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

描述（由申请人提供）：在本提案中，我们将确定人类癌细胞中病毒感染的关键先天免疫反应途径是如何改变的，特别是通过比较原发性先天免疫前哨髓细胞与其转化的对应物。我们将通过炎性小体和NF-κB信号，研究病毒感知途径的机制作用及其与下游先天免疫反应的联系，以确定黏液瘤病毒（MYXV）的趋向性和感知，MYXV是一种兔特异性痘病毒，也表现出感染多种人类癌细胞的能力。我们将研究人髓细胞，特别是单核细胞来源的巨噬细胞和树突状细胞对MYXV的感知和感染。我们将研究含有myxv编码pyrin结构域（PYD）的宿主范围蛋白M013共同调节人类髓细胞两种不同的先天免疫途径的分子机制，特别是炎症小体和NF-κ b介导的炎症信号反应。我们报道了M013结合多种炎性小体复合物的细胞接头蛋白ASC-1（含有CARD的凋亡相关斑点样蛋白）和NF-κB信号通路的NF-κB1/p105。研究这种独特的协同调节将对开发具有靶向多种炎症信号通路能力的药物具有潜在的临床应用价值。最后，我们将利用我们在炎性体和NF-κB通路调控方面的知识，利用髓性白血病模型研究MYXV溶瘤的机制。利用敲除小鼠的关键先天免疫通路和M013位点突变的MYXV构建，我们将研究这些通路在MYXV病毒治疗骨髓癌中的作用。本建议研究的三个重叠领域是：目标1。研究由于人髓细胞转化引起的传感通路的改变及其对MYXV趋向性的影响：我们将确定RLR和TLR信号在诱导I型IFN和促炎细胞因子在允许MYXV的人髓细胞（如THP-1转化骨髓细胞系）或不允许的人髓细胞（如原代人单核/巨噬细胞）中的作用；确定NLRP3炎性小体成分和NF-κB1在MYXV感染反应中前体IL-1ß和IL-18的合成和这些成熟细胞因子的释放中的作用；研究病毒诱导的MAPK的协同上游作用，特别是MEK1/2-ERK1/2激酶的作用，在MYXV感染反应中共同诱导I型IFN和促炎细胞因子信号通路；并明确哪些细胞传感器/传感器组件在转化的人类髓细胞中功能失调。目标2。创建含有pyd的蛋白M013的工程变体，其差异调节炎症小体和NF-κ b信号通路，并在体内caspase 1依赖的大鼠移植血管疾病模型中测试这些M013构建物：我们将生成M013突变体，通过仅与ASC-1或NF-κ b1相互作用选择性抑制炎症小体或NF-κ b通路，并利用这些突变体构建重组MYXVs，以测试其在上述髓细胞中的作用；我们还将测试M013变异，使其能够在大鼠同种异体移植血管疾病（TVD）模型中细胞内渗透，该模型已被证明是由炎症小体介导的caspase 1激活调节的。目标3。阐明宿主炎性小体和NF-κB信号在myxv诱导的髓细胞癌原位溶瘤清除中的作用：我们将比较关键炎性小体和

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.