YERSINIABACTIN INDUCES DISTINCT GENE EXPRESSION PROFILES COMPARED TO <i>E. COLI</i> INFECTION IN MACROPHAGES

Abstract Inflammatory bowel diseases (IBD), particularly Crohn’s disease (CD), are closely linked to colonization by adherent-invasive Escherichia coli (AIEC). Among these, Yersiniabactin (Ybt)-producing AIEC strains are associated with chronic inflammation and intestinal fibrosis. Macrophages play a central role in CD by orchestrating inflammatory and tissue remodeling responses. Our previous work demonstrated that Ybt sequesters zinc from macrophages, thereby activating the hypoxia-inducible factor 1-alpha (HIF-1α) and inducing pro-fibrotic macrophages that activate fibroblasts. Here, we investigated the upstream and downstream regulators of HIF-1α and the full transcriptional repertoire induced by purified Ybt or infection with Ybt-producing AIEC NC101. In this study, we demonstrate that macrophage signaling is profoundly altered by both AIEC infection and exposure to purified Ybt. AIEC infection induced a dominant transcriptional program (Principal Component 1, PC1: 63.9%). Purified Ybt triggered an orthogonal response (PC2: 14.6%) involving alternative pro-fibrotic macrophage activation. AIEC infection led to robust activation of the NF-κB pathway, which, in turn, regulated HIF-1α activation. NF-κB inhibition reduced both HIF-1α activation and the expression of select pro-fibrotic genes in the macrophage–fibroblast co-culture, indicating that NF-κB functions upstream of HIF-1α. Ingenuity Pathway Analysis identified upstream transcriptional regulators strongly associated with AIEC-induced NF-κB, consistent with findings from our IBD mouse model. Interestingly, infection with Ybt-deficient AIEC combined with purified Ybt (ΔYbt +Ybt) produced a unique transcriptional profile, upregulating metallothionein genes involved in zinc homeostasis, supporting our previous findings that Ybt sequesters zinc from macrophages and promotes fibrosis. Ybt altered chemotaxis-related genes, including Ccrl2 and the Cxcr3/Cxcl10 axis, which may contribute to persistent immune cell recruitment, providing a potential mechanism for sustaining chronic inflammation and fibrosis in IBD mouse models and CD patients. Collectively, Ybt and Ybt-producing AIEC modulate macrophage activation via shared and distinct transcriptional programs involving HIF-1α, NF-κB, and additional signaling pathways. These results provide a foundation for future studies to clarify the mechanisms through which Ybt shapes macrophage responses and its possible role in intestinal fibrosis.