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

Full text Figures and data Peer review Side by side Abstract eLife assessment Introduction Results Discussion Materials and methods Data availability References Article and author information Metrics Abstract High-altitude polycythemia (HAPC) affects individuals living at high altitudes, characterized by increased red blood cells (RBCs) production in response to hypoxic conditions. The exact mechanisms behind HAPC are not fully understood. 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 indicate that HH significantly boosts erythropoiesis, leading to erythrocytosis and splenic changes, including initial contraction to splenomegaly over 14 days. A notable decrease in red pulp macrophages (RPMs) in the spleen, essential for RBCs processing, was observed, correlating with increased iron release and signs of ferroptosis. Prolonged exposure to hypoxia further exacerbated these effects, mirrored in human peripheral blood mononuclear cells. Single-cell sequencing showed a marked reduction in macrophage populations, affecting the spleen's ability to clear RBCs and contributing to splenomegaly. Our findings suggest splenic ferroptosis contributes to decreased RPMs, affecting erythrophagocytosis and potentially fostering continuous RBCs production in HAPC. These insights could guide the development of targeted therapies for HAPC, emphasizing the importance of splenic macrophages in disease pathology. eLife assessment This useful study reports that a week or more of hypoxia exposure in mice increases erythropoiesis and decreases the number of iron-recycling macrophages in the spleen, compromising their capacity for red blood cell phagocytosis – reflected by increased mature erythrocyte retention in the spleen. Compared to an earlier version, the study has been strengthened with mouse experiments under hypobaric hypoxia and complemented by extensive ex vivo analyses. Unfortunately, while some of the evidence is solid, the work as it currently stands only incompletely supports the authors' hypotheses. While the study would benefit from additional experiments that more directly buttress the central claims, it should be of interest to the fields of hemopoiesis and bone marrow biology and possibly also blood cancer. https://doi.org/10.7554/eLife.87496.4.sa0 About eLife assessments Introduction A plateau is a special environment characterized by low atmospheric pressure and low partial oxygen pressure. Long-term exposure to plateau environments may lead to chronic mountain disease (Pérez-Padilla, 2022). High-altitude polycythemia (HAPC) is a common and widespread chronic mountain sickness characterized by excessive erythrocytosis (Liu et al., 2022). Hypobaric hypoxia (HH) is the main cause of erythrocytosis, which in turn alleviates hypoxic conditions in tissues under high-altitude (HA) exposure (Tymko et al., 2020). In a healthy organism, a balance is maintained between erythropoiesis in the bone marrow (BM) and erythrophagocytosis in the spleen (Dzierzak and Philipsen, 2013). Even though HA/HH exposure can disrupt the equilibrium between RBCs formation and clearance, healthy individuals can swiftly establish a new steady state of RBCs homeostasis in chronic hypoxia (Robach et al., 2018). This adaptation is a critical response to the altered oxygen availability characteristic of HA environments. However, in patients with HAPC, RBCs homeostasis is disrupted, failing to reach a state of equilibrium, which ultimately leads to a persistent increase in RBCs count (Dzierzak and Philipsen, 2013). This deviation from the normative adaptation process implies a pathological deviation from the usual compensatory mechanisms employed under chronic hypoxia conditions (Yang et al., 2023). It is well-established that exposure to HA/HH can induce erythropoiesis, yet the pathogenesis of erythrophagocytosis under these conditions remains poorly understood (Slusarczyk and Mleczko-Sanecka, 2021). The spleen plays a crucial role in maintaining erythropoietic homeostasis by effectively clearing impaired and senescent RBCs from circulation (Qiang et al., 2023). Particularly, red pulp macrophages (RPMs) within the spleen, serving as primary phagocytes, are responsible for clearing senescent, damaged, and abnormal erythrocytes from circulation to recycle iron (Wirth et al., 2020). RPMs initiate the process of RBCs endocytosis and lysosomal digestion into heme, following the recognition of the RBCs earmarked for removal via their cell surface signals (Slusarczyk et al., 2023; Vahedi et al., 2020). Subsequently, heme oxygenase-1 (HO-1) decomposes the heme into biliverdin, carbon monoxide, and iron (Nemeth and Ganz, 2021). Most of the iron recycled from heme