Toll-like receptor 7 and MyD88 knockdown by lentivirus-mediated RNA interference to porcine dendritic cell subsets.
Sensing of viruses by dendritic cell (DC) pathogen recognition receptors (PRRs) represents a critical event during innate antiviral immune responses. Identification of these PRRs has often posed a problem due to difficulties in performing gene function studies in the naturally targeted hosts. Consequently, we developed a lentivirus (LV)-based strategy for specific gene knockdown in porcine DC. Short hairpin RNAs (shRNAs) were designed, targeting toll-like receptor 7 (TLR7) and the adaptor protein MyD88. As cellular targets, monocyte-derived DC (MoDC) and Flt3 ligand-induced DC (Flt3L-DC), DC precursors including monocytes and haematopoietic stem cells (HSCs) as well as plasmacytoid DCs (pDCs) were employed. Transduction efficiencies ranged from 40 to 95%. The LV-mediated shRNA delivery was functionally active, reducing TLR7 and MyD88 mRNA in MoDC and conventional Flt3L-DC, and blunting the responsiveness to TLR7 ligands in Flt3L-DC. Although infection of MoDC by the LV did neither influence MHC class II and CD80/86 expressions, nor cytokine responses, the infection of Flt3L-DC induced a phenotypic maturation. Furthermore, the interaction of the LV with pDC induced high levels of interferon-alpha. Taken together, these studies characterize the interaction of the LV with different DC subsets and demonstrate the suitability of LV-mediated small interfering RNA delivery for targeting PRR knockout for MoDC and conventional Flt3L-DC.
Alves
M P
MP
Institute of Virology and Immunoprophylaxis, Mittelhäusern, Switzerland.
Neuhaus
V
V
Guzylack-Piriou
L
L
Ruggli
N
N
McCullough
K C
KC
Summerfield
A
A
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D008565:Membrane Proteins / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D051199:Toll-Like Receptor 7 / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods; D014162:Transfection / Q000379:methods
D008565:Membrane Proteins; D053594:Myeloid Differentiation Factor 88; D034741:RNA, Small Interfering; C495348:TLR7 protein, human; D051199:Toll-Like Receptor 7; C084881:flt3 ligand protein