Masashi Muroi

N-Linked Glycosylations at Asn26 and Asn114 of Human MD-2 Are Required for Toll-Like Receptor 4-Mediated Activation of NF-κB by Lipopolysaccharide

MD-2 is physically associated with Toll-like receptor 4 (TLR4) and is required for TLR4-mediated LPS signaling. Western blotting analysis revealed the presence of three forms of human (h)MD-2 with different electrophoretic mobilities. After N-glycosidase treatment of the cellular extract prepared from cells expressing hMD-2, only a single form with the fastest mobility was detected. Mutation of either one of two potential glycosylation sites (Asn26 and Asn114) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn26 and Asn114. Although these mutants were expressed on the cell surface and maintained its ability to associate with human TLR4, these mutations or tunicamycin treatment substantially impaired the ability of MD-2 to complement TLR4-mediated activation of NF-κB by LPS. LPS binding to cells expressing CD14, TLR4, and MD-2 was unaffected by these mutations. These observations demonstrate that hMD-2 undergoes N-linked glycosylation at Asn26 and Asn114, and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS.

MD-2 Is Necessary for the Toll-Like Receptor 4 Protein To Undergo Glycosylation Essential for Its Translocation to the Cell Surface

ABSTRACT MD-2 has been reported to be required for the translocation of the Toll-like receptor 4 (TLR4) to the cell surface. However, the mechanism by which MD-2 promotes TLR4 translocation is unknown. We identified the presence of two forms of TLR4 with different molecular masses (approximately 110 and 130 kDa) when TLR4 was expressed together with MD-2. Expressing TLR4 alone produced only the 110-kDa form. Using a membrane-impermeable biotinylation reagent, we found that only the 130-kDa form of TLR4 was expressed on the cell surface. When a cellular extract prepared from cells expressing TLR4 and MD-2 was treated with N-glycosidase, the two forms of TLR4 converged into a single band whose size was smaller than the 110-kDa form of TLR4. Mutation of TLR4 at Asn526 or Asn575 resulted in the disappearance of the 130-kDa form and prevented TLR4 from being expressed on the cell surface without affecting the ability of TLR4 to associate with MD-2. These results indicate that TLR4 is able to undergo multiple glycosylations without MD-2 but that the specific glycosylation essential for cell surface expression requires the presence of MD-2.

The Polysaccharide Portion Plays an Indispensable Role in Salmonella Lipopolysaccharide-Induced Activation of NF-κB through Human Toll-Like Receptor 4

ABSTRACT The lipid A portion has been identified as the active center responsible for lipopolysaccharide (LPS)-induced macrophage activation. However, we found that Salmonella (Salmonella enterica serovars Abortusequi, Minnesota, and Typhimurium) lipid A is inactive in human macrophages, despite its LPS being highly active. Thus we investigated the critical role of polysaccharide in Salmonella LPS-induced activation of NF-κB. In human monocytic cell line THP-1, Salmonella lipid A and synthetic Salmonella-type lipid A (516) did not induce NF-κB-dependent reporter activity up to 1 μg/ml, whereas strong activation was observed in response to Salmonella LPS. The difference in activity between this lipid A and LPS was further examined by using 293 cells expressing human CD14/Toll-like receptor 4 (TLR4)/MD-2, and similar results were obtained in these cells as well. A polysaccharide preparation obtained from Salmonella LPS was inactive in 293 cells expressing human CD14/TLR4/MD-2 even in combination with 516. Salmonella enterica serovar Minnesota Re LPS, whose structure consists of lipid A and two molecules of 2-keto-3-deoxyoctonic acid, but not its lipid A exhibited strong activity in THP-1 cells and 293 cells expressing human CD14/TLR4/MD-2. These results indicate that the polysaccharide portion covalently bound to lipid A plays the principal role in Salmonella LPS-induced activation of NF-κB through human CD14/TLR4/MD-2.

Regions of the Mouse CD14 Molecule Required for Toll-like Receptor 2- and 4-mediated Activation of NF-κB