is transported out of the cell through a protein called ferroportin (Fpn). This iron then binds to transferrin (Tf), a plasma protein that transports iron in the blood. The iron-transferrin complex interacts with the transferrin receptor (TfR) on the cell membrane, contributing to the formation of RBCs in the erythroid compartment (Hidalgo et al., 2021). A portion of the released iron is loaded into cellular ferritin (Ft; including Ft-H and Ft-L). In the presence of oxygen, the Ft-H facilitates the oxidation of Fe2+ (ferrous ion) to Fe3+ (ferric ion). Subsequently, the Ft-L stores this Fe3+ (Wang et al., 2013). The Ft-L features a nucleation site, consisting of a cluster of cavity-exposed carboxyl residues, which readily bind to Fe3+, thereby simplifying the storage process (Finazzi and Arosio, 2014). When the iron metabolism is vigorous, nuclear receptor coactivator 4 (NCOA4) can recognize Ft-H, bring Ft into the lysosomal pathway for degradation, and release iron in Ft for iron utilization in the body (Bellelli et al., 2016). However, some iron exists in cells in the form of ferrous iron (Fe2+), which is well known to be an important initiator of free radical oxidation (Yang et al., 2023). Moreover, the accumulation of large amounts of Fe2+ in cells may cause ferroptosis (von Krusenstiern et al., 2023). Despite the extensive study of erythropoiesis under HA/HH conditions, the precise effects of HA/HH on erythrophagocytosis within the spleen remain largely unexplored. Considering the significant role of the spleen in RBC processing and the crucial function of RPMs in iron recycling from RBC clearance, we evaluated the impacts of HA exposure on the spleen/splenic macrophages using an HH-exposed mouse model (simulating 6000 m exposure conditions). In the current study, we sought to further investigate whether HA/HH can influence the erythrocyte disposal and iron recycling processes in macrophages. More specifically, we aimed to determine the potential impact of HA/HH exposure on the integrity of the erythrophagocytosis process. We discovered that exposure to HH triggered ferroptosis in the spleen, particularly in macrophages. This led to a reduction in macrophage numbers, which was subsequently followed by disruptions in erythrophagocytosis and iron recycling within the spleen. These findings may hold clinical significance, particularly in the context of continuous pathological erythrocytosis and the progression of HAPC under HA exposure. Results HH exposure promotes erythrocytosis in mice To mimic 6000 m HA exposure, we placed C58BL/6 mice in an animal hypobaric oxygen chamber and detected the blood indices in the blood of the mice after HH exposure for different times. The blood smear showed that the number of RBCs was increased from 3 to 14 days after HH exposure compared with the NN group (Figure 1A). Routine blood tests further confirmed the results in blood smears, which showed that the RBC number (Figure 1B), HGB content (Figure 1C), and HCT value (Figure 1D) were all increased significantly to varying degrees, while MCH was not changed after HH exposure (Figure 1E). We further performed flow cytometry using TO staining to detect reticulocytes after 3, 7, and 14 days of HH treatment. The results showed that the proportion of reticulocytes increased after 7 and 14 days of HH exposure (Figure 1F-G), and the number of RBCs reached the peak after 7 days of HH exposure. These results suggested that the HH-treated mouse model effectively mimics HA exposure, which promotes erythropoiesis and results in erythrocytosis in mice following HH exposure. Figure 1 Download asset Open asset HH exposure promotes the induction of erythrocytosis in mice. C57BL/6 mice were subjected to either normobaric normoxia (NN) or hypobaric hypoxia (HH) conditions for durations of 3, 7, and 14 days. After these treatments, blood samples were collected for a comprehensive analysis. (A) Morphological evaluation of RBCs was conducted via blood smear examination (Wright staining). Routine hematological assessments were performed, encompassing RBCs counts (B), hemoglobin (HGB) levels (C), hematocrit (HCT) percentages (D), and mean corpuscular hemoglobin (MCH) content (E). (F) Flow cytometric analysis was employed to identify TO-positive cells, indicative of reticulocytes, in the whole blood samples. (G) The proportions of TO-positive RBCs in the blood were depicted in bar graphs. The data are presented as means ± SEM for each group (n=5 per group). Statistical significance is denoted by * p<0.05, ** p<0.01, *** p<0.001, relative to the NN group or as specified. Spleen inhibits the immoderate increase in RBCs under HH conditions To determine the roles of the spleen in RBC homeostasis under HA/HH, we investigated