Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells

In the present report we have analyzed whether human normal cord blood-derived mast cells (CBMC) could interact with bacterial products, especially lipopolysaccharide (LPS) from Escherichia coli and peptidoglycan (PGN) from Staphylococcus aureus, known as Toll-like receptor (TLR) 4 and TLR2 agonists, respectively. We found that both LPS and PGN induced significant release of not only tumor necrosis factor-alpha (TNF-alpha), but also IL-5, IL-10 and IL-13 by human mast cells (MC). We also established that the stimulation of CBMC with LPS or with PGN is mediated through interactions with TLR4 or with TLR2, respectively. Thus, our data indicate that activation of either TLR2 or TLR4 pathway may lead to a pro-Th2 immune response. However, the release of TNF-alpha induced by LPS, conversely to PGN, required the priming of CBMC by IL-4 and the presence of serum components, in particular soluble CD14. Of interest, stimulation by PGN, but not by LPS, induced release of histamine by human MC. Altogether, these findings provide the first evidence that human MC differentially respond towards bacterial components, and that their responses depend on TLR pathways and reveal human specificities in the pattern of cytokine production.     

Caspase-dependent and -independent apoptosis of mast cells induced by withdrawal of IL-3 is prevented by Toll-like receptor 4-mediated lipopolysaccharide stimulation

IL-3-dependent mucosal-like mast cells undergo apoptosis upon withdrawal of IL-3. Generally, the apoptosis is mediated by the activation of caspases and inhibited by addition of the pan-caspase inhibitors z-VAD-FMK or BOC-D-FMK. However, DNA fragmentation, a typical characteristic of apoptosis, is not inhibited by z-VAD-FMK or BOC-D-FMK in mast cell apoptosis. In this study, we demonstrate that the apoptosis of mast cells is mediated by both caspase-dependent and -independent mechanisms. The caspase-independent apoptosis is mediated by the translocation of endonuclease G from mitochondria into nuclei. Withdrawal of IL-3 caused down-regulation of Bcl-xL, resulting in a drop in mitochondrial membrane transition potential followed by the release of cytochrome c and endonuclease G from mitochondria. However, stimulation of mast cells through Toll-like receptor 4 (TLR4) by lipopolysaccharide prevented mast cell apoptosis by inducing expression of Bcl-xL. Moreover, the activation of mast cells by LPS is enhanced in the presence of IFN-gamma, which up-regulates the expression of cell surface TLR4. Taken together, these observations provide evidence that mast cells play important roles not only in allergic reactions but also in innate immunity recognizing enterobacteria through TLR4, and are regulated differently from allergic inflammation by Th1 cytokines.     

Increased Toll-like receptor 9 expression indicates adverse prognosis in oesophageal adenocarcinoma

Kauppila J H, Takala H, Selander K S, Lehenkari P P, Saarnio J & Karttunen T J
(2011) Histopathology 59 , 643–649 Increased Toll‐like receptor 9 expression indicates adverse prognosis in oesophageal adenocarcinoma Aims: Toll‐like receptor 9 (TLR‐9) is a cellular DNA receptor that has been linked previously to invasion in various cancers. The aim of this study was to investigate TLR‐9 expression and its possible association with prognosis in oesophageal adenocarcinoma. Methods and results: Immunohistochemical TLR‐9 expression was graded in clinical specimens (n = 76) of oesophageal adenocarcinoma. The TLR‐9 immunostaining intensity was compared with tumour grade, stage and indicators of proliferation, apoptosis and tumour vascular supply. High TLR‐9 expression correlated with advanced tumour stage, tumour unresectability, poor differentiation and high proliferation. Strong immunoreactivity of TLR‐9 also indicated poor overall survival. Conclusions: High TLR‐9 expression is associated with poor differentiation, a high proliferation rate and disseminated disease. Accordingly, increased TLR‐9 expression may contribute to the growth and metastatic properties of oesophageal adenocarcinoma.     

Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components

Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-kappaB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components.     

