Individual Variation of the Bovine Dermal Fibroblast Methylome and its Potential Role in Modifying the Innate Immune Response

The innate ability of dairy cows to respond to pathogens is important in defense against mastitis. Determining the epigenetic variation between animals and its role in the innate immune response is a step towards understanding differences in disease susceptibility among individuals. Dermal fibroblast cultures were established from 15 heifers and challenged with LPS for 36 hr. Animals were ranked by IL-8 secretion, and 4 high (HR) and 4 low (LR) responding cultures were selected for a second challenge in the absence or presence of a combination of DNA demethylating (5-aza-2'-deoxycytidine) and hyper- acetylating (trichostatin A) agents (AZA-TSA). The AZA-TSA treatment increased IL-8 secretion in both HR and LR cultures and abolished the previous difference between them, suggesting the presence of an underlying epigenetic effect. Subsequently, a genome-wide approach was taken to determine differential expression and differential methylation status between high and low responding cattle. The Affymetrix custom bovine array was utilized to analyze three AZA-TSA treated LR cultures following LPS exposure (0, 2, 4, 8 hr) and revealed genes with a higher induction to LPS due to AZA- TSA. Next-generation sequencing based-methylated-CpG island recovery assay (MIRA- Seq) was performed on DNA from 3 LR and 3 HR cultures. Sequence reads, averaging 27.6 million per animal, were aligned to the UMD3.1 reference genome using NextGENe. Methylation peaks were identified using stringent criteria. Further analysis of the differentially methylated peaks (306 greater in HR; 47 greater in LR) may provide insight into the contribution of the methylome to variation of innate immune responses between individuals.