Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression

Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-kappaB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.     

Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein.

Flagellin, the monomeric subunit of flagella, is an inducer of proinflammatory mediators. Bacterial flagellin genes have conserved domains (D1 and D2) at the N terminus and C terminus and a middle hypervariable domain (D3). To identify which domains induced proinflammatory activity, r6-histidine (6HIS)-tagged fusion constructs were generated from the Salmonella dublin (SD) fliC flagellin gene. A full-length r6HIS SD flagellin (6HIS flag) induced IkappaBalpha loss poststimulation and NF-kappaB activation in Caco-2BBe cells and was as potent as native-purified SD flagellin. IFN-gamma-primed DLD-1 cells stimulated with 1 microg/ml of 6HIS flag induced high levels of NO (60 +/- 0.95 microM) comparable to the combination of IL-1beta and IFN-gamma (77 +/- 1.2) or purified native SD flag (66.3 +/- 0.98). Selected rSD flagellin proteins representing the D1, D2, or D3 domains alone or in combination were tested for proinflammatory properties. Fusion proteins representing the D3, amino, or carboxyl regions alone did not induce proinflammatory mediators. The results with a recombinant protein containing the amino D1 and D2 and carboxyl D1 and D2 separated by an Escherichia coli hinge (ND1-2/ECH/CD2) indicated that D1 and D2 were bioactive when coupled to an ECH element to allow protein folding. This chimera, but not the hinge alone, induced IkappaBalpha degradation, NF-kappaB activation, and NO and IL-8 production in two intestinal epithelial cell lines. ND1-2/ECH/CD2-1 also induced high levels of TNF-alpha (900 pg/ml) in human monocytes comparable to native SD flagellin (991.5 pg/ml) and 6HIS flag (987 pg/ml). The potent proinflammatory activity of flagellin, therefore, resides in the highly conserved N and C D1 and D2 regions.     

Flagellin/TLR5 responses in epithelia reveal intertwined activation of inflammatory and apoptotic pathways

Flagellin, the primary structural component of bacterial flagella, is recognized by Toll-like receptor 5 (TLR5) present on the basolateral surface of intestinal epithelial cells. Utilizing biochemical assays of proinflammatory signaling pathways and mRNA expression profiling, we found that purified flagellin could recapitulate the human epithelial cell proinflammatory responses activated by flagellated pathogenic bacteria. Flagellin-induced proinflammatory activation showed similar kinetics and gene specificity as that induced by the classical endogenous proinflammatory cytokine TNF-alpha, although both responses were more rapid than that elicited by viable flagellated bacteria. Flagellin, like TNF-alpha, activated a number of antiapoptotic mediators, and pretreatment of epithelial cells with this bacterial protein could protect cells from subsequent bacterially mediated apoptotic challenge. However, when NF-kappaB-mediated or phosphatidylinositol 3-kinase/Akt proinflammatory signaling was blocked, flagellin could induce programmed cell death. Consistently, we demonstrate that flagellin and viable flagellate Salmonella induces both the extrinsic and intrinsic caspase activation pathways, with the extrinsic pathway (caspase 8) activated by purified flagellin in a TLR5-dependant fashion. We conclude that interaction of flagellin with epithelial cells induces caspase activation in parallel with proinflammatory responses. Such intertwining of proinflammatory and apoptotic signaling mediated by bacterial products suggests roles for host programmed cell death in the pathogenesis of enteric infections.     

Induction of macrophage nitric oxide production by Gram-negative flagellin involves signaling via heteromeric Toll-like receptor 5/Toll-like receptor 4 complexes

The induction of cytokine synthesis by flagellin is mediated by a Toll-like receptor 5 (TLR5) signaling pathway. Although flagellin activation of the IL-1R-associated kinase and induction of TNF-alpha synthesis are dependent on TLR5 and not TLR4, we have found that flagellin stimulates NO in macrophages via a pathway that requires TLR5 and TLR4. Flagellin induced NO synthesis in HeNC2 cells, a murine macrophage cell line that expresses wild-type TLR4, but not in TLR4-mutant or -deficient GG2EE and 10ScNCr/23 cells. Flagellin stimulated an increase in inducible NO synthase (iNOS) mRNA and activation of the iNOS promoter. TLR5 forms heteromeric complexes with TLR4 as well as homomeric complexes. IFN-gamma permitted GG2EE and 10ScNCr/23 cells to produce NO in response to flagellin. Flagellin stimulated IFN-beta synthesis and Stat1 activation. The effect of flagellin on iNOS gene expression was inhibited by a Stat1 mutant protein. Taken together, these results support the conclusions that flagellin induces distinct patterns of inflammatory mediators depending on the nature of the TLR5 signaling complex and that the induction of NO by flagellin involves signaling via TLR5/TLR4 complexes.     

