Disordini della regolazione di epcidina e dell'omeostasi del ferro: meccanismi, diagnosi e trattamenti innovativi.

Different environmental and immune signals polarize macrophages to classic pro-inflammatory (M1) or alternative (M2) program; the latter is associated with regulation of adaptive immunity and control of cell growth and tissue repair. Interestingly, tumor associated macrophages (TAM) acquire an M2-like phenotype. These distinct cell populations strongly differ in the expression of immunoregulatory genes, but we have shown that they are also characterized by a differential regulation of genes involved in iron homeostasis (1). In particular, M2 cells display high ferroportin (Fpn) expression and iron release. Inflammatory macrophages use iron sequestration as a bacteriostatic mechanism and play a key role in body iron homeostasis in inflammation. Moreover, recent data suggest that iron accumulation in macrophages can favor M1 polarization, thus playing a role in the pathogenesis of chronic inflammatory disease. Conversely, the relevance of the Fpn-dependent iron release-prone phenotype of M2 macrophages for their tissue repair and immune regulatory properties (and tumor growth for TAM) is largely unknown.



We therefore propose a research program that characterizes the role of Fpn in iron traffic in M2 polarized cells. To this purpose, our first aim is to establish an in vivo model of macrophage-specific Fpn inactivation. Two mouse lines with macrophage-specific ablation of the Fpn gene will be generated by breeding mice carrying a floxed Fpn allele to mice expressing Cre recombinase under the control of macrophage–specific promoters (lysM and csf1r).



As we have reported that the conditioned medium of human M2 macrophages sustains faster growth of malignant and non malignant cell lines (1), we want to test in vivo the hypothesis that TAM support tumour growth through increased iron bioavailability, and thus we plan to evaluate whether the lack of macrophage Fpn slows down tumour growth. Experimentally, we will evaluate the growth and characteristics of chemically-induced tumours in knockout mice and their floxed littermates. Conversely, the increased extracellular iron availability supported by M2 cells could influence the growth rate of adjacent fibroblasts and collagen biosynthesis (prolyl hydroxylases are iron-dependent enzymes) during the repair phase, thus playing a beneficial role in wound healing. Therefore, the wound repair process after incisional skin damage will be evaluated in the same animal models.



As we found higher levels of Fpn mRNA in M2 than in M1 macrophages (1), mechanistic correlates to the in vivo studies reported above will be obtained by molecular biology experiments that will characterize the mechanisms underlying the transcriptional control of Fpn in response to different biologic inducers of M2 polarization. This may serve valuable information for reappraising the role of hepcidin-independent regulation of Fpn expression in pathophysiological situations.



Evidence in literature supports the notion that the decreased intracellular iron availability in M2 macrophages could have an anti-inflammatory outcome (2). Therefore, another goal of this project is to understand how impaired iron release due to Fpn ablation may modify the capacity of macrophages to undergo M2 differentiation.



The project is strengthened by collaborations with scientists participating to this proposal, who will exploit the mouse model of macrophage-restricted Fpn inactivation; moreover, our project will benefit of the help of several external collaborators. As such, it offers an opportunity for combining clinical investigation with molecular and cellular biology, according to an investigational philosophy that fits well in this call for projects.



We believe that the original approach and novel methodological and technical advances of this project will provide relevant information for the characterization of macrophage iron homeostasis, thus highlighting pathophysiological mechanisms helpful to solve well-defined clinical problems.