Heme oxygenase-1 expression and function is protective against innate immune responses to the enteric microbiota

Abstract

We have previously demonstrated that carbon monoxide (CO) suppresses chronic intestinal inflammation in IL-10−/− mice through a heme oxygenase 1 (HO-1) dependent pathway (Hegazi RA, J Exp Med. 2005). Here, we elucidate mechanisms through which HO-1 expression and function impact innate immune responses to the enteric microbiota. Wild type (WT) and colitis-prone IL-10−/− mice (n=5) raised in a gnotobiotic environment (GF) were transitioned to conventionalized specific pathogen free housing (SPF). HO-1 mRNA and protein were not detected in the colons of GF WT or IL- 10−/− mice, as determined by real time RT-PCR, Western immunoblotting and immunohistochemistry. However, increased HO-1 mRNA and protein were observed in colons from WT SPF-transitioned but not in colons from IL-10−/− mice. In addition, in SPF-transitioned WT and IL-10−/− mice, colonic HO-1 expression inversely correlated with expression of IL-12, IL-23 and IL-17. We further explored the role of IL-10 in the regulation of HO-1 in murine macrophages. IL-10−/− bone marrow-derived macrophages (BMMs) showed significantly reduced LPS-stimulated HO-1 mRNA and protein expression compared to WT BMMs. Furthermore, the addition of IL-10 restored HO-1 expression in IL-10−/− BMMs and enhanced expression in WT BMMs. Defects in intracellular bactericidal activity in macrophages have been implicated in the pathogenesis of IBD. To assess the role of CO and HO-1 in intracellular killing of enteric bacteria, BMMs were cultured with E. coli (NC101, K-12) and a gentamicin protection assay was used to assess anti-microbial responses. CO-exposed BMMs demonstrated increased HO-1 expression and enhanced bacterial killing compared to air-exposed BMMs. CO-induced enhanced bactericidal activity correlated with reduced secretion of IL- 12 p40. To specifically study the role of HO-1, retrovirus transduced RAW 264.7 macrophages that overexpress HO-1 were cultured with E. coli (NC101). Enhanced bacterial killing was demonstrated in RAW 264.7 macrophages over-expressing HO-1. We also studied the regulation of HO-1 in zebra fish (danio rerio). Zebra fish born and raised germ free and colonized with conventionalized microbiota resulted in four fold increase in zebra fish HO-1 mRNA expression when compared to germ free fish. Nuclear factor erythroid-2 related factor 2 (NRF2) regulates HO-1 expression. Next, we used morpholino oligomers specic for NRF2 to knock down its expression in germ free zebra sh prior to conventionalization. This resulted in marked reduction in HO-1 expression in zebra sh on conventionalization. Zebra fish were also exposed to a pharmacological inducer of HO-1, cobalt proptoporphyrin (CoPP) for 48 hrs. A 60 fold increase expression of HO-1 mRNA (Hmox1) was observed in the fish with a dose response curve in Hmox1expression. The enteric microbiota regulates HO-1 expression in the murine colon through IL-10 dependent mechanisms. Macrophage HO-1 expression enhances enteric bactericidal activity. HO-1 is also regulated in other experimental models, including zebra fish. Further work will explore the link between HO-1 expression, innate responses to the enteric microbiota, and intestinal inflammation in IBD.