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In vitro polarization of primary human blood derived macrophages

ABSTRACT: Purpose: Generate a benchmark to assess extent of macrophage polarization in disease or infection models. Method: Monocytes were isolated from blood donors who gave informed written consent (Ethics approval number: 161/17, Philipps University Marburg, Germany) by MACS CD14 positive selection. Monocytes were cultured for 7 days in the presence of 1% human off-the-clot AB serum. On day 7, macrophage morphology was assessed by microscope, and cells were left untreated (ctrl), stimulated with 100ng/ml LPS + 20 ng/ml IFNgamma (M1) or 20 ng/ml IL4 + 20 ng/ml IL-13 for 18h. Additionally, a separate set of cells was infected with Legionella Pneumophila Corby wild type and flagellin deficient at MOI1 for 18 h. Then RNA was isolated by phenol chloroform extraction, sample quality was assessed by Bioanalyzer, and samples with a RNA integrity > 8 were sequenced on an Illumina HiSeq 1500.

ORGANISM(S): Homo sapiens

PROVIDER: GSE157182 | GEO | 2022/08/25

REPOSITORIES: GEO

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Project description:OBJECTIVE: To determine whether macrophages, a type of cell implicated in the pathogenesis of ankylosing spondylitis (AS), exhibit a characteristic gene expression pattern. METHODS: Macrophages were derived from the peripheral blood of 8 AS patients (median disease duration 13 years [range <1-43 years]) and 9 healthy control subjects over 7 days with the use of granulocyte-macrophage colony-stimulating factor. Cells were stimulated for 24 hours with interferon-gamma (IFNgamma; 100 units/ml), were left untreated for 24 hours, or were treated for 3 hours with lipopolysaccharide (LPS; 10 ng/ml). RNA was isolated and examined by microarray and real-time quantitative reverse transcription-polymerase chain reaction analysis. RESULTS: Microarray analysis revealed 198 probe sets detecting the differential expression of 141 unique genes in untreated macrophages from AS patients compared with healthy controls. Clustering and principal components analysis clearly distinguished AS patients and controls. Of the differentially expressed genes, 78 (55%) were IFN-regulated, and their relative expression indicated a reverse IFN signature in AS patient macrophages, where IFNgamma-up-regulated genes were underexpressed and down-regulated genes were overexpressed. Treatment of macrophages with exogenous IFNgamma normalized the expression of these genes between patients and controls. In addition, the messenger RNA encoded by the IFNgamma gene was approximately 2-fold lower in AS patient macrophages at baseline (P = 0.004) and was poorly responsive to LPS (P = 0.018), as compared with healthy controls. CONCLUSIONS: Our findings reveal consistent differences in gene expression in macrophages from AS patients, with evidence of a striking reverse IFN signature. Together with poor expression and responsiveness of the IFNgamma gene, these results suggest that there may be a relative defect in IFNgamma gene regulation, with autocrine consequences and implications for disease pathogenesis. Experiment Overall Design: Macrophages were derived from the peripheral blood of 8 AS patients (median disease duration 13 years [range <1â??43 years]) and 9 healthy control subjects over 7 days with the use of granulocyteâ?? macrophage colony-stimulating factor. Cells were stimulated for 24 hours with interferon- (IFN; 100 units/ ml), were left untreated for 24 hours, or were treated for 3 hours with lipopolysaccharide (LPS; 10 ng/ml). RNA was isolated and examined by microarray and real-time quantitative reverse transcriptionâ??polymerase chain reaction analysis.

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In vitro polarization of primary human blood derived macrophages

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