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（pDCs）是免疫系统的专业干扰素（IFN）产生细胞3-8，专门通过内体TLR7和TLR9识别病毒RNA和DNA。在与MyD88相关的tlr中，TLR7和TLR9严格依赖于MyD88进行信号转导，并与UNC93B和gp96联合存在于内质网（ER）中。早期核内体中CpG-A寡核苷酸（ODN）参与TLR9优先触发TRAF3/IRAK1/IKK1/PI3K/IRF7信号级联，导致1型IFN应答，而晚期核内体中CpG-B ODN参与TLR9优先触发TRAF6/BTK/IRAK4/TAK1/IRF5/NF-kB信号级联，导致促炎细胞因子TNF和IL-6的产生。然而，tlr9介导的早期内体IFN反应与晚期内体促炎细胞因子反应特异性的分子机制尚不清楚。我们发现PACSIN1是PACSIN（神经元中蛋白激酶C和酪蛋白激酶底物）家族的一员，在免疫系统中被pDCs特异性表达。我们的初步研究表明，敲低人pDCs中的PACSIN1表达或敲除小鼠pDCs中的PACSIN1基因，可导致早期内体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，tlr9介导的早期和晚期内体对DNA的不同反应的分子基础是由使用不同的接头分子决定的。第一个目标是通过shRNA稳定地敲低PACSIN1表达的人pDC细胞系和来自PACSIN1缺陷小鼠的小鼠pDC细胞系，研究其在CpG ODN运输中的潜在作用。第二部分将重点通过酵母双杂交筛选和协同ip结合质谱法鉴定pacsin1结合蛋白。第三个目标将集中在进一步表征三个潜在的pacsin1相互作用分子，包括CD2AP， MEKK4和TRAF4。

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.