The Role Of Toll Like Receptor-5and Adherent Invasive Escherichia Coli LF82in The Pathogenesis Of Inflammatory Bowel Disease

【Background and aims】The incidence of inflammatory bowel disease (IBD) is increasing all over the world.However, it is rather difficult to treat IBD because its etiological factors and pathogenesisare still not clear. The pathogenesis of IBD is complex and consists of three interactingelements: genetic susceptibility factors, priming by the enteric microflora, andimmunemediated tissue injury. Identification of adherent-invasive Escherichia coli (AIEC)strains in IBD patients offers an opportunity to characterize the pathogenesis ofmicrobial-induced intestinal inflammation. In which E.coli LF82is the most typical AIECstrain.Although bacteria are implicated in the pathogenesis of chronic IBD, mechanisms ofintestinal injury and immune activation remain unclear. TLRs are membrane-boundantigen-recognition receptors that directly recognize highly conserved molecularstructures in pathogens, they play a key role in innate immunity by eliciting the first lineof defense against invading pathogens. Of the TLRs, toll-like receptor5(TLR5) binding to bacterial flagellin activates NF-κB signaling and triggers an innate immune response tothe invading pathogen. But the exact mechanisms of TLR5involved in inflammationremains to be further studied. At present, the role and mechanism of AIEC and TLR5inIBD still unknown. Therefore we evaluated the role of AIEC strain E.coli LF82in vivoand in vitro, investigated the role if TLR5in this process, and stuied the possible role ofthe molecular mechanisms. So as to clarify the specific types of intestinal E. coli in IBDpathogenesis.【Methods】1. Caco-2cells were plated on transwell filters in order to establish in vitro iniestinalepithelial barrier model. The cell monolayers are polarized epithelial cells that formapical junctional complex (AJCs), resulting in high electrical resistance. Themorphology was monitored regularly by visualization with an inverted microscope andby epithelial resistance measurements. The TEER (trans epithelial electrical resistance)of the filter-grown Caco-2in intestinal monolayers was measured by using themillicell-ERS with or without infected with E.coli LF82. Mucosal-to-serosal flux rateof the established paracellular marker Lucifer yellow was also assessed with orwithout infected with E.coli LF82. The interepithelial tigh tjunction protein weredetected by immunofluorescence and western blot. The cytokine in the cell culturesupernatant was assayed by enzyme-linked immunosorbent assay (ELISA).2. C57BL/10C mice with dextran sulfate sodium (DSS)-injured colon were orallychallenged daily with109CFU E.coli LF82before using DSS for one week. Theseverity of colitis was assessed by determining disease activity index, colonichistological score, and myeoloperoxidase activity. The ultrastructure and cytoskeletonwere detected by transmission electron microscope (TEM) and immunofluorescence.The colon tissues were also stained by periodic acid-schiff stain and sirius red stain toassay the mucin-producing and fibration of colon. Cytokine expression was measuredby bio-plex in serum and by reverse-transcriptase polymerase chain reaction(RT-PCR)/western blot in colonic tissue. The expression level of flagellin receptor TLR5was measured by RT-PCR, immunohistochemistry and western blot in colonictissue.3. TLR5KO C57BL/10C mice were orally challenged daily with109bacteria beforeusing DSS for one week. Then the severity of colitis and cytokine expression weremeasure same as WT mice. The severity of colitis was assessed by determining diseaseactivity index, colonic histological score, and myeoloperoxidase activity. Theultrastructure and cytoskeleton were detected by transmission electron microscope(TEM) and immunofluorescence. The colon tissues were also stained by periodicacid-schiff stain and sirius red to assay the mucin-producing and fibration of colon.Cytokine expression was measured by bio-plex in serum and by reverse-transcriptasepolymerase chain reaction (RT-PCR)/western blot in colonic tissue.【Results】1. E.coli LF82infection decreased TEER and increased mucosal-to-serosal flux rate ofLucifer yellow in Caco-2epithelial cell monolayers. E.coli LF82infection altered theexpression and distribution of tight junction protein ZO-1and claudin-1. E.coli LF82infection also induced the secreteion of cytokine such as TNF-α in cell culturesupernatant.2. E.coli LF82exacerbated colitis in DSS-treated mice, substantially reducing survivalrate, greatly lowering stool consistency, inducing marked weight loss and increasedrectal bleeding, and significantly increasing erosive lesions and mucosal inflammation.E.coli LF82destroied the ultrastructure and cytoskeleton in mice colon, and survivalin colon epithelium. E.coli LF82infection decreased the loss of mucin-producingepithelial cells and increased the fibration in DSS-induced colitis, which was revealedwith PAS staining and sirius red stain. Administration of E.coli LF82significantlyincreased mRNAs levels of some cytokine in serum and colonic specimens. E.coliLF82also induced the increasing of phosphorylation-NF-κB P65andphosphorylation-P38in colon tissue. Furthermore, E.coli LF82infection stronglyenhanced TLR5mRNA and protein levels. 3. Compare to wt mice, E.coli LF82aggravate the clinical symptoms of colitis in injuredcolonic model in TLR5KO mice. Administration of E.coli LF82significantly increasedmRNAs levels of some cytokine in serum and colonic specimens. Thephosphorylation-P38level in colon tissue also increased after infected with E.coliLF82, but the expression of phosphorylation-NF-κB P65didn’t differ from the micewithout infected with E.coli LF82.【Conclusions】Our study indicated that E.coli LF82increase intestinale epithelial paracellularepermeability and disrupts the interepithelial tigh tjunction in vitro iniestinal epithelialbarrier model. E.coli LF82can exacerbate DSS-colitis in mice, the flagella//TLR5/MAPKand NF-κB pathway maybe involved in the mechanism of the action. Since E.coli LF82can also exacerbate DSS-colitis in TLR5KO mice. We presumed there are some othermechanisms participate in the pathogenesis of IBD.