Toll-like receptor 2 and 6 agonist fibroblast-stimulating lipopeptide increases expression and secretion of CXCL1 and CXCL2 by uveal melanocytes.

Fibroblast-stimulating lipopeptide (FSL-1) can activate Toll-like receptor 2 and 6 (TLR2/6), which recognize relevant molecules from gram-positive pathogens, fungus, and mycoplasma, and elevates the expression of CXCL1 and CXCL2, neutrophil chemoattractants, in certain types of cells. This effect has not previously been reported in the uveal melanocytes (UM). This study was designed to test the hypothesis that FSL-1 can induce the expression and secretion of CXCL1 and CXCL2 via activation of TLR2/6 in cultured human UM and producing an acute non-infectious uveitis reaction in the mouse. Flow cytometry and fluorescent immunostaining were used to measure the effect of FSL-1 on the expression of TLR2/6 in UM. Real time PCR and ELISA analysis were used to assess the ability of FSL-1 to elevate CXCL1/CXCL2 levels in cell lysates and conditioned media of UM, respectively. Flow cytometry measured phosphorylated MAPK and activated NF-κB signals in UM, with and without FSL-1 treatment. ELISA analysis tested the impact of various signal inhibitors (NF-κB, p38 MAPK, JNK1/2 and ERK1/2) and TLR2/6 antagonists on FSL-1-induced CXCL1/CXCL2 levels in cultured UM. The effects of neutralizing antibodies to TLR2 on FSL-1-induced mouse uveitis were tested in an experimental animal model. FSL-1 induced the expression of TLR2/6 proteins in cultured UM. FSL-1 significantly elevated the CXCL1 and CXCL2 proteins and mRNA levels in cultured UM time- and dose-dependently. FSL-1 mainly activated NF-κB, JNK, and expression of TLR2. FSL-1-induced expression of CXCL1 and CXCL2 was blocked by NF-κB, JNK, ERK inhibitors and TLR2 antagonists. Intravitreal injection of FSL-1 induced acute non-infectious mouse uveitis, which was significantly reduced in severity by a TLR2 antagonist. These results suggest that UM may play a role in the immune reaction, which targets invading pathogens, especially gram-positive bacteria. On the other hand, an excessive reaction to molecules from gram-positive bacteria may promote an inflammatory state of non-infectious uveitis.

Association of the TLR1 variant rs5743557 with susceptibility to tuberculosis.

BACKGROUND: Toll-like receptor 1 (TLR1) and TLR6 play important roles in the innate immune response against Mycobacterium tuberculosis (M.TB) via interactions with TIR domain-containing adaptor protein (TIRAP) and myeloid differentiation primary response 88 (MYD88). The aim of this study was to investigate the relationship of TLR1, TLR6, MYD88 and TIRAP polymorphisms with susceptibility to latent tuberculosis infection (LTBI) and tuberculosis (TB). METHODS: In total, 204 uninfected healthy controls (HC), 201 individuals with LTBI and 209 TB patients were enrolled. Two interferon-γ release assays were used to differentiate individuals with LTBI from uninfected controls. TagSNPs of the four genes were genotyped by the SNPscanTM Kit. The Haploview 4.2 and SHEsis software packages were combined to perform linkage disequilibrium (LD) and haplotype analyses. Multifactor dimensionality reduction (MDR) software was used to investigate gene-gene interaction. The Stata 12.0 software was used to perform meta-analysis of the relationship between rs5743557 and TB susceptibility. RESULTS: The AA genotype of rs5743557 was associated with reduced TB risk (P=0.006) and the AA/GA genotypes of TLR1 rs5743604 were associated with increased TB risk (P=0.017) when the LTBI group was compared with the TB group. The frequency of TLR1 haplotype rs4833095-rs5743604 CG was significantly higher in the LTBI group than in the TB group (P=0.019877). However, only the relationship between rs5743557 and TB susceptibility remained significant after 1000-fold permutation testing (P=0.023). The meta-analysis suggested that rs5743557_A was associated with decreased TB risk in the Chinese adult population (P<0.001, OR 0.80, 95% CI: 0.72-0.88). No significant gene-gene interactions were found. CONCLUSIONS: The results of our study suggest that the tagSNP rs5743557 of TLR1 is associated with the risk of TB.

