Effect of herbal melanin on IL-8: a possible role of Toll-like receptor 4 (TLR4)

The production of IL-8 can be induced by LPS via TLR4 signaling pathway. In this study, we tested the effect of a herbal melanin (HM) extract, from black cumin seeds (Nigella sativa L.), on IL-8 production. We used HM and LPS in parallel to induce IL-8 production by THP-I, PBMCs, and TLR4-transfected HEK293 cells. Both HM and LPS induced IL-8 mRNA expression and protein production in THP-1 and PBMCs. On applying similar treatment to HEK293 cells that express TLR4, MD2, and CD14, both HM and LPS significantly induced IL-8 protein production. We have also demonstrated that HM and LPS had identical effects in terms of IL-8 stimulation by HEK293 transfected with either TLR4 or MD2-CD14. Melanin extracted from N. sativa L. mimics the action of LPS in the induction of IL-8 by PBMC and the other used cell lines. Our results suggest that HM may share a signaling pathway with LPS that involves TLR4.     

Cutting edge: cells that carry A null allele for toll-like receptor 2 are capable of responding to endotoxin.

Toll-like receptor (TLR) 2 and TLR4 have been implicated in the responses of cells to LPS (endotoxin). CD14-transfected Chinese hamster ovary (CHO)-K1 fibroblasts (CHO/CD14) are exquisitely sensitive to endotoxin. Sequence analysis of CHO-TLR2, compared with human and mouse TLR2, revealed a single base pair deletion. This frameshift mutation resulted in an alternative stop codon, encoding a protein devoid of transmembrane and intracellular domains. CHO-TLR2 cDNA failed to enable LPS signaling upon transient transfection into human epithelial kidney 293 cells. Site-directed mutagenesis of CHO-TLR2 enabled expression of a presumed full-length hamster TLR2 that conferred LPS responsiveness in human epithelial kidney 293 cells. Genomic TLR2 DNA from primary hamster macrophages also contained the frameshift mutation found in CHO fibroblasts. Nevertheless, hamster peritoneal macrophages were found to respond normally to LPS, as evidenced by the induction of cytokines. These results imply that expression of TLR2 is sufficient but not essential for mammalian responses to endotoxin.     

Sialyl residues modulate LPS-mediated signaling through the Toll-like receptor 4 complex

We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFκB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation.     

Membrane-anchored forms of lipopolysaccharide (LPS)-binding protein do not mediate cellular responses to LPS independently of CD14

Inflammatory responses of myeloid cells to LPS are mediated through CD14, a glycosylphosphatidylinositol-anchored receptor that binds LPS. Since CD14 does not traverse the plasma membrane and alternatively anchored forms of CD14 still enable LPS-induced cellular activation, the precise role of CD14 in mediating these responses remains unknown. To address this, we created a transmembrane and a glycosylphosphatidylinositol-anchored form of LPS-binding protein (LBP), a component of serum that binds and transfers LPS to other molecules. Stably transfected Chinese hamster ovary (CHO) fibroblast and U373 astrocytoma cell lines expressing membrane-anchored LBP (mLBP), as well as separate CHO and U373 cell lines expressing membrane CD14 (mCD14), were subsequently generated. Under serum-free conditions, CHO and U373 cells expressing mCD14 responded to as little as 0.1 ng/ml of LPS, as measured by NF-kappaB activation as well as ICAM and IL-6 production. Conversely, the vector control and mLBP-expressing cell lines did not respond under serum-free conditions even in the presence of more than 100 ng/ml of LPS. All the cell lines exhibited responses to less than 1 ng/ml of LPS in the presence of the soluble form of CD14, demonstrating that they are still capable of LPS-induced activation. Taken together, these results demonstrate that mLBP, a protein that brings LPS to the cell surface, does not mediate cellular responses to LPS independently of CD14. These findings suggest that CD14 performs a more specific role in mediating responses to LPS than that of simply bringing LPS to the cell surface.     

