Intravenous Iron Promotes Low-Grade Inflammation in Anemic Patients By Triggering Macrophage Activation
Authors: Vinchi Francesca , Castagna Annalisa , Costa da Silva Milene , Busti Fabiana , Marchi Giacomo , Sparla Richard , Muckenthaler Martina U. , Girelli Domenico 
Published: February 2020

Patients with iron deficiency anemia (IDA) who have intolerance or an unsatisfactory response to oral iron or present with chronic kidney disease often receive intravenous (iv) iron formulations to improve their anemic condition. Here we investigated whether iv iron induces inflammation in anemic patients and the underlying cellular and molecular mechanisms.
We analyzed plasma samples of anemic patients before and 3 weeks after receiving a single-bolus treatment with the iv formulation iron-carboxymaltose and measured circulating inflammatory cytokines. To understand the inflammatory action of iron, we studied the effect of iron-carboxymaltose and non-transferrin bound-like iron (NTBI) source exposure both in an in vitro and in vivo model system.
Anemic patients show an increase in circulating cytokine levels still 3 weeks after iron-carboxymaltose injection, compared to their steady-state condition. We found elevated IL-6, TNFα, CCL2 and INFγ. Importantly, patients with more pronounced increase in circulating inflammatory cytokines show higher serum ferritin as well as hepcidin levels, likely induced by inflammation. These observations suggest that iv iron exerts a pro-inflammatory action in anemic patients. Similarly, treatment of wild-type as well as iron-deficient mice with iron-carboxymaltose induces inflammation by triggering cytokine release in the circulation.
Since iron formulations might first target macrophages to allow iron recycling, we analyzed the phenotype and production of cytokines induced by iron in these cells both in vitro and in vivo. Iron-carboxymaltose triggers a pro-inflammatory phenotypic switching of bone marrow-derived macrophages (BMDMs), hallmarked by increased expression of the M1 markers MHCII, CD86, iNOS and inflammatory cytokines (e.g. TNFα, IL-6, IL-1β) and reduced expression of the M2 markers CD206, Arg-1, Ym1 and IL-10. This effect is directly due to the presence of iron in the formulation, as exposure of BMDMs to NTBI-like iron (ferric-nitrilotriacetate) induces the same polarization shift, which is prevented by the iron carrier transferrin and the iron chelator deferoxamine. Consistently, inflammatory polarization of reticulo-endothelial (RES) macrophages of the liver and the spleen is observed in mice, either in steady-state and IDA condition, after treatment with NTBI-like iron sources (e.g. ferric-ammonium citrate, ferric-nitrate) as well as iron-carboxymaltose. RES macrophages from treated mice show iron deposition and increased expression of iron-related genes (ferroportin, ferritin, HO-1), together with high cytokine production. Transferrin significantly attenuates iron-mediated macrophage activation in mice, leading to an overall reduction of circulating inflammatory cytokines. These results indicate that iron-activated macrophages contribute to inflammatory cytokine release after administration of intravenous iron. We further confirmed that this process occurs with other iron formulations, including iron-sucrose and iron-dextran. Mechanistically, iron affects macrophage plasticity by triggering reactive oxygen species (ROS) production. Application of anti-oxidant molecules such as N-acetyl-cysteine prevents iron-mediated macrophage polarization both in BMDMs and mice.
Collectively, these results indicate that iv iron exerts a clear inflammatory effect in anemic patients through the pro-inflammatory activation of macrophages. Our findings have potential implications for IDA patients who might develop a chronic low-grade inflammation in response to treatment with iv iron formulations. We suggest that inflammation in this context might have a further detrimental effect on the underlying anemia, through a multifactorial action resulting from (i) erythropoiesis suppression, due to the inhibitory action of inflammatory cytokines and (ii) hepcidin induction, which leads to further reduction in iron absorption. In addition, alteration in the inflammatory status might be responsible for commonly reported iv iron-related adverse events, including headache, dizziness, nausea, abdominal pain, constipation, diarrhea, rash and injection-site reactions. We propose that the co-administration of transferrin or anti-oxidants with iv iron in IDA patients is of benefit to counteract iron-induced inflammation, thus improving anemia more effectively and preventing adverse effects.

Disclosures
Vinchi: Vifor Pharma: Research Funding; Pharmanutra: Research Funding; Silence Therapeutics: Consultancy; Novartis: Research Funding. Muckenthaler:Novartis: Research Funding; Silence Therapeutics: Consultancy. Girelli:Vifor Pharma: Other: honoraria for lectures; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees; La Jolla Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.