Background: Loss of the suppressive function of helper T regulatory cells (Tregs) can promote uncontrolled inflammation in Crohn’s Disease (CD). We have previously shown that Tregs of CD patients aberrantly express proinflammatory genes downstream of TNFα signaling, but the mechanisms that regulate this transcriptional change in Tregs of CD patients remain unknown. Distinct chromatin landscapes in human T helper cells were recently reported to regulate gene programs such as tissue repair and pathogenicity. Therefore, targeting chromatin-based features controlling transcription in Tregs could revolutionize CD therapy. The aim of this study was to identify an actionable target to sustain Treg suppressive function and control inflammation in CD.
Methods: To identify upstream regulators of Treg dysfunction, we used a multiomic approach including RNA-seq and assay for transposase-accessible chromatin (ATAC-seq) in different stages of differentiation of Tregs and proinflammatory Th17 cells. We used multiple bioinformatic pipelines to identify epigenetic factors that drive the activation of open chromatin regions associated with proinflammatory gene networks in Tregs. We then validated the enrichment of these factors at these regions by Chromatin immunoprecipitation (ChIP-seq). CRISPR-mediated gene editing in primary human Tregs was performed to test the effect of targeting these factors on sustaining Treg function in the presence of inflammatory stimuli such as TNFα.
Results: We identified open chromatin regions in late-stage differentiated Tregs that are predominantly activated in early-stage differentiation of the proinflammatory Th17 cells. Characterization of gene networks associated with these regions uncovered multiple proinflammatory gene programs that are expressed in Th17 but not expressed in functional Tregs at any differentiation stage. We found that when these regions are inactive during the early stages of Tregs differentiation, TNFα does not affect their activity or the expression of the associated genes. However, it increases the enrichment of the epigenetic regulator, Bromodomain containing protein 4 (BRD4). Knockout of BRD4 in Tregs decreased the activation of proinflammatory genes in Tregs.
Conclusions: This study identifies a proinflammatory Th17-like chromatin landscape that is actively silenced in functional Tregs. Aberrant activation of this program can attenuate Treg function and perpetuate uncontrolled inflammation in CD patients. In this study, we uncover an actionable target (BRD4) to sustain the silencing of the Th17-like program with the goal of controlling inflammation in CD patients.

Patrick J. Fehrenbach

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