the effects of HH on the morphology, volume, and weight of the spleen as well as erythrocyte indices. As shown in Figure 2, the spleen volume and weight were decreased significantly after HH exposure for 1 day compared to NN treatment (Figure 2A-C). However, the spleen was obviously enlarged from 2 to 14 days after HH exposure (Figure 2A-C). The results indicated that the spleen contracted, the stored RBCs in the spleen were released into the blood at 1 day, and the RBCs were produced and/or retained in the spleen from 2 to 14 days after HH exposure. In our research, we also examined the influence of the spleen on RBCs homeostasis under HH conditions. We investigated whether the role of spleen in RBCs clearance under HH conditions could be compensated by the liver or other components of the mononuclear macrophage system. To conduct this, we performed splenectomies on mice and subsequently exposed them to HH conditions for 14 days. This allowed us to monitor RBCs counts and blood deposition. Our findings indicated that, in comparison to both the splenectomized mice under NN conditions and the sham-operated mice exposed to HH, erythrocyte deposition (Figure 2D) and counts (Figure 2E), as well as HGB (Figure 2F) and HCT (Figure 2G) levels, significantly increased 14 days post-splenectomy under HH conditions. Meanwhile, MCH levels remained stable (Figure 2H). These indices did not vary in mice, regardless of whether they had undergone a splenectomy, under NN conditions (Figure 2D and E). These results indicate that in the splenectomized group under NN conditions, erythrophagocytosis is substantially compensated for by functional macrophages in other tissues. However, under HH conditions, our data also suggest that the spleen plays an important role in managing erythrocyte turnover, as indicated by the significant impact of splenectomy on erythrophagocytosis and subsequent RBCs dynamics. Figure 2 Download asset Open asset 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 was determined. (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 leads to a decrease in splenic macrophages Considering macrophages as the primary cell type responsible for processing RBCs within the spleen under physiological conditions, we subsequently investigated the population and activity of these macrophages after exposure to HH for 7 or 14 days, employing flow cytometry and single-cell sequencing techniques. Calcein/PI double staining, examined via flow cytometry, revealed a significant decrease in viable splenic cells and a concomitant increase in dead cells following 7 and 14 days of HH exposure (Figure 3A–C). This observation was further substantiated by single-cell sequencing, which elucidated a pronounced reduction in the population of splenic macrophages after 7 days of HH exposure (Figure 3D–F). Additionally, flow cytometry results indicated a marked decrease in the population of RPMs, identified as F4/80hiCD11blo, in the spleen post 7 days of HH exposure (Figure 3G). Complementary to these findings, immunofluorescence detection demonstrated a significant diminution in RPMs, characterized by F4/80hiCD11blo and F4/80hiCD68hi cell populations, after both 7 and 14 days of HH exposure (Figure 3—figure supplement 1). Furthermore, our single-cell sequencing analysis of the spleen under NN conditions revealed a predominant association of RPMs with Cluster 0. Intriguingly, HH exposure led to a notable reduction in the abundance of RPMs within this cluster. Pseudo-time series analysis provided insights into the transitional dynamics of spleen RPMs, indicating a shift from Cluster 2 and Cluster 1 towards Cluster 0 under NN conditions. However, this pattern was altered under HH exposure, where a shift from Cluster 0 and Cluster 1 towards Cluster 2 was observed (Figure 3—figure supplement 2). Figure 3 with 2 supplements see all Download asset Open asset 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 analysis. (A) Calcein/PI double staining was analysed by flow cytometry to assess cell viability. (B and C) The bar graphs represent the proportions of cell death in total splenic cell population. (D) Uniform Manifold Approximation and Projection (UMAP) provided a visualization of spleen cell clusters, with each color representing a unique cluster characterized by specific gene expression profiles. (E) Comparative analysis of the proportions of distinct macrophage clusters in spleens from NN- and HH-treated mice. (F) Presentation of individual macrophages from spleens of mice treated with either NN or HH. (G) Analysis of F4/80 and CD11b expression in splenic cells, conducted via flow cytometry. (H) qPCR analysis evaluated Ccl2 and Ccl7 gene expression in the spleen. (I) qPCR analysis of Csf1 