Surface-expressed TLR6 participates in the recognition of diacylated lipopeptide and peptidoglycan in human cells

Recognition of microbial components by TLR2 requires cooperation with other TLRs. TLR6 has been shown to be required for the recognition of diacylated lipoproteins and lipopeptides derived from mycoplasma and to activate the NF-kappaB signaling cascade in conjunction with TLR2. Human TLR2 is expressed on the cell surface in a variety of cells, including monocytes, neutrophils, and monocyte-derived, immature dendritic cells (iDCs), whereas the expression profile of TLR6 in human cells remains obscure. In this study we produced a function-blocking mAb against human TLR6 and analyzed TLR6 expression in human blood cells and cell lines and its participation in ligand recognition. TLR6 was expressed, although at a lower level than TLR2, on the cell surface in monocytes, monocyte-derived iDCs, and neutrophils, but not on B, T, or NK cells. Confocal microscopic analysis revealed that TLR6 was colocalized with TLR2 at the plasma membrane of monocytes. Importantly, TLR2/6 signaling did not require endosomal maturation, and anti-TLR6 mAb inhibited cytokine production in monocytes and iDCs stimulated with synthetic macrophage-activating lipopeptide-2 or peptidoglycan, indicating that TLR6 recognized diacylated lipopeptide and peptidoglycan at the cell surface. In addition, TLR2 mutants C30S and C36S (Cys(30) and Cys(36) in TLR2 were substituted with Ser), which were expressed intracellularly in HEK293 cells, failed to induce NF-kappaB activation upon macrophage-activating lipopeptide-2 stimulation even in the presence of TLR6. Thus, coexpression of TLR2 and TLR6 at the cell surface is crucial for recognition of diacylated lipopeptide and peptidoglycan and subsequent cellular activation in human cells.     

Integrin-nucleated Toll-like receptor (TLR) dimerization reveals subcellular targeting of TLRs and distinct mechanisms of TLR4 activation and signaling

Toll-like receptors (TLRs) are activated by microbial structures. To investigate the mechanisms of TLR activation, the 10 human TLRs were expressed as chimeras with the integrin alphav and beta5 subunits. Co-expression of the alphav-TLR and beta5-TLR chimeras in 293T cells generated 10 TLR homodimers, but only TLR4/4 could effectively activate NF-kappaB. TLR4 monomers also activated NF-kappaB but it was enhanced upon homodimerization. The TLR homodimers showed differential surface/intracellular expression. In TLR heterodimers, only TLR2/1 and TLR2/6 were potent in NF-kappaB activation. NF-kappaB activation by TLR2/1, TLR2/6 and the TLR4 monomer, but not TLR4/4, was completely inhibited by dominant negative MyD88, suggesting that TLR4 homodimers and monomers could activate NF-kappaB through different mechanisms.     

Lipoproteins of Listeria monocytogenes are critical for virulence and TLR2-mediated immune activation

Numerous cell surface components of Listeria influence and regulate innate immune recognition and virulence. Here, we demonstrate that lipidation of prelipoproteins in Listeria monocytogenes is required to promote NF-kappaB activation via TLR2. In HeLa cells transiently expressing TLR2, L. monocytogenes and Listeria innocua mutants lacking the prolipoprotein diacylglyceryl transferase (lgt) gene are unable to induce TLR2-dependent activation of NF-kappaB, a property intrinsic to their isogenic parental strains. TLR2-dependent immune recognition is directed to secreted, soluble lipoproteins as evidenced by the sensitivity of the response to lipoprotein lipase. Studies of bone marrow-derived macrophages of C57BL/6 wild-type and TLR2-deficient mice infected with wild-type and lgt mutant strains indicate that the absence of host TLR2 receptor signaling has consequences similar to those of the absence of the bacterial TLR2 ligand, i.e., a delay in cellular immune responses directed toward the bacterium. Infection studies with the wild-type and TLR2(-/-) mice indicated attenuation of the lgt deletion mutant in both mouse strains, implying multiple roles of lipoproteins during infection. Further characterization of the Delta lgt mutant indicated that it is impaired for both invasion and intracellular survival and exhibits increased susceptibility to cationic peptides. Our studies identify lipoproteins as the immunologically active ligand of TLR2 and assign a critical role for this receptor in the recognition of these bacteria during infection, but they also reveal the overall importance of the lipoproteins for the pathogenicity of Listeria.     

Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human Toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans

S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-kappaB reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2(DeltaS40-I64) (a TLR2 mutant with a deletion of the region of Ser(40) to Ile(64)) failed to activate NF-kappaB in response to FSL-1. The deletion mutant TLR2(DeltaC30-S39) induced NF-kappaB reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu(178) to Ala (TLR2(E178A)), TLR2(E180A), TLR2(E190A), and TLR2(L132E) induced NF-kappaB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2(L107E), TLR2(L112E) (a TLR2 point mutant with a substitution of Leu(112) to Glu), and TLR2(L115E) failed to induce NF-kappaB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2(L115E), TLR2(L112E), and TLR2(DeltaS40-I64) were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2(E190A) were. In addition, these mutants, except for TLR2(E180A), functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser(40)-Ile(64) and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans.     

Cutting edge: TNFR-associated factor (TRAF) 6 is essential for MyD88-dependent pathway but not toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta (TRIF)-dependent pathway in TLR signaling

Signaling pathways from TLRs are mediated by the Toll/IL-1R (TIR) domain-containing adaptor molecules. TNF receptor-associated factor (TRAF) 6 is thought to activate NF-kappaB and MAPKs downstream of these TIR domain-containing proteins to induce production of inflammatory cytokines. However, the precise role of TRAF6 in signaling from individual TLRs has not been appropriately addressed. We analyzed macrophages from TRAF6-deficient mice and made the following observations. In the absence of TRAF6, 1) ligands for TLR2, TLR5, TLR7, and TLR9 failed to induce activation of NF-kappaB and MAPKs or production of inflammatory cytokines; 2) TLR4 ligand-induced cytokine production was remarkably reduced and activation of NF-kappaB and MAPKs was observed, albeit with delayed kinetics; and 3) in contrast with previously reported findings, TLR3 signaling was not affected. These results indicate that TRAF6 is essential for MyD88-dependent signaling but is not required for TIR domain-containing adaptor-inducing IFN-beta (TRIF)-dependent signaling.     

Syntenin negatively regulates TRAF6-mediated IL-1R/TLR4 signaling

Toll-like receptors are involved in host defense against invading pathogens. The two members of this superfamily, IL-1R and TLR4, activate overlapping NF-kappaB activate signaling pathway mediated by TRAF6. In this study, we identified syntenin as a negative regulator of IL-1R and TLR4 mediated NF-kappaB activation. Overexpressed syntenin inhibited IL-1- or LPS-, but not TNF- induced NF-kappaB activation and IL-8 mRNA expression in a dose dependent manner. Syntenin specifically interacted with TRAF6 in human 293 cells, and inhibited TRAF6 induced NF-kappaB and AP-1 activation. Syntenin also associated with TRAF6 under physiological condition, and dissociated from TRAF6 upon IL-1 stimulation. This might be due to a competition between syntenin and IRAK1, as overexpression of IRAK1 disrupted the interaction of syntenin with TRAF6, and rescued syntenin induced reduction of TRAF6 ubiquitination. Moreover, knockdown of syntenin potentiated IL-1- or LPS- triggered NF-kappaB activation and IL-8 mRNA expression. These findings suggest that syntenin is a physiological suppressor of TRAF6 and plays an inhibitory role in IL-1R- and TLR4- mediated NF-kappaB activation pathways.     

