Transcriptional effects of siRNA-mediated AhR knockdown on TLR7 activation of M-CSF-derived macrophages

Analysis of the role of AhR on the transcriptional signature of TLR7-activated human M-CSF-dependent monocyte-derived macrophages. Methods: Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from buffy coats from donors over a Lymphoprep gradient according to standard procedures. Monocytes were purified from PBMC by magnetic cell sorting using anti-CD14 microbeads (>95% CD14+ cells). Monocytes from three independent donors (0.5 x 106 cells/ml, >95% CD14+ cells) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2 and 21% O2 for 5 days in the presence of 1000 U/ml M-CSF, with cytokine addition every two days. Then, monocyte-derived macrophages were transfected with AhR-specific siRNA or control siRNA, kept in culture for 48 hours, and then stimulated (or not) with the TLR7 ligand CL264 (1 microgram/ml). After 4 hours, cells were lysed and RNA isolated for transcriptional analysis. Overall design: mRNA profiles of human M-CSF-derived macrophages activated with the TLR7 ligand CL264 after downregulation of AhR expession