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ObjectiveSustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises 2-arachidonoylglycerol, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury.DesignC57BL/6J mice and mice with global invalidation of MAGL (MAGL-/-), or myeloid-specific deletion of either MAGL (MAGLMye-/-), ATG5 (ATGMye-/-) or CB2 (CB2Mye-/-), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl4) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells.ResultsMAGL-/- or MAGLMye-/- mice exposed to CCl4 or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6Clow macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGLMye-/- BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGLMye-/- mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor 2, as they were preserved in CB2Mye-/- mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5Mye-/- BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited.ConclusionMAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.

Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis.

Garlic is regularly consumed and is known to have diverse biologic activities, particularly due to its antioxidant properties. In this study, we hypothesized that crude garlic can prevent iron-mediated oxidative stress in a rat model of nutritional iron overload, and we used an in vitro model to confirm the results. For the in vivo studies, rats received a basal diet supplemented with or without carbonyl iron (3%) and were fed distilled water or garlic solution (1g/kg body weight) by gavage for 3 weeks. The presence of both garlic and iron led to a 2-fold increase in plasma iron and a 50% increase in liver iron as compared with iron alone. However, garlic did not offer any protection against iron-induced oxidative stress. Duodenal divalent metal transporter-1 mRNA expression was fully repressed by iron and by the combined treatments but was also reduced by garlic alone. To confirm these data, we tested the effect of diallyl disulfide, one of the active components in garlic, in vitro on polarized Caco-2 cells. A 24-hour treatment decreased iron uptake at the apical side of Caco-2 cells but increased the percentage of iron transfer at the basolateral side. This probably resulted from a modest induction of ferroportin mRNA and protein expression. These results suggest that garlic, when given in the presence of iron, enhances iron absorption by increasing ferroportin expression. The presence of garlic in the diet at the dose studied does not seem to protect against iron-mediated oxidative stress.

BACKGROUND: Senescent red blood cells (RBC) are recognized, phagocytosed and cleared by tissue macrophages. During this erythrophagocytosis (EP), RBC are engulfed and processed in special compartments called erythrophagosomes. We previously described that following EP, heme is rapidly degraded through the catabolic activity of heme oxygenase (HO). Extracted heme iron is then either exported or stored by macrophages. However, the cellular localization of the early steps of heme processing and iron extraction during EP remains to be clearly defined. METHODOLOGY/PRINCIPAL FINDINGS: We took advantage of our previously described cellular model of EP, using bone marrow-derived macrophages (BMDM). The subcellular localization of both inducible and constitutive isoforms of HO (HO-1 and HO-2), of the divalent metal transporters (Nramp1, Nramp2/DMT1, Fpn), and of the recently identified heme transporter HRG-1, was followed by fluorescence and electron microscopy during the earliest steps of EP. We also looked at some ER [calnexin, glucose-6-phosphatase (G6Pase) activity] and lysosomes (Lamp1) markers during EP. In both quiescent and LPS-activated BMDM, Nramp1 and Lamp1 were shown to be strong markers of the erythrophagolysosomal membrane. HRG-1 was also recruited to the erythrophagosome. Furthermore, we observed calnexin labeling and G6Pase activity at the erythrophagosomal membrane, indicating the contribution of ER in this phagocytosis model. In contrast, Nramp2/DMT1, Fpn, HO-1 and HO-2 were not detected at the membrane of erythrophagosomes. CONCLUSIONS/SIGNIFICANCE: Our study highlights the subcellular localization of various heme- and iron-related proteins during early steps of EP, thereby suggesting a model for heme catabolism occurring outside the phagosome, with heme likely being transported into the cytosol through HRG1. The precise function of Nramp1 at the phagosomal membrane in this model remains to be determined.

