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Toll-like receptor 7 and MyD88 knockdown by lentivirus-mediated RNA interference to porcine dendritic cell subsets

Gene Therapy volume 14, pages 836–844 (2007)Cite this article

Abstract

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-α. 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.

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Acknowledgements

This work was supported by the Swiss Federal Office for Education and Science (Project 3100-068237/1). We thank Peter Zulliger and Daniel Brechbühl for care of the animals and their regular bleeding.

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  1. Institute of Virology and Immunoprophylaxis, Mittelhäusern, Switzerland

    M P Alves, V Neuhaus, L Guzylack-Piriou, N Ruggli, K C McCullough & A Summerfield

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  1. M P Alves
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  2. V Neuhaus
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  3. L Guzylack-Piriou
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Correspondence to A Summerfield.

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Alves, M., Neuhaus, V., Guzylack-Piriou, L. et al. Toll-like receptor 7 and MyD88 knockdown by lentivirus-mediated RNA interference to porcine dendritic cell subsets. Gene Ther 14, 836–844 (2007). https://doi.org/10.1038/sj.gt.3302930

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