and Csf2 expression levels in the spleen. (J) Flow cytometry facilitated the detection of CD11b and Ly6C double-stained cells in BM and spleen. (K–L) Bar graphs represent the proportions of CD11bhiLy6Chi cells in the BM, while (M–N) delineate those in the spleen. (O) HO-1 and F4/80 expression levels in the spleen were monitored after 0 (NN), 7, and 14 days of HH exposure. (P) The relative fluorescence intensities of HO-1 and (Q) F4/80, as outlined in (O), were quantitatively assessed. Data are expressed as means ± SEM for each group (n=3 per group). Statistical significance is indicated by * p<0.05, ** p<0.01, *** p<0.001 when compared to the NN group or the indicated group. To further elucidate the migration and differentiation of monocytes from the bone marrow to the spleen, we analysed the expression of chemokines Ccl2, Ccl7, Csf1, and Csf2 in the spleen using qPCR and determined the number of monocytes in the bone marrow (BM) and spleen via flow cytometry. Our results indicated a significant reduction in the expression of Ccl2, Ccl7 (Figure 3H), Csf1, and Csf2 (Figure 3I) in the spleen after 7 and 14 days of HH exposure. Furthermore, the number of monocytes (Ly6C+/CD11b+) in the bone marrow (Figure 3J–L) and spleen (Figure 3J and M–N) also exhibited a decline after 7 and 14 days of HH exposure. We evaluated the depletion of macrophages in the spleen under HH conditions by examining the expression and distribution of HO-1 and F4/80. There was a decrease in both HO-1 and F4/80, predominantly within the splenic red pulp following HH exposure (Figure 3O–Q). Together, these findings indicate a reduction in the number of splenic macrophages after HH exposure, which could impair the spleen's capacity to process erythrocytes. HH exposure suppresses erythrophagocytosis of RPMs in spleen We investigated the influence of HH exposure on erythrocyte phagocytosis and heme iron recycling within splenic macrophages. To this end, we implemented a dual approach: administering NHS-biotin intravenously to mice, followed by HH exposure, and subsequently introducing PKH67-labelled RBCs into the mice, also followed by HH treatment in vivo. This methodology allowed us to monitor the clearance of RBCs by tracking the retention of both biotin-labelled and PKH67-labelled RBCs using flow cytometry in the blood and spleen of the HH-treated mice. Our findings (as detailed in Figure 4) indicated a pronounced decrease in the presence of both biotin-labelled and PKH67-labelled RBCs in the blood and spleen at 7- and 14 day intervals post HH exposure. Notably, while a natural decline in labelled RBCs over time was expected, the rate of decay was significantly more acute in the NN exposure group as compared to the HH group. This observation was consistent across both blood (Figure 4A and C; Figure 4E and G) and spleen samples (Figure 4B and D; Figure 4F and H). Furthermore, our analysis revealed that the phagocytic capacity of mouse spleen macrophages toward RBCs was notably diminished following HH exposure, particularly on the 14th day (Figure 4A–D; Figure 4E–H). To confirm these findings, we conducted additional assessments of the PKH67-positive RPMs cell population through both flow cytometry and immunofluorescence detection in the spleen post HH exposure. The findings revealed that both the population of splenic RPMs (F4/80hiCD11blo; Figure 5A–B) and the PKH67-positive macrophages (Figure 5A and C; D-E) consistently demonstrated a substantial reduction after 7 or 14 days of HH exposure, further reinforcing the impact of HH on the erythrophagocytic function of splenic macrophages. Figure 4 Download asset Open asset HH exposure decreases RBCs clearance both in the blood and spleen. (A and B) Flow cytometry measurements illustrated the proportions of FITC-stained RBCs in blood and spleen, respectively, after 7 and 14 days of HH exposure. (C and D) Bar graphs depicted the quantified proportions of FITC-positive RBCs in both blood and spleen, respectively. Further, (E and F) presented flow cytometry assessments of the proportions of PKH67-labelled RBCs in blood and spleen following 7 and 14 days of HH exposure. The corresponding proportions of PKH67-positive RBCs in blood (G) and spleen (H) were also shown in bar graph format. The data are expressed as means ± SEM for each experimental group (n=3 per group). Statistical significance is denoted with * p<0.05, ** p<0.01, *** p<0.001, relative to the NN group or as specified in the graph legends. This compilation of data underlines the impact of HH exposure on the reduction of RBCs clearance in both blood and splenic compartments. Figure 5 Download asset Open asset HH exposure reduces erythrophagocytosis in the spleen. Mice were administered PKH67-labeled