ALBERT

Description: 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.

Description: Abstract 2000 Poster Board I-1022 Hepcidin is a 25 amino acid peptide that is the central mediator of iron metabolism. Iron excess, deficiency and maldistribution have been implicated in the etiology of many diseases including atherosclerosis, diabetes, neurodegeneration and the anemia of inflammation. Determination of hepcidin levels may be useful in diagnosis and treatment decisions for some or all of these diseases. Serum hepcidin measurement has so far been limited to a prohepcidin (60 amino acid hepcidin precursor) ELISA, mass spectrometry (MS)-based assays or competition ELISAs using polyclonal anti-hepcidin antibodies. The current work describes the generation of a sandwich ELISA using monoclonal antibodies to detect human hepcidin (hHepc) and optimization of assay conditions to resolve inconsistencies between MS- and ELISA-based detection. The ability of two anti-hHepc antibodies to sandwich (bind simultaneously) with hHepc was demonstrated using a rabbit polyclonal antibody preparation from hHepc-immunized animals. The same polyclonal antibody preparation was used for both hHepc capture and detection. The limit of detection achieved with this assay was O.D.4501000 hybridoma supernatants identified three classes of antibodies: classes 1 and 2 each recognized epitopes found in both full length mature hHepc (hHepc 25) and a shorter version (hHepc 20); class 3 bound a different epitope and demonstrated an increased affinity for hHepc 25 over hHepc 20. The majority of antibodies characterized were in class 1 while antibodies in classes 2 and 3 were rare (∼1% of antibody panel) highlighting the difficulty in achieving a sandwiching event. Antibodies 19D12 (class 1) and 23F11 (class 2) were identified as the optimal sandwich pair with a detection range of approximately 0.2-1000 ng/ml using synthetic hHepc. Initial comparisons of data generated using the sandwich ELISA and a fully-quantitative MS-based assay demonstrated a lack of consistent agreement. This issue was somewhat addressed by introduction of an alkaline treatment step to dissociate any protein/hHepc complexes in serum. Subsequent comparison of the two assays using sera from several different patient populations (anemia of cancer, chemotherapy-induced anemia, kidney disease) as well as healthy donors demonstrated good correlation (R2 range = 0.83-0.92; n=237). This sandwich ELISA may represent a tool for aligning the MS and ELISA-generated results in a format that has the potential to be high throughput and widely available. Disclosures: Arvedson: Amgen: Employment. Doellgast:Amgen: Employment. Salimi-Moosavi:Amgen: Employment. King:Amgen: Employment. Foltz:Amgen: Employment. Chen:Amgen: Employment. Li:Amgen: Employment. Sasu:Amgen: Employment.

Description: Key Points Investigation of the distinct roles of hepcidin and interleukin 6 on iron metabolism and inflammation in the onset and resolution of AI.

Description: Iron maldistribution has been implicated in multiple diseases, including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino acid peptide. Hepcidin is induced by inflammation, causes iron to be sequestered, and thus, potentially contributes to AI. Human hepcidin (hHepc) overexpression in mice caused an iron-deficient phenotype, including stunted growth, hair loss, and iron-deficient erythropoiesis. It also caused resistance to supraphysiologic levels of erythropoiesis-stimulating agent, supporting the hypothesis that hepcidin may influence response to treatment in AI. To explore the role of hepcidin in inflammatory anemia, a mouse AI model was developed with heat-killed Brucella abortus treatment. Suppression of hepcidin mRNA was a successful anemia treatment in this model. High-affinity antibodies specific for hHepc were generated, and hHepc knock-in mice were produced to enable antibody testing. Antibody treatment neutralized hHepc in vitro and in vivo and facilitated anemia treatment in hHepc knock-in mice with AI. These data indicate that antihepcidin antibodies may be an effective treatment for patients with inflammatory anemia. The ability to manipulate iron metabolism in vivo may also allow investigation of the role of iron in a number of other pathologic conditions.

Description: 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-/SSClow/F4/80hi) 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

Description: Abstract 2100 Although enzyme-linked immunosorbent assays (ELISA) exist for measuring human hepcidin, no commercially available antibodies to mouse hepcidin are validated for this purpose. Thus, we developed an ELISA to measure mouse hepcidin using low plasma volumes to allow for repeat testing of individual mice over time. To this end, human hepcidin knock-in mice (Hep2 mice) were used to generate a panel of anti mouse hepcidin antibodies. A sandwich ELISA using these antibodies was validated. Pooled plasma from iron-deficient (combining diet and phlebotomy), iron-replete, and inflamed (injecting sub-clinical amounts of lipopolysaccharide) FVB mice was used to prepare frozen aliquots of low, medium, and high reagent control plasma. These samples were tested on 5 different days (n=8 wells/sample/day). Mean hepcidin levels for the high, medium, and low reagent were 791, 504, and 117 ng/mL, respectively. The intra-assay coefficient of variation (CV) was 10, 4.5, and 15.6% for the high, medium, and low samples, respectively. Inter-assay CV was 4.7, 4.0, and 11.1% for the high, medium, and low samples, respectively. Analytical sensitivity was 0.7 ng/mL and the assay was linear (Pearson r=0.99) to 500 ng/mL at a 1:100 dilution of sample to blocking buffer (i.e. only 1 uL of sample needed per test). With 20 plasma samples spanning the range of expected hepcidin levels, there was high correlation between hepcidin determined by ELISA and the sum of hepcidin-1 and hepcidin-2 by time-of-flight mass spectrometry (Pearson r = 0.99; P

Description: Abstract 626 Iron maldistribution has been associated with multiple diseases including atherosclerosis, diabetes, neurodegenerative disorders and the anemia of inflammation (AI). Hepcidin is a 25aa peptide that is the central mediator of iron homeostasis. It is produced in response to inflammation and is implicated in limiting the response to erythropoietin (EPO) even in the presence of adequate body iron stores. To further explore the role of hepcidin in inflammatory anemia, a novel EPO-resistant model of AI was developed in mice and the effects of a high affinity fully human anti-hepcidin antibody (12B9m; 2pM affinity) were examined. Since antibody 12B9m had low affinity for mouse hepcidin (10-20nM), human hepcidin (hHepc) knock-in mice were created. Strains were generated with either the mouse hepc1 gene or both hepc1 and 2 genes replaced with the single hHepc gene (HAMP). To induce inflammation and establish AI, mice were injected intraperitoneally with heat-killed Brucella abortus (BA: 3×108 particles/mouse): a procedure adapted from an inflammatory fatigue model. BA treatment led to increased serum levels of IL-6 (3818 ± 548 pg/ml versus

Description: Key Points Fully human anti-hepcidin Abs have been generated for use as a potential therapeutic to treat AI. The mechanism of action was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization.