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.     

The CD14 region spanning amino acids 57-64 is critical for interaction with the extracellular Toll-like receptor 2 domain

CD14 has been shown to enhance Toll-like receptor 2 (TLR2)-mediated signaling in response to peptidoglycan. Anti-CD14 monoclonal antibody MEM-18, whose epitope was located at the amino acid residues 57-64, blocked the binding of sCD14 to the recombinant soluble form of the extracellular TLR2 domain (sTLR2). The deletion mutant sCD14Delta57-64 lacking the amino acid residues 57-64 failed to bind to sTLR2. Cotransfection of wild type mCD14 but not mCD14Delta57-64 with TLR2 enhanced NF-kappaB activation in response to peptidoglycan. These results indicate that the CD14 region spanning amino acids 57-64 is critical for interacting with TLR2 and enhancing TLR2-mediated peptidoglycan signaling.     

Oxidized phospholipid inhibition of toll-like receptor (TLR) signaling is restricted to TLR2 and TLR4: roles for CD14, LPS-binding protein, and MD2 as targets for specificity of inhibition

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysaccharide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor-alpha production, IkappaBalpha degradation, p38 MAPK phosphorylation, and NF-kappaB-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I.C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from approximately 30 microm. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.     

MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex

We have demonstrated previously that tetra-acylated LPS derived from the oral bacterium, Porphyromonas gingivalis, and penta-acylated msbB LPS derived from a mutant strain of Escherichia coli can antagonize the ability of canonical hexa-acylated E. coli LPS to signal through the TLR4 signaling complex in human endothelial cells. Activation of the TLR4 signaling complex requires the coordinated function of LPS binding protein (LBP), CD14, MD-2, and TLR4. To elucidate the specific molecular components that mediate antagonism, we developed a recombinant human TLR4 signaling complex that displayed efficient LPS-dependent antagonism of E. coli LPS in HEK293 cells. Notably, changes in the expression levels of TLR4 in HEK293 cells modulated the efficiency of antagonism by P. gingivalis LPS. Both soluble (s) CD14 and membrane (m) CD14 supported efficient P. gingivalis LPS-dependent and msbB LPS-dependent antagonism of E. coli LPS in the recombinant TLR4 system. When cells expressing TLR4, MD-2, and mCD14 were exposed to LPS in the absence of serum-derived LBP, efficient LPS-dependent antagonism of E. coli LPS was still observed indicating that LPS-dependent antagonism occurs downstream of LBP. Experiments using immunoprecipitates of sCD14 or sMD-2 that had been pre-exposed to agonist and antagonist indicated that LPS-dependent antagonism occurs partially at sCD14 and potently at sMD-2. This study provides novel evidence that expression levels of TLR4 can modulate the efficiency of LPS-dependent antagonism. However, MD-2 represents the principal molecular component that tetra-acylated P. gingivalis LPS and penta-acylated msbB LPS use to antagonize hexa-acylated E. coli LPS at the TLR4 signaling complex.     

Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines.

Members of the Toll-like receptor (TLR) family mediate dorsoventral patterning and cellular adhesion in insects as well as immune responses to microbial products in both insects and mammals. TLRs are characterized by extracellular leucine-rich repeat domains and an intracellular signaling domain that shares homology with cytoplasmic sequences of the mammalian IL-1 receptor and plant disease resistance genes. Ten human TLRs have been cloned as well as RP105, a protein similar to TLR4 but lacking the intracellular signaling domain. However, only five TLRs have described functions as receptors for bacterial products (e.g., LPS, lipoproteins). To identify potential sites of action, we used quantitative real-time RT-PCR to examine systematically the expression of mRNAs encoding all known human TLRs, RP105, and several other proteins important in TLR functions (e.g., MD-1, MD-2, CD14, MyD88). Most tissues tested expressed at least one TLR, and several expressed all (spleen, peripheral blood leukocytes). Analysis of TLR expression in fractionated primary human leukocytes (CD4(+), CD8(+), CD19(+), monocytes, and granulocytes) indicates that professional phagocytes express the greatest variety of TLR mRNAs although several TLRs appear more restricted to B cells, suggesting additional roles for TLRs in adaptive immunity. Monocyte-like THP-1 cells regulate TLR mRNA levels in response to a variety of stimuli including phorbol esters, LPS, bacterial lipoproteins, live bacteria, and cytokines. Furthermore, addition of Escherichia coli to human blood ex vivo caused distinct changes in TLR expression, suggesting that important roles exist for these receptors in the establishment and resolution of infections and inflammation.     

