Soluble MD-2 activity in plasma from patients with severe sepsis and septic shock

In this paper, we show that plasma from patients with severe sepsis and septic shock but not normal plasma supports lipopolysaccharide (LPS) activation of epithelial cells expressing Toll-like receptor 4 (TLR4). Recombinant soluble myeloid differentiation protein-2 (MD-2) complemented normal plasma and allowed LPS activation of epithelial cells to levels measured with "septic" plasma, whereas soluble MD-2-depleted plasma lost its effects. The same "MD-2 activity" was found in urine from a patient with septic shock and in lung edema fluids from patients with adult respiratory distress syndrome (ARDS). Recombinant soluble MD-2 enabled LPS-dependent activation of epithelial cells bearing TLR4. LPS-binding protein (LBP) and soluble CD14 increased the sensitivity of TLR4-expressing epithelial cells to LPS but were not able to mediate LPS activation of these cells in the absence of soluble MD-2. An anti-MD-2 monoclonal antibody blocked LPS activation of TLR4-expressing cells only in the presence of septic plasma or septic urine. These results suggest that septic plasma containing soluble MD-2 leaking into the extravascular space supports LPS activation of TLR4-expressing epithelial cells. We therefore propose that soluble MD-2 is an important mediator of organ inflammation during sepsis.     

Ligand-dependent Toll-like receptor 4 (TLR4)-oligomerization is directly linked with TLR4-signaling

Toll-like receptor 4 (TLR4) and MD-2 recognize lipid A, the active moiety of microbial lipopolysaccharide (LPS). Little is known about mechanisms for LPS recognition by TLR4/MD-2. We here showed, by using in vitro transfectants, ligand-induced TLR4-oligomerization, which required both membrane CD14 and MD-2. We previously reported that lipid IVa, a lipid A precursor, is agonistic on mouse TLR4/MD-2 but antagonistic on human TLR4/MD-2 and chimeric mouse TLR4/human MD-2. Lipid IVa triggered oligomerization of mouse TLR4/MD-2 but not human TLR4/MD-2 or chimeric mouse TLR4/human MD-2. Further, lipid IVa inhibited lipid A-dependent oligomerization of chimeric mouse TLR4/human MD-2. These results demonstrate that ligand-induced TLR4-oligomerization is directly linked with TLR4-signaling and suggest that MD-2 has an important role in regulating TLR4-oligomerization.     

Toll-like receptor 4 siRNA attenuates LPS-induced secretion of inflammatory cytokines and chemokines by macrophages

Toll-like receptor 4 (TLR4) is critical for activation of macrophages by Lipopolysaccharide (LPS). In this study, we investigated the silencing effects of TLR4-specific 21-nt small interfering RNAs (siRNA) on TLR4 expression in RAW264.7 cells. It was found that treatment with TLR4 siRNA down-regulated the TLR4 mRNA and protein expression in macrophage RAW264.7 cells, and reduced the sensitivity of the cells to LPS stimulation. Our findings also demonstrate that treatment with TLR4 siRNA significantly decreased the tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) expression induced by LPS. TLR4 siRNA treatment also impaired the signalling of mitogen-activated protein kinases (MAPK) induced by LPS in RAW264.7 cells. These data suggest that inhibition of TLR4 expression by TLR4 siRNA may be therapeutically beneficial in controlling the overall responses of immune cells to LPS.     

