An efficient method for gene silencing in human primary plasmacytoid dendritic cells: silencing of the TLR7/IRF-7 pathway as a proof of concept

Nikaïa Smith (1) , Pierre-Olivier Vidalain (1) , Sébastien Nisole (2) , Jean-Philippe Herbeuval (1)

1 LCBPT - UMR 8601 - Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques
2 IRIM - Institut de Recherche en Infectiologie de Montpellier

Plasmacytoid dendritic cells (pDC) are specialized immune cells that produce massive levels of type I interferon in response to pathogens. Unfortunately, pDC are fragile and extremely rare, rendering their functional study a tough challenge. However, because of their central role in numerous pathologies, there is a considerable need for an efficient and reproducible protocol for gene silencing in these cells. In this report, we tested six different methods for siRNA delivery into primary human pDC including viral-based, lipid-based, electroporation, and poly-ethylenimine (PEI) technologies. We show that lipid-based reagent DOTAP was extremely efficient for siRNA delivery into pDC, and did not induce cell death or pDC activation. We successfully silenced Toll-Like Receptor 7 (TLR7), CXCR4 and IFN regulatory factor 7 (IRF-7) gene expression in pDC as assessed by RT-qPCR or cytometry. Finally, we showed that TLR7 or IRF-7 silencing in pDC specifically suppressed IFN-α production upon stimulation, providing a functional validation of our transfection protocol.

Domaines: Sciences du Vivant [q-bio]; Biologie cellulaire; Biochimie, Biologie Moléculaire; Biotechnologies

Nikaïa Smith, Pierre-Olivier Vidalain, Sébastien Nisole, Jean-Philippe Herbeuval. An efficient method for gene silencing in human primary plasmacytoid dendritic cells: silencing of the TLR7/IRF-7 pathway as a proof of concept. Scientific Reports, 2016, 6 (1), ⟨10.1038/srep29891⟩. ⟨hal-02438269⟩