The extracellular toll-like receptor 2 domain directly binds peptidoglycan derived from Staphylococcus aureus

Toll-like receptor 2 (TLR2) has been recognized to mediate cell signaling in response to peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria. The mechanism by which TLR2 recognizes PGN is unknown. It is not even clear whether TLR2 directly binds to PGN. In this study, we generated a soluble form of recombinant TLR2 (sTLR2) possessing only its putative extracellular domain by using the baculovirus expression system to examine the direct interaction between sTLR2 and PGN. sTLR2 bound avidly to insoluble PGN (iPGN) from Staphylococcus aureus coated onto microtiter wells in a concentration-dependent manner. In contrast, sTLR2 exhibited a very weak binding to lipopolysaccharide. iPGN cosedimented sTLR2 after the mixture of iPGN and sTLR2 had been incubated and centrifuged. sTLR2 partially attenuated the iPGN-induced NF-kappaB activation in TLR2-transfected HEK 293 cells and the iPGN-induced IL-8 secretion in U937 cells. One of anti-human TLR2 monoclonal antibodies, which blocked iPGN-induced NF-kappaB activation in TLR2-transfected cells, inhibited the binding of sTLR2 to iPGN. In addition, we found that sCD14 interacted with sTLR2 and increased the binding of sTLR2 to iPGN. From these results, we conclude that the extracellular TLR2 domain directly binds to PGN.     

Meningococcal porin PorB binds to TLR2 and requires TLR1 for signaling

TLR2 plays a key role in the initiation of the cellular innate immune responses by a wide range of bacterial products. TLRs signaling, including TLR2 and its coreceptors TLR1 and TLR6, is mediated by a number of specific ligands. Although many of the TLR-mediated cell signaling pathways have been elucidated in the past few years, the molecular mechanisms that lead to cell activation are still poorly understood. In this study, we investigate the interaction of PorB from Neisseria meningitidis with TLR2 and describe the direct binding of a bacterial protein to TLR2 for the first time. Using labeled PorB, we demonstrate its binding to TLR2 both in its soluble form in vitro, and when it is over-expressed on the surface of human embryonic kidney 293 cells. We also show that TLR2-mediated binding of PorB is directly related to cellular activation. In addition, using 293 cells expressing the chimeric TLR2/TLR1 and TLR2/TLR6 complexes, we report the selectivity of PorB binding to the TLR2/TLR1 heterodimer, which is required for initiating signaling in transfected 293 cells and in murine B cells. Together, these data provide new evidence that TLR2 recognizes PorB through direct binding, and that PorB-induced cell activation is mediated by a TLR2/TLR1 complex.     

Identification by surface plasmon resonance of the mycobacterial lipomannan and lipoarabinomannan domains involved in binding to CD14 and LPS-binding protein

The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), regulate host defence mechanisms through their interaction with pattern recognition receptors such as Toll-like receptors (TLRs). We have developed a surface plasmon resonance assay to analyse the molecular basis for the recognition of Mycobacterium kansasii LM or LAM, by immobilized CD14 and LPS-binding protein (LBP) both being capable to promote presentation of bacterial glycolipids to TLRs. The affinity of either LM/LAM was higher to CD14 than to LBP. Kinetic and Scatchard analyses were consistent with a model involving a single class of binding sites. These interactions required the lipidic anchor, but not the carbohydrate domains, of LM or LAM. We also provide evidence that addition of recombinant LBP enhanced the stimulatory effect of LM or LAM on matrix metalloproteinase-9 expression and secretion in macrophages, through a TLR1/TLR2-dependent mechanism.     

Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2.

Invasive infection with Gram-positive and Gram-negative bacteria often results in septic shock and death. The basis for the earliest steps in innate immune response to Gram-positive bacterial infection is poorly understood. The LPS component of the Gram-negative bacterial cell wall appears to activate cells via CD14 and Toll-like receptor (TLR) 2 and TLR4. We hypothesized that Gram-positive bacteria might also be recognized by TLRs. Heterologous expression of human TLR2, but not TLR4, in fibroblasts conferred responsiveness to Staphylococcus aureus and Streptococcus pneumoniae as evidenced by inducible translocation of NF-kappaB. CD14 coexpression synergistically enhanced TLR2-mediated activation. To determine which components of Gram-positive cell walls activate Toll proteins, we tested a soluble preparation of peptidoglycan prepared from S. aureus. Soluble peptidoglycan substituted for whole organisms. These data suggest that the similarity of clinical response to invasive infection by Gram-positive and Gram-negative bacteria is due to bacterial recognition via similar TLRs.     

Soluble forms of Toll-like receptor (TLR)2 capable of modulating TLR2 signaling are present in human plasma and breast milk

Dysregulation of the initial, innate immune response to bacterial infection may lead to septic shock and death. Toll-like receptors (TLRs) play a crucial role in this innate immune response, and yet the regulatory mechanisms controlling microbial-induced TLR triggering are still to be fully understood. We have therefore sought specific regulatory mechanisms that may modulate TLR signaling. In this study, we tested for the possible existence of a functionally active soluble form of TLR2. We demonstrated the existence of natural soluble forms of TLR2 (sTLR2), which we show to be capable of modulating cell activation. We found that blood monocytes released sTLR2 constitutively and that the kinetics of sTLR2 release increased upon cell activation. Analysis of cells expressing the human TLR2 cDNA or its c-myc-tagged version indicated that sTLR2 resulted from the posttranslational modification of the TLR2 protein in an intracellular compartment. Moreover, an intracellular pool of sTLR2 is maintained. sTLR2 was found naturally expressed in breast milk and plasma. Milk sTLR2 levels mirrored those of the TLR coreceptor soluble CD14. Depletion of sTLR2 from serum resulted in an increased cellular response to bacterial lipopeptide. Notably, serum sTLR2 was lower in tuberculosis patients. Coimmunoprecipitation experiments and computational molecular docking studies showed an interaction between sTLR2 and soluble CD14 in plasma and milk. These findings suggest the existence of a novel and specific innate immune mechanism regulating microbial-induced TLR triggering, and may lead to new therapeutics for the prevention and/or treatment of severe infectious diseases.     

Toll-like receptor 4 region Glu24-Lys47 is a site for MD-2 binding: importance of CYS29 and CYS40

Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS), but its interaction with MD-2 is required for efficient responses to LPS. Previous studies with deletion mutants indicate a critical role of the amino-terminal TLR4 region in interaction with MD-2. However, it is uncertain which region in the TLR4 molecule directly binds to MD-2. The purpose of this study was to determine a critical stretch of primary sequence in the TLR4 region that directly binds MD-2 and is critical for LPS signaling. The synthetic TLR4 peptide corresponding to the TLR4 region Glu(24)-Lys(47) directly binds to recombinant soluble MD-2 (sMD-2). The TLR4 peptide inhibited the binding of a recombinant soluble form of the extracellular TLR4 domain (sTLR4) to sMD-2 and significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild type TLR4-transfected cells. Reduction and S-carboxymethylation of sTLR4 abrogated its association with sMD-2. The TLR4 mutants, TLR4(C29A), TLR4(C40A), and TLR4(C29A,C40A), were neither co-precipitated with MD-2 nor expressed on the cell surface and failed to transmit LPS signaling. These results demonstrate that the TLR4 region Glu(24)-Lys(47) is a site for MD-2 binding and that Cys(29) and Cys(40) within this region are critical residues for MD-2 binding and LPS signaling.     

