Functional characterization of chicken TLR5 reveals species-specific recognition of flagellin

Mammalian Toll-like receptor 5 (TLR5) senses flagellin of several bacterial species and activates the innate immune system. The avian TLR repertoire exhibits considerable functional diversity compared to mammalian TLRs and evidence of a functional TLR5 in the avian species is lacking. In the present study we cloned and successfully expressed chicken TLR5 (chTLR5) in HeLa cells, as indicated by laser confocal microscopy. Infection of chTLR5 transfected cells with Salmonella enterica serovar Enteritidis activated NF-kappaB in a dose- and flagellin-dependent fashion. Similar NF-kappaB activation was observed with recombinant bacterial flagellin. Targeted mutagenesis of the proline residue at position 737 in the chTLR5-TIR domain was detrimental to chTLR5 function, confirming that the observed effects were conferred via chTLR5 and the MyD88 signaling pathway. Comparison of human, mouse and chicken TLR5 activation by flagellin of S. enterica serovar Typhimurium revealed that chTLR5 consistently yielded stronger responses than human but not mouse TLR5. This species-specific reactivity was not observed with flagellin of serovar Enteritidis. The species-specific TLR5 response was nullified after targeted mutagenesis of a single amino acid (Q89A) in serovar Typhimurium flagellin, while L415A and N100A substitutions had no effect. These results show that chickens express a functional TLR5 albeit with different flagellin sensing qualities compared to human TLR5. The finding that single amino acid substitutions in bacterial flagellin can alter the species-specific TLR5 response may influence the host range and susceptibility of infection.     

Toll-like receptors in bony fish: from genomics to function

Receptors that recognize conserved pathogen molecules are the first line of cellular innate immunity defense. Toll-like receptors (TLRs) are the best understood of the innate immune receptors that detect infections in mammals. Key features of the fish TLRs and the factors involved in their signaling cascade have high structural similarity to the mammalian TLR system. However, the fish TLRs also exhibit very distinct features and large diversity which is likely derived from their diverse evolutionary history and the distinct environments that they occupy. Six non-mammalian TLRs were identified in fish. TLR14 shares sequence and structural similarity with TLR1 and 2, and the other five (TLR19, 20, 21, 22 and 23) form a cluster of novel TLRs. TLR4 was lost from the genomes of most fishes, and the TLR4 genes found in zebrafish do not recognize the mammalian agonist LPS and are likely paralogous and not orthologous to mammalian TLR4 genes. TLR6 and 10 are also absent from all fish genomes sequenced to date. Of the at least 16 TLR types identified in fish, direct evidence of ligand specificity has only been shown for TLR2, TLR3, TLR5M, TLR5S and TLR22. The common carp TLR2 was shown to recognize the synthetic triacylated lipopeptide Pam₃CSK₄ and lipopeptides from gram positive bacteria. The membrane-bound TLR5 (TLR5M) signaling in response to flagellin in rainbow trout is amplified through interaction with the soluble form (TLR5S) in a positive loop feedback. In Fugu, TLR3 is localized to the endoplasmic reticulum (ER) and recognizes relatively short dsRNA, while TLR22 has a surveillance function like the human cell-surface TLR3. Genome and gene duplications have been major contributors to the teleost's rich evolutionary history and genomic diversity. Duplicate or multi-copy TLR genes were identified for TLR3 and 7 in common carp, TLR4b, 5, 8 and 20 in zebrafish, TLR8a in rainbow trout and TLR22 in rainbow trout and Atlantic salmon. The main task for current and near-future fish TLRs research is to develop specificity assays to identify the ligands of all fish TLRs, which will advance comparative immunology research and will contribute to our understanding of disease resistance mechanisms in fish and the development of new adjuvants and/or more effective vaccines and therapeutics.     

