Gene expression profile of primary human monocyte subsets. Gene expression profile of primary human monocyte subsets

Human primary monocytes are composed of a minor, more mature CD16+(CD14low/neg) population and a major CD16neg(CD14+) subset. The specific functions of CD16+ vs. CD16neg monocytes in steady state or inflammation remain poorly understood. In previous work we found that IL-12 is selectively produced by the CD16+ subset in response to the protozoan pathogen, Toxoplasma gondii. Here we demonstrated that this differential responsiveness correlates with the presence of an IFN-induced transcriptional signature in CD16+ monocytes already at baseline. Consistent with this observation, we found that in vitro IFN-γ-priming overcomes the defect in IL-12 response of the CD16neg subset. We have anayzed the gene expression profile of primary human CD16+ and CD16neg monocyte subsets cultured for without or with IFN-gamma Overall design: Monocytes from three healthy donors were purified from elutriated preparations iwith MACS MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). For monocyte subset isolation, elutriated preparations were first depleted of granulocytes and NK cells with a mixture of anti-CD15 and CD56 MicroBeads, followed by CD16+ monocytes purification with a CD16pos Monocyte Isolation Kit, and CD16neg monocytes isolation from flow throw using CD14 MicroBeads. Purity of all populations was >95%. Total RNA was extracted from monocytes cultured for 8 hours with or without IFN-γ, and with or without stimulation with Toxoplasma gondii tachyzoytes, by adding Trizol reagent (Life Technologies) and employing the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. For broad assessment of gene expression, we used the nCounter® Myeloid Innate Immunity Panel (NanoString Technologies, Seatttle, WA). Total RNA (170 ng) from each sample was hybridized with the probes for 770 genes. Data were processed with nSolver Analysis software (NanoString Technologies) which included assessment of quality of the runs and differential gene expression assessed. The false discovery rate (FDR) was calculated using Benjamini-Yekutieli procedure.