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

描述(申请人提供)：浆细胞样树突状细胞(PDCs)是免疫系统3-8中产生干扰素的专业细胞，它们分别通过内体TLR7和TLR9识别病毒RNA和DNA。在与MyD88相关的TLRs中，TLR7和TLR9严格依赖于MyD88进行信号转导，并与uc93B和gp96共同存在于内质网(ER)。CpG-A寡脱氧核苷酸(ODN)与TLR9结合优先触发TRAF3/IRAK1/IKK1/PI3K/IRF7信号级联反应，导致1型干扰素应答；而CpG-B ODN结合TLR9则优先触发TRAF6/BTK/IRAK4/TAK1/IRF5/NF-kB信号级联反应，导致促炎细胞因子TNF和IL-6的产生。然而，TLR9介导的早期内体干扰素反应与晚期内体促炎细胞因子反应特异化的分子机制尚不清楚。我们发现PACSIN1是PACSIN(蛋白激酶C和酪蛋白激酶底物在神经元中)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，PACSIN1在人pDC中的表达下调或Pacsin1基因在小鼠pDC中的敲除导致早期内体干扰素对CpG-A ODN的应答显著降低，而不影响晚期内体促炎细胞因子对CpG-B ODN的应答。我们的中心假设是，TLR9介导的对DNA的不同早期和晚期内体反应的分子基础是由不同的接头分子决定的。第一个目标将集中在CpG ODN运输中的潜在作用，通过使用稳定下调PACSIN1表达的人PDC细胞系和来自PACSIN缺陷小鼠的小鼠pDC。第二个重点是通过酵母双杂交筛选和共IP-MS鉴定PACSIN1结合蛋白。第三个目标将集中在三个潜在的PACSIN1相互作用分子的进一步表征上，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.