EXPRESSION OF IL6 mRNA IS INDEPENDENT WITH EXPRESSION OF TLRS mRNA IN LIPOPOLYSACCHARIDE-TREATED MYOTUBES

Obesity-related metabolic disorders such as insulin resistance and type 2 diabetes are raising as critical health problems of the modern world. Obesity induces an increased plasma level of lipopolysaccharide (LPS) that contributing to system chronic inflammation. This response has been shown as a marked factor linking obesity and its related metabolic disorders. In the present study, we use LPS to treat C2C12 skeletal muscle cells and observe the expression of several inflammatory makers. Our results show that the expression of inflammatory cytokine IL6 mRNA is strongly induced in the LPS-treated C2C12 cells compared to the control cells. Surprisingly, the expression of the upper controller of inflammation TLR2 mRNA in the LPS-treated C2C12 cells is similar to that in the control cells. Consistent with this expression mRNA level of TLR4, another upper regulator of inflammation does not differ between the two group cells. Taken together, our current data suggest that the LPS induced expression of IL6 mRNA in C2C12 cells is not dependent on the regulation of neither TLR2 nor TLR4

Evaluation of Toll-like Receptor 2 Gene Expression in Rheumatoid Arthritis and Correlation with the Disease Activity

Background: Rheumatoid Arthritis (RA) is an autoimmune, chronic, and systematic disease. It affects joints and bones. The exact etiology of RA is still unclear. Varied genetic and environmental factors have been associated with the increased risk for RA. Overactivation of Toll-Like Receptors (TLRs) could initiate the development of autoimmune diseases including RA. Objective: The aim of the study was to evaluate TLR2 gene expression in rheumatoid arthritis patients and investigate its correlation with the disease activity. Materials and Methods: This study included 60 patients and 20 healthy individuals. The patients were diagnosed with RA according to the 2010 American College of Rheumatology/ European League Against Rheumatism criteria (ACR/EULAR). All included subjects did not have any joint disorders and /or autoimmune diseases. RA disease activity was determined by the disease activity score of 28 joints. Whole blood was collected from all participants. Total RNA extraction was done. TLR2 mRNA expression was assessed by reverse transcription-PCR (RT-PCR). Results: TLR2 mRNA expression was found to be significantly higher in RA patients compared to healthy controls. Also, a strong positive correlation was found between TLR2 expression level and the disease activity score. A non significant positive correlation was found between TLR2 expression and serum Rheumatoid Factor (RF) level. Conclusion: TLR2 pathway may have an important role in RA pathogenesis and could be a new biomarker for diagnosis and monitoring disease activity.

Candida albicans induces TLR2/MyD88/NF-κB signaling and inflammation in oral lichen planus-derived keratinocytes

Introduction: The risk of oral lichen planus (OLP), a chronic inflammatory oral mucosal disease, becoming malignant increases by 21-fold in patients with fungal infection. This study examined the impact of Candida albicans exposure on Toll-like receptor (TLR) signaling in primary keratinocyte cultures obtained from OLP patients. Methodology: Following co-culture of primary OLP keratinocyte cultures with C. albicans for 24 hours, inflammatory cytokine concentrations were determined by ELISA. TLR2, MyD88, and NF-κBp65 mRNA and protein expression were assessed using quantitative RT-PCR and Western blot analyses, respectively. Keratinocyte apoptosis was also determined by flow cytometry. Results: IL-10, IL-8, IL-2, and TNF-ɑ levels were significantly higher following co-culture with C. albicans (all p ≤ 0.034). MyD88, NF-κB p65, and TLR2 mRNA (all p < 0.001) and protein (all p ≤ 0.004) expression levels were significantly higher in OLP keratinocytes following C. albicans exposure. Finally, the apoptosis rates of OLP keratinocytes were 21.2%, 29.4%, and 25.4% for the control cells and 3.9%, 5.6%, and 4.4% for those exposed to C. albicans, suggesting that co-culture with C. albicans inhibits the apoptosis of OLP keratinocytes. Conclusions: C. albicans activates the TLR2/MyD88/NF-κB signaling pathway in OLP keratinocytes, resulting in increased cytokine expression and decreased keratinocyte apoptosis. Two key events in the pathogenesis of OLP and its progression to malignancy, namely increased inflammation and decreased apoptosis, were induced by exposure to C. albicans. Thus, targeting this signaling pathway may represent a novel therapeutic strategy to prevent OLP malignant transformation.

Effect of deoxynivalenol on the levels of toll-like receptors 2 and 9 and their mRNA expression in enterocytes in the porcine large intestine: a preliminary study

Deoxynivalenol (DON), one of the most prevalent mycotoxins in the world, and is capable of inducing immune disorders in humans and animals. The aim of this study was to determine the effect of feed contaminated with DON on the number of TLR2- and TLR9-positive cells and their mRNA expression in the porcine large intestine. The experiment was conducted on two equal groups of pigs (n=4). The experimental group (E) was administered feed contaminated with DON (1008 μg/kg of feed) for 6 weeks, and the control group (C) was administered non-contaminated feed over the same period of time. A decrease in the expression of TLR2 mRNA was noted in the cecum. The percentage of TLR9-positive enterocytes increased in the ascending colon and decreased in the cecum. The results of this study indicate that DON can modify the local immune response by changing the expression of TLRs.

Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

ABSTRACTThe migration of vascular smooth muscle cells (VSMCs) from the media to the intima is proposed to be a key event in the development of atherosclerosis. Recently, we reported thatChlamydia pneumoniaeinfection is involved in VSMC migration. However, the exact mechanisms forC. pneumoniaeinfection-induced VSMC migration are not yet well elucidated. In this study, we examined the role of the Toll-like receptor 2 (TLR2) activation-related signaling pathway in VSMC migration induced byC. pneumoniaeinfection. An Affymetrix-based gene expression array was conducted to identify the changes of gene expression in rat primary VSMCs (rVSMCs) infected withC. pneumoniae. Both the microarray analysis and quantitative real-time reverse transcription (RT)-PCR revealed that TLR2 mRNA expression was strongly upregulated 12 h afterC. pneumoniaeinfection. RT-PCR and Western blot analysis further showed that the expression levels of TLR2 mRNA and protein significantly increased at the different time points after infection. Immunocytochemical analysis suggested a TLR2 recruitment to the vicinity ofC. pneumoniaeinclusions. Cell migration assays showed that the TLR2-neutralizing antibody could significantly inhibitC. pneumoniaeinfection-induced rVSMC migration. In addition,C. pneumoniaeinfection stimulated Akt phosphorylation at Ser 473, which was obviously suppressed by the PI3K inhibitor LY294002, thereby inhibiting rVSMC migration caused byC. pneumoniaeinfection. Furthermore, both the infection-induced Akt phosphorylation and rVSMC migration were suppressed by the TLR2-neutralizing antibody. Taken together, these data suggest thatC. pneumoniaeinfection can promote VSMC migration possibly through the TLR2-related signaling pathway.

Regulatory Effect of Peptidoglycan on the Expression of Toll-Like Receptor 2 mRNA and Proteins in Trophoblast Cell Line TEV-1 Cells

Objective. To investigate the regulatory effect of peptidoglycan on the expression of human Toll-like receptors 2 (TLR2) mRNA and proteins in the human extravillous trophoblast cell line (TEV-1). Methods. TEV-1 cells were incubated with different doses of peptidoglycan. The expression of TLR2 mRNA and protein was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry SP staining. Results. TLR2 was expressed in TEV-1 cells and localized to both the cytoplasm and plasma membrane. Compared with the untreated control, TEV-1 cells incubated with 30 g/ml peptidoglycan significantly upregulated the expression of TLR2 mRNA and protein after 12 hours of treatment (). However, the expression of TLR2 mRNA and protein was decreased but had no significant difference compared with the control () after 24 hours of treatment. On the other hand, 10 g/ml peptidoglycan did not seem to have regulatory effect on mRNA and protein expression of TLR2 (). Conclusion. Peptidoglycan has a role in regulating the expression of TLR2 mRNA and protein in TEV-1 cells. It suggests that the trophoblast cells may play important role in the immune response at the fetal-maternal interface and affect the result of pregnancy by expressing TLR2.

Toll-like receptor 2 is upregulated by hog confinement dust in an IL-6-dependent manner in the airway epithelium

Hog confinement workers are at high risk to develop chronic bronchitis as a result of their exposure to organic dust. Chronic bronchitis is characterized by inflammatory changes of the airway epithelium. A key mediator in inflammation is Toll-like receptor 2 (TLR2). We investigated the role of TLR2 in pulmonary inflammation induced by hog confinement dust. Normal human bronchial epithelial cells (NHBE) were grown in culture and exposed to hog confinement dust extract. Hog confinement dust upregulated airway epithelial cell TLR2 mRNA in a concentration- and time-dependent manner using real-time PCR. There was a similar increase in TLR2 protein at 48 h as shown by Western blot. TLR2 was upregulated on the surface of airway epithelial cells as shown by flow cytometry. A similar upregulation of pulmonary TLR2 mRNA and protein was shown in a murine model of hog confinement dust exposure. Hog confinement dust is known to stimulate epithelial cells to produce IL-6. To determine whether TLR2 expression was being regulated by IL-6, the production of IL-6 was blocked using an IL-6-neutralizing antibody. This resulted in attenuation of the dust-induced upregulation of TLR2. To further demonstrate the importance of IL-6 in the regulation of TLR2, NHBE were directly stimulated with recombinant human IL-6. IL-6 alone was able to upregulate TLR2 in airway epithelial cells. Hog confinement dust upregulates TLR2 in the airway epithelium through an IL-6-dependent mechanism.

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

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