TLR signaling at the intestinal epithelial interface

The intestinal epithelium provides a critical interface between lumenal bacteria and the mucosal immune system. Whereas normal commensal flora do not trigger acute inflammation, pathogenic bacteria trigger a potent inflammatory response. Our studies emanate from the hypothesis that the intestinal epithelium is normally hyporesponsive to commensal pathogen-associated molecular patterns (PAMPs) such as LPS. Our data demonstrate that normal human colonic epithelial cells and lamina propria cells express low levels of TLR4 and its co-receptor MD-2. This expression pattern is mirrored by intestinal epithelial cell (IEC) lines. Co-expression of TLR4 and MD-2 is necessary and sufficient for LPS responsiveness in IEC. Moreover, LPS sensing occurs along the basolateral membrane of polarized IEC in culture. Expression of MD-2 is regulated by IFN-gamma. Cloning of the MD-2 promoter demonstrates that promoter activity is increased by IFN-gamma and blocked by the STAT inhibitor SOCS3. We conclude from our studies that the intestinal epithelium down-regulates expression of TLR4 and MD-2 and is LPS unresponsive. The Th1 cytokine IFN-gamma up-regulates expression of MD-2 in a STAT-dependent fashion. The results of our studies have important implications for understanding human inflammatory bowel diseases.     

Toll-like receptor 3 ligand attenuates LPS-induced liver injury by down-regulation of toll-like receptor 4 expression on macrophages

This study demonstrates that pretreatment with polyinosinic-polycytidylic acid (poly I:C) significantly decreased the mortality and liver injury caused by injection of lipopolysaccharide (LPS) in the presence of d-galactosamine (d-GalN) in C57BL/6 mice. Depletion of natural killer, natural killer T, and T cells did not change the protective effect of poly I:C on LPS/d-GalN-induced liver injury in vivo. However, depletion of macrophages abolished LPS/d-GalN-induced fulminant hepatitis, which could be restored by adoptive transfer of macrophages but not by transfer of poly I:C-treated macrophages. Treatment with poly I:C down-regulated the expression of the toll-like receptor 4 (TLR4) on macrophages and reduced the sensitivity of macrophages (Kupffer cells and peritoneal macrophages from C57BL/6 mice, or RAW264.7 cells) to LPS stimulation. Poly I:C pretreatment also impaired the signaling of mitogen-activated protein kinases and NF-kappaB induced by LPS in RAW264.7 cells. Blockade of TLR3 with a TLR3 antibody abolished poly I:C down-regulation of TLR4 expression and LPS stimulation of TNF-alpha production in RAW264.7 cells. Taken together, our findings suggest that activation of TLR3 by its ligand, poly I:C, induced LPS tolerance by down-regulation of TLR4 expression on macrophages.     

Involvement of TLR4/MD-2 complex in species-specific lipopolysaccharide-mimetic signal transduction by Taxol

Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice, but not in humans. The LPS-mimetic activity of Taxol is not observed in LPS-hyporesponsive C3H/HeJ mice which possess a point mutation in Toll-like receptor 4 (TLR4); therefore, TLR4 appears to be involved in both Taxol and LPS signaling. In addition, TLR4 was recently shown to physically associate with MD-2, a molecule that confers LPS-responsiveness on TLR4. Here we examined whether or not TLR4/MD-2 complex mediates a Taxol-induced signal by using transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, both mouse TLR4 and mouse MD-2, and both mouse MD-2 and mouse TLR4 lacking the cytoplasmic portion. Our results demonstrated that co-expression of mouse TLR4 and mouse MD-2 was required for Taxol responsiveness, and that the TLR4/MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice. We also found that mouse MD-2, but not human MD-2, is involved in Taxol signaling, suggesting that MD-2 is responsible for the species-specific responsiveness to Taxol.     

Selective priming to Toll-like receptor 4 (TLR4), not TLR2, ligands by P. acnes involves up-regulation of MD-2 in mice

