CN115286701A - Target for regulating intestinal mucosa homeostasis or inflammatory bowel disease of mammals and application thereof - Google Patents

Abstract

The invention discloses a target for regulating intestinal mucosa homeostasis or inflammatory bowel disease of mammals and application thereof. The target is Fas related death domain FADD protein, and the sequence of the FADD protein is an amino acid sequence shown in SEQ ID No. 1. The method is realized by simulating the expression of protein corresponding to FADD non-phosphorylated gene, and the sequence of the gene is the nucleotide sequence shown in SEQ ID No. 2. The protein corresponding to the gene simulating the non-phosphorylation of the FADD is obtained by mutating the 194 th serine of the wild-type human FADD protein to alanine, or mutating the 191 th serine of the mouse FADD protein to alanine. The mock non-phosphorylated FADD (S191A) mice of the present invention exhibited more severe intestinal inflammation.

Description

Target for regulating intestinal mucosa homeostasis or inflammatory bowel disease of mammals and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a target for regulating intestinal mucosa homeostasis or inflammatory bowel disease of mammals and application thereof.

Background

Intestinal intraepithelial lymphocytes (IELs) are the vast majority of immune cells in the intestinal mucosal immune system, with approximately 90% of IELs expressing T Cell Receptors (TCRs) (Olivares-Villagomez D et al, trends immunol.2018,39 (4): 264-275). TCR is a characteristic marker of the surface of all T cells, and binds to CD3 non-covalently, forming a TCR-CD3 complex. TCRs are divided into two categories: TCR1 and TCR2; TCR1 consists of two chains, γ and δ, and TCR2 consists of two chains, α and β. Based on its TCR expression profile, TCR ⁺ IELs can be divided into two major subgroups: one is thymus-dependent conventional T cell, which expresses TCR α β together with the CD4 or CD8 α β co-receptor; another is non-conventional IELs that are independent of thymus development, co-express TCR α β or TCR γ δ with CD8 α α homodimers (Lambolez F et al, J Exp Med.2002,195 (4): 437-449 Peaudeerf L et al, mucosal Immunol.2011,4 (1): 93-101 McDonald BD et al, nat Rev Immunol.2018,18 (8): 514-525). These different subgroups contribute differently to intestinal mucosal homeostasis: unconventional IELs (TCR. Alpha. Beta. Beta.) ⁺ CD8αα ⁺ And TCR γ δ ⁺ CD8αα ⁺ ) Protection of the intestinal epithelium against auto-ligands and inflammatory attacks (Qiu Y et al, dig Dis Sci.2016,61 (6): 1451-1460); conventional TCR α β ⁺ IELs respond to both external attacks and microbial loads (Ishibashi H et al, diagn Pathol.2016,11, mayassi T et al, mucosal Immunol.2018,11 (5): 1281-1289). The dysregulated population partitioning of IELs has been implicated in Inflammatory Bowel Disease (IBD), such as Crohn's Disease (CD) and Ulcerative Colitis (UC) (Vivinus-Nebot M et al, gut.2014,63 (5): 744-75218,11 (2): 357-368; regner EH et al, arthritis Res ther.2018,20 (1): 149).

Traditional treatment drugs for IBD mainly include three main classes of salicylic acid preparations, glucocorticoids, and immunosuppressive drugs. According to the research that the relevant factors involved in pathogenesis include interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin (interleukin) -12 (IL-12) and IL-18, and the anti-inflammatory factors include IL-10 and transforming growth factor beta (TGF-beta), etc., the effect of treating IBD can be achieved by inhibiting the inflammatory factors and up-regulating the expression of the immune regulator to restore the steady-state balance of the cytokines.

In fact, the healthy intestinal mucosa shows many features of a chronic inflammatory response due to the presence of a large number of effector lymphocytes in the tissue. Human small and large intestine expressed as TCR alpha beta ⁺ IELs predominate, as well as in mice. More microbial load in the large intestine leads to more adaptively induced TCR α β production ⁺ IELs derived from peripherally activated CD8 ⁺ T cells, then migrate to the intestinal epithelium (Beagley KW et al, J Immunol.1995,154 (11): 5611-5619). Recent studies have reported that the antigen undergoes TCR α β ⁺ CD8αβ ⁺ The phenotype of IELs and the CD8 seen by antigens present in peripheral lymphoid organs (e.g., spleen) ⁺ T cells differ in phenotype (McDonald BD et al, nat Rev Immunol.2018,18 (8): 514-525). However, regulatory factors for the development of these IELs remain to be investigated.

Despite the various treatment modalities available for IBD patients, the results have not been satisfactory to date. Recent studies have shown that Fas associated death domain binding protein (FADD) plays an important role in embryogenesis, innate immunity, T cell activation and proliferation. FADD is an adaptor protein required for apoptosis induced by all known Death Receptors (DR), and T cell-specific FADD knockout mice (lck-cre or CD 4-creFADD) show that peripheral FADD deficient T cells fail to respond to TCR-stimulation-induced proliferation.

Cell proliferation induced by a TCR modulated by FADD signalling is dependent on phosphorylation of its C-terminal S191 (mouse) or S194 (human) (Hua ZC et al, immunity.2003,18 (4): 513-52)1; zhang J et al, 2004,56 (7): 395-401). FADD (S191D) transgenic mice in FADD ^-/- Mutations to Asp at the Ser191 site in the background present a number of immune development problems, including defects in thymocyte development. FADD (S191D) is similar to FADD deficient, resulting in a proliferation deficiency of T cells. Interestingly, FADD (S191A) mice are in FADD ^-/- In the background, mutations to Ala at Ser191 indicate a normal number of thymocytes (Hua ZC et al, immunity.2003,18 (4): 513-521).

Currently, there is a lack of a target for regulating intestinal mucosal homeostasis or inflammatory bowel disease in mammals and uses thereof.

Disclosure of Invention

In order to solve the problems of the prior art, the invention provides a target point for regulating intestinal mucosa homeostasis or inflammatory bowel disease of mammals and application thereof, namely CD8 ⁺ Inflammatory bowel disease with T cell dysfunction provides a new treatment.

The invention compares the difference of the intestinal intraepithelial lymphocyte (IELs) population of Wild Type (WT) mice and FADD (S191A) (FADD non-phosphorylated) mice, and finds the influence of the FADD non-phosphorylated or phosphorylated level on the intestinal mucosa homeostasis of mammals; a second objective of the present invention is to explore the effects of FADD (S191A) on the proliferation and expansion of different populations of IELs; it is a third object of the present invention to explore the effect of FADD (S191A) -induced populations of IELs on the development of IBD.

