Comparación con otras alternativas

The overarching aim of this research is to investigate how ω-3 PUFAs and other fatty acids within the blood interact with inflammation, thus a critical component of this work was the identification of target genes and specific CpG sites within them. The current research into the interaction between fatty acids and DNA methylation, described in Chapter 1, has particularly focused on fatty acid metabolism genes and genome-wide methylation analysis. The methylation genome-wide association studies (meth- GWAS) research has identified gene pathways enriched for DMRs which are linked to fatty acids and include pathways involved in initiating and regulating inflammation. Therefore, the decision was made to investigate genes that have been associated with fatty acids or where their transcripts have been altered by fatty acid consumption. Inflammatory diseases are often characterised with chronic activation of NFκB transcription factor and release of inflammatory cytokines (Epstein, Barnes and Karin, 1997) and therefore an important target for changes in DNA methylation by ω-3 PUFAs. Associations between dietary fats and DNA methylation in the NFκB pathway, measured using the 450k array, were observed in the Greek preadolescent cross- sectional cohort (Voisin et al., 2015), and therefore may provide valuable targets within intervention studies. The transcription factor NFκB is a protein complex encoded by multiple genes including NFKB1 and NFKB2. Furthermore, methylation of these genes have been associated with dietary intake of milk post-exercise and to exercise

interventions (Y. Zhang et al., 2015; Masuki et al., 2017). These two genes warrant further investigation as to their interactions with fatty acids.

Considering the cytokines within the NFκB pathways, the inflammatory cytokine tumor necrosis factor alpha (TNFa), encoded by the TNF gene, primarily mediates the acute- phase response in the inflammatory process (Sullivan et al., 2007). TNFα is associated with a wide range of inflammatory disorders, and therefore is regularly a therapeutic target with anti-TNFα drugs (Bazzani et al., 2009). When a macrophage cell line has been stimulated with LPS in vitro, TNFα secretion is decreased by the treatment of ω-3 PUFAs EPA and DHA (Mullen, Loscher and Roche, 2010). EPA treatment does not affect the stability of TNF mRNA in cytoplasm and therefore reductions in TNF mRNA as a result of EPA treatment is likely to be the result of decreased formation rather than degradation of the cytokine (Zhao et al., 2004). The influence of ω-3 PUFAs on TNF expression may be the result of alterations in epigenetic mechanisms such as DNA methylation. Epigenetic regulation plays an important role in the control of TNF expression (Kochanek et al., 1991; Takei et al., 1996; Sullivan et al., 2007). In a cell line that does not produce TNFα in response to LPS or phorbol myristate acetate (PMA), the promotor and exon-1 of TNF had higher methylation levels than TNFα expressing cells (THP-1 macrophages) which had a demethylated promotor and exon-1 (Sullivan et al., 2007). Specifically, TNF has been shown to be hypomethylated in Crohn’s disease patients (Nimmo et al., 2012), and that CpG methylation interacts with histone methylation to silence TNF expression during endotoxin tolerance (Gazzar et al., 2008). Although some previous research has not observed associations between DNA methylation of TNF and fatty acids (Lee et al., 2013; Bollati et al., 2014); other research has found environmental factors, including diet and fatty acids, that do have relationships with TNF methylation (Campión et al., 2009; Hermsdorff et al., 2013; Bollati et al., 2014; Gómez-Uriz et al., 2014). For this reason, it is worth considering cytokine genes as potential targets for further investigation.

The expression of IL6 is also decreased after the treatment of ω-3 Fatty acids (Cawood et al., 2010). There is previous evidence for the regulation of gene expression through alterations in the methylation of the IL6 promotor (Poplutz et al., 2014). The occurrence of DNA methylation of four CpG sites in the IL6 promoter is

significantly higher in cancer cell lines and associated with lower IL6 mRNA and methylation of the IL6 promoter was inversely related to mRNA levels (Tekpli et al., 2013). In addition, when macrophages are stimulated with LPS, increased mRNA is associated with lower IL6 promotor methylation (Nile et al., 2008). Measures of adiposity have been found to associate with IL6 promotor methylation (Na et al., 2015). A cross sectional study comparing ω-3 PUFA in erythrocyte membranes with IL6 methylation of a single CpG site within the IL6 gene found that DHA and total ω-3 fatty acid was negatively associated with the methylation level. Furthermore, methylation of this site was correlated with IL6 expression and plasma IL6 concentration (Ma et al., 2016). A separate CpG site in the same study found no relationship between IL6 expression and plasma IL6 concentration (Ma et al., 2016), indicating the importance of site selection. In addition, this study used 450k array measurements of the methylation at each of these sites, demonstrating that although 450k arrays sample a high number of sites, it depends if those individual CpGs are biologically relevant to be altered or have associations with fatty acids.

A third cytokine, for which evidence of alteration by of ω-3 PUFAs exists, is IL1β (Allam-Ndoul et al., 2016). There is also evidence of transcriptional regulation of this cytokine through methylation of the promotor (Hashimoto et al., 2009; Aoi et al., 2011; Tekpli et al., 2013). In cancer cells, CpG sites near the transcription start site (TSS) of IL1B “interleukin 1 beta” promoter were significantly more methylated and associated with lower IL1B mRNA levels (Tekpli et al., 2013). Methylation of the IL1B promoter was inversely related to mRNA levels in cultured human lung cells (Tekpli et al., 2013). Two CpG sites (-299 and -256bp from Exon 1) in the IL1B promoter were differentially methylated between control samples and cytokine-treated chondrocytes (Hashimoto et al., 2009). There is currently no evidence on the effects of ω-3 PUFAs on the methylation levels of IL1B and therefore will investigate this further in this thesis. The following genes were considered for assay design: NFKB1, NFKB2 (failed assay design), TNF, IL6 and IL1B.