks;

[0109] D: PCoA plot to analyze the differences in fecal flora between cohoused Flox mice and cohoused cGAS CD4-KO mice fed a HFD for 12 weeks;

[0110] E: Venn diagram to determine the intersection of differential flora in three 16S sequencing datasets.

[0111] Figure 12 Results graph of the study on the effect of Alistipes bacteria on body weight and metabolism in Example 8 of the present invention, where

[0112] A: Effect of oral administration of Alistipes dispar strain on the body weight of mice;

[0113] B: Effect of oral administration of Alistipes dispar strain on the glucose tolerance of mice;

[0114] C: Effects of oral administration of Alistipes dispair on insulin resistance in mice;

[0115] D: Relative mRNA levels of Alistipes in feces of people with different BMIs. Detailed implementation manners

[0116] The present invention will be further described through the following examples and drawings, but the protection scope of the present invention is not limited to the following examples only.

[0117] Those skilled in the art know that CD4+ T cells are a type of biological cells. The reduction in the number of CD4+ T cells is an obvious feature during AIDS, and it is also the main target attacked by the HIV virus. Treg, that is, regulatory T cells (Regulatory cells, abbreviated as Tregs), is a subset of T cells that control the autoimmune reactivity in the body. In the early stage, it was also called suppressor T cells. Regulatory T cells can be divided into naturally occurring natural regulatory T cells (n T-regs) and induced adaptive regulatory T cells (a T-regs or i T-regs), such as Th3, Tr1. In addition, there are also CD8 Treg, NKT cells, etc., which are closely related to the occurrence of autoimmune diseases, and their abnormal expression can lead to autoimmune diseases. Among them, Tr1 cells secrete IL-10; Th3 cells secrete TGF-β.

[0118] For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified in the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0119] In the following examples of the present invention, the mice are placed in a specific pathogen-free animal facility, with a temperature of (23 ± 1) °C, a relative humidity of 50 - 60%, and a light / dark cycle of 12 hours. The mice can freely access food and water and eat at will throughout the experiment. At the end of the study, the mice are euthanized by bleeding through a neck incision and then cervical dislocation. Then the organs are quickly frozen in liquid nitrogen for future analysis. All animal studies are carried out according to the protocol approved by the Animal Care and Use Committee of Central South University, and all relevant ethical regulations for animal testing and research are complied with.

[0120] The collection and use of human samples in the following examples of the present invention have been approved by the Review Committee of the Second Xiangya Hospital of the People's Republic of China and comply with all ethical regulations applicable to research involving human participants. This research is carried out in accordance with the principles of the Declaration of Helsinki. In addition, this research follows the review and approval guidelines for human genetic resources formulated by the Ministry of Science and Technology.

[0121] Unless otherwise specified, the data shown in the following embodiments of the present invention are the results of three independent experiments, representing the mean ± SEM. Statistical analysis was performed using GraphPad Prism 7 (GraphPad software). The statistical analysis of the data was carried out by using unpaired t-tests or ANOVA (two-way ANOVA with single repeated measures ANOVA or post hoc Bonferroni test). In the result graphs, * indicates a significance level of p ≤ 0.05, ** indicates a significance level of p < 0.01, *** indicates a significance level of p < 0.001, and ns indicates a significance level of p > 0.05. The present invention uses Prism 8 (GraphPad) or Excel (Microsoft) software for statistical analysis.

[0122] Unless otherwise specified, the different groups of mice used for testing and data comparison in the following examples are of the same age and sex. Unless otherwise specified, the cGAS CD4-KO mice and Flox control mice used for testing and data comparison are not co-housed and fed. Unless otherwise specified, the mice are fed a high-fat diet (HFD). Unless otherwise specified, the intestine or colon or gut used in the examples to detect immune cells and cGAS expression refers to the respective lamina propria.

[0123] The mouse CD4 used in the following embodiments of the present invention + The mouse CD4 T cell isolation kit was purchased from Miltenyi, Auburn, USA; the insulin ultrasensitive enzyme immunoassay kit was purchased from Alpco Diagnostics, Slemm, NH; the UCP1 and Actin antibodies were purchased from Sigma; the PGC1α antibody was purchased from Abcam; the cGAS antibody was purchased from CST; the Minispec body composition analyzer LF50 was purchased from Bruker, Germany; the bomb calorimeter was purchased from IKA, Germany; TRIzol reagent was purchased from Life Technologies; ampicillin, neomycin, and erythromycin in the broad-spectrum antibiotics were purchased from Fisher Scientific, and gentamicin was purchased from GoldBio; the QIAamp Fast DNA Stool Mini Kit was purchased from QIAGEN, Venlo, the Netherlands, Cat# 51604; the MiSeq PE300 platform was from Shanghai OE Biotech Co., purchased from Illumina, California, USA; the QIIME software package version was 1.8.0; the Silva database version was 123.