In order to solve the above problems and achieve the above objects, the present invention adopts the following technical solutions based on the comparison between a mimic non-phosphorylated FADD (S191A) protein and a wild type FADD with phosphorylation: the target for regulating intestinal mucosa homeostasis or inflammatory bowel disease of mammals is Fas-related death domain FADD protein, and the sequence of the FADD protein is an amino acid sequence (Ser at the No. 191 site) shown in SEQ ID No. 1.

The method for regulating the intestinal mucosa homeostasis or the target of inflammatory bowel disease of mammals is realized by regulating the non-phosphorylation or phosphorylation level of FADD of cells derived from mammals or mammals.

Furthermore, the method is realized by simulating the expression of protein corresponding to the FADD non-phosphorylated gene, and the sequence of the gene is the nucleotide sequence shown in SEQ ID No. 2.

Furthermore, the protein corresponding to the gene simulating the non-phosphorylation of the FADD is a protein obtained by mutating the 194 th serine of the wild-type human FADD protein to alanine, or mutating the 191 th serine of the mouse FADD protein to alanine.

Furthermore, the modulation of FADD non-phosphorylation of cells of mammalian or mammalian origin is achieved by treating and acting on cells of mammalian or mammalian origin with a substance that effects FADD non-phosphorylation of cells of mammalian or mammalian origin or that reduces the level of phosphorylation of FADD of cells of mammalian or mammalian origin.

Further, the mammal is a mouse or a human.

Further, screening for substances capable of achieving non-phosphorylation of or reducing the level of phosphorylation of FADD in cells of mammalian or mammalian origin includes macromolecular substances capable of achieving non-phosphorylation of or reducing the level of phosphorylation of FADD in cells of mammalian or mammalian origin: protein, nucleic acid or polysaccharide, or small molecule substance: fatty acids, vitamins, natural products, chemically synthesized or engineered products, small organic or inorganic molecules, or nanomolecules.

The invention regulates intestinal mucosa homeostasis of mammals or regulates TCR alpha beta of mammals ⁺ Application of target point and regulation method for rapid amplification of intestinal intraepithelial lymphocyte IELs (intestinal epithelial cells) in regulation of intestinal mucosa homeostasis or inflammatory bowel disease, wherein the target point and regulation method promote TCR alpha beta ⁺ The rapid amplification of type intestinal intraepithelial lymphocyte IELs, the rapid amplification of TCR alpha beta + type intestinal intraepithelial lymphocyte IELs is TCR alpha beta ⁺ CD8 ⁺ Type intestinal intraepithelial lymphocyte IELs subgroup, the intestinal intraepithelial lymphocyte IELs subgroup of TCR alpha beta + CD8+The population is rapidly amplified into TCR alpha beta + CD8 alpha + type IELs and TCR alpha beta + CD8 alpha beta + type IELs subgroups.

Further, decreased unphosphorylation or phosphorylation of FADD results in more severe intestinal inflammation in colitis model mice; partial phosphorylation of the FADD relatively reduces intestinal inflammation of a colitis model mouse, and the FADD and phosphorylation thereof can become therapeutic targets of colitis.

Further, non-phosphorylated or reduced phosphorylated Fas-related death domain proteins result in significantly elevated levels of IFN- γ, TNF- α, IL-1, and IL-6 inflammatory factors in the serum of colitis model mice; partial phosphorylation of the FADD will relatively improve the levels of IFN-gamma, TNF-alpha, IL-1 and IL-6 inflammatory factors in the serum of a mouse model of colitis.

Has the advantages that: the FADD is a target for regulating the intestinal mucosa homeostasis of mammals or regulating the rapid amplification of TCR alpha beta + type intestinal intraepithelial lymphocyte IELs of the mammals, wherein the rapid amplification of the TCR alpha beta + type intestinal intraepithelial lymphocyte IELs is the rapid amplification of TCR alpha beta + CD8+ type intestinal intraepithelial lymphocyte IELs subsets, and the rapid amplification of TCR alpha beta + CD8+ intestinal intraepithelial lymphocyte IELs subsets is the rapid amplification of TCR alpha beta + CD8 alpha + type IELs and TCR alpha beta + CD8 alpha beta + type IELs subsets. The results of the mouse model for colitis show that: mock non-phosphorylated FADD (S191A) mice exhibited more severe intestinal inflammation; FADD phosphorylated (wild type FADD) mice then exhibit reduced intestinal inflammation and their relatively low water IFN-. Gamma., TNF-. Alpha., IL-1 and IL-6 inflammatory factors in serum. Thus proving that: intestinal inflammation can be reduced or treated by regulating the phosphorylation level of FADD.

Drawings

FIG. 1 shows the results of thymus, spleen and lymph node cell counts of wild type FADD and FADD (S191A) mice of 6-8 weeks of age according to the present invention.

FIG. 2 is a graph showing the percentage of CD4 and CD8 cells in thymus, spleen and lymph nodes of mice of the present invention. The numbers indicate the percentage of cells in each gate, and FADD-a is an abbreviation for FADD (S191A) mice.

FIG. 3 is a graph of total LPL and IEL counts in the intestinal tract of 8-week-old WT and FADD (S191A) mice of the present invention.

FIG. 4 shows FACS analysis (flow cytometric fluorescence sorting) of the WT and FADD (S191A) mouse intestinal IELs of the present invention. Staining IELs against TCR α β and TCR γ δ subsets ⁺ Gating of IELs further analyzed the expression of CD4, CD8 α and CD8 β, CD8 α cells gated to CD8 α ⁺ CD8β ^- 。

FIG. 5 shows the intestinal TCR α β of WT and FADD (S191A) mice of the present invention ⁺ IELs、TCRγδ ⁺ IELs、CD4 ⁺ 、CD8 ⁺ 、CD8αβ ⁺ 、CD8αα ⁺ The number of cells.

FIG. 6 shows TCR α β in intestinal sections of WT and FADD (S191A) mice of the present invention ⁺ And TCR γ δ ⁺ Representative images (40-fold) of immunofluorescent staining of IELs and their counting results.

FIG. 7 shows total IELs and LPLs in the intestinal tract of NCG mice (hyperimmune deficient mice deficient in T cells, B cells and NK cells) that received WT or FADD (S191A) mouse bone marrow cell transplantation according to the present invention.

FIG. 8 is a flow cytometric analysis of WT/NCG and FADD-A/NCG mouse IELs cells of the present invention.