Differential interactions of fimbriae and lipopolysaccharide from Porphyromonas gingivalis with the Toll-like receptor 2-centred pattern recognition apparatus

The lipopolysaccharide (LPS) and fimbriae of Porphyromonas gingivalis play important roles in periodontal inflammation and pathogenesis. We investigated fimbriae and LPS from several P. gingivalis strains in terms of relative dependence on Toll-like receptor (TLR) signalling partners or accessory pattern-recognition molecules mediating ligand transfer to TLRs, and determined induced assembly of receptor complexes in lipid rafts. Fimbriae could utilize TLR1 or TLR6 for cooperative TLR2-dependent activation of transfected cell lines, in contrast to LPS and a mutant version of fimbriae which displayed preference for TLR1. Whether used to activate human cell lines or mouse macrophages, fimbriae exhibited strong dependence on membrane-expressed CD14 (mCD14), which could not be substituted for by soluble CD14 (sCD14). In contrast, sCD14 efficiently substituted for mCD14 in LPS-induced cellular activation. LPS-binding protein was more important for LPS- than for fimbria-induced cell activation, whereas the converse was true for CD11b/CD18. Cell activation by LPS or fimbriae required lipid raft function and formation of heterotypic receptor complexes (TLR1-2/CD14/CD11b/CD18), although wild-type fimbriae additionally recruited TLR6. In summary, TLR2 activation by P. gingivalis LPS or fimbriae involves differential dependence on accessory signalling or ligand-binding receptors, which may differentially influence innate immune responses.     

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.     

Response to Neisseria gonorrhoeae by cervicovaginal epithelial cells occurs in the absence of toll-like receptor 4-mediated signaling

Toll-like receptors (TLRs) have recently been identified as fundamental components of the innate immune response to bacterial pathogens. We investigated the role of TLR signaling in immune defense of the mucosal epithelial cells of the lower female genital tract. This site provides first line defense against microbial pathogens while remaining tolerant to a complex biosystem of resident microbiota. Epithelial cells derived from normal human vagina, ectocervix, and endocervix expressed mRNA for TLR1, -2, -3, -5, and -6. However, they failed to express TLR4 as well as MD2, two essential components of the receptor complex for LPS in phagocytes and endothelial cells. Consistent with this, endocervical epithelial cells were unresponsive to protein-free preparations of lipooligosaccharide from Neisseria gonorrhoeae and LPS from Escherichia coli. However, they were capable of responding to whole Gram-negative bacteria and bacterial lysates, as demonstrated by NF-kappaB activation and proinflammatory cytokine production. The presence of soluble CD14, a high-affinity receptor for LPS and other bacterial ligands, enhanced the sensitivity of genital tract epithelial cells to both low and high concentrations of bacteria, suggesting that soluble CD14 can act as a coreceptor for non-TLR4 ligands. These data demonstrate that the response to N. gonorrhoeae and other Gram-negative bacteria at the mucosal surface of the female genital tract occurs in the absence of endotoxin recognition and TLR4-mediated signaling.     

Membrane sorting of toll-like receptor (TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic associations with CD36 and intracellular targeting

Toll-like receptors (TLRs) are receptors of the innate immune system responsible for recognizing pathogen-associated molecular patterns. TLR2 seems to be the most promiscuous TLR receptor able to recognize the most diverse set of pathogen-associated patterns. Its promiscuity has been attributed to its unique ability to heterodimerize with TLRs 1 and 6 and, most recently, to its association with CD36 in response to diacylated lipoproteins. Thus, it seems that TLR2 forms receptor clusters in response to different microbial ligands. In this study we investigated TLR2 cell surface heterotypic interactions in response to different ligands as well as internalization and intracellular trafficking. Our data show that TLR2 forms heterodimers with TLR1 and TLR6 and that these heterodimer pre-exist and are not induced by the ligand. Upon stimulation by the specific ligand, these heterodimers are recruited within lipid rafts. In contrast, heterotypic associations of TLR2/6 with CD36 are not preformed and are ligand-induced. All TLR2 receptor clusters accumulate in lipid rafts and are targeted to the Golgi apparatus. This localization and targeting is ligand-specific. Activation occurs at the cell surface, and the observed trafficking is independent of signaling.     

