HH exposure promotes erythrocytosis in mice. The C57BL/6 mice were treated with normobaric normoxia (NN) and hypobaric hypoxia (HH) for 3, 7 and 14 days, and blood was collected for the following tests. (A) Observation of the number and morphology of RBCs by blood cell smear. Routine blood analysis of RBC counts (B) and HGB (C), HCT (D), and MCH content (E). (F) Representative dot plots (one of three experiments) of TO-positive cells (reticulocytes) in total populations of spleen. (G) CD47 staining in splenic cells was analysed by flow cytometry. Representative dot plots (one of three experiments) of CD47 expression after HH for 7 (H) and 14 (I) days in total populations of the spleen. Data were expressed as the means ± SEM (n = 5 per group); * P < 0.05, ** P < 0.01, *** P < 0.001 versus the NN group or the indicated group.

The spleen plays an important role in suppressing the immoderate increase in RBCs under HH conditions. C57BL/6 mice with or without splenectomy were treated with NN and HH for different numbers of days, and the spleen and blood were collected for subsequent analyses. (A) Morphological observation, (B) spleen volume and (C) spleen weight detection after HH exposure. (D-H) Blood observation and hematological index detection. Data were expressed as the means ± SEM (n = 9 per group); * P < 0.05, ** P < 0.01, *** P < 0.001 versus the NN group or the indicated group.

HH exposure causes a decrease in the number of macrophages in spleen. The C57BL/6 mice were treated with NN and HH for 7 or 14 days, and the spleen and blood were collected for subsequent detection. (A) Visualization of spleen cell clusters using uniform manifold approximation and projection (UMAP). Each color represents a cluster of specific cells of the spleen, which was identified by a defined gene expression profile. (B) The proportions of different macrophage clusters derived from either NN-or HH-treated mouse spleens were analysed. (C) Individual macrophages derived from either NN-or HH-treated mouse spleens are denoted. (D) Calcein/PI double staining was analysed by flow cytometry. (E and F) Cell death proportions in total splenic cells are given as bar graphs. (G and J) Western blot detecting CD16 and CD206 protein expression in the spleen. (H-I and K-L) Statistical analysis of CD16 and CD206 expression in H and K. (M) F4/80 staining with CD11b, CD86 or CD206 in splenic cells was analysed by flow cytometry. (N) qPCR analysis of CCL2 and CCL7 expression in the spleen. (O) qPCR analysis of Csf1 and Csf2 expression in the spleen. (P) Flow cytometry detection of CD111b and Ly6C double-stained cells in the BM and spleen. CD11bhiLy6Chi cell proportions in the BM (Q-R) and spleen (S-T) are given as bar graphs. (U) HO-1 and F4/80 expression in the spleen after HH exposure for 7 and 14 days. Data were expressed as the means ± SEM (n = 3 per group); * P < 0.05, ** P< 0.01, *** P < 0.001 versus the NN group or the indicated group.

HH exposure decreases erythrophagocytosis and iron processing capacity in the spleen. (A) Ecoil was labelled with Cy5.5 dye and photographed under a fluorescence microscope. (B) The phagocytic activity of splenic cells for Cy5.5-labelled Ecoil in vitro. (C) Autologous erythrocytes were labelled with biotin followed by NIH-FITC detection and photographed under a fluorescence microscope. (D) Experimental diagram of phagocytic activity in splenic cells after HH exposure in vivo. Flow cytometry detection of FITC-stained cells in the blood (E) and spleen (F) after HH exposure for 7 and 14 days. The FITC-positive cell proportions in the blood (G) and spleen (H) are given as bar graphs. (I) Experimental design of C57Bl6 mice (n=3 per group) treated at days 7 and 11 with a single intravenous injection of Tuftsin (1.5 mg/kg), followed by HH exposure until day 14 for isolation of the spleen for F4/80 immunohistochemistry and iron staining. (J) F4/80 and iron staining in the spleen after HH exposure for 14 days with Tuftsin treatment. (K) Quantitative results of F4/80 expression in the spleen described in (J). (L) Semiquantitative iron levels in the spleen described in (J). Data were expressed as the means ± SEM (n = 3 per group); * P < 0.05, ** P < 0.01, *** P < 0.001 versus the NN group or the indicated group.