RBCs, followed by exposure to HH for durations of 7 and 14 days. (A) Flow cytometry was employed to analyze the proportions of RPMs (identified as F4/80+CD11b-) and PKH67-positive RPMs in the spleen post-exposure. (B and C) Bar graphs represent the quantified proportions of RPMs (F4/80+CD11b-) and PKH67-positive RPMs in the spleen, respectively. (D) The spleens were subjected to immunofluorescence analysis to detect F4/80 in conjunction with PKH67 fluorescence post-perfusion at 7 and 14 days following HH exposure. (E) Subsequent quantification of the number of PKH67-positive F4/80hi cells in the spleen, as depicted in (D), was conducted. The data derived from these analyses are expressed as means ± SEM for each group (n=3 per group). Statistical significance is denoted with * p<0.05, and *** p<0.001, relative to the indicated group in the graph legends. Reduced erythrophagocytosis leads to RBCs retention in the spleen under HH exposure Based on the reduced RPMs and erythrophagocytosis caused by HH exposure in spleen, we next investigated whether RBCs were retained in spleen after HH exposure. Initial examination involved detecting RBCs in the spleen post HH exposure, both with and without perfusion. HE staining (Figure 6A–B) and Band 3 immunostaining in situ (Figure 6C–D and G–H) revealed a significant increase in RBCs numbers in the spleen following 7 and 14 days of HH exposure. Furthermore, we quantified RBCs retention by employing Wright-Giemsa composite staining on single splenic cells post-perfusion at both 7- and 14 days post HH exposure. The results consistently indicated a substantial elevation in RBC counts within the spleen (Figure 6E–F). To enhance the specificity of our investigation, we labelled RBCs in vitro with PKH67 and then administered them to mice. Following HH exposure for 7 and 14 days, spleen sections were analyzed without perfusion to detect retained PKH67-labeled RBCs. Fluorescence detection techniques was utilized, revealing a marked increase in PKH67-labeled RBCs post HH exposure (Figure 6I–J). These results collectively confirm a pronounced impairment in erythrophagocytosis within the spleen under HH conditions. This is evidenced by the observed increase in RBCs deformation and retention following 7 and 14 days of HH exposure. The data thus suggest that HH exposure leads to an increase in RBCs retention in the spleen, highlighting significant alterations in splenic function and RBCs dynamics under hypoxic conditions. Figure 6 Download asset Open asset HH exposure increases RBCs retention in the spleen. (A) HE staining was utilized to examine the presence of RBCs in the spleen post-perfusion at 7 and 14 days following HH exposure. (B) The number of RBCs present in the spleen, as observed in (A), was quantitatively assessed. (C and G) Immunohistochemical and immunofluorescent analyses using Band 3 staining were conducted to evaluate RBCs content in the spleen post-perfusion at 7 and 14 days of HH exposure. (D and H) The expression levels of Band 3 in the spleen, as shown in (C and G), were quantified. (E) Wright-Giemsa composite staining was performed on splenic cells following HH exposure for durations of 0 (NN), 7, and 14 days. (F) The proportion of RBCs within the perfused splenic cell population was determined. (I) Detection of PKH67 fluorescence in the spleen, without perfusion, was conducted after administering PKH67-labeled RBCs and subjecting them to HH exposure for 7 and 14 days. (J) The number of PKH67-positive RBCs within the spleen, as outlined in (I), was quantified. Data presented in this figure are expressed as means ± SEM for each experimental group (n=3 per group). Statistical significance is indicated by * p<0.05, ** p<0.01, *** p<0.001 when compared to the NN group or as specified. HH induced reduction in erythrophagocytosis leads to a decrease in the capacity of iron processing in the spleen To investigate the hypothesis that increased retention of RBCs in the spleen due to HH exposure is a result of impaired erythrophagocytosis by RPMs, we employed a series of experiments. Initially, RBCs were labelled with PKH67 cell linker and then injected into mice. Subsequently, Tuftsin was administered to stimulate the phagocytic activity of macrophages. After 1 hr, 1 day, 3 days, 5 days, or 7 days of NN exposure, PKH67 fluorescence was analyzed via flow cytometry. The findings demonstrated a gradual reduction in PKH67 fluorescence over the course of NN exposure; however, a significant decrease in PKH67 fluorescence was noted in the spleen following Tuftsin administration compared to the NN group. This observation suggested that RBC lifespan is diminished following enhanced erythrophagocytosis (as presented in Figure 7A–B). Further investigation