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Towards understandingTissue Mϕ heterogeneity and functions using quantitative proteomics

ABSTRACT: Resident macrophages are important for maintaining tissue homeostasis and for defence against infections, but their precise functions in different tissues are not fully elucidated.We have used high resolution quantitative proteomics to investigate the functions of splenic red pulp macrophages and peritoneal cavity macrophages in the steady-state. The validation of several proteins at cellular expression levels by the flow cytometry analysis was consistently in agreement with the proteomics data. Peritoneal macrophages were shown to be enriched in a number of key enzymes and metabolic pathways normally associated with the liver, such as metabolism of fructose, detoxification, nitrogen homeostasis and the urea cycle. Supporting observations in proteomics, we find that PM are able to utilise glutamine and glutamate which are rich in peritoneum for urea generation. In comparison, splenic red pulp macrophages were enriched in proteins important for adaptive immunity such as antigen presenting MHC molecules, in addition to proteins required for erythrocyte homeostasis and iron turnover. We also show that these tissue macrophages may utilise carbon and nitrogen substrates for different metabolic fates to support distinct tissue-specific roles. This study provides a valuable resource for biologists interested in the functions of tissue macrophages.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Spleen, Peritoneal Macrophage, Macrophage

SUBMITTER: Manjula Nagala

LAB HEAD: Professor Paul Crocker

PROVIDER: PXD019362 | Pride | 2020-06-24

REPOSITORIES: Pride

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Project description:Siglec-E is a murine CD33-related siglec that functions as an inhibitory receptor and is expressed mainly on neutrophils and macrophage populations. Recent studies have suggested that siglec-E is an important negative regulator of LPS-TLR4 signalling and one report (Wu et al 2016) claimed that siglec-E is required for TLR4 endocytosis following uptake of Escherichia coli by macrophages and dendritic cells (DCs). Our attempts to reproduce these observations using cells from wildtype (WT) and siglec E deficient mice were unsuccessful. We used a variety of assays to determine if siglec-E expressed by different macrophage populations can regulate TLR-4 signalling in response to LPS, but found no consistent differences in cytokine secretion in vitro and in vivo, comparing 3 different strains of siglec-E-deficient mice with matched WT controls. No evidence was found that the siglec-E deficiency was compensated by expression of siglecs-F and –G, the other murine inhibitory CD33-related siglecs. Quantitative proteomics was used as an unbiased approach and provided additional evidence that siglec-E does not suppress inflammatory TLR4 signaling. Interestingly, proteomics revealed a siglec E dependent alteration in macrophage phenotype that could be relevant to functional responses in host defence. In support of this, siglec-E-deficient mice exhibited enhanced growth of Salmonella enterica serovar Typhimurium in the liver following intravenous infection, but macrophages lacking siglec-E did not show altered uptake or killing of bacteria in vitro. Using various cell types including bone marrow derived DCs (BMDC), splenic DCs and macrophages from WT and siglec-E-deficient mice, we showed that siglec-E is not required for TLR4 endocytosis following E.coli uptake or LPS challenge. We failed to see expression of siglec-E by BMDC even after LPS-induced maturation, but confirmed previous studies that splenic DCs express low levels of siglec-E. Taken together, our findings do not support a major role of siglec-E in regulation of TLR4 signalling functions or TLR4 endocytosis in macrophages or DCs. Instead, they reveal that induction of siglec-E by LPS can modulate the phenotype of macrophages, the functional significance of which is currently unclear.

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.

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Towards understandingTissue Mϕ heterogeneity and functions using quantitative proteomics

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