Inclusion of Flagellin during Vaccination against Influenza Enhances Recall Responses in Nonhuman Primate Neonates.

UNLABELLED: Influenza virus can cause life-threatening infections in neonates and young infants. Although vaccination is a major countermeasure against influenza, current vaccines are not approved for use in infants less than 6 months of age, in part due to the weak immune response following vaccination. Thus, there is a strong need to develop new vaccines with improved efficacy for this vulnerable population. To address this issue, we established a neonatal African green monkey (AGM) nonhuman primate model that could be used to identify effective influenza vaccine approaches for use in young infants. We assessed the ability of flagellin, a Toll-like receptor 5 (TLR5) agonist, to serve as an effective adjuvant in this at-risk population. Four- to 6-day-old AGMs were primed and boosted with inactivated PR8 influenza virus (IPR8) adjuvanted with either wild-type flagellin or inactive flagellin with a mutation at position 229 (m229), the latter of which is incapable of signaling through TLR5. Increased IgG responses were observed following a boost, as well as at early times after challenge, in infants vaccinated with flagellin-adjuvanted IPR8. Inclusion of flagellin during vaccination also resulted in a significantly increased number of influenza virus-specific T cells following challenge compared to the number in infants vaccinated with the m229 adjuvant. Finally, following challenge infants vaccinated with IPR8 plus flagellin exhibited a reduced pathology in the lungs compared to that in infants that received IPR8 plus m229. This study provides the first evidence of flagellin-mediated enhancement of vaccine responses in nonhuman primate neonates. IMPORTANCE: Young infants are particularly susceptible to severe disease as a result of influenza virus infection. Compounding this is the lack of effective vaccines for use in this vulnerable population. Here we describe a vaccine approach that results in improved immune responses and protection in young infants. Incorporation of flagellin during vaccination resulted in increased antibody and T cell responses together with reduced disease following virus infection. These results suggest that flagellin may serve as an effective adjuvant for vaccines targeted to this vulnerable population.

Enhanced antigen processing of flagellin fusion proteins promotes the antigen-specific CD8+ T cell response independently of TLR5 and MyD88.

Flagellin is a highly effective adjuvant for CD4(+) T cell and humoral immune responses. However, there is conflicting data in the literature regarding the ability of flagellin to promote a CD8(+) T cell response. In this article, we report that immunization of wild-type, TLR5(-/-), and MyD88(-/-) adoptive transfer recipient mice revealed the ability of flagellin fusion proteins to promote OVA-specific CD8(+) T cell proliferation independent of TLR5 or MyD88 expression by the recipient animal. Wild-type and TLR5(-/-) APCs were able to stimulate high levels of OVA-specific CD8(+) T cell proliferation in vitro in response to a flagellin fusion protein containing full-length OVA or the SIINFEKL epitope and 10 flanking amino acids (OVAe), but not to OVA and flagellin added as separate proteins. This effect was independent of the conserved regions of flagellin and occurred in response to OVAe alone. Comparison of IFN-γ production by CD8(+) effector cells revealed higher levels of SIINFEKL peptide-MHC I complexes on the surface of APCs that had been pulsed with OVAe-flagellin fusion proteins than on cells pulsed with OVA. Inhibition of the proteasome significantly reduced Ag-specific proliferation in response to OVAe fusion proteins. In summary, our data are consistent with the conclusion that flagellin-OVA fusion proteins induce an epitope-specific CD8(+) T cell response by facilitating Ag processing and not through stimulatory signaling via TLR5 and MyD88. Our findings raise the possibility that flagellin might be an efficient Ag carrier for Ags that are poorly processed in their native state.

Flagellin as an adjuvant: cellular mechanisms and potential.

