anti-TLR4 mAb Research Articles

Uncarinic acid C plus IFN-γ generates monocyte-derived dendritic cells and induces a potent Th1 polarization with capacity to migrate

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DC used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferon’s (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to either URC alone, or in combination with TNF-α, IFN-α or IFN-γ, and thereafter co-cultured with naïve T cells. We found that the expression levels of CD1a, CD83 and HLA-DR on URC-primed DC were influenced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. Moreover, the production of IL-12p70 by URC-primed DC was enhanced by IFN-γ. IL-12p70 production by URC-primed DC alone was influenced following treatment with anti-TLR4 mAb, but not DC differentiated with URC plus IFN-γ. URC plus IFN-γ-primed DC induced a substantial increase in the secretion of IFN-γ by T cells, which is dependent on IL-12 secretion. DC maturated with URC plus IFN-γ had an intermediate migratory capacity towards CCL19 and CCL21. In addition, the expression levels of CCR7 on URC-primed DC were enhanced by IFN-γ. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α, and IFN-α. These results suggest that the enhancement of Th1 cells polarization to URC-primed DC induced by IFN-γ depends on the activation of IL-12p70 and independent on TLR4. DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy.

The involvement of TLR2 in cytokine and reactive oxygen species (ROS) production by PBMCs in response toLeishmania majorphosphoglycans (PGs)

In the present study, we show for the first time that lipophosphoglycan (LPG) stimulated cytokine production by human peripheral blood mononuclear cells is also mediated via Toll-like receptor (TLR2). In addition, in order to verify if TLR2 is involved in recognition of the purified PGs, neutralizing mAbs against TLR2 and TLR4 were used to treat the cells before being stimulated with PGs. We found strong Th1-promoting cytokines induced by sLPG but not by mLPG which was blocked by presence of anti-TLR2 mAb. This finding reveals a mechanism by which the first encounter and recognition of L. major promastigotes by mLPG after interaction with TLR2 provides a cytokine milieu for consequent Th2 differentiation. Moreover, having shown the strong induction of Th1-promoting cytokines and low production of IL-10 in response to sLPG might have vaccine implication since it is recognized by TLR2 providing signals to professional antigen presenting cells that reside in the skin to promote effective T cell responses against Leishmania infection. In addition, it was shown that purified mLPG and sLPG activate reactive oxygen species (ROS) production which is also blocked by anti-TLR2 but not by anti-TLR4. However, no inhibition was seen in PPG-induced cytokine and ROS production in the presence of anti-TLR2 and anti-TLR4, implying involvement of other receptors.

Legionella lipoprotein activates toll-like receptor 2 and induces cytokine production and expression of costimulatory molecules in peritoneal macrophages

Legionella bacterium, an intracellular pathogen of mononuclear phagocytes, causes acute fatal pneumonia, especially in patients with impaired cellular immune responses. Until recently, however, the toll-like receptor (TLR) engagement of bacterial proteins derived from Legionella is uncertain. We previously showed that a 19-kDa highly conserved peptidoglycan-associated lipoprotein (PAL) of Legionella pneumophila induced the PAL-specific B cell and T cell responses in mice. In this study, we observed that the rPAL antigen of L. pneumophila, as an effector molecule, activated murine macrophages via TLR2 and produced proinflammatory cytokines such as IL-6 and TNF-alpha. In both BALB/c and TLR4-deficient C3H/HeJ mice, pretreatment of macrophages with anti-TLR2 mAb showed severely impaired cytokine production in response to the rPAL. In addition, in vitro the rPAL treatment increased the cell surface expression of CD40, CD80, CD86 and MHC I/II molecules. We further showed that the synthetic CpG-oligodeoxynucleotides (CpG ODN) coadministered with the rPAL enhanced IL-12 and IL-6 production and expression of CD40, CD80 and MHC II compared to the rPAL treatment alone. In conclusions, these results indicate that Legionella PAL might activate macrophages via a TLR2-dependent mechanism which thus induce cytokine production and expression of costimulatory and MHC molecules.