[0124] Isolation of primary mouse adipocytes: Fat tissue was carefully excised, minced, digested with 1.5 g / L type II collagenase (Sigma-Aldrich) at 37 °C for 10 minutes with shaking. The digested cells were filtered through a 300-mesh nylon sieve, washed, and centrifuged for 5 minutes to separate preadipocytes from floating mature adipocytes. The preadipocytes were then differentiated into mature adipocytes using an adipogenic induction differentiation agent and further processed.

[0125] GTT and ITT experiments: Glucose tolerance test (GTT) was performed by injecting glucose (2 g / kg body weight i.p.) into overnight-fasted mice. Blood was drawn from the tail vein at 0, 15, 30, 60, and 120 minutes after glucose injection, and blood glucose levels in mice were measured using a glucometer (One Touch; Bionime Corp.). Insulin tolerance test (ITT) was performed by injecting human insulin (0.75 U / kg body weight i.p.) into 4-hour-fasted mice. Blood was drawn from the tail vein at 0, 15, 30, 60, and 90 minutes after insulin injection, and serum insulin levels were measured using an ultrasensitive insulin enzyme immunoassay (Alpco Diagnostics, Salem, NH).

[0126] 16S sequencing experiment: Fecal samples of experimental mice were collected before the experiment and stored at -80 °C. Genomic DNA of the samples was extracted using the QIAamp Fast DNA Stool Mini Kit (Cat#51604, QIAGEN, Venlo, Netherlands). The purity and concentration of the extracted DNA were detected by agarose gel electrophoresis. The V3-V4 region of bacterial DNA was amplified using primers (5'-TACGGRAGGCAGCAG-3', 5'-GGGTATCTAATCCT-3'). DNA was sequenced using the MiSeq PE300 platform of Shanghai OE Biotech Co., Ltd. (Illumina, California, USA). The raw data were processed using the QIIME software package (version 1.8.0). Then, the representative sequences of OTUs were analyzed in the Silva database (version 123). Alpha diversity and beta diversity were analyzed using the QIIME software package (version 1.8.0).

[0127] Metabolomics: For non-targeted metabolite analysis, samples were analyzed by liquid chromatography-mass spectrometry. UPLC-Q-TOF / MS (ACQUITY UPLC I-Class, Waters, Massachusetts, USA) and ESI-QTOF / MS (Xevo G2-SQ-TOF, Waters) were used. The chromatographic column was an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 100 mm, Waters). Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. The gradient elution was 1-5% mobile phase B (0-1 min), 5-30% mobile phase B (1-2 min), 30-60% mobile phase B (2-3.5 min), 60-90% mobile phase B (3.5-7.5 min), 90-100% mobile phase B (7.5-9.5 min), 100% mobile phase B (9.5-12.5 min), 100-1% mobile phase B (12.5-12.7 min), 1% mobile phase B (12.7-16 min). The spectral signals of the samples were obtained by electrospray ionization in positive and negative ionization modes. The data were preprocessed using progenesis QI (Waters), and then SIMCA software (version 14.0, Umetrics, ume <s:1>, Sweden) for multivariate statistical analysis. The KEGG database (http: / / www.genome.jp / KEGG / pathway.html) and R (version 3.4.1) were used for enrichment pathway analysis of the altered metabolites. Metabolites contributing to the clustering were identified based on a VIP value greater than 1.5 and a p-value less than 0.05. Four metabolite databases, namely HMDB (http: / / www.hmdb.ca / ), Lipid MAPS (http: / / www.lipidmaps.org), METLIN (http: / / metlin.scripps.edu / ), and KEGG (http: / / www.genome.jp / kegg / ), were used for the identification of potential differential metabolites.

[0128] Measurement of body weight, body composition, food intake, and intestinal absorption: The body weight of the mice was monitored weekly. The body composition of the mice was measured using the Minispec LF50 body composition analyzer from Bruker, Germany. The mice were individually housed for 4 consecutive days, and the food intake and fecal output were measured. According to the manufacturer's instructions, the feces were dried and ground into a fine powder, and then placed in a bomb calorimeter (IKA, Germany). The calorie excretion was calculated by multiplying the feces produced by the calorie content per gram of feces.