Roles of N-linked glycans in the recognition of microbial lipopeptides and lipoproteins by TLR2

Details of roles of carbohydrates attached to Toll-like receptors (TLRs) in the recognition of pathogen-associated molecular patterns and in the formation of the functional receptor complex still remain unknown. This study was designed to determine whether the glycans linked at Asn114, Asn199, Asn414 and Asn442 residues of TLR2 ectodomain were involved in the recognition of diacylated lipopeptide and lipoprotein. Single and multiple mutants were transfected into human embryonic kidney (HEK) 293 cells together with a NF-kappaB luciferase reporter plasmid. All of these mutants were expressed on the surface. SDS-PAGE of the transfectants demonstrated that these mutants migrated lower than wild-type TLR2 and their molecular masses decreased as the number of mutated Asn residues increased. TLR2(N114A), TLR2(N199A) and TLR2(N414A) as well as wild-type TLR2 induced NF-kappaB activation when stimulated with these ligands, whereas TLR2(N442A) failed to induce NF-kappaB activation. All of triple and quadruple mutants failed to induce NF-kappaB activation, but were associated with both wild-type TLR2 and TLR6 in the transfectants. TLR2(N114A,N199A), TLR2(N114A,N414A) and, to a lesser extent, TLR2(N114A,N442A), in which two N-linked glycans are speculated to be exposed to the concave surface of TLR2 solenoid, not only induce NF-kappaB activation but also are associated with wild-type TLR2 and TLR6. These results suggest that the glycan at Asn442 and at least two N-linked glycans speculated to be exposed to the concave surface of TLR2 solenoid are involved in the recognition of ligands by TLR2 and/or in formation or maturation of a functional TLR2 receptor complex.     

Lipoproteins are critical TLR2 activating toxins in group B streptococcal sepsis

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Deltalgt and the Deltalsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Deltalgt and Deltalsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Deltalgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Deltalgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice

The Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor-alpha and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2(-/-)) or TLR1 (TLR1(-/-)) produced low titers of antibodies against OspA. Notably, macrophages from TLR2(-/-) mice were unresponsive to OspA and PGN, whereas those from TLR1(-/-) mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.     

Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and TLR4 agonists in murine macrophages: effects of TLR "homotolerance" versus "heterotolerance" on NF-kappa B signaling pathway components

In this study, tolerance induction by preexposure of murine macrophages to Toll-like receptor (TLR)2 and TLR4 agonists was revisited, focusing on the major signaling components associated with NF-kappaB activation. Pretreatment of macrophages with a pure TLR4 agonist (protein-free Escherichia coli (Ec) LPS) or with TLR2 agonists (Porphyromonas gingivalis LPS or synthetic lipoprotein Pam3Cys) led to suppression of TNF-alpha secretion, IL-1R-associated kinase-1, and IkappaB kinase (IKK) kinase activities, c-jun N-terminal kinase, and extracellular signal-regulated kinase phosphorylation, and to suppression of NF-kappaB DNA binding and transactivation upon challenge with the same agonist (TLR4 or TLR2 "homotolerance," respectively). Despite inhibited NF-kappaB DNA binding, increased levels of nuclear NF-kappaB were detected in agonist-pretreated macrophages. For all the intermediate signaling elements, heterotolerance was weaker than TLR4 or TLR2 homotolerance with the exception of IKK kinase activity. IKK kinase activity was unperturbed in heterotolerance. TNF-alpha secretion was also suppressed in P. gingivalis LPS-pretreated, Ec LPS-challenged cells, but not vice versa, while Pam3Cys and Ec LPS did not induce a state of cross-tolerance at the level of TNF-alpha. Experiments designed to elucidate novel mechanisms of NF-kappaB inhibition in tolerized cells revealed the potential contribution of IkappaBepsilon and IkappaBxi inhibitory proteins and the necessity of TLR4 engagement for induction of tolerance to Toll receptor-IL-1R domain-containing adapter protein/MyD88-adapter-like-dependent gene expression. Collectively, these data demonstrate that induction of homotolerance affects a broader spectrum of signaling components than in heterotolerance, with selective modulation of specific elements within the NF-kappaB signaling pathway.     