FIG. 9 shows the number of total IELs in the mixed bone marrow chimeras of the present invention. Mixed bone marrow chimeras were prepared by mixing bone marrow from BoyJ mice (CD 45.1) and FADD (S191A) mice (CD 45.2) or WT mice (CD 45.2) into NCG mice.

FIG. 10 is a graph of the percentage and number of IELs from mixed bone marrow cells of CD45.1 or CD45.2 of the present invention.

FIG. 11 is CD45.1 from a mixed chimera of the invention ⁺ Or CD45.2 ⁺ Flow cytometry analysis of the markers specified in IELs.

Fig. 12 shows the results of BrdU staining of WT or FADD (S191A) mice.

FIG. 13 is a graph of the number of total IELs and LPLs in 3 to 10 week old WT or FADD (S191A) mice of the present invention.

FIG. 14 is a TCR α β of total IELs in 3 to 10 week old WT or FADD (S191A) mice of the present invention ⁺ And TCR γ δ ⁺ Number of T cells

FIG. 15 shows the number of IELs in WT and FADD (S191A) mice treated with antibiotics of the present invention.

FIG. 16 shows WT or FADD (S191A) mouse primary CD8 of the present invention ⁺ Results of CFSE experiments on T cells.

FIG. 17 shows WT or FADD (S191A) mouse primary CD8 of the present invention ⁺ Western blot analysis of T cells.

FIG. 18 is a graph of the change in FADD phosphorylation during T cell activation of the present invention.

FIG. 19 shows a CD8 of the present invention ⁺ FACS analysis of p-FADD (S191) in T cells.

FIG. 20 is a photograph of the colon of WT and FADD (S191A) mice of the present invention. Sham was Sham-operated control group; TNBS-colitis is a mouse model group for colitis.

FIG. 21 shows the results of colon length quantification of WT and FADD (S191A) mice of the present invention.

FIG. 22 is a graph showing the body weight change of WT and FADD (S191A) mice of the present invention.

FIG. 23 shows the results of H & E staining of colon sections of WT and FADD (S191A) mice of the present invention.

FIG. 24 shows the levels of production of inflammatory cytokines TNF- α, IFN- γ, IL-1 β and IL-6 in the serum of WT and FADD (S191A) mice of the present invention.

Detailed Description

The invention will be better understood from the following examples. However, it is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating the present invention and should not be construed as limiting the present invention as detailed in the claims.

The target for regulating intestinal mucosa homeostasis or inflammatory bowel disease of mammals is Fas-related death domain FADD protein, and the sequence of the FADD protein is an amino acid sequence (Ser at the No. 191 site) shown in SEQ ID No. 1.

The method for regulating the intestinal mucosa homeostasis or the target of inflammatory bowel disease of mammals is realized by regulating the non-phosphorylation or phosphorylation level of FADD of cells derived from mammals or mammals.

The method is realized by simulating the expression of protein corresponding to the FADD non-phosphorylated gene, and the sequence of the gene is the nucleotide sequence shown in SEQ ID No. 2.

The protein corresponding to the gene simulating the non-phosphorylation of the FADD is obtained by mutating the 194 th serine of the wild-type human FADD protein to alanine, or mutating the 191 th serine of the mouse FADD protein to alanine.

The modulation of FADD non-phosphorylation of cells of mammalian or mammalian origin is achieved by treating and acting on cells of mammalian or mammalian origin with a substance capable of effecting FADD non-phosphorylation of cells of mammalian or mammalian origin or capable of reducing the level of phosphorylation of FADD of cells of mammalian or mammalian origin.

The mammal is a mouse or a human.

Screening for substances capable of achieving non-phosphorylation or reducing the phosphorylation level of a FADD in a cell of mammalian or mammalian origin, including macromolecular substances capable of achieving non-phosphorylation or reducing the phosphorylation level of a FADD in a cell of mammalian or mammalian origin: protein, nucleic acid or polysaccharide, or small molecule substance: fatty acids, vitamins, natural products, chemically synthesized or engineered products, small organic or inorganic molecules, or nanomolecules.

The invention regulates intestinal mucosa homeostasis of mammals or regulates TCR alpha beta of mammals ⁺ Application of target point and regulation method for rapid amplification of intestinal intraepithelial lymphocyte IELs (intestinal epithelial cells) in regulation of intestinal mucosa homeostasis or inflammatory bowel disease, wherein the target point and regulation method promote TCR alpha beta ⁺ The rapid amplification of type intestinal intraepithelial lymphocyte IELs, the rapid amplification of TCR alpha beta + type intestinal intraepithelial lymphocyte IELs is TCR alpha beta ⁺ CD8 ⁺ And the intestinal intraepithelial lymphocyte IELs subgroup of TCR alpha beta + CD8+ is rapidly amplified into TCR alpha beta + CD8 alpha + IELs and TCR alpha beta + CD8 alpha beta + IELs.

Non-phosphorylation or decreased phosphorylation of FADD results in colitis model mice exhibiting more severe intestinal inflammation; partial phosphorylation of the FADD relatively reduces intestinal inflammation of a colitis model mouse, and the FADD and phosphorylation thereof can become therapeutic targets of colitis.

Non-phosphorylation or decreased phosphorylation of Fas-associated death domain proteins results in significantly elevated levels of IFN-gamma, TNF-alpha, IL-1 and IL-6 inflammatory factors in the serum of colitis model mice; partial phosphorylation of FADD relatively improved the levels of IFN-. Gamma., TNF-. Alpha., IL-1 and IL-6 inflammatory factors in the serum of colitis model mice.

Example 1

FADD (S191A) mouse model construction and whole body immune tissue analysis

1.1 Experimental materials

1.1.1 mice

FADD (S191A) mice were constructed by the inventors at the Aster Winoto laboratory at the university of california, berkeley, usa. NCG mice (NODprkdc) ^-/- IL-2Rg ^-/- Genotyped hyperimmune deficient mice) were purchased from the research center for model animal genetics at the university of Nanjing (Nanjing, china). BoyJ mice are professor donations Wang Xiao at the university of medical, nanjing. All animal experiments were approved by the university of Nanjing animal Care and use Committee (NJU-ACUC).

1.1.2 reagents

Fetal calf serum FCS (total calf serum) from Thermo; dithiothreitol DTT is purchased from Biyun Tian; CMFDA from Invitrogen;10 × PBS was purchased from Shengxing biology of Nanjing; RPMI 1640; a suspension chip;

percoll mother liquor; thioglycolates were purchased from beijing solibao; trypan blue dye; FITC-bound TCR α β, APC-bound TCR γ δ, PE-bound CD8 α, percpcy5.5-bound CD8 β, PE/Cy 7-bound CD4, FITC-bound CD45.1, and PercpCy 5-bound CD45.2.