Toll-like receptors in bony fish: From genomics to function

Receptors that recognize conserved pathogen molecules are the first line of cellular innate immunity defense. Toll-like receptors (TLRs) are the best understood of the innate immune receptors that detect infections in mammals. Key features of the fish TLRs and the factors involved in their signaling cascade have high structural similarity to the mammalian TLR system. However, the fish TLRs also exhibit very distinct features and large diversity which is likely derived from their diverse evolutionary history and the distinct environments that they occupy. Six non-mammalian TLRs were identified in fish. TLR14 shares sequence and structural similarity with TLR1 and 2, and the other five (TLR19, 20, 21, 22 and 23) form a cluster of novel TLRs. TLR4 was lost from the genomes of most fishes, and the TLR4 genes found in zebrafish do not recognize the mammalian agonist LPS and are likely paralogous and not orthologous to mammalian TLR4 genes. TLR6 and 10 are also absent from all fish genomes sequenced to date. Of the at least 16 TLR types identified in fish, direct evidence of ligand specificity has only been shown for TLR2, TLR3, TLR5M, TLR5S and TLR22. The common carp TLR2 was shown to recognize the synthetic triacylated lipopeptide Pam₃CSK₄ and lipopeptides from gram positive bacteria. The membrane-bound TLR5 (TLR5M) signaling in response to flagellin in rainbow trout is amplified through interaction with the soluble form (TLR5S) in a positive loop feedback. In Fugu, TLR3 is localized to the endoplasmic reticulum (ER) and recognizes relatively short dsRNA, while TLR22 has a surveillance function like the human cell-surface TLR3. Genome and gene duplications have been major contributors to the teleost's rich evolutionary history and genomic diversity. Duplicate or multi-copy TLR genes were identified for TLR3 and 7 in common carp, TLR4b, 5, 8 and 20 in zebrafish, TLR8a in rainbow trout and TLR22 in rainbow trout and Atlantic salmon. The main task for current and near-future fish TLRs research is to develop specificity assays to identify the ligands of all fish TLRs, which will advance comparative immunology research and will contribute to our understanding of disease resistance mechanisms in fish and the development of new adjuvants and/or more effective vaccines and therapeutics.     

AsialoGM1 and TLR5 cooperate in flagellin-induced nucleotide signaling to activate Erk1/2

Bacterial flagellin can interact with both Toll-like receptor 5 (TLR5) and the cell surface glycolipid, asialoGM1, to activate an innate immune response. The induction of mucin by flagellin in human lung epithelial cells (NCIH292) is dependent on asialoGM1 ligation, ATP receptor signaling, Ca2+ mobilization, and Erk1/2 activation. Conversely, the activation of NF-kappaB by flagellin is dependent on signaling through TLR5. These results prompted us to ask whether the flagellin-induced TLR5 signaling pathway was intersecting with or mutually independent of the nucleotide receptor pathway activated downstream of asialoGM1. Herein, we demonstrate that the release of ATP induced by flagellin is dependent on a Toll signaling cascade. Although Toll was able to activate NF-kappaB in the absence of extracellular ATP, Toll required ATP to activate Erk1/2. These results suggest interdependence between the asialoGM1 and TLR5 pathways and reveal a previously unsuspected role for autocrine extracellular ATP signaling in TLR signaling.     

Ligand-induced differential cross-regulation of Toll-like receptors 2, 4 and 5 in intestinal epithelial cells

Toll-like receptors (TLR) 2, TLR4 and TLR5 are primary mucosal sensors of microbial patterns. Dissection of the cross-talk between TLRs in intestinal cells has thus far been hampered by the lack of functional TLR2 and TLR4 in in vitro model systems. Here we report that the mouse intestinal epithelial cell line mIC(cl2) expresses these TLRs and that receptor expression and function are regulated by environmental TLR stimuli. Our results show that stimulation of TLR5 by bacterial flagellin resulted in upregulated TLR2 and TLR4 mRNA and concomitant sensitization of the cells for subsequent TLR2 (Pam(3)CSK(4)) and TLR4 (LPS) stimulation. Exposure to low amounts of either Pam(3)CSK(4) or LPS in turn downregulated TLR5 mRNA and attenuated subsequent flagellin-mediated NF-kappaB activation, pointing to a negative feedback mechanism. Pam(3)CSK(4) and LPS also downregulated TLR4 mRNA but upregulated TLR2 mRNA and sensitized cells for subsequent TLR2 stimulation. Inhibition of the phosphatidyl-inositol-3-kinase/Akt pathway only affected LPS-mediated TLR cross-talk indicating that differential TLR cross-regulation was conferred via different mechanisms. Together, our results demonstrate that the expression and function of TLR in intestinal cells are highly dynamic and tightly regulated in response to encountered bacterial stimuli.     

