Interaction of omega-3 fatty acids and cytokine gene DNA methylation

Abstract

It is well established that inflammation is associated with many non-communicable diseases and that when there is a failure in the resolution process acute inflammation becomes chronic with many undesired consequences. Treatment of chronic inflammation with pharmacological anti-inflammatory drugs can have detrimental effects on the body and can lead to an immunocompromised state. It is therefore prudent to consider whether other interventions can combat chronic inflammation.It is possible that omega-3 polyunsaturated fatty acids (\uf077-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), high levels of which are found in fatty fish, can influence the inflammatory process in a beneficial way and promote the resolution of inflammation thus facilitating a return to homeostasis. The mechanisms through which this might be achieved are not fully understood but the answer may lie within the epigenetic changes that are provoked by the presence of the fatty acids in the diet.Epigenetics is a rapidly developing field of study which investigates heritable chemical modifications to the genome which are independent of the DNA sequence. These chemical changes are vital for normal cellular development, cellular processes, and cell-specific gene expression profiles; and if \uf077-3 PUFAs (EPA and DHA) could influence them, they could provide a target for therapeutic interventions. Research to date has demonstrated that \uf077-3 PUFA (EPA and DHA) supplementation is associated with changes to markers of inflammation and changes in gene expression. Few studies have researched the effect of dietary \uf077-3 PUFA (EPA and DHA) supplementation on the epigenome and none have focused specifically on the inflammatory pathways associated with chronic inflammation in adult humans. The doctoral research reported in this thesis aims to elucidate the interaction between dietary fatty acids and the epigenome particularly as regards inflammation and its resolution.Focusing on one epigenetic marker, DNA methylation, this work has employed a hypothesis driven targeted approach with candidate genes selected after a review of relevant literature. Custom designed pyrosequencing assays were developed to facilitate the measurement of DNA methylation at specific cytosine bases in identified biologically relevant stretches of DNA. We performed a cross sectional study to investigate the relationship between methylation levels of the gene which encodes tumor necrosis factor alpha (TNFα), and levels of fatty acids within the blood. It was found that many of the fatty acids included in the study were not correlated with methylation levels of TNF but some significant correlations were discovered. By analysing the results by sex it was also discovered that some correlations are sex-specific. Specifically, for the males within the study, significant correlations were observed between TNF methylation and \uf077-3 PUFA DHA, as well as \uf077-6 PUFA arachidonic acid (AA). The relationship between blood fatty acid levels and DNA methylation of cytokine genes was further investigated using a four-week ω-3 PUFA (EPA and DHA) supplementation intervention. The genes included in this study were TNF, interleukin 6 (IL6) and interleukin 1 beta (IL1B). Peripheral blood mononuclear cells were isolated from blood samples before and after supplementation and these were analysed for fatty acid levels in the cell membranes in order to explore the timeframe over which incorporation occurred. Despite seeing an increase in \uf077-3 PUFAs EPA and DHA in PBMCs, there were no changes in methylation of the candidate genes over the intervention period and therefore postulated that this might be because an inflammatory stimulus was absent in this cohort of healthy males.In the final study of this thesis methylation of candidate genes at biologically relevant sites cytokines and RNA expression were measured before and after an inflammatory stimuli, and either side of fatty acid supplementation. In this randomised double blind, repeated measures supplementation study the inflammation stimulus was provided by eccentric exercise and one group was supplemented with ω-3 PUFA (EPA and DHA) and the other with olive oil. Baseline measurements, made before the inflammatory stimulus or supplementation, showed that methylation levels of TNF were negatively associated with the expression of TNF mRNA, however there was no association with TNFα cytokine levels. There was a negative relationship between TNF and IL1B methylation levels at baseline.The inflammatory stimulus induced a decrease in TNF and IL6 methylation levels and an increase of serum cytokines coded by these genes. In contrast, an increase in methylation in the measured region of IL1B was observed. The results clearly demonstrated the effect of a repeated bout of exercise in which decreased inflammation (cytokines and muscle damage markers) is observed after the second bout. There was no difference in the decrease as a consequence of the dietary supplementation. In conclusion it was found that as a result of the cross-sectional study, that some but not all fatty acids in the blood affect the methylation levels of certain CpG sites in exon1 of TNF. Positive and negative associations were observed, with many of the associations being sex specific. In the second study it was established that a four-week period of dietary \uf077-3 PUFA (EPA and DHA) supplementation does not significantly change the methylation levels of TNF, IL6 or IL1B and, in the final study, muscle damage was induced through eccentric exercise and this effected a transient epigenetic change, but that fatty acid supplementation had no significant effect over the period of the study. Although DNA methylation of TNF, IL6 and IL1B at the sites studied is not altered by a change in blood fatty acids over a short period this may not be the case over longer periods of intervention and this, together with exploration of the interaction with other environmental factors, such as muscle induced inflammatory stimulus, present opportunities for future research.