1.1.3 instruments and consumables

Flow cytometry was purchased from BD; the oscillator is purchased from Lin Beier of Jiangsu province; automated cytometers were purchased from Invitrogen; the autoclave was purchased from Nanjing Yipuyida; fluorescence microscopy was purchased from Carl Zeiss AG, germany, model Zeiss AX10; centrifuge was purchased from semer feishel corporation; FACS analysis was performed on a FACS Calibur flow cytometer equipped with Cell Quest software (BD Biosciences, franklin Lakes, N.J.).

1.1.4 solutions

CMF solution: 100mL 10 × HBSS (without calcium and magnesium); 100mL 10 XHEPES-sodium bicarbonate buffer, 20mL FBS plus water to 1L, filter sterilization. Can be stored at 4 ℃ for two weeks. Only 800mL of 4 samples are needed.

CMF/FCS/DTT: add 8mL FCS to 92mL CMF, add 15.4mg DTT (dithiothreitol) (ready to use) 4 samples, only 200mL.

1 × Percoll stock (ready to use): 90mL Percoll was added to 10mL 10 XPBS.

44% Percoll solution: 44mL of 1 XPercoll stock was added 56mL of RPMI 1640.

67% Percoll solution: 67mL of 1 XPercoll stock was added 33mL of RPMI 1640. The pH was adjusted to 7.2 with HCl and placed on ice for use. All solutions used must be maintained at 4 ℃ or in an ice bath.

1.2 Experimental methods

1.2.1 construction of FADD (S191A) mouse model

To assess the applicability of FADD (S191A) mice to intestinal mucosal immunity studies, the present invention first constructed a FADD (S191A) mouse model and analyzed whether it has a normal immune system. FADD (S191A) mice were generated from transgenic mice carrying mutant FADD and FADD ^+/- Mice were generated by mating for at least two generations, so FADD (S191A) mice only expressed mutant FADD to mimic mice bearing FADD ^-/- Constitutive unphosphorylated FADD of alleles.

1.2.2 extraction of intestinal epithelial lymphocytes

(1) Mice were sacrificed, midline ventral incisions, and skin was strained.

(2) The top end of the intestine is clamped by forceps, the colon is slowly pulled out of the abdominal cavity, the lower end of the colon is cut at the appendix, and the colon is pulled out to the anus to be cut.

(3) A20 mL syringe was filled with the 4 ℃ CMF solution, the colon was grasped with forceps and placed in a large beaker, and the 18-G or 20-G needle was inserted into the bowel lumen and pressure was slowly applied to the syringe barrel (5 mL per minute). If a leak or blockage occurs during the flush, the forceps are moved to the problem site, the syringe is reinserted, and the perfusion of the CMF is continued. The entire intestine was rinsed with 40mL of 4 ℃ CMF.

(4) The intestinal tissue was placed on CMF soaked filter paper and the remaining fat and connective tissue were removed with two forceps. The forceps are horizontally placed on the surface of the intestine and gently pressed along the outer surface of the intestine to remove mucus in the intestinal cavity. The intestine was cut longitudinally and cut laterally into pieces of about 0.5 cm. The Pe-Shi node is cut off from the small intestine by scissors, and the Pe-Shi node is not arranged on the colon and does not need to be cut off.

(5) The intestinal tissue pieces were placed into a 50mL conical centrifuge tube and 40mL of 4 ℃ CMF solution was added. The tube was inverted several times until the tissue settled, and the supernatant was decanted. Three replicates were performed until the supernatant was relatively clear.

(6) The tissue was placed in a 50mL conical flask containing 20mL of CMF/FCS/DTT solution, sealed with a sealing membrane and incubated on a shaker at 37 ℃ and 220rpm for 20min.

(7) The solution and tissue were transferred to a 50mL conical centrifuge tube and vortexed for 15s with maximum intensity. The tissue was allowed to settle and the supernatant was transferred to another 50mL conical centrifuge tube by filtration through a cell sieve.

(8) To the intestinal tissue mass, 20mL of CMF/FCS/DTT solution was added and step 7 was repeated. Supernatants from both runs were pooled. The debris formed was removed and the supernatant was stored on ice.

(9) The tissue pieces were returned to the 50mL Erlenmeyer flask, and the 6 to 8 steps were repeated, and the supernatants were combined. Centrifuge at 400 Xg for 5min at 4 ℃. The pellet was resuspended in 5mL of 4 ℃ precooled RPMI 1640 and stored on ice.

(10) Centrifuge at 400 Xg for 5min at 4 ℃. Resuspend cells in 44% Percoll at 8mL room temperature.

(11) Several tubes of 17X 100mm were prepared, the tubes were rinsed sequentially with FCS, and the FCS was discarded.

(12) Percoll was added at rate of 5mL 67% per tube, and 8mL of 44% Percoll cell suspension was slowly added. Centrifuge at 600 Xg for 30min at 4 ℃.

(13) Half of the liquid in the upper layer of the gradient was aspirated off until about 2cm from the phase interface (cell layer). The interphase was collected and the cells were diluted with 4 ℃ RPMI 1640 at 1:3. Centrifuging at 400 Xg for 5min at 4 deg.C to precipitate and agglomerate.

(14) Viable cells were counted with a Typan blue.

(15) After incubation of the antibodies, the analysis was performed by flow cytometry.

1.3 Experimental results and analysis

When FADD (S191A) mice were analyzed for intestinal IELs population, FADD (S191A) mice were not phenotypically different in thymus, spleen and lymph nodes compared to Wild Type (WT) mice (fig. 1). Flow cytometric analysis of CD4 and CD8 profiles also showed that T cell populations of thymocytes, splenocytes, and lymph nodes of FADD (S191A) mice were similar to WT mice (fig. 2). Therefore, FADD (S191A) mice have normal systemic immune tissues and can be used for studies on intestinal mucosal immunity.

Example 2

FADD (S191A) causes intestinal IEL dysplasia

2.1 Experimental materials

2.1.1 mice

FADD (S191A) mice were constructed by the inventors at the Aster Winoto laboratory at the university of california, berkeley, usa. The BoyJ mouse is a professor donation of Wang Xiao of the university of medical science of Nanjing, china. All animal experiments were approved by the university of Nanjing animal Care and use Committee (NJU-ACUC).

