In Vitro and ex Vivo Activation of the TLR5 Signaling Pathway in Intestinal Epithelial Cells by a Commensal Escherichia coli Strain

Abstract

The capacity of non-pathogenic enteric bacteria to induce a pro-inflammatory response is under debate in terms of its effect on the symbiosis between the mammalian host and its commensal gut microflora. Activation of NF-κB and induction of interleukin-8 (IL-8) and CCL-20 by the commensal Escherichia coli strain MG1655 were first studied in vitro in the human intestinal epithelial cell (IECs) lines HT29-19A and Caco-2, transfected or not with plasmids encoding dominant negative Toll-like receptor (TLR) 5 and myeloid differentiation factor-88 (MyD88) adaptor protein. The response of enterocytes in situ was then assessed using murine ileal biopsies mounted in Ussing chambers. Commensal E. coli induced NF-κB DNA binding, NF-κB transcriptional activity, CCL-20 expression, and IL-8 secretion in the human IEC lines. E. coli MG1655 flagellin was necessary and sufficient to trigger this pro-inflammatory pathway via its interaction with TLR5 and the subsequent recruitment of the adaptor protein MyD88. Following epithelial cell polarization, signaling could be induced by live E. coli and flagellin on the apical side of HT29-19A. The in vivo relevance of our findings was confirmed, because immunohistochemical staining of murine ileum demonstrated expression of TLR5 in the apical part of enterocytes in situ. Furthermore, flagellin added on the mucosal side of murine ileal biopsies mounted in Ussing chambers induced a basolateral production of KC, a functional murine homolog of human IL-8. These findings provide strong evidence that flagellin released by flagellated commensal bacteria in the intestinal lumen can induce a pro-inflammatory response in enterocytes in vivo. The capacity of non-pathogenic enteric bacteria to induce a pro-inflammatory response is under debate in terms of its effect on the symbiosis between the mammalian host and its commensal gut microflora. Activation of NF-κB and induction of interleukin-8 (IL-8) and CCL-20 by the commensal Escherichia coli strain MG1655 were first studied in vitro in the human intestinal epithelial cell (IECs) lines HT29-19A and Caco-2, transfected or not with plasmids encoding dominant negative Toll-like receptor (TLR) 5 and myeloid differentiation factor-88 (MyD88) adaptor protein. The response of enterocytes in situ was then assessed using murine ileal biopsies mounted in Ussing chambers. Commensal E. coli induced NF-κB DNA binding, NF-κB transcriptional activity, CCL-20 expression, and IL-8 secretion in the human IEC lines. E. coli MG1655 flagellin was necessary and sufficient to trigger this pro-inflammatory pathway via its interaction with TLR5 and the subsequent recruitment of the adaptor protein MyD88. Following epithelial cell polarization, signaling could be induced by live E. coli and flagellin on the apical side of HT29-19A. The in vivo relevance of our findings was confirmed, because immunohistochemical staining of murine ileum demonstrated expression of TLR5 in the apical part of enterocytes in situ. Furthermore, flagellin added on the mucosal side of murine ileal biopsies mounted in Ussing chambers induced a basolateral production of KC, a functional murine homolog of human IL-8. These findings provide strong evidence that flagellin released by flagellated commensal bacteria in the intestinal lumen can induce a pro-inflammatory response in enterocytes in vivo. Intestinal epithelial cells (IECs) 1The abbreviations used are: IECs, intestinal epithelial cells; -CM, -conditioned medium; LPS, lipopolysaccharide; IL, interleukin; NF-κB, nuclear factor-κB; TLR, toll-like receptor; MyD88, myeloid differentiation factor 88; WT, wild-type; PBS, phosphate-buffered saline; PAMPs, pathogenic associated molecular patterns; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; BSA, bovine serum albumin; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; ABC, avidin-biotin complex; EMSA, electrophoretic mobility shift assay; Dn, dominant negative. 1The abbreviations used are: IECs, intestinal epithelial cells; -CM, -conditioned medium; LPS, lipopolysaccharide; IL, interleukin; NF-κB, nuclear factor-κB; TLR, toll-like receptor; MyD88, myeloid differentiation factor 88; WT, wild-type; PBS, phosphate-buffered saline; PAMPs, pathogenic associated molecular patterns; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; BSA, bovine serum albumin; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; ABC, avidin-biotin complex; EMSA, electrophoretic mobility shift assay; Dn, dominant negative. serve as a barrier to bacteria colonizing the gut. Rather than a passive barrier, the intestinal epithelium is now considered to be an integral and essential component of the innate mucosal immune system of the host (1Strober W. Ann. N. Y. Acad. Sci. 1998; 859: 37-45Crossref PubMed Scopus (40) Google Scholar). IECs can respond to enteric pathogens (e.g. Salmonella species, Yersinia enterocolitica, and enteropathogenic Escherichia coli) either by the release of molecules directly endowed with bactericidal properties (2Hecht G. Am. J. Physiol. 