Lipopolysaccharide (LPS) triggers cytokine production through Toll-like receptor 4 (TLR4), which shares downstream signaling pathways with TLR2. We investigated the roles of TLR2 and TLR4 in Propionibacterium acnes (P. acnes)-primed, LPS-induced liver damage using selective TLR ligands. Stock LPS induced interleukin 8 in both TLR4- and TLR2-expressing human embryonic kidney (HEK) 293 cells. Purified LPS (TLR4 ligand) activated HEK/TLR4 cells, while peptidoglycan and lipoteichoic acid (TLR2 ligands) activated HEK/TLR2 cells, respectively. In mice, P. acnes priming resulted in increased liver messenger RNA (mRNA) and serum levels of tumor necrosis factor alpha, interleukin 12, and interferon gamma (IFN-gamma) by both stock LPS and purified LPS challenges compared with nonprimed controls. In contrast, P. acnes failed to sensitize to TLR2 ligands (peptidoglycan + lipoteichoic acid). In the liver, P. acnes-priming was associated with up-regulation of TLR4 and MD-2 proteins, and subsequent LPS challenge further increased MD-2 and CD14 mRNA levels. The lack of sensitization to TLR2 ligands by P. acnes correlated with no increase in hepatic TLR1 or TLR6 mRNA. In vitro, P. acnes pretreatment desensitized RAW macrophages to a secondary stimulation via both TLR2 and TLR4. However, IFN-gamma could selectively prevent desensitization to TLR4 but not to TLR2 ligands. Furthermore, P. acnes induced production of IFN-gamma in vivo as well as in isolated splenocytes. In vitro, P. acnes-primed Hepa 1-6 hepatocytes but not RAW macrophages produced increased MD-2 and CD14 mRNA levels after an LPS challenge. In conclusion, P. acnes priming to selective TLR4-mediated liver injury is associated with up-regulation of TLR4 and MD-2 and is likely to involve IFN-gamma and prevent TLR4 desensitization by P. acnes.     

Roles for accessory molecules in microbial recognition by Toll-like receptors

The Toll family of receptors recognizes a variety of microbial products and triggers immune responses. Recent progress has revealed a requirement for accessory molecules in microbial recognition by Toll-like receptors. Lipopolysaccharide (LPS) recognition requires LPS binding protein (LBP), CD14, and MD-2. MD-2 is directly involved in ligand-binding and subsequent receptor activation, whereas LBP and CD14 control ligand presentation to the receptor complex, Toll-like receptor (TLR4)/MD-2. CD14 and LBP influence the amplitude of LPS responses and LPS-induced type I interferon production. TLR2 is also reported to require similar accessory molecules. Innate immune responses to microbial products driven by TLRs are controlled by accessory molecules working upstream of TLRs.     

Overexpression of CD14, TLR4, and MD-2 in HEK 293T cells does not prevent induction of in vitro endotoxin tolerance

TLR4 and MD-2 are necessary for conferring cellular responsiveness to LPS. Prior exposure to LPS induces a transient state of cell refractoriness to subsequent LPS re-stimulation, known as 'endotoxin tolerance'. While induction of LPS tolerance has been reported to correlate with down-regulation of cell surface expression of TLR4/MD-2, other mechanisms of LPS tolerance have been revealed that target intracellular intermediates downstream of the TLR4/MD-2 complex. In this study, we sought to examine whether endotoxin tolerance could be induced under conditions where expression of TLR4 and MD-2 proteins is not affected by LPS. Human HEK 293T cells are completely unresponsive to LPS, but acquire high LPS sensitivity following transient transfection with CD14, TLR4, and MD-2 (293T/CD14/TLR4/MD-2 cells), as judged by NF-kappaB activation, ERK 1/2 phosphorylation, and TNF-alpha gene expression. Prior exposure of 293T/CD14/TLR4/MD-2 cells to LPS resulted in a significant decrease of LPS-mediated responses, yet failed to affect expression levels of TLR4 and MD-2. Thus, altered expression and/or function of intracellular mediators downstream of the TLR4/MD-2 complex play an important role in mediating LPS tolerance.     