[0129] The CD4-Cre mice used in the following embodiments of the present invention are from Jackson Laboratory, numbered 017336. The anti-fc receptor used in flow cytometry in the following format examples of the present invention is purchased from Biolegend, San Diego, CA; the transcription factor buffer is purchased from BD Biosciences, San Jose, CA; PMA (50 ng / mL) is purchased from Baoteng, Shanghai, China; ionomycin (750 ng / mL) is purchased from Millipore, Darmstadt, Germany; Brefeldin A (10 μg / ml) is purchased from Beyotime, Shanghai, China; the fixation / permeabilization solution kit is purchased from BD Biosciences, San Jose, USA; FVS520, APC-Cy7-CD45, Percp-CD4, APC-CD62L, PE-Cy7-CD44, Alexa fluor 647-GATA3, FITC-γδTCR antibodies are purchased from BD Biosciences, San Jose, USA; Zombie NIR, PE-Cy7-CD45, PE-CD3, PE-CD4, PE-Cy7-CD8, PE-CD8, PE-CD25, APC-CD8, APC-IFN-γ, Alexa fluor 647-Foxp3, Percp-Cy5.5-CD11b, APC-F4 / 80, FITC-cd206 antibodies are purchased from Biolegend, San Diego, USA; pe-siglece-f, Alexa fluor 647-Foxp3, FITC-γδTCR antibodies are purchased from ebioscience, CA, USA; data analysis is performed using FlowJo V10 (BD Biosciences, San Jose, USA) software.

[0130] Unless otherwise specified, the immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, recombinant DNA, etc. used in the present invention are conventional skills in the art.

[0131] Example 1: Effect of high-fat diet on intestinal CD4 + T cell and cGAS expression

[0132] 1. Effect of high-fat diet on intestinal CD4 + T cells

[0133] In this example, C57 mice fed a normal diet (ND) and a high-fat diet (HFD) for 16 weeks were used to study the effect of a high-fat diet on intestinal CD4 + Effect of T cells. CD4 T cells in different tissues of C57 mice fed ND and HFD for 16 weeks were isolated and counted respectively. In this example, mouse CD4 T cell isolation kit was used to isolate CD4 T cells from each tissue through magnetic bead separation system. + T cells. + T cells were isolated from each tissue using the mouse CD4 + T cell isolation kit through the magnetic bead separation system.

[0134] The results of isolation and counting showed that the number of CD4 + T cells in the small intestine (Intestine) and colon (Colon) was significantly reduced under HFD feeding compared with ND feeding (Figure A in Figure 1 , Figure B in Figure 1 ). Correspondingly, no obvious difference was found in the spleen (Spleen) and thymus (Thymus) tissues (Figure C in Figure 1 , Figure D in Figure 1 ). Further experiments using flow cytometry found that CD4 + T cells rather than CD8 + T cells in the intestinal lamina propria were significantly reduced under HFD feeding (Figure E in Figure 1 , Figure F in Figure 1 ).

[0135] 2. Study on the effect of high-fat diet on the expression level of cGAS

[0136] In this example, Western blot experiment was used to study the expression level of cGAS protein in different tissues of C57 mice and the change of cGAS expression level under HFD feeding.

[0137] The results showed that the expression of cGAS protein was significantly higher in the small intestine Intestine than in other tissues (Figure G in Figure 1 ). As can be seen from Figure G, the expression of cGAS protein was significantly higher in the intestine Intestine including duodenum Duo, jejunum Jet and ileum ile. CD4 + T cells in different immune tissues were further isolated to detect the expression of cGAS protein in them (the schematic diagram of the isolation process is shown in Figure H in Figure 1 ). It was found that among CD4 + T cells and CD4-T cells in the spleen, thymus, intestinal lamina propria, colonic lamina propria and intestinal lymph nodes (PP), the cGAS of CD4 + T cells in the intestine and colonic lamina propria was significantly highly expressed (Figure I in Figure 1 ). Further comparing the cGAS expression levels of CD4 + T cells in different tissues under ND and HFD feeding conditions, the results showed that only the cGAS of CD4 + T cells showed a significant decrease in the expression level of cGAS protein under HFD feeding ( Figure 1 Figure J in Figure 1 , and in the spleen ( Figure 1 Figure L in Figure 1 ) and thymus ( + Figure M in Figure 1 ), there was no significant change in the cGAS expression of CD4

[0138] T cells under HFD feeding conditions. In addition, this example also found that the cGAS protein expression in the intestinal lamina propria of mice was significantly higher than that in the epithelial layer ( Figure 1 Figure N in

[0138] ).

