Role of Toll-Like Receptor 5 (TLR5) in Experimental Melioidosis.

The Gram-negative intracellular pathogen Burkholderia pseudomallei is the causative agent of melioidosis, an important cause of sepsis in Southeast Asia. Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is essential for an appropriate immune response during pathogen invasion. In patients with melioidosis, TLR5 is the most abundantly expressed TLR, and a hypofunctional TLR5 variant has been associated with improved survival. Here, we studied the functional role of TLR5 and its ligand flagellin in experimental melioidosis. First, we observed differential TLR5 expression in the pulmonary and hepatic compartments upon infection with B. pseudomallei Next, we found that B. pseudomallei-challenged TLR5-deficient (Tlr5-/- ) mice were more susceptible to infection than wild-type (WT) mice, as demonstrated by higher systemic bacterial loads, increased organ injury, and impaired survival. Lung bacterial loads were not different between the two groups. The phenotype was flagellin independent; no difference in in vivo virulence was observed for the flagellin-lacking mutant MM36 compared to the wild-type B. pseudomallei strain 1026b. Tlr5-/- mice showed a similar impaired antibacterial defense when infected with MM36 or 1026b. Ex vivo experiments showed that TLR5-deficient macrophages display markedly impaired phagocytosis of B. pseudomallei In conclusion, these data suggest that TLR5 deficiency has a detrimental flagellin-independent effect on the host response against pulmonary B. pseudomallei infection.

Contributions of MyD88-dependent receptors and CD11c-positive cells to corneal epithelial barrier function against Pseudomonas aeruginosa.

Previously we reported that corneal epithelial barrier function against Pseudomonas aeruginosa was MyD88-dependent. Here, we explored contributions of MyD88-dependent receptors using vital mouse eyes and confocal imaging. Uninjured IL-1R (-/-) or TLR4 (-/-) corneas, but not TLR2 (-/-), TLR5 (-/-), TLR7 (-/-), or TLR9 (-/-), were more susceptible to P. aeruginosa adhesion than wild-type (3.8-fold, 3.6-fold respectively). Bacteria adherent to the corneas of IL-1R (-/-) or TLR5 (-/-) mice penetrated beyond the epithelial surface only if the cornea was superficially-injured. Bone marrow chimeras showed that bone marrow-derived cells contributed to IL-1R-dependent barrier function. In vivo, but not ex vivo, stromal CD11c+ cells responded to bacterial challenge even when corneas were uninjured. These cells extended processes toward the epithelial surface, and co-localized with adherent bacteria in superficially-injured corneas. While CD11c+ cell depletion reduced IL-6, IL-1ß, CXCL1, CXCL2 and CXCL10 transcriptional responses to bacteria, and increased susceptibility to bacterial adhesion (>3-fold), the epithelium remained resistant to bacterial penetration. IL-1R (-/-) corneas also showed down-regulation of IL-6 and CXCL1 genes with and without bacterial challenge. These data show complex roles for TLR4, TLR5, IL-1R and CD11c+ cells in constitutive epithelial barrier function against P. aeruginosa, with details dependent upon in vivo conditions.

The anti-microbial peptide TP359 attenuates inflammation in human lung cells infected with Pseudomonas aeruginosa via TLR5 and MAPK pathways.

Pseudomonas aeruginosa infection induces vigorous inflammatory mediators secreted by epithelial cells, which do not necessarily eradicate the pathogen. Nonetheless, it reduces lung function due to significant airway damage, most importantly in cystic fibrosis patients. Recently, we published that TP359, a proprietary cationic peptide had potent bactericidal effects against P. aeruginosa, which were mediated by down-regulating its outer membrane biogenesis genes. Herein, we hypothesized that TP359 bactericidal effects could also serve to regulate P. aeruginosa-induced lung inflammation. We explored this hypothesis by infecting human A549 lung cells with live P. aeruginosa non-isogenic, mucoid and non-mucoid strains and assessed the capacity of TP359 to regulate the levels of elicited TNFα, IL-6 and IL-8 inflammatory cytokines. In all instances, the mucoid strain elicited higher concentrations of cytokines in comparison to the non-mucoid strain, and TP359 dose-dependently down-regulated their respective levels, suggesting its regulation of lung inflammation. Surprisingly, P. aeruginosa flagellin, and not its lipopolysaccharide moiety, was the primary inducer of inflammatory cytokines in lung cells, which were similarly down-regulated by TP359. Blocking of TLR5, the putative flagellin receptor, completely abrogated the capacity of infected lung cells to secrete cytokines, underscoring that TP359 regulates inflammation via the TLR5-dependent signaling pathway. Downstream pathway-specific inhibition studies further revealed that the MAPK pathway, essentially p38 and JNK are necessary for induction of P. aeruginosa elicited inflammatory cytokines and their down-regulation by TP359. Collectively, our data provides evidence to support exploring the relevancy of TP359 as an anti-microbial and anti-inflammatory agent against P. aeruginosa for clinical applications.

