Effects of flagellin on innate and adaptive immunity (original) (raw)
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The Journal of …, 2001
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-B 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.
Nature Immunology, 2003
Flagellated bacteria cause a broad range of serious gastrointestinal, urinary tract and respiratory tract infections. In models of mucosal infection, the production of functional flagella is a bacterial virulence factor, which is required for colonization and tissue invasiveness and can elicit the recruitment of host inflammatory cells . Flagella also facilitate the infection of macrophages and epithelial cells 3,8-10 . The host targets flagella through antibodies, T cell recognition and the innate immune system 11-13 . Through TLR5, flagellin activates macrophages, dendritic cells, neutrophils and intestinal epithelial cells to produce inflammatory mediators . In addition to animals, plants and insects also have evolved innate immune recognition systems for bacterial flagellin . Plants recognize bacterial flagellin through FLS2, the flagellin sensitivity locus 2 gene product and an apparent functional equivalent of TLR5, and this recognition event triggers a systemic disease resistance response .
Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa
Trends in microbiology, 2004
Flagella contribute to 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 is constituted of a conserved domain widespread in bacterial species dedicated to filament polymerization. Conversely, mammalian hosts detect the conserved domain on flagellin monomers through the Toll-like receptor (TLR) 5 and trigger pro-inflammatory and adaptive immune responses. This review describes the relation among flagellin molecular structure, bacterial virulence, and host defences with special emphasis on mucosal tissues.-88 words-Teaser: Mucosal pathogens use flagella for invasion of host surfaces whereas the host detects and induces defences to pathogen through Toll-like receptor 5 that detects flagellin, the subunit of flagellum.
The Journal of Immunology, 2008
TLRs trigger immunity by detecting microbe-associated molecular patterns (MAMPs). Flagellin is a unique MAMP because it harbors 1) an antigenic hypervariable region and 2) a conserved domain involved in TLR5-dependent systemic and mucosal proinflammatory and adjuvant activities. In this study, the contribution of the flagellin domains in TLR5 activation was investigated. We showed that TLR5 signaling can be neutralized in vivo by flagellin-specific Abs, which target the conserved domain. However, deletions of flagellin's hypervariable region abrogated the protein's intrinsic ability to trigger the production of neutralizing Abs. The fact that MAMP-specific Abs block TLR-mediated responses shows that this type of neutralization is a novel mechanism for down-regulating innate immunity. The stimulation of mucosal innate immunity and adjuvancy to foreign Ag was not altered by the hypervariable domain deletions. In contrast, this domain is essential to trigger systemic innate immunity, suggesting that there are distinct mechanisms for TLR5 activation in systemic and mucosal compartments. In summary, specific MAMP determinants control the production of neutralizing Abs and the compartmentalization of innate responses.
TLR5 functions as an endocytic receptor to enhance flagellin‐specific adaptive immunity
European journal of …, 2011
Innate immune activation via TLR induces dendritic cell maturation and secretion of inflammatory mediators, generating favorable conditions for naïve T-cell activation. Here, we demonstrate a previously unknown function for TLR5, namely that it enhances MHC class-II presentation of flagellin epitopes to CD4 1 T cells and is required for induction of robust flagellin-specific adaptive immune responses. Flagellin-specific CD4 1 T cells expanded poorly in TLR5-deficient mice immunized with flagellin, a deficiency that persisted even when additional TLR agonists were provided. Flagellin-specific IgG responses were similarly depressed in the absence of TLR5. In marked contrast, TLR5deficient mice developed robust flagellin-specific T-cell responses when immunized with processed flagellin peptide. Surprisingly, the adaptor molecule Myd88 was not required for robust CD4 1 T-cell responses to flagellin, indicating that TLR5 enhances flagellin-specific CD4 1 T-cell responses in the absence of conventional TLR signaling. A requirement for TLR5 in generating flagellin-specific CD4 1 T-cell activation was also observed when using an in vitro dendritic cell culture system. Together, these data uncover an Myd88-independent function for dendritic cell TLR5 in enhancing the presentation of peptides to flagellin-specific CD4 1 T cells.
