Nitric Oxide Dependent Metabolism Regulates the Proliferation and Differentiation of Stress Erythroid Progenitors
Authors: Ruan Baiye , Chen Yuanting , Koo Imhoi , Cai Jingwei , Mcguigan John , Hall Molly , Patterson Andrew , Paulson Robert 
Published: November 2021

Abstract
Infection and tissue damage induce inflammation, which increases myelopoiesis at the expense of steady state erythropoiesis. Stress erythropoiesis is induced to compensate for the loss of erythroid output until the inflammation is resolved and bone marrow erythropoiesis can resume. Steady state erythropoiesis constantly produces erythrocytes, while stress erythropoiesis generates a bolus of new erythrocytes through the rapid expansion of immature progenitor cells which is followed by the synchronous differentiation of progenitors. We hypothesized that the proliferation of early progenitor cells and their transition to differentiation is regulated by changes in metabolism.
Metabolomics and isotope tracing analysis was performed to assess the intracellular metabolic profiles in proliferating progenitors isolated from in vitro stress erythropoiesis cultures. We observed an active engagement of glucose metabolism in glycolysis and anabolic biosynthesis, while the levels of TCA intermediates suggested that TCA cycle and mitochondrial respiration were blocked. Concomitantly, inducible nitric oxide synthase (iNOS) was induced in progenitor cells to increase the production of nitric oxide (NO), which was demonstrated to be crucial for proliferating progenitor metabolism. Inhibition or genetic mutation of iNOS decreased NO levels resulting in the suppression of progenitor proliferation in vitro and in vivo. As evaluated by RNA-seq, inhibition of iNOS suppressed cell proliferation-related pathways including cell cycle and nucleotide metabolism, while upregulating erythroid differentiation genes. These data suggest that iNOS-derived NO production establishes a metabolism that promotes the proliferation of progenitor cells while inhibiting their differentiation.
Notably, proliferating progenitor cells displayed low levels of the metabolite itaconate and decreased expression of Immunoresponsive gene 1 (Irg1), the enzyme that catalyzes itaconate synthesis from cis-aconitate. Further analysis showed that the addition of 4-Octyl itaconate (OI), a cell-permeable itaconate derivative, inhibited iNOS-derived NO production by activating nuclear factor erythroid 2-related factor 2 (Nrf2), which in turn impaired progenitor expansion. These results indicate that itaconate production is inhibited to enable the accumulation of NO and the NO dependent metabolism required for progenitor cell proliferation during the initial expansion stage of stress erythropoiesis.
In contrast, the transition to differentiation is marked by elevated itaconate synthesis, Nrf2 activation, and attenuated iNOS expression. We hypothesized that the inhibition of NO production alters metabolism and in concert with new cell signaling removes the NO-dependent inhibition of erythroid program, which allows the differentiation of progenitor cells. We tested this mechanism by examining the effects of iNOS inhibitors and mutants in iNOS, Irg1 and Nrf2 on progenitor cells isolated from differentiation cultures. iNOS deficiency led to the activation of erythroid transcriptional program, and increased numbers of mature progenitors as well as stress BFU-Es. In contrast, Irg1 and Nrf2 mutants showed impaired transition to erythroid differentiation, while they had elevated iNOS expression and NO production. Further analysis showed that treatment with either OI or iNOS inhibitor inhibited NO production in Irg1 and Nrf2 deficient progenitors, and consequently rescued the defects in erythroid differentiation.
These data support a model in which inflammation inhibits steady state erythropoiesis, while at the same time promoting stress erythropoiesis to maintain homeostasis. Our work reveals a dynamic and tight coordination between pro-inflammatory signals and progenitor cell metabolism in regulating the proliferation and differentiation of stress erythroid progenitors, and highlights the therapeutic potential of targeting metabolic and inflammatory signaling pathways in inflammatory anemia.

Disclosures
Paulson: Forma Therapeutics: Consultancy.