Toll-like receptor 6-independent signaling by diacylated lipopeptides

Bacterial lipopeptides are strong immune modulators that activate early host responses after infection as well as initiating adjuvant effects on the adaptive immune system. These lipopeptides induce signaling in cells of the immune system through Toll-like receptor 2 (TLR2)-TLR1 or TLR2-TLR6 heteromers. So far it has been thought that triacylated lipopeptides, such as the synthetic N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3)-CSK4, signal through TLR2-TLR1 heteromers, whereas diacylated lipopeptides, like the macrophage-activating lipopeptide from Mycoplasma fermentans (MALP2) or S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam2)-CGNNDESNISFKEK, induce signaling through TLR2-TLR6 heteromers. Using new synthetic lipopeptide derivatives we addressed the contribution of the lipid and, in particular, the peptide moieties with respect to TLR2 heteromer usage. In contrast to the current model of receptor usage, not only triacylated lipopeptides, but also diacylated lipopeptides like Pam2CSK4 and the elongated MALP2 analog Pam2CGNNDESNISFKEK-SK4 (MALP2-SK4) induced B lymphocyte proliferation and TNF-alpha secretion in macrophages in a TLR6-independent manner as determined with cells from TLR6-deficient mice. Our results indicate that both the lipid and the N-terminal peptides of lipoproteins contribute to the specificity of recognition by TLR2 heteromers and are responsible for the ligand-receptor interaction on host cells.     

Increased expression of Toll-like receptor 4 in peripheral blood leucocytes and serum levels of some cytokines in patients with ankylosing spondylitis

Toll-like receptor 4 (TLR4) is a member of the Toll-like receptor family, which can bridge innate and adaptive immune responses. Activation of the TLR4 signalling pathway may induce the release of proinflammatory cytokines such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-12, which was considered to play an important role in pathogenesis of immune-mediated diseases. Ankylosing spondylitis (AS) is an immune-mediated disease whose aetiology remains unknown. The aim of the study was to investigate the expression of TLR4 and serum TNF-alpha, IL-12 and soluble tumour necrosis factor-related apoptosis-inducing ligand (sTRAIL) level in AS patients. The results indicated that TLR4 protein and mRNA levels were significantly higher in AS patients than in healthy controls; however, there was no significant difference between human leucocyte antigen (HLA)-B27-positive and -negative AS patients, as well as serum levels of TNF-alpha, IL-12 and sTRAIL. In addition, in HLA-B27-positive AS patients, TLR4 level showed close associations with the cytokines and laboratory parameters of disease activity [erythrocyte sedimentation rate (ESR) and plasma C-reactive protein (CRP)], respectively. Similarly, the strong associations between the cytokines or between IL-12 and ESR or CRP were observed in HLA-B27-positive AS patients. Interestingly, in HLA-B27-positive AS patients, TNF-alpha correlated significantly with ESR, but did not with CRP. In contrast, sTRAIL correlated with CRP, but did not with ESR. Among HLA-B27-negative patients, no close correlation was found. In our study, it was suggested that the abnormal activation of TLR4 signalling and serum TNF-alpha, IL-12 and sTRAIL may play a key role in the development and progression of AS, which may be dependent on the status of HLA-B27 antigen.     

Deviation from major codons in the Toll-like receptor genes is associated with low Toll-like receptor expression

Microbial structures activate Toll-like receptors (TLRs) and TLR-mediated cell signalling elicits and regulates host immunity. Most TLRs are poorly expressed but the underlying expression mechanism is not clear. Examination TLR sequences revealed that most human TLR genes deviated from using major human codons. CD14 resembles TLRs in sequence but its gene preferentially uses major codons. Indeed, CD14 expression on monocytes was higher than expression of TLR1 and TLR2. The TLR9 gene is abundant in major codons and it also showed higher expression than TLR1, TLR2 and TLR7 in transfected 293T cells. Change of the 5'-end 302 base pairs of the TLR2 sequence into major human codons markedly increased TLR2 expression, which led to increased TLR2-mediated constitutive nuclear factor-kappaB activation. Change of the 5'-end 381 base pairs of the CD14 sequence into prevalent TLR codons markedly reduced CD14 expression. These results collectively show that the deviation of TLR sequences from using major codons dictates the low TLR expression and this may protect the host against excessive inflammation and tissue damages.     