Regions of mouse CD14 required for Toll-like receptor 2 (TLR2)- and TLR4-mediated activation of NF-κB were studied in transiently transfected 293 cells. Wild-type CD14 enhanced lipopolysaccharide (LPS)-induced NF-κB-dependent reporter activity in cells expressing TLR4/MD-2, and deletion of amino acid regions 35–44, 144–153, 235–243, and 270–275 impaired the TLR4-mediated activation. Unlike human CD14, mouse CD14 truncated at amino acid 151 lost the activity. Deletion of amino acids 35–44 or 235–243 also abrogated TLR2-mediated activation of NF-κB, whereas mutants lacking 144–153 and 270–275 retained the activity. Deletion and alanine substitution experiments revealed that amino acids 151–153 and 273–275 were required for the TLR4-mediated activation. Both deletion mutants lacking amino acids 35–44 and 235–243 and alanine substitution mutants in regions 151–153 and 273–275 were expressed on the cell surface and retained the ability to associate with TLR4. A cross-linking study with photoreactive LPS showed that the labeling intensities to CD14 mutants/TLR4/MD-2 were paralleled by the ability of CD14 mutants to increase TLR4-mediated activation. These results indicate that different regions of mouse CD14 are required for TLR4- and TLR2-mediated activation of NF-κB and suggest that amino acids 35–44, 151–153, 235–243, and 273–275 of mouse CD14 play an important role in LPS binding and its transfer to TLR4/MD-2.

Structural Regions of MD-2 That Determine the Agonist-Antagonist Activity of Lipid IVa

A cell surface receptor complex consisting of CD14, Toll-like receptor (TLR4), and MD-2 recognizes lipid A, the active moiety of lipopolysaccharide (LPS). Escherichia coli-type lipid A, a typical lipid A molecule, potently activates both human and mouse macrophage cells, whereas the lipid A precursor, lipid IVa, activates mouse macrophages but is inactive and acts as an LPS antagonist in human macrophages. This animal species-specific activity of lipid IVa involves the species differences in MD-2 structure. We explored the structural region of MD-2 that determines the agonistic and antagonistic activities of lipid IVa to induce nuclear factor-κB activation. By expressing human/mouse chimeric MD-2 together with mouse CD14 and TLR4 in human embryonic kidney 293 cells, we found that amino acid regions 57–79 and 108–135 of MD-2 determine the species-specific activity of lipid IVa. We also showed that the replacement of Thr57, Val61, and Glu122 of mouse MD-2 with corresponding human MD-2 sequence or alanines impaired the agonistic activity of lipid IVa, and antagonistic activity became evident. These mutations did not affect the activation of nuclear factor-κB, TLR4 oligomerization, and inducible phosphorylation of IκBα in response to E. coli-type lipid A. These results indicate that amino acid residues 57, 61, and 122 of mouse MD-2 are critical to determine the agonist-antagonist activity of lipid IVa and suggest that these amino acid residues may be involved in the discrimination of lipid A structure.

MD-2, a Novel Accessory Molecule, Is Involved in Species-Specific Actions of Salmonella Lipid A

ABSTRACT Salmonella lipid A is inactive in human macrophages despite being potently active in murine macrophages. We investigated the molecular basis for this species-specific action of Salmonella lipid A. When murine CD14 (mCD14), mTLR4, and mMD-2 were all expressed in human monocytic THP-1 cells, these cells were capable of responding to Salmonella lipid A. Expressing each of these proteins separately did not impart such responsiveness. Expression of mTLR4 plus mMD-2, but not mCD14 plus mTLR4 or mCD14 plus mMD-2, conferred this responsiveness. In THP-1 cells expressing mCD14, mTLR4, and mMD-2, replacing mCD14 with human CD14 had no effect on responsiveness to Salmonella lipid A or synthetic Salmonella-type lipid A (compound 516). When mTLR4 was replaced with human TLR4, the responses to these lipid A preparations were decreased to half, and the replacement of mMD-2 decreased responsiveness to one-third, although the responses to Escherichia coli lipid A or synthetic E. coli-type lipid A (compound 506) were not affected. These results indicate that both TLR4 and MD-2 participate in the species-specific action of Salmonella lipid A.

Prolonged Toll-like receptor stimulation leads to down-regulation of IRAK-4 protein

Interleukin‐1 receptor‐associated kinase (IRAK)‐4 is a key mediator in the Toll‐like receptor (TLR) signaling. We found that stimulation of TLR2, TLR4, or TLR9, but not TLR3, caused a decrease in IRAK‐4 protein without affecting its mRNA level in a mouse macrophage cell line, RAW 264. The decrease in IRAK‐4 was accompanied by the appearance of a smaller molecular weight protein (32 kD), which was recognized by an anti‐IRAK‐4 antibody raised against the C‐terminal region. The decrease in IRAK‐4 and the appearance of the 32‐kD protein occurred with slower kinetics than the activation of IRAK‐1 and were suppressed by inhibitors of the proteasome, inducible inhibitor of κBα phosphorylation or protein synthesis, but not by caspase inhibitors. These results indicate that prolonged stimulation of TLR2, TLR4, or TLR9 causes a down‐regulation of IRAK‐4 protein, which may be mediated through cleavage of IRAK‐4 by a protease induced by the activation of nuclear factor‐κB.

