Pro-inflammatory Cytokine IFNγ and Microbiome-Derived Metabolites Dictate Epigenetic Switch between FOXP3 Isoforms in Celiac Disease

Abstract

Celiac disease (CD) is an autoimmune enteropathy triggered by gluten and characterized by a strong Th1/Th17 immune response in the small intestine. Treg cells are CD4+ CD25++ cells that regulate the immune response and maintain immune homeostasis. Given its role in controlling the differentiation and function of Treg cells, FOXP3 has been considered their master transcription factor. Two main isoforms of FOXP3 have been described. Conversely to its counterpart FOXP3 full length (FL), the alternatively spliced isoform FOXP3 ∆2 cannot properly down-regulate Th17 driven immune response. Since the active state of CD has been associated with impairments in Treg cell function, we aimed at determining whether imbalances between the FOXP3 isoforms may be associated with the development of the disease. Intestinal biopsies from patients with active CD showed increased expression of the less functional FOXP3 ∆2 isoform over FL, while both isoforms were similarly expressed in non-celiac control subjects (HC). Conversely to what we saw in the intestine, peripheral blood mononuclear cells (PBMC) from HC subjects did not show the same balance between the two isoforms. We therefore hypothesized that the intestinal microenvironment may play a role in modulating the alternative splicing of FOXP3 isoforms. The pro-inflammatory intestinal microenvironment of active patients have been reported to be enriched in butyrate producing bacteria, while high concentrations of lactate have been shown to characterize the preclinical stage of the disease. We show that the combination of IFNγ (the main pro-inflammatory cytokine produced in the intestine of active CD patients) and butyrate triggers the balance between FOXP3 isoforms in HC subjects, while the same does not occur in CD patients. Furthermore we report that lactate significantly increases both isoforms in CD patients when compared to control subjects. Collectively these findings highlight the importance of the ratio between FOXP3 isoforms in CD and, for the first time, mechanistically associate the alternative splicing process with microbial derived metabolites.