OP012. Epigenetic control of colonic inflammation via the methyl-binding protein Mbd2
G.-R. Jones1, P. Cook2, A. MacDonald2, 1University of Edinburgh, Institute of Immunology and infection research, Edinburgh, United Kingdom, 2Manchester Collaborative Centre for Inflammation Research, Faculty Life Sciences, Manchester, United Kingdom
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 but display a dysregulated T cell cytokine response. The impact of Mbd2 deficiency on innate immune cell function and the immunological phenotype in the GI tract is unknown.
Mbd2−/−, CD11cCre and littermate Mbd2+/+ or Cre negative control (WT) mice were given 2% Dextran Sulphate Sodium (DSS) in drinking water for 7–10 d.
0.5×106 Mbd2−/− or WT CD4+ CD25− CD45RbHi T cells were adoptively transferred by intra-peritoneal injection into Rag−/− in a T cell model of chronic enterocolitis. Both models were assessed for weight loss, symptom score, colon histology score and mRNA expression of selected cytokines. Single cell suspension of colon lamina propria (LP) leucocytes were isolated and assessed by FACS for CD4+, FoxP3, CD8, CD25, CD11b, CD11c, CD103, CD64, MertK, F4/80, Ly6C, MHC-II, SiglecF, Ly6G. Intracellular cytokines (IL1b, TNF-alpha, IL-10, IL-17 and IFN-gamma) were assessed by FACS post 4hrs ex vivo Golgistop or PMA/ionomycin incubation.
Mbd2−/− and CD11cCre mice had equivalent proportions of all myeloid subsets in the LP compared to WT in the steady state. Mbd2−/− displayed significantly worse colitis post-DSS compared to WT, with greater weight loss, histology score, and mRNA expression of IFN-gamma and IL-17. CD11cCre mice similarly displayed significantly greater weight loss with increased proportion of LP neutrophils and Ly6C+ MHC-II +/− inflammatory monocytes after 8 days of DSS compared to WT. CD11cCre DSS treated LP inflammatory monocytes also had significantly greater TNF and IL-1b producing ability than WT as assessed by FACS suggesting for the first time epigenetic processes can regulate this cell type, which is previously shown to be the dominant pathogenic lineage in this model.
Mbd2−/− adoptively transferred T cells produced a significantly greater colitis with increased weight loss, histology score and mRNA expression of IL-17 and IFN-gamma. Mbd2−/− CD4+ LP T cells also had significantly greater proportion of double positive IL-17 and IFN-gamma producing cells demonstrating epigenetic regulation of T cells is critical in this established Th17 model of colitis.
These data reveal for the first time that Mbd2 is of critical importance in the orchestration of a T cell dependent and independent model of murine colitis in vivo. They also identify methyl-binding proteins and/or genes that they regulate as exciting new targets for therapeutic modulation of GI inflammation.