Introduction Methyl-CpG binding protein domain protein-2 (Mbd2) is a transcriptional co-repressor that binds to methylated DNA. Mbd2 can recruit a nucleosome remodelling complex which contains chromatin remodelling and histone deacetylase properties. Mbd2 deficient mice are viable and fertile. However, they display a dysregulated immune phenotype with an aberrant T cell cytokine response and susceptibility to intestinal helminth infection (1). This immunological phenotype has not been explored in the GI tract.
Aim To assess the impact of Mbd2 deficiency on the activation status and cytokine production of naïve Mbd2-/- leucocytes isolated from murine small intestine (SI) and large intestine (LI) lamina propria (LP).
Methods All mice were bred in specific-pathogen free facilities at the University of Edinburgh. Mbd2-/- mice were produced as described previously (1). Single cell suspension of SI and LI LP were isolated as previously described, n = 4 in each group, minimum 2 experiments (2). Controls were wildtype (WT) age and sex matched littermates.
Cells were first stained with LiveDead blue (Life Technologies), FcR-Block and subsequently; CD11c, Ly6C, CD80, B220, CD11b, MHC(II), Ly6G, CD103, F4/80, CD40 and CD45. 4hr incubation with PMA/ionomycin/GolgiStop (BD bioscience) was performed for intracellular cytokine analysis with IFNgamma, IL-4, IL-13, IL-17 and TNFalpha. Samples were acquired using an LSRII and analysed with FlowJo (TreeStar), student t test was used with Prism (GraphPad) in statistical analyses.
Results Mbd2-/- mice showed significantly greater IFNgamma, TNFalpha and IL-17 but not IL-13 or IL-4 production in CD4+ and CD8+ lymphocytes isolated from SI and LI LP compared to WT controls. In addition surface activation markers CD80 and CD86 were significantly greater in Mbd2-/- CD103+CD11b+CD11c+ Dendritic cells and CD11b+CD11c+F4/80 macrophages from SI LP.
Conclusion These results show for the first time that epigenetic processes can regulate both antigen presenting cell activation status and T cell production of potentially damaging inflammatory cytokines at the mucosal-environmental barrier. They also identify methyl-binding proteins and/or genes that they regulate as exciting new targets for therapeutic modulation of GI inflammation.
Disclosure of Interest None Declared
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