Comparative transcriptomic analysis of human macrophages during Mycobacterium avium versus Mycobacterium tuberculosis infection
Buffy coats were collected from healthy anonymous Dutch adult donors after written informed consent (Sanquin Blood Bank, Amsterdam, The the Netherlands). CD14+ monocytes were isolated from peripheral blood mononuclear cells using density gradient centrifugation with Ficoll (Pharmacy, LUMC, the Netherlands) and subsequently magnetic-activated cell sorting (MACS) with anti-CD14-coated microbeads (Miltenyi Biotec, Auburn, CA, USA). Total RNA was extracted with TRIzol reagent and the Direct-zol RNA miniPrep kit according to the manufacturer's protocol (Zymo Research, Leiden, the Netherlands). Isolated CD14+ monocytes from healthy human donors were cultured for 6 days in RPMI + 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 ng/ml macrophage colony-stimulating factor at 37°C, 5% CO2. After 6 days, the differentiated human monocyte-derived macrophages (hMDM) with M2 phenotype were harvested using trypsin-EDTA and reseeded in cell culture medium without antibiotics. The cells were cultured for  24 h at 37°C, 5% CO2. hMDM were infected with M. tuberculosis strain H37Rv or M. avium strain MAC101 at MOI 10, or left uninfected. After 1 h, the cells were treated with 30 μg/ml gentamicin-containing medium for 10 min to inactivate and remove residual extracellular bacteria, after which the medium was refreshed with medium containing 5 μg/mL gentamicin sulfate before cells were incubated at 37°C/5% CO2 until indicated timepoints. Total RNA was extracted with TRIzol reagent and the Direct-zol RNA miniPrep kit according to the manufacturer's protocol (Zymo Research, Leiden, the Netherlands). Library preparation was performed by GenomeScan (Leiden, the Netherlands) using the NEBNext Ultra Directional RNA Library prep kit (New England Biolabs, Ipswich, MA, USA) for Illumina according to the manufacturers protocol