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，我们将确定病毒感染的关键先天免疫应答途径在人类癌细胞中是如何改变的，特别是通过将原发性先天免疫哨兵骨髓细胞与其转化的对应细胞进行比较。我们将研究病毒感应途径的机制作用及其通过炎性小体和NF-κB信号传导与下游先天免疫应答的联系，以确定粘液瘤病毒（MYXV）的嗜性和感应，MYXV是一种兔特异性痘病毒，也具有感染广谱人类癌细胞的能力。我们将研究MYXV传感和人类骨髓细胞，特别是单核细胞衍生的巨噬细胞和树突状细胞的感染。我们将研究MYXV编码的含有Pyrin结构域（PYD）的宿主范围蛋白M013共同调节人类骨髓细胞两种不同的先天免疫途径的分子机制，特别是由炎性小体和NF-κ B介导的炎症信号应答介导的机制。我们已经报道了M013结合多种炎性小体复合物的细胞衔接蛋白ASC-1和NF-κB信号通路的NF-κB1/p105。对这种独特的共调节的研究对于开发具有靶向多种炎症信号通路的能力的药物具有潜在的临床实用性。最后，我们将利用我们对炎症体和NF-κB通路调节的知识，使用髓系白血病模型研究MYXV的溶瘤机制。使用敲除小鼠的关键先天免疫途径和MYXV构建突变的M013位点，我们将研究这些途径在MYXV病毒治疗骨髓癌的作用。本提案中要调查的三个重叠领域是：目标1。研究由于人髓系细胞转化引起的传感途径的改变及其对MYXV嗜性的影响：我们将确定RLR和TLR信号传导在诱导人髓系细胞中I型IFN和促炎细胞因子中的作用，这些细胞允许MYXV（例如转化的骨髓细胞系，如THP-1）或非允许的（例如，原代人单核细胞/巨噬细胞）;确定NLRP 3炎性体组分和NF-κB1在前体IL-1 β和IL-18的合成以及这些成熟细胞因子响应于MYXV感染的释放中的作用;研究病毒诱导的MAPK的协调上游作用，特别是MEK 1/2-ERK 1/2激酶在响应MYXV感染共诱导I型IFN和促炎细胞因子信号传导途径中的作用;并具体鉴定哪些细胞传感器/换能器组件在转化的人骨髓细胞中变得功能障碍。目标2.创建差异调节炎性体和NF-κB信号通路的含PYD蛋白M013的工程化变体，并在体内半胱天冬酶1依赖性大鼠移植血管疾病模型中测试这些M013构建体：我们将产生M013突变体，其通过仅与ASC-1或NF-κB相互作用而选择性地抑制炎性体或NF-κB途径。κB1，并利用这些突变体构建重组MYXVs，以测试它们在上述骨髓细胞中的作用;我们还将在大鼠同种异体移植血管疾病（TVD）模型中测试经工程改造以允许细胞内渗透的M013变体，所述模型已显示受炎性小体调节，介导的半胱天冬酶1的活化。目标3。阐明宿主炎性小体和NF-κB信号在MYXV诱导的髓样癌原位溶瘤清除中的作用：我们将比较关键炎性小体和NF-κB信号在MYXV诱导的髓样癌原位溶瘤清除中的作用。 NF-κB组分（即ASC-1、caspase-1、NLRP 3和NF-κB1）在MYXV诱导的抗肿瘤反应和人肿瘤细胞清除中的作用（例如THP-1）或鼠（例如RAW426.7）分别在NSG免疫受损小鼠和基于C57 BL/6的免疫活性小鼠模型中的骨髓性白血病;使用M013 KO与野生型MYXV，以及仅选择性抑制炎性小体或NF-κB途径的表达M013变体的病毒，研究M013变体在选择性敲除小鼠中的作用。

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.