Distinct Roles For Hepcidin and Interleukin 6 In The Recovery From Anemia Following Administration Of Heat-Killed Brucella Abortus

Anemia of inflammation (AI) is a complex condition commonly observed in chronic inflammatory states and associated with poor health outcomes and low quality of life. Increased hepcidin (Hamp) expression triggered by interleukin-6 (IL6) is a hallmark of AI and is responsible for iron-restricted erythropoiesis and anemia. Several translational studies have been aimed at neutralizing Hamp overexpression as a therapeutic strategy against AI. However, additional Hamp-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis.

In our study, we investigated the distinct roles of iron metabolism and inflammation triggered by Hamp and IL6 in AI. We utilized Hamp-knockout (Hamp-KO), IL6-KO, and wild-type (WT) mice in which we induced inflammation with an intraperitoneal injection of heat-killed Brucella abortus (HKBA). In these mice, we characterized erythropoiesis with weekly CBC, FACS, and red blood cell (RBC) lifespan analysis. In addition, we measured cytokine expression, iron and transferrin (Tf) saturation in sera as indicators of inflammation and iron availability reflecting of iron stores.

CBC analysis showed that hemoglobin (Hb) reached a nadir 2 weeks after HKBA injection in WT mice (6.0±1.1 g/dl), and 1 week after HKBA injection in IL6-KO (8.8±1.8 g/dl), and Hamp-KO mice (11.3±1.0 g/dl) (N=10/group), demonstrating that knockout mice were partially protected against HKBA-induced anemia. Additionally, knockout mice fully recovered within 3-4 weeks, whereas WT mice required more than 6 weeks. Mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and reticulocyte hemoglobin content (CHr) were decreased in both WT and IL6-KO mice after HKBA, although values were higher in the latter. Interestingly, Hamp-KO mice showed the highest levels of serum iron, Tf-saturation, MCH, MCV and CHr, indicating that the mild anemia induced by HKBA in these mice was a consequence of superior hemoglobinization of their reticulocytes and RBCs. Iron-depleted Hamp-KO mice were still less sensitive to HKBA administration, suggesting that lack of Hamp expression was contributing to protect these mice from anemia, rather than merely iron overload.

FACS analysis performed 3 days after HKBA injection showed that BM erythropoiesis was disrupted in all treated mice. After 1 week, only IL6-KO mice were recovering, although their marrow erythroid progenitors were still reduced compared to PBS-treated mice. The faster recovery in IL6-KO mice was supported by the increased production of reticulocytes compared to Hamp-KO and WT mice. After 1 week, we observed stress erythropoiesis in the spleen of all mice, which was most significant in Hamp-KO, who also showed the highest levels of erythropoiesis in the spleen at steady state. Further investigation of stress erythropoiesis in phlebotomized WT mice injected with HKBA demonstrated that spleen erythropoiesis was partially spared from the inflammatory insult.

We showed that a shortened RBC lifespan was contributing to anemia in HKBA-treated mice, and that impaired production of RBCs was accompanied by an increased rate of elimination. In fact, by FACS analysis we characterized macrophages (Gr1^-/CD115^-/SSC^low/F4/80^hi) engulfing erythroid cells (Ter119⁺) by discriminating multiplets vs. single cells (SSC-H vs. SSC-A), and observed that hemophagocytosis was occurring in HKBA-treated mice.

Finally, we analyzed 58 inflammatory cytokines (RodentMap, Myriad RBM) from mouse sera and showed that HKBA administration changed the level of expression of inflammatory molecules more substantially in Hamp-KO than in IL6-KO compared to WT mice (26 cytokines changed in Hamp-KO mice, with fold change > or <1.5 vs. 1 changed in IL6-KO mice; P<.001). This indicated that lack of Hamp influences cytokine expression more significantly than lack of IL6 after HKBA injection. Further analyses are in progress to define the function and pathways that correlate the different cytokines.

In conclusion, we showed that both IL6 and Hamp ablation offered protection against AI and that IL6-KO and Hamp-KO mice exhibited different patterns in the development and resolution of anemia. We believe that our data support the notion that not only IL6 and Hamp play distinct roles in modulating erythropoiesis in AI, but also that cytokines could be targeted as a therapeutic approach to improve marrow erythropoiesis in AI.</abstract>