Influential Role of Macrophages in Hepcidin Regulation in Apo-Transferrin Treated β-Thalassemic Mice

Iron is a crucial component of the oxygen carrying molecules hemoglobin and myoglobin, and its recycling, primarily through macrophages, is essential to enable sufficient hemoglobin production to offset senescent red blood cell loss. The peptide hormone hepcidin, chiefly synthesized in the liver by hepatocytes, is the principle negative regulator of iron homeostasis and functions through binding and down-regulating ferroportin, the only known iron exporter on cells. IL-6 is an inflammatory cytokine secreted by macrophages, known to induce hepcidin expression via Stat3 signaling (Nemeth et al 2003; Wrighting and Andrews 2006). The disease β-thalassemia is characterized by a decrease in β-globin chain production that ultimately leads to a combination of anemia, ineffective erythropoiesis, and iron overload. We have shown that exogenous apo-transferrin (apoTf) treatment ameliorates ineffective erythropoiesis and reduces iron overload by increasing hepcidin expression in β-thalassemic mice (Li et al 2010). In addition to changes in MEK/ERK signaling (Chen et al 2016), our preliminary data reveals enhanced Stat3 signaling in hepatocytes from apoTf-treated mice. We hypothesize that apoTf functions in part through altering the function of macrophages and explore mechanisms of macrophage-induced hepcidin regulation in this setting. First, we examined the direct effect of apoTf on macrophages in vitro and demonstrate increased macrophage cell number and increasing IL-6, MCP-1, and IL-10 concentrations in supernatant of apoTf-treated macrophages. The addition of apoTf to cultured peritoneal macrophages from iron dextran treated mice resulted in increased iron mobilization to the supernatant without increasing ferroportin expression. Second, we explored the possibility that apoTf alters the polarization of macrophages or in another way influences the proportion of different types of macrophages. For example, two distinct macrophage populations have been identified in the peritoneal cavity, large peritoneal macrophages (LPM) and small peritoneal macrophages (SPM), that are functionally and developmentally distinct (Ghosn et al 2010). To assess the in vivo effect of exogenous apoTf on macrophages, we performed intraperitoneal apoTf injections of wild type and β-thalassemic mice and demonstrate a significantly increased proportion of SPM to LPM compared to PBS injected controls in both mice strains (Fig 1). Third, since apoTf upregulates hepcidin expression in vivo and shifts the population of peritoneal macrophages, we sought to test the role of macrophages in hepcidin regulation. To test the in vitro effect of macrophages on hepcidin expression, we co-cultured primary hepatocytes and macrophages. Peritoneal macrophages co-cultured with hepatocytes activated Stat3 signaling. Increasing the number of macrophages co-cultured with hepatocytes also correspondingly caused increased hepcidin expression. Furthermore, liver specific macrophages (Kupffer cells) and peritoneal macrophages both significantly upregulated hepcidin expression in hepatocyte co-culture and showed no difference in hepcidin induction ability between the two macrophage subsets (Fig 2). In addition, macrophages in co-culture with hepatocytes further increased hepcidin expression in the presence of additional apoTf, findings that were not recapitulated with addition of holo-transferrin in place of apoTf to co-culture (Fig 3). Lastly, mice were either treated with a low iron diet or intraperitoneally injected with iron dextran to induce iron loading to assess peritoneal macrophages' effect on hepcidin regulation in different iron conditions. Iron loaded macrophages induced the lowest hepcidin expression in hepatocyte co-culture while low iron diet macrophages caused the highest expression (Fig 4). IL-6 excretion was also highest in the low iron condition. Taken together, our data delineate the role of macrophages in hepcidin regulation in apoTf-treated, iron overloaded, and iron restricted conditions and suggest that apoTf functions through directly altering macrophage function and possibly sub-population to induce hepcidin expression in a paracrine manner in the liver.

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