Investigating the Role of Cytokines and Hepcidin in Anemia of Inflammation
Authors: Gardenghi Sara , Renaud Thomas M. , Meloni Alessandra , Ramos Pedro , Casu Carla , Cooke Keegan S , Sasu Barbra J , Giardina Patricia , Grady Robert W , Rivella Stefano 
Published: October 2019

Abstract
Abstract 1046
Anemia of inflammation (AI) is a widespread multi-factorial form of anemia characterized by hepcidin-induced iron restricted erythropoiesis as well as direct cytokine effects on the bone marrow, blunted erythropoietin production and efficacy, and shortened red blood cell (RBC) lifespan. Our aim is to perform an in depth study of AI, identifying the components and mechanisms associated with its pathophysiology.
We generated a mouse model of AI using a single intraperitoneal injection of heat-killed Brucella abortus (HKBA). In this model we explored the role played by interleukin-6 and hepcidin in the onset of anemia. We utilized wild-type (WT), interleukin-6 knockout (IL-6 KO) and hepcidin knockout (Hamp KO) mice (n ≥ 6/group) injected with HKBA, and conducted weekly CBC's for 7 weeks to follow the progression and resolution of anemia.
Anemia started developing one week after HKBA administration and reached a nadir after 2 weeks in all mice. Hemoglobin values from WT mice were lowest 2 weeks after injection (6.4 ± 1.2 g/dl) but slowly recovered over 7 weeks. Initially, IL-6 KO mice were equally affected with similar hemoglobin values at 2 weeks (6.9 ± 1.3 g/dl). However, these mice recovered after 3 weeks. Hamp KO mice were less anemic throughout the course of the study, with hemoglobin values of 10.3 ± 0. 9 g/dl at 2 weeks and resolution after 4 weeks. These data demonstrate that while both interleukin-6 and hepcidin contribute to AI, lack of either molecule alone is not sufficient to prevent AI. Therefore, additional factors likely play an important role in the etiology of AI.
In order to rule out the effect of iron overload on the reduced severity of anemia observed in Hamp KO mice injected with HKBA, 1 week-old mice were fed an iron-deficient diet in order to first deplete their iron stores, and then returned to the normal diet before HKBA injection. We observed that iron-depleted Hamp KO mice were still less sensitive to HKBA administration, suggesting that this effect was independent of iron overload and dependent on the intrinsic lack of hepcidin expression.
We further investigated the erythropoiesis in WT, IL-6 KO, and Hamp KO mice one week after HKBA injection. We performed FACS analyses of BM and spleen using CD44 and Ter119 antibodies. Both the mature RBCs (CD44−/Ter119+) and erythroid progenitor cells (CD44+/Ter119+) were dramatically reduced in the BM of HKBA-treated WT mice compared to controls (CD44+/Ter119+ cells diminished from 35.5 ± 0.2% to 2.8 ± 0.8%; CD44−/Ter119+ cells from 17.2 ± 0.2% to 8.2 ± 0.8%). The reduction of erythroid cells was attenuated in HKBA-treated IL-6 KO mice (CD44+/Ter119+ cells diminished from 32.8 ± 0.1% to 7.5 ± 6.0%; CD44−/Ter119+ cells from 22.1 ± 0.5% to 10.4 ± 3.8%). Hamp KO mice, on the other hand, showed a dramatic reduction of the CD44+/Ter119+ population in their BM (from 24.1 ± 2.5% to 1.8 ± 0.3%), while mature CD44−/Ter119+ cells were less affected (from 15.4 ± 2.3% to 14.1 ± 2.6%).
Erythropoiesis was altered in the spleen as well. However, while the CD44+/Ter119+ cells were reduced in all the mice strains, the CD44−/Ter119+ population was increased one week after HKBA injection. This profile was more similar to ineffective erythropoiesis than iron-restricted erythropoiesis. Splenomegaly was also observed in all HKBA-treated mice. In addition, we measured increased apoptosis and production of reactive oxygen species (ROS) in the reticulocytes and orthochromatic erythroblasts of the spleen and BM of all mice. Overall, these data suggest that, in addition to iron restricted-erythropoiesis, an acute inflammatory effect on erythropoiesis is occurring in the HKBA model of AI, affecting erythroid cell survival and/or proliferation. Further analyses aimed at determining the RBC life span and survival in these mice are in progress.
Moreover, we are analyzing iron-related gene expression in all groups of mice, along with measurement of their serum iron levels, iron stores, and serum cytokine levels, at different time points. Preliminary data indicate that numerous cytokine mRNAs (including IL-1α, IL-1β, TNF-α, INF-γ) are elevated in the spleen of WT mice 6 hours after HKBA injection. We are investigating the role that these cytokines might have on erythropoiesis, and the anemia observed in IL-6 KO and Hamp KO mice after injection of HKBA.

Disclosures:
Cooke: Amgen: Employment. Sasu:Amgen: Employment.