Flagellin is a potent activator of a broad range of cell types involved in innate and adaptive immunity. An increasing number of studies have demonstrated the effectiveness of flagellin as an adjuvant, as well as its ability to promote cytokine production by a range of innate cell types, trigger a generalized recruitment of T and B lymphocytes to secondary lymphoid sites, and activate TLR5(+)CD11c(+) cells and T lymphocytes in a manner that is distinct from cognate Ag recognition. The plasticity of flagellin has allowed for the generation of a range of flagellin-Ag fusion proteins that have proven to be effective vaccines in animal models. This review summarizes the state of our current understanding of the adjuvant effect of flagellin and addresses important areas of current and future research interest.

Protection against lethal vaccinia virus challenge by using an attenuated matrix protein mutant vesicular stomatitis virus vaccine vector expressing poxvirus antigens.

Recombinant vesicular stomatitis viruses (VSV) are excellent candidate vectors for vaccination against human diseases. The neurovirulence of VSV in animal models requires the attenuation of the virus for use in humans. Previous efforts have focused on attenuating virus replication. Studies presented here test an alternative approach for attenuation that uses a matrix (M) protein mutant (rM51R) VSV as a vaccine vector against respiratory infection. This mutant is attenuated for viral virulence by its inability to suppress the innate immune response. The ability of rM51R VSV vectors to protect against lethal respiratory challenge was tested using a vaccinia virus intranasal challenge model. Mice immunized intranasally with rM51R vectors expressing vaccinia virus antigens B5R and L1R were protected against lethal vaccinia virus challenge. A single immunization with the vectors provided protection against vaccinia virus-induced mortality; however, a prime-boost strategy reduced the severity of the vaccinia virus-induced disease progression. Antibody titers measured after the prime and boost were low despite complete protection against lethal challenge. However, immunized animals had higher antibody titers during the challenge, suggesting that memory B-cell responses may be important for the protection. Depletion experiments demonstrated that B cells but not CD8 T cells were involved in the protection mediated by rM51R vaccine vectors that express B5R and L1R. These results demonstrate the potential of M protein mutant VSVs as candidate vaccine vectors against human diseases.

Stimulation of human dendritic cells by wild-type and M protein mutant vesicular stomatitis viruses engineered to express bacterial flagellin.

Vesicular stomatitis viruses (VSVs) containing wild-type (wt) or mutant matrix (M) proteins are being developed as candidate vaccine vectors due to their ability to induce innate and adaptive immunity. Viruses with wt M protein, such as recombinant wild-type (rwt) virus, stimulate maturation of dendritic cells (DC) through Toll-like receptor 7 (TLR7) and its adaptor molecule MyD88. However, M protein mutant viruses, such as rM51R-M virus, stimulate both TLR7-positive and TLR7-negative DC subsets. The goal of this study was to determine whether the ability of rwt and rM51R-M viruses to induce maturation of human DC can be enhanced by engineering these vectors to express bacterial flagellin. Flagellin expressed from the rwt virus genome partially protected human DC from VSV-induced shutoff of host protein synthesis and promoted the production of interleukin 6 (IL-6) and IL-1ß. In addition, DC infected with rwt virus expressing flagellin were more effective at stimulating gamma interferon (IFN-γ) production from CD8(+) allogeneic T cells than DC infected with rwt virus. Although rM51R-M virus effectively stimulated human DC, flagellin expressed from the rM51R-M virus genome enhanced the production of cytokines. Furthermore, mice immunized with both rwt and rM51R-M viruses expressing flagellin had enhanced anti-VSV antibody responses in vivo. Therefore, rwt and rM51R-M viruses expressing flagellin may be promising vectors for the delivery of foreign antigen due to their potential to stimulate DC function.

Direct stimulation of tlr5+/+ CD11c+ cells is necessary for the adjuvant activity of flagellin.