A bacterial flagellin, Vibrio vulnificus FlaB, has a strong mucosal adjuvant activity to induce protective immunity

Flagellin, the structural component of flagellar filament in various locomotive bacteria, is the ligand for Toll-like receptor 5 (TLR5) of host cells. TLR stimulation by various pathogen-associated molecular patterns leads to activation of innate and subsequent adaptive immune responses. Therefore, TLR ligands are considered attractive adjuvant candidates in vaccine development. In this study, we show the highly potent mucosal adjuvant activity of a Vibrio vulnificus major flagellin (FlaB). Using an intranasal immunization mouse model, we observed that coadministration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific immunoglobulin A (IgA) responses in both mucosal and systemic compartments and IgG responses in the systemic compartment. The mice immunized with TT plus FlaB were completely protected from systemic challenge with a 200x minimum lethal dose of tetanus toxin. Radiolabeled FlaB administered into the nasal cavity readily reached the cervical lymph nodes and systemic circulation. FlaB bound directly to human TLR5 expressed on cultured epithelial cells and consequently induced NF-kappaB and interleukin-8 activation. Intranasally administered FlaB colocalized with CD11c as patches in putative dendritic cells and caused an increase in the number of TLR5-expressing cells in cervical lymph nodes. These results indicate that flagellin would serve as an efficacious mucosal adjuvant inducing protective immune responses through TLR5 activation.     

Isolation and characterization of rainbow trout C-reactive protein

An acute phase serum component, C-reactive protein (CRP), was isolated from the sera of rainbow trout (Salmo gairdneri). The isolation was based on its calcium-dependent binding affinity for pneumococcal C-polysaccharide (CPS) according to the isolation procedure of human C-reactive protein. In SDS-PAGE, the nonreduced CRP showed two subunits with molecular weights of 43,700 and 26,600, respectively, at a molar ratio of 1:1. The reduced CRP showed a single subunit of 26,600. The molecular weight of the native protein was estimated as 66,000 by native gradient PAGE and 81,400 by sedimentation equilibrium analysis using ultracentrifugation. The antigenic determinant on CPS-reactive site was destroyed by periodate oxidation, indicating that rainbow trout CRP is a glycoprotein. CRP levels in rainbow trout serum measured by the CPS-ELISA procedure showed that the rainbow trout CRP could behave as an acute phase reactant, following experimental infection with the fish pathogen Vibrio anguillarum.     

Lipopolysaccharides from Periodontopathic Bacteria Porphyromonas gingivalis and Capnocytophaga ochracea Are Antagonists for Human Toll-Like Receptor 4

Toll-like receptors (TLRs) 2 and 4 have recently been identified as possible signal transducers for various bacterial ligands. To investigate the roles of TLRs in the recognition of periodontopathic bacteria by the innate immune system, a Chinese hamster ovary (CHO) nuclear factor-kappaB (NF-kappaB)-dependent reporter cell line, 7.7, which is defective in both TLR2- and TLR4-dependent signaling pathways was transfected with human CD14 and TLRs. When the transfectants were exposed to freeze-dried periodontopathic bacteria, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Capnocytophaga ochracea, and Fusobacterium nucleatum, and a non-oral bacterium, Escherichia coli, all species of the bacteria induced NF-kappaB-dependent CD25 expression in 7.7/huTLR2 cells. Although freeze-dried A. actinomycetemcomitans, F. nucleatum, and E. coli also induced CD25 expression in 7.7/huTLR4 cells, freeze-dried P. gingivalis did not. Similarly, lipopolysaccharides (LPS) extracted from A. actinomycetemcomitans, F. nucleatum, and E. coli induced CD25 expression in 7.7/huTLR4 cells, but LPS from P. gingivalis and C. ochracea did not. Furthermore, LPS from P. gingivalis and C. ochracea attenuated CD25 expression in 7.7/huTLR4 cells induced by repurified LPS from E. coli. LPS from P. gingivalis and C. ochracea also inhibited the secretion of interleukin-6 (IL-6) from U373 cells, the secretion of IL-1beta from human peripheral blood mononuclear cells, and ICAM-1 expression in human gingival fibroblasts induced by repurified LPS from E. coli. These findings indicated that LPS from P. gingivalis and C. ochracea worked as antagonists for human TLR4. The antagonistic activity of LPS from these periodontopathic bacteria may be associated with the etiology of periodontal diseases.     

