P143 HIF1A INHIBITS PRO-INFLAMMATORY CELLULAR PROGRAM IN FOXP3+ CELLS IMPLICATED IN IBD.

Abstract

A perplexing observation in Crohn’s disease has been the detection of increased numbers of FOXP3+ cells within the lesions, a higher proportion of FOXP3+to CD4+ cells within the mucosa of Crohn’s patients and a significantly higher gene count of FOXP3 from CD4+ cells isolated from lesions. These FOXP3+ cells appear to be unable to suppress inflammation in situ, and their role in disease is poorly understood. We have shown that a majority of FOXP3 gene targets in CD4+ lymphocytes isolated from Crohn’s lesions are in fact significantly upregulated and display a Th1/Th17-like gene set enrichment. These findings are incongruent with FOXP3’s ascribed role as a Treg specific transcriptional repressor. The hypoxia-inducible transcription factor HIF1a is upregulated in Tregs. During physiologic hypoxia experienced in the gut mucosa, the abundant HIF1a levels expressed in FOXP3+ Tregs are not driving aerobic glycolysis, as these cells extensively utilize fatty acid oxidative metabolism for their bioenergetic needs. We present data demonstrating the importance of nuclear HIF1a in inhibiting the FOXP3 activator complex, which leads to pro-inflammatory pathological FOXP3+ cells, and instead promotes the FOXP3 repressor complex, resulting in anti-inflammatory suppressor FOXP3+ cells. FOXP3 complex composition +/- HIF1a or IL-6 was assessed by immuno-precipitation and PLA. FOXP3 gene target promoter histone and histone modifying enzyme status was assessed by ChIP. A FOXP3-cre driven, EZH2fl/fl animal model was utilized to assess FOXP3 activator function/pro-inflammatory phenotype with HIF1a modulation on colitis. The FOXP3/p300 activation complex was more abundant in the absence of HIF1a, whereas the FOXP3/EZH2 repressor complex was reciprocally more abundant in the presence of HIF1a. Furthermore, IL-6 disrupts FOXP3/HIF1a association and the FOXP3/EZH2 repressor complex, resulting in increased FOXP3/p300 complex. Using IFNg and TNFa as a model genes for FOXP3 transcriptional regulation, we demonstrated that HIF1a regulates FOXP3 dependent gene activation by histone modifications at the IFNg and TNFa promoters. We demonstrated that HIF1a restores repression of the master lineage markers TBET and RORgT in in-vitro derived Tregs lacking FOXP3/EZH2 repressor function. Finally, the use of a HIF1a activator rescues colitis through HIF1a stabilization in the FOXP3 driven EZH2 null animals. FOXP3 acts as a binary switch, recruiting repressor complexes, or activator complexes to key Treg gene networks. We demonstrate HIF1a to be a key regulator of the FOXP3 switch mechanism directing program execution towards a repressed or anti-inflammatory state.