Brucella lipoproteins mimic dendritic cell maturation induced by Brucella abortus

Infection with Brucella abortus induces a pro-inflammatory response that drives T cell responses toward a Th1 profile. The mechanism by which this bacterium triggers this response is unknown. Dendritic cells (DC) are crucial mediators at the host-pathogen interface and are potent Th1-inducing antigen-presenting cells. Thus, we examined the mechanism whereby B. abortus stimulate human DC maturation. B. abortus-infected DC increased the expression of CD86, CD80, CCR7, CD83, MHCII, MHCI and CD40 and induced the production of TNF-α, IL-6, IL-10 and IL-12. Both phenomena were not dependent on bacterial viability since they were also induced by heat-killed B. abortus (HKBA). B. abortus LPS was unable to induce markers up-regulation or cytokine production. We next investigated the capacity of the outer membrane protein 19 (Omp19) as a B. abortus lipoprotein model to induce DC maturation. Lipidated Omp19 (L-Omp19), but not its unlipidated form, increased the expression of cell surface markers and the secretion of cytokines. L-Omp19-matured DC also have decreased endocytic activity and displayed enhanced T cell stimulatory activity in a MLR. Pre-incubation of DC with anti-TLR2 mAb blocked L-Omp19-mediated cytokine production. These results demonstrate that B. abortus lipoproteins can stimulate DC maturation providing a mechanism by which these bacteria generate a Th1-type immune response.

TLR4-induced IFN-γ production increases TLR2 sensitivity and drives Gram-negative sepsis in mice

Gram-negative bacterial infection is a major cause of sepsis and septic shock. An important inducer of inflammation underlying both syndromes is the cellular recognition of bacterial products through pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). We identified a novel antagonistic mAb (named 1A6) that recognizes the extracellular portion of the TLR4–MD-2 complex. If applied to mice before infection with clinical isolates of Salmonella enterica or Escherichia coli and subsequent antibiotic therapy, 1A6 prevented otherwise fatal shock, whereas application of 1A6 after infection was ineffective. In contrast, coapplication of 1A6 and an anti-TLR2 mAb up to 4 h after infection with Gram-negative bacteria, in combination with the start of antibiotic therapy (mimicking clinical conditions), provided robust protection. Consistent with our findings in mice, dual blockade of TLR2 and TLR4 inhibited TNF-α release from human peripheral blood mononuclear cells upon Gram-negative bacterial infection/antibiotic therapy. Both murine splenocytes and human PBMCs released IFN-γ in a TLR4-dependent manner, leading to enhanced surface TLR2 expression and sensitivity for TLR2 ligands. Our results implicate TLR2 as an important, TLR4-driven sensor of Gram-negative bacterial infection and provide a rationale for blockade of both TLRs, in addition to antibiotic therapy for the treatment of Gram-negative bacterial infection.

Differential Regulation of DC Function by Siphonodiol

Siphonodiol is a polyacetylene diol isolated from marine sponges Callyspongia sp. We demonstrate that the effect of Siphonodiol on the phenotypic and functional maturation of human monocyte derived DC in vitro. Human monocytes were exposed to Siphonodiol alone, or in combination with LPS and thereafter co-cultured with naïve T cells. The expression levels of CD1a, CD80, CD83, CD86 and HLA-DR on LPS-primed DC were partially enhanced by Siphonodiol. Siphonodiol augmented the T cell stimulatory capacity in an allo MLR to LPS-primed DC. Siphonodiol dose-dependently enhanced the production of IL-12p70 by LPS-primed DC and this cytokine production was inhibited by anti-TLR4 mAb. IFN-γ secretion from naïve T cells co-cultured with DC differentiated with LPS was augmented by Siphonodiol. These results suggest that the enhancement of Th1 cells polarization to LPS-primed DC induced by Siphonodiol depends on TLR4 and via the activation of IL-12p70.

Introduction of Zwitterionic Motifs into Bacterial Polysaccharides Generates TLR2 Agonists Able to Activate APCs

It was shown previously that bacterial polysaccharides (PS), which naturally contain both positive and negative charges, are able to activate T cells and APCs. However, the vast majority of bacterial PS are anionic and do not have these properties. In this study, we show that chemical introduction of positive charges into naturally anionic bacterial PS confers to the resulting zwitterionic PS (ZPS) the ability to activate pure human monocytes, monocyte-derived dendritic cells, and mouse bone marrow-derived dendritic cells, as do natural bacterial ZPS. Cells are induced to up-regulate MHC class II and costimulatory molecules and to produce cytokines. In mixed monocyte-T cell cocultures, ZPS induce MHC II-dependent T cell proliferation and up-regulation of activation markers. These stimulatory qualities of ZPS disappear when the positive charge is chemically removed from the molecules and thus the zwitterionic motif is destroyed. The ability of natural and chemically derived ZPS to activate APCs can be blocked by anti-TLR2 mAbs, and TLR2 transfectants show reporter gene transcription upon incubation with ZPS. In conclusion, the generation of a zwitterionic motif in bacterial PS confers the ability to activate both APCs and T cells. This finding has important implications for the design of novel polysaccharide vaccines.

