Evaluation of DNA methylation status of toll-like receptors 2 and 4 promoters in Behcet's disease.

BACKGROUND: Altered innate immune function plays an important role in the initiation of inflammatory response in Behcet's disease (BD). Toll-like receptors (TLRs) are the master regulators of the innate immune system. Because the role of TLRs remains unknown in the pathogenesis of BD, the present study aimed to evaluate the expression levels and methylation status of the TLR2 and TLR4 promoters in patients with BD. METHODS: In the present study, Iranian Azeri BD patients (n = 47) with an active (n = 22) and inactive (n = 25) period, and healthy controls (n = 61), were matched according to age, sex and ethnicity. TLR2 and TLR4 genes promoter CpG islands were predicted with the Eukaryotic Promoter Database (https://epd.vital-it.ch). Methylated DNA immunoprecipitation (MeDIP) was conducted. RESULTS: The results showed that mRNA of TLR4 was significantly increased in the peripheral blood mononuclear cells (PBMCs) of BD patients with an active phase compared to the control group. Differences in mRNA of TLR4 between the inactive BD and control groups were not significant. Differences in TLR2 mRNA levels in the PBMCs of the active and inactive phase BD and control groups were not significant. The methylation rate of TLR4 gene promoter was significantly lower in the active and inactive BD groups compared to the control group. The difference between the active and inactive BD groups was not significant. There was no significant difference in the methylation rates of the TLR2 gene between studied groups. CONCLUSIONS: Our preliminary findings suggest that the hypomethylation of TLR4 gene may be involved in the pathogenesis of BD via increasing TLR4 expression.

Determination of mir-155 and mir-146a expression rates and its association with expression level of TNF-α and CTLA4 genes in patients with Behcet's disease.

INTRODUCTION: MicroRNAs (miRNAs) are involved in the pathogenesis of inflammatory diseases. MiR-146 and miR-155 emerged as key regulators of the immune response. This study designed to analyze the miR-146a and miR-155 expression in patients with Behcet's disease (BD) and investigated their association with the expression of tumor necrosis factor-alpha (TNF-α) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4) genes. METHODOLOGY: In a case-control study, 47 Iranian Azeri BD patients and 61 age- and sex matched healthy controls recruited to the study. Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA blood tubes by Ficoll density-gradient centrifugation. Genomic DNA samples of BD and healthy controls were extracted using the rapid genomic DNA extraction method from the peripheral blood collected in tubes containing EDTA. Total RNA was extracted from the PBMCs according to the TRIzol protocol. MiR-146a, miR-155, TNF-α and CTLA-4 expression were studied using real-time PCR. RESULTS: MiR-155 and TNF-α expression was significantly increased, whilst CTLA-4 expression was significantly decreased in the PBMCs of BD patients. There was no significant difference in the miR-146a expression rate between BD patients and controls. A positive correlation between miR-155 and TNF-α expression and negative correlation between miR-155 and CTLA-4 expression were observed. No significant association was observed between the expression of miR-155, miR-146a, TNF-α and CTLA-4 genes with BD activity. MiR-155 and miR-146a expression rate were significantly higher in patients with uveitis and phlebitis, respectively. DISCUSSION AND CONCLUSION: The expression of miR-155 increased in BD and associated with upregulation of TNF-α and downregulation of CTLA-4 genes.