Differential Recovery of Macrophage Iron Deficiency By Oral Iron Formulations Determines Cell Inflammatory Response in Conditions of Iron Deficiency Anemia

Introduction: Sucrosomial iron (SI) is an innovative oral iron formulation which is absorbed intact through para and trans-cellular intestinal routes as well as via M-cells. Once in the circulation SI is internalized intact by reticulo-endothelial macrophages, which likely play a major role in iron recycling from the sucrosome shell and release into the bloodstream where iron joins the transferrin (Tf)-bound iron pool. By contrast, the common oral iron salt iron sulfate (FeSO4) is absorbed via the canonical inorganic iron pathway involving DMT1 and FPN and directly joins the Tf-bound iron pool. FeSO4 administration generates higher levels of free non-transferrin-bound iron (NTBI) to which macrophages can be exposed, leading to cell iron accumulation. Thus iron provided both as SI or FeSO4 can target macrophages. Recently, macrophage iron overload has been implicated in sterile inflammation through ROS-dependent cell pro-inflammatory activation. Here we investigated and compared how SI and FeSO4 shape the polarization status of macrophages and affect inflammation in conditions of iron deficiency anemia. Methods: To this aim, iron-deficient wild-type mice administered a low iron diet (<10ppm) were repeatedly treated with SI or FeSO4 every day for 2 weeks to correct their anemia, and hepatic and splenic macrophages were analyzed in term of cell iron status and inflammatory response. Results: Iron-deficient mice treated with SI showed an efficient but slightly slower recovery of anemia as monitored by blood parameters (e.g. Hb, RBC, HCT) compared to FeSO4-treated animals. The gradual anemia recovery by SI likely reflects the additional macrophage-dependent processing needed to recycle iron from the intact SI shell, which is not required following FeSO4 absorption. Indeed, Tf saturation and NTBI were more elevated in FeSO4-treated than SI-treated mice. Consistent with these observations, FeSO4 corrected intracellular iron deficiency in macrophages more rapidly than SI, as indicated by a more pronounced TfR1 suppression and bigger labile iron pool. This triggered more elevated ROS levels and increased apoptosis in macrophages from FeSO4-treated compared to SI-treated mice. Importantly, the faster cell iron deficiency recovery was associated with TNFα, IL1β and IL-6 release in macrophages from FeSO4-treated mice, which remained almost negligible in cells from SI-treated animals. In vivo findings are fully recapitulated in vitro in iron-deficient bone marrow-derived macrophages (BMDM) exposed to SI or FeSO4. FeSO4 corrected macrophage iron status faster than SI, as suggested by a quick rise in labile iron pool and suppression of TfR1 after 2h of treatment. By contrast, SI showed a slower and progressive ability to improve cell iron deficiency, modulating LIP and TfR1 after 5h of treatment, in agreement with a longer recycling process of iron from the SI shell. While FeSO4 exposure caused a massive increase in ROS levels and a significant elevation of inflammatory cytokines, SI minimally affected ROS and inflammatory cytokine production in BMDMs. Conclusions: Our data indicate that the gradual cell iron deficiency correction by SI exerts a protective effect in macrophages against iron-mediated inflammatory activation by limiting ROS formation, suggesting that the kinetic of cell iron accumulation determines macrophage inflammatory response. Overall, these studies show that SI is a superior oral iron formulation than iron salts in terms of reduced pro-oxidant and inflammatory action, with relevance for iron deficiency anemia treatment in individuals with pre-existing inflammatory conditions, including chronic kidney disease, anemia of cancer, inflammatory bowel disease, ulcerative colitis, post-operative anemia, celiac disease and obesity.