1999; 277: C351-C358Crossref PubMed Google Scholar) or by the secretion of pro-inflammatory mediators (3Fierer J. Eckmann L. Kagnoff M. Infect Agents Dis. 1993; 2: 255-258PubMed Google Scholar, 4Eckmann L. Jung H.C. Schurer-Maly C. Panja A. Morzycka-Wroblewska E. Kagnoff M.F. Gastroenterology. 1993; 105: 1689-1697Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 5Eckmann L. Kagnoff M.F. Fierer J. Infect. Immun. 1993; 61: 4569-4574Crossref PubMed Google Scholar, 6Witthoft T. Eckmann L. Kim J.M. Kagnoff M.F. Am. J. Physiol. 1998; 275: G564-G571PubMed Google Scholar) and the expression of adhesion molecules (7Huang G.T. Eckmann L. Savidge T.C. Kagnoff M.F. J. Clin. Invest. 1996; 98: 572-583Crossref PubMed Scopus (151) Google Scholar), which permit the recruitment of immune cells and the induction of a protective inflammatory response able to eradicate pathogens. Many studies have demonstrated that the response of mammalian cells to pathogens is orchestrated through the activation of the nuclear transcription factor κB (NF-κB) (8Elewaut D. DiDonato J.A. Kim J.M. Truong F. Eckmann L. Kagnoff M.F. J. Immunol. 1999; 163: 1457-1466PubMed Google Scholar, 9Philpott D.J. Yamaoka S. Israel A. Sansonetti P.J. J. Immunol. 2000; 165: 903-914Crossref PubMed Scopus (214) Google Scholar) following recognition of specific prokaryote motifs named PAMPs (pathogenic associated molecular patterns) by cellular receptors. Toll-like receptors (TLRs), some of which are expressed by enterocytes, form the best characterized family of mammalian receptors to PAMPs (10Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4731) Google Scholar). TLRs recognize an array of prokaryote motifs, including unmethylated CpG DNA motifs, lipopolysaccharide (LPS), lipoproteins, peptidoglycan, and flagellin (10Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4731) Google Scholar, 11Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Crossref PubMed Scopus (3925) Google Scholar, 12Athman R. Philpott D. Curr. Opin. Microbiol. 2004; 7: 25-32Crossref PubMed Scopus (112) Google Scholar), shared by both pathogenic and commensal bacteria, suggesting that either type of bacteria may have the potential to initiate innate immune host responses in IECs. Accordingly, Haller et al. (13Haller D. Russo M.P. Sartor R.B. Jobin C. J. Biol. Chem. 2002; 277: 38168-38178Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) have recently shown that, following recognition of LPS by TLR4, the non-pathogenic commensal enteric bacteria Bacteroides vulgatus activates NF-κB nuclear translocation and thereafter proinflammatory gene expression in IEC lines as well as in primary enterocytes. In contrast, Neish et al. (14Neish A.S. Gewirtz A.T. Zeng H. Young A.N. Hobert M.E. Karmali V. Rao A.S. Madara J.L. Science. 2000; 289: 1560-1563Crossref PubMed Scopus (747) Google Scholar) showed that a nonpathogenic Salmonella strain not only failed to activate epithelial cells but could also prevent the pro-inflammatory response induced by a pathogenic strain of Salmonella (14Neish A.S. Gewirtz A.T. Zeng H. Young A.N. Hobert M.E. Karmali V. Rao A.S. Madara J.L. Science. 2000; 289: 1560-1563Crossref PubMed Scopus (747) Google Scholar). In addition, a recent study has shown that Bacteroides thetaiotaomicron, a prevalent anaerobe commensal of the human intestine, attenuates pro-inflammatory cytokine expression induced by a pathogenic Salmonella strain in IECs (15Kelly D. Campbell J.I. King T.P. Grant G. Jansson S. S. Nat. Immunol. 2004; PubMed Scopus Google Scholar). the capacity of commensal bacteria to activate a pro-inflammatory response in IECs In this have in vitro and vivo to the capacity of the E. coli MG1655 commensal strain G. V. M. J. J. D.J. Y. Science. 277: PubMed Scopus Google Scholar) to induce a pro-inflammatory response in enterocytes. strain was as a of flagellated commensal Furthermore, E. coli is the first of the human and serve to intestinal by that are on in In vitro studies to the of this commensal strain to activate the NF-κB pathway in IEC lines and to this NF-κB induction to the interaction of flagellin with TLR5 in enterocytes. vivo studies using Ussing chambers were used to that this pro-inflammatory pathway can also be in vivo in murine ileal enterocytes. and E. coli MG1655 strain as the of commensal non-pathogenic E. has human and in with only of the and and its is G. V. M. J. J. D.J. Y. Science. 