Manipulating the Erythroid Iron Deprivation Response Affects Inflammatory Signaling in Anemia of Chronic Inflammation

Abstract 179

Impaired erythropoiesis in anemia of chronic inflammation (ACI) results from the inhibitory effects of inflammatory cytokines and iron restriction. Key inflammatory cytokines implicated in ACI include tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). Iron restriction has been shown to inhibit erythropoiesis through the inactivation of aconitase enzymes, which normally convert citrate to isocitrate. In previously presented data (Richardson et al., ASH 2010), iron deprivation markedly sensitized cultured erythroblasts to the inhibitory effects of IFNγ, and this sensitization was abrogated by inclusion of isocitrate in the medium. Strikingly, intraperitoneal (IP) injections of isocitrate at 200 mg/kg/day × 3 days completely and durably reversed anemia in a rat model of ACI involving chronic arthritis induction by Streptococcal cell wall peptidoglycan-polysaccharide (PG-PS).

The current studies have further characterized the in vivo effects of isocitrate on ACI in the rat model of chronic arthritis. In addition to correcting anemia in animals with arthritis, isocitrate treatment also diminished neutrophilia and arthritis severity. Histologic assessment of spleens harvested on day 36 post induction of arthritis showed less granulomatous inflammation and less iron deposition in isocitrate-treated animals. Measurement of hepcidin expression showed significantly diminished HAMP mRNA levels in day 36 liver tissue from the isocitrate-treated animals. Thus, in addition to correcting anemia, isocitrate treatment promoted iron mobilization and diminished systemic inflammation. To assay for potential immunosuppressive effects, isocitrate was administered in 3 different murine models of autoimmune inflammation (spontaneous autoimmune orchitis, orchitis induction by Treg depletion plus vasectomy, neonatal autoimmune oophoritis), with no effects observed on disease development. Based on these findings, we propose a mechanism in which isocitrate acts primarily on the marrow to stimluate erythropoiesis, leading in turn to diminished hepcidin expression followed by iron mobilization out of splenic macrophages. Based on recent findings of Sindrilaru et al. that iron overloading polarizes macrophages toward an M1 proinflammatory phenotype (JCI, 121:985, 2011), isocitrate stimulation of erythropoiesis is proposed to break a vicious cycle of macrophage iron loading followed by pro-inflammatory cytokine secretion.

To further study the effects of iron restriction on interferon signaling ex vivo in erythroblasts, human CD34+ cells in erythroid medium underwent IFNγ stimulation −/+ iron deprivation and −/+ isocitrate treatment. As previously reported, neither iron restriction nor isocitrate treatment had any effect on IFNγ induction of STAT1 expression or STAT1 phosphorylation on Y701 and S727. However, iron restriction markedly impaired IFNγ induction of the interferon response factor, IRF9 at the level of protein expression. IRF9 normally functions as a transcriptional cofactor for STAT1 and STAT2 in the activation of a subset of interferon response genes. Iron restriction had no effect on IFNγ induction of the related interferon response factor IRF8. The block in IFNγ induction of IRF9 caused by iron restriction was reversed by the addition of isocitrate. The mechanism for this block appeared to occur at the level of translation, as iron restriction did not affect IFNγ induction of IRF9 mRNA. siRNA knockdowns of IRF9 addressed its role in sensitizing erythroid cells to inhibition by IFNγ. Notably, knockdown of IRF9, in the absence of IFNγ, did not impair erythroid viability or differentiation. Similarly, IFNγ in the absence of IRF9 knockdown had no inhibitory effects on erythropoiesis. However, the combination of IRF9 knockdown and IFNγ treatment inhibited both differentiation and viability. These findings suggest that a translational block in IRF9 upregulation caused by iron restriction may contribute to sensitizing erythroid progenitors to IFNγ-mediated inhibition. As IRF9 assembly with STAT1-associated transcriptional complexes dictates the transcriptional output of interferon signaling, the IRF9 deficiency caused by iron deprivation is predicted to reconfigure the IFNγ transcriptional program.