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.     

Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4

Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-kappaB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-kappaB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-kappaB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-kappaB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.     

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.     

PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN^fl/fl mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3 kinase inhibitors reverse the lymphoproliferative phenotype in vivo.     

Neisseria meningitidis can induce pro-inflammatory cytokine production via pathways independent from CD14 and toll-like receptor 4

Fulminant meningococcal sepsis (FMS) is considered the prototypical Gram-negative sepsis. Lipopolysaccharide (LPS) is thought to be the main toxic element that induces pro-inflammatory cytokine production after interaction with CD14 and toll-like receptor 4 (TLR4). However, there is increasing evidence that LPS is not the sole toxic element of meningococci. The aim of the present study was to determine the role of CD14 and TLR4 in pro-inflammatory cytokine induction by meningococci. To this end, cytokine induction by isolated meningoccal LPS, wild-type N. meningitidis H44/76 (LPS+-meningococci) matched for concentrations of LPS and LPS-deficient N. meningitidis H44/76lpxA (LPS - -meningococci) was studied in human PBMCs and murine peritoneal macrophages (PMs). Pre-incubation of PBMCs with WT14, a monoclonal antibody against CD14, abolished TNF-alpha and IL-1beta induction by E. coli LPS, while cytokine induction by meningococcal LPS was only partially inhibited. When LPS+- and LPS - -meningococci at higher concentrations were used as stimuli, anti-CD14 had a minimal effect. In C3H/HeJ murine PMs, devoid of a functional TLR4, minimal IL-1alpha, IL-6 and TNF-alpha production was seen after stimulation with 10 ng/mL E. coli or meningococcal LPS. However, at higher concentrations (1000 ng LPS/mL) the production of TNF-alpha, but not IL-1alpha or IL-6, occurred also independently of TLR4. The expression of a functional TLR4 in murine PMs had no effect on the cytokine induction by LPS+- or LPS - -meningococci. It is concluded that pro-inflammatory cytokine induction by N. meningitidis can occur independently of CD14 and TLR4.     

Lipopolysaccharides with Acylation Defects Potentiate TLR4 Signaling and Shape T Cell Responses

Lipopolysaccharides or endotoxins are components of Gram-negative enterobacteria that cause septic shock in mammals. However, a LPS carrying hexa-acyl lipid A moieties is highly endotoxic compared to a tetra-acyl LPS and the latter has been considered as an antagonist of hexa-acyl LPS-mediated TLR4 signaling. We investigated the relationship between the structure and the function of bacterial LPS in the context of human and mouse dendritic cell activation. Strikingly, LPS with acylation defects were capable of triggering a strong and early TLR4-dependent DC activation, which in turn led to the activation of the proteasome machinery dampening the pro-inflammatory cytokine secretion. Upon activation with tetra-acyl LPS both mouse and human dendritic cells triggered CD4⁺ T and CD8⁺ T cell responses and, importantly, human myeloid dendritic cells favored the induction of regulatory T cells. Altogether, our data suggest that LPS acylation controlled by pathogenic bacteria might be an important strategy to subvert adaptive immunity.     

Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction

Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4- adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

Differences in the expression of LPS-receptors are not responsible for the sex-specific immune response after trauma and hemorrhagic shock

Several studies demonstrated a sex-specific cytokine secretion by macrophages following trauma-hemorrhage (T-H) and incubation with lipopolysaccharide A (LPS). Although LPS is known to act via the receptors CD14 and TLR4 on macrophages, it remains unknown whether differences in LPS receptor expression in males and females may be responsible for the gender-specific LPS induced cytokine response following (T-H). To study this, male and proestrus female mice (C3H/HeN) were subjected to trauma (laparotomy) followed by hemorrhage or sham operation. At 2 h thereafter, SMphi and PMphi were harvested and cultured for 2 h. The expression of CD14 and TLR4 was measured by flow cytometry on unstimulated SMphi and PMphi as well as after LPS stimulation. The results indicate that the expression of CD14 and TLR4 on SMphi and PMphi from female and male mice was similar in sham-operated animals and after (T-H). Incubation of macrophages with LPS did not alter CD14 and TLR4 expression in the study groups. Thus, the sex specific LPS induced cytokine secretion after (T-H) is not caused by differences in LPS receptor expression on Mphi of male and female mice.     

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.     

The Asp299Gly polymorphism alters TLR4 signaling by interfering with recruitment of MyD88 and TRIF

