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; Pierre-Olivier Vidalain; Sébastien Nisole; Jean-Philippe Herbeuval

doi:10.1038/srep29891

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

Sci Rep. 2016 Jul 14:6:29891. doi: 10.1038/srep29891.

Authors

Nikaïa Smith^{1

2}, Pierre-Olivier Vidalain^{1

2}, Sébastien Nisole^{2

3}, Jean-Philippe Herbeuval^{1

2}

Affiliations

¹ Equipe Chimie et Biologie, Modélisation &Immunologie pour la Thérapie (CBMIT), CNRS UMR8601, Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, CICB-Paris (FR 3567), Centre Universitaire des Saints-Pères, 45 rue des Saints Pères, 75006, Paris, France.
² Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
³ INSERM UMR-S 1124, 45 rue des Saints-Pères, 75006 Paris, France.

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Dendritic Cells / cytology
Dendritic Cells / metabolism*
Electroporation / methods
Fatty Acids, Monounsaturated / chemistry*
Gene Silencing
Gene Transfer Techniques*
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Humans
Interferon Regulatory Factor-7 / antagonists & inhibitors*
Interferon Regulatory Factor-7 / genetics
Interferon Regulatory Factor-7 / metabolism
Interferon-alpha / antagonists & inhibitors
Interferon-alpha / metabolism
Lentivirus / genetics
Lentivirus / metabolism
Polyethyleneimine / chemistry
Primary Cell Culture
Quaternary Ammonium Compounds / chemistry*
RNA, Small Interfering / genetics
RNA, Small Interfering / metabolism
Receptors, CXCR4 / antagonists & inhibitors*
Receptors, CXCR4 / genetics
Receptors, CXCR4 / metabolism
Toll-Like Receptor 7 / antagonists & inhibitors*
Toll-Like Receptor 7 / genetics
Toll-Like Receptor 7 / metabolism

Substances

CXCR4 protein, human
Fatty Acids, Monounsaturated
Interferon Regulatory Factor-7
Interferon-alpha
Quaternary Ammonium Compounds
RNA, Small Interfering
Receptors, CXCR4
TLR7 protein, human
Toll-Like Receptor 7
Polyethyleneimine
1,2-dioleoyloxy-3-(trimethylammonium)propane