Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.     

Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii

GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-alpha production by macrophages exposed to T. gondii GPIs. Importantly, TNF-alpha response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88(-/-) mice were more susceptible to death than wild-type (WT), TLR2(-/-), TLR4(-/-), TLR2/4(-/-), and CD14(-/-) mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4(-/-), but not TLR2(-/-), TLR4(-/-), and CD14(-/-), mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-gamma production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2(-/-) and TLR2/4(-/-) mice produced more IFN-gamma than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.     

Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses

Leukocyte responsiveness to LPS is dependent upon CD14 and receptors of the Toll-like receptor (TLR) family. Neutrophils respond to LPS, but conflicting data exist regarding LPS responses of eosinophils and basophils, and expression of TLRs at the protein level in these granulocyte lineages has not been fully described. We examined the expression of TLR2, TLR4, and CD14 and found that monocytes expressed relatively high levels of cell surface TLR2, TLR4, and CD14, while neutrophils also expressed all three molecules, but at low levels. In contrast, basophils expressed TLR2 and TLR4 but not CD14, while eosinophils expressed none of these proteins. Tested in a range of functional assays including L-selectin shedding, CD11b up-regulation, IL-8 mRNA generation, and cell survival, neutrophils responded to LPS, but eosinophils and basophils did not. In contrast to previous data, we found, using monocyte depletion by negative magnetic selection, that neutrophil responses to LPS were heavily dependent upon the presence of a very low level of monocytes, and neutrophil survival induced by LPS at 22 h was monocyte dependent. We conclude that LPS has little role in the regulation of peripheral blood eosinophil and basophil function, and that, even in neutrophils, monocytes orchestrate many previously observed leukocyte LPS response patterns.     

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.     

Type 1 fimbriae deliver an LPS- and TLR4-dependent activation signal to CD14-negative cells

Fimbriae target bacteria to different mucosal surfaces and enhance the inflammatory response at these sites. Inflammation may be triggered by the fimbriae themselves or by fimbriae-dependent delivery of other host activating molecules such as lipopolysaccharide (LPS). Although LPS activates systemic inflammation through the CD14 and Toll-like receptor 4 (TLR4) pathways, mechanisms of epithelial cell activation by LPS are not well understood. These cells lack CD14 receptors and are unresponsive to pure LPS, but fimbriated Escherichia coli overcome this refractoriness and trigger epithelial cytokine responses. We now show that type 1 fimbriae can present an LPS- and TLR4-dependent signal to the CD14-negative epithelial cells. Human uroepithelial cells were shown to express TLR4, and type 1 fimbriated E. coli strains triggered an LPS-dependent response in those cells. A similar LPS- and fimbriae-dependent response was observed in the urinary tract of TLR4-proficient mice, but not in TLR4-defective mice. The moderate inflammatory response in the TLR4-defective mice was fimbriae dependent but LPS independent. The results demonstrate that type 1 fimbriae present LPS to CD14-negative cells and that the TLR4 genotype determines this response despite the absence of CD14 on the target cells. The results illustrate how the host "sees" LPS and other microbial products not as purified molecules but as complexes, and that fimbriae determine the molecular context in which LPS is presented to host cells.     

Role for CD14, TLR2, and TLR4 in bacterial product-induced anorexia

The cell surface component CD14 and the toll-like receptors 2 and 4 (TLR2 and TLR4) are important in mediating the immune responses to bacterial products in mammals. Using mice genetically deficient in CD14, TLR2, or TLR4, we studied the role of these molecules in the anorectic effects of LPS and muramyl dipeptide (MDP). CD14 or TLR2 knockout (KO) and TLR4-deficient (TLR4-DEF) mice as well as corresponding wild-type (WT) colittermates were injected intraperitoneally at dark onset with LPS (2 microg/mouse), MDP (10 mg/kg), interleukin-1 beta (IL-1 beta, 150 ng/mouse), or vehicle, and food intake was recorded. LPS and MDP reduced food intake in WT mice of all genotypes tested. The anorectic effect of LPS was attenuated (P < 0.04) in CD14-KO and TLR4-DEF mice but not in TLR2-KO (P > 0.05). The anorectic effect of MDP was blunted in CD14-KO and TLR2-KO (P < 0.02) mice but not in TLR4-DEF mice. IL-1 beta reduced food intake similarly in all genotypes tested. These results indicate that CD14 is involved in mediating the anorectic effects of both LPS and MDP. Furthermore, TLR4 and TLR2 are specifically involved in mediating the anorectic effects of LPS and MDP, respectively. The results are consistent with the hypothesis that TLR4 functions as the true LPS receptor and that TLR2 is involved in recognition of gram-positive bacterial products.     