TLR9 is localized in the endoplasmic reticulum prior to stimulation

In mammals, 10 TLRs recognize conserved pathogen-associated molecular patterns, resulting in the induction of inflammatory innate immune responses. One of these, TLR9, is activated intracellularly by bacterial DNA and synthetic oligodeoxynucleotides (ODN), containing unmethylated CpG dinucleotides. Following treatment with CpG ODN, TLR9 is found in lysosome-associated membrane protein type 1-positive lysosomes, and we asked which intracellular compartment contains TLR9 before CpG exposure. Surprisingly, we found by microscopy and supporting biochemical evidence that both transfected and endogenously expressed human TLR9 is retained in the endoplasmic reticulum. By contrast, human TLR4 trafficked to the cell surface, indicating that endoplasmic reticulum retention is not a property common to all TLRs. Because TLR9 is observed in endocytic vesicles following exposure to CpG ODN, our data indicate that a special mechanism must exist for translocating TLR9 to the signaling compartments that contain the CpG DNA.     

The scaffold MyD88 acts to couple protein kinase Cepsilon to Toll-like receptors

Mice lacking protein kinase Cepsilon (PKCepsilon) are hypersensitive to both Gram-positive and Gram-negative bacterial infections; however, the mechanism of PKCepsilon coupling to the Toll-like receptors (TLRs), responsible for pathogen detection, is poorly understood. Here we sought to investigate the mechanism of PKCepsilon involvement in TLR signaling and found that PKCepsilon is recruited to TLR4 and phosphorylated on two recently identified sites in response to lipopolysaccharide (LPS) stimulation. Phosphorylation at both of these sites (Ser-346 and Ser-368) resulted in PKCepsilon binding to 14-3-3beta. LPS-induced PKCepsilon phosphorylation, 14-3-3beta binding, and recruitment to TLR4 were all dependent on expression of the scaffold protein MyD88. In mouse embryo fibroblasts and activated macrophages from MyD88 knock-out mice, LPS-stimulated PKCepsilon phosphorylation was reduced compared with wild type cells. Acute knockdown of MyD88 in LPS-responsive 293 cells also resulted in complete loss of Ser-346 phosphorylation and TLR4/PKCepsilon association. By contrast, MyD88 overexpression in 293 cells resulted in constitutive phosphorylation of PKCepsilon. A general role for MyD88 was evidenced by the finding that phosphorylation of PKCepsilon was induced by the activation of all TLRs tested that signal through MyD88 (i.e. all except TLR3) both in RAW cells and in primary human macrophages. Functionally, it is established that phosphorylation of PKCepsilon at these two sites is required for TLR4- and TLR2-induced NFkappaB reporter activation and IkappaB degradation in reconstituted PKCepsilon(-/-) cells. This study therefore identifies the scaffold protein MyD88 as the link coupling TLRs to PKCepsilon recruitment, phosphorylation, and downstream signaling.     

Contributions of Unique Intracellular Domains to Switchlike Biosensing by Toll-like Receptor 4