277: PubMed Scopus Google Scholar). E. coli MG1655 and were with in were by with and to a of of bacteria of E. coli was to a Acad. Sci. S. A. 2000; PubMed Scopus Google Scholar). of the recognition gene the were with that a or to or of the gene to the and MG1655 E. coli that and in and was by with the which the by by the in the were on with and to the of the The of the the gene was by using or to the or of the gene and The was then with the which a the was of the and the gene was by between the recognition the in an in the gene to The was by The of the was in by of with were and E. coli as in to that of a in Dulbecco's modified Eagle's bacteria were by were through and In some were with and to was added first the to was added to the a was to and the were by and the were through that molecules than were and of the and staining a of with which was in the E. and The of flagellin in the was using the because this not a of in the E. flagellin was used and of human IEC lines HT29-19A and were in with and to in in 5 and an in FCS, cell were with either live E. coli or a of of bacteria LPS E. coli flagellin or prevent in of IECs with live E. was added of HT29-19A and cells were on and a of in with FCS, then a of intestinal cell were as and were the of the and and cells were as and nuclear were as G. Israel A. Biol. PubMed Google Scholar). cells were in PBS, the and and the cell were in on was added a of and were 5 were and nuclear were with and in with were 5 were and were in and of and of DNA to the κB to the were on a in The was to a and with the NF-κB or were added of nuclear with the DNA of IL-8 and were using enzyme-linked immunosorbent were with or and were by with bovine serum to bacteria, and were then added and the was were added with and used as a were and cytokine were by with of of cells with an was using and to was then with and of the with including and in an system CCL-20 and and and of and were to expression of and of HT29-19A and NF-κB E. G. Israel A. J. PubMed Google Scholar) and the dominant negative expression were by D. J. Philpott with of was used to the was using to the and cells were and in a of of cell was with of the of the and of or of encoding either dominant negative TLR5 or dominant negative MyD88, and was then a and DNA cell and were cells were with of with and a of cells cells were with bacteria, as or with either or flagellin were then in and was using the system were on and were using in and and then with Salmonella in and with of was with and to of in Ussing were the of in with the of the of were by and was were the part of the as a with and on a and were mounted 5 of in Ussing chambers a of were on side with of the following and The was and by a and The mucosal and were by to of the potential and to of the were 5 and the in was used to the were flagellin LPS or to the of of were added in the and a the mucosal and the were and by of of were in and in were in BSA, and with a of or serum with a of and then with avidin-biotin to were in BSA, and were in this between was using the to the were with and mounted with staining was following of with TLR5 a of was using the to the Commensal E. coli MG1655 a in a commensal E. coli can induce a pro-inflammatory response in IECs, NF-κB was assessed by electrophoretic mobility shift in the HT29-19A and human IEC lines that with E. coli MG1655 a of of bacteria shown in by bacteria induced a strong in NF-κB in both cell lines which used as a induced only a in NF-κB activity, which was in than in HT29-19A with an the in a shift of the NF-κB this was in the of the suggesting that the NF-κB induced by commensal E. coli both and NF-κB is to the activation of pro-inflammatory in human IECs, including IL-8 (8Elewaut D. DiDonato J.A. Kim J.M. Truong F. Eckmann L. Kagnoff M.F. J. Immunol. 1999; 163: 1457-1466PubMed Google Scholar, A. G. Am. J. Physiol. PubMed Google Scholar). IL-8 was in by of shown in E. coli to induced the secretion of IL-8 in the IEC lines that NF-κB in the induction of cytokine LPS of the commensal bacteria vulgatus was recently shown to activate the NF-κB signaling pathway in IECs (13Haller D. Russo M.P. Sartor R.B. Jobin C. J. Biol. Chem. 2002; 277: 38168-38178Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The pro-inflammatory effect of E. coli MG1655 was with of E. and E. coli E. induced NF-κB and IL-8 secretion in HT29-19A and cells as as bacteria, LPS effect a of Furthermore, of to the E. not its its was with to the of a The by Commensal E. coli by and Activation of the of E. and staining a protein with an molecular of which was to that of E. coli flagellin The of the was by its with an Salmonella flagellin and the of this protein in an E. coli of the commensal E. coli MG1655 in the E. coli as in to which The that the strain was was shown to induce IL-8 secretion and CCL-20 expression in in vitro of IECs F. A. D. Acad. Sci. S. A. 2001; 98: PubMed Scopus Google Scholar, A.T. Neish A.S. Madara J.L. J. Clin. Invest. 