The Role Of Dock2 In The Host Defenseagainst Citrobacter Roderntium Infection

Background:The relationship between digestive tract microecology and digestive tract inflammation and tumorigenesis and development has gradually gained importance in recent years.Gastrointestinal inflammation and tumorigenesis are thought to be involved in a variety of factors,including genetic defects,immune dysfunction,and intestinal flora imbalance.However,the exact pathogenesis remains unclear.Dock2 is a member of the evolutionarily conserved Dock protein family,an atypical guanine exchange factor(GEFs)that specifically activates the small G protein Rac1 by mediating GTP-GDP exchange.Formation of the cytoskeleton.In this study,we established a model of colitis induced by Citrobacter rodentium.We mainly conducted in-depth research on immune cells and intestinal flora that may be affected by Dock2 defects.To study the molecular signaling pathway of IBD pathogenesis and to provide an important theoretical basis for the treatment strategy of IBD with Dock2 as the target.In addition,we established a cell model of Eca109 and KYSE510 infected by Porphyromonas gingivalis in vitro to investigate the effects of P.gingivalis on the proliferation and migration of Eca109 and KYSE510 through NF-?B pathway.Methods:(1)We firstly measured the expression of Dock2 in intestinal epithelial cells and various immune cells including macrophages by qRT-PCR;(2)We compared the susceptibility of WT,Dock2-/-mice,and Rag1-/-mice that do not have adaptive immunity,to C.rodentium infection;(3)Wild type(WT)and Dock2-/-mice were infected with C.rodentium and the body weight changes were monitored.Sacrificed the mice at different time points, colon lengths,spleen weights and fecal C.rodentium numbers were measured;(4)ELISA assay detected the proinflammatory cytokine levels in WT and Dock2-/-mice at 21 days of C.rodentium infection;immunohistochemistry and flow cytological measured inflammatory cells and T cell infiltration in WT and Dock2-/-mice;flow cytometry analyzed the total number and percentage of thymocytes in WT and Dock2-/-mice were analyzed by cytometry.(5)T cell and serum transfer assays observed the changes in the severity of host colitis induced by C.rodentium.(6)We established a co-cage experimental model of WT and Dock2-/-mice,compared the changes of intestinal flora of the two groups after co-caged by16S rRNA high-throughput sequencing.(7)Cell models of Eca109 and KYSE510 cells infected with Porphyromonas gingivalis were established,MTT assay,colony formation assay,migration and wound scratch assay,and the effects of cell proliferation on proliferation and migration of Eca109 and KYSE510 cells were determined.(8)PCR,Western blot analysis and luciferase assay were used to detect the effects of P.gingivalis infection on Eca109 and KYSE510 cells on key molecules of NF-?B pathway.Results:(1)Dock2 were expressed in immune cells of various intestinal tracts other than intestinal epithelial cells;(2)Compared with Rag1-/-mice,Dock2-/-mice had both the defect in adaptive immunity and in innate immunity during C.rodentium infection(3)Dock2-/-mice were more susceptible to C.rodentuim infection than WT mice during infection;Dock2 was required for intestinal bacterial clearance after C.rodentium infection;(4)Compared with WT mice after infection,Dock2-/-mice had significantly more pro-inflammatory cytokine production and lymphocyte infiltration.However,compared to WT mice,Dock2-/-mice had significantly reduced spleen T cells,B cell count and serum antibody production.(5)T cell and serum transfer experiment had an inhibitory effect on C.rodentium-induced colitis.(6)After co-caged,the intestinal flora of WT mice and Dock2-/-mice changed significantly,with segmental filamentous bacteria(SFB)decreased,while Prevotella increased significantly.(7)P.gingivalis may promote the proliferation and movement of Eca109 and KYSE510 cells by activating NF-?B signaling.Conclusion:Dock2 can not only regulate adaptive immune response,but also regulate innate immune response during C.rodentium infection.Dock2 plays a key role in lymphocyte function,which may play an important role in the host's clearance of intestinal pathogens.In addition,the gut microbiota may affect the host susceptibility of Dock2-/-mice to C.rodentium-induced colitis.P.gingivalis may promote the proliferation and movement of Eca109 and KYSE510 cells by activating NF-?B signaling.