Flagellin is a highly effective adjuvant, but the cellular mechanism underlying this activity remains uncertain. More specifically, no consensus exists as to whether flagellin activates dendritic cells (DC) directly or indirectly. Intramuscular immunization with flagellin-OVA fusion protein resulted in enhanced in vivo T cell clustering in draining lymph nodes and IL-2 production by OVA-specific CD4(+) T cells. Immunization with flagellin-OVA also triggered greater levels of Ag-specific CD4(+) T cell proliferation than immunization with flagellin and OVA as separate proteins. To determine whether flagellin, in the context of a fusion protein with OVA, was acting directly on DC, we used a combination of CD4(+) T cell adoptive transfers and bone marrow chimera mice in which the presence or absence of potential tlr5(+/+) CD11c(+) cells was controlled by injection of diphtheria toxin. The Ag-specific CD4(+) T cell response in mice with CD11c(+) cells from a tlr5(-/-) background and mixed populations of all other hematopoietic cells was dramatically reduced in comparison to mice that had DC from tlr5(-/-) and wild-type backgrounds. Immunization of MyD88(-/-)tlr5(+/+) mice revealed that the enhanced response following immunization with flagellin-OVA is dependent on signaling via the TLR5-MyD88 pathway as well as enhanced Ag uptake and processing resulting from Ag targeting via TLR5. In summary, our data are consistent with the conclusion that direct stimulation of tlr5(+/+) CD11c(+) cells is necessary for the adjuvant activity of a flagellin fusion protein and that this adjuvant effect requires signaling through TLR5.

A fusion protein vaccine containing OprF epitope 8, OprI, and type A and B flagellins promotes enhanced clearance of nonmucoid Pseudomonas aeruginosa.

Although chronic Pseudomonas aeruginosa infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients, there is no approved vaccine for human use against P. aeruginosa. The goal of this study was to establish whether a multivalent vaccine containing P. aeruginosa type A and B flagellins as well as the outer membrane proteins OprF and OprI would promote enhanced clearance of P. aeruginosa. Intramuscular immunization with flagellins and OprI (separate) or OprI-flagellin fusion proteins generated significant antiflagellin immunoglobulin G (IgG) responses. However, only the fusions of OprI with type A and type B flagellins generated OprI-specific IgG. Immunization with a combination of OprF epitope 8 (OprF(311-341)), OprI, and flagellins elicited high-affinity IgG antibodies specific to flagellins, OprI, and OprF that individually promoted extensive deposition of C3 on P. aeruginosa. Although these antibodies exhibited potent antibody-dependent complement-mediated killing of nonmucoid bacteria, they were significantly less effective with mucoid isolates. Mice immunized with the OprF(311-341)-OprI-flagellin fusion had a significantly lower bacterial burden three days postchallenge and cleared the infection significantly faster than control mice. In addition, mice immunized with the OprF(311-341)-OprI-flagellin fusion had significantly less inflammation and lung damage throughout the infection than OprF-OprI-immunized mice. Based on our results, OprF(311-341)-OprI-flagellin fusion proteins have substantial potential as components of a vaccine against nonmucoid P. aeruginosa, which appears to be the phenotype of the bacterium that initially colonizes CF patients.

Engineered expression of the TLR5 ligand flagellin enhances paramyxovirus activation of human dendritic cell function.

The paramyxovirus simian virus 5 (SV5) is a poor activator of human dendritic cell (DC) maturation pathways in vitro, and infected DC do not upregulate cell surface costimulatory proteins or secretion of immunomodulatory cytokines. We evaluated the hypothesis that activation of SV5-infected DC would be enhanced by engineering SV5 to express a Toll-like-receptor (TLR) ligand. To test this hypothesis, a novel virus was engineered such that the gene encoding an intracellular form of the TLR5 ligand flagellin was expressed from the genome of wild-type (WT) SV5 (SV5-flagellin). Cells infected in vitro with the flagellin-expressing virus released low levels of biologically active flagellin, which was capable of stimulating TLR5 signaling. Infection of human peripheral blood mononuclear cell-derived immature DC with SV5-flagellin resulted in enhanced levels of interleukin-6 (IL-6) and IL-12 compared to infection with DC with the parental virus, WT SV5. In contrast to cytokine induction, the flagellin-expressing virus did not appreciably increase DC surface expression of the costimulatory molecule CD80 or CD86 above the level seen with WT SV5 alone. In mixed-culture assays, DC infected with the flagellin-expressing virus were more effective at activating gamma interferon secretion from both CD8(+) and CD4(+) allogeneic T cells than DC infected with WT SV5. Our results with SV5-directed intracellular expression of flagellin may be applicable to other vectors or pathogenic viruses where overcoming impairment of DC activation could contribute to the development of safer and more effective vaccines.

