The Relationship Between Th17 Cells And Gastrointestinal Epithelial Barrier Integrity In HIV Infection

CD4~+T cell loss and body sustained immune activation is the immune pathological features of HIV infection.Virus infection leads to damage of the structure and function of the intestinal immune system,destruction of mucosal barrier,microbial translocation,causing the body chronic immune activation,which may be an important mechanism of HIV-1 pathogenicity.Although Th17 cell had changes and loss in the course of HIV infection,the loss of Th17 cell effect on immune system has not been studied.The role in damaged mucosal barrier of the Thl7 cell is still being explored when HIV infection happened.In this study,we used SIV/SHIV infected rhesus to analysis the relationship between gastrointestinal mucosal CD4~+T Th 17 cell subsets and gastrointestinal mucosal barrier structure;application of Caco-2 cells was used to construct cell monolayer in vitro and to explore IL-17 and the SHIV/SIV protein components such as gp160 and Tat effect on epithelial cell barrier.Then this study will provide new ideas for repairing gastrointestinal mucosal barrier.In the in vivo study,we analysised rhesus gastrointestinal mucosal CD4~+T Th17 cell subsets and explored the impact of infection on the gastrointestinal mucosal barrier structure.We used flow cytometry to detect CD3+CD4~+T cells,IL-17+CD3+CD4~+T cells,IL-10+CD3+CD4~+T cells,Foxp3+CD3+CD4~+T cells,IL-4+CD3+CD4~+T cells and IFNy+CD3+CD4~+T cells and to analysis SIV/SHIV infected animal gastrointestinal mucosal CD4~+T cell sub-group.Real Time RT-PCR was used to detect IL-17A,RORyt,TGF-beta,GATA-3,T-bet and Foxp3 mRNA expression levels in normal animals and virus infected animals.In the aspect mucosal barrier structure research,gastrointestinal mucosal epithelium of normal animals and infected animals were observed by transmission electron microscopy,using real time RT-PCR detected tight junction gene(claudin-1,claudin-3,occludin and ZO-1)transcription level,western blot and immunofluorescence staining to detect tight junction related protein claudin-1,claudin-3,occludin and ZO-1 expression level.To test and verify the protective effect of IL-17,we used embedded transwell to culture Caco-2 cells,constructed cell monolayer in vitro,explored IL-17 and the SHIV/SIV protein components gp160 and Tat impact on epithelial barrier.In this study,by measuring transepithelial resistance and permeability to assess the effect of gp160,Tat and IL-17 on epithelial barrier;the application of real time RT-PCR to detect gene transcription levels of tight junction(claudin-1,claudin-3,and ZO-1)in cell monolayer.The study found that,the analysis of cell subsets,in the SIV/SHIV infected animal digestive tract mucosa cells,CD3+CD4~+T cell proportion compared to normal animals were decreased;the proportion of IFNy+CD3+CD4~+T cells and IL-4+CD3+CD4~+T cells compared to uninfected animals increased;to detect Thl cells and Th2 nuclear transcription factor T-bet and GATA-3 transcription levels compared to uninfected animals having increasing;intestinal mucosa in the infected animals,in addition to the ileum,IL-17+CD3+CD4~+T cell proportion compared with the normal group reduced,three groups of infected animals digestive tract mucosa compared to normal animals IL-17 transcription level both decreased;detection of IL-10+CD3+CD4~+T cells and Foxp3+CD3+CD4~+T cells,SIV/SHIV infected animals in addition to the colon of Foxp3+CD3+CD4~+T cell ratio reduce than normal animals,the proportion of these two types of cells in the rest of the digestive tract mucosa were higher than normal animals;real time RT-PCR analysis of Foxp3,except Gr4 group jejunum,cecum and colon Foxp3 transcription level decreased than normal animals,Foxp3 transcription level of the other two groups SHIV infected animals gastrointestinal mucosa was higher compared to normal animals.Ultrastructure of epithelial barrier in the digestive tract of animals,the Gr4 group and Gr 19 animal small intestine and large intestinal epithelial cells observed by transmission electron microscopy compared with normal animals,visible epithelial cell tight junctions widened,intercellular gap increased,microvilli scattered,thinning,sparse,showing lodging like,part of the fracture,fall off;expansion in the endoplasmic reticulum,swelling of mitochondria,vacuoles in the cytoplasm.At the genetic level,claudin-1 transcription level of infected animals duodenum,jejunum,ileum,cecum and colon was lower than the normal group;claudin-1 transcription level of rectum Grl9 was reduced when compared with the normal group;at protein level,Claudin-1 of infected animals jejunum,cecum and colon mucosa were decreased;confocal laser microscopy observed virus infected animals gastrointestinal mucosa,distribution of Claudin-1 and protein expression level were affected.Claudin-3 at the gene level,duodenum and jejunum of the infected animals transcriptional level lower than the normal group;ileum,colon and rectum in Gr19 claudin-3 transcription levels were lower than the normal group;protein Claudin-3 expression level of infected animals jejunum,colon and rectal mucosa was reduced;protein Claudin-3 expression level of duodenum in Gr15 group was decreased;Claudin-3 protein level of cecal in Gr19 and Gr15 were lower than the normal group.Real-time RT PCR detected infected animals' duodenum,jejunum and rectum,we found that occludin transcription level was lower than the normal group;detection of the intestinal mucosa Occludin protein levels by western blot showed colon of Gr19 and Gr4 group reduced.Real-time RT PCR detected infected animals' duodenum and jejunum,we found that ZO-1 transcriptional level was lower than the normal group;confocal microscopy observed animal gastrointestinal mucosa immunofluorescence staining and found that virus infection animal gastrointestinal mucosal tissue distribution of ZO-1 and protein expression levels were affected,discontinuous protein distribution and fluorescence reduced.In vitro study,we found that we can establish a stable Caco-2 cells in vitro research system.Tat(14ng/ml)had immediate effect on the cell monolayer effect that increased the permeability of the cell monolayer after 2h and reduced TER;specific concentration Tat and gp160 had little effect on tight junctions gene expression of Caco-2 cell monolayers;IL-17(100ng/ml)had a certain protection with cell monolayer.In a word,the ratio of gastrointestinal mucosal CD3+CD4~+T cells and IL-17+CD3+CD4~+T cell in SIV/SHIV infected rhesus monkeys were decreased.The transcription level of tight junction associated genes(claudin-1,claudin-3,occludin and ZO-1)and the expression level of the protein had varying degrees of decline.Tat(14ng/ml)had immediate effect on cell monolayers;IL-17(100ng/ml)had a certain degree of protection on cell monolayers.This study played a important role in discovering and exploring new treatments policy or new ways to reduce microbial translocation and immune activation.