[0138] 3. cGAS expression levels in different BMI populations

[0139] In this example, intestinal tissues of people with different weights and BMIs were collected, and the expression of cGAS was analyzed by immunofluorescence experiments. The results showed that in the intestinal tissues of overweight (24 ≤ BMI < 28) and obese (BMI ≥ 28) people, the expression level of cGAS in CD4 + T cells was significantly reduced ( Figure 1 Figure O in

[0140] ).

[0140] Based on the above experimental results and analysis, the research in this example shows that there is a potential link between CD4 + T cells in intestinal immunity and obesity caused by high-fat diet, and cGAS protein may play a key role in this process.

[0141] Example 2: Construction and verification of cGAS knockout mice (cGASCD4-KO)

[0142] This example provides the construction and verification of CD4 + T cell-specific cGAS knockout mice (cGASCD4-KO). The specific construction method is as follows:

[0143] (1) cGASf / f mice (cGAS f / f, Flox mice) were crossed with CD4-Cre mice to obtain CD4 + T cell-specific cGAS knockout mice (cGASCD4-KO). The cGAS gene of cGAS f / f mice (Flox mice) has a loxP site on each side. In cells where Cre protein exists, Cre will recognize the two loxP sites, and ultimately the sequence between the two loxP sites will be deleted, thereby achieving the knockout of cGAS.

[0144] (2) cGAS f / f mice were crossed with CD4 - Cre mice. This mouse has CD4 + Cre recombinase is specifically expressed in T cells, so the resulting mice after hybridization can express Cre recombinase specifically in CD4 + T cells. Under the action of Cre recombinase, only in CD4 + T cells, the cGAS gene locus between the two loxP sites will be excised, resulting in the knockout of the cGAS gene. This mouse is called a cGAS CD4-KO mouse.

[0145] (3) After hybridization, the genotypes of all offspring mice are identified by PCR, sequencing or Western blot, and then obtained after screening. Note: Only those mice that carry both cGAS f / f and Cre in their genotypes can be considered as successfully generated CD4 + T cell-specific cGAS knockout mice (cGAS CD4-KO).

[0146] Among them, the Western blot experiment was performed on the above constructed CD4 + T cell-specific cGAS knockout mice (cGAS CD4-KO), and the verification results are shown in Figure 2 A as shown, indicating that almost no cGAS protein was detected in CD4 + T cells isolated from the spleen, indicating that the present invention successfully constructed CD4 + T cell-specific cGAS knockout mice. Figure 2 The content in B-I is used in Example 3 below.

[0147] Example 3: Study on the effect of CD4 + T cell-specific cGAS deficiency on HFD-induced obesity and metabolic syndrome

[0148] 1. Study on the effect on the number of immune cells

[0149] In this example, the number of immune cells in cGAS CD4-KO mice was first studied. cGAS CD4-KO mice were used as the experimental group, and cGAS f / f mice (Flox mice) were used as the control group. The methods of cell isolation and counting and flow cytometry were used to explore the levels of immune cell numbers in their thymus, spleen and intestine.

[0150] The results showed that CD4 + T cell-specific cGAS knockout had no effect on the number of thymocytes ( Figure 3 Figure A in + T and CD8 + T cell populations in the spleen and thymus ( Figure 3 Figure B in + CD44 - ) and memory (CD62L - CD44 + ) The proportion of T cell populations ( Figure 3 Figure C in), the development of thymic regulatory T (Treg) cells ( Figure 3 Figure D in) were not significantly affected. These results indicate that cGAS deficiency does not affect the overall development of T cells in the spleen.

[0151] Due to the presence of different naive CD4 + T and effector CD4 + T cell populations ( Figure 3 Figure E in), this example further studied different types of CD4 + T cells in the intestines of Flox control mice and cGAS CD4-KO mice.

[0152] It was found that, different from the spleen, the number level of naive CD4 + T cells in the small intestinal lamina propria (siLP) of cGAS CD4-KO mice was significantly higher than that of Flox control mice, while the number level of effector CD4 + T cells was significantly lower ( Figure 3 Figure F in), and the same trend was maintained in the epithelium (siLEL) ( Figure 3 Figure G in).

[0153] 2. Study on the effects on obesity and metabolic syndrome

[0154] In this example, cGAS CD4-KO mice and Flox control mice were used for ND feeding and HFD feeding for 12 weeks. By measuring and studying obesity and metabolic parameters such as the body weight, blood glucose level, insulin level, fat mass, and glucose tolerance of the mice, the effects of CD4 + T cell-specific cGAS deficiency on HFD-induced obesity and metabolic syndrome were further studied.