Epithelial-specific Toll-like Receptor (TLR)5 Activation Mediates Barrier Dysfunction in Experimental Ileitis.

BACKGROUND: A large body of evidence supports a central role of TLR5 and its natural ligand, flagellin, in Crohn's disease (CD), with the precise mechanism(s) still unresolved. METHODS: We investigated the role of flagellin/TLR5 in SAMP1/YitFc (SAMP) mice, a spontaneous model of Crohn's disease-like ileitis. RESULTS: Ileal Tlr5 and serum antiflagellin IgG antibodies were increased in SAMP before the onset of inflammation and during established disease; these trends were abrogated in the absence of colonizing commensal bacteria. Irradiated SAMP receiving either wild-type (AKR) or SAMP bone marrow (BM) developed severe ileitis and displayed increased ileal Tlr5 compared with AKR recipients of either SAMP or AKR bone marrow, neither of which conferred ileitis, suggesting that elevated TLR5 in native SAMP is derived primarily from a nonhematopoietic (e.g., epithelial) source. Indeed, ileal epithelial TLR5 in preinflamed SAMP was increased compared with age-matched AKR and germ-free SAMP. TLR5-specific ex vivo activation of SAMP ileal tissues decreased epithelial barrier resistance, indicative of increased permeability, and was accompanied by altered expression of the tight junction proteins, claudin-3, occludin, and zonula occludens-1. CONCLUSIONS: Our results provide evidence that aberrant, elevated TLR5 expression is present in the ileal epithelium of SAMP mice, is augmented in the presence of the gut microbiome, and that TLR5 activation in response to bacterial flagellin results in a deficiency to maintain appropriate epithelial barrier integrity. Together, these findings represent a potential mechanistic pathway leading to the exacerbation and perpetuation of chronic gut inflammation in experimental ileitis and possibly, in patients with Crohn's disease.

Inhibition of Interleukin-10 Signaling Induces Microbiota-dependent Chronic Colitis in Apolipoprotein E Deficient Mice.

BACKGROUND: Apolipoprotein E (ApoE) mediates potent antiinflammatory and immunomodulatory properties in addition to its roles in regulating cholesterol transport and metabolism. However, its role in the intestine, specifically during inflammation, is largely unknown. METHODS: Mice (C57BL/6 or ApoE-deficient [ApoE-KO] mice) were administered either single or 4 injections (weekly) of anti-interleukin (IL)-10 receptor monoclonal antibody (1.0 mg/mouse; intraperitoneally) and euthanized 1 week after the last injection. 16S rRNA sequencing was performed in fecal samples to analyze the gut bacterial load and its composition. Microbiota was ablated by administration of broad-spectrum antibiotics in drinking water. IL-10KO mice were cohoused with ApoE-KO mice or their wild-type littermates to monitor the colitogenic potential of gut microbiota harbored in ApoE-KO mice. RESULTS: ApoE-KO mice developed severe colitis upon neutralization of IL-10 signaling as assessed by every parameter analyzed. 16S rRNA sequencing revealed that the ApoE-KO mice display elevated and altered gut microbiota that were accompanied with impaired production of intestinal antimicrobial peptides. Interestingly, microbiota ablation ameliorates colitis development in ApoE-KO mice. Exacerbated and accelerated colitis was observed in IL-10KO mice when cohoused with ApoE-KO mice. CONCLUSIONS: Our study highlights a novel interplay between ApoE and IL-10 in maintaining gut homeostasis and that such crosstalk may play a critical role in the pathogenesis of inflammatory bowel disease. Gut sterilization and the cohousing experiment suggest that microbiota play a pivotal role in the development of inflammatory bowel disease in mice lacking ApoE.