Mucosal adjuvant activity of flagellin in aged mice.

We evaluated the ability of flagellin, a highly effective mucosal adjuvant in mice and non-human primates, to promote mucosal innate and adaptive immunity in aged mice. We found that intratracheal instillation of flagellin induced a stronger respiratory innate response in aged mice than in young mice, and that intranasal instillation of flagellin was equally effective at triggering recruitment of T and B lymphocytes to the draining lymph nodes of young and aged mice. Intranasal immunization of aged mice with flagellin and the Yersinia pestis protein F1 promoted specific IgG and IgA production, but at lower levels and lower avidities than in young mice. Although intranasal instillation of flagellin and F1 antigen increased germinal center formation and size in young mice, it did not do so in aged mice. Our findings are consistent with the conclusion that flagellin can promote adaptive immune responses in aged mice, but at a less robust level than in young mice.

A phase I safety and immunogenicity dose escalation trial of plague vaccine, Flagellin/F1/V, in healthy adult volunteers (DMID 08-0066).

INTRODUCTION: Intentional aerosolization of Yersinia pestis may result in pneumonic plague which is highly fatal if not treated early. METHODS: We conducted a phase 1 randomized, double blind (within each group), placebo controlled, dose escalation trial to evaluate a plague vaccine, Flagellin/F1/V, in healthy adults aged 8 through 45years. Vaccine was administered intramuscularly on Days 0 and 28 at a dose of 1, 3, 6 or 10mcg. Subjects were observed for 4h after vaccination for cytokine release syndrome. Reactogenicity and adverse events (AE) were collected for 14 and 28days, respectively, after each vaccination. Serious AE were collected for the entire study. ELISA antibody and cytokines were measured at multiple time points. Subject's participation lasted 13months. RESULTS: Sixty healthy subjects were enrolled; 52% males, 100% non-Hispanic, 91.7% white and mean age 30.8years. No severe reactogenicity events occurred; most AE were mild. No serious AE related to vaccine occurred. A dose response effect was observed to F1, V and flagellin. The peak ELISA IgG antibody titers (95% CI) after two 10mcg doses of vaccine were 260.0 (102.6-659.0) and 983.6 (317.3-3048.8), respectively, against F1 and V antigens. The 6mcg dose group provided similar titers. Titers were low for the placebo, 1mcg and 3mcg recipients. A positive antibody dose response was observed to F1, V and flagellin. Vaccine antigen specific serum IgE was not detected. There were no significant rises in serum or cellular cytokine responses and no significant IgG increase to flagellin after the second dose. CONCLUSION: The Flagellin/F1/V vaccine exhibited a dose dependent increase in immunogenicity and was well tolerated at all doses. Antibody specific responses to F1, V and flagellin increased as dose increased. Given the results from this trial, testing higher doses of the vaccine may be merited.

Effects of flagellin on innate and adaptive immunity.

