Regulatory T (Treg) cells expressing the transcription factor FOXP3 play a pivotal role in maintaining immunologic self-tolerance. We and others have shown previously that EZH2 is recruited to the FOXP3 promoter and its targets in Treg cells. To further address the role for EZH2 in Treg cellular function, we have now generated mice that lack EZH2 specifically in Treg cells (EZH2FOXP3). We find that EZH2 deficiency in FOXP3 T cells results in lethal multiorgan autoimmunity. We further demonstrate that EZH2FOXP3 T cells lack a regulatory phenotype in vitro and secrete proinflammatory cytokines. Of special interest, EZH2FOXP3 mice develop spontaneous inflammatory bowel disease. Guided by these results, we assessed the FOXP3 and EZH2 gene networks by RNA sequencing in isolated intestinal CD4 T cells from patients with Crohn's disease. Gene network analysis demonstrates that these CD4 T cells display a Th1/Th17-like phenotype with an enrichment of gene targets shared by FOXP3 and EZH2. Combined, these results suggest that the inflammatory milieu found in Crohn's disease could lead to or result from deregulation of FOXP3/EZH2-enforced T cell gene networks contributing to the underlying intestinal inflammation.

Background & AimsForkhead box protein 3 (FOXP3)+ regulatory T cell (Treg) dysfunction is associated with autoimmune diseases; however, the mechanisms responsible for inflammatory bowel disease pathophysiology are poorly understood. Here, we tested the hypothesis that a physical interaction between transcription factor FOXP3 and the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is essential for gene co-repressive function.MethodsHuman FOXP3 mutations clinically relevant to intestinal inflammation were generated by site-directed mutagenesis. T lymphocytes were isolated from mice, human blood, and lamina propria of Crohn’s disease (CD) patients and non-CD controls. We performed proximity ligation or a co-immunoprecipitation assay in FOXP3-mutant+, interleukin 6 (IL6)-treated or CD-CD4+ T cells to assess FOXP3–EZH2 protein interaction. We studied IL2 promoter activity and chromatin state of the interferon γ locus via luciferase reporter and chromatin-immunoprecipitation assays, respectively, in cells expressing FOXP3 mutants.ResultsEZH2 binding was abrogated by inflammatory bowel disease–associated FOXP3 cysteine 232 (C232) mutation. The C232 mutant showed impaired repression of IL2 and diminished EZH2-mediated trimethylation of histone 3 at lysine 27 on interferon γ, indicative of compromised Treg physiologic function. Generalizing this mechanism, IL6 impaired FOXP3–EZH2 interaction. IL6-induced effects were reversed by Janus kinase 1/2 inhibition. In lamina propria–derived CD4+T cells from CD patients, we observed decreased FOXP3–EZH2 interaction.ConclusionsFOXP3–C232 mutation disrupts EZH2 recruitment and gene co-repressive function. The proinflammatory cytokine IL6 abrogates FOXP3–EZH2 interaction. Studies in lesion-derived CD4+ T cells have shown that reduced FOXP3–EZH2 interaction is a molecular feature of CD patients. Destabilized FOXP3–EZH2 protein interaction via diverse mechanisms and consequent Treg abnormality may drive gastrointestinal inflammation.

Background The aetiology of Crohn’s disease [CD] involves immune dysregulation in a genetically susceptible individual. Genome-wide association studies [GWAS] have identified 200 loci associated with CD, ulcerative colitis, or both, most of which fall within non-coding DNA regions. Long non-coding RNAs [lncRNAs] regulate gene expression by diverse mechanisms and have been associated with disease activity in inflammatory bowel disease. However, disease-associated lncRNAs have not been characterised in pathogenic immune cell populations. Methods Terminal ileal samples were obtained from 22 CD patients and 13 controls. RNA from lamina propria CD4+ T cells was sequenced and long intergenic non-coding RNAs [lincRNAs] were detected. Overall expression patterns, differential expression [DE], and pathway and gene enrichment analyses were performed. Knockdown of novel lincRNAs XLOC_000261 and XLOC_000014 was performed. Expression of Th1 or Th17-associated transcription factors, T-bet and RORγt, respectively, was assessed by flow cytometry. Results A total of 6402 lincRNAs were expressed, 960 of which were novel. Unsupervised clustering and principal component analysis showed that the lincRNA expression discriminated patients from controls. A total of 1792 lincRNAs were DE, and 295 [79 novel; 216 known] mapped to 267 of 5727 DE protein-coding genes. The novel lincRNAs were enriched in inflammatory and Notch signalling pathways [p <0.05]. Furthermore, DE lincRNAs in CD patients were more frequently found in DNA regions with known inflammatory bowel disease [IBD]-associated loci. The novel lincRNA XLOC_000261 negatively regulated RORγt expression in Th17 cells. Conclusions We describe a novel set of DE lincRNAs in CD-associated CD4+ cells and demonstrate that novel lincRNA XLOC_000261 appears to negatively regulate RORγt protein expression in Th17 cells.

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T cell lineage decisions are critical for the development of proper immune responses to pathogens as well as important for the resolution of inflammatory responses. This differentiation process relies on a combination of intrinsic and extrinsic factors converging upon epigenetic regulation of transcriptional networks relevant to specific T cell lineages. As these biochemical modifications represent therapeutic opportunities in cancer biology and autoimmunity, implications of writers and readers of epigenetic marks to immune cell differentiation and function are highly relevant. Given the ready adoption of histone methyltransferase inhibitors in the clinic, we focus this review on the role of three histone modifying complexes: PRC-1, PRC-2, and G9A in modulating T cell fate decisions. Furthermore, we explore the role of long non-coding RNAs in regulating these processes, and discuss recent advances and challenges of implementing epigenetic therapies into clinical practice.