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.

Brilli:PharmaNutra S.p.A.: Current Employment. Tarantino:PharmaNutra S.p.A.: Current Employment. Vinchi:PharmaNutra S.p.A: Research Funding; Vifor (International) Ltd: Research Funding; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.