2010
Introduction: Systemic inflammation in sepsis is initiated by interactions between pathogen molecular motifs and specific host receptors, especially toll-like receptors (TLRs). Flagellin is the main flagellar protein of motile microorganisms and is the ligand of TLR5. The distribution of TLR5 and the actions of flagellin at the systemic level have not been established. Therefore, we determined TLR5 expression and the ability of flagellin to trigger prototypical innate immune responses and apoptosis in major organs from mice. Methods: Male Balb/C mice (n = 80) were injected intravenously with 1-5 μg recombinant Salmonella flagellin. Plasma and organ samples were obtained after 0.5 to 6 h, for molecular investigations. The expression of TLR5, the activation state of nuclear factor kappa B (NFB) and mitogen-activated protein kinases (MAPKs) [extracellular related kinase (ERK) and c-jun-NH2 terminal kinase (JNK)], the production of cytokines [tumor necrosis alpha (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6), macrophage inhibitory protein-2 (MIP-2) and soluble triggering receptor expressed on myeloid cells (TREM-1)], and the apoptotic cleavage of caspase-3 and its substrate Poly(ADP-ribose) polymerase (PARP) were determined in lung, liver, gut and kidney at different time-points. The time-course of plasma cytokines was evaluated up to 6 h after flagellin. Results: TLR5 mRNA and protein were constitutively expressed in all organs. In these organs, flagellin elicited a robust activation of NFB and MAPKs, and induced significant production of the different cytokines evaluated, with slight interorgan variations. Plasma TNFα, IL-6 and MIP-2 disclosed a transient peak, whereas IL-1β and soluble TREM-1 steadily increased over 6 h. Flagellin also triggered a marked cleavage of caspase-3 and PARP in the intestine, pointing to its ability to promote significant apoptosis in this organ. Conclusions: Bacterial flagellin elicits prototypical innate immune responses in mice, leading to the release of multiple pro-inflammatory cytokines in the lung, small intestine, liver and kidney, and also activates apoptotic signalling in the gut. Therefore, this bacterial protein may represent a critical mediator of systemic inflammation and intestinal barrier failure in sepsis due to flagellated microorganisms .
Expression, purification, and functional characterisation of Flagellin, a TLR5-ligand
Flagellin, a Toll-like receptor 5 (TLR5)-ligand, is known for its activities like adjuvant, induction of pro-inflammatory cytokines and innate immunity. In this context, fliC gene of Salmonella Typhimurium was cloned into pET32a expression plasmid using in-house designed gene specific primers. The frame and orientation of the inserted fliC gene was confirmed upon colony PCR, restriction enzyme analysis and sequencing. Sequence analysis of fliC revealed proper orientation of the gene and had 1,485 nucleotides. Following transformation of pET-fliC plasmid into Escherichia coli BL21 (DE3) cells, the gene was expressed after inducing with IPTG (Isopropylβ-D-1-thiogalactopyranoside). The polyHis-tag-fliC was ~70kDa as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The identity/authenticity of the recombinant-fliC was confirmed by its specific reactivity with commercial anti-fliC MAb of S. Typhimurium. Further, the antigenic and functional properties of recombinant-fliC were determined espousing its ability to induce antigen specific antibodies in G pigs and increased m-RNA expression of certain pro-inflammatory mediators like TNF-α and GM-CSF in vitro.
Involvement of Toll-like receptor 5 in the recognition of flagellated bacteria
Proceedings of the National Academy of Sciences, 2006
Toll-like receptors (TLRs) are key components of the immune system that detect microbial infection and trigger antimicrobial host defense responses. TLR5 is a sensor for monomeric flagellin, which is a component of bacterial flagella known to be a virulence factor. In this study we generated TLR5-deficient mice and investigated the role of TLR5 signaling in the detection of flagellin and antibacterial immune responses to Salmonella typhimurium and Pseudomonas aeruginosa. We found that TLR5 is essential for the recognition of bacterial flagellin both in vivo and ex vivo. TLR5 contribution to antibacterial host response to i.p. infection with S. typhimurium or intranasal administration of P. aeruginosa may be masked by TLR4 or other sensing mechanisms. By using radiation bone marrow chimera, we showed that upon i.p. injection of flagellin immune responses are mediated by lymphoid cells, whereas resident cells are required for the initiation of response upon intranasal flagellin administration. These results suggest that flagellin recognition in different organs is mediated by distinct TLR5-expressing cells and provide insights into the cooperation of the TLR5 and TLR4 signaling pathways used by the innate immune system in the recognition of bacterial pathogens.