Flagellin-induced tolerance of the Toll-like receptor 5 signaling pathway in polarized intestinal epithelial cells

Salmonella typhimurium is a gram-negative enteric pathogen that invades the mucosal epithelium and is associated with diarrheal illness in humans. Flagellin from S. typhimurium and other gram-negative bacteria has been shown to be the predominant proinflammatory mediator through activation of the basolateral Toll-like receptor 5 (TLR5). Recent evidence has shown that prior exposure can render immune cells tolerant to subsequent challenges by TLR ligands. Accordingly, we examined whether prior exposure to purified flagellin would render human intestinal epithelial cells insensitive to future contact. We found that flagellin-induced tolerance is common to polarized epithelial cells and prevents further activation of proinflammatory signaling cascades by both purified flagellin and Salmonella bacteria but does not affect TNF-alpha stimulation of the same pathways. Flagellin tolerance is a rapid process that does not require protein synthesis, and that occurs within 1 to 2 h of flagellin exposure. Prolonged flagellin exposure blocks activation of the NF-kappaB, MAPK, and phosphoinositol 3-kinase signaling pathways and results in the internalization of a fraction of the basolateral TLR5 without affecting the polarity or total expression of TLR5. After removal of flagellin, cells require more than 24 h to fully recover their ability to mount a normal proinflammatory response. We have found that activation of phosphoinositol 3-kinase and Akt by flagellin has a small damping effect in the early stages of flagellin signaling but is not responsible for tolerance. Our study indicates that inhibition of TLR5-associated IL-1 receptor-associated kinase-4 activity occurs during the development of flagellin tolerance and is likely to be the cause of tolerance.     

Luminal-applied flagellin is internalized by polarized intestinal epithelial cells and elicits immune responses via the TLR5 dependent mechanism

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn's lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.     

Characterization of flagellin expression and its role in Listeria monocytogenes infection and immunity

Flagellin is the structural component of flagella produced by many pathogenic bacteria and is a potent proinflammatory molecule that mediates these effects through Toll-like receptor (TLR) 5. In Listeria monocytogenes (LM), flagellin expression is regulated by temperature and has been described as being shut off at 37 degrees C. In this study, we demonstrate that TLR5-mediated cell activation and flagellin expression is maintained at 37 degrees C in some laboratory-adapted strains and in approximately 20% of LM clinical isolates. To determine the role of flagellin in LM infection, a targeted mutation in the structural gene for flagellin (flaA) was generated in a parental LM strain that expressed flagellin under all conditions examined. In vitro studies demonstrated that this deltaflaA mutant was (i). non-motile; (ii). not able to activate TLR5-transfected HeLa cells; and (iii). induced tumour necrosis factor (TNF)-alpha production in approximately 50% fewer CD11b+ cells in splenocytes from normal mice compared with the parental strain. However, there was no significant alteration in virulence of the deltaflaA mutant after either intravenous or oral murine infection. Similarly, there was no difference in the generation of LM-specific CD8 or CD4 T cells after intravenous or oral infection. These data indicate that flagellin is not essential for LM pathogenesis or for the induction of LM-specific adaptive immune responses in normal mice.     

Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa

Flagella contribute to the virulence of pathogenic bacteria through chemotaxis, adhesion to and invasion of host surfaces. Flagellin is the structural protein that forms the major portion of flagellar filaments. Thus, flagellin consists of a conserved domain that is widespread in bacterial species and is dedicated to filament polymerization. Conversely, mammalian hosts detect the conserved domain on flagellin monomers through Toll-like receptor (TLR) 5, which triggers proinflammatory and adaptive immune responses. This review describes the relationships among flagellin molecular structure, bacterial virulence and host defenses, with special emphasis on mucosal tissues.     

Identification of conserved domains in Salmonella muenchen flagellin that are essential for its ability to activate TLR5 and to induce an inflammatory response in vitro

The bacterial surface protein flagellin is widely distributed and well conserved among distant bacterial species. We and other investigators have reported recently that purified flagellin from Salmonella dublin or recombinant flagellin of Salmonella muenchen origin binds to the eukaryotic toll receptor TLR5 and activates the nuclear translocation of NF-kappaB and mitogen-activated protein kinase, resulting in the release of a host of pro-inflammatory mediators in vitro and in vivo. The amino acid sequence alignment of flagellins from various Gram-negative bacteria shows that the C and N termini are well conserved. It is possible that sequences within the N and C termini or both may regulate the pro-inflammatory activity of flagellin. Here we set out to map more precisely the regions in both termini that are required for TLR5 activation and pro-inflammatory signaling. Systematic deletion of amino acids from either terminus progressively reduced eukaryotic pro-inflammatory activation. However, deletion of amino acids 95-108 (motif N) in the N terminus and 441-449 (motif C) in the C terminus abolished pro-inflammatory activity completely. Site-directed mutagenesis analysis provided further evidence for the importance of motifs N and C. We also present evidence for the functional role of motifs N and C with the TLR5 receptor using a reporter assay system. Taken together, our results demonstrate that the pro-inflammatory activity of flagellin results from the interaction of motif N with the TLR5 receptor on the cell surface.     

