ABSTRACT: The incidence of inflammatory bowel disease, including Crohn’s disease (CD), continues to increase worldwide, however, the contribution of CD4+ cell populations remains to be elucidated. Emerging evidence suggested increased infiltration of potentially dysfunctional FOXP3+ regulatory T (Treg) cells in inflammatory lesions. In this study, we aimed to provide an in-depth transcriptional assessment of CD4+ cells driving chronic ileal inflammation. For this purpose, ileal biopsies were isolated from five CD patients as well as six healthy individuals and CD4+ cells were purified for subsequent single cell RNA-sequencing (scRNA-seq). Our data revealed the presence of five distinct Treg subpopulations and that Tregs isolated from healthy control subjects represent the origin of pseudotemporal development into inflammation-associated subtypes. These pro-inflammatory Tregs isolated from CD patients displayed a unique responsiveness to TNFα signaling resulting in the upregulation of Treg-specific genes induced in Crohn’s (TRICs). We were able to establish an in vitro model of these pro-inflammatory FOXP3-positive cells which displayed an upregulation of TRICs, impaired suppressive activity as well as an elevated cytokine response in a novel organoid co-culture system. Intriguingly, based on our scRNA-seq data, an in silico drug prediction tool identified the histone deacetylase (HDAC) inhibitor (HDACi) Vorinostat as a putative regulator of the gene expression patterns from CD Tregs compared to control Tregs. Indeed, Vorinostat treatment resulted in a normalization of gene expression patterns and rescued the suppressive function of FOXP3-positive cells in vitro. Together, we provide insight into the role of CD4+ cells in CD lesions and have demonstrate a pathophysiologic role for TNFα-exposed Treg cells, underlying transcriptome-wide changes as well as potential novel therapeutic approaches. The incidence of inflammatory bowel disease, including Crohn’s disease (CD), continues to increase worldwide, however, the contribution of CD4+ cell populations remains to be elucidated. Emerging evidence suggested increased infiltration of potentially dysfunctional FOXP3+ regulatory T (Treg) cells in inflammatory lesions. In this study, we aimed to provide an in-depth transcriptional assessment of CD4+ cells driving chronic ileal inflammation. For this purpose, ileal biopsies were isolated from five CD patients as well as six healthy individuals and CD4+ cells were purified for subsequent single cell RNA-sequencing (scRNA-seq). Our data revealed the presence of five distinct Treg subpopulations and that Tregs isolated from healthy control subjects represent the origin of pseudotemporal development into inflammation-associated subtypes. These pro-inflammatory Tregs isolated from CD patients displayed a unique responsiveness to TNFα signaling resulting in the upregulation of Treg-specific genes induced in Crohn’s (TRICs). We were able to establish an in vitro model of these pro-inflammatory FOXP3-positive cells which displayed an upregulation of TRICs, impaired suppressive activity as well as an elevated cytokine response in a novel organoid co-culture system. Intriguingly, based on our scRNA-seq data, an in silico drug prediction tool identified the histone deacetylase (HDAC) inhibitor (HDACi) Vorinostat as a putative regulator of the gene expression patterns from CD Tregs compared to control Tregs. Indeed, Vorinostat treatment resulted in a normalization of gene expression patterns and rescued the suppressive function of FOXP3-positive cells in vitro. Together, we provide insight into the role of CD4+ cells in CD lesions and have demonstrate a pathophysiologic role for TNFα-exposed Treg cells, underlying transcriptome-wide changes as well as potential novel therapeutic approaches.
