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Transcriptomic Profiling of Splenic Red Pulp Macrophages in Response to Dietary Iron Deficiency

ABSTRACT: This study investigates how dietary iron deficiency affects the transcriptional profile of spleen-derived red pulp macrophages (RPMs) in mice. Female C57BL/6J mice were divided into two groups: Iron-deficient (ID) group: fed a low-iron diet (<5 ppm Fe) from 4 to 9 weeks of age. Iron-balanced (IB) group: maintained on a standard iron-sufficient diet (200 ppm Fe) over the same period. At 9 weeks of age, mice were sacrificed, and spleens were harvested. RPMs were FACS-sorted using F4/80 and TREML4 markers, approx. 100 000 RPMs were sorted/sample. Sorting was performed to TRIzol, and RNA was isolated using Direct-zol columns. For all samples RIN was at least 7. Sequencing was conducted on the Illumina NextSeq 500 platform at the EMBL Genomics Core Facility (Heidelberg, Germany) using 75 bp single-end reads. The dataset consists of 8 samples: 4 from iron-deficient mice (S1_DEF to S4_DEF) 4 from control mice (S5_BAL to S8_BAL) This RNA-seq dataset enables comparison of gene expression changes in red pulp macrophages under conditions of iron deficiency, contributing to our understanding of iron homeostasis and macrophage adaptation.

ORGANISM(S): Mus musculus

PROVIDER: GSE312948 | GEO | 2025/12/13

REPOSITORIES: GEO

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Project description: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.

Project description:Tissue iron (Fe) overload is a frequent pathologic finding in multiple disease states including non-alcoholic fatty liver disease (NAFLD), neurodegenerative disorders, cardiomyopathy, diabetes, and some forms of cancer. The role of iron, as a cause or consequence of disease progression and observed phenotypic manifestations, remains controversial. In addition, the impact of genetic variation on iron overload related phenotypes is unclear, and the identification of genetic modifiers is incomplete. Here, our aim was to characterize the genetic architecture of dietary iron overload and pathology using the Hybrid Mouse Diversity Panel (HMDP), a panel of over 100 genetically distinct mouse strains optimized for genome-wide association studies and systems genetics. Dietary iron overload was induced by feeding male mice (114 strains, 6-7 mice per strain on average) a high iron diet for six weeks, and then tissues were collected at 10-11 weeks of age. Liver metal levels and gene expression were measured by ICP-MS and RNASeq, and lipids were measured by colorimetric assays. FaST-LMM was used for genetic mapping, and Metascape, WGCNA, and Mergeomics were used for pathway, module, and key driver bioinformatics analyses. Mice on the high Fe diet accumulated Fe in the liver, with a 6.5-fold difference across strain means. The iron loaded diet also led to a spectrum of copper deficiency and anemia, with liver copper levels highly correlated with red blood cell count, hemoglobin, and hematocrit. Hepatic steatosis of various severity was observed histologically, with 52.5-fold variation in triglyceride (TG) levels across the strains. TG and Fe mapped most significantly to an overlapping locus on chromosome 7 that has not been previously associated with either trait. Based on network modeling, significant key drivers for both Fe and TG accumulation are involved in cholesterol biosynthesis and oxidative stress management. To make the full data set accessible and useable by others, we have made our data and analyses available on a resource website.

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Transcriptomic Profiling of Splenic Red Pulp Macrophages in Response to Dietary Iron Deficiency

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