High-altitude hypoxia exposure inhibits erythrophagocytosis by inducing macrophage ferroptosis in the spleen

High-altitude polycythemia (HAPC) is a chronic condition prevalent in individuals residing at high altitudes (HA), characterized by an augmented production of red blood cells (RBCs) due to hypoxic conditions. Despite its prevalence, the pathophysiological basis and molecular mechanisms driving HAPC remain largely unelucidated. In our study, we utilized a mouse model exposed to hypobaric hypoxia (HH), replicating the environmental conditions experienced at 6000 m above sea level, coupled with in vitro analysis of primary splenic macrophages under 1% O2 to investigate these mechanisms. Our findings revealed that HH exposure significantly stimulates erythropoiesis, leading to erythrocytosis. This was accompanied by a notable sequence of splenic changes, initially characterized by splenic contraction, which subsequently progressed to splenomegaly over 14 days. A key observation was the impairment on the capacity of spleen to process RBCs, primarily attributed to a reduction in splenic macrophages located in the red pulp. Extended HH treatment over 7 and 14 days precipitated increased iron mobilization and the onset of ferroptosis within the spleen. This was evidenced by altered expression levels of iron metabolism and ferroptosis-related proteins, paralleling gene expression patterns observed in human peripheral blood mononuclear cells. Single-cell sequencing of splenic tissue post-HH exposure demonstrated a marked decrease in macrophage populations after 7 days. Our study further disclosed a significant increase in RBCs retention in the spleen following HH exposure, likely a consequence of diminished red pulp macrophages (RPMs) and erythrophagocytosis. This hypothesis was corroborated through comprehensive analyses involving flow cytometry, histological staining, and immunostaining, which collectively provided a detailed understanding of RBCs dynamics under HH conditions. In vitro analyses substantiated the decline in primary splenic macrophages and induction of ferroptosis under hypoxic treatment, effects that were relieved by pre-treatment with the ferroptosis inhibitor ferrostatin-1. Collectively, our data suggest that HH exposure initiates splenic ferroptosis, leading primarily to a decrease in RPMs. This decrease potentially impacts erythrophagocytosis, contributing to increased RBCs retention and subsequent splenomegaly. Such changes could potentially foster continuous RBCs production and accelerate the progress of HAPC. In conclusion, our study highlights the important role of the spleen and splenic macrophages in the pathogenesis of HAPC, providing crucial insights into the disease progression and potential therapeutic targets. C57BL/6J mice were exposed to a hypobaric hypoxia (HH) chamber for 7 days to establish an animal model simulating high altitude exposure at 6000 meters, while the control group animals were kept in a similar chamber under normobaric normoxia (NN) conditions for 7 days. Spleen tissues were surgically extracted and preserved in MACS Tissue Storage Solution (Miltenyi Biotec, Bergisch Gladbach, Germany) until further processing. Single-cell dissociation was performed by researchers at the GENECHEM laboratory (Shanghai, China). Dissociated single cells were then stained with Calcein-AM (BD Biosciences, USA) and Draq7 (BD Biosciences) for viability assessment. The BD Rhapsody system was utilized to capture transcriptomic information from individual cells. Library quantification was conducted using a High Sensitivity D1000 ScreenTape and High Sensitivity D1000 Reagents on a 4150 TapeStation System (Agilent, Palo Alto, CA, USA), as well as the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific). Sequencing of libraries was carried out on an Illumina sequencer (Illumina Nova Seq 6000, San Diego, CA) through a paired-end run of 150 bp.