Effect of DNA Methylation on LPS-Induced Expression of Tumour Necrosis Factor Alpha (TNF-α) in Bovine Mammary Epithelial Cells

Background: Cow mastitis is a major disease that affects dairy industry worldwide. Although the inflammatory response induced by lipopolysaccharide (LPS) in bovine mammary epithelial cells (BMECs) is similar to the response to pathogenic bacteria, the underlying regulatory mechanisms remain unclear. This study aimed to clarify the effect of DNA methylation on LPS-induced expression of tumour necrosis factor alpha (TNF-α) in BMECs. Methods: The mammary epithelial cells were treated with LPS and DNA methylation inhibition 5-Aza-2’-deoxycytodine (5Aza). Expression of TNF-α, IL-6, BNBD-5, DNA methyltransferases (DNMT1, DNMT2, DNMT3A and DNMT3B) and DNA methylation at TNF-α regions, were analyzed using quantitative realtime PCR (qRT-PCR), Elisa and bisulfite sequencing PCR (BSP). Result: Our results showed that LPS significantly increased the expression of inflammatory factors, including interleukin-6 (IL-6), bovine neutrophil beta-defensins (BNBD-5) and TNF-α. Further, we observed that the DNA methylation inhibitor, 5-Aza-2'-deoxycytosine (5-Aza), enhanced LPS-induced TNF-α mRNA expression. In addition, we found that LPS treatment significantly decreased the methylation levels of specific CpG sites in the TNF-α promoter (at -245 and -323) and inhibited the expression of DNA methyl transferases (DNMT1, DNMT2, DNMT3A and DNMT3B). Our results indicate that LPS promotes the expression of TNF-α in BMECs by inhibiting DNA methylation in the gene promoter region.