Interleukin-18 secretion and Th1-like cytokine responses in human peripheral blood mononuclear cells under the influence of the toll-like receptor-5 ligand flagellin

Flagellin is the major protein component of the flagella from motile bacteria and was identified as the ligand for toll-like receptor (TLR)-5. Whereas its effects on epithelial cells have been studied in detail, activation of human peripheral blood mononuclear cells (PBMC) by flagellin is characterized only partially. By using the recombinant protein of Salmonella muenchen we confirm the proinflammatory nature of flagellin as detected by nuclear factor-kappaB activation and interleukin (IL)-8 production. Aim of the current study was to elucidate in PBMC effects of flagellin on IL-18 and Th1-like cytokine responses. We report that flagellin in pathophysiologically relevant concentrations augmented release of mature IL-18 by THP-1 monocytes, PBMC, and whole blood stimulated with nigericin or by ATP-mediated P2X7 purinergic receptor activation. Further key functions of the IL-18/IL-12/interferon-gamma (IFNgamma) pathway were upregulated by flagellin. Flagellin synergized with IL-12 for production of IFN-gamma and augmented secretion of interferon-inducible protein-10, a CXC-chemokine that is key to the generation of Th1-type responses. In contrast, neither IL-18-binding protein nor IL-4 was affected. Taken together, the present data demonstrate for the first time that flagellin at concentrations that are detectable in the blood compartment during sepsis efficiently enhances the IL-18/IL-12/IFNgamma pathway and thus Th1-like cytokine responses in PBMC.     

Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide

The lumenal surface of the colonic epithelium is continually exposed to Gram-negative commensal bacteria and LPS. Recognition of LPS by Toll-like receptor (TLR)-4 results in proinflammatory gene expression in diverse cell types. Normally, however, commensal bacteria and their components do not elicit an inflammatory response from intestinal epithelial cells (IEC). The aim of this study is to understand the molecular mechanisms by which IEC limit chronic activation in the presence of LPS. Three IEC lines (Caco-2, T84, HT-29) were tested for their ability to activate an NF-kappaB reporter gene in response to purified, protein-free LPS. No IEC line responded to LPS, whereas human dermal microvessel endothelial cells (HMEC) did respond to LPS. IEC responded vigorously to IL-1beta in this assay, demonstrating that the IL-1 receptor signaling pathway shared by TLRs was intact. To determine the reason for LPS hyporesponsiveness in IEC, we examined the expression of TLR4 and MD-2, a critical coreceptor for TLR4 signaling. IEC expressed low levels of TLR4 compared with HMEC and none expressed MD-2. To determine whether the low level of TLR4 expression or absent MD-2 was responsible for the LPS signaling defect in IEC, the TLR4 or MD-2 gene was transiently expressed in IEC lines. Transient transfection of either gene individually was not sufficient to restore LPS signaling, but cotransfection of TLR4 and MD-2 in IEC led to synergistic activation of NF-kappaB and IL-8 reporter genes in response to LPS. We conclude that IEC limit dysregulated LPS signaling by down-regulating expression of MD-2 and TLR4. The remainder of the intracellular LPS signaling pathway is functionally intact.     

Gram-negative flagellin-induced self-tolerance is associated with a block in interleukin-1 receptor-associated kinase release from toll-like receptor 5

Flagellin from a number of Gram-negative bacteria induces cytokine and nitric oxide production by inflammatory cell types. In view of the evidence that flagellin responsiveness is subject to modulation, we explored the possibilities that a prior exposure to flagellin might result in a state of reduced flagellin responsiveness or tolerance and that lipopolysaccharide (LPS) and flagellin may induce a state of cross-tolerance to each other. Our results demonstrate that a prior exposure to flagellin results in a subsequent state of flagellin tolerance in human monocytes, THP1 cells, Jurkat cells, and COS-1 cells. Tolerance occurs within 2 h after addition of flagellin and does not require protein synthesis. Flagellin did not induce tolerance to LPS in monocytes and THP1 cells; however, LPS treatment of monocytes and THP1 cells resulted in a state of flagellin cross-tolerance. Flagellin-induced self-tolerance is not the result of a decrease in the steady-state level of toll-like receptor 5 (TLR5) or interleukin-1 receptor associated kinase (IRAK), but it is associated with a block in the release of IRAK from the TLR5 complex in flagellin-tolerant cells. Release is essential for IRAK activity because the TLR5-associated IRAK lacks kinase activity. LPS-induced cross-tolerance to flagellin is also associated with a block in IRAK release from TLR5. These results provide evidence for a novel mechanism of TLR signaling control.