Understanding the early and late endosomal TLR9-mediated responses to viral DNA.

描述（由申请人提供）：浆细胞样树突状细胞（pDC）是免疫系统3 -8的专职干扰素（IFN）产生细胞，其专门分别通过内体TLR 7和TLR 9识别病毒RNA和DNA。在与MyD 88相关的TLR中，TLR 7和TLR 9严格依赖于MyD 88进行信号转导，并且与UNC 93 B和gp 96相关地存在于内质网（ER）中。早期内体中CpG-A寡脱氧核苷酸（ODN）与TLR 9的接合优先触发TRAF 3/IRAK 1/IKK 1/PI 3 K/IRF 7信号级联，导致1型IFN应答，而晚期内体中CpG-B ODN与TLR 9的接合优先触发TRAF 6/BTK/IRAK 4/TAK 1/IRF 5/NF-kB信号级联，导致促炎细胞因子TNF和IL-6的产生。然而，TLR 9介导的早期内体IFN应答相对于晚期内体促炎细胞因子应答的特化的分子机制尚不清楚。我们发现，PACSIN 1，PACSIN（神经元中的蛋白激酶C和酪蛋白激酶底物）家族的成员，在免疫系统中由pDC特异性表达。我们的初步研究表明，敲低人pDCs中PACSIN 1的表达或敲低小鼠pDCs中Pacsin 1的基因导致早期内体IFN对CpG-A ODN的反应大大降低，而不影响晚期内体促炎细胞因子对CpG-B ODN的反应。我们的中心假设是，差异TLR 9介导的早期和晚期内体对DNA的反应的分子基础是通过使用不同的接头分子来确定的。第一个目标将集中在CpG ODN运输的潜在作用，通过使用具有稳定敲低PACSIN 1表达的shRNA的人pDC细胞系和来自PACSIN缺陷小鼠的小鼠pDC。第二个将集中于通过酵母双杂交筛选和co-IP随后通过质谱鉴定PACSIN 1结合蛋白。第三个目标将集中在进一步表征三个潜在的PACSIN 1相互作用分子，包括CD 2AP，MEKK 4和TRAF 4。

DESCRIPTION (provided by applicant): Plasmacytoid dendritic cells (pDCs) are professional interferon (IFN)-producing cells of the immune system3-8 that are specialized in recognizing viral RNA and DNA via endosomal TLR7 and TLR9, respectively. Among the TLRs that associate with MyD88, TLR7 and TLR9 strictly depend on MyD88 for signal transduction, and reside in the endoplasmic reticulum (ER) in association with UNC93B and gp96. Engagement of TLR9 by CpG-A oligodeoxynucleotide (ODN) in the early endosomes preferentially triggers the TRAF3/IRAK1/IKK1/PI3K/IRF7 signal cascade leading to type 1 IFN responses, whereas engagement of TLR9 by CpG-B ODN in the late endosomes preferentially triggers the TRAF6/BTK/IRAK4/TAK1/IRF5/NF-kB signal cascade leading to the production of proinflammatory cytokines TNF and IL-6. However, the molecular mechanisms underlying the specialization of TLR9-mediated early endosomal IFN responses versus late endosomal proinflammatory cytokine responses are unknown. We discovered that PACSIN1, a member of the PACSIN (protein kinase C and casein kinase substrate in neurons) family, was specifically expressed by pDCs within the immune system. Our preliminary studies showed that knockdown of PACSIN1 expression in human pDCs or knockout of the Pacsin1 gene in mouse pDCs led to a great reduction in the early endosomal IFN response to CpG-A ODN without affecting the late endosomal proinflammatory cytokine response to CpG-B ODN. Our central hypothesis is that the molecular basis for the differential TLR9-mediated early and late endosomal responses to DNA is determined by the use of different adaptor molecules. The first aim will focus on the potential role of in CpG ODN trafficking by using a human pDC cell line with stable knockdown of PACSIN1 expression by shRNA and mouse pDCs derived from PACSIN-deficient mice. The second will focus on identification of PACSIN1-binding proteins by yeast two-hybrid screening and co-IP followed by mass spectrometry. The third aim will focus on further characterization of three potential PACSIN1-interacting molecules including CD2AP, MEKK4 and TRAF4.