Targeting the Erythroid Iron Deprivation Response as a Therapeutic Approach In Anemia Not Caused by Iron Deficiency

Abstract 166

The erythroid iron deprivation response results from lineage-selective inactivation of aconitase enzymes, causing diminished erythropoietin (Epo) responsiveness in erythroid progenitors. Provision of exogenous isocitrate in either cell culture or murine models of iron deficiency restores Epo responsiveness and abrogates the erythropoietic block characteristic of the iron deprivation response. Although isocitrate administration can restore erythropoiesis in iron deficient mice, the response is transient, and iron administration is required for sustained correction. In anemias other than those due to iron deficiency, inappropriate activation of this iron deprivation pathway might also contribute to suppression of erythropoiesis. Such anemias are predicted to respond to isocitrate administration. A major area of clinical controversy is the degree to which iron restriction plays a role anemia of chronic inflammation (ACI). In support of such a role, inflammatory stimuli promote increased hepcidin production by the liver, and diminished serum iron represents a consistent finding in patients with ACI. Challenging such a role, a recently published series of elderly patients with ACI showed no evidence of increased hepcidin production (Ferrucci et al., Blood 115:3810, 2010). Furthermore, ACI usually manifests as a normochromic, normocytic anemia in contrast to the microcytic, hypochromic anemia of iron deficiency. Finally, administration of anti-TNF antibody to patients with rheumatoid arthritis corrected their associated anemia, suggesting a role for direct cytokine repression of erythropoiesis (Papadaki et al., Blood 100:474, 2002). To experimentally examine the role of the erythroid iron deprivation response in ACI, we determined the effects of isocitrate administration in a classic rat arthritis model of ACI. 6 week-old female Lewis rats received a single IP injection (15 μ g rhamnose/g) of Streptococcal cell wall peptidoglycan-polysaccharide (PG-PS). Normochromic, normocytic anemia developed at 2 weeks post injection. Specifically, the mean red cell count (RBC) in rats receiving PG-PS was 7.18 ± 0.22 × 10⁶ cells/μ l vs 8.65 ± 0.39 × 10⁶ cells/μ l in rats not receiving PG-PS (P = 0.014). At this time, the anemic rats underwent daily IP injections with either trisodium isocitrate (200 mg/kg/day) or with equivalent volumes of saline. Rats receiving isocitrate showed correction of anemia after 3 days of treatment, with a mean RBC of 8.14 ± 0.06 × 10⁶ cells/μ l as opposed to a mean RBC in the control group of 6.42 ± 0.45 × 10⁶ cells/μ l (P = 0.018). This correction was maintained after 5 additional days of treatment: RBC in isocitrate-treated group of 8.36 ± 0.24 × 10⁶ cells/μ l versus RBC in saline-treated group of 7.01 ± 0.19 × 10⁶ cells/μ l (P = 0.004). No differences in neutrophil or platelet counts were observed at any point in rats receiving isocitrate vs saline. These results support a role for the erythroid iron deprivation response in the impaired erythropoiesis associated with ACI. These results also support our previous in vitro findings that iron deprivation sensitizes erythroid progenitors to inhibition by inflammatory cytokines (i.e. IFNγ or TNFα) (Richardson et al., ASH 2009 #159). More recent in vitro studies on the mechanisms underlying this sensitization have addressed whether iron restriction synergizes with inflammatory cytokines in promoting aconitase inactivation. Using a gel-based enzymatic assay, we confirmed the inhibitory effect of iron deprivation on both cytosolic and mitochondrial aconitase isoforms, but could find no inhibitory effects associated with either IFNγ or TNFα treatment. In subsequent experiments, a functional screen for elements integrating the erythroid iron deprivation response with inflammatory signaling implicated a calmodulin-regulated factor. Specifically, the calmodulin inhibitor, KN62 reversed the cell death observed with the combination of iron deprivation plus inflammatory cytokines but had minimal effects on viability of cells subjected to either iron deprivation or inflammatory cytokines separately (n = 4). These data thus delineate an iron-regulated signaling element downstream of aconitase which employs calmodulin to modulate erythroid responsiveness to inflammatory cytokines. Pharmacologic targeting of this element, as with isocitrate administration, may provide a new avenue for clinical management of ACI.