Manipulation of inflammasomes and NF-kB signaling in human myeloid cells by Myxom

描述(申请人提供)：在这项提案中，我们将确定病毒感染的关键先天免疫反应途径是如何在人类癌细胞中改变的，特别是通过比较原代先天免疫哨兵髓系细胞和它们转化的同类细胞。我们将研究病毒感知途径的机制作用及其与下游天然免疫反应的联系，通过炎性小体和NF-κB信号来确定粘液瘤病毒(MYXV)的嗜性和感知。粘液瘤病毒是一种兔特异性痘病毒，也显示出感染广泛范围的人类癌细胞的能力。我们将研究MYXV对人类髓系细胞的感知和感染，特别是单核细胞来源的巨噬细胞和树突状细胞。我们将研究MYXV编码的比林结构域(PYD)宿主范围蛋白M013共同调节人类髓系细胞两条不同的天然免疫途径的分子机制，特别是通过炎症体和NF-κB介导的炎症信号反应。我们已经报道，M013结合了多种炎症体复合体的细胞适配器蛋白Asc-1(含一张卡片的凋亡相关斑点样蛋白)和NF-κB信号通路中的NF-κB1/p105。对这种独特的协同调节的研究将对开发具有针对多个炎症信号通路的能力的药物具有潜在的临床实用价值。最后，我们将利用我们对炎症体和NF-κB通路的调控知识，利用髓系白血病模型来研究MYXV的溶瘤机制(S)。利用MYXV基因敲除小鼠作为关键的天然免疫通路，构建MYXV基因突变的M013位点，我们将研究这些通路在MYXV病毒治疗髓系癌中的作用。目的1.研究人髓系细胞转化后感觉通路的改变及其对MYXV趋向性的影响：我们将确定RLR和TLR信号在诱导MYXV允许的人髓系细胞(如THP-1转化的髓系细胞)或不允许的人髓系细胞(如原代人单核/巨噬细胞)中诱导I型干扰素和促炎细胞因子的作用；确定NLRP3炎症体成分和NF-κB1在MYXV感染后合成前体IL-1β和IL-18以及释放这些成熟细胞因子中的作用；研究病毒诱导的MAPK的上游协调作用，特别是MEK1/2-ERK1/2激酶在共同诱导I型干扰素和促炎细胞因子信号通路方面的作用，以响应MYXV感染；并具体识别在转化的人髓系细胞中哪些细胞传感器/转导组件功能失调。目的：建立含有PYD的M013蛋白的工程变体，对炎症体和NF-κB信号通路进行差异调控，并在依赖caspase1的大鼠移植血管疾病体内模型中测试这些M013结构：我们将产生M013突变体，它们只与Asc-1或NF-κB1相互作用，选择性地抑制炎症体或NF-κB通路，并利用这些突变体构建重组MYXV，以测试其在上述髓系细胞中的作用；我们还将在大鼠同种异体移植血管疾病模型中测试M013变异体，该模型已被证明受炎症体介导的Caspase1的激活调节。目的3.阐明宿主炎症体和NF-κB信号在MyXV诱导的原位髓系癌溶瘤清除中的作用：我们将比较关键的炎症体和 分别在NSG免疫低下小鼠和C57BL/6免疫活性小鼠模型中，研究MYXV诱导的抗肿瘤反应和人(例如THP-1)或小鼠(例如RAW426.7)髓系白血病的清除作用；利用M013KO vs野生型MYXV，研究M013变体在选择性基因敲除小鼠中的作用；利用M013KO vs野生型MYXV，研究M013变体在选择性抑制炎症或NF-κB通路中的作用。

DESCRIPTION (provided by applicant): In this proposal, we will determine how key innate immune response pathways to viral infection are altered in human cancer cells, particularly by comparing primary innate immune sentinel myeloid cells to their transformed counterparts. We will investigate the mechanistic roles of virus sensing pathways and their linkage to downstream innate immune responses via inflammasomes and NF-κB signaling in determining the tropism and sensing of myxoma virus (MYXV), a rabbit specific poxvirus that also exhibits the capacity to infect a wide spectrum of human cancer cells. We will investigate MYXV sensing and infection of human myeloid cells, especially monocyte-derived macrophages and dendritic cells. We will examine the molecular mechanisms used by MYXV-encoded pyrin domain (PYD)-containing host range protein M013 to co-regulate two distinct innate immune pathways of human myeloid cells, particularly as mediated by inflammasomes and by the NF-κB-mediated inflammatory signaling responses. We have reported that M013 binds the cellular adaptor protein ASC-1 (apoptosis-associated speck-like protein containing a CARD) of various inflammasome complexes and NF-κB1/p105 of NF-κB signaling pathway. Investigation of this unique co-regulation will have potential clinical utility for developing drugs having the capacity o target multiple inflammatory signaling pathways. Finally, we will take advantage of our knowledge of regulation of the inflammasome and NF-κB pathways to study the mechanism(s) of oncolysis by MYXV using myeloid leukemia models. Using knock-out mice for key innate immune pathways and MYXV constructed mutated at the M013 locus, we will study the role of these pathways in MYXV virotherapy for myeloid cancer. The three overlapping areas to be investigated in this proposal are: Aim 1. Study the alteration of sensing pathways due to transformation of human myeloid cells and its impact on MYXV tropism: We will determine the role of RLR and TLR signaling in inducing type I IFN and pro-inflammatory cytokines in human myeloid cells that are permissive for MYXV (eg transformed myeloid cell lines like THP-1) or nonpermissive (eg primary human monocytes/macrophages); determine the role of NLRP3 inflammasome components and NF-κB1 in the synthesis of precursor IL-1ß and IL-18 and release of these mature cytokines in response to MYXV infection; study the co-ordinating upstream role of virus-induced MAPK, in particular the role of MEK1/2-ERK1/2 kinases, in co-inducing both the type I IFN and pro-inflammatory cytokine signaling pathways in response to MYXV infection; and identify specifically which cellular sensor/transducer components become dysfunctional in transformed human myeloid cells. Aim 2. Create engineered variants of the PYD-containing protein M013 that differentially regulate the inflammasome and NF-κB signaling pathways and test these M013 constructs in an in vivo caspase 1- dependent rat transplant vascular disease model: We will generate M013 mutants that selectively inhibit either the inflammasome or NF-κB pathways by interaction with only ASC-1 or NF-κB1 and exploit these mutants to construct recombinant MYXVs to test their effects in the myeloid cells described above; we will also test M013 variants engineered to allow intracellular penetration in a rat allograft transplant vascular disease (TVD) model that has been shown to be regulated by inflammasome-mediated activation of caspase 1. Aim 3. Elucidate the roles of host inflammasomes and NF-κB signaling in MYXV-induced oncolytic clearance of myeloid cancer in situ: We will compare the roles of key inflammasome and NF-κB components (ie ASC-1, caspase-1, NLRP3 and NF-κB1) in MYXV-induced anti-tumor responses and clearance of human (eg THP-1) or murine (eg RAW426.7) myeloid leukemias in NSG immunocompromised mice and in C57BL/6-based immunocompetent mouse models, respectively; study the role of M013 variants in selective knockout mice using the M013KO vs wild-type MYXV, and the M013 variant-expressing viruses that selectively inhibit only the inflammasome or NF-κB pathways.