Transcriptomic profile of macrophages activation state in healthy controls and MS patients

CD14+CD16- monocytes from MS patients and healthy controls (HC) were activated in vitro to obtain homeostatic-like, pro-inflammatory and pro-regenerative macrophages. Macrophage activation profiles were assessed through RNA sequencing Blood was sampled from all participants in acid citrate dextrose (ACD) tubes. From blood samples, peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll Paque Plus (GE Healthcare Life Sciences) and centrifugation (2200 rpm, 20 min). Cells were washed in PBS (2x10 min at 1500 rpm) and RPMI 1640 + 10% fetal bovine serum (FBS) (5 min at 1500 rpm) (all products from ThermoFisher). Monocytes were isolated with anti-CD14 microbeads (Miltenyi) and plated in 12-well plates (500 000 cells/well) or in 24-well plates (200 000 cells/well) in RPMI 1640 + 10% FBS and granulocyte macrophage colony-stimulating factor (GM-CSF) (500 U/ml, ImmunoTools). After 72h, media was replaced with fresh media and one of the following: GM-CSF (500 U/ml); IL4 (1000 U/ml, ImmunoTools); or combined IFNγ (200 U/ml, ImmunoTools) and ultra-pure LPS (10 ng/ml, InvivoGen). Macrophage samples were processed 24h post-activation for RNA extraction using Nucleospin RNA extraction kit. Transcriptome sequencing was performed on 28 patients and 11 HC using Truseq stranded mRNA kit on a NextSeq 500 sequencer. Raw data quality was assessed with FastQC and trimmed with Fastp. Reads were aligned to hg38 genome using Star v2.5.3a, and gene abundances were quantified with RSEM 1.2.28.