Molecular Mechanisms Of Intestinal Epithelial Tight Junction Abnormalities Caused By HIV-1 Infection

BackgroundMucosa is known as the main portal for most pathogens including human immunodeficiency virus.The mucosal innate immune system is a natural barrier against mucosal infections and has a broad spectrum of anti-infective effects.As the most important component of the intestinal mucosal barrier,the tight junction of the intestinal epithelium plays an important role in preventing the penetration of pathogenic microorganisms in the intestinal lumen from entering into the mucosal tissue and the circulatory system.Once the tight junction of the intestine is destroyed,it will cause the infiltration of harmful substances and microbial components in the intestinal lumen,which will cause inflammation and disorder of the mucosal system,eventually leading to the activation of the intestinal and even the systemic immune system.Although long-term combined antiretroviral therapies(cART)can inhibit HIV-1 to undetectable levels in the infected individuals,gut barrier damage recovers only partially and low levels of systemic immune activation persist,complicating the discovery of functional cures for HIV/AIDS.Understanding the molecular mechanisms of intestinal tight junction abnormalities in HIV-1 infection is important for the prevention and repair of intestinal barrier damage in HIV/AIDS.It is known that the expression,distribution and regulation of tight junction proteins such as CLDN1,CLDN3,OCLN and ZO-1 in the intestinal mucosa are abnormal in both HIV-1 and SIV infection.However,the changes of many of the other tight junction proteins which play important roles in the maintenance of barrier function have not been reported.The molecular mechanism of HIV/AIDS-related tight junction abnormalities is unclear.As major extracellular regulators of tight junction,IL-17A and TNF-a are abnormally expressed in the intestinal mucosa in HIV-1/SIV infection.The IL-17/IL-17R pathway plays a crucial role in maintaining mucosal immune homeostasis,intestinal mucosal barrier integrity,and the normal composition and function of the gut microbiota.Although the expression of IL-17A in intestinal mucosa is correlated with multiple tight junction genes,the changes of other factors of IL-17 family have not been reported in HIV/AIDS.The massive loss of Th17 cells in the intestinal tract of HIV-1/SIV infection,the activation of related inflammatory processes,the generation of microbial translocation and the presence of microbial components may all be important factors leading to abnormal function of the intestinal mucosal barrier.However,the effects of inflammatory factors such as IL-17 and TNF-a,microbial translocation,and the combination of these factors on intestinal barrier function are still poorly understood.This study systematically observed changes in the expression of multiple tight junction-associated genes in SHIV/SIV infection and their correlation with IL-17 family cytokine expression;established an in vitro intestinal epithelial cell barrier model to study the effect of IL-17A and IL-17F on HIV-1 gp140-mediated intestinal epithelial barrier injury;investigated the effects of IL-17A/IL-17F on the barrier function of epithelial cells and the expression of tight junction-associated proteins in the presence of the inflammatory factor TNF-a and the marker of microbial translocation LPS.Methods1.Real-time quantitative RT-PCR was established to detect the mRNA levels of various tight junction-associated genes including CLDN2,4,5,7,8,11,12,14,15 in the gut of normal and SHIV/SIV-infected Macaca mulatta(animal model for HIV-1 infection),and the mRNA levels of IL-17A/IL-17F and its receptors were also detected.Statistical methods were used to analyze the correlation between the mRNA levels of tight junction-associated genes and IL-17A/IL-/17F/IL-17R.2.Construction of an in vitro epithelial cells barrier model(Caco-2 cells)using transwell.The barrier function of Caco-2 cells in the presence of HIV-1 gp140 was evaluated by measuring the barrier monolayer transmembrane electronical resistance and FITC-Dextran flux,and the mRNA levels of tight junction-associated genes in the presence of HIV-1 gp140 were detected by Real-time quantitative RT-PCR.Intestinal epithelial barrier function and mRNA levels of tight junction-associated genes induced by IL-17A,IL-17F and HIV-1 gp 140 alone or in combination were detected by Real-time quantitative RT-PCR and Western blot.3.Real-time quantitative RT-PCR and Western blot were used to detect the tight junction-associated gene levels of Caco-2 cells treated with IL-17A,IL-17F and HIV-1 gp140 alone or in combination.Western blot was used to detect the expression of total protein and phosphorylated protein of NF-?B p65 and MAPK p38 induced by IL-17A and IL-17F.Signaling pathways involved in IL-17A and IL-17F regulation of tight junction-associated gene expression were determined using Act1 specific siRNA interference and BAY11-7082(NF-?B selective inhibitor)and U0126(MAPK selective inhibitor).4.Real-time quantitative RT-PCR was used to detect the levels of TNF-a mRNA in the intestinal mucosa of normal and SHIV/SIV-infected Macaca mulatta and the level of 16S rRNA gene in mesenteric lymph nodes.Correlations between TNF-a mRNA levels and viral load or IL-17R mRNA levels were analyzed using statistical methods.Real-time