In addressing factors that suppress erythropoiesis in anemia of chronic inflammation (ACI), we previously showed that iron restriction sensitizes cultured human erythroblasts to the inhibitory effects of inflammatory cytokines including interferon γ (IFNγ) (Richardson et al., ASH 2010). This sensitizing effect was reversed by addition of isocitrate to cultures, and in a rat arthritis ACI model intraperitoneal injections of isocitrate completely and durably reversed anemia through in vivo stimulation of erythropoiesis (Richardson et al, ASH 2011). New studies using cultures of human hematopoietic progenitor cells (huHPC) have explored the signaling mechanisms by which iron and isocitrate modulate erythroid responsiveness to IFNγ. No impact of iron restriction or isocitrate treatment could be seen on IFNγ activation of STAT1 phosphorylation on tyrosine 701 or on serine 727. Similarly, iron restriction and isocitrate had no effects on IFNγ-mediated upregulation of STAT1 protein, STAT2 protein, IRF8 protein, or IRF9 mRNA levels. These findings suggest that iron and isocitrate do not affect the classical JAK1-STAT1-IRF1 or the alternative GATE-IRF9 pathways. We then examined expression of the transcription factor PU.1, a master regulator whose levels dictate myeloid versus erythroid cell fate in hematopoietic progenitors. Libregts et al., recently demonstrated that IFNγ upregulated PU.1 erythroblasts via IRF1 (Blood 2011;118(9):2578–2588). Our results showed that iron restriction potently augmented IFNγ induction of PU.1, by 2–3-fold, and also induced PU.1 on its own to a lesser degree. Importantly, isocitrate abrogated the upregulation of PU.1 caused by iron restriction. Furthermore, qRT-PCR on sorted erythroblasts from rat marrows showed increased PU.1 expression in animals with ACI and normalization of erythroid PU.1 expression in association with isocitrate treatment. Pop et al., have recently shown that downregulation of PU.1 early in erythropoiesis constitutes a key step in lineage commitment (PLoS Biol 2010;8(9):e1000484). Therefore we examined the kinetics of PU.1 expression in the huHPC model system. As expected, huHPC downregulated PU.1 during the initial 2–4 days of standard erythroid culture. Similar downregulation occurred in the presence of IFNγ, under iron replete conditions. However, with the combination of IFNγ and iron restriction, PU.1 levels remained high the entire culture period and showed minimal downregulation. Several experimental approaches addressed the erythroid developmental stages affected by iron restriction and IFNγ. Flow cytometry with intracellular staining showed that iron and isocitrate influenced IFNγ induction of PU.1 at an early CD34+ CD36+ stage. These findings were corroborated by immunoblot analysis of sorted progenitors showing iron restriction and isocitrate to affect PU.1 levels within CD36+ GPA- erythroid progenitors; the later CD36+ GPA+ progenitors showed extinction of PU.1 expression regardless of culture conditions. Finally, using purified CD36+ cells as a starting population, iron restriction and IFNγ again cooperated in induction of PU.1, with isocitrate reversing this effect. Prior studies from our lab have shown that erythroid iron restriction results in hyperactivation of PKCα/β. In addition PKCα/β is known to directly phosphorylates and activates PU.1. We therefore sought to determine whether PKC contributes to cooperative upregulation of PU.1 in erythroid progenitors subjected to iron restriction and IFNγ. Supporting this notion, the pan-PKC inhibitor BIM abrogated the effects of iron restriction plus IFNγ on PU.1 upregulation. Importantly, the dosage of BIM employed had no effect on viability or differentiation. Our findings thus define a pathway in which iron restriction and IFNγ act in a cooperative manner on early erythroid progenitors to increase PU.1 expression and interfere with its normal downregulation. Iron restriction and isocitrate exert their influences, at least in part, through alteration of PKC activation. We propose a model of ACI in which iron restriction and inflammatory signaling are both required to attain a critical threshold of erythroid PU.1, which may then interfere with early stages of lineage commitment. Through its reversal of PKC activation by iron restriction, isocitrate may act to keep PU.1 levels below this critical threshold.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.