Alterations In The Foxp3-Ezh2 Pathway Associates With Increased Susceptibility To Colitis In Both Mice And Human

Abstract Crohnu0027s disease is a common intestinal inflammatory disorder of uncertain etiology and incomplete treatment options. It is characterized by lesions infiltrated by inflammatory CD4 lymphocytes; yet the mechanism of CD4 mediated pathophysiology is unclear. Through whole genome approaches on clinical cohorts of CD patients, combined with functional in-vivo and in-vitro murine data, we sought to identify and evaluate aberrant transcriptional gene networks in disease-associated CD4 cells. We comparatively studied the expression profile in CD4 lymphocytes isolated from the Ileum of 21 CD-affected individuals and 12 age/gender matched control individuals. Utilizing RNA-seq, we conducted upstream target analysis to identify top disease associated regulatory networks. Potential coordinated function between FOXP3 and EZH2 was identified, and common gene targets were interrogated for differential expression and pathway analysis.We crossed FOXP3-driven CRE recombinase expressing mice (B6129S-Tg(Foxp3-EGFP/cre)1aJbs/J) with EZH2fl/fl animals to better define the effect of EZH2 ablation on Treg function. Splenocytes were analyzed by cell surface markers for T cell distribution and phenotype. EZH2Δ/Δ FOXP3+ cell function was determined by in-vitro Treg suppressor function assay. Cytokine analyses were performed on supernatants of stimulated EZH2Δ/ΔFOXP3+ splenocytes and serum from 14 to 17 days old FOXP3-CRE;EZH2Δ/Δ mice. FOXP3-CRE;EZH2Δ/Δ or animals treated with EZH2 inhibitors received 3% DSS in an acute model of colitis. Furthermore, RB45high transfer utilizing WT or EZH2Δ/ΔTregs was used in a chronic model of colitis. Differential expression analysis between CD and CTRL samples revealed 5328 statistically significant Differentially Expressed Genes (DEGs). Principle component analysis, and hierarchical clustering, separated patients from control subjects. Among the DEGs, 83 were well-defined transcription factors (TFs) identified by Master Regulatory Analysis. The transcription factor FOXP3 and its associated regulatory network emerged as a highly differentially expressed pathway in diseased samples by Master Regulatory Analysis, Ingenuity Pathway Analysis, and Gene Set Enrichment Analysis. Additionally, common gene targets for both FOXP3 and EZH2 were concurrently up regulated in disease. Genetic ablation of EZH2 in mouse FOXP3+ cells resulted in Treg cells converted to a TH1/TH17- like effector phenotype, a pattern shared by disease associated CD4 T cells. These cells exhibited pro-inflammatory cell surface markers, cytokine expression, and reduced in-vitro suppressive capacity. EZH2Δ/Δ Tregs failed to mitigate both DSS and T cell mediated colitis, and mice treated with the EZH2 inhibitor DZNep were also more susceptible to DSS colitis. In conclusion, we compare EZH2/Foxp3 co-regulation of gene networks between Crohnu0027s lesions and controls, and in multiple mouse models, to clarify the role of epigenetic regulation in this disease process. Multiple colitis disease models show us that interfering with EZH2 by genetic or pharmaceutical methods results in increased susceptibility to colitis. We postulate the inflammatory milieu found in IBD modulates EZH2 function, thereby making it less able to co-repress non-Treg differentiation programs even in the presence of upregulated FOXP3. Further investigation into EZH2 mutations, signaling cascades and relevant post-translation modifications to the FOXP3-EZH2 gene network may assist to elucidate and predict disease progression and/or severity.