HH exposure promotes iron mobilization and induces ferroptosis in the spleen. The C57BL/6 mice were treated with NN and HH for 7 days, and the spleen was collected for subsequent detection. (A) Western blot detection of HO-1, Ft-L, Ft-H, NCOA4, Fpn and TfR protein expression in spleen. (B) Statistical analysis of HO-1, Ft-L, Ft-H, NCOA4, Fpn and TfR protein expression in A. (C) GEO data analysis of HMOX1, FTL, FTH1, NCOA4, SLC40A1, and TFRC mRNA expression in PMBCs before and after climbing to high altitude (n=98). (D) Western blot detection of ACSL4, GPX4 and xCT protein expression in the spleen. (E) Statistical analysis of ACSL4, GPX4 and xCT protein expression in D. (F) GEO data analysis of ACSL4, GPX4 and SLC7A11 mRNA expression in PMBCs before and after climbing to high altitude (n=98). (G) The content of Fe2+ in the spleen was detected using the FerroFarRed probe by flow cytometry. (H) The level of lipid ROS in the spleen was detected using the C11-BODIPY probe by flow cytometry. The MDA (I), Cys (J) and GSH (K) levels in the spleen were detected using biochemical detection kits. Data were expressed as the means ± SEM (n = 3 per group); * P < 0.05, ** P < 0.01, *** P < 0.001 versus the NN group or the indicated group.

Hypoxia promoted primary peritoneal macrophage ferroptosis. Primary peripheral macrophages were cultured under 1% hypoxia for different times (0, 12, 24, 48 h) with or without pre-treatment with 10 µM Fer-1 for 1 h, and then the cells were collected for subsequent detection. (A) Immunofluorescence detection of Fe2+ levels in macrophages after hypoxia exposure for 24 h using the FerroFarRed probe under a fluorescence microscope. (B) Quantitative results of fluorescence intensity in the macrophages described in A. (C) Flow cytometry detection of Fe2+ levels in macrophages exposed to hypoxia for 24 h using the FerroFarRed probe. (D) Immunofluorescence detection of lipid ROS levels in macrophages exposed to hypoxia for 24 h using the C11-BODIPY probe under a fluorescence microscope. (E) Quantitative results of mean fluorescence intensity in the macrophages described in D. (F) Western blot detection of ACSL4, GPX4 and xCT protein expression in macrophages under hypoxia for different times. (G) Calcein/PI double staining in macrophages after 24 h of exposure to hypoxia was analysed by flow cytometry. (H) Cell death proportions in macrophages are given as bar graphs. (I) Macrophage phagocytic activity of Cy5.5-labelled Ecoil after 24 h of hypoxia exposure by flow cytometry in vitro. (J) Immunofluorescence detection of Fe2+ levels in macrophages pre-treated with Fer-1 followed by 24 h of hypoxia exposure using the FerroFarRed probe. (K) Quantitative results of fluorescence intensity in the macrophages described in J. (L) Western blot detection of ACSL4, GPX4 and xCT protein expression in macrophages pre-treated with Fer-1 followed by 24 h of hypoxia exposure. (M and N) Statistical analysis of ACSL4, GPX4 and xCT protein expression in L. MDA (P), Cys (Q), and GSH (R) levels in macrophages pre-treated with Fer-1 followed by 24 h of hypoxia exposure were detected using kits by biochemical methods. Data were expressed as the means ± SEM (n = 3 per group); * P < 0.05, ** P < 0.01, *** P <0.001 versus the NN group or the indicated group.

High altitude/hypobaric hypoxia exposure inhibits erythrophagocytosis by inducing ferroptosis in macrophages in the spleen. HA/HH exposure induces iron mobilization, especially increased Fe2+ levels, which are further increased in the spleen/macrophages of mice. On the other hand, HA/HH exposure increased ACSL4 expression and inhibited GSH production by suppressing xCT expression and GSH production. The above results enhance lipid peroxidation and further macrophage ferroptosis. Meanwhile, HA/HH exposure decreases monocyte migration and differentiation from the BM to the spleen. Taken together, HA/HH exposure inhibits erythrophagocytosis and further promotes erythrocytosis, which may aggravate HAPC development.

HH exposure promotes iron mobilization and induces ferroptosis in the spleen. The C57BL/6 mice were treated with NN and HH for 14 days, and the spleen was collected for subsequent detection. (A) Western blot detection of HO-1, Ft-L, Ft-H, NCOA4, Fpn and TfR protein expression in spleen. (B) Statistical analysis of HO-1, Ft-L, Ft-H, NCOA4, Fpn and TfR protein expression in A. (C) Western blot detection of ACSL4, GPX4 and xCT protein expression in the spleen. (D) Statistical analysis of ACSL4, GPX4 and xCT protein expression in C. (E) The content of Fe2+ in the spleen was detected using the FerroFarRed probe by flow cytometry. (F) Quantitative results of the mean fluorescence intensity in E. (G) The level of lipid ROS in the spleen was detected using the C11-BODIPY probe by flow cytometry. (H) Quantitative results of the mean fluorescence intensity in G. (I) MDA, (J) Cys, and (K) GSH levels in the spleen were detected using kits by biochemical methods. Data were expressed as the means ± SEM (n = 3 per group); * P < 0.05, ** P < 0.01, *** P < 0.001 versus the NN group or the indicated group.