Immune activation of type I IFNs by Listeria monocytogenes occurs independently of TLR4, TLR2, and receptor interacting protein 2 but involves TNFR-associated NF kappa B kinase-binding kinase 1

Type I IFNs are well established antiviral cytokines that have also been shown to be induced by bacteria. However, the signaling mechanisms regulating the activation of these cytokines during bacterial infections remain poorly defined. We show that although Gram-negative bacteria can activate the type I IFN pathway through TLR4, the intracellular Gram-positive bacterium Listeria monocytogenes (LM) can do so independently of TLR4 and TLR2. Furthermore, experiments using genetic mutants and chemical inhibitors suggest that LM-induced type I IFN activation occurs by an intracellular pathway involving the serine-threonine kinase TNFR-associated NF-kappaB kinase (TANK)-binding kinase 1 (TBK1). Interestingly, receptor-interacting protein 2, a component of the recently discovered nucleotide-binding oligomerization domain-dependent intracellular detection pathway, was not involved. Taken together, our data describe a novel signal transduction pathway involving TBK1 that is used by LM to activate type I IFNs. Additionally, we provide evidence that both the LM- and TLR-dependent pathways converge at TBK1 to activate type I IFNs, highlighting the central role of this molecule in modulating type I IFNs in host defense and disease.     

Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products

Toll-like receptors (TLRs) 2 and 4 are signal transducers for lipopolysaccharide, the major proinflammatory constituent in the outer membrane of Gram-negative bacteria. We observed that membrane lipoproteins/lipopeptides from Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans activated cells heterologously expressing TLR2 but not those expressing TLR1 or TLR4. These TLR2-expressing cells were also stimulated by living motile B. burgdorferi, suggesting that TLR2 recognition of lipoproteins is relevant to natural Borrelia infection. Importantly, a TLR2 antibody inhibited bacterial lipoprotein/lipopeptide-induced tumor necrosis factor release from human peripheral blood mononuclear cells, and TLR2-null Chinese hamster macrophages were insensitive to lipoprotein/lipopeptide challenge. The data suggest a role for the native protein in cellular activation by these ligands. In addition, TLR2-dependent responses were seen using whole Mycobacterium avium and Staphylococcus aureus, demonstrating that this receptor can function as a signal transducer for a wide spectrum of bacterial products. We conclude that diverse pathogens activate cells through TLR2 and propose that this molecule is a central pattern recognition receptor in host immune responses to microbial invasion.     

Bacterial lipoprotein TLR2 agonists broadly modulate endothelial function and coagulation pathways in vitro and in vivo

TLR2 activation induces cellular and organ inflammation and affects lung function. Because deranged endothelial function and coagulation pathways contribute to sepsis-induced organ failure, we studied the effects of bacterial lipoprotein TLR2 agonists, including peptidoglycan-associated lipoprotein, Pam3Cys, and murein lipoprotein, on endothelial function and coagulation pathways in vitro and in vivo. TLR2 agonist treatment induced diverse human endothelial cells to produce IL-6 and IL-8 and to express E-selectin on their surface, including HUVEC, human lung microvascular endothelial cells, and human coronary artery endothelial cells. Treatment of HUVEC with TLR2 agonists caused increased monolayer permeability and had multiple coagulation effects, including increased production of plasminogen activator inhibitor-1 (PAI-1) and tissue factor, as well as decreased production of tissue plasminogen activator and tissue factor pathway inhibitor. TLR2 agonist treatment also increased HUVEC expression of TLR2 itself. Peptidoglycan-associated lipoprotein induced IL-6 production by endothelial cells from wild-type mice but not from TLR2 knockout mice, indicating TLR2 specificity. Mice were challenged with TLR2 agonists, and lungs and plasmas were assessed for markers of leukocyte trafficking and coagulopathy. Wild-type mice, but not TLR2 mice, that were challenged i.v. with TLR2 agonists had increased lung levels of myeloperoxidase and mRNAs for E-selectin, P-selectin, and MCP-1, and they had increased plasma PAI-1 and E-selectin levels. Intratracheally administered TLR2 agonist caused increased lung fibrin levels. These studies show that TLR2 activation by bacterial lipoproteins broadly affects endothelial function and coagulation pathways, suggesting that TLR2 activation contributes in multiple ways to endothelial activation, coagulopathy, and vascular leakage in sepsis.     

Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex

Bacterial lipoproteins are the most potent microbial agonists for the Toll-like receptor 2 (TLR2) subfamily, and this pattern recognition event induces cellular activation, leading to host immune responses. Triacylated bacterial lipoproteins coordinately bind TLR1 and TLR2, resulting in a stable ternary complex that drives intracellular signaling. The sensitivity of TLR-expressing cells to lipoproteins is greatly enhanced by two lipid-binding serum proteins known as lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14); however, the physical mechanism that underlies this increased sensitivity is not known. To address this, we measured the ability of LBP and sCD14 to drive ternary complex formation between soluble extracellular domains of TLR1 and TLR2 and a synthetic triacylated lipopeptide agonist. Importantly, addition of substoichiometric amounts of either LBP or sCD14 significantly enhanced formation of a TLR1·TLR2 lipopeptide ternary complex as measured by size exclusion chromatography. However, neither LBP nor sCD14 was physically associated with the final ternary complex. Similar results were obtained using outer surface protein A (OspA), a naturally occurring triacylated lipoprotein agonist from Borrelia burgdorferi. Activation studies revealed that either LBP or sCD14 sensitized TLR-expressing cells to nanogram levels of either the synthetic lipopeptide or OspA lipoprotein agonist. Together, our results show that either LBP or sCD14 can drive ternary complex formation and TLR activation by acting as mobile carriers of triacylated lipopeptides or lipoproteins.     

Lipopolysaccharide activates NF-kappaB by TLR4-Bcl10-dependent and independent pathways in colonic epithelial cells

In colonic epithelium, one of the pathways of lipopolysaccharide (LPS) activation of NF-kappaB and IL-8 is via Toll-like receptor (TLR)4, MyD88, IRAK1/4, and B-cell CLL/lymphoma 10 (Bcl10). However, this innate immune pathway accounts for only approximately 50% of the NF-kappaB activation, so additional mechanisms to explain the LPS-induced effects are required. In this report, we identify a second pathway of LPS-induced stimulation, mediated by reactive oxygen species (ROS), in human colonic epithelial tissue cells in tissue culture and in ex vivo mouse colonic tissue. Measurements of IL-8, KC, Bcl10, phospho-IkappaBalpha, nuclear NF-kappaB, and phosphorylated Hsp27 were performed by ELISA. The TLR4-Bcl10 pathway was inhibited by Bcl10 siRNA and in studies with colonic tissue from the TLR4-deficient mouse. The ROS pathway was inhibited by Tempol, a free radical scavenger, or by okadaic acid, an inhibitor of Hsp27 dephosphorylation by protein phosphatase 2A (PP2A). The ROS pathway was unaffected in the TLR4-deficient tissue or by silencing of Bcl10. The combination of exposure to the free radical scavenger Tempol and of TLR4 or Bcl10 suppression was required to completely inhibit the LPS-induced activation. The ROS pathway was associated with dephosphorylation of Hsp27. LPS appears to activate both the regulatory component of the IkappaBalpha-kinase (IKK) signalosome through Bcl10 interaction with Nemo (IKKgamma) and the catalytic component through Hsp27 interaction with IKKbeta. Since LPS exposure is associated with septic shock and the systemic inflammatory response syndrome, distinguishing between these two pathways of LPS activation may facilitate new approaches to prevention and treatment.