2001; PubMed Scopus Google Scholar, J.A. J.A. E. Infect Immun. 2003; PubMed Scopus Google Scholar). E. coli induced the secretion of IL-8 and the of in both HT29-19A and cells and effect was and a of was sufficient a the of flagellin E. coli was to that with of flagellin Salmonella not a the E. not induce the pro-inflammatory response induced by E. coli MG1655 in our cell lines could be to the pro-inflammatory properties of the live of the commensal E. coli shown in the E. coli was to activate NF-κB DNA and failed to induce IL-8 secretion and CCL-20 expression in either IEC The of flagellin was demonstrated by the signaling pathway by E. coli MG1655 in IECs. Activation of NF-κB by flagellin enteropathogenic bacteria as S. and E. coli its to and the subsequent recruitment of the adaptor protein F. A. Akira S. A. 2001; PubMed Scopus Google Scholar). In addition, to TLR5 a on flagellin that is between M.F. A. G. S. K. S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, E. F. K. A. Nat. Immunol. 2004; Scopus Google Scholar). the of TLR5 in both HT29-19A and cells not the of TLR5 to NF-κB activation by E. coli HT29-19A and cells were transfected with encoding dominant negative of either TLR5 or adaptor protein with an NF-κB shown in both flagellin E. and live E. but not the induced the activation of NF-κB, expressed in with HT29-19A cells our by In contrast, HT29-19A cells transfected with TLR5 showed a and in with cells transfected with with flagellin or live E. were in HT29-19A cells transfected with with and in with flagellin and live E. NF-κB was in the of but was not in the of TLR5 the of the were with the IECs not that flagellin E. was the only of the pro-inflammatory of E. coli MG1655 in the IEC lines IECs IL-8 in to Commensal E. coli MG1655 or E. coli bacteria the intestinal and only with IECs via apical Accordingly, has that the of TLR5 the basolateral of IECs may be the between commensal and pathogenic flagellated with only flagellated pathogens able to the cells and to induce a pro-inflammatory response the basolateral side A.T. Neish A.S. Madara J.L. J. Clin. Invest. 2001; PubMed Scopus Google Scholar, A.T. S. P.J. Madara J.L. J. Immunol. 2001; PubMed Scopus Google Scholar, H. Y. Y. Madara J.L. Gewirtz A.T. Neish A.S. J. Immunol. 2003; PubMed Scopus Google Scholar). the in vivo pro-inflammatory of E. coli or of its E. the of on HT29-19A and which on in was by that the of IECs mounted in Ussing chambers was IL-8 secretion in response to added flagellin or and in than in cells not the of this cell to apical basolateral In contrast, not IL-8 secretion in the HT29-19A cell which was used this part of the shown in added the apical side of but not the E. coli strain induced a basolateral secretion of IL-8. of HT29-19A cells by than basolateral also in a strong secretion of IL-8 to that with E. coli of the was the of the the that some added flagellin or released by live to the basolateral through between epithelial in to by E. coli flagellin released by commensal bacteria is to activate epithelial cells in TLR5 expression was studied by in ileal using a with murine TLR5 staining was in the part of the and in the apical part of enterocytes staining was because the was following of the with a TLR5 expressed TLR5 is to the effect of flagellin was assessed in murine ileal biopsies mounted in Ussing because this to the side of the of the ileal biopsies was the to epithelial barrier of was as the because studies have demonstrated that is of to T. C. C. A. C. N. M. Gastroenterology. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). were by flagellin E. coli added to the apical of KC, a murine cytokine that functional properties with human IL-8 J. J.L. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar), was in the the mucosal and of the shown in this flagellin E. coli induced a in production in the basolateral with In contrast, LPS E. a to that in the of flagellin by the not secretion the of E. not induce that flagellin was the only the pro-inflammatory is that induction of an intestinal proinflammatory response is a of pathogenic intestinal bacteria, commensal bacteria not induce this response as this of between the bacteria and mammalian cells the gut have recently to be a in the of the of host proinflammatory responses to bacteria via expression of receptors is that intestinal epithelial cells are to commensal bacteria to the expression of receptors apical R. Philpott D. Curr. Opin. Microbiol. 