Asp(299)Gly (D299G) and, to a lesser extent, Thr(399)Ile (T399I) TLR4 polymorphisms have been associated with gram-negative sepsis and other infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of the D299G and T399I polymorphisms on TLR4 expression, interactions with myeloid differentiation factor 2 (MD2), LPS binding, and LPS-mediated activation of the MyD88- and Toll/IL-1R resistance domain-containing adapter inducing IFN-β (TRIF) signaling pathways. Complementation of human embryonic kidney 293/CD14/MD2 transfectants with wild-type (WT) or mutant yellow fluorescent protein-tagged TLR4 variants revealed comparable total TLR4 expression, TLR4-MD2 interactions, and LPS binding. FACS analyses with anti-TLR4 Ab showed only minimal changes in the cell-surface levels of the D299G TLR4. Cells transfected with D299G TLR4 exhibited impaired LPS-induced phosphorylation of p38 and TANK-binding kinase 1, activation of NF-κB and IFN regulatory factor 3, and induction of IL-8 and IFN-β mRNA, whereas T399I TLR4 did not cause statistically significant inhibition. In contrast to WT TLR4, expression of the D299G mutants in TLR4(-/-) mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-β mRNA. Coimmunoprecipitation revealed diminished LPS-driven interaction of MyD88 and TRIF with the D299G TLR4 species, in contrast to robust adapter recruitment exhibited by WT TLR4. Thus, the D299G polymorphism compromises recruitment of MyD88 and TRIF to TLR4 without affecting TLR4 expression, TLR4-MD2 interaction, or LPS binding, suggesting that it interferes with TLR4 dimerization and assembly of intracellular docking platforms for adapter recruitment.     

Proinflammatory phenotype of vascular smooth muscle cells: role of efficient Toll-like receptor 4 signaling

Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerotic lesions. In this study, we tested whether TLR4 signaling promotes a proinflammatory phenotype in human and mouse arterial smooth muscle cells (SMC), characterized by increased cytokine and chemokine synthesis and increased TLR expression. Human arterial SMC were found to express mRNA encoding TLR4 and the TLR4-associated molecules MD-2 and CD14 but not TLR2 mRNA. Mouse aortic SMC, on the other hand, expressed both TLR2 and TLR4 mRNA constitutively. Human SMC derived from the coronary artery, but not those from the pulmonary artery, were found to express cell surface-associated CD14. Low concentrations (ng/ml) of Escherichia coli LPS, the prototypical TLR4 agonist, markedly stimulated extracellular regulated kinase 1/2 (ERK1/2) activity, induced release of monocyte-chemoattractant protein-1 (MCP-1) and interleukin (IL)-6, and stimulated IL-1alpha expression in human aortic SMC, and exogenous CD14 enhanced these effects. Expression of a dominant negative form of TLR4 in human SMC attenuated LPS-induced ERK1/2 and MCP-1 release. LPS was a potent inducer of NF-kappaB activity, ERK1/2 phosphorylation, MCP-1 release, and TLR2 mRNA expression in wild-type mice but not in TLR4-signaling deficient mouse aortic SMC. These studies show that TLR4 signaling promotes a proinflammatory phenotype in vascular smooth muscle cells (VSMC) and suggest that VSMC may potentially play an active role in vascular inflammation via the release of chemokines, proinflammatory cytokines, and increased expression of TLR2.     

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.     

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.     

PECAM-1 ligation negatively regulates TLR4 signaling in macrophages

Uncontrolled TLR4 signaling may induce excessive production of proinflammatory cytokines and lead to harmful inflammation; therefore, negative regulation of TLR4 signaling attracts much attention now. PECAM-1, a member of Ig-ITIM family, can mediate inhibitory signals in T cells and B cells. However, the role and the mechanisms of PECAM-1 in the regulation of TLR4-mediated LPS response in macrophages remain unclear. In this study, we demonstrate that PECAM-1 ligation with CD38-Fc fusion protein negatively regulates LPS-induced proinflammatory cytokine TNF-alpha, IL-6, and IFN-beta production by inhibiting JNK, NF-kappaB, and IFN regulatory factor 3 activation in macrophages. In addition, PECAM-1 ligation-recruited Src homology region 2 domain-containing phosphatase 1 (SHP-1) and Src homology region 2 domain-containing phosphatase 2 (SHP-2) may be involved in the inhibitory effect of PECAM-1 on TLR4 signaling. Consistently, silencing of PECAM-1 enhances the macrophage response to LPS stimulation. Taken together with the data that PECAM-1 is constitutively expressed in macrophages and its expression is up-regulated by LPS stimulation, PECAM-1 might function as a feedback negative regulator of LPS inflammatory response in macrophages. This study may provide a potential target for intervention of inflammatory diseases.     

Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression

Monocytes/macrophages exposed to LPS show reduced responses to second stimulation with LPS, which is termed LPS tolerance. In this study, we investigated molecular mechanism of LPS tolerance in macrophages. Mouse peritoneal macrophages pre-exposed to LPS exhibited reduced production of inflammatory cytokines in a time- and dose-dependent manner. Activation of neither IL-1 receptor-associated kinase nor NF-kappaB was observed in macrophages that became tolerant by LPS pretreatment, indicating that the proximal event in Toll-like receptor 4 (TLR4)-MyD88-dependent signaling is affected in tolerant macrophages. Although TLR4 mRNA expression significantly decreased within a few hours of LPS pretreatment and returned to the original level at 24 h, the surface TLR4 expression began to decrease within 1 h, with a gradual decrease after that, and remained suppressed over 24 h. A decrease in inflammatory cytokine production in tolerant macrophages well correlates with down-regulation of the surface TLR4 expression, which may explain one of the mechanisms for LPS tolerance.