Two Different Mechanisms Of Reg??/? Expression Induced By Pathogenic Bacteria And Symbioti Bacteria In Intestinal Epithelial Cells Of Mice

There are a large number of diverse intestinal commensal flora and pathogenic bacteria taken through oral cavity in the human intestine.The host has formed a complete defense mechanism against commensal bacteria and pathogen microbial invasion in the long-term evolution process.The first line of defense against host symbiotic flora and exogenous pathogens is the intestinal mucosal barrier,which is tightly jointed with the intestinal epithelium and protected by the intestinal mucus layer containing a large number of antimicrobial peptides.Among all the antimicrobial peptides,Reg III?and Reg III?have an efficient bactericidal action in the process of anti-bacterial infection.Studies have shown that the secretion of antimicrobial peptides depends not only on IL-22 secreted by intestinal type 3 innate lymphoid cells(ILC3),but also on the My D88 signal transduction pathway of the intestinal epithelium activated by the intestinal flora.However,the mechanism of these two modes and the timing of their effects are still confusing.This study will observe how the intestines use these two modes to regulate the secretion of antimicrobial peptides under the conditions of intestinal flora disorder and pathogen infection.In this study,flow cytometric sorting and FQ-PCR techniques were used to detect changes in the expression levels of Reg III?and Reg III?in intestinal epithelial cells respectively under the condition of antibiotic treatment-resulted loss of intestinal flora and infection by Salmonella and Listeria.Using mice whose intestinal epithelial cells defect My D88 and mice with systemic deletion of My D88,we investigated the role of My D88 in the secretion and expression of Reg III?and Reg III?in intestinal epithelial cells under steady state and infection status.Using Villin^creMy D88^fl/fl mice(Selective knockout of My D88 molecules in intestinal epithelial cells)and Zbtb46-DTR mice(Selective removal of classical dendritic cells)to construct experimental models of systemic DC-deficiency and My D88-deletion in intestinal epithelium,we investigated the effection and mech-anisms of My D88 and DC on the secretion of Reg III?and Reg III?in intestinal epithelium during bacterial infection with flow cytometry and FQ-PCR techniques.Finally we achieved the following results:1.To study the effect of symbiotic bacteria on the expression of My D88 molecules and antimicrobial peptides in the intestinal tract under steady state,we used antibiotics to treat the intestinal tract of Wild type(WT)C57BL/6 mice to eliminate commensal flora.Results show that the My D88,antibacterial peptides Reg III?and Reg III?expressed by the intestinal epithelial cells of the antibiotics treated mice decreased significantly,indica-ting that the intestinal commensal flora can stimulate the expression of My D88,antibac-terial peptide Reg III?and Reg III?in intestinal epithelial cells.2.To study the effect of bacterial infection on the expression of My D88,Reg III?and Reg III?in intestinal epithelium,we used pathogenic bacteria Salmonella typhimurium and Listeria to infect WT mice,3 days later we found that expression of My D88 in intestinal epithelial cells were on a declining trend,while the expression of intestinal epithelial antibacterial peptides such as Reg III?and Reg III?was significantly increased.3.To investigate whether the expression of Reg III?and Reg III?in the infected state is dependent on the My D88 signaling pathway,we used My D88^-/-mice(systemic knocking-out of My D88)and found that the expression of the antibacterial peptides Reg III?and Reg III?in intestinal epithelial cells was significantly decreased after infection with Salmonella.It indicates that My D88 molecule plays an important role in promoting the expression of Reg III?and Reg III?.4.To study the effetion of My D88 in intestinal epithelial cells on the expression of Reg III?and Reg III?under the steady state and bacterial infection state,we constructed mice selectively-lacking My D88 signaling molecule in intestinal epithelium(Villin^creMy D88^fl/fl).After deletion of My D88 in intestinal epithelial cells,the expression of anti-bacterial peptides Reg III?and Reg III?under stady state was significantly decreased.After infection with Villin^creMy D88^fl/fl by Salmonella,it was found that although the levels of Reg III?and Reg III?expressed in the intestinal epithelium of Villin^creMy D88^fl/flmice were significantly lower than those of infected WT mice,they were still significantly higher than those of uninfected Villin^creMy D88^fl/flmice.These results show that expression of Reg III?and Reg III?after Salmonella infection may not depend on the My D88 signaling pathway of intestinal epithelium.5.To investigate the effects of intestinal c DCs on the expression of Reg III?and Reg III?in the intestinal epithelium under infected state,we transferred bone marrow cells of Zbtb46-DTR mice to deathly irradiated Villin^creMy D88^fl/fl mice by intravenous injection to construct bone marrow chimeric mice with systemic deficiency of c DCs and selectively intestinal epithelial defection of My D88 were constructed.c DCs were cleared after diph-theria toxin(DT)treatment and then Salmonella typhimurium infection was applied.With the same infected Salmonella typhimurium,compared with Villin^creMy D88^fl/fl mice with-out c DCs deficiency,expression of antibacterial peptides such as Reg III?and Reg III?in Villin^creMy D88^fl/fl mice with c DCs deficiency was significantly decreased.These results show that the expression of the antibacterial peptides Reg III?and Reg III?induced by the infection of Salmonella depends on c DCs.In the infected state,c DCs play an important role in promoting the expression of Reg III?and Reg III?.Based on the above results,that is the expression of Reg III?and Reg III?is dependent on the intestinal epithelium-expressing My D88 under steady state,while under infected state,the My D88 is dependent on My D88,but not on the My D88 signal of the intestinal epithelium.And because of the reason that c DCs are important"receptor cells"that can alert pathogen invasion,c DCs play an important role in promoting the expression of Reg III?and Reg III?in the intestinal epithelium under infection.Therefore,we hypothesized that My D88 expressed by certain immune cells including c DCs may play an important promoting role in the expression of Reg III?and Reg III?in the infected state.But these require further study.To sum up,we conclude that:There are different mechanisms for regulating the expression of Reg III?and Reg III?in the intestinal epithelium under steady state and infection state.There are two different expression regulation modes,which play different roles.Specifically speaking,in the steady state,the intestinal flora induces the expression of Reg III?and Reg III?in the intestinal epithelium through the My D88 signaling pathway expressed by the intestinal epithelium;and when the intestinal epithelium is invaded by pathogens,it is a“My D88-not-from-intestinal epithelium”mode that regulates expression of Reg III?and Reg III?The above studies preliminary revealed the induction mechanism of Reg III?and Reg III?expression in intestinal epithelium.It helps people understand the interaction between the body and the symbiotic flora besides between the body and pathogens,and it provids a theoretical basis for the prevention and treatment of intestinal bacterial infection.