A Double Knock out of Hepcidin and IL6 Demonstrates Independent Roles of the Two Genes in Anemia of Inflammation

Inflammatory states seen in infection and other chronic disorders are often characterized by a condition called anemia of inflammation (AI). The iron deficiency in AI is predominantly due to an altered balance of the cytokine-interleukin-6 (IL6) and the hormone hepcidin (Hamp). IL6 has been implicated in inducing expression of hepcidin, which degrades the iron exporter ferroportin. We have previously shown that lack of IL6 or hepcidin in knockout mouse models (IL6-KO and Hamp-KO) injected with the heat-killed pathogen Brucella abortus (BA) results in improved recovery from anemia. This recovery was different in IL6-KO and Hamp-KO mice, suggesting that the two proteins contribute independently to AI. Here, we formally validated the independent role of IL6 and Hamp in AI by generating a double-knockout (DKO) mouse model lacking the expression of both. The DKO mice showed the most ameliorated phenotype following BA administration. BA-treated-DKO mice showed an increased number of erythroblasts in the bone marrow (BM) and spleen as seen by flow cytometry, in comparison to IL6-KO and Hamp-KO. Concurrently, compared to WT, Hamp-KO and IL6-KO animals, in DKO mice the reticulocyte count was already increased by week-2. The anemia induced by the pathogen by week-1 was less severe in DKO mice. Moreover, both hemoglobin and RBC values measured at week-2 were the highest in DKO, followed by Hamp-KO and then IL6-KO. We also investigated RBC lifespan in these animals by measuring the turnover of biotinylated RBC over time. The turnover of the biotinylated RBC occurred in two phases. In the initial phase the percentage of biotinylated and non-biotinylated RBCs in the BA-treated animals remained the same, while in the second phase it decreased, indicating production of new RBCs. In Hamp-KO and the IL6-KO mice the first phase lasted for 7 days and 4 days respectively, while in DKO animals the percentage of biotinylated RBC had already started to decrease by day 4, indicating an accelerated production of new RBCs compared to single KO mice. Additionally, we used the RodentMAP®-v.3.1 (MyriadRBM) to quantify 51 serum inflammatory biomarkers, and Ingenuity Pathway Analysis to identify pathways activated in single KO as well as DKO mice. Of the top 10 pathways activated in all three models, 4 present in the IL6-KO were also activated in the DKO model, and the remaining 6 were unique. Five of the pathways that came up in Hamp-KO were also activated in DKO mice and the remaining 5 were unique. For instance, both DKO and Hamp-KO mice showed activation of Hmgb1 signaling, suggesting a response to limit inflammation and reduce tissue damage.  Moreover, both the DKO and IL6-KO models showed activation of granulocyte adhesion and diapedesis, the former suggesting an inflammatory response associated with the infection while the latter possibly indicating mobilization of cells in response to the infection. A unique pathway activated in DKO mice was that associated with increased production of pluripotent stem cells, likely triggered by the damage observed to the BM and anemia and potentially responsible for the accelerated recovery observed in these animals. In conclusion, these results suggest that the absence of both IL6 and Hamp not only is associated with activation of pathways in common with the single KO, but also with unique features triggered by the concurrent depletion of the two genes. Potential clinical implications will be discussed.