Reviewer #1 (Public Review): High-altitude hypoxia exposure inhibits erythrophagocytosis by inducing macrophage ferroptosis in the spleen

This study investigates the role of the spleen and splenic macrophages in the development of high-altitude polycythemia (HAPC), a condition characterized by an imbalance between erythropoiesis and eryptosis imbalance, typically observed in high-altitude (HA) environments. We employed a mouse model subjected to hypobaric hypoxia (HH) to simulate the conditions of a 6000 m HA exposure. For in vitro examination, primary splenic macrophages were treated with 1% hypoxia. Our findings revealed that the HH treatment enhanced erythropoiesis, resulting in erythrocytosis, with marked splenic contraction evident, later progressing to splenomegaly over a 14-day period. HH exposure also impaired the ability of the spleen to process red blood cells (RBCs), predominantly due to a decrease in splenic macrophages within the red pulp. Furthermore, the application of HH treatment over 7 and 14-day intervals resulted in increased iron mobilization and onset of ferroptosis within the spleen, as corroborated by the expression of iron metabolism-related and ferroptosis-related proteins. The expression levels of these proteins mirrored gene expression levels in human peripheral blood mononuclear cells. Subsequent single-cell sequencing of the spleen demonstrated a substantial decrease in macrophages 7 days post-HH exposure. In vitro investigations confirmed the decline in primary splenic macrophages and induction of ferroptosis following hypoxic treatment, which were reversed by pre-treatment with the ferroptosis inhibitor ferrostatin-1. In summary, the data suggested that HH exposure instigates splenic ferroptosis, predominantly in the red pulp, thereby hampering the RBCs clearance in the spleen. This leads to increased 46 RBCs retention within the spleen, triggering splenomegaly, which may potentially foster continuous RBCs production and accelerate HAPC progression. The major conclusion from this study elucidates the critical role of spleen and splenic macrophages in the pathogenesis of HAPC.