Membrane cholesterol plays an important role in enteropathogen adhesion and the activation of innate immunity via flagellin-TLR5 signaling.

Lipid rafts are cholesterol- and sphingolipid-rich ordered microdomains distributed in the plasma membrane that participates in mammalian signal transduction pathways. To determine the role of lipid rafts in mediating interactions between enteropathogens and intestinal epithelial cells, membrane cholesterol was depleted from Caco-2 and IPEC-J2 cells using methyl-ß-cyclodextrin. Cholesterol depletion significantly reduced Escherichia coli and Salmonella enteritidis adhesion and invasion into intestinal epithelial cells. Complementation with exogenous cholesterol restored bacterial adhesion to basal levels. We also evaluated the role of lipid rafts in the activation of Toll-like receptor 5 signaling by bacterial flagellin. Depleting membrane cholesterol reduced the ability of purified recombinant E. coli flagellin to activate TLR5 signaling in intestinal cells. These data suggest that both membrane cholesterol and lipid rafts play important roles in enteropathogen adhesion and contribute to the activation of innate immunity via flagellin-TLR5 signaling.

Toll-like receptor 5 in obesity: the role of gut microbiota and adipose tissue inflammation.

OBJECTIVE: This study aimed at establishing bacterial flagellin-recognizing toll-like receptor 5 (TLR5) as a novel link between gut microbiota composition, adipose tissue inflammation, and obesity. METHODS: An adipose tissue microarray database was used to compare women having the highest (n = 4, H-TLR) and lowest (n = 4, L-TLR) expression levels of TLR5-signaling pathway genes. Gut microbiota composition was profiled using flow cytometry and FISH. Standard laboratory techniques were used to determine anthropometric and clinical variables. In vivo results were verified using cultured human adipocytes. RESULTS: The H-TLR group had higher flagellated Clostridium cluster XIV abundance and Firmicutes-to-Bacteroides ratio. H-TLR subjects had obese phenotype characterized by greater waist circumference, fat %, and blood pressure (P < 0.05 for all). They also had higher leptin and lower adiponectin levels (P < 0.05 for both). Six hundred and sixty-eight metabolism- and inflammation-related adipose tissue genes were differentially expressed between the groups. In vitro studies confirmed that flagellin activated TLR5 inflammatory pathways, decreased insulin signaling, and increased glycerol secretion. CONCLUSIONS: The in vivo findings suggest that flagellated Clostridium cluster XIV bacteria contribute to the development of obesity through distorted adipose tissue metabolism and inflammation. The in vitro studies in adipocytes show that the underlying mechanisms of the human findings may be due to flagellin-activated TLR5 signaling.

Viral infection. Prevention and cure of rotavirus infection via TLR5/NLRC4-mediated production of IL-22 and IL-18.

Activators of innate immunity may have the potential to combat a broad range of infectious agents. We report that treatment with bacterial flagellin prevented rotavirus (RV) infection in mice and cured chronically RV-infected mice. Protection was independent of adaptive immunity and interferon (IFN, type I and II) and required flagellin receptors Toll-like receptor 5 (TLR5) and NOD-like receptor C4 (NLRC4). Flagellin-induced activation of TLR5 on dendritic cells elicited production of the cytokine interleukin-22 (IL-22), which induced a protective gene expression program in intestinal epithelial cells. Flagellin also induced NLRC4-dependent production of IL-18 and immediate elimination of RV-infected cells. Administration of IL-22 and IL-18 to mice fully recapitulated the capacity of flagellin to prevent or eliminate RV infection and thus holds promise as a broad-spectrum antiviral agent.

Molecular cloning and functional analysis of duck Toll-like receptor 5.