Flagella are locomotive organelles present on a wide range of bacteria and are important for the pathogenesis of many species. Cells of the innate immune system lack memory per se, but recognize conserved pathogen-associated molecular patterns (PAMPs) through a family of type I membrane receptors known as Toll-like receptors (TLRs). Flagellin, the major structural component of flagella, is a highly conserved protein recognized in hosts by TLR5. Signaling of flagellin via TLR5/TLR4 heteromeric complexes enhances the diversity of the response, likely by engaging MyD88-independent adaptors to activate the interferon pathway. Flagellin is a potent immune activator, stimulating diverse biologic effects that mediate both innate inflammatory responses as well as the development of adaptive immunity. Binding of flagellin to the extracellular domain of TLR5 rapidly induces a signal cascade that culminates in the production of proinflammatory mediators such as cytokines, chemokines, and costimulatory molecules. This review focuses on the mechanisms of action of flagellin and its effects on both innate and adaptive immunity.

Parainfluenza virus 5-based vaccine vectors expressing vaccinia virus (VACV) antigens provide long-term protection in mice from lethal intranasal VACV challenge.

To test the potential for parainfluenza virus 5 (PIV5)-based vectors to provide protection from vaccinia virus (VACV) infection, PIV5 was engineered to express secreted VACV L1R and B5R proteins, two important antigens for neutralization of intracellular mature (IMV) and extracellular enveloped (EEV) virions, respectively. Protection of mice from lethal intranasal VACV challenge required intranasal immunization with PIV5-L1R/B5R in a prime-boost protocol, and correlated with low VACV-induced pathology in the respiratory tract and anti-VACV neutralizing antibody. Mice immunized with PIV5-L1R/B5R showed some disease symptoms following VACV challenge such as loss of weight and hunching, but these symptoms were delayed and less severe than with unimmunized control mice. While immunization with PIV5 expressing B5R alone conferred at least some protection, the most effective immunization included the PIV5 vector expressing L1R alone or in combination with PIV5-B5R. PIV5-L1R/B5R vectors elicited protection from VACV challenge even when CD8+ cells were depleted, but not in the case of mice that were defective in B cell production. Mice were protected from VACV challenge out to at least 1.5 years after immunization with PIV5-L1R/B5R vectors, and showed significant levels of anti-VACV neutralizing antibodies. These results demonstrate the potential for PIV5-based vectors to provide long lasting protection against complex human respiratory pathogens such as VACV, but also highlight the need to understand mechanisms for the generation of strong immune responses against poorly immunogenic viral proteins.

A recombinant flagellin-poxvirus fusion protein vaccine elicits complement-dependent protection against respiratory challenge with vaccinia virus in mice.

Bacterial flagellin is a potent adjuvant that enhances adaptive immune responses to a variety of protein antigens. The vaccinia virus antigens L1R and B5R are highly immunogenic in the context of the parent virus, but recombinant forms of the proteins are only weakly immunogenic. Therefore we evaluated the humoral response to these antigens in mice when flagellin was used as an adjuvant. Flagellin-L1R and flagellin-B5R fusion proteins were more potent than flagellin, L1R, and B5R as separate proteins. At least three immunizations with flagellin-L1R and flagellin-B5R fusion proteins were required to confer protection in mice against challenge with vaccinia virus. Immune mice exhibited only limited signs of disease following challenge. Additionally, virus neutralization titers correlated with protection. Depletion of complement using cobra venom factor resulted in a marked decrease in the survival of immunized mice after challenge with vaccinia virus. Our results are consistent with the conclusion that flagellin-L1R and flagellin-B5R fusion proteins are effective in eliciting protective immunity against vaccinia virus that is dependent, in large part, on complement.

Immunization of young African green monkeys with OprF epitope 8-OprI-type A- and B-flagellin fusion proteins promotes the production of protective antibodies against nonmucoid Pseudomonas aeruginosa.