Molecular cloning, characterization and expression of goose Toll-like receptor 5

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that are vital to activation of the innate immune system in response to invading pathogens through their recognition of pathogen-associated molecular patterns (PAMPs). TLR5 is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the goose TLR5 gene using rapid amplification of cDNA ends (RACE). The open reading frame (ORF) of goose TLR5 cDNA is 2583 bp in length and encodes an 860 amino acid protein. The entire coding region of the TLR5 gene was successfully amplified from genomic DNA and contained a single exon. The putative amino acid sequence of goose TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR-CT) domain, a transmembrane domain and an intracellular Toll-interleukin-1 receptor (TIR) domain. The amino acid sequence of goose TLR5 shared 50.5% identity with human (Homo sapiens), 49.8% with mouse (Mus musculus) and 82.7% with chicken (Gallus gallus). The goose TLR5 gene was highly expressed in the spleen, liver and brain; moderately expressed in PBMCs, kidney, lung, heart, bone marrow, small intestine and large intestine; and minimally expressed in the cecum. HEK293 cells transfected with goose TLR5 and NF-κB-luciferase containing plasmids significantly responded to flagellin from Salmonella typhimurium indicating that it is a functional TLR5 homologue. In response to infection with S. enterica serovar Enteritidis (SE), the level of TLR5 mRNA significantly increased over the control in PBMCs at 1 d post infection (p.i.) and was slightly elevated in the spleen at 1 d or 3 d p.i. IL-6 was expressed below control levels in PBMCs but was upregulated in the spleen. In contrast to IL-6, an evident decrease in the expression level of IL-8 was observed in both PBMCs and spleens at 1 d or 3 d p.i. SE challenge also resulted in an increase in the mRNA expression of IL-18 and IFN-γ in PBMCs and the spleen. These results imply that the expression of goose TLR5 is differentially regulated in various tissues and may participate in the immune response against bacterial pathogens.     

Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and activates host inflammatory responses. In this study, we examine the nature of the TLR5-flagellin interaction. With deletional, insertional and alanine-scanning mutagenesis, we precisely mapped the TLR5 recognition site on flagellin to a cluster of 13 amino acid residues that participate in intermolecular interactions within flagellar protofilaments and that are required for bacterial motility. The recognition site is buried in the flagellar filament, and monomeric flagellin, but not the filamentous molecule, stimulated TLR5. Finally, flagellin coprecipitated with TLR5, indicating close physical interaction between the molecules. These studies demonstrate the exquisite ability of the innate immune system to precisely target a conserved site on flagellin that is essential for bacterial motility.     

TLR5 functions as an endocytic receptor to enhance flagellin-specific adaptive immunity

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⁺ T cells and is required for induction of robust flagellin‐specific adaptive immune responses. Flagellin‐specific CD4⁺ 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, TLR5‐deficient 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⁺ T‐cell responses to flagellin, indicating that TLR5 enhances flagellin‐specific CD4⁺ T‐cell responses in the absence of conventional TLR signaling. A requirement for TLR5 in generating flagellin‐specific CD4⁺ 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⁺ T cells.     

Over-activation of TLR5 signaling by high-dose flagellin induces liver injury in mice