Direct role of NF-kappaB activation in Toll-like receptor-triggered HLA-DRA expression

Microbial components, such as DNA containing immunostimulatory CpG motifs (CpG-DNA) and lipopolysaccharides (LPS), elicit the cell surface expression of MHC class II (MHC-II) through Toll-like receptor (TLR)/IL-1R. Here, we show that CpG-DNA and LPS induce expression of the HLA-DRA in the human B cell line, RPMI 8226. Ectopic expression of the dominant negative mutant of CIITA and RNA interference targeting the CIITA gene indicate that CIITA activation is not enough for the maximal MHC-II expression induced by CpG-DNA and LPS. Additionally, nuclear factor (NF)-kappaB activation is required for the CpG-DNA-activated and LPS-activated HLA-DRA expression, whereas IFN-gamma-induced MHC-II expression depends on CIITA rather than on NF-kappaB. Comprehensive mutant analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays, reveal that the functional interaction of NF-kappaB with the promoter element is necessary for the TLR-mediated HLA-DRA induction by CpG-DNA and LPS. This novel mechanism provides the regulation of MHC-II gene expression with complexity and functional diversity.     

Binding specificity of Toll-like receptor cytoplasmic domains

MyD88 participates in signal transduction by binding to the cytoplasmic Toll/IL-1 receptor (TIR) domains of activated Toll-like receptors (TLR). Yeast two-hybrid experiments reveal that the TIR domains of human TLR differ in their ability to associate with MyD88: The TIR of TLR2 binds to MyD88 but the TIR of the closely related TLR1, 6, or 10 do not. Using chimeric TIR domains, we define the critical region responsible for differential MyD88 binding, and use a computational analysis of the critical region to reveal the amino acids that differ between MyD88 binders and non-binders. Remarkably, a single missense mutation created in TLR1 (N672D) confers on it the ability to bind MyD88, without affecting its association with other proteins. Mutations identified as critical for MyD88 binding also affect signaling of TLR pairs in mammalian cells. To investigate the difference between MyD88 binders and non-binders, we identify novel interacting proteins for each cytoplasmic domain of TLR1, 2, 6, and 10. For example, heat shock protein (HSP)60 binds to TLR1 but not to TLR2, and HSP60 and MyD88 appear to bind the same region of the TIR domain. In summary, interactions between the TLR, MyD88, and novel associated proteins have been characterized.     

Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-gamma and provide a mechanism for enhanced synovial chemokine levels in septic arthritis

The CXC chemokine IFN-gamma-inducible protein-10 (IP-10/CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T cells and natural killer cells. Peripheral blood mononuclear cells (PBMC) produce low but significant amounts of IP-10/CXCL10 protein upon stimulation with double-stranded (ds) RNA, the Toll-like receptor 3 (TLR3) ligand. IFN-gamma is a superior IP-10/CXCL10inducer. The bacterial TLR4 and TLR2 ligands, LPS and peptidoglycan (PGN), inhibit IFN-gamma- or dsRNA-dependent IP-10/CXCL10 production in PBMC, whereas IL-8/CXCL8 production was enhanced. In fibroblasts a different picture emerges with IFN-gamma inducing moderate and dsRNA provoking strong IP-10/CXCL10 production. Furthermore, treatment of fibroblasts with IFN-gamma in combination with bacterial LPS or PGN results in a synergistic production of IP-10/CXCL10 and IL-8/CXCL8. The synergistic induction of IP-10/CXCL10 in fibroblasts is reflected by significantly enhanced IP-10/CXCL10 concentrations in synovial fluids of septic compared to osteoarthritis patients to reach on average higher levels than those of IL-8/CXCL8. These high amounts of IP-10/CXCL10 produced by connective tissue fibroblasts not only attract CXCR3 expressing activated Th1 cells and natural killer cells to sites of infection but may also antagonize the CCR3 dependent attraction of Th2 lymphocytes and exert CXCR3-independent, defensin-like antibacterial activity.     