Toll-like receptors (TLRs) mediate immune recognition of both microbial infections and tissue damage. Aberrant TLR signaling promotes disease; thus, understanding the regulation of TLR signaling is of medical relevance. Although downstream mediators of TLR signaling have been identified, the detailed mechanism by which ligand binding-mediated dimerization induces downstream signaling remains poorly understood. Here, we investigate this question for TLR4, which mediates responsiveness to bacterial LPS and drives inflammatory disease. TLR4 exhibits structural and functional features that are unique among TLRs, including responsiveness to a wide variety of ligands. However, the connection between these structural features and the regulation of signaling is not clear. Here, we investigated how the unique intracellular structures of TLR4 contribute to receptor signaling. Key conclusions include the following. 1) The unique intracellular linker of TLR4 is important for achieving LPS-inducible signaling via Toll/IL-1 receptor (TIR) domain-containing adapter-inducing interferon-β (TRIF) but less so for signaling via myeloid differentiation primary response 88 (MyD88). 2) Membrane-bound TLR4 TIR domains were sufficient to induce signaling. However, introducing long, flexible intracellular linkers neither induced constitutive signaling nor ablated LPS-inducible signaling. Thus, the initiation of TLR4 signaling is regulated by a mechanism that does not require tight geometric constraints. Together, these observations necessitate refining the model of TLR4 signal initiation. We hypothesize that TLR4 may interact with an inhibitory partner in the absence of ligand, via both TIR and extracellular domains of TLR4. In this speculative model, ligand binding induces dissociation of the inhibitory partner, triggering spontaneous, switchlike TIR domain homodimerization to initiate downstream signaling.     

Lipopolysaccharide and double-stranded RNA up-regulate toll-like receptor 2 independently of myeloid differentiation factor 88

Toll-like receptor 2 (TLR2) is a signaling receptor for a variety of microbial products, including bacterial lipoproteins and peptidoglycan, and is central in initiating immune responses toward Gram-positive bacteria, spirochetes, and mycobacteria. The mechanisms behind regulation of TLR2 protein expression are still not well understood. By using a newly developed monoclonal antibody against mouse TLR2, we detected TLR2 protein expression on macrophages, neutrophils, and dendritic cells. Endogenous macrophage TLR2 localized mostly to the cell membrane, with particular accumulation around phagosomes containing zymosan. Treatment of macrophages with the TLR2 antibody diminished cellular response to lipoproteins and down-regulated membrane TLR2. Marked up-regulation of surface TLR2 was observed on macrophages in response to whole bacteria, lipoproteins, lipopolysaccharide, poly(I-C) (double-stranded RNA), R848, and CpG DNA, and this up-regulation appeared to be a very sensitive marker for the presence of microbial products. Up-regulation of TLR2 in response to stimuli correlated with an increased response to secondary lipoprotein exposure following a low concentration of primary lipoprotein challenge. By comparison, exposure to a larger primary challenge induced a hyporeactive state. Most interestingly, lipopolysaccharide- and double-stranded RNA-induced up-regulation of surface TLR2 in macrophages was found to be MyD88-independent, whereas the up-regulation in response to lipoproteins, R848, and CpG DNA was absent in MyD88-deficient cells. We conclude that complex mechanisms regulate expression and signaling via TLR2. Up-regulation of TLR2 in the presence of low, yet clinically relevant amounts of microbial products may be an important mechanism by which the immune system boosts its response to a beginning infection.     

Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling

TLRs have been implicated in recognition of pathogen-associated molecular patterns. TLR4 is a signaling receptor for LPS, but requires MD-2 to respond efficiently to LPS. The purposes of this study were to examine the interactions of the extracellular TLR4 domain with MD-2 and LPS. We generated soluble forms of rTLR4 (sTLR4) and TLR2 (sTLR2) lacking the putative intracellular and transmembrane domains. sTLR4 consisted of Glu(24)-Lys(631). MD-2 bound to sTLR4, but not to sTLR2 or soluble CD14. BIAcore analysis demonstrated the direct binding of sTLR4 to MD-2 with a dissociation constant of K(D) = 6.29 x 10(-8) M. LPS-conjugated beads precipitated MD-2, but not sTLR4. However, LPS beads coprecipitated sTLR4 and MD-2 when both proteins were coincubated. The addition of sTLR4 to the medium containing the MD-2 protein significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild-type TLR4-expressing cells. These results indicate that the extracellular TLR4 domain-MD-2 complex is capable of binding LPS, and that the extracellular TLR4 domain consisting of Glu(24)-Lys(631) enables MD-2 binding and LPS recognition to TLR4. In addition, the use of sTLR4 may lead to a new therapeutic strategy for dampening endotoxin-induced inflammation.