[0155] Among them, the method of glucose tolerance test (GTT) was: by injecting glucose (2 g / kg body weight i.p.) into overnight-fasted mice. At 0, 15, 30, 60, and 120 minutes after glucose injection, blood was drawn from the tail vein, and the blood glucose of the mice was detected with a blood glucose meter.

[0156] The method of insulin tolerance test (ITT) was: by injecting human insulin (0.75 U / kg body weight i.p.) into 4-hour-fasted mice. At 0, 15, 30, 60, and 90 minutes after insulin injection, blood was drawn from the tail vein, and the serum insulin level was measured by insulin ultrasensitive enzyme immunoassay.

[0157] Body weight, body composition, food intake, and intestinal absorption were measured as follows: The body weight of mice was monitored weekly; body composition of mice was determined using a Minispec body composition analyzer LF50. Mice were housed individually every day for 4 consecutive days, and food intake and feces production were measured; feces were dried and ground into a fine powder, which was then placed in an oxygen bomb calorimeter to calculate calorie excretion by multiplying the feces produced by the calorie content per gram of feces.

[0158] The results showed that under ND feeding conditions, the body weight of cGASCD4-KO mice and Flox control mice ( Figure 4 A), fat and lean mass ( Figure 4 Figure B), fasting blood glucose level ( Figure 4 Figure C), insulin levels ( Figure 4 D in Figure ) and food intake ( Figure 4 However, under HFD-fed conditions, cGASCD4-KO mice showed more weight gain than Flox control mice ( Figure 2 ), significantly more fat gain and less lean body mass loss ( Figure 2 C in Figure ), and cGASCD4-KO mice showed impaired glucose tolerance ( Figure 2 D in Figure ) and decreased insulin sensitivity ( Figure 2 Correspondingly, the volume and ratio of adipose tissue to body weight in cGASCD4-KO mice were significantly larger than those in Flox control mice ( Figure 2 F), including inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and brown adipose tissue (BAT). Moreover, the adipocyte size of iWAT and eWAT in cGASCD4-KO mice was significantly larger than that in Flox control mice ( Figure 2 In addition, the present invention also found that cGASCD4-KO mice showed increased liver fibrosis ( Figure 2 G in Figure ), fasting insulin levels are elevated ( Figure 2 H in the figure) and significantly increased blood sugar levels ( Figure 2 Figure I in Figure 1).

[0159] Based on the above experimental results and analysis, the research in this example shows that: CD4 + cGAS deficiency in T cells exacerbates diet-induced obesity and metabolic dysfunction. These findings highlight the important role of cGAS in regulating metabolic homeostasis and suggest that it plays a key role in CD4 + Deficiencies in T cells contribute to the development of metabolic disorders associated with obesity.

[0160] Example 4: CD4 + Study on the Effects of T Cell-Specific cGAS Deficiency on Energy Expenditure and BAT Thermogenesis

[0161] 1. Study on the Effects on Energy Metabolism

[0162] In this example, cGAS CD4-KO mice and Flox control mice were fed with HFD for 5 weeks, and the energy expenditure, oxygen consumption, expression levels of thermogenic genes under cold exposure, and body temperature of the mice were studied to investigate the effects of CD4 + T cell-specific cGAS deficiency on the energy metabolism of mice.

[0163] Among them, the cold exposure experimental method was as follows: Experimental mice and their control mice were individually housed in a non-littered cage at 6°C and provided with food and water. At the end of the experiment, the mice were sacrificed, and adipose tissue was isolated for gene and protein expression analysis.

[0164] The results showed that the oxygen consumption ( Figure 5 Figure A in Figure 5 ) and energy expenditure ( Figure 5 ) of cGAS CD4-KO mice were decreased compared with those of Flox control mice. Under cold exposure conditions, the mRNA levels of thermogenic genes such as Ucp1, Ppargc1a, Prdm16, Dio2, and Cidea in BAT of cGAS CD4-KO mice were significantly lower than those of Flox control mice ( Figure 5 Figure D in Figure 6 ), and the experimental results under room temperature conditions showed the same trend ( Figure 6 Figure A in Figure 5 ), while there was no significant difference in subcutaneous white adipose tissue (sWAT) ( Figure 5 Figure B in Figure 5 ). Western blot experiments further confirmed that the protein expression of UCP1 in BAT of cGAS CD4-KO mice was significantly decreased ( + Figure E in

[0165] ), while there was no change in sWAT (

[0166] Figure E in

[0167] It was found that the intestinal length of cGAS CD4-KO mice ( Figure C in Figure 6 ), total fecal calories ( Figure D in Figure 6 ), intestinal villi ( Figure E in Figure 6 ), and the serum concentration of an index of intestinal absorption - orally administered FITC-dextran ( Figure F in Figure 6 ) showed no significant differences compared with those of Flox control mice. These results indicate that the deteriorated metabolic phenotype observed in cGAS CD4-KO mice is not due to altered intestinal absorption. Additionally, cGAS CD4-KO mice showed significantly higher levels of total serum cholesterol (TC) ( Figure G in Figure 6 ) and triglyceride (TG) ( Figure H in Figure 6 ).

