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Investigating innate immune response differences between Angus and Holstein cattle with the dermal fibroblast model
Individual immune responses to pathogens can be variable, depending on environmental, genetic, and possibly epigenetic influences. Holstein and Angus cattle are selectively bred and managed for different traits, which could impact disease susceptibility between these breeds. A dermal fibroblast model was used to investigate potential genetic and epigenetic influences on the innate immune response in each breed. Skin biopsies were collected from the shoulder area of 5 Holstein and 12 Angus 19-month heifers. Fibroblasts were isolated by collagenase digestion and cryopreserved. Revived cells were challenged with LPS (100ng/ml; 24h) and levels of secreted IL-8 were determined. The Holstein cultures produced approximately 3 times more (P < 0.01; unpaired t-test) IL-8 then the Angus cultures (2190 ± 600 vs. 650 ± 370 pg/ml, respectively). Total RNA was collected from four Angus and four Holstein cultures that were cultured in parallel and challenged with LPS for 0, 2, and 8 h. Whole transcriptome analysis was performed by RNA-seq with an average of 46 million reads/sample aligned to the UMD 3.1 reference genome by NextGENe software. Breed differences in gene expression were determined with the edgeR statistical package. Between breeds there were 849, 1014, and 751 genes differentially expressed genes (FDR < 0.05, fold change > 2) at hours 0, 2, and 8 post-LPS, respectively. Immune response genes such as TNF-a at 2h, and IL-8 and CCL20 at 8h were induced 6.9, 4.5, and 8.6-fold more in Holsteins compared to Angus; while expression at 2h of CXCL12 and TRAIP, an inhibitor of TRAF2-mediated NF-kB activation, were 7.4 and 2.7-fold higher in Angus. Additionally, a semi-quantative assessment of global DNA methylation was performed by methylated CpG island recovery assay (MIRA-seq) on genomic DNA extracted from these cultures. Read alignment (44 million reads/sample) and differential methylation region (DMR) analysis was performed similarly to RNA-seq. The genome was analyzed in consecutive 3kb regions and revealed 51 DMR (FDR < 0.1, fold change > 2). Of these, 35 were more methylated in Angus and 16 were more methylated in Holsteins. Relationships among these DMRs and the differential gene expression are not readily apparent but are being further investigated. Our results reveal breed differences in the LPS response of dermal fibroblasts isolated from Angus and Holstein heifers. Given that the cells were cultured side-by-side in controlled environmental conditions, the observed differences are likely due to a combination of genetic and epigenetic factors.
Keywords: Variation, Epigenetics