2.1.2 reagents

Fetal calf serum FCS (total calf serum) from Thermo; dithiothreitol DTT was purchased from Byunyan; CMFDA from Invitrogen;10 × PBS was purchased from Shengxing biology of Nanjing; RPMI 1640; a suspension chip;

percoll mother liquor; thioglycolates were purchased from beijing solibao; trypan blue dye; FITC-bound TCR α β, APC-bound TCR γ δ, PE-bound CD8 α, percpcy5.5-bound CD8 β, PE/Cy 7-bound CD4, FITC-bound CD45.1, and PercpCy 5-bound CD45.2. Collagenase type VIII was purchased from Sigma; DNase I was purchased from an organism;

2.1.3 instruments

Cryomicrotomes were purchased from Leica; flow cytometry was purchased from BD; the oscillator is purchased from Lin Beier of Jiangsu province; fluorescence microscopy was purchased from Carl Zeiss AG, germany, model Zeiss AX10; centrifuge was purchased from semer feishel corporation; FACS analysis was performed on a FACS Calibur flow cytometer equipped with Cell Quest software (BD Biosciences, franklin Lakes, NJ).

2.2 Experimental methods

Separation of IEL and LPL:

mucosal lymphocytes were isolated and prepared according to previously published methods. The cut small intestine sections were washed and then incubated for 30 minutes at 37 ℃ with vigorous shaking at 220rpm in CMF solution containing 10% FCS and 1mM DTT. The suspension was filtered through a nylon mesh to remove debris and centrifuged through a 40-70% discontinuous gradient Percoll (Solarbio, beijing, china) at 600g for 20 minutes. 40-70% of the alternate IEL was collected. For LPL isolation, the tissue was digested in RPMI-1640 supplemented with 300U/ml collagenase type VIII (Sigma-Aldrich, st Louis, MO, USA), 0.1mg/ml DNase I (Sangon Biotech, shanghai, china) and 10% FCS at 220rpm, 37 ℃ for 30 minutes, then fractionated at 44% -67% by Percoll density centrifugation. These cells were washed once in PBS and used for FACS analysis experiments.

Fluorescence immunohistochemistry:

the frozen intestinal tissue was cut into sections of 10 μm with a cryomicrotome (Leica, CM1950, germany). After 5% normal goat serum blocking, the frozen sections were incubated with FITC-labeled antibodies to TCR α β (553171, BD pharmingen) or TCR γ δ (11-5811-82, eBioscience) at 4 ℃ for 3 hours in the dark, and finally counterstained using DAPI for fluorescence microscopy (Zeiss AX10, carl Zeiss AG, germany).

2.3 Experimental results and analysis

In the intestinal tract, effector cells are present in two major compartments, namely Lamina Propria Lymphocytes (LPLs) and intraepithelial lymphocytes (IELs), which are phenotypically and functionally similar to peripheral lymphocytes. Analysis of differences between intestinal LPLs and IELs of FADD (S191A) mice and wild type FADD mice by flow cytometry revealed that: the cell counts of LPLs from FADD (S191A) mice were similar to wild-type FADD mice (fig. 3). Almost a 10-fold increase in abnormal IELs was observed in FADD (S191A) mice compared to wild-type FADD mice (fig. 3).

Using a set of TCR α β and TCR γ δ indices vs TCR γ δ in IEL ⁺ And TCR α β ⁺ The subset ratio changes were analyzed and the results showed that the FADD (S191A) mice were TCR γ δ in FADD ⁺ And TCR α β ⁺ The subset ratio changes were different from wild type FADD mice (fig. 4). Gated TCR α β ⁺ A subset was used to analyze CD4 and CD8 profiles, and the results showed TCR α β ⁺ CD8 ⁺ The FADD (S191A) IEL of the subpopulation was increased. To refine the CD8 subset, TCR α β was further analyzed using CD8 α and CD8 β ⁺ Subpopulations. TCR α β observed in FADD (S191A) IEL ⁺ CD8αα ⁺ A significant increase in the ratio (fig. 4). However, cell counts indicated FADD (S191A) mice have normal numbers of TCR γ δ ⁺ IEL, and the increased IEL population is primarily TCR α β ⁺ IEL at TCR α β ⁺ CD8αβ ⁺ And TCR α β ⁺ CD8αα ⁺ Middle (fig. 5). To confirm TCR γ δ in vivo ⁺ And TCR α β ⁺ Difference in IEL numbers, the present invention performed fluorescent immunohistochemical staining of intestinal sections of FADD (S191A) mice and wild type FADD mice. A greater number of TCR α β positive staining was observed in the villi of FADD (S191A) mice, with TCR γ δ positive staining unchanged in these mice (fig. 6).

Example 3

FADD (S191A) induces TCR α β in mice ⁺ IEL over-amplification

3.1 Experimental materials

3.1.1 mice

FADD (S191A) mice were constructed by the inventors at the Aster Winoto laboratory at the university of california, berkeley, usa. The Boy mouse is a professor donation of Wang Xiao of the university of medical science of Nanjing, china. All animal experiments were approved by the university of Nanjing animal Care and use Committee (NJU-ACUC).

3.2 Experimental methods

Bone Marrow (BM) chimera transplantation:

for BM transplantation, NCG mice received 2Gy of gamma irradiation and intravenous injection. Injection of 2X 10 from WT or FADD (S191A) mice ⁶ Total Bone Marrow Cells (BMC). Generation of bone marrow chimeras with mixing according to the previously described procedureSomatic mice. Briefly, mice from donor small FADD (S191A) or wild type FADD (CD 45.2) ⁺ ) And BoyJ mice (CD 45.1) ⁺ ) The bone marrow cells of (1) were mixed in PBS buffer at a ratio of 1:1. A total of 2X 10 in 200. Mu.l PBS ⁶ Mixed BMs were transplanted intravenously into irradiated (2.5 Gy) NCG mice. Recipient mice were sacrificed 28 days post-transplantation and intestinal IELs were prepared for FACS analysis.

3.3 Experimental results and analysis

To exclude potential factors caused by systemic expression of FADD (S191A) in mice, bone Marrow Cells (BMCs) were isolated from wild-type FADD or FADD (S191A) mice and injected into NCG recipient mice (hyperimmune deficient mice and NK cells lacking T cells, B cells). At 28 days post-injection, total IEL from FADD (S191A)/NCG mice was significantly increased (-8 fold) compared to wild type FADD/NCG mice in the IEL chamber, while total LPL was similar (fig. 7), consistent with the phenotype of FADD (S191A) mice. Similar to FADD (S191A) mice, TCR α β ⁺ A significant increase in the proportion of subpopulations and cell number, FADD (S191A)/NCG mice TCR α β ⁺ CD8αα ⁺ The ratio of (a) also increases significantly (fig. 8).