[0168] Based on the above experimental results and analysis, the study of this example shows that: CD4 + T cell cGAS deficiency exacerbates obesity by impairing thermogenesis and reducing energy expenditure, rather than due to altered intestinal absorption.

[0169] Example 5: Study on the effect of CD4 + T cell-specific cGAS deletion on intestinal immune homeostasis

[0170] 1. Effect on the number of Tregs

[0171] In this example, the abundances of germinal center-T follicular helper cells (GC-Tfh) and germinal center-B (GC-B) cells related to T cell deficiency-induced obesity in the Peyer's patches and mesenteric lymph nodes of cGAS CD4-KO mice and Flox control mice fed a HFD for 17 weeks were studied (the gating strategies for the GC-Tfh cells and GC-B cells are as shown in Figure 7 ).

[0172] It was found that there were no significant differences in the frequencies of GC-Tfh cells ( Figure A in Figure 8 ) and GC-B cells ( Figure B in Figure 8 ) between cGAS CD4-KO mice and Flox control mice. However, cGAS deficiency in CD4 + T cells led to a significant reduction in the number of regulatory T (Treg) cells in the small intestinal lamina propria (siLP) ( Figure C in Figure 8 ), intestinal Paneth knots ( Figure D in Figure 8 ), and colonic lamina propria (cLP) ( Figure E in Figure 8 ) of mice, while the number of Th1 or Th17 cells did not show a significant reduction.

[0173] 2. Relationship between T cell dysfunction and obesity

[0174] In this example, by detecting the proportions of three T cell subsets in the intestines of cGAS CD4-KO mice and Flox control mice, the relationship between T cell dysfunction and obesity was further explored.

[0175] The results showed that in the siLP ( Figure 8 Figure F therein) and cLP ( Figure 8 Figure G therein), the proportion of Treg in cGAS CD4-KO mice was significantly lower, but the proportions of Th1 or Th17 cells did not decrease; for Treg-related cytokines such as IL-10 and TGFβ, they were significantly reduced in the siLP ( Figure 8 Figure H therein) and cLP ( Figure 8 Figure I therein) of cGAS CD4-KO mice. Additionally, in vitro Treg culture of naive CD4 + T cells isolated from the spleens of cGAS CD4-KO mice and control mice also showed that cGAS in CD4 + T cells promoted Treg polarization ( Figure 8 Figure J therein).

[0176] Based on the above experimental results and analysis, the research in this example showed that knockout of cGAS in CD4 + T cells led to the imbalance of intestinal inflammatory homeostasis, and the main reason for this effect was that cGAS affected the number of Tregs and the expression levels of Treg-related cytokines.

[0177] Example 6: Exploration of the Causes of Obesity Caused by Specific Deletion of cGAS in CD4 + T Cells

[0178] In this example, it was studied whether the metabolic phenotype changes induced by cGAS deficiency were mediated by the gut microbiota. cGAS CD4-KO mice and Flox control mice were co-housed and fed a high-fat diet (HFD), and parameters such as weight gain, blood glucose, and insulin levels were measured, and intervention studies were conducted using broad-spectrum antibiotics (ABX).

[0179] The method for the broad-spectrum antibiotic treatment experiment was as follows: Mice drank water containing 0.5 mg / mL ampicillin, neomycin, erythromycin, and gentamicin for 1 week to reduce the endogenous microbiota, and then were re-colonized by fecal transfer.