MALP-2, an agonist of TLR6, promotes the immune status without affecting the differentiation capacity of umbilical cord mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are increasingly used in cell-based therapy due to their multiple differentiation capacity, low expression of co-stimulatory factors and immunosuppressive effect. However, accumulating studies reported the recognition and rejection of engrafted MSCs, which eventually led to the fail of clinical trials. Toll-like receptors (TLRs) are important in mediating the immune response. In the present study, macrophage-activated lipopeptide-2 (MALP-2) was introduced to activate the TLR6 pathway in umbilical cord MSCs (UCMSCs). PBLs isolated from healthy volunteers were co-cultured with UCMSCs to measure whether activation of TLR6 of UCMSCs could stimulate immune responses. Reverse transcription-quantitative polymerase chain reaction and immunohistochemistry were performed to detect pro-inflammatory molecules and differentiation status of UCMSCs, respectively. The results indicated that activation of TLR6 in UCMSCs increased the proliferation of peripheral blood leukocytes (PBLs) and enhanced the release of lactate dehydrogenase in damaged UCMSCs, which confirmed the role of TLR6 in promoting the immunogenicity of UCMSCs. Furthermore, quantitative polymerase chain reaction demonstrated that the expression of proinflammatory molecules (including IL-1ß, IL-6, IL-8, IL-10, CCL1 and CCL4) was induced, whereas the expression of stem cell markers (Klf4 and Nanog) was inhibited. The differentiation results indicated that activation of TLR6 had no effect on the differentiation capacity of UCMSCs. All these findings suggest that stimulation of TLR6 pathway may increase the immunogenicity of UCMSCs in in vitro detections. In conclusion, the results of the current study indicated a new role of TLR6 in regulating the biological function of UCMSCs.

TLR-6 SNP P249S is associated with healthy aging in nonsmoking Eastern European Caucasians - A cohort study.

BACKGROUND: To investigate mechanisms that determine healthy aging is of major interest in the modern world marked by longer life expectancies. In addition to lifestyle and environmental factors genetic factors also play an important role in aging phenotypes. The aged immune system is characterized by a chronic micro-inflammation, known as inflamm-aging, that is suspected to trigger the onset of age-related diseases such as cardiovascular disease, Alzheimer's disease, cancer, and Diabetes Mellitus Type 2 (DMT2). We have recently shown that a Toll-like receptor 6 variant (P249S) is associated with susceptibility to cardiovascular disease and speculated that this variant may also be associated with healthy aging in general by decreasing the process of inflamm-aging. RESULTS: Analyzing the PolSenior cohort we show here that nonsmoking S allele carriers are significantly protected from age-related diseases (P = 0.008, OR: 0.654). This association depends not only on the association with cardiovascular diseases (P = 0.018, OR: 0.483) for homozygous S allele carriers, but is also driven by a protection from Diabetes Mellitus type 2 (P = 0.010, OR: 0.486) for S allele carriers. In addition we detect a trend but no significant association of this allele with inflamm-aging in terms of baseline IL-6 levels. CONCLUSION: We confirm our previous finding of the TLR-6 249S variant to be protective regarding cardiovascular diseases. Furthermore, we present first evidence of TLR-6 249S being involved in DMT2 susceptibility and may be in general associated with healthy aging possibly by reducing the process of inflamm-aging.

Toll-like receptors 1, 2, 4 and 6 in esophageal epithelium, Barrett's esophagus, dysplasia and adenocarcinoma.

BACKGROUND: Toll-like receptors (TLRs) recognize microbial and endogenous ligands and have already shown to play a role in esophageal cancer. In this study, we evaluated especially TLRs that sense bacterial cell wall components in Barrett's esophagus, dysplasia and esophageal adenocarcinoma. METHODS: TLRs 1, 2, 4 and 6 were stained immunohistochemically and assessed in esophageal specimens from patients with esophageal dysplasia (n = 30) or adenocarcinoma (n = 99). Structures and lesions were evaluated including normal esophagus (n = 88), gastric (n = 67) or intestinal metaplasia (n = 51) without dysplasia, and low-grade (n = 42) or high-grade dysplasia (n = 37), and esophageal adenocarcinoma (n = 99). RESULTS: We found TLR1, TLR2, TLR4 and TLR6 expression in all lesions. TLR expression increased in Barrett's mucosa and dysplasia. There was profound increase of TLR expression from gastric- to intestinal-type columnar epithelium. In cancers, high nuclear and cytoplasmic staining of TLR4 associated with metastatic disease and poor prognosis. CONCLUSIONS: TLR1, TLR2, TLR4 and TLR6 are upregulated during malignant changes of esophageal columnar epithelium. Increased TLR4 expression associates with advanced stage and poor prognosis in esophageal adenocarcinoma.

Toll-like receptor-6 (TLR6) deficient mice are protected from myocardial fibrosis induced by high fructose feeding through anti-oxidant and inflammatory signaling pathway.