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the single-exon TLR5 gene of the Maya breed of Common Shelduck (Tadorna tadorna). The TLR5 open reading frame is 2580 bp in length and encodes an 859-amino acid protein. The putative amino acid sequence of duck TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat domains, a leucine-rich repeat C-terminal domain, a transmembrane domain, and an intracellular Toll-interleukin-1 receptor domain. The duck TLR5 gene was highly expressed in the lung, bone marrow, spleen, and liver; moderately expressed in kidney, small intestine, large intestine, and brain. A plasmid expressing duck TLR5 was constructed and transfected into HEK293T cells, and expression was confirmed by indirect immunofluorescence assay. HEK293T cells transfected with duck TLR5- and NF-κB-luciferase-containing plasmids significantly responded to flagellin from Salmonella typhimurium, indicating that it is a functional TLR5 homolog.

Functional characterisation of bovine TLR5 indicates species-specific recognition of flagellin.

Mammalian toll-like receptor 5 (TLR5) senses flagellin of several bacterial species and has been described to activate the innate immune system. To assess the role of bovine TLR5 (boTLR5) in the cattle system, we cloned and successfully expressed boTLR5 in human embryonic kidney (HEK) 293 cells, as indicated by quantitative PCR and confocal microscopy. However, in contrast to huTLR5-transfected cells, exposure of boTLR5-transfected cells to flagellin neither activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nor CXCL8 production. Subsequent comparison of the flagellin response induced in human and bovine primary macrophages revealed that flagellin did not lead to phosphorylation of major signalling molecules. Furthermore, the CXCL8 and TNFα response of primary bovine macrophages stimulated with flagellin was very low compared to that observed in human primary macrophages. Our results indicate that cattle express a functional TLR5 albeit with different flagellin sensing qualities compared to human TLR5. However, boTLR5 seemed to play a different role in the bovine system compared to the human system in recognizing flagellin, and other potentially intracellular expressed receptors may play a more important role in the bovine system to detect flagellin.

Increased Toll-like receptor 5 expression indicates esophageal columnar dysplasia.

Toll-like receptor 5 (TLR5) is an immune receptor, which recognizes bacterial flagellin. Increased expression has been reported in various premalignant and malignant lesions indicating a role in carcinogenesis. We assessed the expression of TLR5 in normal esophageal squamous epithelium, Barrett's esophagus with and without dysplasia, and in esophageal adenocarcinoma. Specimens with normal esophagus (n=93), gastric (n=75) or intestinal metaplasia (n=53) without dysplasia, and low-grade (n=56) or high-grade dysplasia (n=33) and esophageal adenocarcinoma (n=94) were studied. TLR5 immunohistochemical stainings were analyzed for the proportion of positive cells and the intensity of expression. In normal squamous epithelium, only the basal third showed TLR5 expression. In esophageal gastric or intestinal metaplasia, expression was present in majority of the cells but significantly weaker (p<0.001) than in dysplastic epithelium. In dysplasia, expression extended to the apical cytoplasm, contrasting basolateral expression in non-dysplastic columnar epithelium. Receiver operating characteristic curve analysis showed that moderate to high expression intensity of TLR5 indicates low-grade dysplasia with 86 % sensitivity and 83 % specificity. Carcinomas showed increased expression in comparison with non-dysplastic columnar epithelium, but there was no association with prognosis. Our results indicate that the esophageal columnar dysplasia is associated with clear increase of TLR5 expression and dissolution of regular polarized expression. TLR5 staining provides a possible biomarker for the recognition of low-grade dysplasia. In addition, the findings suggest a role for abnormal expression of TLR5 in the pathogenesis of esophageal adenocarcinoma and suggest importance of altered microbiome in the pathogenesis of complications of Barrett's esophagus.

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 (Isopropylß-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.

PTEN regulates TLR5-induced intestinal inflammation by controlling Mal/TIRAP recruitment.

