The Mechanism Of Increased Escherichia Coli Crossing Intestinal Tight Junction Barrier Induced By Tumor Necrosis Factor α

ObjectiveIn addition to being the organ responsible for digestion and absorption of nutrients, the intestine serves a barrier function that is a critical component of the innate immune system.Only a single layer of epithelial cells separates the luminal contents from effector immune cells in the lamina propria and the internal milieu of the body. Breakdown of the barrier is implicated in bacterial translocation,leading to sepsis,and in the pathogenesis of several illnesses such as inflammatory bowel disease,liver failure,acute severe pancreases and multiple organ system failure.Studies in rodents show that tumor necrosis factorα(TNFα) can lead to increased ileal permeability,and anti-TNFαantibody can prevent the intestinal permeability disorders.The mechanisms of TNFα-related epithelial breakdown are unclear,but regulation of paracellular pathways,especially via the interepithelial tight junction(TJ) proteins, and subsequent stimulation of highly immunoreactive submucosal cells are likely to play a significant role.Therefore,we detect the effect of TNFαon intestinal permeability,bacteria translocation and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells,and study the mechanisms involved in tight junction induced by TNFα.Materials and methods1,Cell culturesCaco-2 cells were grown in a culture medium composed of RPMI 1640 with 50IU/ml penicillin,50μg/ml streptomycin and 15%FBS.In order to establish in vitro intestinal epithelial barrier model,Caco-2 cells were plated on Transwell filters and monitored regularly by visualization with an inverted microscope and by epithelial resistance measurements. 2,Determination of epithelial monolayer resistance,paracellular permeability and the clone forming units per milliliter of E coli K1.E44The electrical resistance of the filter-grown Caco-2 intestinal monolayers was measured by using the millicell-ERS as previously reported.For determination of the effect of TNFαon Caco-2 monolayer paracellular permeability,the filter-grown Caco-2 cells by 3 wk postplating were incubated with TNFα(0,10,50 or 100ng/ml) for 24 hours or with TNFα100ng/ml for indicated times(2,4,8,24h).The transepithelial resistance,mucosal-to-serosal flux rate of the established paracellular marker lucifer yellow and bacterial translocation was assessed with or without pretreatment with anti-TNFRⅠmonoclonal antibody or anti-TNFRⅡmonoclonal antibody.3,Transmission electron microscopyCells were treated with TNFα(0,50 or 100ng/ml) for 24 hours and ultrathin sections were made and stained with saturated uranyl acetate and Reynold's lead citrate. Ultrastructure of junctional complexes was observated by transmission electron microscopy.4,Localization and expression of ZO-1 proteinCells were treated with TNFα(0,10,50 or 100ng/ml) for 24 hours and the localization and expression of ZO-1 protein were detected by immunofluorescence and Western blot analysis.SYBR-Green-based real-time PCR was used to measure the expression of ZO-1 mRNA.Results1,TNFαincrease intestinal epithelial paracellular permeability and bacterial translocationTNFαtreatment of filter-grown Caco-2 monolayers(0-100ng/ml) produced a concentration and time-dependent drop in Caco-2 transepithelial electrical resistance (TEER) during the 24-h experimental period.The TNFαeffect on Caco-2 paracellular permeability examined by using the paracellular markers lucifer yellow indicated that TNFαproduced a progressive concentration and time-dependent increase in transepithelial permeability to lucifer yellow.The enhanced bacterial translocation was observed when confluent Caco-2 cell monolayers were exposed to TNFα.Blocking antibodies showed that both anti-TNFRⅠand TNFRⅡmonoclonal antibodies decreased intertinal epithelial paracellular permeability and bacterial translocation.2,TNFαdisrupts the interepithelial tight junctionNormal tight junctions were visible at the apical portion of the lateral cell membrane in Caco-2 cells,showing as the high electron-dense material.In cells without TNFα,the TJ displayed an intact structure.When the cells were incubated with TNFα(50,100ng/ml) for 24 hours,the TJ complex appeared reduced and contained less electron-dense material.3,TNFαcauses tight junction protein ZO-1 disturbanceIn the control Caco-2 monolayers,ZO-1 proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders.TNFα(100ng/ml) caused a progressive disturbance in the continuity of ZO-1 localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contacts.Anti-TNFRⅠor anti-TNFRⅡmonoclonal antibody prevented the TNFα-induced location and expression disturbance of ZO-1.4,TNFαdecreases the expression of ZO-1 proteinThe effect of TNFαon ZO-1 protein expression was examined by Western blot analysis.TNFαproduced a progressive decrease in ZO-1 protein expression over the 24h treatment period.The maximal drop in ZO-1 protein level occurred at TNFα100ng/ml and at the 24h time point.Either anti-TNFRⅠor TNFRⅡmonoclonal antibodies prevented the TNFα-induced decrease in ZO-1 protein level.5,TNFαdoes not affect the expression of ZO-1 mRNATNFαdoes not cause a significant decrease in ZO-1 mRNA production compared with controls considering the difference of the concentration and time of TNFαtreatment.Conclusion1,TNFαdisrupted the interepithelial tight junction and increases Caco-2 intestinal epithelial permeability.2,TNFαcaused a disturbance in the continuity of ZO-1 localization at the cellular borders and produced a decrease in ZO-1 protein expression.3,TNFαdidn't induced a significant decrease in ZO-1 mRNA production.4,Anti-TNFRⅠor TNFRⅡmonoclonal antibodie attenuated the effect of TNFαon the intestinal epithelial permeability,the tight junction constructure and tight junction protein expression.