Diabetic cardiomyopathy is an essential complication of diabetes and characterized by persistent diastolic dysfunction, leading to myocardial fibrosis. Oxidative stress and inflammation lead to cell damage and are implicated in many disease states. In our study, we evaluated the effects of toll-like receptor 6 (TLR6) in cardiac remodeling. We established a mouse model of myocardial fibrosis with diabetes using 30% fructose. In comparison to HF-feeding control mice, TLR6 deficient mice developed less myocardial fibrosis with lower myocardial injury marker enzymes and AngII and aldosterone (ALD). In addition, Collagen type I/III, alpha smooth muscle-actin (α-SMA) and FSP-1, as typical markers of myocardial fibrosis formation, were found to be reduced due to TLR6 knockout in HF-induced mice. HF-feeding mice developed myocardial fibrosis with lower SOD activity, high level of MDA, O2(-) and H2O2 and increased serum pro-inflammatory cytokines, whereas TLR6 deficient mice after HF-administration were protected from myocardial fibrosis progression significantly. HF-feeding mice also displayed lower Nrf2 and higher XO levels, which was not observed in TLR6 deficient mice after HF-feeding. Furthermore, NF-κB pathway was inactivated for TLR6 knockout compared with HF-feeding mice. In vitro, fructose directly up-regulated α-SMA, TGF-ß1, Collagen type I/III and FSP-1 via ROS production and NF-κB phosphorylation as well as pro-inflammatory cytokines releasing, which were inhibited for TLR6 deficiency. Taken together, TLR6 contributed to myocardial fibrosis progression, at least partly, through oxidative stress and inflammatory response, providing a potential therapeutic strategy for myocardial fibrosis treatment.

Association between toll-like receptor 6 expression and auxiliary T cells in the peripheral blood of pediatric patients with allergic purpura.

The aim of the present study was to investigate the correlations between toll-like receptor 6 (TLR6) expression in the peripheral blood mononuclear cells (PBMCs) and auxiliary T cells of children with purpura. A total of 42 children with acute Henoch-Schönlein purpura (HSP) were selected for the study, and a further 30 healthy children were selected as a control group. Enzyme-linked immunosorbent assays were performed to detect the levels of plasma interferon (IFN)-γ, interleukin (IL)-4 and IL-17, and flow cytometry was performed to detect the TLR6 protein expression levels in PBMCs. The plasma levels of IL-4, IFN-γ and IL-17 in the HSP group were significantly higher compared with those in the normal control group. TLR6 protein expression was significantly increased in the PBMCs of the HSP patients. The TLR6 protein expression levels in the monocytes of the HSP group significantly positively correlated with the serum IL-4 and IL-17 levels, but not with the serum levels of IFN-γ. Therefore, the results of the present study suggest that the activation of TLR6 may be involved in the immunopathogenesis of HSP, and that the activated TLR6 may mediate this process by upregulating the immune responses of type 2 T helper (Th2) and Th17 cells.

Toll-like receptor 2 and 6 interdependency in the erosive stage of Staphylococcus aureus induced septic arthritis mediated by IFN-γ and IL-6--A possible involvement of IL-17 in the progression of the disease.

Staphylococcus aureus induced septic arthritis has emerged as a potent disabling and life threatening disease; hence combating this malady has become an imperative need of medical science. Role of TLR-2 in innate recognition of S. aureus and activation of inflammatory cascade by the interplay of some proinflammatory cytokines, resulting in joint inflammation has been established. Variation in the reports suggesting both functional dependency and independency of TLR-2 on its heterodimeric partner TLR-6 in response to ligands exists, thus this study was postulated to observe the expression pattern of TLR-6 in synovial tissue and lymphoid organs after inducing septic arthritis by S. aureus in Swiss albino mouse model and the instigated cytokine profile could affirm its plausible role in SA. The functional relation of TLR-2 and 6 was verified by simulating an in vitro study design on synovial mononuclear cells, blocking TLR-2 and 6, and it was found that they are required to co-express for generating cytokine, NO and H2O2 on infection. IFN-γ, IL-6 and IL-17 were identified to play a distinguished role in SA from their secretion pattern in both in vivo and in vitro study. IFN-γ and IL-6 remained high throughout the infection possibly by the shift of response from Th1 to Th2 and Th17 and contribute in various converging pathways of inflammation. IL-17 increased with the onset of the disease but reduced on the late period. Hence IFN-γ, IL-6, IL-17 along with TLR-6 can be a potent target for therapeutic approach because of their significant contribution in SA.

7α-Hydroxycholesterol Elicits TLR6-Mediated Expression of IL-23 in Monocytic Cells.