Defective IL-10 allele is a risk factor for intestinal inflammation. Indeed, IL-10(-/-) mice are predisposed to spontaneous colitis in the presence of intestinal microbiota, indicating that microbial factors contribute to developing intestinal inflammation. By recognizing flagellin, TLR5 plays a quintessential role in microbial recognition in intestinal epithelial cells. Here, we treated flagellin (1.0 µg/mouse/d) in mouse colon and found that it elicited colonic inflammation in IL-10(-/-) mice, characterized with tissue hypertrophy, inflamed epithelium, and enhanced cytokine production in the colon (MPO, KC, IL-6; ≥2-fold; P < 0.05). These inflammatory effects were dramatically inhibited in TLR5(-/-);IL-10(-/-) mice. Intestinal epithelium specific PTEN deletion significantly attenuated flagellin-promoted colonic inflammation in IL-10(-/-) mice. As a molecular mechanism that PTEN deletion inhibited TLR5-elicited responses, we hypothesized that PTEN regulated TLR5-induced responses by controlling the involvement of Mal in TLR5 engagement. Mal interacted with TLR5 on flagellin, and Mal deficiency inhibited flagellin-induced responses in intestinal epithelial cells. Similarly, Mal(-/-);IL-10(-/-) mice showed reduced flagellin-promoted responses. Furthermore, PTEN deletion disrupted Mal-TLR5 interaction, resulting in diminished TLR5-induced responses. PTEN deletion impeded Mal localization at the plasma membrane and suppressed Mal-TLR5 interaction. These results suggest that, by controlling Mal recruitment, PTEN regulates TLR5-induced inflammatory responses.

Airway epithelial expression of TLR5 is downregulated in healthy smokers and smokers with chronic obstructive pulmonary disease.

The TLRs are important components of the respiratory epithelium host innate defense, enabling the airway surface to recognize and respond to a variety of insults in inhaled air. On the basis of the knowledge that smokers are more susceptible to pulmonary infection and that the airway epithelium of smokers with chronic obstructive pulmonary disease (COPD) is characterized by bacterial colonization and acute exacerbation of airway infections, we assessed whether smoking alters expression of TLRs in human small airway epithelium, the primary site of smoking-induced disease. Microarrays were used to survey the TLR family gene expression in small airway (10th to 12th order) epithelium from healthy nonsmokers (n = 60), healthy smokers (n = 73), and smokers with COPD (n = 36). Using the criteria of detection call of present (P call) ≥ 50%, 6 of 10 TLRs (TLRs 1-5 and 8) were expressed. Compared with nonsmokers, the most striking change was for TLR5, which was downregulated in healthy smokers (1.4-fold, p < 10⁻¹°) and smokers with COPD (1.6-fold, p < 10⁻¹¹). TaqMan RT-PCR confirmed these observations. Bronchial biopsy immunofluorescence studies showed that TLR5 was expressed mainly on the apical side of the epithelium and was decreased in healthy smokers and smokers with COPD. In vitro, the level of TLR5 downstream genes, IL-6 and IL-8, was highly induced by flagellin in TLR5 high-expressing cells compared with TLR5 low-expressing cells. In the context that TLR5 functions to recognize pathogens and activate innate immune responses, the smoking-induced downregulation of TLR5 may contribute to smoking-related susceptibility to airway infection, at least for flagellated bacteria.

TLR5, a novel and unidentified inflammatory mediator in rheumatoid arthritis that correlates with disease activity score and joint TNF-α levels.

The innate immune system plays an important role in rheumatoid arthritis (RA) pathogenesis. Previous studies support the role of TLR2 and 4 in RA and experimental arthritis models; however, the regulation and pathogenic effect of TLR5 is undefined in RA. In this study, we show that TLR5 is elevated in RA and osteoarthritis ST lining and sublining macrophages and endothelial cells compared with normal individuals. Furthermore, expression of TLR5 is elevated in RA synovial fluid macrophages and RA peripheral blood monocytes compared with RA and normal peripheral blood in vitro-differentiated macrophages. We also found that TLR5 on RA monocytes is an important modulator of TNF-α in RA synovial fluid and that TLR5 expression on these cells strongly correlates with RA disease activity and TNF-α levels. Interestingly, TNF-α has a feedback regulation with TLR5 expression in RA monocytes, whereas expression of this receptor is regulated by IL-17 and IL-8 in RA macrophages and fibroblasts. We show that RA monocytes and macrophages are more responsive to TLR5 ligation compared with fibroblasts despite the proinflammatory response being mediated through the same signaling pathways in macrophages and fibroblasts. In conclusion, we document the potential role of TLR5 ligation in modulating transcription of TNF-α from RA synovial fluid and the strong correlation of TLR5 and TNF-α with each other and with disease activity score in RA monocytes. Our results suggest that expression of TLR5 may be a predictor for RA disease progression and that targeting TLR5 may suppress RA.