Membrane-anchored CD14 is required for LPS-induced TLR4 endocytosis in TLR4/MD-2/CD14 overexpressing CHO cells

Lipopolysaccharide (LPS) induces inflammatory activation through TLR4 (toll-like receptor-4)/MD-2 (myeloid differentiation-2)/CD14 (cluster of differentiation-14) complex. Although optimal LPS signaling is required to activate our innate immune systems against gram-negative bacterium, excessive amount of LPS signaling develops a detrimental inflammatory response in gram-negative bacterial infections. Downregulation of surface TLR4 expression is one of the critical mechanisms that can restrict LPS signaling. Here, we found that membrane-anchored CD14 is required for LPS-induced downregulation of TLR4 and MD-2 in CHO cells. Moreover, pretreatment of the cells with sterol-binding agent filipin reduced LPS-induced TLR4 downregulation, suggesting the involvement of caveolae-mediated endocytosis pathway. Involvement of caveolae in LPS-induced TLR4 endocytosis was further confirmed by immunoprecipitation. Thus, our data indicate that caveolae-dependent endocytosis pathway is involved in LPS-induced TLR4 downregulation and that this is dependent on membrane-anchored CD14 expression.     

CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation

Toll‐like receptor 4 (TLR4) is responsible for the immediate response to Gram‐negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal‐MyD88 [Mal (MyD88‐adaptor‐like) ‐ MYD88 (Myeloid differentiation primary response gene (88))] is recruited to the plasma membrane to initiate the signaling cascade leading to production of pro‐inflammatory cytokines while TRAM‐TRIF [TRAM (TRIF‐related adaptor molecule)‐TRIF (TIR‐domain‐containing adapter‐inducing interferon‐β)] is recruited to early endosomes to initiate the subsequent production of type I interferons. We have investigated the dynamics of TLR4 and TRAM during lipopolysaccharide (LPS) stimulation. We found that LPS induced a CD14‐dependent immobile fraction of TLR4 in the plasma membrane. Total internal reflection fluorescence microscopy (TIRF) revealed that LPS stimulation induced clustering of TLR4 into small punctate structures in the plasma membrane containing CD14/LPS and clathrin, both in HEK293 cells and the macrophage model cell line U373‐CD14. These results suggest that laterally immobilized TLR4 receptor complexes are being formed and prepared for endocytosis. RAB11A was found to be involved in localizing TRAM to the endocytic recycling compartment (ERC) and to early sorting endosomes. Moreover, CD14/LPS but not TRAM was immobilized on RAB11A‐positive endosomes, which indicates that TRAM and CD14/LPS can independently be recruited to endosomes.      

Escherichia coli P fimbriae utilize the Toll-like receptor 4 pathway for cell activation

Fimbriae mediate bacterial attachment to host cells and provide a mechanism for tissue attack. They activate a host response by delivery of microbial products such as lipopolysaccharide (LPS) or through direct fimbriae-dependent signalling mechanisms. By coupling to glycosphingolipid (GSL) receptors, P fimbriae trigger cytokine responses in CD14 negative host cells. Here we show that P fimbriae utilize the Toll-like receptor 4 (TLR4)-dependent pathway to trigger mucosal inflammation. Escherichia coli strains expressing P fimbriae as their only virulence factor stimulated chemokine and neutrophil responses in the urinary tract of TLR4 proficient mice, but TLR4 defective mice failed to respond to infection. Mucosal cells were CD14 negative but expressed several TLR species including TLR4, and TLR4 protein was detected. Infection with P fimbriated bacteria stimulated an increase in TLR4 mRNA levels. The activation signal did not involve the LPS-CD14 pathway and was independent of lipid A myristoylation, as shown by mutational inactivation of the msbB gene. Co-staining experiments revealed that TLR4 and the GSL receptors for P fimbriae co-localized in the cell membrane. The results demonstrate that P fimbriae activate epithelial cells by means of a TLR4-dependent signalling pathway, and suggest that GSL receptors for P fimbriae can recruit TLR4 as co-receptors.     

