Abstract
BackgroundDifferences in DNA methylation are known to contribute to the development of immune-related disorders in humans but relatively little is known about how methylation regulates immune function in cattle. Utilizing whole-transcriptome analyses of bovine dermal fibroblasts, we have previously identified an age and breed-dependent up-regulation of genes within the toll-like receptor 4 (TLR4) pathway that correlates with enhanced fibroblast production of IL-8 in response to lipopolysaccharide (LPS). Age-dependent differences in IL-8 production are abolished by treatment with 5-aza-2-deoxycytidine and Trichostatin A (AZA-TSA), suggesting epigenetic regulation of the innate response to LPS. In the current study, we performed reduced representation bisulfite sequencing (RRBS) on fibroblast cultures isolated from the same animals at 5- and 16-months of age to identify genes that exhibit variable methylation with age. To validate the role of methylation in gene expression, six innate response genes that were hyper-methylated in young animals were assessed by RT-qPCR in fibroblasts from animals at different ages and from different breeds.ResultsWe identified 14,094 differentially methylated CpGs (DMCs) that differed between fibroblast cultures at 5- versus 16-months of age. Of the 5065 DMCs that fell within gene regions, 1117 were located within promoters, 1057 were within gene exons and 2891 were within gene introns and 67% were more methylated in young cultures. Transcription factor enrichment of the promoter regions hyper-methylated in young cultures revealed significant regulation by the key pro-inflammatory regulator, NF-κB. Additionally, five out of six chosen genes (PIK3R1, FES, NFATC1, TNFSF13 and RORA) that were more methylated in young cultures showed a significant reduction in expression post-LPS treatment in comparison with older cultures. Two of these genes, FES and NFATC1, were similarly down-regulated in Angus cultures that also exhibit a low LPS response phenotype.ConclusionsOur study has identified immune-related loci regulated by DNA methylation in cattle that may contribute to differential cellular response to LPS, two of which exhibit an identical expression profile in both low-responding age and breed phenotypes. Methylation biomarkers of differential immunity may prove useful in developing selection strategies for replacement cows that are less susceptible to severe infections, such as coliform mastitis.
Highlights
Differences in DNA methylation are known to contribute to the development of immune-related disorders in humans but relatively little is known about how methylation regulates immune function in cattle
While it is clear that the response elicited by Tolllike receptor (TLR) and other Pattern recognition receptor (PRR) are crucial to the defense against a variety of bacterial, viral and eukaryotic pathogens [4,5,6], an exacerbated or dysregulated innate response contributes to infection related inflammatory disorders, such as in antibody dependent enhancement and the subsequent “cytokine storm” characteristic of Dengue hemorrhagic fever [7]
Overall, the current study has shown that many sites differ in their methylation status in young versus old cultures, with the majority of sites becoming less methylated as the animals increased in age
Summary
Differences in DNA methylation are known to contribute to the development of immune-related disorders in humans but relatively little is known about how methylation regulates immune function in cattle. Utilizing whole-transcriptome analyses of bovine dermal fibroblasts, we have previously identified an age and breed-dependent up-regulation of genes within the toll-like receptor 4 (TLR4) pathway that correlates with enhanced fibroblast production of IL-8 in response to lipopolysaccharide (LPS). Innate immune cells are characterized by their expression of germ-line encoded pattern recognition receptors (PRRs) which recognize conserved molecular patterns (PAMPs), such as lipopolysaccharide found on the outside of Gram-negative bacteria and viral nucleic acids, and elicit the appropriate defense response upon recognition. While it is clear that the response elicited by TLRs and other PRRs are crucial to the defense against a variety of bacterial, viral and eukaryotic pathogens [4,5,6], an exacerbated or dysregulated innate response contributes to infection related inflammatory disorders, such as in antibody dependent enhancement and the subsequent “cytokine storm” characteristic of Dengue hemorrhagic fever [7]. The innate response is a key contributor to a number of sterile pro-inflammatory conditions, such as rheumatoid arthritis and asthma [9, 10]
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