Genetics of inflammatory mediators, in particular interleukin 6 and selenoprotein S, that have a role in coronary artery disease.

Coronary artery disease (CAD) is inflammatory and caused by genetic/environmental factors. Interleukin (IL) 6 is a pro-inflammatory cytokine implicated in CAD. Selenoprotein S (SelS) has recently been implicated in inflammation and endoplasmic reticulum (ER) stress. Several IL6 and SelS polymorphisms are associated with increased CAD risk. The aim of this project was to investigate the role of polymorphisms of IL6 (-6331 T/C,-597 G/A ,-572 G/C), IL-6R (rs8192284A/C, Asp358Ala) and SelS (-1500 A/C,+3705 G/A,+5227 C/T, and +9000 C/G) on the expression of their mRNA/proteins. This will help us understand the role of these SNPs in risk of CAD/ Myocardial infarction (MI). Flow cytometry was used to assess the surface expression of the macrophage differentiation-specific markers CD14 and CD11c, and IL-6R by peripheral blood mononuclear cell (PBMC)- derived macrophages. Messenger RNA levels were determined by quantitative PCR (QPCR) in lipopolysaccharide (LPS) and IL-1β-stimulated THP-1 cells and in PBMC-derived macrophages from healthy donors. SelS protein expression was analysed by Western blotting. Cell culture supernatants were assayed for cytokine production by flow cytometry (Cytokine Bead Array). Gene expression (mRNA and protein) was compared to IL6 and SelS genotype and haplotype. CD11c-hi, CD14-lo expression on 8-day cultured PBMCs confirmed their differentiation state as macrophage. The effect of LPS or IL-1β at 8 hours on SelS mRNA expression of SelS combined data did not show a significant increase but SelS mRNA was upregulated in LPS-induced +CATG SelS haplotype in PBMC-derived macrophages. +CATG SelS Haplotype did not increase the SelS protein concentration in either LPS or IL-1β stimulated PBMC-derived macrophages at 8 hours time point. SelS-1500 CC genotype was associated with high SelS fold induction in PBMC-derived macrophages treated by LPS. IL6 mRNA was significantly up-regulated in LPS-stimulated THP-1. IL-1β- and LPS-induced +TGG, +TAG and +CGG haplotypes in PBMC-derived macrophages. IL6 protein production was also significantly increased in the LPS-induced +TAG and +CGG haplotypes. IL6-597A allele increased the IL6 mRNA fold induction. Interestingly, PBMCs from people with the IL6 TAG haplotype (or –597A allele) have higher IL6 and in contrast, lower SelS at baseline and IL-1β. CD126 (IL-6R) expression was very low on PBMC-derived macrophages, suggesting that classical IL6 signalling may not occur in these PBMCs. In conclusion, SelS mRNA was up-regulated in LPS-induced +CATG SelS haplotype in PBMC-derived macrophages. +CATG SelS Haplotype did not increase the SelS protein concentration in either LPS or IL-1β stimulated PBMC-derived macrophages at 8 hours time point. SelS-1500 CC genotype was associated with high SelS fold induction in PBMC-derived macrophages treated by LPS. The individuals with +TAG IL6 haplotype (or IL6-597 A allele) show greater induction with pro-inflammatory stimuli. In contrast, PBMCs from people with the IL6 TAG haplotype (or –597A allele) have lower SelS at baseline and following IL-1β (and probably also LPS) stimulation.