The equine TLR4/MD-2 complex mediates recognition of lipopolysaccharide from Rhodobacter sphaeroides as an agonist

Lipopolysaccharide (LPS) antagonists inhibit the response of inflammatory cells to LPS, presumably by competitive inhibition, and may be of therapeutic value in the treatment of endotoxemia and sepsis. The inhibitory effects of some LPS antagonists are restricted to certain host species, however, as the same molecules can have significant endotoxic activity in other species. This species-specific recognition appears to be mediated by Toll-like receptor 4 (TLR4) and/or MD-2. We have shown previously that LPS from Rhodobacter sphaeroides ( RsLPS) is an LPS antagonist in human cells but an agonist (or LPS mimetic) in equine cells. In the present study, HEK293 cells were transfected with combinations of human and equine CD14, TLR4 and MD-2, and incubated with either RsLPS or with LPS from Escherichia coli as an endotoxin control. NF-kappaB activation was measured in a dual luciferase assay as an indicator of cellular activation. Our results indicate that E. colic LPS activated NF-kappaB in cells transfected with all combinations of the three receptor proteins, whereas RsLPS activated NF-kappaB only in cells expressing the single combination of equine TLR4 and equine MD-2. We conclude that the TLR4/MD-2 complex is responsible for recognition of RsLPS as an agonist in equine cells.     

Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4

Toll-like receptor 4 (TLR4), the principal signaling receptor for lipopolysaccharide (LPS) in mammals, requires the binding of MD-2 to its extracellular domain for maximal responsiveness. MD-2 contains a leader sequence but lacks a transmembrane domain, and we asked whether it is secreted into the medium as an active protein. As a source of secreted MD-2 (sMD-2), we used culture supernatants from cells stably transduced with epitope-tagged human MD-2. We show that sMD-2 exists as a heterogeneous collection of large disulfide-linked oligomers formed from stable dimeric subunits and that concentrations of sMD-2 as low as 50 pM enhance the responsiveness of TLR4 reporter cells to LPS. An MD-2-like activity is also released by monocyte-derived dendritic cells from normal donors. When coexpressed, TLR4 indiscriminately associates in the endoplasmic reticulum/cis Golgi with different-sized oligomers of MD-2, and excess MD-2 is secreted into the medium. We conclude that normal and transfected cells secrete a soluble form of MD-2 that binds with high affinity to TLR4 and that could play a role in regulating responses to LPS and other pathogen-derived substances in vivo.     

Molecular basis for lipopolysaccharide mimetic action of Taxol and flavolipin

We previously reported that Taxol, which mimics the action of LPS on murine macrophages, induces signals via mouse TLR4/MD-2, but not via human TLR4/MD-2. Here we investigated the molecular basis for this species-specific action of Taxol. Expression of mouse MD-2 conferred both LPS and Taxol responsiveness on HEK293 cells expressing mouse TLR4, whereas expression of human MD-2 conferred LPS responsiveness alone, suggesting that MD-2 is responsible for the species-specificity of Taxol responsiveness. Furthermore, mouse MD-2 mutants, in which Gln-22 was changed to other amino acids, showed dramatically reduced ability to confer Taxol responsiveness, although their ability to confer LPS responsiveness was not affected. These results indicated that Gln-22 of mouse MD-2 is essential for Taxol signaling, but not for LPS signaling. In this study, we also found that the TLR4/MD-2 complex, together with CD14, mediated signal transduction induced by flavolipin, an amino acid-containing lipid unique to Flavobacterium meningosepticum.     

Soluble MD-2 is an acute-phase protein and an opsonin for Gram-negative bacteria

Myeloid differentiation factor-2 (MD-2) is a lipopolysaccharide (LPS)-binding protein usually coexpressed with and binding to Toll-like receptor 4 (TLR4), conferring LPS responsiveness of immune cells. MD-2 is also found as a soluble protein. Soluble MD-2 (sMD-2) levels are markedly elevated in plasma from patients with severe infections, and in other fluids from inflamed tissues. We show that sMD-2 is a type II acute-phase protein. Soluble MD-2 mRNA and protein levels are up-regulated in mouse liver after the induction of an acute-phase response. It is secreted by human hepatocytic cells and up-regulated by interleukin-6. Soluble MD-2 binds to Gram-negative but not Gram-positive bacteria, and sMD-2 secreted by hepatocytic cells is an essential cofactor for the activation of TLR4-expressing cells by Gram-negative bacteria. Soluble MD-2 opsonization of Gram-negative bacteria accelerates and enhances phagocytosis, principally by polymorphonuclear neutrophils. In summary, our results demonstrate that sMD-2 is a newly recognized type II acute-phase reactant, an opsonin for Gram-negative bacteria, and a cofactor essential for the activation of TLR4-expressing cells. This suggests that sMD-2 plays a key role in the host innate immune response to Gram-negative infections.     