We investigated the question of whether 7-oxygenated cholesterol derivatives could affect inflammatory and/or immune responses in atherosclerosis by examining their effects on expression of IL-23 in monocytic cells. 7α-Hydroxycholesterol (7αOHChol) induced transcription of the TLR6 gene and elevated the level of cell surface TLR6 protein in THP-1 monocytic cells. Addition of an agonist of TLR6, FSL-1, to TLR6-expressing cells by treatment with 7αOHChol resulted in enhanced production of IL-23 and transcription of genes encoding the IL-23 subunit α (p19) and the IL-12 subunit ß (p40). However, treatment with 7-ketocholesterol (7K) and 7ß-hydroxycholesterol (7ßOHChol) did not affect TLR6 expression, and addition of FSL-1 to cells treated with either 7K or 7ßOHChol did not influence transcription of the genes. Pharmacological inhibition of ERK, Akt, or PI3K resulted in attenuated transcription of TLR6 induced by 7αOHChol as well as secretion of IL-23 enhanced by 7αOHChol plus FSL-1. Inhibition of p38 MAPK or JNK resulted in attenuated secretion of IL-23. These results indicate that a certain type of 7-oxygenated cholesterol like 7αOHChol can elicit TLR6-mediated expression of IL-23 by monocytic cells via PI3K/Akt and MAPKs pathways.

Toll-like receptor 2 subfamily gene polymorphisms are associated with Bacillus Calmette-Guérin osteitis following newborn vaccination.

AIM: Toll-like receptor (TLR) 1, 2, 6 and 10, the TLR2 subfamily, are known to be associated with immunity against tuberculosis. We evaluated whether polymorphisms in genes encoding TLR1, TLR2 and TLR6 were associated with osteitis in infants who received the Bacillus Calmette-Guérin (BCG) vaccination soon after birth. METHODS: Blood samples from 132 adults aged 21-49 who had BCG osteitis in early childhood were analysed in a controlled study for TLR1 T1805G (rs5743618), TLR2 G2258A (rs5743708) and TLR6 C745T (rs5743810) gene single nucleotide polymorphisms. RESULTS: The frequencies of the variant genotypes differed between the cases and controls: 11.4% versus 5.7% for TLR2 G2258A (p = 0.033) and 77.3% versus 61.6% for TLR6 C745T (p = 0.001). The TLR2 and TLR6 variant genotypes were associated with a higher risk of BCG osteitis, with adjusted odds ratios (aOR) of 2.154 (95%CI 1.026-4.521) and 1.907 (95%CI 1.183-3.075), respectively. The frequency of the TLR1 T1805G variant genotype was 19.7% in the cases and 33.6% in the controls (p = 0.003). The TLR1 variant genotype was associated with a lower risk of BCG osteitis (aOR 0.554, 95%CI 0.336-0.911). CONCLUSION: Gene polymorphisms that regulate the function of the TLR2 subfamily play a role in the development of BCG osteitis in vaccinated infants.

7alpha-Hydroxycholesterol Elicits TLR6-Mediated Expression of IL-23 in Monocytic Cells

We investigated the question of whether 7-oxygenated cholesterol derivatives could affect inflammatory and/or immune responses in atherosclerosis by examining their effects on expression of IL-23 in monocytic cells. 7alpha-Hydroxycholesterol (7alphaOHChol) induced transcription of the TLR6 gene and elevated the level of cell surface TLR6 protein in THP-1 monocytic cells. Addition of an agonist of TLR6, FSL-1, to TLR6-expressing cells by treatment with 7alphaOHChol resulted in enhanced production of IL-23 and transcription of genes encoding the IL-23 subunit alpha (p19) and the IL-12 subunit beta (p40). However, treatment with 7-ketocholesterol (7K) and 7beta-hydroxycholesterol (7betaOHChol) did not affect TLR6 expression, and addition of FSL-1 to cells treated with either 7K or 7betaOHChol did not influence transcription of the genes. Pharmacological inhibition of ERK, Akt, or PI3K resulted in attenuated transcription of TLR6 induced by 7alphaOHChol as well as secretion of IL-23 enhanced by 7alphaOHChol plus FSL-1. Inhibition of p38 MAPK or JNK resulted in attenuated secretion of IL-23. These results indicate that a certain type of 7-oxygenated cholesterol like 7alphaOHChol can elicit TLR6-mediated expression of IL-23 by monocytic cells via PI3K/Akt and MAPKs pathways.

Preliminary X-ray crystallographic studies of the TIR domain of human Toll-like receptor 6.

Toll-like receptor (TLR) proteins have been identified and shown to play a role in the innate immune response. TLR6 associated with TLR2 can recognize diacylated lipoprotein. In this study, the human TLR6 TIR domain corresponding to amino acids 640-796 was overexpressed in Escherichia coli using engineered C-terminal His tags. The TLR6 TIR domain was then purified to homogeneity and crystallized at 20°C. Finally, X-ray diffraction data were collected to a resolution of 2.2 Šfrom a crystal belonging to space group C2, with unit-cell parameters a = 127.60, b = 44.20, c = 75.72 Å, ß = 118.89°