Loss of nitric oxide signaling compromises the development of the stress erythropoiesis niche Free

Steady-state erythropoiesis is a homeostatic process that constantly produces new erythrocytes in the bone marrow to replace senescent cells removed by the spleen. Inflammation caused by infection or tissue damage skews bone marrow hematopoiesis towards myelopoiesis and disrupts this process. In response to this loss of erythroid production, an extramedullary pathway known as stress erythropoiesis is initiated, which maintains erythroid homeostasis until the source of inflammation is resolved.

Stress erythropoiesis differs significantly from steady-state erythropoiesis. Inflammation suppresses steady-state erythropoiesis by increasing erythroid turnover, which elevates pro-inflammatory signals (such as TNFα and IFNγ) and initiates stress erythropoiesis. This triggers the recruitment of short-term hematopoietic stem cells (ST-HSCs) and monocytes from the bone marrow to extramedullary sites, such as the spleen, where they interact with the splenic microenvironment to form stress erythroid progenitors (SEPs) and the stress erythropoiesis niche.

Erythroblastic islands (EBIs) are specialized microenvironmental structures, composed of monocytes and macrophages, that supply SEPs with essential nutrients and growth factors for their development. Unlike steady-state erythropoiesis, where EBIs are constantly present, stress erythropoiesis EBIs develop only in response to inflammatory stress. Preliminary work from our lab supports a model in which the development of the stress erythropoiesis niche occurs in discrete stages.

Previous work from our lab has shown that the monocyte/macrophage niche develops in concert with the SEPs. We have shown that the initial development of the stress erythropoiesis niche relies on pro-inflammatory signals, such as TNFα and nitric oxide (NO). These signals lead to the homing of Ly6c⁺monocytes to the spleen, where they phagocytose erythrocytes. This is followed by heme-dependent SpiC signaling, which drives the development of these monocytes into macrophages that serve as the EBI macrophages supporting stress erythropoiesis. Defects in this process lead to an impaired niche, which compromises stress erythropoiesis.

Our recent data shows that inhibiting inducible nitric oxide synthase (NOS2)-dependent NO production decreased SEP proliferation and delayed recovery from inflammatory anemia. NO also regulated SEP metabolism, promoting proliferation and inhibiting differentiation. Using heat-killed Brucella abortus to induce inflammatory anemia, Nos2^-/- mice exhibited greater splenomegaly, the resolution of anemia was delayed, SEP proliferation in the spleen was reduced, and fewer BFU-Es were generated. In addition, approximately 40% of the Nos2^-/- mice failed to survive. In addition to SEP defects, defective niche development was also observed, with Ly6C⁺ cells accumulating in the spleen but failing to differentiate into macrophages. These monocytes exhibited increased erythrophagocytosis compared to wild-type controls, suggesting that despite heme signaling, they were unable to form a functional stress erythropoiesis niche.

These findings aligned with our preliminary observations in sickle cell anemia. In human samples, sickle cell patients — but not normal controls — had Kit⁺CD34⁺ cells and monocytes in the peripheral blood, which were able to generate SEPs when cultured in vitro. In sickle cell mice samples, spleens were enlarged, recruitment of Ly6C⁺ monocytes to the spleen were increased, and BFU-E numbers were reduced. This phenotype closely mirrored our observations in Nos2^-/- mice, further supporting a role for NO-dependent signaling in stress erythropoiesis. Because low NO bioavailability is also a hallmark of sickle cell disease, these findings highlight a role for NOS2–NO signaling in regulating stress erythropoiesis niche in sickle cell disease and identify a potential new target for improving erythropoiesis and reducing anemia in sickle cell and other inflammatory conditions.