Neisseria meningitidis type C capsular polysaccharide inhibits lipooligosaccharide-induced cell activation by binding to CD14.

Encapsulated Neisseria meningitidis can invade mucosal barriers and cause systemic diseases. Activation of the innate immune system by conserved meningococcal molecules such as lipooligosaccharides (LOS) is essential for the generation of an effective host immune response. Here we show that the type C capsular polysaccharide of N. meningitidis (MCPS) inhibited LOS-induced interleukin-6 and TNF-alpha secretion from monocytes, and blocked the maturation of dendritic cells induced by LOS, while the capsular polysaccharide from group B streptococcus type III and t(4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll had no such effect. MCPS also inhibited the LOS-induced NF-kappaB activation and phosphorylation of signalling molecules such as ERK1/2, p38 and Jun N-terminal kinase. In a direct binding assay, MCPS manifested a concentration-dependent binding to recombinant lipoprotein binding protein and CD14, the two members of the LOS receptor complex. In addition, the binding of LOS to CD14 and lipopolysaccharide binding protein was inhibited by MCPS. We established that MCPS binding to CD14 is responsible for the inhibition of LOS-mediated cell activation because MCPS inhibition of LOS was reversed when access amounts of CD14 were added to culture media of HEK293 cells expressing TLR4 and MD-2, and the magnitude of recovery in LOS stimulation correlated with the increase in CD14 concentration. These results suggest a new virulence property of meningococcal capsular polysaccharides.

Induction of dendritic cell maturation by pertussis toxin and its B subunit differentially initiate Toll-like receptor 4-dependent signal transduction pathways.

OBJECTIVE: Pertussis toxin (PT) has the capacity to activate dendritic cells (DCs) for the augmentation of cell-mediated immune responses. To investigate the mechanism(s) by which PT activates DCs, we investigated the effects of PT and its B-oligomer (PTB) on the maturation of human and mouse DCs and determined whether PT could act as a pathogen-associated molecular pattern to activate one of the Toll-like receptors (TLRs). METHODS: The effects of PT and PTB on the maturation of human and mouse DCs were analyzed in terms of surface marker expression, cytokine production, antigen-presenting capacity, and intracellular signaling. The participation of TLR4 in PT-induced signaling was determined by comparing the effect of PT on DCs derived from TLR4-deficient and wild-type mice, as well as by measuring PT-induced NF-kappaB activation in HEK293 cells transiently transfected to express various TLRs. RESULTS: Although both promoted phenotypic and functional maturation DCs, however, unlike PT that induced DC production of interleukin (IL)-6, tumor necrosis factor-alpha, IL-12, and interferon-inducible protein, PTB was capable of stimulating the production of interferon-inducible protein. Bone marrow-derived DCs from C3H/HeJ mice with defective TLR-4 alleles were unresponsive to PT and PTB, whereas DCs from C3H/HeN mice responded. In addition, PT induced NF-kappaB activation and IL-8 production in HEK293 cells transfected with a combination of TLR4 and MD2 but not in nontransfected or TLR2-transfected HEK293 cells. Comparison of the patterns of cytokine induction and intracellular signaling events in DCs treated by PT and PTB revealed that although PT, like lipopolysaccharide, triggered both MyD88-dependent and -independent pathways, PTB preferentially triggered MyD88-independent pathways. Interestingly, mouse splenocyte proliferation in response to PT and PTB was only partially dependent on TLR4. CONCLUSION: The data identify PT as another pathogen-associated molecular pattern that induces DC maturation in a TLR4-dependent manner. Unlike PT, which triggers both MyD88-dependent and -independent pathways, PTB only triggers the MyD88-independent pathway in DCs.

Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling.

TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in the extracellular domain of TLR4 (Asp(299)Gly and Thr(399)Ile) with decreased LPS responsiveness. To analyze the molecular basis for diminished responsiveness, site-specific mutations (singly or coexpressed) were introduced into untagged and epitope (Flag)-tagged wild-type (WT) TLR4 expression vectors to permit a direct comparison of WT and mutant signal transduction. Coexpression of WT TLR4, CD14, and MD-2 expression vectors in HEK293T cells was first optimized to achieve optimal LPS-induced NF-kappaB reporter gene expression. Surprisingly, transfection of cells with MD-2 at high input levels often used in the literature suppressed LPS-induced signaling, whereas supraoptimal CD14 levels did not. Under conditions where WT and polymorphic variants were comparably expressed, significant differences in NF-kappaB activation were observed in response to LPS and two structurally unrelated TLR4 agonists, chlamydial heat shock protein 60 and RSV F protein, with the double, cosegregating mutant TLR4 exhibiting the greatest deficiency. Overexpression of Flag-tagged WT and mutant vectors at input levels resulting in agonist-independent signaling led to equivalent NF-kappaB signaling, suggesting that these mutations in TLR4 affect appropriate interaction with agonist or coreceptor. These data provide new insights into the importance of stoichiometry among the components of the TLR4/MD-2/CD14 complex. A structural model that accounts for the diminished responsiveness of mutant TLR4 polymorphisms to structurally unrelated TLR4 agonists is proposed.

