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) Analysis of the number and morphology of RBCs via a blood cell smear. Routine blood analysis was performed for RBC counts (B), as well as HGB (C), HCT (D), and MCH content (E). (F) Representative dot plots (from three experiments) indicate TO-positive cells (reticulocytes) in whole blood. Data are expressed as 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 varying durations, and the spleen and blood were collected for subsequent analyses. (A) Morphological observation, (B) spleen volume, and (C) spleen weight were determined post-HH exposure. (D-H) Blood observation and hematological index detection followed. Data are 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 results in a decrease in the number of splenic macrophages.

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) Calcein/PI double staining was analysed by flow cytometry. (B and C) The bar graphs represent cell death proportions in total splenic cells. (D) Uniform manifold approximation and projection (UMAP) visualized spleen cell clusters. Each color represents a cluster of specific spleen cells, identified by a distinct gene expression profile. (E) The proportions of different macrophage clusters derived from either NN-or HH-treated mouse spleens were analysed. (F) Individual macrophages derived from either NN-or HH-treated mouse spleens are denoted. (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 CD11b 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 0 (NN), 7, and 14 914 days of HH exposure. (V) The relative fluorescence of HO-1 and (W) F4/80 915 expression in the spleen as described in (U) was quantified. Data are 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.

Flow cytometry revealed the proportion of FITC-stained cells in the blood (A) and spleen (B) after 7 and 14 days of HH exposure. The proportions of these FITC-positive cells in the blood (C) and spleen (D) are presented as bar graphs. (E) Wright staining of splenic cells after HH exposure for 0 (NN), 7 and 14 days. (F) The proportion of retention RBCs within the spleen cells. (G) Illustrating the experimental design, in which C57BL6 mice (n=3 per group) were treated on days 7 and 11 with a single intravenous Tuftsin injection (1.5 mg/kg), followed by HH exposure until day 14 for spleen isolation and subsequent F4/80 immunohistochemistry and iron staining. (H) F4/80 and iron staining in the spleen after HH exposure for 14 days with Tuftsin treatment. (I) Quantitative the relative fluorescence of F4/80 expression in the spleen described in (H). (J) Semiquantitative iron levels in the spleen described in (H). Data are 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. (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). Western blot detection of ACSL4, GPX4, and xCT protein expression in the spleen is shown in (D) and its statistical analysis in (E). (F) GEO data analysis of ACSL4, GPX4 and SLC7A11 mRNA expression in PMBCs before and after reaching 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, respectively. (L) Lillie staining of Fe2+ in the spleen after 7 days of HH exposure. (M) Fe2+ deposition in the spleen as described in (L) was quantified. Data are 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 enhances primary peritoneal macrophage ferroptosis.

Primary peripheral macrophages were cultured under 1% hypoxia for varying durations (0, 12, 24, 48 h), either with or without a pre-treatment of 10 µM Fer-1 for 1 h, before being collected for further examination. (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) Flow cytometry was employed to analyze Calcein/PI double staining in macrophages following a 24 h hypoxia exposure. (H) Cell death proportions in macrophages are given as bar graphs. (I) Macrophage phagocytic activity against Cy5.5-labelled E.coli following 24 h of hypoxia exposure was assessed using flow cytometry in vitro. (J) Fe2+ levels in macrophages pre-treated with Fer-1, then exposed to 24 h of hypoxia, were detected via the FerroFarRed probe in immunofluorescence. (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, then followed by 24 h of hypoxia exposure. (M-O) Statistical analysis of ACSL4, xCT, and GPX4 protein expression in L. MDA (P), Cys (Q), and GSH (R) levels in macrophages pre-treated with Fer-1 and subsequently exposed to 24 h of hypoxia were detected using kits and biochemical methods. Data are 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 suppresses erythrophagocytosis by inducing macrophages ferroptosis 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 increases ACSL4 expression, suppressing xCT expression and GSH production, which escalates lipid peroxidation and subsequent 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.