Peptidoglycan and mannose-based molecular patterns trigger the arachidonic acid cascade in human polymorphonuclear leukocytes

The release of arachidonic acid (AA) in response to microorganism-derived products acting on pattern recognition receptors (PRR) was assayed in human polymorphonuclear leukocytes (PMN). Peptidoglycan (PGN) and mannan were found to be strong inducers of AA metabolism, as they produced the release of AA at a similar extent to that produced by agonists of pathophysiological relevance such as complement-coated zymosan particles and IgG immune complexes. In sharp contrast, lipoteichoic acid, LPS, muramyldipeptide, and the bacterial lipoprotein mimic palmitoyl-3-cysteine-serine-lysine-4 failed to do so. Leukotriene B4 and PGE2 were synthesized in response to mannan and PGN, thus suggesting that the lipoxygenase and the cyclooxygenase routes are operative in human PMN in response to pathogen-associated molecular patterns (PAMP). Analysis of the lipid extracts of supernatants and cell pellets as well as pharmacological studies with the calpain inhibitor calpeptin and the cytosolic phospholipase A2 (PLA2) inhibitor pyrrolidine-1 showed the dependence of AA release on cytosolic PLA2-catalyzed reactions. The effect of PGN was not inhibited by previous treatment with anti-TLR2 mAb, thus suggesting a nonarchetypal involvement of the TLR2 signaling route and/or participation of other receptors. Because of the abundance of mannose-based and PGN-containing PAMP in fungi and bacteria and the wide array of PRR in human PMN, these finding disclose a role of prime importance for PAMP and PRR in AA metabolism in the inflammatory response mediated by PMN.

A Mechanism for Exacerbation of Chronic ITP by Infection: Toll-Like Receptor 4 (TLR4) Activation Enhances Antibody-Mediated Platelet Phagocytosis by Human Macrophages.

Infection often triggers an exacerbation of chronic ITP. The precise mechanism for the decreased platelet count associated with infection is not completely understood. However, interference with megakaryopoiesis is often invoked, and increased production of autoantibodies is also possible. We hypothesized that stimulation of innate immunity could enhance phagocytosis in the reticuloendothelial system and thus contribute to an exacerbation of chronic ITP in the setting of infection. The innate immune response involves activation of Toll-like receptors (TLRs) that initiate intracellular signaling through MyD88 kinase as well as through other incompletely characterized MyD88-independent pathways. We have previously shown that Syk is a crucial signaling tyrosine kinase involved in Fcγ receptor-mediated platelet phagocytosis and that Syk-specific dsRNA inhibits antibody-mediated platelet phagocytosis. Furthermore, the ligand binding requirements for Syk are not stringently limited to classical tandem immunoreceptor tyrosine-based activation motifs. We speculated that TLR4 might employ Syk tyrosine kinase intracellular signaling in human macrophages as a mechanism to enhance immune platelet phagocytosis. The present study was undertaken to address the following questions: Does lipopolysaccharide (LPS) stimulation, an in vitro model for innate immunity triggered by gram-negative bacterial infection, augment antibody-dependent phagocytosis of platelets? Does TLR4 associate with Syk tyrosine kinase in macrophages? Does LPS stimulation induce phosphorylation of Syk?Methods: Antibody-mediated phagocytosis of platelets by THP-1 (human macrophage) cells in culture was assayed by co-incubation of THP-1 cells with or without LPS stimulation at 37°C with platelets that were labeled with 5-(and 6-) carboxyfluorescein diacetate mixed isomers (CFDA) in the presence of human serum containing antihuman GPIIIa IgG antibody. Association of TLR4 with the tyrosine kinase, Syk, was studied in THP-1 cells by immunoprecipitation and immunoblotting after ligation with anti-TLR4 monoclonal antibody (mAb) or stimulation with LPS. Phosphorylation of TLR4-associated Syk after LPS stimulation was studied by immunoprecipitation and immunodetection with anti-TLR4, antiphosphotyrosine (PY99), and anti-Syk mAbs.Results: Antibody-mediated platelet phagocytosis by THP-1 cells was enhanced by 50% after LPS exposure. In five experiments a mean of 21.8% ± 4.1% CFDA-labeled platelets were ingested by THP-1 cells upon antiplatelet antibody exposure in the absence of LPS, while 32.8% ± 6.3% were phagocytosed after LPS (p<0.001.) Syk protein was coimmunoprecipitated with TLR4 from THP-1 cell lysates by anti-TLR4 mAb. Furthermore, intracellular Syk became phosphorylated upon LPS stimulation of THP-1 cells.Conclusions: These studies support the hypothesis that activation of TLR4 in the setting of infection contributes to enhanced antibody-mediated platelet phagocytosis by macrophages and thus exacerbates chronic ITP. Syk becomes phosphorylated upon TLR4 stimulation of macrophages and may play a role in signal transduction triggered by the innate immune system through TLR4. These novel findings may have an impact on the understanding and management of immune thrombocytopenia in the setting of infectious diseases and offer a new therapeutic target for chronic ITP.