Human conjunctival epithelial cells lack lipopolysaccharide responsiveness due to deficient expression of MD2 but respond after interferon-gamma priming or soluble MD2 supplementation.

Inflammatory responses to Gram-positive and Gram-negative bacterial cell wall components are initiated by Toll-like receptor 2 (TLR2) and TLR4, respectively. Therefore, the existence of functionally active TLR2 and TLR4 in human conjunctival epithelial cells (HCEC) are critical for the effective host defense against bacterial infections in the eye. We examined the ability of HCEC to respond to TLR4 ligand, lipopolysaccharide (LPS), or TLR2 ligands, lipoteichoic acid (LTA) and peptidoglycan (PGN) using the Chang conjunctival epithelial cell line and the primary conjunctival epithelial cell line (IOBA-NHC) as in vitro models. Incubation of Chang cells with LPS (1 to 1,000 ng/ml) failed to stimulate IL-6 production where as stimulation with LTA or PGN resulted in marked increases in IL-6 production. Semi-quantitative RT-PCR and immunofluorescence analyses showed that Chang cells express TLR2 and TLR4 mRNA and proteins. However, these cells expressed little or no mRNA encoding MD2, an accessory molecule required for TLR4 signaling. Incubation of Chang epithelial cells with interferon-gamma (IFNgamma), but not TNF-alpha, stimulated MD2 mRNA expression and restored LPS responsiveness. In addition, when Chang cell cultures were supplemented with soluble MD2, LPS was able to stimulate IL-6 production. The lack of LPS response, deficient expression of MD2, and induction of MD2 expression and LPS response after IFNgamma priming, were also evident in IOBA-NHC cells. These results demonstrate that HCEC lack LPS responsiveness due to deficient expression of MD2 and that the response can be restored by IFN-gamma priming or MD2 supplementation.

TLR9 is localized in the endoplasmic reticulum prior to stimulation.

In mammals, 10 TLRs recognize conserved pathogen-associated molecular patterns, resulting in the induction of inflammatory innate immune responses. One of these, TLR9, is activated intracellularly by bacterial DNA and synthetic oligodeoxynucleotides (ODN), containing unmethylated CpG dinucleotides. Following treatment with CpG ODN, TLR9 is found in lysosome-associated membrane protein type 1-positive lysosomes, and we asked which intracellular compartment contains TLR9 before CpG exposure. Surprisingly, we found by microscopy and supporting biochemical evidence that both transfected and endogenously expressed human TLR9 is retained in the endoplasmic reticulum. By contrast, human TLR4 trafficked to the cell surface, indicating that endoplasmic reticulum retention is not a property common to all TLRs. Because TLR9 is observed in endocytic vesicles following exposure to CpG ODN, our data indicate that a special mechanism must exist for translocating TLR9 to the signaling compartments that contain the CpG DNA.

A complex of soluble MD-2 and lipopolysaccharide serves as an activating ligand for Toll-like receptor 4.

MD-2, a glycoprotein that is essential for the innate response to lipopolysaccharide (LPS), binds to both LPS and the extracellular domain of Toll-like receptor 4 (TLR4). Following synthesis, MD-2 is either secreted directly into the medium as a soluble, active protein, or binds directly to TLR4 in the endoplasmic reticulum before migrating to the cell surface. Here we investigate the function of the secreted form of MD-2. We show that secreted MD-2 irreversibly loses activity over a 24-h period at physiological temperature. LPS, but not lipid A, prevents this loss in activity by forming a stable complex with MD-2, in a CD14-dependent process. Once formed, the stable MD-2.LPS complex activates TLR4 in the absence of CD14 or free LPS indicating that the activating ligand of TLR4 is the MD-2.LPS complex. Finally we show that the MD-2.LPS complex, but not LPS alone, induces epithelial cells, which express TLR4 but not MD-2, to secrete interleukin-6 and interleukin-8. We propose that the soluble MD-2.LPS complex plays a crucial role in the LPS response by activating epithelial and other TLR4(+)/MD-2(-) cells in the inflammatory microenvironment.

