Differential Effects of Monoferric Forms of Transferrin on Mouse Models of Anemia of Inflammation and Congenital Anemia

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), iron bound to the N-lobe, the C-lobe or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric (Dirusso et al., 1985). The TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. TF also interacts with TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and other cells of the bone marrow and is thought to primarily influence cellular signaling events regulating hepcidin expression and erythropoiesis. In erythrocytes, loss of TFR2 results in abnormally high red blood cell production under conditions of iron-deficiency (Artuso et al., 2018), suggesting it functions as an iron sensor. Additional evidence suggests TFR2 modulates erythropoiesis via the erythropoietin (EPO) receptor (EPOR) (Forejtnikovà et al., 2010).

To understand the role of monoferric TF forms in vivo, we previously generated N-lobe blocked (Tf^N-bl) or C-lobe blocked (Tf^C-bl) TF mutant mice. Characterization of these mice demonstrated dramatic differences in RBC levels, hepcidin expression relative to iron status, and EPO sensitivity (Parrow et al., 2019). More recently, we have become interested in characterizing the immune compartment of these mice. Recent studies point to iron being an important regulator of immune responses, which has important implications worldwide in understanding how to treat iron-deficient patients without exacerbating latent infections. Cells belonging to the immune branch have also been shown to express TFR2 and EPOR (Cantarelli et al., 2019; Roetto et al., 2011; Kawabata et al., 2001), however their function is largely unexplored.

Therefore, in this work, we investigate the role of the two monoferric forms of TF on ineffective erythropoiesis (IE) using a mouse model of β-thalassemia (BT) intermedia (Hbb^th3/+) and chronic anemia of inflammation using heat-killed Brucella Abortus (HKBA).

Based on observations in BT mice treated with exogenous TF (Li et al., 2010), which led to reduced erythroid iron intake, reduced EPO requirement (as in Tf^C-bl mutant mice) and improved anemia, we hypothesized that Hbb^th3/+ mice crossed with Tf^C-bl would demonstrate improved erythropoietic and iron parameters compared with Hbb^th3/+Tf^N-bl. We measured effects on hematologic parameters, IE, and expression of relevant iron regulatory serum proteins. Hbb^th3/+Tf^C-bl mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology, decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of IE. Additionally, serum erythroferrone (ERFE) was significantly reduced and hepcidin levels were increased in Hbb^th3/+Tf^C-bl relative to Hbb^th3/+Tf^+/+ controls. However, hematological parameters from Hbb^th3/+Tf^N-bl mice did not show these improvements.

In our studies examining the immune compartments of Tf^C-bl and Tf^N-bl mice we found striking differences in their expression of TFR1 and response to immunological challenge by HKBA. Our preliminary data shows that expression levels of TFR1 were elevated in both TF mutant mice relative to control at baseline on lymphocyte, monocyte and granulocyte populations of the bone marrow. When challenged with a single intraperitoneal injection of HKBA, Tf^C-bl-HKBA mice suffered a more severe anemic phenotype with a 30% probability of survival following HKBA treatment. On the contrary, Tf^N-bl-HKBA mice displayed a blunted response to the immunological challenge, with faster recovery and no death.

Taken together, our work provides additional evidence that TF is not only delivering iron cargo to cells, but also functioning as a signaling molecule via the lobe that is bound to iron. Despite each mouse only possessing a TF capable of binding one atom of iron, the two mouse models show profound differences in the pathophysiology of BT and AI. We hypothesize that the monoferric TF forms exert their influence via TFR2. We are currently characterizing a new loxp flanked TFR2-FLAG mouse model generated by our lab, which has been crossed to the TF monoferric mutant mice to test this hypothesis. Additionally, we are also exploring the translation potential of the two TF forms to treat anemia in BT and AI.

Parrow:Protagonist: Consultancy. Ginzburg:Ionis: Consultancy; Takeda: Consultancy; Dexcel: Consultancy; Repare: Research Funding; Protagonist: Consultancy, Research Funding. Fleming:Protagonist Therapeutics: Consultancy; Silence Therapeutics: Consultancy; Ultragenyx: Research Funding. Rivella:BVF Partners L.P: Consultancy; Cambridge Healthcare Res: Consultancy; Celgene: Consultancy; Disc Medicine: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; First Manhattan Co: Consultancy; FORMA Therapeutics: Consultancy; Ghost Tree Capital: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Keros Therapeutics: Consultancy; MeiraGTx: Membership on an entity's Board of Directors or advisory committees; Noble insight: Consultancy; Protagonist Therapeutics: Consultancy; Rallybio, LLC: Consultancy; Sanofi Aventis U.S: Consultancy; Slingshot Insight: Consultancy; Techspert.io: Consultancy; venBio Select LLC: Consultancy; Vifor: Membership on an entity's Board of Directors or advisory committees.