Porphyromonas gingivalis fimbriae induce CD14+CD16+ dendritic cell phenotype via TLR2

Dendritic cells (DCs) play a critical role in activation of T cells as well as shaping immune responses. DCs express a pattern of Toll-like receptors (TLRs) to recognize invading pathogens and to initiate specific immune responses. In this study, we investigated the effect of Porphyromonas gingivalis fimbriae on the phenotype and function of human peripheral blood DCs (PBDCs) in comparison to TLR2 and TLR4 ligand, peptidoglycan (PGN) from Staphylococcus aureus and Escherichia coli O55:B5 LPS, respectively. P. gingivalis fimbriae and PGN preferentially upregulated CD14 and CD16 expression on immature PBDCs (CD14−CD16−), although E. coli LPS did not alter the expression of these molecules. Fimbriae-stimulated DCs also exhibited a different profile of cytokine production and surface molecule expression as compared with E. coli LPS-stimulated DCs. Pretreatment of PBDCs with anti-TLR2 monoclonal antibody (mAb), but not with anti-TLR4 mAb, abrogated the upregulation of CD14 and CD16 on fimbriae and PGN-treated DCs. These results indicate that different TLR signaling affects the mature DC phenotype and function and is thus crucial to the regulation of immunity to the pathogen.

The Secreted Peptidyl Prolyl cis,trans-Isomerase HP0175 of Helicobacter pylori Induces Apoptosis of Gastric Epithelial Cells in a TLR4- and Apoptosis Signal-Regulating Kinase 1-Dependent Manner

Apoptosis contributes to the pathology of gastric epithelial cell damage that characterizes Helicobacter pylori infection. The secreted peptidyl prolyl cis, trans-isomerase of H. pylori, HP0175 executed apoptosis of the gastric epithelial cell line AGS in a dose- and time-dependent manner. The effect of HP0175 was confirmed by generating an isogenic mutant of H. pylori disrupted in the HP0175 gene. The apoptosis-inducing ability of this mutant was impaired compared with that of the wild type. The effect of HP0175 was mediated through TLR4. Preincubation of the gastric epithelial cell line AGS with anti-TLR4 mAb inhibited apoptosis induced by HP0175. Downstream of TLR4, apoptosis signal-regulating kinase 1 activated MAPK p38, leading to the caspase 8-dependent cleavage of Bid, its translocation to the mitochondria, mitochondrial pore formation, cytochrome c release, and activation of caspases 9 and 3. We show for the first time that a secreted bacterial Ag with peptidyl prolyl cis,trans-isomerase activity signals through TLR4, and that this Ag executes gastric epithelial cell apoptosis through a signaling pathway in which TLR4 and apoptosis signal-regulating kinase 1 are central players.

Study on the relationship between heat shock protein 70 and toll-like receptor-4 of monocytes.