CC chemokine receptor 4 modulates Toll-like receptor 9-mediated innate immunity and signaling

The present study addressed the modulatory role of CC chemokine receptor 4 (CCR4) in Toll-like receptor (TLR) 9-mediated innate immunity and explored the underlying molecular mechanisms. Our results demonstrated that CCR4-deficient mice were resistant to both septic peritonitis induced by cecal ligation and puncture (CLP) and CpG DNA/D-galactosamine-induced shock. In bone marrow-derived macrophages (BMMPhi) from CLP-treated CCR4-deficient mice, TLR9-mediated pathways of MAPK/AP-1, PI3K/Akt, and IkappaB kinase (IKK)/NF-kappaB were impaired compared to wild-type (WT) cells. While TLR9 expression was not altered, the intensity of internalized CpG DNA was increased in CCR4-deficient macrophages when compared to WT macrophages. Pharmacological inhibitor studies revealed that impaired activation of JNK, PI3K/Akt, and/or IKK/NF-kappaB could be responsible for decreased proinflammatory cytokine expression in CCR4-deficient macrophages. Interestingly, the CCR4-deficient BMMPhi exhibited an alternatively activated (M2) phenotype and the impaired TLR9-mediated signal transduction responses in CCR4-deficient cells were similar to the signaling responses observed in WT BMMPhi skewed to an alternatively activated phenotype. These results indicate that macrophages deficient in CCR4 impart a regulatory influence on TLR9-mediated innate immunity.     

The role of Toll-like receptor 4 versus interleukin-12 in immunity to respiratory syncytial virus

Toll-like receptors (TLR) and IL-12 represent key elements of innate immunity. Using C57BL/10 ScCr mice it was shown that TLR4 is important for control of infection with respiratory syncytial virus (RSV). Since these mice have an additional defect in the IL-12R, we reinvestigated immunity to RSV in several C57BL/10 and BALB/c mouse strains lacking a functional TLR4, a functional IL-12-IL-12R interaction or both. In the absence of a functional IL-12 axis, early virus control was impaired in C57BL/10 mice, but not in BALB/c mice. By contrast, TLR4 had no impact on RSV elimination. Pulmonary NK cell recruitment was impaired in IL-12 deficient BALB/c mice and NK cytotoxicity was reduced in IL-12/IL-12R-deficient mice of both genetic backgrounds. Absence of TLR4 had no impact on NK cell recruitment or NK activity nor on recruitment of other pulmonary inflammatory cells. Activation of RSV-specific T cell immunity, including T cell mediated immunopathology, was normal in all mutant strains. These findings clearly argue against a significant role for TLR4 and define a limited role for IL-12 in primary murine RSV infection.     

Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens

Rapid overproduction of proinflammatory cytokines are characteristic of sepsis. CD14(dim)CD16(+) monocytes are thought to be major producers of cytokine and have been shown to be elevated in septic patients. Toll-like receptors (TLR) are pattern recognition receptors important in mediating the innate immune response and their activation can lead to production of cytokines. Using whole blood culture and flow cytometry we have investigated TLR2 and TLR4 regulation after stimulation with sepsis-relevant antigens [lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB) and peptidoglycan (PGN)]. The percentage of CD14(dim)CD16(+) monocyte population expanded at 20 h post-stimulation, after a rise in tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 at 2 h. A strong positive correlation between the percentage of CD14(dim)CD16(+) monocytes and secreted TNF-alpha was demonstrated (r = 0.72). Furthermore, we were able to induce expansion of the CD14(dim)CD16(+) population to approximately 35% of all monocytes with the addition of recombinant TNF-alpha to the whole blood culture. TLR4 was found to be expressed 2.5 times higher on CD14(dim)CD16(+) compared to CD14(+) CD16(-) monocytes, while TLR2 expression was similar in both subpopulations. The CD14(dim)CD16(+) and CD14(+) CD16(-) monocyte populations were different in their response to various antigens. LPS down-regulated TLR4 by 4.9 times in CD16(+) monocytes compared to only 2.3 times in CD16(-) monocytes at 2 h. LPS was able to up-regulate TLR2 by 6.2 times after 2 h, with no difference between the subpopulations. LPS further up-regulated TLR2 by 18.4 times after 20 h only in the CD14(+) CD16(-) population. PGN and SEB induced no significant changes in TLR2 or TLR4 expression. We hypothesize that following exposure to bacterial antigens, subsequent TNF-alpha drives a differentiation of monocytes into a CD14(dim)CD16(+) subpopulation.     