Contribution of Toll-like receptors to the innate immune response to Gram-negative and Gram-positive bacteria.

Innate recognition of bacteria is a key step in the activation of inflammation and coagulation, and it is dependent on pathogen-associated molecular pattern (PAMP) ligation to Toll-like receptors (TLRs) and CD14. The dominant receptors activated when cells encounter a whole bacterium, which express several PAMPs, are poorly defined. Herein, we have stimulated various human cells with prototypic Gram-negative and Gram-positive bacteria. Receptor-dependent responses to whole bacteria were assessed using both TLR-transfected cells and specific monoclonal antibodies against TLRs, MD-2, and CD14. Enterobacteria-activated leukocytes and endothelial cells in a TLR4/MD-2-dependent manner, most likely via lipopolysaccharide (LPS). TLR2 activation was observed with a high bacterial inoculum, and in epithelial cells expressing TLR2 but not TLR4. Pseudomonas aeruginosa stimulated cells by both TLR2 and TLR4/MD-2. Gram-positive bacteria activated cells only at high concentrations, in a partially TLR2-dependent but TLR4/MD-2-independent manner. Either TLR or CD14 neutralization blocked activation to all bacterial strains tested with the exception of some Gram-positive strains in whole blood in which partial inhibition was noted. This study identifies dominant TLRs involved in responses to whole bacteria. It also validates the concept that host cell activation by bacterial pathogens can be therapeutically reduced by anti-TLR4, -TLR2, and -CD14 mAbs.     

Lipopolysaccharide (LPS) directly suppresses growth hormone receptor (GHR) expression through MyD88-dependent and -independent Toll-like receptor-4/MD2 complex signaling pathways

INTRODUCTION: Sepsis is associated with growth hormone (GH) insensitivity and in the intact animal the major surface component of the bacterial cell wall, lipopolysaccharide (LPS), inhibits GH receptor (GHR) gene expression. The prevailing explanation for LPS-induced effects on the GHR promoter is that this effect is indirect via generation of cytokines. Our recent studies demonstrate that saturated free fatty acids (FFAs) inhibit the activity of the murine GHR promoter. Saturated FFAs are an essential component of the lipid A moiety of LPS required for biological activity of LPS. HYPOTHESIS: LPS directly modulates the activity of the dominant GHR promoter via interaction with Toll-like receptor(s) (TLR)/MD2 complex and activation of cognate signaling pathway(s). RESULTS: In transient transfection experiments with RAW 264.7 cells which express endogenous TLR4 and MD2, LPS treatment inhibited GHR promoter activity. Co-transfection of dominant negative TLR4 abrogated this effect on GHR promoter activity. In HEK 293T cells, which are devoid of endogenous TLR4 or MD2, ectopic expression of TLR4 and MD2 resulted in LPS-induced inhibition of the GHR promoter activity. The inhibition of GHR promoter activity was demonstrable by 5-6h after exposure to LPS and persisted at 24h. Fatty-acid free LPS failed to elicit a similar effect on the GHR promoter and the effect of LPS was abrogated by Polymyxin B. The essential role of the cofactor MD2 on the effect of LPS on the GHR promoter was established in experiments using ectopic expression of wild type and mutant MD2. Cotransfection of CD14 in these cells failed to alter the effect of LPS on the activity of the GHR promoter. Analysis of cell culture supernatant excluded the possibility that the effect of LPS was secondary to release of cytokines from the transfected cells. The effect of LPS on the endogenous GHR promoter activity and protein expression was confirmed in F442A preadipocyte cells. In HEK 293T cells, ectopic expression of mutant MyD88 or mutant TRIF abrogated the effect of LPS on the GHR promoter, suggesting that the effect of LPS on the GHR promoter was via both MyD88-dependent and -independent pathways. CONCLUSIONS: LPS acts through both MyD88-dependent and -independent TLR4 signaling pathways to directly inhibit GHR gene expression. Our results establish a novel cytokine-independent mechanism for decrease in GHR expression in bacterial sepsis.     