To explore the relation between human heat shock protein 70 (hsp70) and TLR4 in human monocytes in vitro, human monocytes were stimulated with various concentrations of HSP70, and TNF-alpha production in supernatants was measured by ELISA. Pre-incubated with or without anti-TLR4 mAb, and stimulated with hsp70 (5.0 microg/ml), NF-kappaB p65 of human monocytes in different time points were detected by immunohistochemistry and monocyte surface expression of TLR4 was measured by flow cytometry. After the human monocytes were pre-incubated with various concentrations of anti-TLR4 and stimulated with hsp70 (5.0 microg/ml), TNF-alpha production in supernatants was measured. The results showed that hsp70 enhanced NF-kappaB activation, which was clearly inhibited by anti-TLR4, with the positive cell ratios being 67.44%, 39.17%, 31.56% and 28.05 %, respectively. TLR4 was rapidly down-regulated in the presence of hsp70. MFI of TLR4 on monocytes in different time points were 87.77 +/- 5.38, 78.16 +/- 6.01 and 45.17 +/- 4.97 (P<0.05), 26.98 +/- 5.83 (P<0.01), respectively. Moreover, hsp70-induced TNF-alpha production by human monocytes was inhibited by anti-TLR4. It is suggested that TLR4 is involved in the hsp70-mediated activation of innate immunity.

Staphylococcal enterotoxins condition cells of the innate immune system for Toll-like receptor 4 stimulation

In this report we examined overlap between superantigen (SAg) and Toll-like receptor 4 (TLR4) stimulation of the innate immune system. Before in vivo stimulation we found that mouse splenic DCs expressed unexpectedly low levels of surface TLR4 compared to macrophages. In response to LPS, TLR4 gene expression in fractionated spleen cells was downregulated. By comparison, surface TLR4 staining with the Sa15-21 mAb showed little downregulation, and the anti-TLR4 MTS510 mAb showed decreased staining, suggesting that LPS was bound to TLR4 at the time points examined. Interestingly, SAg stimulation induced decreased TLR4 staining as measured by the MTS510 mAb, even though the TLR4 gene was not downregulated. Nevertheless, LPS potently induced DCs to produce TNF and IL-12, but SAg did not, even though they efficiently activated DCs. Notwithstanding, in vivo stimulation with staphylococcal enterotoxin SAg conditioned the innate immune system to hyper-respond to various pathogen-associated molecular patterns (PAMPs). Specifically, pre-priming with SAg enhanced LPS-mediated DC synthesis of TNF and IL-12. Thus, SAgs may exert their pathogenesis on the host by conditioning DCs, in a T cell activation dependent manner to potentiate responses to PAMPs.

Expression of the unspliced tenascin-C isoform (Tn-C L) in urothelial carcinoma of the urinary bladder (UC)

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DC used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferon’s (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to either URC alone, or in combination with TNF-α, IFN-α or IFN-γ, and thereafter co-cultured with naïve T cells. We found that the expression levels of CD1a, CD83 and HLA-DR on URC-primed DC were influenced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. Moreover, the production of IL-12p70 by URC-primed DC was enhanced by IFN-γ. IL-12p70 production by URC-primed DC alone was influenced following treatment with anti-TLR4 mAb, but not DC differentiated with URC plus IFN-γ. URC plus IFN-γ-primed DC induced a substantial increase in the secretion of IFN-γ by T cells, which is dependent on IL-12 secretion. DC maturated with URC plus IFN-γ had an intermediate migratory capacity towards CCL19 and CCL21. In addition, the expression levels of CCR7 on URC-primed DC were enhanced by IFN-γ. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α, and IFN-α. These results suggest that the enhancement of Th1 cells polarization to URC-primed DC induced by IFN-γ depends on the activation of IL-12p70 and independent on TLR4. DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy.

204 Impaired toll-like receptor expression in chronic hepatitis B

Dendritic cells (DCs) play a critical role in activation of T cells as well as shaping immune responses. DCs express a pattern of Toll-like receptors (TLRs) to recognize invading pathogens and to initiate specific immune responses. In this study, we investigated the effect of Porphyromonas gingivalis fimbriae on the phenotype and function of human peripheral blood DCs (PBDCs) in comparison to TLR2 and TLR4 ligand, peptidoglycan (PGN) from Staphylococcus aureus and Escherichia coli O55:B5 LPS, respectively. P. gingivalis fimbriae and PGN preferentially upregulated CD14 and CD16 expression on immature PBDCs (CD14−CD16−), although E. coli LPS did not alter the expression of these molecules. Fimbriae-stimulated DCs also exhibited a different profile of cytokine production and surface molecule expression as compared with E. coli LPS-stimulated DCs. Pretreatment of PBDCs with anti-TLR2 monoclonal antibody (mAb), but not with anti-TLR4 mAb, abrogated the upregulation of CD14 and CD16 on fimbriae and PGN-treated DCs. These results indicate that different TLR signaling affects the mature DC phenotype and function and is thus crucial to the regulation of immunity to the pathogen.

