{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Mathieu VETTER"],"organism":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14408"],"description":["Fever, a cardinal sign of inflammation, has been shown to modulate macrophage functions. Here, we wonder whether fever affects macrophage efferocytosis. This process is critical for the resolution of inflammation and the return to homeostasis with the reprogramming of macrophages toward a pro-resolving phenotype. Using primary mouse bone marrow-derived macrophages (BMDM) stimulated by LPS and IFN-γ (M1-like BMDM), we first validated that exposure to febrile-range temperature (39.5°C) induced a heat shock protein response. This was done by RNA sequencing (RNAseq), quantitative RT-PCR (RT-qPCR), and intracellular flow cytometry (FC). Then, we observed that febrile-range temperature decreased pro-inflammatory macrophage efferocytosis assessed by two different FC-based assays and by IncuCyte® live-cell imaging. This reduced efferocytic capacity of macrophages exposed to febrile-range temperature resulted from a decrease capacity to interact with apoptotic cells and to internalise these dying cells. This real-time image-based efferocytosis assay showed also that the heat reduced cell motility of macrophages in response to apoptotic cells. In our RNAseq dataset, we found an upregulation of the Adam17 gene and confirmed this increase by RT-qPCR analysis. Since this gene encodes a protease shedding the efferocytic receptor Mer, we determined Mer expression by FC and soluble Mer in the culture supernatants by ELISA. Febrile-range hyperthermia induced the cleavage of Mer from the cell surface of pro-inflammatory macrophages. Overall, decreased efferocytosis induced by fever-range hyperthermia may result from reduced Mer expression. This suggests that fever, by acting on Mer expression, decreases the efferocytic capacity of macrophages to prolong the pro-inflammatory state."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - Total RNA from pro-inflammatory macrophages cultured at 37°C or at 39.5°C for 4, or 8 hours was extracted with RNeasy© mini kit (74106, Qiagen) according to the manufacturer’s recommendations. DNA were eliminated with DNase I (79254, Qiagen) during extraction. Purity of mRNA was assessed by determining the ratio of absorbance readings at 260 nm and 280 nm, and sample quality was verified with the NanoDrop 2000 spectrophotometer (ThermoFisher scientific). The stability of mRNA was determined with TapeStation 4150 (Agilent) with RNA ScreenTape kits (Agilent).","Library Construction - Library construction and sequencing were performed by the Beijing Genomics Institute (BGI, Shenzhen, China).","Sample Collection - Adult male C57BL/6 Lys5.1 mice (Charles River) were used to generate BMDM. After euthanasia, femurs and tibias were recovered and were put in Roswell Park Memorial Institute medium (RPMI, 72400, Gibco) containing 1% penicillin/streptomycin (P/S, CABPES01-0U, Eurobio). Bone marrow cells were flushed from bones, and then separated with a 25-gauge needle. After centrifugation, red cells were lysed using a hypotonic solution (150 mM NH4Cl, 0.1 mM EDTA disodium salt and 1 mM KHCO3). After bone marrow cell centrifugation, cells pellets were suspended in complete Iscove’s Modified Dulbecco’s Medium (IMDM, L0190, Biowest) supplemented with 1% P/S, 1X non-essential amino acids (NEAA, M7145, Sigma-Aldrich), 1 mM sodium pyruvate (S8636, Sigma-Aldrich) and 10% foetal bovine serum (FBS, 10270106, Gibco) and passed through a filter with 100 μm pores (130-098-463, Miltenyi Biotec). Bone marrow cells were concentrated at 1 millions cells/ml in complete IMDM supplemented with 10 ng/ml of M-CSF (130-101-706, Miltenyi Biotec) and cultured at 37°C and 5% CO2 during 7 days. At day 7, medium was removed and replaced with a pro-inflammatory polarisation medium consisting in complete IMDM with 100 ng/ml of LPS (Escherichia coli O55:B5, L6529, Sigma-Aldrich) and 50 ng/ml of IFN-γ (130-105-774, Miltenyi Biotec) for two days. Pro-inflammatory polarisation medium were replaced with RPMI medium containing 1% P/S, 1% NEAA, 1% sodium pyruvate and 10% FBS.","Sample Treatment - Cells were placed at 37°C or at 39.5°C under 5% CO2 during 4 and 8 hours.","Sequencing - RNA sequencing were performed using the DNBSEQ Sequencing platform (sequencing length, PE150)."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - Alignment of reads sequence was performed by BGI (Beijing Genomics Institute)","Data Transformation - FPKM RAW data were normalised with Biomex software with normal normalisation"],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["DNBSEQ-G400"],"study_type":["RNA-seq of coding RNA"],"species":["Mus musculus"],"pubmed_authors":["Philippe SAAS","Mathieu VETTER"],"additional_accession":[]},"is_claimable":false,"name":"Fever-range temperature alters continual efferocytosis mediated by mouse pro-inflammatory macrophages","description":"Fever, a cardinal sign of inflammation, has been shown to modulate macrophage functions. Here, we wonder whether fever affects macrophage efferocytosis. This process is critical for the resolution of inflammation and the return to homeostasis with the reprogramming of macrophages toward a pro-resolving phenotype. Using primary mouse bone marrow-derived macrophages (BMDM) stimulated by LPS and IFN-γ (M1-like BMDM), we first validated that exposure to febrile-range temperature (39.5°C) induced a heat shock protein response. This was done by RNA sequencing (RNAseq), quantitative RT-PCR (RT-qPCR), and intracellular flow cytometry (FC). Then, we observed that febrile-range temperature decreased pro-inflammatory macrophage efferocytosis assessed by two different FC-based assays and by IncuCyte® live-cell imaging. This reduced efferocytic capacity of macrophages exposed to febrile-range temperature resulted from a decrease capacity to interact with apoptotic cells and to internalise these dying cells. This real-time image-based efferocytosis assay showed also that the heat reduced cell motility of macrophages in response to apoptotic cells. In our RNAseq dataset, we found an upregulation of the Adam17 gene and confirmed this increase by RT-qPCR analysis. Since this gene encodes a protease shedding the efferocytic receptor Mer, we determined Mer expression by FC and soluble Mer in the culture supernatants by ELISA. Febrile-range hyperthermia induced the cleavage of Mer from the cell surface of pro-inflammatory macrophages. Overall, decreased efferocytosis induced by fever-range hyperthermia may result from reduced Mer expression. This suggests that fever, by acting on Mer expression, decreases the efferocytic capacity of macrophages to prolong the pro-inflammatory state.","dates":{"release":"2025-12-19T00:00:00Z","modification":"2025-12-19T02:02:29.614Z","creation":"2024-09-03T10:32:48.913Z"},"accession":"E-MTAB-14408","cross_references":{"ENA":["ERP163738"],"EFO":["EFO_0002944","EFO_0004170","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184","EFO_0003969"]}}