Heat-killed Brucella abortus induces TNF and IL-12p40 by distinct MyD88-dependent pathways: TNF, unlike IL-12p40 secretion, is Toll-like receptor 2 dependent.

Cattle and humans are susceptible to infection with the Gram-negative intracellular bacterium Brucella abortus. Heat-killed B. abortus (HKBA) is a strong Th1 adjuvant and carrier. Previously, we have demonstrated that dendritic cells produce IL-12 in response to HKBA stimulation. In the present study, we use knockout mice and in vitro reconstitution assays to examine the contribution of signaling by Toll-like receptors (TLRs) and their immediate downstream signaling initiator, myeloid differentiation protein MyD88, in the activation following stimulation by HKBA. Our results show that HKBA-mediated induction of IL-12p40 and TNF is dependent on the adapter molecule MyD88. To identify the TLR involved in HKBA recognition, we examined HKBA responses in TLR2- and TLR4-deficient animals. TNF responses to HKBA were TLR4 independent; however, the response in TLR2-deficient mice was significantly delayed and reduced, although not completely abolished. Studies using Chinese hamster ovary/CD14 reporter cell lines stably transfected with either human TLR2 or human TLR4 confirmed the results seen with knockout mice, namely TLR2, but not TLR4, can mediate cellular activation by HKBA. In addition, human embryonic kidney 293 cells, which do not respond to HKBA, were made responsive by transfecting TLR2, but not TLR4 or TLR9. Taken together, our data demonstrate that MyD88-dependent pathways are crucial for activation by HKBA and that TLR2 plays a role in TNF, but not IL-12p40 pathways activated by this microbial product.

The role of disulfide bonds in the assembly and function of MD-2.

MD-2 is a secreted glycoprotein that binds to the extracellular domain of Toll-like receptor 4 (TLR4) and is required for the activation of TLR4 by lipopolysaccharide (LPS). The protein contains seven Cys residues and consists of a heterogeneous collection of disulfide-linked oligomers. To investigate the role of sulfhydryls in MD-2 structure and function, we created 17 single and multiple Cys substitution mutants. All of the MD-2 mutant proteins, including one totally lacking Cys residues, were secreted and stable. SDSPAGE analyses indicated that most Cys residues could participate in oligomer formation and that no single Cys residue was required for oligomerization. Of the single Cys substitutions, only C95S and C105S failed to confer LPS responsiveness on TLR4 when mutant and TLR4 were cotransfected into cells expressing an NF-kappaB reporter plasmid. Surprisingly, substitution of both C95 and C105 partially restored activity. Structural analyses revealed that C95 and C105 formed an intrachain disulfide bond, whereas C95 by itself produced an inactive dimer. In contrast to the cotransfection experiments, only WT MD-2 conferred responsiveness to LPS when secreted proteins were added directly to TLR4 reporter cells. Our data are consistent with a model in which most, possibly all sulfhydryls lie on the surface of a stable MD-2 core structure where they form both intra- and interchain disulfide bridges. These disulfide bonds produce a heterogeneous array of oligomers, including some species that can form an active complex with TLR4.

Cutting edge: histamine inhibits IFN-alpha release from plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (DC) are professional APC and a major source of type I IFN following viral infection. We previously showed that histamine alters the cytokine profiles of maturing monocyte-derived DC resulting in a change from Th1 to Th2 in their T cell polarizing function. In this study, we show that human plasmacytoid DC, activated by either CpG oligodeoxynucleotides or viral infection, also respond to histamine through H2 receptors, leading to a marked down-regulation of IFN-alpha and TNF-alpha and a moderate switch in their capacity to polarize naive T cells. Our findings provide an explanation for low levels of type I IFN frequently observed in atopic individuals.

IL-4-induced arginase 1 suppresses alloreactive T cells in tumor-bearing mice.

We previously demonstrated that a specialized subset of immature myeloid cells migrate to lymphoid organs as a result of tumor growth or immune stress, where they suppress B and T cell responses to Ags. Although NO was required for suppression of mitogen activation of T cells by myeloid suppressor cells (MSC), it was not required for suppression of allogenic responses. In this study, we describe a novel mechanism used by MSC to block T cell proliferation and CTL generation in response to alloantigen, which is mediated by the enzyme arginase 1 (Arg1). We show that Arg1 increases superoxide production in myeloid cells through a pathway that likely utilizes the reductase domain of inducible NO synthase (iNOS), and that superoxide is required for Arg1-dependent suppression of T cell function. Arg1 is induced by IL-4 in freshly isolated MSC or cloned MSC lines, and is therefore up-regulated by activated Th2, but not Th1, cells. In contrast, iNOS is induced by IFN-gamma and Th1 cells. Because Arg1 and iNOS share L-arginine as a common substrate, our results indicate that L-arginine metabolism in myeloid cells is a potential target for selective intervention in reversing myeloid-induced dysfunction in tumor-bearing hosts.

Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2.

beta-Defensins are small antimicrobial peptides of the innate immune system produced in response to microbial infection of mucosal tissue and skin. We demonstrate that murine beta-defensin 2 (mDF2beta) acts directly on immature dendritic cells as an endogenous ligand for Toll-like receptor 4 (TLR-4), inducing up-regulation of costimulatory molecules and dendritic cell maturation. These events, in turn, trigger robust, type 1 polarized adaptive immune responses in vivo, suggesting that mDF2beta may play an important role in immunosurveillance against pathogens and, possibly, self antigens or tumor antigens.

Toll-like receptor 1 inhibits Toll-like receptor 4 signaling in endothelial cells.

Toll-like receptor 4 (TLR4) is the signal-transducing component of the LPS recognition complex and is essential for LPS-induced septic shock. Here we demonstrate that TLR1 has the capacity to abrogate TLR4 signaling. Human microvascular endothelial cells express TLR4 but not TLR1 and respond to LPS through TLR4. The ability of these cells to respond to LPS was lost, however, when they were transfected with TLR1. Inhibition was specific for TLR1 because TL5 failed to block TLR4 function. Moreover, TLR1 had no effect upon TNF-alpha signaling, indicating that TLR1 operated at a step upstream of the convergence between the two pathways. Inhibition of TLR4 signaling was mediated by the extracellular, but not cytoplasmic domain of TLR1. In addition, TLR1 physically associated with TLR4 in co-precipitation experiments. These findings suggest that TLR1 might restrain potentially dangerous innate response to LPS by binding to TLR4 and preventing the formation of active signaling complexes.

Brain macrophage surface marker expression with HIV-1 infection and drug abuse: a preliminary study.

GOAL: To determine the heterogeneity of surface marker expression of macrophages in the temporal lobe of patients who died with AIDS who were also Drug Abusers (DAs). We studied the expression of macrophage surface markers CD11c, CD14, CD68, and HLA-DR and T cell surface markers CD4, and CD8. BACKGROUND: The macrophage is the prime locus for HIV-1-associated pathology, is the most frequently infected cell in the brain, and has the highest virus load compared to other cells. We previously described the heterogeneity of macrophage surface marker expression and performed morphometric analysis in peripheral nerves of patients who died from AIDS compared to HIV-1 negative individuals. We showed that the HIV-related neuropathy in AIDS is a multifocal process. It is similarly important to determine the expression of macrophage surface markers in brain. Temporal lobe tissue was selected for this preliminary study because we previously found elevated HIV-1 proviral DNA load and inflammatory processes in this neuroanatomic location for subjects who died with AIDS. There is a high prevalence of Drug Abuse in Miami, Florida, associated with AIDS that may interactively affect HIV-associated pathology. METHODS: Temporal lobe tissue was examined from 17 HIV-1-seropositive patients (4 with Drug Abuse and 13 without Drug Abuse) and 11 HIV-seronegative individuals (5 with Drug Abuse and 6 without Drug Abuse). Standard immunohistochemistry utilized alkaline phosphatase conjugate secondary antibody and fuchsin substrate. RESULTS: We found that HIV-1 infection and the interaction of HIV-1 infection and Drug Abuse produced changes in macrophage surface marker expression. Macrophage surface markers, CD11c, CD14, CD68, and HLA-DR, and T-cell marker CD4 were increased with statistical significance due to HIV-1 infection (all p < .001) whereas CD8 remained unchanged. Changes due to Drug Abuse alone were not significant. Interaction of Drug Abuse and HIV-infected individuals showed increased expression of CD68 (p = .011), HLA-DR (p = .001), CD4 (p = .027), and CD8 (p = .016). CONCLUSION: Drug Abuse and HIV-1 infection are factors that differentially and interactively result in multiple macrophages surface marker effects. In HIV-1 infected individuals, Drug Abuse stimulates surface marker expression. Since brain macrophage surface makers do not change uniformly as a result of Drug Abuse and HIV infection, these cells may be heterogeneous and contain sub-types (sub-sets). It remains to be determined which macrophage sub-types may be most pathognomic for pathology.