TRAF6 distinctively mediates MyD88-and IRAK-1-induced activation of NF-κB

MyD88 and IL‐1R‐associated kinase 1 (IRAK‐1) play crucial roles as adaptor molecules in signal transduction of the TLR/IL‐1R superfamily, and it is known that expression of these proteins leads to the activation of NF‐κB in a TNFR‐associated factor 6 (TRAF6)‐dependent manner. We found in this study, however, that a dominant‐negative mutant of TRAF6, lacking the N‐terminal RING and zinc‐finger domain, did not inhibit IRAK‐1‐induced activation of NF‐κB in human embryonic kidney 293 cells, although the TRAF6 mutant strongly suppressed the MyD88‐induced activation. The dominant‐negative mutant of TRAF6 did not affect the IRAK‐1‐induced activation, regardless of the expression level of IRAK‐1. In contrast, small interfering RNA silencing of TRAF6 expression inhibited MyD88‐induced and IRAK‐1‐induced activation, and supplementation with the TRAF6 dominant‐negative mutant did not restore the IRAK‐1‐induced activation. Expression of IRAK‐1, but not MyD88, induced the oligomerization of TRAF6, and IRAK‐1 and the TRAF6 dominant‐negative mutant were associated with TRAF6. These results indicate that TRAF6 is involved but with different mechanisms in MyD88‐induced and IRAK‐induced activation of NF‐κB and suggest that TRAF6 uses a distinctive mechanism to activate NF‐κB depending on signals.

Role of Toll‐Like Receptor 4 in Mediating Multiorgan Dysfunction in Mice With Acetaminophen Induced Acute Liver Failure

Strategies for the prevention of multiorgan dysfunction (MOD) in acetaminophen (APAP)‐induced acute liver failure (ALF) are an unmet need. Our study tested the hypothesis that sterile inflammation induced by APAP in a mouse model would activate toll‐like receptor 4 (TLR4) in the liver and extrahepatic organs and lead to the progression of ALF and MOD and that the administration of the novel TLR4 antagonist STM28 (a peptide formed of 17 amino‐acids) would prevent liver injury and associated MOD. ALF and, subsequently, MOD were induced in TLR4‐knockout (KO) mice (B6.B10ScN‐Tlr4 lpsdel /JthJ) and wild‐type (WT) mice (C57BL/6) with APAP (500 mg/kg). A second set of experiments was conducted to evaluate the effects of a pretreatment with a novel TLR4 antagonist, STM28, on APAP‐induced MOD in CD1 mice. Animals were sacrificed at the coma stage, and plasma, peripheral blood cells, liver, kidneys, and brain were collected. Biochemistry values and cytokines were measured. Liver and kidneys were studied histologically and were stained for TLR4 and activated Kupffer cells, and the expression of nuclear factor kappa B–p65 was quantified with western blotting. Brain water was measured in the frontal cortex. After APAP administration, TLR4‐KO (NFkBp65) mice were relatively protected from liver necrosis and end‐organ dysfunction and had significantly better survival than WT controls (P < 0.01). STM28 attenuated liver injury and necrosis, reduced creatinine levels, and delayed the time to a coma significantly. The increases in cytokines in the plasma and liver, including TLR4 expression and the activation of Kupffer cells, after APAP administration were reduced significantly in the STM28‐treated animals. The increased number of circulating myeloid cells was reduced significantly after STM28 treatment. In conclusion, these data provide evidence for an important role of the TLR4 antagonist in the prevention of the progression of APAP‐induced ALF and MOD. Liver Transpl 19:751–761, 2013..

Deoxynivalenol and nivalenol inhibit lipopolysaccharide-induced nitric oxide production by mouse macrophage cells.

Deoxynivalenol (DON) and nivalenol (NIV), trichothecene mycotoxins, are secondary metabolites produced by Fusarium fungi. Trichothecene mycotoxins cause immune dysfunction, thus leading to diverse responses to infection. The present study evaluated the effect of DON and NIV on nitric oxide (NO) production by RAW264 cells stimulated with lipopolysaccharide (LPS). LPS-induced NO production was reduced in the presence of these toxins. The transcriptional activation and expression of inducible NO synthase (iNOS) by LPS were also repressed by these toxins. DON or NIV inhibited LPS-induced expression of interferon-beta (IFN-beta), which plays an indispensable role in LPS-induced iNOS expression. These results indicate that DON and NIV inhibit the LPS-induced NO and IFN-beta production, which both play an important role for host protection against invading pathogens, and suggests that the inhibition of these factors may be involved in the immunotoxic effects of these mycotoxins.