Fc γR-TLR Cross-Talk Enhances TNF Production by Human Monocyte-Derived DCs via IRF5-Dependent Gene Transcription and Glycolytic Reprogramming

This study was done according to the ethical guidelines of the Academic Medical Center and human material was obtained in accordance with the AMC Medical Ethics Review Committee according to the Medical Research Involving Human Subjects Act. Buffy coats obtained after blood donation (Sanquin blood supply) are not subjected to informed consent, which is according to the Medical Research Involving Human Subjects Act and the AMC Medical Ethics Review Committee. All samples were handled anonymously. Ethical review and approval was not required for this study in accordance with the local legislation. Monocytes were isolated from buffy coats by density gradient centrifugation on Lymphoprep (Nycomed) and Percoll (Pharmacia). DCs or macrophages were generated by culturing monocytes for 6 days in IMDM (Lonza) containing 5% FBS (Biowest) and 86 μg/mL gentamicin (Gibco), supplemented with 20 ng/mL GM-CSF (Invitrogen) and 2 ng/mL IL-4 (Miltenyi Biotec) for DCs or 50 ng/mL recombinant human M-CSF (BioLegend) for macrophages. At day 2 or 3, half of the medium was replaced by new medium containing cytokines. For silencing at day 3, cells were harvest by resuspending (DCs) or by using TrypLE Select (Invitrogen) (macrophages). Cells were microporated in the presence of 500 nM IRF5 si-RNA or control si-RNA (Dharmacon) and cultured for 3 more days in IMDM without gentamicin with supplemented cytokines. DCs were harvested at day 6 by putting the cells on ice for 30 min and macrophages ...
Source: Frontiers in Immunology - Category: Allergy & Immunology Source Type: research