Anemia in critically ill patients is frequent and consists of chronic disease associated with blood losses. These two mechanisms have opposite effects on iron homeostasis, especially on the expression of the iron regulatory hormone hepcidin. We developed a mouse model mimicking the intensive care anemia to explore iron homeostasis.Experimental study.University-based research laboratory.C57BL/6 mice.Mice received either a single intraperitoneal injection of lipopolysaccharide followed 1 week later by zymosan, or were subjected to repeated phlebotomies by retro-orbital punctures, or both. Several subsets of mice were analyzed over a 14-day period to describe the mouse model of intensive care anemia. Additional mice received erythropoietin injections with or without the zymosan treatment and were killed at day 5, to perform a more detailed analysis.We observed anemia as soon as 5 days after zymosan injection, together with increased messenger RNA (mRNA) levels for interleukin-6 and hepcidin. Phlebotomies alone fully suppressed hepcidin mRNA expression. Interestingly, in mice treated with zymosan and phlebotomies, hepcidin expression was suppressed, despite the persistent increase in interleukin-6. Stimulation of erythropoiesis by erythropoietin injections also led to a decrease in hepcidin mRNA in zymosan-treated mice. In these situations combining inflammation and erythropoiesis stimulation, there was no change in ferroportin, the membrane iron exporter, at the mRNA level, whereas ferroportin protein increased. Macrophage iron stores (assessed by histology using diaminobenzidine staining, or by quantification of nonheme iron and ferritin concentrations) were depleted in the spleen.These results suggest that the erythroid factor dominates over inflammation for hepcidin regulation, and that iron could be mobilized in these situations combining inflammation and erythropoiesis stimulation.

Ferroportin, the only mammalian iron exporter identified to date, is highly expressed in duodenal enterocytes and in macrophages. Several lines of evidence indicate that in enterocytes the iron export mediated by ferroportin occurs and is regulated at the basolateral cell surface, where the transporter is strongly expressed. By contrast, in macrophages, ferroportin has been shown in intracellular vesicles. We used a high-affinity antibody to specify the localization of endogenous ferroportin expressed in primary culture of bone marrow–derived macrophages, in both basal and induced conditions. Our observations indicate that ferroportin is expressed in vesicular compartments that can reach the plasma membrane of macrophages. Of importance, when ferroportin expression was up-regulated through iron treatment or erythrophagocytosis, ferroportin expression was strongly enhanced at the plasma membrane of macrophages. Moreover, hepcidin dramatically reduced macrophage ferroportin protein levels. At the subcellular level, hepcidin was shown to induce rapid internalization and degradation of the macrophage iron exporter. These data are consistent with a direct iron export by ferroportin through the plasma membrane of macrophages and strongly support an efficient posttranscriptional down-regulation of ferroportin by hepcidin in these cells.

Following erythrophagocytosis (EP) of senescent red blood cells (RBCs), heme iron is recycled to the plasma by tissue macrophages. This process is critical for mammalian iron homeostasis but remains elusive. We characterized a cellular model using artificially-aged murine RBCs and murine bone marrow-derived macrophages (BMDMs) and study mRNA and protein expression of HO-1, ferroportin and ferritin after EP. In vitro ageing of RBCs was obtained by raising intracellular calcium concentration. These RBCs exhibit several features of erythrocyte senescence including externalization of phosphatidyl-serine, specific binding and phagocytosis by BMDMs. During the first hours of EP, we observed a rapid increase of HO-1 and ferroportin mRNAs and proteins, whereas ferritin protein expression was progressively induced with no major changes in RNA levels. At later stages after EP, a different pattern of expression was observed with a net decrease of ferroportin, a sustained high level of HO-1, and a strong increase in ferritins. Taken together, these results suggest that after EP, iron is rapidly extracted from heme and exported by ferroportin. Surprisingly, the gene expression profile at late stages after EP, which is indicative of iron storage, is reminiscent of what is observed in inflammation. However, phagocytosis of artificially-aged red blood cells seems to repress the proinflammatory response of macrophages.