Toll-like receptor-mediated tyrosine phosphorylation of paxillin via MyD88-dependent and -independent pathways

Toll-like receptor (TLR)-mediated recognition of pathogens represents one of the most important mechanisms of innate immunity. A proximal signaling event of TLR is the direct binding of an adaptor protein MyD88 to TLR and recruitment of the IL-1R-associated kinase (IRAK). In the present study, we examined the effect of several TLR ligands on protein tyrosine phosphorylation in rat macrophages. Macrophage-activating lipopeptide-2 kDa (MALP2) and lipoarabinomannan were used as activators of TLR2, while lipopolysaccharides (LPS) and lipoteichoic acid were used as TLR4 ligands. All these ligands induced tyrosine phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) and its substrate paxillin, an integrin-associated focal adhesion adaptor protein, in the macrophages. PP2, an inhibitor of Src family tyrosine kinases, prevented the TLR-induced phosphorylation of paxillin and Pyk2 without affecting TLR-induced IRAK activation. MALP2 failed to induce paxillin phosphorylation in the macrophages from MyD88-knockout mice. In contrast, the effect of LPS weakened, but was still observed even in the MyD88-deficient cells. Thus, TLR regulate the function of paxillin in an Src family-dependent mechanism through both MyD88-dependent and MyD88-independent pathways.     

血小板表达TLR4调节LPS刺激血小板释放细胞因子的作用

目的 证实人类血小板表面是否表达Toll样受体4(Toll like receptor 4,TLR4),探讨脂多糖(lipopolysaccharides,LPS)刺激血小板后TLR4表达变化及其对细胞因子白细胞介素-8(interlukine-8,IL-8)、β血小板球蛋白(β-thromboglobulin,β-TG)、可溶性CD40配体(soluble CD40 ligand,sCD40L)释放的凋节作用.方法 流式细胞技术测定不同浓度LPS刺激后血小板TLR4表达情况;ELISA法测定TLR4单克隆抗体封闭或不封闭LPS刺激的人血小板释放的细胞因子IL-8、β-TG、sCD40L的变化.结果 人血小板表达TLR4,LPS刺激后血小板对TLR4表达检出率降低(P<0.01),血小板对sCD40L、β-TG释放显著升高(P<0.001),但LPS在1～5μg/ml内不同浓度之间,sCD40L、β-TG浓度差异无统计学意义(P>0.05),LPS诱发血小板释放sCD40L、β-TG增加效应被TLR4单克隆抗体削弱.LPS刺激前后,反应体系中IL-8浓度无明显变化(P>0.05).结论 血小板TLR4与LPS相互作用诱发血小板大量释放sCD40L、β-TG,而血小板对IL-8的释放不依赖于TLR4-LPS途径.     

Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8 alpha+ DC correlates with unresponsiveness to imidazoquinolines

Toll-like receptors (TLR) recognize microbial and viral patterns and activate dendritic cells (DC). TLR distribution among human DC subsets is heterogeneous: plasmacytoid DC (PDC) express TLR1, 7 and 9, while other DC types do not express TLR9 but express other TLR. Here, we report that mRNA for most TLR is expressed at similar levels by murine splenic DC sub-types, including PDC, but that TLR3 is preferentially expressed by CD8 alpha(+) DC while TLR5 and TLR7 are selectively absent from the same subset. Consistent with the latter, TLR7 ligand activates CD8 alpha(-) DC and PDC, but not CD8 alpha(+) DC as measured by survival ex vivo, up-regulation of surface markers and production of IL-12p40. These data suggest that the dichotomy in TLR expression between plasmacytoid and non-plasmacytoid DC is not conserved between species. However, lack of TLR7 expression could restrict the involvement of CD8 alpha(+) DC in recognition of certain mouse pathogens.     

