E. Coli heme oxygenase modulates murine innate immune response

Abstract

Exposure of mice to the gaseous molecule CO (carbon monoxide) ameliorates experimental colitis through induction of mammalian heme oxygenase-1 (HO-1). Interestingly, enteric bacteria such as Escherichia Coli (EC) and Pseudomonas Aeroginosa express HO-1 homologues (ChuS and BphO, respectively). HO degrades heme into equimolar quantities of iron, biliverdin and CO that provides an alternative iron source for bacteria in low iron conditions. Mammalian HO-1 cloned and overexpressed in bacteria (Lactobacillus lactis) had been shown to decrease mucosal injury in a model of hemorrhagic shock in rats, providing evidence that enteric bacteria can modulate the host mammalian immune response through expression of HO-1 and generation its metabolic products (such as CO) in the intestinal lumen. Clinically, triggers that may result in bacterial HO induction, such as low iron diet or exposure to CO, may suppress the immune response through CO release. We hypothesize that the enteric bacterial biomass serves as a significant source of HO (and consequently CO released into the gut lumen) that may contribute to intestinal immune homeostasis. To begin to test this hypothesis, we studied effects of EC overexpressing ChuS on macrophage inflammatory responses. EC NC101 that constitutively overexpress ChuS (ChuS EC) (1500 fold increase of mRNA compared with WT EC) were generated. Murine bone marrow derived macrophages (BMMs) from C57Bl/6 mice were co-cultured with EC NC101 or ChuS EC (MOI 1:20) in low iron conditions supplemented with hemin as an alternative iron source. Il10 expression was 4.46 higher in BMMs co-cultured with ChuS EC compared to EC NC101 (p=0.007), correlating with increased IL-10 protein levels. Il12b expression and IL-12 p40 protein were lower in BMMs co-cultured with EC ChuS. Gentamicin protection assays demonstrated decreased phagocytosis (45% less) and killing (9.2% vs 30.5%) of ChuS EC compared to EC NC101 (p=0.016). Exposure of mice to exogenous CO for 14 days and analysis of enteric bacterial populations through T-RFLP and qPCR demonstrated an increase in E. Coli spp that express ChuS. ChuS E. Coli induces IL-10 and inhibits IL-12 p40 expression in macrophages. CO exposure results in alterations to the enteric microbiota in mice, with increased numbers of EC spp and increased amounts of ChuS.