LPS-induces IL-6 and IL-8 gene expression in bovine endometrial cells “through DNA methylation”

Abstract

Uterine infection in dairy cows causes great economic loss. In bovine endometrial cells, lipopolysaccharide (LPS)-stimulated increase in interleukin 6 (IL-6) and interleukin 8 (IL-8) mRNA is crucial for the inflammatory response; however, the regulatory mechanisms remain unclear. Here, we investigated the role of DNA methylation in IL-6 and IL-8 mRNA expression following LPS-induction in bovine endometrial cells. IL-6 and IL-8 mRNA expression was evaluated under DNA methylation inhibition using 5-Aza-2′-deoxycytodine (5Aza) following LPS stimulation. Expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), methyl CpG-binding protein 2 (MeCP2) and DNA methylation at IL-6 and IL-8 regions, were analyzed using quantitative real-time PCR (qRT-PCR) and bisulfite sequencing PCR (BSP) following 24 h of LPS treatment. Inhibition of DNA methylation significantly enhanced LPS-induced IL-6 and IL-8 mRNA expression. LPS increased IL-6 and IL-8 mRNA expression, and decreased methylation levels of specific CpG sites at the IL-6 promoter (at −366 and −660) and the IL-8 promoter (at −120 and −48) after 24 h. Furthermore, LPS treatment for 24 h significantly increased DNMT1, DNMT3A, DNMT3B, and MeCP2 mRNA expression. Our results indicate that treating bovine endometrial cells with LPS induces the expression of IL-6 and IL-8 mRNA regulated by IL-6 and IL-8 promoter methylation.