Age dependent changes in heifer fibroblast DNA methylation and LPS-induced gene expression

Abstract Text: To determine how the innate immune response develops in dairy calves, dermal fibroblasts were isolated from 15 heifers at three stages of development (5, 11, and 16 months of age) and challenged with 100 ng/ml of lipopolysaccharide (LPS) for 36h. The secretion of interleukin (IL)-8 protein into media in response to LPS increased significantly (P<0.01) at each age as measured by a paired one-way ANOVA with average IL-8 levels of 300 ± 220, 1340 ± 530, and 1750 ± 560 pg/ml at 5, 11, and 16 months, respectively. To investigate a potential involvement of DNA methylation in this differential responsiveness, cultures from three of these animals obtained when they were young (5 month) and older (16 month) underwent DNA de-methylation through exposure to 10mM 5-aza-2’-deoxycytidine (AZA) for four days prior to 24 h LPS exposure (n=3/group).  The AZA treatment abolished the differential IL-8 response to LPS seen under control conditions (P<0.01) primarily due to an increase in production by the young cultures (control Young vs Old 240 ± 20 vs. 1350 ± 290 pg/ml, respectively; AZA Young vs Old 1580 ± 50 vs. 1690 ± 70 pg/ml, respectively). The role of DNA-methylation in the gene expression response to LPS was further investigated by comparing the findings of methylated-CpG island recovery assay (MIRA-Seq) on DNA from 3 pairs of young and older cultures to RNA-seq findings on the same cultures at 0, 2, and 8 hours post-LPS exposure. The resulting libraries averaged 71 and 49 million reads per sample for RNA-seq and MIRA-seq, respectively. Sequence reads were aligned to the UMD3.1 reference genome using NextGENe software and expression and methylation analysis were performed with EdgeR. The overall response to LPS was robust with 617 and 414 genes displaying differential expression (>2 fold difference; FDR<0.05) at hours 2 and 8, respectively, as compared to hour 0. Older cultures had greater expression than younger cultures of many immune associated genes such as IL-8, IL-6, TNF-a, and CCL20 at 2 hours post-LPS exposure (5.8, 10.5, 10.4, and 18.1-fold, respectively). In addition whole genome MIRA-seq analysis of consecutive 3kb regions identified 20 differentially methylated regions between young and older cultures (>2 fold difference; FDR<0.1). Further analysis of these candidate regions is being conducted to determine how DNA methylation changes within animals over time and it’s potential role in the development of the innate immune response. 

Keywords: epigenetics, methylation, innate immunity