quantitative RT-PCR and Western blot were used to detect the expression levels of IL-17R and tight junction-associated genes and proteins in Caco-2 cells treated with LPS+TNF-a and IL-17A/IL-17F alone and in combination.The level of tight junction protein ZO-1 in the presence of LPS+TNF-? and IL-17A was detected by laser confocal microscopy.Results1.The tight junction-associated genes CLDN5,8,11,12,14 mRNA levels in the intestinal mucosa of SHIV/SIV-infected Macaco mulatta were significantly lower than those in normal animals.The level of CLDN5 and CLDN8 mRNA in the intestinal mucosa of infected animals was half that of normal animals,and the level of CLDN11,12,14 mRNA was 0.4 fold that of normal animals.CLDN4,7,15 mRNA did not change significantly in the intestinal tract of normal and infected animals.There was a significant positive correlation between CLDN5,8,11,12,14 mRNA levels in infected animals.2.The IL-17A mRNA level in the intestinal mucosa of normal animals was 163 fold higher than that in SHIV/SIV infected animals,and there was a positive correlation between IL-17A and CLDN5,11 mRNA in infected animals.The IL-17F mRNA in the intestinal mucosa of infected animals was 2.7 fold higher than that in normal animals;IL-17RA/IL-17RC mRNA was 5?6 fold higher than that in normal animals.There was a negative correlation between IL-17F and CLDN5,8,11,12,14 mRNA levels in infected animals;IL-17RA was positively correlated with CLDN3,5,8,12,14 mRNA;IL-17RC was positively correlated with CLDN1,2,3,4,7,15 mRNA,respectively.3.The Caco-2 transmembrane resistance decreased by 30-40%after treatment with HIV-1 gp140 for 24 h,and the FITC-Dextran transmittance increased by about 50%,accompanied by tight junction-related genes CLDN1,2,3,4,5,7,8,OCLN and ZO-1 down-regulated by 2 to 3.5 fold.IL-17A and IL-17F prevent the damage of intestinal epithelial cell barrier by HIV-1 gp140 by synergistically up-regulating the expression of tight junction proteins such as CLDN1,OCLN and ZO-1.When IL-17A is absent,IL-17F alone has a certain effect on the epithelial barrier function,but it is not significant.When the expression of Act1 was inhibited by siRNA,IL-17A and IL-17F-mediated OCLN and ZO-1 transcription and protein levels were significantly down-regulated compared with siRNA control.The regulation of tight junction-associated genes by IL-17A and IL-17F is accompanied by the activation of NF-?B and MAPK in the downstream signaling pathway of IL-17R.NF-?B selective inhibitor BAY11-7082 inhibited the expression of CLDN1.OCLN and ZO-1,while MAPK kinase MEK1/2 inhibitor U0126 only inhibited the expression of ZO-1.4.Microbial translocation occurs in the intestinal tract of SHIV/SIV-infected Macaca mulatta.The level of TNF-a mRNA in the intestine of infected animals is twice that of normal animals.There was a positive correlation between TNF-a and viral load,IL-17RA and IL-17RC mRNA levels in infected animals.LPS+TNF-a in combination with IL-17A/IL-17F promotes the expression of IL-17RA and IL-17RCin Caco-2 cells.HIV-1 gp140 alone can inhibit the expression of IL-17R,however,in the presence of LPS+TNF-a and IL-17A/IL-17F,the combination of HIV-1 gp140 with them can significantly up-regulate the expression of IL-17RA and IL-17RC.5.LPS+TNF-a slightly up-regulated the expression of CLDN1,2,3,4,7,11 and ZO-1 mRNA in Caco-2 cells,but did not affect the expression of CLDN12,14,15 and OCLN.In addition,LPS+TNF-a has a certain inhibitory effect on the expression of CLDN5,8 mRNA.In the presence of IL-17A,LPS+TNF-a promoted the intestinal epithelial barrier function and the expression of tight junction proteins CLDN1,CLDN3 and ZO-1 more than the sum of the effects alone.In the presence of IL-17F,LPS+TNF-a was not able to significantly promote intestinal barrier function in the intestinal epithelium.The process by which LPS,TNF-a,and IL-17A synergistically promote the expression of tight junction-associated proteins is accompanied by activation of NF-?B and MAPK.NF-?B selective inhibitor BAY 11-7082 significantly inhibited the expression of CLDN1,CLDN3 and ZO-1.MAPK kinase MEK1/2 inhibitor U0126 significantly inhibited the expression of CLDN3 and ZO-1,but had no significant effect on the expression of CLDN1.ConclusionIn the gut of SHIV/SIV-infected Macaca mulatta,CLDN5,8.11.12.14 and other tight junction-associated genes were significantly down-regulated.and HIV-1 gp140 directly disrupted the integrity of the intestinal epithelial cell barrier.The presence of the virus and its protein components may be the direct cause of the destruction of the intestinal barrier function.LPS+TNF-a and IL-17A have a synergistic effect on the promotion of intestinal epithelial barrier function and tight junction protein expression.Although HIV/AIDS infection is accompanied by the occurrence of intestinal microbial translocation(the presence of LPS)and up-regulation of cytokines such as IL-17F and TNF-?,these factors only have a weak effect on the recovery of intestinal barrier function.Deletion of Th17 cells in the intestinal mucosa and decreased levels of IL-17A may be important causes of barrier function damage and difficulty in repair.These results extend the understanding of the mechanisms of intestinal barrier damage in HIV/AIDS,suggested that additional treatments targeting the loss of Th17 in the gut of HIV-1 infected individuals might facilitate the restoration of gut barriers.