There is currently no approved vaccine against Pseudomonas aeruginosa, the major cause of morbidity and mortality in cystic fibrosis (CF) patients and a major pathogen in ventilated and burn patients. In a previous study, we demonstrated the immunization of mice with OprF(311-341)-OprI-type A- and B-flagellin fusion proteins dramatically enhanced clearance of nonmucoid P. aeruginosa. The goal of the current study was to evaluate the ability of OprF(311-341)-OprI-flagellins to elicit the production of protective IgG in young (4-6 months old) African green monkeys. Intramuscular immunization of African green monkeys with 1, 3, 10, or 30mug of OprF(311-341)-OprI-flagellins generated robust antigen-specific IgG responses. In addition, immunization with OprF(311-341)-OprI-flagellins elicited high-affinity anti-flagellins, OprI, and OprF IgG that individually promoted extensive deposition of complement component C3 on P. aeruginosa and synergized to facilitate maximal C3 deposition. Passive immunization of mice with plasma from OprF(311-341)-OprI-flagellins immunized monkeys significantly reduced lung bacterial burden three days post-challenge compared to mice that received pre-immunization plasma. Based on our results, OprF(311-341)-OprI-A- and B-flagellin fusion proteins are highly effective in mice and nonhuman primates and thus merit additional development as a potential vaccine for use in humans.

Flagellin-F1-V fusion protein is an effective plague vaccine in mice and two species of nonhuman primates.

A number of studies have clearly demonstrated that flagellin is a potent adjuvant that promotes robust immune responses when it is given with a protein antigen. In view of the potential biological and practical benefits of a recombinant protein vaccine composed of a single fusion protein containing flagellin and antigen, we have evaluated the efficacy of a fusion protein composed of flagellin and two protective antigens of Yersinia pestis (F1 and V) in eliciting protection against respiratory challenge with Y. pestis. Flagellin-F1-V was produced and purified in high yield under good manufacturing practices conditions. The fusion protein retains full Toll-like receptor 5-stimulating activity in vitro. Using a prime-boost immunization protocol, we found that flagellin-F1-V elicits robust antigen-specific humoral immunity in mice and two species of nonhuman primates. Immune mice were fully protected against intranasal challenge with 150 mean tolerated doses of Y. pestis CO92. In immune mice, the bacteria were completely cleared within 3 days after challenge. Flagellin-F1-V exhibited full stability for at least 297 days at 4 degrees C and at least 168 days at 25 degrees C. At between 29 and 84 days at 37 degrees C, the protein exhibited a loss of biological activity that appeared to be associated with a substantial change in protein diameter, possibly due to oligomerization. On the basis of our results, we believe that flagellin-F1-V is an outstanding candidate for evaluation in studies with humans.

Flagellin is an effective adjuvant for immunization against lethal respiratory challenge with Yersinia pestis.

Gram-negative flagellin, a Toll-like receptor 5 (TLR5) agonist, is a potent inducer of innate immune effectors such as cytokines and nitric oxide. In the lung, flagellin induces a localized and transient innate immune response characterized by neutrophil infiltration and the production of cytokines and chemokines. In view of the extraordinary potency of flagellin as an inducer of innate immunity and the contribution of innate responses to the development of adaptive immunity, we evaluated the efficacy of recombinant Salmonella flagellin as an adjuvant in an acellular plague vaccine. Mice immunized intranasally or intratracheally with the F1 antigen of Yersinia pestis and flagellin exhibited dramatic increases in anti-F1 plasma immunoglobulin G (IgG) titers that remained stable over time. In contrast, control mice had low or undetectable antibody responses. The IgG1/IgG2a ratio of antibody titers against F1 in immunized mice is consistent with a Th2 bias. However, no significant antigen-specific IgE production was detected. Interferons, tumor necrosis factor alpha, and interleukin-6 were not essential for the adjuvant effects of flagellin. Preexisting antiflagellin antibodies had no significant effect on the adjuvant activity of flagellin. Importantly, intranasal immunization with flagellin and the F1 antigen was protective against intranasal challenge with virulent Y. pestis CO92, with 93 to 100% survival of immunized mice. Lastly, vaccination of cynomolgus monkeys with flagellin and a fusion of the F1 and V antigens of Y. pestis induced a robust antigen-specific IgG antibody response.