Essential role of MD-2 in B-cell responses to lipopolysaccharide and Toll-like receptor 4 distribution

Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signaling in a variety of cell types. MD-2 is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses in vitro. Moreover, mice lacking MD-2 (MD-2(-/-)) do not respond to LPS, survive endotoxin shock, and are susceptible to Salmonella typhimurium infection. Here, we further show that B cells lacking MD-2 do not up-regulate CD23 in response to LPS. TLR4 predominantly resides in the Golgi apparatus without MD-2. MD-2 is essential for LPS responses in vivo.     

Innate recognition of lipopolysaccharide by Toll-like receptor 4/MD-2 and RP105/MD-1

The Toll family of receptors has been implicated in innate recognition and subsequent activation of defense programs against pathogens such as bacteria and fungi. TLR4, for example, signals the presence of lipopolysaccharide (LPS), a membrane constituent of Gram-negative bacteria. LPS signaling via TLR4 is greatly enhanced by a molecule referred to as MD-2, which is associated with the extracellular domain of TLR4. The TLR4/MD-2 complex, therefore, recognizes LPS. RP105, another member of the Toll family, has a striking similarity to TLR4 in that it is associated with an MD-2-like molecule MD-1. B-cells lacking RP105 are severely impaired in LPS-induced proliferation and antibody production. Studies employing transfectants showed that RP105/MD-1, like MD-2, enhances the LPS signaling via TLR4. RP105/MD-1 thus constitutes an LPS-signaling complex on B-cells. These results suggest that a variety of cell surface molecules regulate LPS recognition/signaling by TLR4.     

MD-2 binds to bacterial lipopolysaccharide

Many LPS binding proteins have been described, but the exact nature of the LPS receptors that signal cells remains unclear. MD-2 is a molecule that is found in association with Toll-like receptor 4, which has been shown to be a receptor for LPS. We have produced human MD-2 in baculovirus and tested it for LPS binding. MD-2 binds the lipid A region of LPS without the need for LPS binding protein. These data suggest that MD-2 may be binding LPS as part of the TLR4 receptor complex.     

Structural basis of species-specific endotoxin sensing by innate immune receptor TLR4/MD-2

