Toll-like receptor 7 and MyD88 knockdown by lentivirus-mediated RNA interference to porcine dendritic cell subsets
Toll-like receptor 7 and MyD88 knockdown by lentivirus-mediated RNA interference to porcine dendritic cell subsets
复制标题
DOI：
10.1038/sj.gt.3302930
复制
发表时间：
2007-05-01
期刊：
影响因子：
5.1
通讯作者：
Summerfield, A.
中科院分区：
文献类型：
--
作者：
Alves, M. P.;Neuhaus, V.;Summerfield, A.
树突状细胞（DC）病原体识别受体（PRRs）对病毒的感知是先天抗病毒免疫反应中的一个关键事件。由于在自然靶宿主中进行基因功能研究的困难，这些PRRs的鉴定经常提出一个问题。因此，我们开发了一种基于慢病毒（LV）的策略来敲除猪DC中的特定基因。设计了短发夹rna (Short hairpin rna, shRNAs)，靶向toll样受体7 （toll-like receptor 7, TLR7）和接体蛋白MyD88。作为细胞靶点，采用单核细胞衍生DC （MoDC）和Flt3配体诱导DC (Flt3L-DC)， DC前体包括单核细胞和造血干细胞（hsc）以及浆细胞样DC （pDCs）。转导效率从40%到95%不等。lv介导的shRNA传递具有功能活性，可降低MoDC和常规Flt3L-DC中TLR7和MyD88 mRNA的表达，减弱Flt3L-DC中TLR7配体的响应性。虽然LV感染MoDC不影响MHC II类和CD80/86表达，也不影响细胞因子反应，但Flt3L-DC感染诱导表型成熟。此外，左室与pDC的相互作用诱导了高水平的干扰素- α。综上所述，这些研究表征了LV与不同DC亚群的相互作用，并证明了LV介导的小干扰RNA递送靶向PRR敲除MoDC和传统Flt3L-DC的适用性。
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.