The study of Glyburide effect on the expression of inflammatory and anti-inflammatory factors in monocyte-derived dendritic cells

Abstract

Tolerogenic dendritic cells (TolDCs) are attractive therapeutic options for autoimmune disorders because they suppress autologous T-cell responses. Dendritic cells (DCs) are equipped with pattern recognition receptors (PRR), including NLRs such as NLRP3. Abnormal NLRP3 activation has been reported to be correlated with the occurrence of autoimmune disorders. Our aim in this study was to treat DCs with glyburide and load a group of them with insulin to induce TolDCs and evaluate their effects on DCs. Consequently, T lymphocytes (T cell) mediated responses ensuing co-culture of them with DC groups were evaluated. Materials and methods After isolation of peripheral blood mononuclear cells (PBMCs) using the Ficoll technique, monocytes were isolated from PBMCs by MACS. Monocytes were converted to immature DCs by adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Glyburide was added to the treatment group and insulin-loaded group. NovoRapid insulin was only added to the third group. After 5 hours, LPS and ATP were added. To investigate glyburide treatment and insulin on moDCs, flow cytometry technique was used. The real-time PCR method was used to investigate the gene expression of inflammatory and anti-inflammatory factors not only in the DC groups but also in cocultured T cells and gene expression of diverse T cells’ related transcription factors were also assessed. Determination of cytokines’ levels in supernatant of DC groups and DC-T cells coculture groups were evaluated by ELISA. Results Our findings illustrated that glyburide treatment of DCs, generates TolDCs with a decreased expression of markers that correlate with DC maturation and Ag presentation and diminished expression level of inflammatory but increased expression of anti-inflammatory cytokines. Even, altered level of cytokines’ production by DCs were confirmed by ELISA. Insulin loading demonstrated more anti-inflammatory functions. In addition, co-cultured T cells showed regulatory or T helper 2 phenotype instead of T helper 1 features by altering the expression level of transcription factors and production of cytokines, confirming the ability of TolDCs induced by our strategy in inducing the differentiation of T cells to Tregs.