Role of TLR in B cell development: signaling through TLR4 promotes B cell maturation and is inhibited by TLR2

The role of TLR4 in mature B cell activation is well characterized. However, little is known about TLR4 role in B cell development. Here, we analyzed the effects of TLR4 and TLR2 agonists on B cell development using an in vitro model of B cell maturation. Highly purified B220(+)IgM(-) B cell precursors from normal C57BL/6 mouse were cultured for 72 h, and B cell maturation in the presence of the TLR agonists was evaluated by expression of IgM, IgD, CD23, and AA4. The addition of LPS or lipid A resulted in a marked increase in the percentage of CD23(+) B cells, while Pam3Cys had no effect alone, but inhibited the increase of CD23(+) B cell population induced by lipid A or LPS. The TLR4-induced expression of CD23 is not accompanied by full activation of the lymphocyte, as suggested by the absence of activation Ag CD69. Experiments with TLR2-knockout mice confirmed that the inhibitory effects of Pam3Cys depend on the expression of TLR2. We studied the effects of TLR-agonists on early steps of B cell differentiation by analyzing IL-7 responsiveness and phenotype of early B cell precursors: we found that both lipid A and Pam3Cys impaired IL-7-dependent proliferation; however, while lipid A up-regulates B220 surface marker, consistent with a more mature phenotype of the IgM(-) precursors, Pam3Cys keeps the precursors on a more immature stage. Taken together, our results suggest that TLR4 signaling favors B lymphocyte maturation, whereas TLR2 arrests/retards that process, ascribing new roles for TLRs in B cell physiology.     

Toll-like receptor 4, but not toll-like receptor 2, is a signaling receptor for Escherichia and Salmonella lipopolysaccharides

Two members of the mammalian Toll-like receptor (TLR) family, TLR2 and TLR4, have been implicated as receptors mediating cellular activation in response to bacterial LPS. Through the use of mAbs raised against human TLR2 and TLR4, we have conducted studies in human cell lines and whole blood to ascertain the relative contribution of these receptors to LPS induced cytokine release. We show that the contribution of TLR2 and TLR4 to LPS-induced cellular activation correlates with the relative expression levels of these two TLRs in a given cell type. In addition, we have found that significant differences in cell stimulatory activity exist between various smooth and rough LPS types that cannot be ascribed to known LPS structural features. These results suggest that impurities in the LPS may be responsible for some of the activity and this would be in agreement with recently published results of others. Upon repurification, none of the commercial LPS preparations activate cells through TLR2, but continue to stimulate cells with comparable activity through TLR4. Our results confirm recent findings that TLR4, but not TLR2, mediates cellular activation in response to LPS derived from both Escherichia coli and Salmonella minnesota. Additionally, we show that TLR4 is the predominant signaling receptor for LPS in human whole blood.     

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.     

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.     

Beta2-integrin-induced p38 MAPK activation is a key mediator in the CD14/TLR4/MD2-dependent uptake of lipopolysaccharide by hepatocytes

The liver is the main organ that clears circulating lipopolysaccharide (LPS), and hepatocytes are a major cell type involved in LPS uptake. Little is known about the mechanisms for LPS internalization in hepatocytes and what signaling pathways are involved. We show here that LPS uptake is initiated after formation of a multi-receptor complex within lipid rafts. We find that essential components for LPS uptake are CD14, TLR4, MD2, and the beta2-integrin CD11b/CD18. Activation of p38 MAPK is also essential for the initiation of LPS uptake, and interestingly, we show that this activation is not through TLR4 signaling by MyD88 but through activation of TIRAP via CD11b/CD18. However, TLR4/MD2 remain essential components at the cell surface as part of the LPS receptor complex. We therefore suggest novel roles for TLR4/MD2, CD11b/CD18, TIRAP, and p38 MAPK in LPS uptake by hepatocytes.     

Stimulation of toll-like receptor 2 by Coxiella burnetii is required for macrophage production of pro-inflammatory cytokines and resistance to infection

Innate and adaptive immune responses are initiated upon recognition of microbial molecules by Toll-like receptors (TLRs). We have investigated the importance of these receptors in the induction of pro-inflammatory cytokines and macrophage resistance to infection with Coxiella burnetii, an obligate intracellular bacterium and the etiological agent of Q fever. By using a Chinese hamster ovary/CD14 cell line expressing either functional TLR2 or TLR4, we determined that C. burnetii phase II activates TLR2 but not TLR4. Macrophages deficient for TLR2, but not TLR4, produced less tumor necrosis factor-alpha and interleukin-12 upon C. burnetii infection. Furthermore, it was found that TLR2 activation interfered with C. burnetii intracellular replication, as macrophages from TLR2-deficient mice were highly permissive for C. burnetii growth compared with macrophages from wild type mice or TLR4-deficient mice. Although LPS modifications distinguish virulent C. burnetii phase I bacteria from avirulent phase II organisms, electrospray ionization-mass spectrometry analysis showed that the lipid A moieties isolated from these two phase variants are identical. Purified lipid A derived from either phase I or phase II LPS failed to activate TLR2 and TLR4. Indeed, the lipid A molecules were able to interfere with TLR4 signaling in response to purified Escherichia coli LPS. These studies indicate that TLR2 is an important host determinant that mediates recognition of C. burnetii and a response that limits growth of this intracellular pathogen.     