was conducted on the ability of the spleen to regulate heme iron recycling under HH conditions through immunofluorescence and iron staining (Figure 7C). The results, as illustrated in Figure 7D, indicated that both F4/80 expression and iron deposition in the red pulp of the spleen were notably reduced following HH exposure, compared to the NN group. Contrastingly, Tuftsin administration under HH conditions resulted in an upsurge in F4/80 expression and iron deposition in the red pulp (Figure 7E–F). These findings collectively suggest that the splenic capacity for erythrocyte phagocytosis and subsequent heme iron recycling is significantly compromised under HH conditions, primarily due to a reduction in RPMs. This reduced erythrophagocytic capacity of the spleen under HH exposure is a critical factor contributing to the decreased efficiency of iron processing and increased RBCs retention in this organ. Figure 7 Download asset Open asset Impaired erythrophagocytosis of RBCs after HH exposure leads to a decrease in iron processing capacity in the spleen. To stimulate phagocytosis of macrophages, Tuftsin was administered immediately following the injection of PKH67-labeled RBCs into mice. Subsequent to various exposure durations under NN conditions (1 hr, 1, 3, 5, or 7 days), (A) PKH67 fluorescence within the spleen was measured using flow cytometry. (B) The percentages of PKH67-positive RBCs in the spleen are represented in bar graph format. (C) The experimental design is depicted, wherein C57BL/6 mice (n=3 per group) were administered a single intravenous dose of Tuftsin (0.15 mg/kg) on days 7 and 11, followed by HH exposure until day 14, culminating in spleen isolation for F4/80 immunohistochemistry and iron staining. (D) Demonstrates F4/80 and iron staining in the spleen after 14 days of HH exposure with Tuftsin treatment. (E) Quantitative analysis of the relative fluorescence intensity of F4/80 expression in the spleen as described in (D). (F) Semi-quantitative assessment of iron levels in the spleen as outlined in (D). Data presented in this figure are articulated as means ± SEM for each group (n=3 per group). Statistical significance is denoted by * p<0.05, ** p<0.01, *** p<0.001 when compared to the NN group or as indicated. HH exposure induces iron mobilization and ferroptosis in the spleen To elucidate the precise mechanisms underlying the macrophage reduction caused by HH, we examined the expression of proteins related to iron metabolism and ferroptosis in mice treated with HH and analyzed the corresponding gene expression in peripheral blood mononuclear cells (PBMCs) from healthy humans acutely exposed to HA conditions using GEO data (No. GSE46480). The results showed that, compared to the NN group, the expression levels of HO-1, Ft-L, Ft-H, NCOA4, and xCT were decreased, while Fpn, TfR, and ACSL4 expressions were significantly increased after 7 and 14 days of HH exposure (Figure 8A–B and D–E; Figure 3—figure supplement 1A–D). Apart from NCOA4, the alterations in gene expressions related to iron metabolism and ferroptosis in PBMCs were consistent with our Western blot results (Figure 8C and F). The GPX4 gene and protein expressions remained largely unchanged in both human PBMCs and mouse spleens. The changes in iron metabolism- and ferroptosis-related genes in PBMCs reflect the protein changes in the spleen under HH exposure. We detected Fe2+ and lipid ROS levels in the spleen by flow cytometry, and quantified MDA, GSH, and Cys levels using biochemical detection kits. As depicted in Figure 8, G and H; Figure 3—figure supplement 1I-K, the Fe2+ (Figure 8G; Figure 3—figure supplement 1E and F) and lipid ROS (Figure 8H; Figure 3—figure supplement 1G and H) levels in the spleen increased significantly after HH exposure. Additionally, the MDA content (Figure 8I; Figure 3—figure supplement 1I) in the spleen increased, whereas Cys (Figure 8J; Figure 3—figure supplement 1J) and GSH (Figure 8K; Figure 3—figure supplement 1K) levels significantly decreased after HH exposure. To ascertain the increased Fe2+ primarily emanated from the red pulp, we detected Fe2+ deposition and distribution in the spleen by Lillie stain following 7 and 14 days of HH exposure (Figure 8L–M; Figure 3—figure supplement 1L–M). These results demonstrated increased Fe2+ primarily deposited in the red pulp of the spleen. Taken together, these findings suggest that iron mobilization in the spleen was enhanced and ferroptosis was induced after 7 days of HH exposure. Figure 8 with 1 supplement see all Download asset Open asset HH exposure enhances 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 HA (n=98). (D) Western blot analysis for ACSL4, GPX4, and xCT protein expression in the spleen, with (E) depi