Toll-like receptor 5 (TLR5), IL-1ß secretion, and asparagine endopeptidase are critical factors for alveolar macrophage phagocytosis and bacterial killing.

A deficit in early clearance of Pseudomonas aeruginosa (P. aeruginosa) is crucial in nosocomial pneumonia and in chronic lung infections. Few studies have addressed the role of Toll-like receptors (TLRs), which are early pathogen associated molecular pattern receptors, in pathogen uptake and clearance by alveolar macrophages (AMs). Here, we report that TLR5 engagement is crucial for bacterial clearance by AMs in vitro and in vivo because unflagellated P. aeruginosa or different mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. In addition, the clearance of PAK (a wild-type P. aeruginosa strain) by primary AMs was causally associated with increased IL-1ß release, which was dramatically reduced with PAK mutants or in WT PAK-infected primary TLR5(-/-) AMs, demonstrating the dependence of IL-1ß production on TLR5. We showed that this IL-1ß production was important in endosomal pH acidification and in inducing the killing of bacteria by AMs through asparagine endopeptidase (AEP), a key endosomal cysteine protease. In agreement, AMs from IL-1R1(-/-) and AEP(-/-) mice were unable to kill P. aeruginosa. Altogether, these findings demonstrate that TLR5 engagement plays a major role in P. aeruginosa internalization and in triggering IL-1ß formation.

Induction of Paneth cell degranulation by orally administered Toll-like receptor ligands.

The secretory activity of Paneth cells is related to the bacterial milieu in the small intestine; however, the molecules involved in inducing Paneth cell secretion of enzymes and antimicrobial peptides are not well-defined. Mice treated orally with CpG-oligodeoxynucleotide (ODN), an agonist of Toll-like receptor (TLR) 9, showed rapid and massive Paneth cell degranulation. CpG-ODN-induced degranulation was not observed in TLR9(-/-) mice or in chimeric TLR9(-/-) mice reconstituted with wild-type (WT) bone marrow, but was observed in WT mice reconstituted with TLR9(-/-) bone marrow, indicating a role for TLR9-expressing gastrointestinal cells in CpG recognition. The TLR3 agonist polyinosinic-polycytidylic acid also induced rapid degranulation, whereas the TLR4 and TLR5 agonists LPS and flagellin, respectively, induced late degranulation mediated by TNF-α. Our evidence that TLR9 and TLR3 agonists induce Paneth cell degranulation points to the need for further studies of the mechanisms underlying Paneth cell function as an avenue toward preventing infection and treating inflammatory bowel diseases.

Toll-like receptor 5 deficiency protects from wasting disease in a T cell transfer colitis model in T cell receptor-ß-deficient mice.

BACKGROUND: Toll-like receptor 5 (TLR5) is implicated in the innate and adaptive immune responses that are associated with inflammatory bowel disease (IBD). In humans TLR5 is expressed on CD4(+) T cells and costimulation with flagellin potentiates effector and regulatory T cell responses. The aim of this study was to determine the role of TLR5 in CD4(+) T cell subsets versus other cells in induction of disease in a model of T cell-dependent colitis. METHODS: TLR5 expression on CD4(+) T cells was assessed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Wildtype (WT) or TLR5-deficient (5-/-) CD4(+) T conventional cells (Tconv) and T regulatory cells (Treg) were compared for their ability to induce and suppress T cell transfer colitis, respectively. In addition, the role of TLR5 expression in recipient mice was analyzed. RESULTS: TLR5 is preferentially expressed on mouse Treg compared to Tconv, although expression levels were low. The colitogenic capacity of WT and 5-/- Tconv was found to be similar and Treg from WT or 5-/- donor animals both prevented T cell transfer colitis in TLR-competent hosts. TLR5 deficiency in recipient mice, however, did affect the disease process, as T cell receptor-ß (TCRß) 5-/- recipients had decreased weight loss compared to TCRß recipient mice when WT Tconv were used. CONCLUSIONS: TLR5 expression on T cells is not required for induction of or protection from T cell-dependent colitis. Expression of TLR5 in non-T cells has a pathogenic role, since TLR5 deficiency in recipient mice protects against weight loss induced by WT T cells.