Mechanism Of FADD Dominant Negative Mutant Affecting The Development Of Intestinal Intraepithelial T Cells

Fas-associated death domain protein(FADD)is a cytoplasmic protein that plays an important adaptor role in the TNF receptor family-induced apoptotic signaling pathway.In addition to mediating the classical apoptotic pathway,FADD is involved in other biological processes.Many studies have shown that FADD participates in the development and proliferation of T cells.However,these studies have focused on the effects of FADD on thymocytes,and the role of FADD in the development of mouse intestinal intraepithelial lymphocytes is poorly studied.In this study,we analyzed the effect of FADD on intestinal intraepithelial lymphocytes,and initially explored the mechanism of FADD affecting intestinal intraepithelial lymphocyte development.In this study,we first analyzed intestinal intraepithelial lymphocytes from WT mice and FADD dominant negative(FADD DN)mice,and found that there was no significant difference in the total number of intestinal intraepithelial lymphocytes,but further analysis of the cell subset found that the percentage of CD8αα+TCRγδ+T cells was significantly reduced in FADD DN mice.To analyze whether the effect of FADD dominant negative on intestinal intraepithelial CD8αα+TCRγδ+T cells was changed over time,we extracted the intestinal intraepithelial lymphocytes of FADD DN mice and WT mice aged 3-8 weeks old.The results showed that the intestinal intraepithelial CD8αα+TCRγδ+T cell subsets of FADD DN mice decreased with time and were always lower than WT mice,indicating that FADD dominant negative can inhibit the development of intestinal intraepithelial CD8αα+TCRγδ+T cells in mice.In the second part of the study,we initially explored the mechanism by which FADD dominant negative inhibits the development of mouse intestinal intraepithelial CD8αα+TCRγδ+T cells.According to the literature,mouse intestinal intraepithelial lymphocytes are developed from two parts of cells:part of the lymphocytes derived from the development of the thymus,and part of the development of the precursor cells in the intestine.We analyzed thymocytes and intestinal intraepithelial lymphocytes after removal of the thymus,and found FADD dominant negative inhibits intestinal-derived CD8αα+TCRγδ+T cell development.To further analyze the mechanism,we studied intestinal-derived precursor T lymphocytes,which are lineage makers negative intestinal lamina propria lymphocytes(Lin-LPLs).The results showed that the proportion of Lin-LPLs in FADD DN mice was significantly higher than that in WT mice,indicating that the development of intestinal intraepithelial lymphocytes in FADD DN mice was arrested in the precursor phase.Notch1 plays an important role in the regulation of precursor cell development.Our analysis showed that the expression of Notch1 in Lin-LPLs of FADD DN mice was significantly down-regulated,and the results was also confirmed in Jurkat cells overexpressing FADD DN in vitro.IL-7R is one of the Notch 1 target genes,and Notch 1 regulates T cell development by regulating the expression of IL-7R on the surface of precursor cells.Our analysis revealed that the expression of IL-7R in Lin-LPLs of FADD DN mice was down-regulated.IL-7 induces the development of intestinal intraepithelial lymphocytes,and immature TCRγδ+T cells must be combined with appropriate ligands for further development.Therefore,the FADD DN Lin-LPLs may not be able to develop normally due to the lack of IL-7R on the cell surface and the inability to bind to IL-7.Many researches have found that intestinal intraepithelial γδ T cells play an immune-protective role in the mouse gut.To investigate whether the significant decrease of γδ T subpopulation cells in FADD DN mice may affect the development of mouse colitis,the third part of this study,we used FADD DN mice as a research object and established a mouse DSS colitis pathology model to better explore the regulation of FADD on mouse intestinal intraepithelial γδ T cells.The results showed that FADD DN mice were more likely to induce colitis than wild-type mice and the degree of of FADD on mouse intestinal intraepithelial γδ T cells.The results showed that FADD DN mice were more likely to induce colitis than wild-type mice and the degree of inflammation of colitis was significantly aggravated.