Flagellin is a potent activator of a broad range of cell types that are involved in innate and adaptive immunity. Therefore, it is a good adjuvant candidate for vaccines, and it might function as a biological protectant against both major acute radiation syndrome during cancer radiotherapy and a mitigator of radiation emergencies. However, accumulating evidence has implicated flagellin in the occurrence of some inflammatory diseases, such as acute lung inflammation, cardiovascular collapse and inflammatory bowel disease. The aim of this study was to elucidate whether only flagellin-TLR5 signaling activation plays a role in the pathophysiology of liver or whether some other flagellin activity also contributes to liver injury either via bacterial infections or during clinical applications. Recombinant flagellin proteins with or without TLR5-stimulating activity were used to evaluate the role of flagellin-TLR5 signaling in liver injury in wild-type and TLR5 KO mice. Gross lesions and large areas of hepatocellular necrosis were observed in liver tissue 12 h after the intraperitoneal administration of 100 or 200 µg flagellin (FliC) in a dose- and time-dependent manner in wild-type mice, but not in TLR5 KO mice. Deletion of the N-terminal or TLR5 binding domain of flagellin inhibited flagellin-induced inflammatory responses and the subsequent acute liver function abnormality and damage. These data confirmed that flagellin is an essential determinant of liver injury and demonstrated that the over-activation of TLR5 signaling by high-dose flagellin caused acute inflammatory responses, neutrophil accumulation and oxidative stress in the liver, which contributes to the progression and severity of flagellin-induced liver injury.     

Cross-talk between toll-like receptors 5 and 9 on activation of human immune responses

The recognition of pathogen-associated molecular patterns by TLRs triggers the activation of innate and adaptive immune responses. Flagellin, the agonist of TLR5, is expressed by prokaryotes and eukaryotes, and DNA sequences containing unmethylated CpG dinucleotides, agonists of TLR9, are present essentially in prokaryotes. To test the potential modulating effects of simultaneous activation of different TLRs on the immune response, we compared the outcomes in different immune cell compartments induced by triggering TLR5 and TLR9 individually and in combination. PBMCs, monocytes, and monocyte-derived DC (MoDC) secreted high levels of IL-10 in response to flagellin, whereas oligodeoxynucleotides (ODN) containing the CpG sequence (CpG-ODN), synthetic ligands of TLR9, did not induce IL-10 secretion in any of the three cell types but synergized with flagellin in this induction. In contrast, PBMC production of IFN-alpha induced by CpG-ODN was strongly inhibited by flagellin. Conversely, CpG-ODN did not enhance the up-regulation of activation markers in MoDC induced to mature in the presence of flagellin. Flagellin-matured, but not CpG-ODN-matured, MoDC stimulated the expansion of allogeneic CD4+CD25+ T cells, and the extent of expansion induced by MoDC, matured in the presence of flagellin and CpG-ODN, was similar to that induced by flagellin-matured MoDC. Moreover, flagellin and CpG-ODN differentially affected NK-mediated cytotoxicity, and flagellin completely abrogated the NK-mediated immune response induced by CpG-ODN stimulation. Together, these results suggest that flagellin inhibits the TLR9-induced cell activation and cytokine production, which favor Th1-type immune responses, possibly because the signals evoked by flagellin to indicate the presence of extracellular pathogens must favor a Th2-polarized response. Thus, TLR5 and TLR9, alerted by the presence of microorganisms, influence each other to mount the more efficient and appropriate immune response to contain the infection of a specific pathogen.     

TLR5 or NLRC4 is necessary and sufficient for promotion of humoral immunity by flagellin

The fact that some TLR-based vaccine adjuvants maintain function in TLR-deficient hosts highlights that their mechanism of function remains incompletely understood. Thus, we examined the ability of flagellin to induce cytokines and elicit/promote murine antibody responses upon deletion of the flagellin receptors TLR5 and/or NLRC4 (also referred to as IPAF) using a prime/boost regimen. In TLR5-KO mice, flagellin failed to induce NF-κB-regulated cytokines such as keratinocyte-derived chemokine (CXCL1) but induced WT levels of the inflammasome cytokine IL-18 (IL-1F4). Conversely, in NLRC4-KO mice, flagellin induced keratinocyte-derived chemokine, but not IL-18, whereas TLR5/NLRC4-DKO lacked induction of all cytokines measured. Flagellin/ovalbumin treatment resulted in high-antibody titers to both flagellin and ovalbumin in WT, TLR5-KO and DKO mice but did not elicit antibodies to either in TLR5/NLRC4-DKO mice. Thus, flagellin's ability to elicit/promote humoral immunity requires a germ-line-encoded receptor capable of recognizing this molecule. Such promotion of adaptive immunity can be effectively driven by either TLR5-mediated activation of NF-κB or NLRC4-mediated activation of the inflammasome.