To better explore the effect of FADD on intestinal IEL population expansion, mixed BM chimeras were generated. BM chimerism is the establishment of a competitive assay by mixing bone marrow of a BoyJ mouse (CD 45.1) and a FADD (S191A) mouse (CD 45.2) or a wild type FADD mouse (CD 45.2) into NCG mice. There was no difference in IEL total number between the two mixed BM chimeras (fig. 9).

Analysis of IEL by CD45.1 and CD45.2 showed a correlation with CD45.1 from WT-BMC ⁺ CD45.2 from donor FADD (S191A) -BMC in contrast to IEL ⁺ IEL increased significantly (-4 fold) (fig. 10). Further analysis of TCR α β and TCR γ δ then demonstrated CD45.2 from FADD (S191A) -BMCs by gating CD45.1 or CD45.2 ⁺ IEL is primarily TCR α β ⁺ Cells (up to 92%), and CD45.1 ⁺ The IEL is completely different (fig. 11). In addition, with CD45.1 ⁺ IEL compares CD45.2 from FADD (S191A) -BMC ⁺ TCR α β in IEL ⁺ CD8αα ⁺ Also increased, consistent with the observations in FADD (S191A) mice (fig. 11).

Example 4

FADD (S191A) promotes TCR α β ⁺ Proliferation of IEL, not TCR γ δ ⁺ IEL

4.1 Experimental materials

4.1.1 mice

FADD (S191A) mice were constructed by the inventors at the Aster Winoto laboratory at the university of california, berkeley, usa. The BoyJ mouse is a professor donation of Wang Xiao of the university of medical science of Nanjing, china. All animal experiments were approved by the university of Nanjing animal Care and use Committee (NJU-ACUC).

4.1.2 reagents

Bromodeoxyuridine (BrdU); brdU flow kit (BD pharmingen); metronidazole (1 g/L), neomycin sulfate (1 g/L), ampicillin (1 g/L), and vancomycin (0.5 g/L) were purchased from Cayman Chemical;

4.2 Experimental methods

BrdU assay:

bromodeoxyuridine (BrdU) is a marker for proliferating cells. BrdU (50 mg/kg) was injected twice daily into the peritoneum of mice at 8-hour intervals for 7 consecutive days. After dissecting the mice, the IELs were collected and stained with BrdU flow kit (BD pharmingen) and analyzed by flow cytometry.

Antibiotic treatment:

according to the reported method, four antibiotics were used to remove commensal bacteria in the intestinal tract. Briefly, mice were provided with sterile water supplemented with metronidazole (1 g/L), neomycin sulfate (1 g/L), ampicillin (1 g/L), and vancomycin (0.5 g/L) (Cayman Chemical, michigan, USA) for 2 weeks starting at 5 weeks of age. Mice were provided with water containing antibiotics and replaced twice weekly.

Thymus gland removal:

wild type FADD mice and FADD (S191A) mice at 8 weeks of age were intraperitoneally injected with 100. Mu.l of 0.1% pentobarbital for anesthesia. The mouse was then shaved gently with a razor, and sterilized with alcohol. The cortex and the muscle layer of the mouse are cut off in sequence by a sterilized scalpel, and the breathing machine catheter is inserted into the trachea of the mouse to assist the mouse to breathe in the operation process. Muscle layers were excised 2cm above the chest of the mice, and 2 or 3 ribs were excised. At this time, the thymus was gently removed with forceps, and then the cortex and muscle layers were sutured in order. Mice with thymus removed were fed for one month.

4.3 Experimental results and analysis

In order to monitor T cell proliferation within the intestinal mucosa, the present invention performs BrdU incorporation in vivo. After 3 days of dosing with BrdU solution, intestinal IELs were collected to determine BrdU incorporation by FACS analysis. TCR γ δ in similar proportions in wild type FADD and FADD (S191A) mice ⁺ IEL is BrdU labeled, but with BrdU positive staining of TCR alpha beta ⁺ The percentage of IEL increased to-86% in FADD (S191A) mice (fig. 12), indicating that IEL overload in FADD (S191A) mice, TCR α β ⁺ IEL significantly accelerated the proliferation results.

To verify FADD (S191A) vs. TCR α β ⁺ Proliferation of IEL development, the present invention tracks IEL development in mice of 3 to 10 weeks of age. Compared to wild-type FADD mice, the total IEL of FADD (S191A) mice increased from week 5 and rapidly increased with age (fig. 13). LPL increased at 5 weeks but gradually returned to normal levels, similar to WT mice (fig. 13). Further analysis of the IEL population revealed TCR α β ⁺ The number of IELs showed an abnormal increase, which is consistent with the trend of total IELs, whereas TCR γ δ in FADD (S191A) mice ⁺ IELs remained normal, similar to wild type FADD mice (FIG. 14). Taking into account TCR α β ⁺ The accumulation of IEL in the gut is likely from food and microbiota, and mice received antibiotic combination therapy to reduce microbiota. As expected, the total IEL was reduced in antibiotic-treated mice, in particular in antibiotic-treated FADD (S191A) mice (fig. 15). Further analysis showed that FADD (S191A) induced excessive TCR α β ⁺ IELs were almost eliminated after antibiotic treatment, but TCR γ δ ⁺ The IELs were unchanged in antibiotic-treated mice (figure 15).

Due to the vast majority of TCR alpha beta ⁺ T-IEL is thymus-derived peripheral T cells that subsequently migrate to the intestinal epithelium, and the present invention detects T-IEL development in thymus-depleted FADD (S191A) mice. Total IEL recovery in thymectomized FADD (S191A) mice one month after thymus excisionReverting to similar numbers as thymectomized wild type FADD mice (fig. 15). The total number of IELs in FADD (S191A) mice (FIG. 8) became normal. Consistently, FADD (S191A) mouse CD8 in the excised thymus ⁺ TCRγδ ⁺ The native T-IEL subpopulation of cells was also reconstituted to resemble the normal phenotype of wild-type FADD mice. This indicates that the intestinal T cells affected by FADD (S191A) originate from the thymus, rather than the local development of T-IELs.

