The Effect of Monoferric Transferrins on Hypoferremia in a Murine Model of Inflammation

Introduction: Modulation of iron metabolism and suppression of erythropoiesis are central components of the acute phase response (APR) and underlie the anemia of inflammation. Hypoferremia is characteristic of the APR and contributes to erythroid suppression by restricting the iron available for erythropoiesis. The role of transferrin (Tf) signaling in the APR has not been explored. We previously generated mice with transferrin mutations that prevent binding of iron to either the N-terminal (N-blocked) or C-terminal (C-blocked) lobe and demonstrated distinct effects on erythropoiesis and the status of the iron regulatory hormone hepcidin. We hypothesize that monoferric transferrins differentially affect modulation of iron metabolism during the APR.

Methods: For a model of acute inflammation, 14 day old wild-type (wt), N-blocked and C-blocked Tf mutant mice were treated with 1mg/kg lipopolysaccharide (LPS) or carrier by intraperitoneal injection and sacrificed 6 hours later. A repeat dose regimen, consisting of seven consecutive daily intraperitoneal injections of 0.3 mg/kg LPS or carrier beginning at seven days of age, was used as a model of chronic inflammation.

Results: In the acute model, wt and C-blocked mice demonstrate the expected decrease in serum iron in response to LPS administration (38 + 7 and 70 +6 ug/dL, respectively). In contrast, N-blocked mice fail to decrease serum iron following LPS administration (164 + 22 ug/dL; p <0.01). Liver and spleen iron concentrations are also significantly higher in N-blocked mice following LPS administration, compared to C-blocked and wild-type mice (LIC: 587 + 28 vs 311 + 53 and 243 + 9 ug Fe/g dry weight; p <0.0001; SIC: 471 + 16 vs 406 + 16 and 336 + 18 ug Fe/g dry weight; p <0.05). LPS administration increases hepcidin all three strains. In contrast, elevated Hamp1 expression is not sustained in the repeat dose model. Nevertheless, serum iron remains elevated in N-blocked mice (201.3 + 30 ug/dL; p <0.05) whereas it returns to baseline in wt and C-blocked mice (76.8 + 39 and 86.6 + 21.4 ug/dL, respectively). Liver and spleen iron concentrations also remain higher in the N-blocked mice relative to C-blocked and wt mice (764.7 + 202 vs 295.7 + 62.7 and 301.3 + 74.8 ug Fe/g dry weight; p <0.05 for liver and 511.9 + 36.2 vs 404.8 + 61.3 and 427.3 + 33.ug Fe/g dry weight; p < 0.05 for spleen). All mice demonstrate marked splenomegaly in response to repeated doses of endotoxin without significant differences between strains. Hemoglobin concentrations are decreased in all strains after repeated endotoxin dosing, with N-blocked mice presenting the most severe anemia (4.1 + .21 vs 5.1 + .42 and 5.3 + .28 g/dL for N-blocked, C-blocked and wt, respectively; p < 0.05).

Conclusions: In LPS-mediated inflammation, mice unable to bind iron on the N-lobe of transferrin fail to develop hypoferremia and have elevated liver iron concentrations, despite increased hepcidin and splenic iron concentrations. We speculate that the iron excess observed in the Tf N-blocked mice arises from a more profound suppression of erythropoiesis, and relatively decreased utilization of iron in hemoglobin production.

Parrow:Protagonist: Consultancy. Ginzburg:Protagonist: Consultancy, Research Funding; Ionis: Consultancy; Takeda: Consultancy; Dexcel: Consultancy; Repare: Research Funding. Rivella:BVF Partners L.P: Consultancy; Cambridge Healthcare Res: Consultancy; Catenion: 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: Consultancy; Ghost Tree Capita: 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: Consultancy; 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. Fleming:Protagonist Therapeutics: Consultancy; Silence Therapeutics: Consultancy; Ultragenyx: Research Funding.