Dynamic alterations in labile heme levels and heme biosynthesis during inflammatory activation of macrophages

Heme is an iron-containing tetrapyrrole with dual biological functions. While it serves as an essential prosthetic group in various hemoproteins, heme is cytotoxic in its 'free', non-protein-bound, form. Labile heme (LH) denotes the intracellular fraction of bioavailable heme that is readily exchangeable for incorporation into hemoproteins. To investigate the regulatory role of this heme fraction in inflammatory activated macrophages, we applied the selective fluorescent small molecule H-FluNox for LH detection in lipopolysaccharide (LPS)-stimulated murine bone marrow-derived macrophages (BMDMs). Studies with H-FluNox and its cell-permeable derivative acetylated (Ac)-H-FluNox revealed a time-dependent decrease of LH levels in living BMDMs upon treatment with LPS. Expression of δ-aminolevulinate synthase 1, the rate-limiting enzyme of heme synthesis, was up-regulated in parallel to decreased LH. Studies in subcellular organelles of BMDMs demonstrated that LH concentrations were markedly higher in mitochondria compared to cytosol and nuclei. Furthermore, expression of inducible nitric oxide synthase (iNOS), a heme-containing pro-inflammatory enzyme, was linked to intracellular LH concentrations. Specifically, LPS-dependent iNOS induction was attenuated in BMDMs displaying decreased LH, either after treatment with pharmacological heme synthesis inhibitors, or with genetic deficiency of the nuclear heme sensor BACH1. By contrast, inducibility of iNOS by LPS was markedly higher in BMDMs exhibiting increased levels of LH following treatment with the heme synthesis substrate δ-aminolevulinate. Finally, pharmacological inhibition of succinate dehydrogenase, which enhances intracellular levels of δ-aminolevulinate and LH, was also associated with higher inducibility of iNOS by LPS. In conclusion, the data indicate that intracellular LH is modulated by inflammatory stimulation in mouse macrophages and is critical for heme incorporation into the hemoprotein iNOS. Thus, heme availability may serve as a regulatory link between metabolic and inflammatory pathways.