Host species-specific usage of the TLR4-LPS receptor complex

Recognition of LPS depends on the interaction of at least three molecules forming the LPS-receptor complex. The most important ones, CD14, MD2 and Toll-like receptor (TLR) 4 share a high degree of homology between species. In the present study, we investigated the importance of species-specific restriction on the recognition of LPS using stably transfected HEK293 cell lines expressing either human or bovine LPS-receptor complex components. Species-specific MD2 appeared to confer LPS recognition, whereas species-specific CD14 only appeared to play a minor role. In addition to the recognition of LPS, there is evidence that the fusion (F) protein of respiratory syncytial virus (RSV), which is the most common viral respiratory pathogen during infancy world-wide, interacts with TLR4, and plays an important role in the initiation of the innate immune response. Our findings suggest that human and bovine RSV may activate human and bovine TLR4 receptors, respectively, in the presence of both MD2 and CD14. However, no clear role for the RSV F protein of either human or bovine RSV alone in stimulating TLR4-dependent NF-kappaB activation was observed.     

MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor.

The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins, CD14, TLR4, and MD-2. Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4. We demonstrate that MD-2 contains 2 N-glycosylated sites at positions Asn(26) and Asn(114), whereas the amino-terminal ectodomain of human TLR4 contains 9 N-linked glycosylation sites. Site-directed mutagenesis studies showed that cell surface expression of MD-2 did not depend on the presence of either N-linked site, whereas in contrast, TLR4 mutants carrying substitutions in Asn(526) or Asn(575) failed to be transported to the cell surface. Using a UV-activated derivative of Re595 LPS (ASD-Re595 LPS) in cross-linking assays, we demonstrated a critical role of MD-2 and TLR4 carbohydrates in LPS cross-linking to the LPS receptor. The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells. The double mutant of MD-2 failed to support LPS-induced activation of an interleukin-8 (IL-8) promoter-driven luciferase reporter to induce IL-8 secretion or to activate amino-terminal c-Jun kinase (JNK). Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites. Surprisingly, the reduction in activation resulting from expression of the Asn mutants of MD-2 and TLR4 can be partially reversed by co-expression with CD14. This suggests that the functional integrity of the LPS receptor depends both on the surface expression of at least three proteins, CD14, MD-2, and TLR4, and that N-linked sites of both MD-2 and TLR4 are essential in maintaining the functional integrity of this receptor.     

The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable haemophilus influenzae from the mouse lung

TLRs are important for the recognition of conserved motifs expressed by invading bacteria. TLR4 is the signaling receptor for LPS, the major proinflammatory component of the Gram-negative cell wall, whereas CD14 serves as the ligand-binding part of the LPS receptor complex. Triggering of TLR4 results in the activation of two distinct intracellular pathways, one that relies on the common TLR adaptor MyD88 and one that is mediated by Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF). Nontypeable Haemophilus influenzae (NTHi) is a common Gram-negative respiratory pathogen that expresses both TLR4 (LPS and lipooligosaccharide) and TLR2 (lipoproteins) ligands. To determine the roles of CD14, TLR4, and TLR2 during NTHi pneumonia, the following studies were performed: 1) Alveolar macrophages from CD14 and TLR4 knockout (KO) mice were virtually unresponsive to NTHi in vitro, whereas TLR2 KO macrophages displayed a reduced NTHi responsiveness. 2) After intranasal infection with NTHi, CD14 and TLR4 KO mice showed an attenuated early inflammatory response in their lungs, which was associated with a strongly reduced clearance of NTHi from the respiratory tract; in contrast, in TLR2 KO mice, lung inflammation was unchanged, and the number of NTHi CFU was only modestly increased at the end of the 10-day observation period. 3) MyD88 KO, but not TRIF mutant mice showed an increased bacterial load in their lungs upon infection with NTHi. These data suggest that the MyD88-dependent pathway of TLR4 is important for an effective innate immune response to respiratory tract infection caused by NTHi.     

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.     

The lipopolysaccharide-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2

We analysed the lipopolysaccharide (LPS)-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2. When TLR4 and CD14 were transiently expressed in HEK293 cells, cell-surface expression of TLR4 was observed, although the expression level was lower than that in cells coexpressing MD-2. We found that membrane CD14-TLR4 complexes were formed in these cells in response to LPS stimulation even in the absence of MD-2 expression, although NF-kappaB-dependent reporter activity was not induced. A strong activation of NF-kappaB was observed when these cells were stimulated with LPS followed by soluble MD-2 in this order, even when excess LPS was removed after formation of the CD14-TLR4 complex by washing cells prior to sMD-2 addition. From these results, we propose an additional LPS-recognition mechanism. In cells expressing TLR4 and CD14 but lacking MD-2, LPS is first transferred to membrane CD14 with the aid of LPS binding protein, which leads to the formation of the TLR4-CD14 complex. Then, the binding of soluble MD-2 to this complex triggers the transmembrane signal transduction. Cells expressing TLR4 and CD14 but lacking MD-2, such as airway epithelial cells, may be activated in response to LPS by this mechanism.     

Elucidation of the MD-2/TLR4 interface required for signaling by lipid IVa

LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57-66 and 82-89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure (Cell 130: 1071-1082) of the ligand-bound TLR1/2 ectodomain heterodimer.