Lipopolysaccharide (LPS), also known as endotoxin, activates the innate immune response through toll-like receptor 4 (TLR4) and its coreceptor, MD-2. MD-2 has a unique hydrophobic cavity that directly binds to lipid A, the active center of LPS. Tetraacylated lipid IVa, a synthetic lipid A precursor, acts as a weak agonist to mouse TLR4/MD-2, but as an antagonist to human TLR4/MD-2. However, it remains unclear as to how LPS and lipid IVa show agonistic or antagonistic activities in a species-specific manner. The present study reports the crystal structures of mouse TLR4/MD-2/LPS and TLR4/MD-2/lipid IVa complexes at 2.5 and 2.7 Å resolutions, respectively. Mouse TLR4/MD-2/LPS exhibited an agonistic “m”-shaped 2:2:2 complex similar to the human TLR4/MD-2/LPS complex. Mouse TLR4/MD-2/lipid IVa complex also showed an agonistic structural feature, exhibiting architecture similar to the 2:2:2 complex. Remarkably, lipid IVa in the mouse TLR4/MD-2 complex occupied nearly the same space as LPS, although lipid IVa lacked the two acyl chains. Human MD-2 binds lipid IVa in an antagonistic manner completely differently from the way mouse MD-2 does. Together, the results provide structural evidence of the agonistic property of lipid IVa on mouse TLR4/MD-2 and deepen understanding of the ligand binding and dimerization mechanism by the structurally diverse LPS variants.Toll-like receptors (TLRs) recognize and respond to diverse pathogenic components of microorganisms and provide the first line of defense against microbial infection (1, 2). Among the microbial components, endotoxic lipopolysaccharide (LPS) from a membrane component of Gram-negative bacteria elicits the potent innate immune response through the receptor complex of TLR4 and MD-2 (3, 4). Excessive exposure to LPS often causes exaggerated signaling via TLR4 and fatal septic shock (5, 6), which is associated with a high mortality (20–30%) and is the most common cause of death in intensive care units (5, 6).The lipid A moiety of LPS, which anchors LPS to the outer membrane of Gram-negative bacteria, is responsible for the immunostimulatory activity of LPS (7, 8). Lipid A consists of a 1,4′-bis-phosphorylated diglucosamine backbone to which variable lengths and numbers of acyl chains are covalently linked (8). The two phosphate groups are also important for the agonistic activity of lipid A because deletion of either phosphate group reduces the endotoxic activity (9, 10).TLR4 is a type I transmembrane protein composed of 22 extracellular leucine-rich repeats (LRRs), a transmembrane domain, and the Toll/IL-1 receptor domain (TIR domain) that is essential for TLR signaling and conserved among members of the Toll receptor family (1). TLR4 alone does not directly bind LPS and requires the coreceptor MD-2 (11). MD-2 is associated with the extracellular domain of TLR4 and is indispensable for LPS recognition (4). A member of the MD-2–related lipid-recognition protein family (12), MD-2 directly binds to LPS in its hydrophobic cavity with high affinity (13).Recently, the crystal structure of human TLR4/MD-2/Ra-LPS (Ra chemotype of Escherichia coli LPS) complex (14) was solved, which revealed that five of the six acyl chains of LPS are buried inside the MD-2 cavity. The sixth acyl chain lies on the surface of MD-2, partially exposed to the solvent. Together with the hydrophobic residues of MD-2, the partially exposed acyl chain constitutes the secondary binding site for the hydrophobic patch on the C-terminal convex face of the horseshoe structure of TLR4, leading to the formation of the “m”-shaped 2:2:2 hTLR4/MD-2/LPS complex. The close proximity of the C terminus of the extracellular domain in the complex induced by binding to LPS may allow for dimerization and signaling by the intracellular TIR domains (15, 16).The number and length of the acyl chains determine the agonistic property of lipid A (17–19). E. coli lipid A is usually hexaacylated and acts as a potent agonist for all mammalian cells. In contrast, tetraacylated lipid IVa, the precursor of E. coli LPS, acts as an agonist only for some mammalian species. In particular, it acts as a weak agonist on mouse and as an antagonist on human cells (20, 21). Although several studies have investigated the species-specific activity of lipid IVa (22–28), these studies primarily used mutational and computational simulation methods. Structural information on the agonistic form of TLR4/MD-2 is limited to the hTLR4/MD-2/LPS complex; no structures of mTLR4/MD-2 complexed with LPS or lipid IVa are currently available. Structural knowledge may provide critical clues regarding the agonistic and antagonistic mechanisms by LPS and lipid IVa ligands that underlie species specificity.Here, we present the two agonistic structures of mouse TLR4/MD-2/Re-LPS (Re chemotype of E. coli LPS) and TLR4/MD-2/lipid IVa complexes at 2.5 and 2.7 Å resolutions, respectively. This structural study will provide better understanding of the LPS recognition and signaling mechanism and will contribute to the development of therapeutic antiseptic shock drugs targeting TLR4/MD-2.