2004; 7: 25-32Crossref PubMed Scopus (112) Google Scholar), bacteria have and can activate expressed or on the basolateral this our study that a commensal enteric E. coli strain can activate an pro-inflammatory response not only in some IEC but also in murine ileal epithelial cells in biopsies mounted in Ussing chambers. of the NF-κB pathway on the interaction of flagellin to flagellated bacteria with TLR5 expressed in situ in the both the and apical of enterocytes. In vitro studies using HT29-19A and IEC lines to that the commensal E. coli MG1655 strain in with pathogenic bacteria as Salmonella (8Elewaut D. DiDonato J.A. Kim J.M. Truong F. Eckmann L. Kagnoff M.F. J. Immunol. 1999; 163: 1457-1466PubMed Google Scholar), D.J. Yamaoka S. Israel A. Sansonetti P.J. J. Immunol. 2000; 165: 903-914Crossref PubMed Scopus (214) Google Scholar), and E. coli A. G. Am. J. Physiol. PubMed Google Scholar), activate the NF-κB pathway and induce expression of endowed with bactericidal properties able to cells of the innate immune These in vitro studies also demonstrated the of the interaction between flagellin and the pro-inflammatory response induced by E. coli was by flagellin E. coli on the was in the of bacteria or the E. coli strain in which the flagellin encoding gene is in with studies that flagellin by pathogens as S. F. A. D. Acad. Sci. S. A. 2001; 98: PubMed Scopus Google Scholar, A.T. Neish A.S. Madara J.L. J. Clin. Invest. 2001; PubMed Scopus Google Scholar), but also E. coli pathogens J.A. J.A. E. Infect Immun. 2003; PubMed Scopus Google Scholar, A. Y. Kagnoff M.F. Microbiol. 2002; PubMed Scopus Google Scholar), can induce an NF-κB the of which TLR5 as a the of mammalian of IEC lines with a encoding dominant negative TLR5 the induction of NF-κB in response to both E. coli flagellin and live E. with the of TLR5 in flagellin induction of NF-κB by live E. coli or flagellin was also in IECs transfected with dominant negative MyD88, the of TLR5 in the NF-κB pathway F. A. Akira S. A. 2001; PubMed Scopus Google Scholar). The of flagellin between flagellated bacteria M.F. A. G. S. K. S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, E. F. K. A. Nat. Immunol. 2004; Scopus Google Scholar) non-pathogenic as well as pathogenic bacteria with the capacity to activate the NF-κB signaling in IECs. recent study also that a commensal bacteria, can activate NF-κB in IECs (13Haller D. Russo M.P. Sartor R.B. Jobin C. J. Biol. Chem. 2002; 277: 38168-38178Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The bacteria are and the pro-inflammatory factor was as LPS, to commensal and pathogenic bacteria, which the NF-κB to (13Haller D. Russo M.P. Sartor R.B. Jobin C. J. Biol. Chem. 2002; 277: 38168-38178Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). In that the effect of LPS was also in primary epithelial but the of LPS to induce this pathway in vivo was not because were on cells the intestine, to that was in situ to LPS released by The to because has on the study of the epithelial cell that its the basolateral of IECs enterocytes to between commensal in the gut lumen and pathogenic able to the basolateral A.T. Neish A.S. Madara J.L. J. Clin. Invest. 2001; PubMed Scopus Google Scholar, A.T. S. P.J. Madara J.L. J. Immunol. 2001; PubMed Scopus Google Scholar, H. Y. Y. Madara J.L. Gewirtz A.T. Neish A.S. J. Immunol. 2003; PubMed Scopus Google Scholar). of IL-8 by apical in and epithelial cell lines in response to flagellin S. and E. F. A. D. Acad. Sci. S. 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Infect Immun. 2000; PubMed Scopus Google Scholar). the in vivo capacity of enterocytes to respond to murine ileal was mounted in Ussing and flagellin was added on the apical vivo induced a basolateral secretion of KC, a murine cytokine that functional properties with human IL-8 J. J.L. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In with murine is under NF-κB transcriptional and can be by proinflammatory including LPS, in cell including epithelial cells Y. J. Immunol. Google Scholar, Y. S. J. Immunol. Google Scholar, F. K. D. J. W. G. E. J. J. Immunol. 1999; Google Scholar). The human IEC lines in the study failed to respond to LPS, as by et al. E. M. J. Immunol. 2001; PubMed Scopus Google Scholar). In contrast, a murine IEC was recently to respond to LPS, following and interaction of LPS and in the T. A. S. A. J. 2002; PubMed Scopus Google Scholar, A. S. J. 2003; PubMed Scopus Google Scholar). Furthermore, by Haller et al. (13Haller D. Russo M.P. Sartor R.B. Jobin C. J. 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