Possible Involvement of Toll-Like Receptor 4 in Endothelial Cell Activation of Larger Vessels in Response to Lipopolysaccharide

Toll-like receptors (TLRs) serve as recognition and signaling elements for bacterial substances. To examine the role of TLRs in endothelial cells of larger vessels in lipopolysaccharide (LPS)-induced signaling, the expression and function of TLRs in human umbilical vein endothelial cells (HUVEC) were analyzed. A high level of TLR4 mRNA expression was found in HUVEC, human peripheral blood mononuclear cells (PBMC) and human monocyte cell line THP-1 cells. Little or no TLR2 mRNA expression was observed in HUVEC. In contrast, strong TLR2 mRNA expression was observed in PBMC and THP-1 cells. Moderate and high levels of TLR1 mRNA expression were found in HUVEC, PBMC and THP-1 cells, respectively. TLR3 mRNA expression was moderate in PBMC but weak in HUVEC and THP-1 cells. Little or no TLR5 and RP105 mRNA expression was observed in HUVEC, whereas a moderate level was detected in PBMC and THP-1 cells. The LPS-induced E-selectin expression in HUVEC was significantly inhibited by pretreatment with an anti-TLR4 mAb. Preincubation of HUVEC with an anti- TLR4 mAb significantly reduced the LPS-induced IL-6 production. LPS induced E-selectin and IL-6 production by HUVEC only in the presence of human serum, suggesting the involvement of soluble CD14. Anti-CD14 mAb strongly inhibited the LPS-induced E-selectin and IL-6 production by HUVEC. The inhibition with the concomitant treatment with anti-TLR4 and anti-CD14 mAbs was stronger than that with anti-CD14 mAb only, although it was slight. These results show that TLR4 in the presence of soluble CD14 plays a major role in the signaling of LPS in endothelial cells of larger vessels.

A polymorphism in the toll-like receptor 2 is associated with IL-12 production from monocyte in lepromatous leprosy.

Toll-like receptor 2 (TLR2) is critical in the immune response to mycobacterial infections, and the mutations in the TLR2 have been shown to confer the susceptibility to infection with mycobacteria. We previously reported the detection of TLR2 Arg677Trp mutation in lepromatous leprosy. Here, the events triggered by TLR2 in response to cell lysate of Mycobacterium leprae(MLL), the causative agent of leprosy, were investigated. Upon stimulation with MLL, monocytes produced TNF-alpha and Interleukin-12 (IL-12), which play a role in the innate immune response to infection. Anti-TLR2 mAb blocked greater than 50% of the MLL-induced production of IL-12. We also performed the functional study on TLR2 by measurement of IL-12 production in serum and monocytes from leprosy patients with TLR2 mutation (Arg677Trp). The monocytes obtained from patients with the TLR2 mutation, in comparison to the wild-type TLR2, is significantly less responsive to MLL. It was also confirmed that patients with TLR2 mutation showed significantly lower serum levels of IL-12, in comparing with TLR2 wild-type. Our results reveal that innate immune response of monocytes against M. lepraeis mediated by TLR2, and suggest that the mutation in the intracellular domain of TLR2 gene is associated with IL-12 production in lepromatous leprosy.

Two lipoproteins extracted from Escherichia coli K-12 LCD25 lipopolysaccharide are the major components responsible for Toll-like receptor 2-mediated signaling.

Toll-like receptor 2 (TLR2)-mediated cell activation induced by commercial preparations of LPS was recently shown to arise from impurities whose identities are not known. In this work, we determined the molecules responsible for TLR2-mediated cell activation in LPS derived from Escherichia coli K-12 strain LCD25. When LCD25 LPS was phenol extracted, two proteins capable of TLR2-mediated cell activation were purified and identified as E. coli lipoproteins. We cloned, expressed, and purified these two lipoproteins, Lip19 and Lip12. Lip19 or Lip12 activated TNF-alpha production from RAW264.7 and THP-1 cells in a TLR2-dependent manner. However, neither Lip19 nor Lip12 activated HUVECs, which lack endogenous TLR2. Additionally, IkappaB kinase beta and c-Jun N-terminal kinase 1 activation in THP-1 cells induced by Lip19 or Lip12 was observed. TLR2 activation by Lip19 and Lip12 in HEK293 cells was blocked by inhibitory anti-TLR2 mAbs. The unlipidated mutants, Lip19-C19S and Lip12-C21S, in which the NH(2)-terminal cysteine was substituted by serine, lost their ability to activate TLR2-transfected HEK 293 cells. Taken together, these results demonstrate that two lipoproteins constitute the major contaminants responsible for TLR2-mediated cell activation in E. coli LCD25 LPS.