[0180] The results showed that co-housing transferred the weight gain of cGAS CD4-KO mice to the control mice ( Figure 9 Figure A therein), and after co-housing and feeding, the fasting blood glucose between cGAS CD4-KO mice and Flox control mice ( Figure 9 in Panel B), insulin levels ( Figure 9 in Panel C), glucose tolerance ( Figure 9 in Panel D), insulin tolerance ( Figure 9 in Panel E), fat pad and liver size ( Figure 9 in Panel F), and weight ( Figure 9 in Panel G) were reduced. Consistent with this, intervention with broad-spectrum antibiotics (ABX) in HFD-fed cGAS CD4-KO mice and Flox control mice showed that ABX treatment had little effect on weight gain in Flox control mice but completely reversed weight gain ( Figure 9 in Panel H) in cGAS CD4-KO mice, making their weight gain significantly faster, and significantly increased fat mass ( Figure 9 in Panel I), significantly reduced lean body mass ( Figure 9 in Panel J), and significantly increased fat size and liver fibrosis ( Figure 9 in Panel K) in ABX-treated cGAS CD4-KO mice.

[0181] In addition, in this example, fecal microbiota transplantation (FMT) of cGAS CD4-KO mice into Flox control mice resulted in weight gain ( Figure 9 in Panel L), impaired glucose tolerance (GTT) ( Figure 9 in Panel M), and insulin tolerance (ITT) ( Figure 9 in Panel N) in recipient mice, and a decrease in core body temperature ( Figure 9 in Panel O).

[0182] Based on the above experimental results and analysis, the study in this example showed that changes in the gut microbiota led to differences in CD4 + T cell cGAS-deficiency-mediated obesity in HFD-fed mice, and CD4 + T cell cGAS deletion may affect metabolism and obesity by influencing gut immune homeostasis and potentially the gut microbiota.

[0183] Example 7: Study on the effect of CD4 + T cell-specific cGAS deletion on the gut microbiota

[0184] 1. 16S ribosomal RNA sequencing

[0185] In this example, feces of cGAS CD4-KO mice and Flox control mice fed an HFD for 5 weeks were subjected to 16S ribosomal RNA (rRNA) gene sequencing to study the microbiota associated with the altered metabolic phenotypes of cGAS CD4-KO mice.

[0186] Among them, the method of 16S sequencing is as follows: Before the experiment, fecal samples of experimental mice were collected and stored at -80°C. The genomic DNA of the samples was extracted using the QIAamp Fast DNA Stool Mini Kit. The purity and concentration of the extracted DNA were detected by agarose gel electrophoresis. The V3-V4 region of bacterial DNA was amplified with primers (the sequences of the forward and reverse primers for the V3-V4 region are 5'-TACGGRAGGCAGCAG-3' and 5'-GGGTATCTAATCCT-3'). The DNA was sequenced using the MiSeq PE300 platform. The raw data was processed using the QIIME software package. Then the representative sequences of OTUs were analyzed in the Silva database. The alpha diversity and beta diversity were analyzed using the QIIME software package.

[0187] It was found that the diversity of the intestinal flora of cGAS CD4-KO mice was significantly lower than that of Flox control mice ( Figure 10 Figure A in Figure 10 Figure B in : principal coordinate analysis PCoA plot, based on weighted UniFrac distance). The microbial community analysis of cGAS CD4-KO mice and Flox control mice is shown in Figure 10 Figure C in . It should be noted that the abundance of Roseburia in the feces of cGAS CD4-KO mice was higher than that of Flox control mice ( Figure 10 Figure D in Figure 10 Figure E in ), Figure 10 Both Figure D and Figure E in represent the top 10 bacterial genera affecting the standard error of random forest linearization of body weight gain and intestinal microbiota. The genera enriched in relative abundance in Flox control mice are shown in red, and the genera enriched in relative abundance in cGAS CD4-KO mice are shown in green.

[0188] 2. Further 16S ribosomal RNA sequencing of co-housed mice

[0189] In this example, fecal samples of cGAS CD4-KO mice, Flox control mice, and cGAS CD4-KO mice and Flox control mice co-housed with HFD for 12 weeks were subjected to 16S sequencing.

[0190] It was found that there were obvious differences in the microbial communities between cGAS CD4-KO mice and Flox mice, but these differences disappeared in the co-housed group ( Figure 11 Figure A in ). The PCoA plot (based on weighted UniFrac distance) showed that there were significant differences in the fecal flora composition between cGAS CD4-KO and Flox control mice after 12 weeks of HFD feeding ( Figure 11 Figure B in), while after cohousing and feeding Flox control mice with cGAS CD4-KO mice, significant changes occurred in the fecal microbiota diversity and distribution of Flox mice ( Figure 11 Figure C in), in contrast, cohoused and fed Flox and cGAS CD4-KO mice showed similar microbiota distribution patterns ( Figure 11 Figure D in), these findings are consistent with the reduction of metabolic parameter differences between cohoused and fed Flox control and cGAS CD4-KO mice found in Example 6.