Example 5

FADD (S191A) promotes TCR α β ⁺ CD8 ⁺ Rapid proliferation of T cells

5.1 Experimental materials

5.1.1 mice

FADD (S191A) mice were constructed by the inventors at the Aster Winoto laboratory at the university of california, berkeley, usa. The BoyJ mouse is a professor donation of Wang Xiao of the university of medical science of Nanjing, china. All animal experiments were approved by the university of Nanjing animal Care and use Committee (NJU-ACUC).

5.1.2 reagents

1 × BD IMag TM buffer; mouse CD4 ⁺ Magnetic microbeads (551539, bd Pharmingen); mouse CD8 ⁺ Magnetic microbeads (551516, bd Pharmingen);

5.2 Experimental methods

Magnetic bead sorting of primary T cells:

mouse primary T cells were prepared from lymph nodes and resuspended in 1 × BD i magtm buffer. 1x10 ⁷ Cells 100ul and 50 ul mouse CD4 ⁺ (551539) or CD8 α ⁺ (551516) magnetic microbeads (BD Pharmingen). The sample tube was placed in a cell separation magnet (IMagnet, BD Pharmingen), and the cells were washed 3 times with 1 × BD IMagTM buffer. The desired cells were eluted according to the manufacturer's recommended protocol.

5.3 Experimental results and analysis

To further examine the modulation of TCR-mediated cell expansion by FADD, the present invention isolated primary T cells from lymph nodes for CFSE assay. TCR α β from FADD (S191A) mice following CD3/CD28 co-stimulation ⁺ CD8 ⁺ T cells showed accelerated proliferation, significantly faster than those from wild typeProliferation of FADD mice (fig. 16). Consistently, in FADD (S191A) CD8 ⁺ Stronger activation of IKK and NF- κ B was observed in T cells (FIG. 17). Considering that FADD (S191A) mimics unphosphorylated FADD, the present invention further detects changes in FADD phosphorylation during T cell activation. Immunoblots indicated the presence of FADD dephosphorylation process in response to TCR activation (fig. 18). Similar results were confirmed by flow cytometry (FACS) analysis and intracellular staining of phosphofadd (fig. 19). Although there was no difference in the levels of FADD proteins between wild-type FADD and FADD (S191A), FADD exists in a state in which a small amount of protein is phosphorylated in wild-type FADD, and as a result, FADD (S191A) CD8 ⁺ NF-. Kappa.B was strongly activated in T cells.

Example 6

FADD (S191A) causes more severe inflammation in TNBS-induced colitis

6.1 Experimental materials

6.1.1 mice

FADD (S191A) mice were constructed by the inventors at the Aster Winoto laboratory at the university of california, berkeley, usa. The BoyJ mouse is a professor donation of Wang Xiao of the university of medical science of Nanjing, china. All animal experiments were approved by the university of Nanjing animal Care and use Committee (NJU-ACUC).

6.2 Experimental methods:

modeling of mouse TNBS enteritis:

(1) Selecting a plurality of mice of 16-18 g, and placing the mice in a metabolism cage for fasting for 24 hours.

(2) Preparing a medicine: 50% absolute ethanol +48% 5% TNBS mother liquor +2% ddH ₂ O。

(3) After the ether is inhaled to anaesthetize the mouse, 0.1mL of the prepared medicine is sucked by the syringe and is connected with an intragastric needle (the inner diameter is 1.2 mm), the mouse is fixed by one hand, the catheter is rotationally guided into the colon which is 5-6 cm away from the anus by the other hand, the TNBS modeling group is slowly injected with 0.1mL of TNBS (total concentration), and the control group is injected with PBS (equivalent amount). When injecting medicine, the head should be low and the tail should be high to prevent the infusion liquid from overflowing, after the liquid is pushed into colon, the intragastric needle is slowly drawn out, and the mouse is hung upside down for 1min after infusion.

(4) Naturally keeping clear-headed and conventionally raising.

(5) Body weight was recorded daily and mice were observed for fecal status, eating status and mental status.

H & E staining

Hematoxylin (H) as a basic dye stains endoribosomes bluish-purple. Eosin (E) is an acid dye that stains the cytoplasm red or reddish.

(1) The colon inflammation part is cut and put into PFA fixing solution to denature and coagulate tissue protein.

(2) Gradient alcohol dehydration → xylene wax immersion embedding.

(3) The tissue blocks were embedded in paraffin and cooled.

(4) The wax block was fixed and cut into 5 μm slices. Ironing in hot water, putting on a glass slide, and drying.

(5) Dyeing: several minutes for hematoxylin solution → several seconds for color separation in acid water and ammonia water → flowing water washing for 1h → a moment for distilled water → 10min for 70% alcohol dehydration → 10min for 90% alcohol dehydration → 3min for staining with alcohol eosin staining solution. And dropping gum seal.

6.3 Experimental results and analysis

To evaluate the effect of FADD (S191A) -induced aberrant IEL populations on the development of IBD, the present invention established a mouse model of TNBS-induced colitis. FADD (S191A) mice exhibited more severe disease, characterized by intestinal bleeding, shortened colon length, and weight loss compared to wild-type FADD mice (fig. 20-22). Pathological changes such as crypt deformation, mucosal injury and necrosis are also observed in immunohistochemical detection of FADD (S191A) mouse colon specimens, suggesting that FADD (S191A) causes colitis to be aggravated (FIG. 23). Significant increases in IFN-. Gamma.and TNF-. Alpha.were detected in the sera of FADD (S191A) mice with colitis (FIG. 24). The important proinflammatory cytokines IL-1 and IL-6 were also elevated in colitis mice, and significantly elevated in FADD (S191A) mice with colitis, also indicating severe inflammation in FADD (S191A) mice (fig. 24). The basal levels of these inflammatory factors were normal in FADD (S191A) mice, similar to those in intestinal bleeding, colon length shortening and weight loss mice (fig. 24), consistent with the absence of spontaneous enteritis in FADD (S191A) mice. Compared with the condition that the FADD is in a non-phosphorylation state in the FADD (S191A) mice, the severity of various indexes of the TNBS-induced colitis is greatly reduced by the small phosphorylation of the FADD protein in the wild type FADD mice, and meanwhile, the level of proinflammatory cytokines related to the colitis is greatly reduced. The full demonstration that the FADD is an intervention target of the colitis is that the effect of treating the colitis can be achieved by regulating the phosphorylation levels of the phosphorylation, the non-phosphorylation and the like of the FADD.