Gangliosides inhibit flagellin signaling in the absence of an effect on flagellin binding to toll-like receptor 5.

A recent study (Ogushi, K., Wada, A., Niidome, T., Okuda, T., Llanes, R., Nakayama, M., Nishi, Y., Kurazono, H., Smith, K. D., Aderem, A., Moss, J., and Hirayama, T. (2004) J. Biol. Chem. 279, 12213-12219) concluded that gangliosides serve as co-receptors for flagellin signaling via toll-like receptor 5 (TLR5). In view of several findings in this study that were inconsistent with a role for gangliosides as co-receptors, we re-examined this important issue. Using TLR5-negative RAW 264.7 cells and a TLR5-enhanced yellow fluorescent protein chimera, we established an assay for specific binding of flagellin to cells. Inhibition of clatherin-mediated internalization of flagellin.TLR5-enhanced yellow fluorescent protein complexes did not impair flagellin activation of IRAK-1. Thus flagellin signal occurs at the cell surface and not intracellularly. Exogenous addition of mixed gangliosides (GM1, GD1a, and GT1b) as well as GD1a itself inhibited flagellin-induced interleukin-1 receptor-associated kinase activation as well as tumor necrosis factor alpha production in HeNC2, THP-1, and RAW 264.7 cells. Gangliosides inhibited flagellin signaling in the absence of an effect on flagellin binding to TLR5. Depletion of gangliosides in RAW 264.7 cells did not alter the concentration dependence or magnitude of flagellin signaling as measured by interleukin-1 receptor-associated kinase activation or tumor necrosis factor alpha production. Our findings are consistent with the conclusions that gangliosides are not essential co-receptors for flagellin and that the inhibitory effect of gangliosides is mediated by at least one mechanism that is distinct from any effect on the binding of flagellin to TLR5.

Mucosal administration of flagellin induces innate immunity in the mouse lung.

Nonsurgical intratracheal instillation of 1 microg of purified, recombinant flagellin in several strains of mice stimulated a transient innate immune response in the lung characterized by the infiltration of neutrophils and the rapid production of tumor necrosis factor alpha, interleukin 6, granulocyte colony-stimulating factor, and the chemokines keratinocyte-derived chemokine, MIP1alpha, and MIP-2.

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.

Identification of a sequence in human toll-like receptor 5 required for the binding of Gram-negative flagellin.

Flagellins from Gram-negative bacteria activate inflammatory cells by a toll-like receptor 5 (TLR5)-dependent signaling pathway. We have examined the interaction between flagellin and TLR5 using an in vitro binding assay. Purified recombinant His-tagged flagellin from Salmonella enteritidis bound to TLR5 in detergent lysates from COS-1 cells transiently transfected with a human TLR5 expression plasmid. Flagellins from Salmonella typhimurium and Escherichia coli also bound to TLR5. The specificity of this interaction was demonstrated by its concentration dependence and lack of TLR5 binding to a biologically inactive form of flagellin or to a His-tagged non-flagellar protein. Flagellin bound to the extracellular domain of TLR5 expressed on the surface of COS-1 cells and to a soluble, monomeric form of the extracellular domain (amino acids 1-636). Although a TLR5 extracellular domain containing amino acids 1-407 retained flagellin binding activity, binding was not evident with a TLR5 peptide encoding residues 1-386. Conversely, a peptide containing amino acid residues 386-636 retained flagellin binding. Thus it is likely that amino acids 386-407 is a binding site for flagellin. This sequence contains a putative leucine-rich repeat. These results support the conclusion that flagellin signaling via TLR5 involves a direct interaction between flagellin and a leucine-rich region in TLR5. We also show that the NH2-terminal 358 amino acids of TLR5 play an important role in its signaling activity. Our results provide, for the first time, a molecular basis for the agonist specificity of a TLR.