Epigenetic Modifications Influence NOD‐Like Receptor Expression and Associated Pro‐Inflammatory Activity

Abstract

BackgroundNOD‐like receptors (NLRs) are a family of intracellular pathogen recognition receptors associated with the innate immune response. NOD1 and NOD2 are the best characterised members of this class of receptors. Upon activation of these receptors by invading bacterial components, NOD1 and NOD2 promote pro‐inflammatory cytokine production. Aberrant NLR expression has been associated with many chronic inflammatory diseases, such as inflammatory bowel disease. Mechanisms underlying NLR expression regulation have yet to be elucidated. We hypothesise that epigenetic modification, such as DNA methylation and histone acetylation, may play a regulatory role for expression of these receptors.ObjectiveThe objective of this study was to quantify basal NOD1/NOD2 expression and pro‐inflammatory activity in response to epigenetic pattern alterations.MethodsMonocytic and intestinal epithelial cell lines were used in this study. Cells were treated with pharmacological agents targeting and interfering with epigenetic patterns including; DNA methyltransferase 1 inhibitors (DNMT1i) and histone deacetylase inhibitors (HDACi), after which basal NOD1/NOD2 expression was quantified. Pro‐inflammatory activity following DNMT1i/HDACi exposure was examined by stimulating pre‐treated cells with iE‐DAP (NOD1 ligand) or MDP (NOD2 ligand) and subsequently quantifying pro‐inflammatory cytokine expression. Gene expression analysis was carried out by quantitative polymerase chain reaction (qPCR). Protein expression was analysed by western blotting. Independent t‐tests, one‐way ANOVA or two‐way ANOVA analysis was carried out where appropriate, followed by suitable post‐hoc tests.ResultsTreatment of the monocytic cell line with a DNMT1i significantly increased NOD1 (mRNA p<0.001; protein p<0.01) and NOD2 (mRNA and protein p<0.001) expression. HDACi treatment had similar effects on NOD2 in these cells, resulting in a significant upsurge in NOD2 (mRNA p<0.001) expression. In the intestinal epithelial cell line, DNMT1i also significantly increased NOD1 (Protein p<0.01) and NOD2 (mRNA p<0.01; protein p<0.05) expression. The HDACi exposure also significantly increased NOD1 and NOD2 (Protein p<0.05) expression. Pre‐treatment of monocytic/intestinal epithelial cell lines with a DNMT1i or HDACi was found to have significantly increase TNF‐α and IL‐6 expression in response to stimulation with NOD1 (p<0.001) or NOD2 (p<0.001) ligands, indicating increased pro‐inflammatory activity following alterations in epigenetic patterns.ConclusionsThis study has uncovered, for the first time, a relationship between epigenetic modifications and NOD‐like receptor expression and activity, suggesting that DNA methyltransferase or histone deacetylase enzymes could play a key role in the regulation of these pathogen recognition receptors. If this is indeed the case, pharmacological targeting of these enzymes could have the potential to modify the innate immune response and serve as novel therapeutics to treat chronic inflammatory diseases, such as inflammatory bowel disease.Support or Funding InformationThis study was supported by funding from the Hardiman Scholarship, NUI Galway.