Macrophage migration inhibitory factor contributes to the development of acute dextran sulphate sodium-induced colitis in Toll-like receptor 4 knockout mice

Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharides, plays an important role in the innate immune response. In this study, we investigated the role of TLR4 in the development of experimental colitis with regard to the biological actions of macrophage migration inhibitory factor (MIF) using TLR4 null ((-/-)) mice. TLR4(-/-) mice were given 2% dextran sulphate sodium (DSS) in drinking water to induce colitis, which was clinically and histologically as severe as that seen in wild-type (WT) mice. The level of tumour necrosis factor (TNF)-alpha in colon tissues was increased in WT mice but unchanged in TLR4(-/-) mice. The level of myeloperoxidase (MPO) activity in colon tissues was increased by DSS administration in both TLR4(-/-) and WT mice. The expression of MIF was up-regulated in the colons of TLR4(-/-) mice with acute DSS-induced colitis. An anti-MIF antibody significantly suppressed colitis and elevation of matrix metalloproteinase-13 in TLR4(-/-) mice. The current results obtained from TLR4(-/-) mice provide evidence that MIF plays a critical role in the development of acute DSS-induced colitis.     

Heat shock up-regulates expression of Toll-like receptor-2 and Toll-like receptor-4 in human monocytes via p38 kinase signal pathway

Summary Heat stress can alert innate immunity by inducing stress proteins such as heat-shock proteins (HSPs). However, it remains unclear whether heat stress affects the activation of antigen-presenting cell (APC) in response to pathogen-associated molecule patterns (PAMPs) by directly regulating pathogen recognition receptors (PRRs). As an important kind of PRRs, Toll-like receptors (TLRs) play critical roles in the activation of immune system. In this study, we demonstrated that heat shock up-regulated the expression of HSP70 as well as TLR2 and TLR4 in monocytes. The induction of TLRs was prior to that of HSP70, which suggesting the up-regulation of TLR2 and TLR4 might be independent of the induction of HSP70. Heat shock activated p38 kinase, extracellular signal-related kinase (ERK) and nuclear factor-kappa B (NF-kappaB) signal pathways in monocytes. Pretreatment with specific inhibitor of p38 kinase, but not those of ERK and NF-kappaB, inhibited heat shock-induced up-regulation of TLR2 and TLR4. This indicates that p38 pathway takes part in heat shock-induced up-regulation of TLR2 and TLR4. Heat shock also increased lipoteichoic acid- or lipopolysaccharide-induced interleukin-6 production by monocytes. These results suggest that the p38 kinase-mediated up-regulation of TLR2 and TLR4 might be involved in the enhanced response to PAMP in human monocytes induced by heat shock.     

Toll-like receptor (TLR)-9 genetics and function in sarcoidosis

Sarcoidosis is a systemic disorder characterized by the formation of non‐caseating granulomas in variable organs. Toll‐like receptor (TLR)‐9 is important in the innate immune response against both Mycobacterium tuberculosis and Propionibacterium acnes, candidate causative agents in sarcoidosis. The aim of our study was to investigate possible genetic and functional differences in TLR‐9 between patients and controls. TLR‐9 single nucleotide polymorphisms were genotyped in 533 patients and divided into a study cohort and validation cohort and 185 healthy controls. Furthermore, part of the promotor as well as the entire coding region of the TLR‐9 gene were sequenced in 20 patients in order to detect new mutations. No genetic differences were found between patients and controls. In order to test TLR‐9 function, peripheral blood mononuclear cells (PBMCs) of 12 healthy controls and 12 sarcoidosis patients were stimulated with a TLR‐9 agonist and the induction of interleukin (IL)‐6, interferon (IFN)‐γ and IL‐23 was measured. Sarcoidosis patients produce significantly less IFN‐γ upon stimulation with different stimuli. Regarding IL‐23 production, a significant difference between patients and controls was found only after stimulation with the TLR‐9 agonist. In conclusion, we did not find genetic differences in the TLR‐9 gene between sarcoidosis patients and controls. Sarcoidosis patients produce less IFN‐γ regardless of the stimulating agent, probably reflecting the anergic state often seen in their peripheral blood T lymphocytes. The differences in TLR‐9‐induced IL‐23 production could indicate that functional defects in the TLR‐9 pathway of sarcoidosis patients play a role in disease susceptibility or evolution.