While the foregoing description shows and describes several preferred embodiments of this invention, it is to be understood, as noted above, that this invention is not limited to the forms disclosed herein, but is not intended to be exhaustive or to exclude other embodiments and may be used in various other combinations, modifications, and variations within the scope of the inventive concept, as may be realized by the teachings set forth above or as may be learned by the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Nanjing Ji Ruikang Biotechnology research institute, inc. of Jiangsu target biomedical research institute, inc

<120> a target for regulating intestinal mucosa homeostasis or inflammatory bowel disease of mammals and application thereof

<130> 2022

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<170> SIPOSequenceListing 1.0

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<213> Artificial sequence (amino acid sequence of FADD protein)

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1 5 10 15

Gly Asn Asp Leu Met Glu Leu Lys Phe Leu Cys Arg Glu Arg Val Ser

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Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Thr Val

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Leu Leu Glu Gln Asn Asp Leu Glu Arg Gly His Thr Gly Leu Leu Arg

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Glu Leu Leu Ala Ser Leu Arg Arg His Asp Leu Leu Gln Arg Leu Asp

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Asp Phe Glu Ala Gly Thr Ala Thr Ala Ala Pro Pro Gly Glu Ala Asp

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Leu Gln Val Ala Phe Asp Ile Val Cys Asp Asn Val Gly Arg Asp Trp

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Leu Lys Val Trp Lys Asn Ala Glu Lys Lys Asn Ala Ser Val Ala Gly

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Leu Val Lys Ala Leu Arg Thr Cys Arg Leu Asn Leu Val Ala Asp Leu

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Val Glu Glu Ala Gln Glu Ser Val Ser Lys Ser Glu Asn Met Ala Pro

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Val Leu Arg Asp Ser Thr Val Ser Ser Ser Glu Thr Pro

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agtggcctgg acctgttcac ggtgctgctg gagcagaacg acctggagcg cgggcacacc 180

gggctgctgc gcgagttgct ggcctcgctg cgccgacacg atctactgca gcgcctggac 240

gacttcgagg cggggacggc gaccgctgcg cccccggggg aggcagatct gcaggtggca 300

tttgacattg tgtgtgacaa tgtggggaga gactggaaaa gactggcccg cgagctgaag 360

gtgtctgagg ccaagatgga tgggattgag gagaagtacc cccgaagtct gagtgagcgg 420

gtaagggaga gtctgaaagt ctggaagaat gctgagaaga agaacgcctc ggtggccgga 480

ctggtcaagg cgctgcggac ctgcaggctg aatctggtgg ctgacctggt ggaagaagcc 540

caggaatctg tgagcaagag tgagaatatg gcccccgtac taagggattc aactgtgtct 600

tcctcagaaa caccctga 618

Claims (10)

1. A target for modulating gut mucosal homeostasis or inflammatory bowel disease in a mammal, comprising: the target is Fas related death structural domain FADD protein, and the sequence of the FADD protein is an amino acid sequence shown in SEQ ID No. 1.

2. The method of modulating gut mucosal homeostasis or a target of inflammatory bowel disease in a mammal of claim 1, wherein: by regulating the non-phosphorylation or phosphorylation level of FADD of a cell of mammalian or mammalian origin.

3. The method of modulating gut mucosal homeostasis or a target of inflammatory bowel disease in a mammal according to claim 2, wherein: the method is realized by simulating the expression of protein corresponding to the FADD non-phosphorylated gene, and the sequence of the gene is the nucleotide sequence shown in SEQ ID No. 2.

4. The method of modulating gut mucosal homeostasis or a target of inflammatory bowel disease in a mammal according to claim 3, wherein: the protein corresponding to the gene simulating the non-phosphorylation of the FADD is obtained by mutating the 194 th serine of the wild-type human FADD protein to alanine, or mutating the 191 th serine of the mouse FADD protein to alanine.

5. The method of modulating gut mucosal homeostasis or a target of inflammatory bowel disease in a mammal according to claim 3, wherein: the modulation of FADD non-phosphorylation of cells of mammalian or mammalian origin is achieved by treating and acting on cells of mammalian or mammalian origin with a substance capable of effecting FADD non-phosphorylation of cells of mammalian or mammalian origin or capable of reducing the level of phosphorylation of FADD of cells of mammalian or mammalian origin.

6. The method of modulating gut mucosal homeostasis or a target of inflammatory bowel disease in a mammal according to claim 1, wherein: the mammal is a mouse or a human.

7. The method of modulating gut mucosal homeostasis or a target of inflammatory bowel disease in a mammal according to claim 1, wherein: screening for substances capable of achieving non-phosphorylation or reducing the phosphorylation level of a FADD in a cell of mammalian or mammalian origin, including macromolecular substances capable of achieving non-phosphorylation or reducing the phosphorylation level of a FADD in a cell of mammalian or mammalian origin: protein, nucleic acid or polysaccharide, or small molecule substance: fatty acids, vitamins, natural products, chemically synthesized or chemically engineered products, small organic or inorganic molecules, or nano-molecules.

8. The method of any one of claims 1 to 7 for modulating gut mucosal homeostasis or modulating TCR α β in a mammal ⁺ The application of the target point and the regulation method of the intestinal intraepithelial lymphocyte IELs for rapid amplification or inflammatory bowel disease in the regulation of intestinal mucosa homeostasis or inflammatory bowel disease is characterized in that: the target and the regulation method promote TCR alpha beta ⁺ The rapid amplification of type intestinal intraepithelial lymphocyte IELs, the rapid amplification of TCR alpha beta + type intestinal intraepithelial lymphocyte IELs into TCR alpha beta ⁺ CD8 ⁺ And the intestinal intraepithelial lymphocyte IELs subgroup of TCR alpha beta + CD8+ is rapidly amplified into TCR alpha beta + CD8 alpha + IELs and TCR alpha beta + CD8 alpha beta + IELs.

9. Use of the target of and the method of modulating the intestinal mucosal homeostasis in a mammal or modulating the rapid expansion of TCR α β + intestinal intraepithelial lymphocytes (IELs) in a mammal, or inflammatory bowel disease, according to any one of claims 1 to 7, for modulating the intestinal mucosal homeostasis, or inflammatory bowel disease, wherein: the non-phosphorylation or the reduction of the phosphorylation level of the FADD causes the colitis model mouse to show more serious intestinal inflammation, and can become a therapeutic target of colitis.

10. Use according to claim 9, characterized in that: non-phosphorylation or decreased phosphorylation of Fas-associated death domain proteins results in significantly elevated levels of IFN-gamma, TNF-alpha, IL-1 and IL-6 inflammatory factors in the serum of mice model colitis.

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