ZINC RESTRICTION BY INTESTINAL E. COLI - PRODUCED YERSINIABACTIN ACTIVATES HIF-1α IN MACROPHAGES TO PROMOTE CD-ASSOCIATED FIBROSIS

Abstract

Abstract Intestinal fibrosis is a significant complication in Crohn’s disease (CD) patients, causing severe intestinal strictures and obstructions that are ultimately relieved by surgical bowel resection. Mechanisms driving CD-associated fibrosis are poorly understood but implicate the microbiome, especially adherent-invasive E. coli (AIEC). Previously, our work demonstrated that AIEC production of the metallophore yersiniabactin (Ybt) promotes fibrosis in the well-established gnotobiotic Il10-/- CD mouse model. These pro-fibrotic effects do not require bacterial uptake/utilization of Ybt, suggesting Ybt directly targets the host. Here we show that CD11b+ F4/80+ MHCIIlow macrophages are abundant in the submucosa and muscularis of fibrotic lesions in our mouse model. Similarly, CD68+ macrophages are abundant in strictures, not adjacent margins, of ileocolonic CD patients. We hypothesized that Ybt targets host macrophages to drive fibrosis. A critical metal-dependent pathway for immune cell function during inflammation is HIF-1α. We demonstrate that AIEC infection activates HIF-1α in macrophages, which is dependent upon Ybt production. Importantly, in fibrotic lesions of CD mouse models and resected strictures of ileocolonic CD patients, HIF-1α is activated and localized in nuclei of F4/80+ or CD68+ macrophages. We identified that zinc sequestration by Ybt is responsible for HIF-1α activation, a novel mechanism of HIF-1α stabilization. Furthermore, Ybt-producing AIEC induce potent pro-fibrotic gene expression in macrophage:fibroblast co-cultures, a process dependent on macrophage HIF-1α. This implies that Ybt induces pro-fibrotic macrophages, activating adjacent fibroblasts and fostering intestinal fibrosis. In summary, our findings suggest that as intestinal macrophages encounter AIEC, Ybt sequesters zinc, causes an accumulation of nuclear HIF-1α, and this imbalance of metal homeostasis drives pro-fibrotic macrophage and fibroblast activation. Ultimately, effective fibrotic remodeling fails and leads to intestinal fibrosis in CD patients.