Expression of a Porphyromonas gingivalis lipid A palmitylacyltransferase in Escherichia coli yields a chimeric lipid A with altered ability to stimulate interleukin-8 secretion

In Escherichia coli the gene htrB codes for an acyltransferase that catalyses the incorporation of laurate into lipopolysaccharide (LPS) as a lipid A substituent. We describe the cloning, expression and characterization of a Porphyromonas gingivalis htrB homologue. When the htrB homologue was expressed in wild-type E. coli or a mutant strain deficient in htrB, a chimeric LPS with altered lipid A structure was produced. Compared with wild-type E. coli lipid A, the new lipid A species contained a palmitate (C16) in the position normally occupied by laurate (C12) suggesting that the cloned gene performs the same function as E. coli htrB but preferentially transfers the longer-chain palmitic acid that is known to be present in P. gingivalis LPS. LPS was purified from wild-type E. coli, the E. coli htrB mutant strain and the htrB mutant strain expressing the P. gingivalis acyltransferase. LPS from the palmitate bearing chimeric LPS as well as the htrB mutant exhibited a reduced ability to activate human embryonic kidney 293 (HEK293) cells transfected with TLR4/MD2. LPS from the htrB mutant also had a greatly reduced ability to stimulate interleukin-8 (IL-8) secretion in both endothelial cells and monocytes. In contrast, the activity of LPS from the htrB mutant bacteria expressing the P. gingivalis gene displayed wild-type activity to stimulate IL-8 production from endothelial cells but a reduced ability to stimulate IL-8 secretion from monocytes. The intermediate activation observed in monocytes for the chimeric LPS was similar to the pattern seen in HEK293 cells expressing TLR4/MD2 and CD14. Thus, the presence of a longer-chain fatty acid on E. coli lipid A altered the activity of the LPS in monocytes but not endothelial cell assays and the difference in recognition does not appear to be related to differences in Toll-like receptor utilization.     

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.     

Human MD-2 discrimination of meningococcal lipid A structures and activation of TLR4

MD-2, a eukaryotic accessory protein, is an essential component for the molecular pattern recognition of bacterial endotoxins. MD-2 interacts with lipid A of endotoxins [lipopolysaccharide (LPS) or lipooligosaccharide (LOS)] to activate human toll-like receptor (TLR) 4. The structure of lipid A influences the subsequent activation of human TLR4 and the immune response, but the basis for the discrimination of lipid A structures is unclear. A recombinant human MD-2 (rMD-2) protein was produced in the Pichia pastoris yeast expression system. Human embryonic kidney (HEK293) cells were transfected with human TLR4 and were stimulated with highly purified LOS (0.56 pmol) from Neisseria meningitidis or LPS from other structurally defined bacterial endotoxins in the presence or absence of human rMD-2. Human rMD-2 restored, in a dose-dependent manner, interleukin (IL-8) responsiveness to LOS or LPS in TLR4-transfected HEK293 cells. The interaction of endotoxin with human rMD-2 was then assessed by enzyme-linked immunosorbent assays. Wild-type meningococcal LOS (Wt m LOS) bound human rMD-2, and binding was inhibited by an anti-MD-2 antibody to MD-2 dose-dependently (P < 0.005). Wt m LOS or meningococcal KDO(2)-lipid A had the highest binding affinity for human rMD-2; unglycosylated meningococcal lipid A produced by meningococci with defects in the 3-deoxy-d-manno-2-octulosonic acid (KDO) biosynthesis pathway did not appear to bind human rMD-2 (P < 0.005). The affinity of meningococcal LOS with a penta-acylated lipid A for human rMD-2 was significantly less than that for hexa-acylated LOS (P < 0.05). The hierarchy in the binding affinity of different lipid A structures for human rMD-2 was directly correlated with differences in TLR4 pathway activation and cytokine production by human macrophages.     

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.     

Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance

In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse DeltaTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IkappaB-alpha degradation, and activation of NF-kappaB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-kappaB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes.     

Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells

Leptospira interrogans is a spirochete that is responsible for leptospirosis, a zoonotic disease. This bacterium possesses an unusual LPS that has been shown to use TLR2 instead of TLR4 for signaling in human cells. The structure of its lipid A was recently deciphered. Although its overall hexa-acylated disaccharide backbone is a classical feature of all lipid A forms, the lipid A of L. interrogans is peculiar. In this article, the functional characterization of this lipid A was studied in comparison to whole parental leptospiral LPS in terms of cell activation and use of TLR in murine and human cells. Lipid A from L. interrogans did not coagulate the Limulus hemolymph. Although leptospiral lipid A activated strongly murine RAW cells, it did not activate human monocytic cells. Results obtained from stimulation of peritoneal-elicited macrophages from genetically deficient mice for TLR2 or TLR4 clearly showed that lipid A stimulated the cells through TLR4 recognition, whereas highly purified leptospiral LPS utilized TLR2 as well as TLR4. In vitro experiments with transfected human HEK293 cells confirmed that activation by lipid A occurred only through murine TLR4-MD2 but not through human TLR4-MD2, nor murine or human TLR2. Similar studies with parental leptospiral LPS showed that TLR2/TLR1 were the predominant receptors in human cells, whereas TLR2 but also TLR4 contributed to activation in murine cells. Altogether these results highlight important differences between human and mouse specificity in terms of TLR4-MD2 recognition that may have important consequences for leptospiral LPS sensing and subsequent susceptibility to leptospirosis.     

Negative Regulation of Toll-like Receptor-4 Signaling through the Binding of Glycosylphosphatidylinositol-anchored Glycoprotein,CD14, with the Sialic Acid-binding Lectin,CD33

When monocyte-derived immature dendritic cells (imDCs) were stimulated with LPS in the presence of anti-CD33/Siglec-3 mAb, the production of IL-12 and phosphorylation of NF-κB decreased significantly. The cell surface proteins of imDCs were chemically cross-linked, and CD33-linked proteins were analyzed by SDS-PAGE and immunoblotting. It was CD14 that was found to be cross-linked with CD33. A proximity ligation assay also indicated that CD33 was colocalized with CD14 on the cell surface of imDCs. Sialic acid-dependent binding of CD33 to CD14 was confirmed by a plate assay using recombinant CD33 and CD14. Three types of cells (HEK293T cells expressing the LPS receptor complex (Toll-like receptor (TLR) cells), and the LPS receptor complex plus either wild-type CD33 (TLR/CD33WT cells) or mutated CD33 without sialic acid-binding activity (TLR/CD33RA cells)) were prepared, and then the binding and uptake of LPS were investigated. Although the level of LPS bound on the cell surface was similar among these cells, the uptake of LPS was reduced in TLR/CD33WT cells. A higher level of CD14-bound LPS and a lower level of TLR4-bound LPS were detected in TLR/CD33WT cells compared with the other two cell types, probably due to reduced presentation of LPS from CD14 to TLR4. Phosphorylation of NF-κB after stimulation with LPS was also compared. Wild-type CD33 but not mutated CD33 significantly reduced the phosphorylation of NF-κB. These results suggest that CD14 is an endogenous ligand for CD33 and that ligation of CD33 with CD14 modulates with the presentation of LPS from CD14 to TLR4, leading to down-regulation of TLR4-mediated signaling.     

The CD14 ligands lipoarabinomannan and lipopolysaccharide differ in their requirement for Toll-like receptors

Mammalian Toll-like receptor (TLR) proteins are new members of the IL-1 receptor family that participate in activation of cells by bacteria and bacterial products. Several recent reports indicate that TLR proteins mediate cellular activation by bacterial LPS via a signaling pathway that is largely shared by the type I IL-1 receptor. We previously showed that Chinese hamster ovary (CHO) fibroblasts engineered to express CD14 (CHO/CD14) were responsive to LPS, but not to a distinct CD14 ligand, mycobacterial lipoarabinomannan (LAM). These CHO/CD14 cells were subsequently found to possess a frame-shift mutation within the TLR2 gene which resulted in their inability to express functional TLR2 protein. Thus, we hypothesized that TLR2, but not TLR4, was necessary for LAM signaling. In this paper we show that CHO/CD14 cells engineered to express functional TLR2 protein acquired the ability to be activated by LAM. Similarly, overexpression of TLR2 in murine macrophages conferred enhanced LAM responsiveness. Together, our data demonstrate that the distinct CD14 ligands LAM and LPS utilize different TLR proteins to initiate intracellular signals. These findings suggest a novel receptor signaling paradigm in which the binding of distinct ligands is mediated by a common receptor chain, but cellular activation is initiated via distinct signal-transducing chains that confer ligand specificity. This paradigm contrasts with many cytokine receptor complexes in which receptor specificity is conferred by a unique ligand-binding chain but cellular activation is initiated via shared signal-transducing chains.     

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.     

Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B

LPS, the primary constituent of the outer membrane of Gram-negative bacteria, is recognized by TLR4. Binding of TLR4 to LPS triggers various cell signaling pathways including NF-kappaB activation and reactive oxygen species (ROS) production. In this study, we present the data that LPS-induced ROS generation and NF-kappaB activation are mediated by a direct interaction of TLR4 with (NAD(P)H oxidase 4 (Nox) 4), a protein related to gp91phox (Nox2) of phagocytic cells, in HEK293T cells. Yeast two hybrid and GST pull-down assays indicated that the COOH-terminal region of Nox4 interacted with the cytoplasmic tail of TLR4. Knockdown of Nox4 by transfection of small interference RNA specific to the Nox4 isozyme in HEK293T cells expressing TLR4 along with MD2 and CD14 resulted in inhibition of LPS-induced ROS generation and NF-kappaB activation. Taken together, these results indicate that direct interaction of TLR4 with Nox4 is involved in LPS-mediated ROS generation and NF-kappaB activation.     

Neisseria meningitidis lipid A mutant LPSs function as LPS antagonists in humans by inhibiting TLR 4-dependent cytokine production

Lipopolysaccharide is a major constituent of the outer membrane of Gram-negative bacteria and important in the induction of pro-inflammatory responses. Recently, novel LPS species derived from Neisseria meningitidis H44/76 by insertional inactivation of the lpxL1 and lpxL2 genes have been created with a lipid A portion consisting of five (penta-acylated lpxL1) or four (tetra-acylated lpxL2) fatty acids connected to the glucosamine backbone instead of six fatty acids in the wild-type LPS. We show that these mutant LPS-types are poor inducers of cytokines (tumor-necrosis factor-α, IL-1β, IL-10, IL-RA) in human mononuclear cells. Both penta- and tetra-acylated meningococcal LPSs were able to inhibit cytokine production by wild-type Escherichia coli or meningococcal LPS. Binding of FITC-labelled E. coli LPS TLR4 transfected Chinese hamster ovary (CHO) cells was inhibited by both mutant LPS-types. Experiments with CHO fibroblasts transfected with human CD14 and TLR4 showed that the antagonizing effect was dependent on the expression of human TLR4. In contrast to the situation in humans, lpxL1 LPS has agonistic activity for cytokine production in peritoneal macrophages of DBA mice, and exacerbated arthritis in murine collagen induced arthritis model. N. meningitidis lipid A mutant LPSs lpxL1 and lpxL2 function as LPS antagonists in humans by inhibiting TLR4-dependent cytokine production but have agonistic activity in mice.     

Essential role of Toll-like receptor 2 in macrophage activation by glycogen

We prepared enzymatically synthesized glycogen (ESG) with the same characteristics as natural glycogen and investigated whether the macrophage-stimulating activity of glycogen was related to Toll-like receptors (TLRs), which are important receptors for innate immunity. ESG induced no nuclear factor-kappa B (NF-κB) activity in TLR4/MD-2/CD14-expressed human embryonic kidney 293 (HEK293) reporter cells, whereas this polysaccharide did activate peritoneal exude cells (PECs) derived from TLR4-deficient mice at the same level as those from wild-type (WT) mice. Similarly, ESG did not activate HEK293 cells expressing TLR3, 5, 7, 8 or 9, suggesting that these TLRs were irrelevant to the activity of ESG. In contrast, ESG enhanced the NF-κB activity of TLR2-expressed HEK293 reporter cells in a concentration-dependent manner. Furthermore, the cell-stimulating activity of ESG was remarkably lower for PECs from TLR2-deficient mice compared with those from WT mice. The activity of ESG completely disappeared after treatment with a glycogen-degrading enzyme, indicating that the activity derived from ESG itself and not from contamination with canonical TLR2 ligands such as bacterial lipopeptides. Moreover, it was clarified by ELISA that ESG was directly bound to TLR2. Taken together, these results demonstrated that TLR2 directly recognizes glycogen and that the recognition activates immunocytes such as macrophages to enhance the production of nitric oxide and inflammatory cytokines. In addition, it was suggested that TLR2 could be involved in the glycogen activity in vivo. We propose that glycogen act as an activator to potentiate the host defense through TLR2 on the macrophage.