Dependence of bacterial protein adhesins on toll-like receptors for proinflammatory cytokine induction.

Toll-like receptors (TLRs) are important signal transducers that mediate inflammatory reactions induced by microbes through pattern recognition of virulence molecules such as lipopolysaccharide (LPS) and lipoproteins. We investigated whether proinflammatory cytokine responses induced by certain bacterial protein adhesins may also depend on TLRs. In differentiated THP-1 mononuclear cells stimulated by LPS-free recombinant fimbrillin (rFimA) from Porphyromonas gingivalis, cytokine release was abrogated by monoclonal antibodies (MAbs) to CD14 and TLR4 but not to TLR2. Similar experiments using anti-beta2 integrin MAbs suggested that beta2 integrins (CD11/CD18) also play a role in cytokine induction by rFimA or native fimbriae. Minor fimbriae (distinct from the fimA-encoded major fimbriae) of P. gingivalis induced proinflammatory cytokine release in a CD14- and TLR2-dependent mode. Cytokine induction by BspA, a leucine-rich repeat protein from Bacteroides forsythus, depended heavily on CD14 and TLR2. We also found that the ability of the streptococcal protein AgI/II to stimulate cytokine release depended partially on CD14 and TLR4, and the AgI/II segment that possibly interacts with these receptors was identified as its N-terminal saliva-binding region. When THP-1 cells were exposed to rFimA for 24 h, surface expression of CD14 and CD18 was decreased and the cells became hyporesponsive to cytokine induction by a second challenge with rFimA. However, tolerance induction was abolished when the THP-1 cells were pretreated with rFimA in the presence of either anti-CD14 MAb or anti-TLR4 MAb. Induction of cross-tolerance between rFimA and LPS correlated with downregulation of the pattern recognition receptors involved. Our data suggest that the CD14-TLR2/4 system is involved in cytokine production and tolerance induction upon interaction with certain proinflammatory bacterial protein adhesins.

Human gingival CD14(+) fibroblasts primed with gamma interferon increase production of interleukin-8 in response to lipopolysaccharide through up-regulation of membrane CD14 and MyD88 mRNA expression.

Gamma interferon (IFN-gamma)-primed human gingival fibroblasts (HGF) have been shown to produce higher levels of interleukin-8 (IL-8) upon stimulation with bacterial products and inflammatory cytokines than nonprimed controls. In this study, we examined whether priming of HGF with IFN-gamma up-regulates IL-8 production by the cells in response to purified lipopolysaccharide (LPS). The priming effect of IFN-gamma was clearly observed in the high-CD14-expressing (CD14(high)) HGF but not in the low-CD14-expressing (CD14(low)) HGF. The CD14(high) HGF were most effectively primed with IFN-gamma (1,000 IU/ml) for 72 h. To elucidate the mechanism of the priming effects of IFN-gamma for the LPS response by HGF, we examined whether IFN-gamma regulated expression of CD14, Toll-like receptor 2 (TLR2), TLR4, MD-2, and MyD88, all of which are molecules suggested to be associated with LPS signaling. In CD14(high) HGF, IFN-gamma markedly up-regulated CD14 and MyD88 but not TLR4 protein and MD-2 mRNA expression, while in CD14(low) HGF, IFN-gamma slightly increased MyD88 and scarcely affected CD14, TLR4 protein, and MD-2 mRNA levels. LPS-induced IL-8 production by IFN-gamma-primed CD14(high) HGF was significantly inhibited by monoclonal antibodies (MAbs) against CD14 and TLR4, but not by an anti-TLR2 MAb. These findings suggested that IFN-gamma primed CD14(high) HGF to enhance production of IL-8 in response to LPS through augmentation of the CD14-TLR system, where the presence of membrane CD14 was indispensable for the response of HGF to LPS.