[0191] 3. Search for key differential microorganisms

[0192] Due to the obvious differences in the microbiota composition of HFD-fed Flox control and cGAS CD4-KO mice at 5 weeks and 12 weeks, as well as the differences between Flox and cage-mate fed Flox mice, this example also aims to determine the most critical differential microorganisms by identifying the common elements among these three 16S sequencing datasets (the differential microbiota of HFD-fed for 12 weeks is named Set1, the differential microbiota of cage-mate cohoused and HFD-fed for 12 weeks is named Set2, and the differential microbiota of HFD-fed for 5 weeks is named Set3), and determine the intersection through a Venn diagram at the genus level of the microbiota. The result shows that the genus Alistipes is the only bacterial genus that shows changes in all three 16S sequencing comparisons ( Figure 11 Figure E in), suggesting that Alistipes is an important differential microbiota that regulates the obese phenotype of cGAS CD4-KO mice.

[0193] Based on the above experimental results and analysis, the research in this example shows that CD4 + Specific deletion of cGAS in T cells has a greater impact on the diversity and community composition of the intestinal microbiota of mice. After cohousing and feeding, the differences in the intestinal microbiota between cGAS CD4-KO mice and Flox control mice can be reduced, and it is also found that CD4 + Specific deletion of cGAS in T cells causes a decrease in the content of the genus Alistipes in the intestinal microbiota, indicating that the genus Alistipes may be an important differential microbiota that regulates the obese phenotype of cGAS CD4-KO mice.

[0194] Example 8: Study on the effects of Alistipes bacteria on body weight and metabolism

[0195] 1. Effects of Alistipes bacteria on body weight and metabolism of mice

[0196] Based on the findings in Example 7, this example studies the effect of orally administering the strain Alistipes dispair to C57 mice on reducing body weight and promoting thermogenesis.

[0197] It was found that under the HFD feeding condition, the weight gain of mice fed with food supplemented with the Alistipes dispair strain was significantly inhibited compared with that of mice supplemented with the PBS control after 7 weeks ( Figure 12 Figure A in Figure 12 ), and its glucose tolerance (GTT) was also inhibited ( Figure 12 Figure B in

[0198] 2. Relationship between Alistipes bacteria and human BMI

[0199] In this implementation, feces of people with different BMIs were also collected, and the content of the Alistipes dispair strain was detected. The relative mRNA levels of the Alistipes dispair strain in the feces of normal (18.5 ≤ BMI < 24), overweight (24 ≤ BMI < 28), and obese (BMI ≥ 28) people were measured. It was found that the Alistipes dispair strain in the feces of normal people was higher, showing a very significant difference from that in overweight and obese people ( Figure 12 Figure D in

[0200] Based on the above experimental results and analysis, the research in this example shows that the content of the Alistipes dispair strain in the intestine is related to human overweight or obesity. The lower the content, the more likely people are to be overweight or obese; moreover, oral administration of the Alistipes dispair strain helps to enhance thermogenic metabolism, reduce weight gain, and achieve the effect of weight loss.

[0201] In summary, through a series of experimental designs and experimental data analyses, the present invention found that intestinal immune balance is the key to controlling fat thermogenesis. In particular, there is a potential link between CD4 + T cells in intestinal immunity and obesity caused by a high-fat diet, and cGAS plays a key role therein; cGAS in CD4 + T cells is a key factor regulating intestinal immunity. Knockout of cGAS in CD4 + T cells will lead to a decrease in the number of Tregs and a decrease in the expression levels of Treg-related cytokines, thus resulting in intestinal immune disorders; the lack of cGAS in CD4 + T cells will also exacerbate diet-induced obesity and metabolic dysfunction, by reducing thermogenesis and energy consumption, thus exacerbating obesity. In addition, the present invention also demonstrated that CD4 + T cell cGAS deficiency affects intestinal immune balance, which in turn affects metabolism and obesity by influencing the intestinal microbiota. Among them, the Alistipes dispair species may be the most important microorganism regulating the obese phenotype of cGAS CD4-KO mice, and the Alistipes dispair species is closely related to overweight and obesity in the human population. Oral administration of the Alistipes dispair species helps to enhance thermogenic metabolism, reduce weight gain, and achieve weight loss effects.

[0202] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions and substitutions can be made, which should all be regarded as belonging to the protection scope of the present invention.

Claims

1.cGAS antibody is prepared through the small intestine and large intestine lamina propria CD4 + The invention relates to a product for diagnosing overweight or obesity by reducing the expression of cGAS protein in T cells, wherein the cGAS is cyclic GMP-AMP synthase.

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