The data presented here support

The data presented here support a dynamic and complex interplay among labile heme, BACH1 and HO-1. It appears also that metabolites of heme degradation may participate in the mechanistic regulation of the various factors examined. Our results point to the possibility that the HO-1 pathway restricts BACH1 protein since its silencing enhances the levels of this repressor and the HO-1 product CO, released from CORM-401, led to significant inhibition of BACH1 in unstimulated and LPS-challenged cells. In addition, CORM-401 not only counteracted, but even over-compensated LPS-dependent down-regulation of HO-1 gene Alda 1 in hMDM. These effects are likely dependent on increased mobilization of labile heme observed after treatment with CO. Interestingly, enhanced cytosolic and nuclear labile heme mobilization has been reported also in Saccharomyces cerevisiae after exposure to the gaseous molecule NO [23]. Clearly, further experimental studies are required for a better understanding of the molecular mechanisms that are implicated in the interaction of CO, labile heme and BACH1 in macrophages. Macrophages are key regulators of immune homeostasis and inflammatory responses [56,57]. They exhibit phenotypical alterations ranging from inflammatory (also called M1) to anti-inflammatory (M2) macrophages as extremes of a continuous spectrum of activation in inflammation [58,59]. Notably, up-regulation of HO-1 has been associated with anti-inflammatory polarization of macrophages [60,61]. Thus, our current findings suggest that regulation of the cellular labile heme pool is critically involved in macrophage polarization via BACH1-dependent regulation of HO-1. Interestingly, loss of BACH1 in a mouse model of genetic BACH1 deficiency has previously been linked with M2 macrophage polarization in an in vivo model of colitis [62]. Moreover, administration of liposome-packed exogenous heme has been shown to provide protective therapeutic effects in a mouse model of myocardial infarction via reversing the M1 phenotype of inflammatory macrophages into anti-inflammatory M2 macrophages [63] and similar observations have recently also been reported in a dextran sodium sulfate-induced colitis model [64]. Finally, cellular levels of labile heme in macrophages may also be affected by alterations of extracellular heme in various in vivo situations such as hemolysis and/or tissue damage [27,65,66].
Acknowledgments We would like to thank Anette Sarti and Sylvie Manin for technical assistance. This work was supported by funding from a PHC Procope/DAAD Exchange Program between France and Germany (Project 57317676). Work in SI's laboratory is supported by the Deutsche Forschungsgemeinschaft (IM 20/4-1) and the European Union and the State of Niedersachsen project EFRE ZW6-85007634. RM and RF are supported by funding from INSERM and University Paris Est Créteil.
The HO family is a group of cytoprotective enzymes which catalyzes the regiospecific degradation of heme with the consequent formation of an equal amount of CO, ferrous iron (Fe2+) and BV, then reduced to BR [1]. Three different isoforms have been discovered so far, but most representatives are HO-1 and HO-2. The third isoform (HO-3), devoid of enzymatic activity, has been reported only in rats and it is currently poorly investigated [2]. HO-1 (32 kDa), also referred as heat shock protein 32 (Hsp32), is highly inducible by a wide variety of factors, such as the same substrate heme, oxidative stress, heavy metals, UV radiations, toxins, oxidizing agents, and xenobiotics. Dissimilarly, HO-2 (36.5 kDa) is constitutively expressed, mainly in brain and testis, and it is involved in modulation of germ and neuronal cells production. Low levels of HO-2 have also been reported in endothelial, kidney and hepatic cells [3]. HO-1 and HO-2 are encoded by two distinct genes (HMOX1 and HOMX2) that show nearly 40% homology of the amino acid sequence and a 55% homology in the highly conserved catalytic core [4,5].