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

Myxom 操纵人骨髓细胞中的炎症小体和 NF-kB 信号传导

• 批准号: 8501735
• 负责人: Grant McFadden
• 金额: $ 37.25万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8501735
• 关键词: Adaptor Signaling Protein; Address; Allografting; Apoptosis; Area; Binding; Carcinoma in Situ; Caspase-1; Cell Line; Cells; Clinical; Complex; Cytokine Signaling; Dendritic Cells; Disease; Disease model; Engineering; European; Exhibits; Family; Family member; Human; Immune; Immune response; Immunocompetent; Immunocompromised Host; In Situ; Individual; Infection; Inflammatory; Interferons; Interleukin-18; Investigation; Knockout Mice; Knowledge; Laboratories; Lagomorpha; MAP Kinase Gene; MAP2K1 gene; MAPK3 gene; Malignant Neoplasms; Mediating; Modeling; Molecular; Multiple Myeloma; Mus; Mutate; Myelogenous; Myeloid Cells; Myeloid Leukemia; Myxoma virus; NF-kappa B; Nature; Oncolytic; Oncolytic viruses; Oryctolagus cuniculus; Pathway interactions; Penetration; Pharmaceutical Preparations; Phosphotransferases; Play; Poxviridae; Process; Protein Binding; Proteins; Rattus; Recombinants; Regulation; Reporting; Role; Sentinel; Signal Pathway; Signal Transduction; TNF gene; Testing; Therapeutic; Transducers; Transplantation; Transplanted tissue; Tropism; Variant; Vascular Diseases; Viral; Viral Proteins; Virus; Virus Diseases; Xenograft Model; base; cancer cell; cell type; cellular engineering; cytokine; in vivo; macrophage; marenostrin; monocyte; mouse model; mutant; novel therapeutics; oncolysis; public health relevance; receptor; response; sensor; tool; tumor; virus tropism

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-?B 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-?B -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-?B1/p105 of NF-?B 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-?B 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-?B1 in the synthesis of precursor IL-1¿ 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-?B 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-?B pathways by interaction with only ASC-1 or NF-?B1 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-?B signaling in MYXV-induced oncolytic clearance of myeloid cancer in situ: We will compare the roles of key inflammasome and NF-?B components (ie ASC-1, caspase-1, NLRP3 and NF-?B1) 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-?B pathways.

描述（由申请人提供）：在本提案中，我们将确定人类癌细胞中针对病毒感染的关键先天免疫反应途径如何改变，特别是通过将原代先天免疫前哨骨髓细胞与其转化的对应细胞进行比较，我们将研究其机制作用。病毒传感途径及其通过炎症小体和 NF-κB 信号传导与下游先天免疫反应的联系，以确定粘液瘤病毒 (MYXV) 的向性和传感，粘液瘤病毒是一种兔特异性痘病毒，也我们将研究 MYXV 感知和感染人类骨髓细胞，特别是单核细胞源性巨噬细胞和树突状细胞的能力。我们将研究 MYXV 编码的热蛋白结构域 (PYD) 使用的分子机制。 -含有宿主范围蛋白M013，共同调节人骨髓细胞的两种不同的先天免疫途径，特别是由炎症体和炎症小体介导的NF-κB 介导的炎症信号反应，我们报道了 M013 结合各种炎症小体复合物的细胞衔接蛋白 ASC-1（含有 CARD 的凋亡相关斑点样蛋白）和 NF-κB 的 NF-κB1/p105。 B 信号通路的研究对于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实用性。炎症小体和 NF-κB 途径，使用髓系白血病模型研究 MYXV 的溶瘤机制。使用关键先天免疫途径的敲除小鼠和在 M013 位点构建突变的 MYXV，我们将研究这些途径的作用。该提案要研究的三个重叠领域是： 目标 1. 研究人类转化引起的传感途径的改变。骨髓细胞及其对 MYXV 趋向性的影响：我们将确定 RLR 和 TLR 信号传导在诱导 MYXV 允许的人类骨髓细胞（例如 THP-1 等转化的骨髓细胞系）或促炎细胞因子中诱导 I 型 IFN 和促炎细胞因子的作用。不允许（例如原代人单核细胞/巨噬细胞）；确定NLRP3炎性体成分和NF-κB1在前体合成中的作用IL-1??和 IL-18 以及这些成熟细胞因子响应 MYXV 感染而释放；研究病毒诱导的 MAPK 的上游协调作用，特别是 MEK1/2-ERK1/2 激酶在共同诱导这两种类型中的作用； I 响应 MYXV 感染的 IFN 和促炎细胞因子信号传导通路；并明确哪些细胞传感器/转导元件在转化的人类骨髓细胞中变得功能障碍。含有 PYD 的蛋白 M013 差异调节炎症小体和 NF-κB 信号通路，并在体内 caspase 1 依赖性大鼠移植血管疾病模型中测试这些 M013 构建体：我们将生成选择性抑制炎症小体或 NF-κB 信号通路的 M013 突变体。 ?B 途径仅与 ASC-1 或 NF-?B1 相互作用，并利用这些突变体构建重组 MYXV 以测试其效果在上述骨髓细胞中；我们还将测试 M013 变体，该变体被设计为允许在大鼠同种异体移植血管疾病 (TVD) 模型中进行细胞内渗透，该模型已被证明受到炎症小体介导的 caspase 1 激活的调节。宿主炎症体和 NF-κB 信号传导在 MYXV 诱导的原位骨髓癌溶瘤清除中的作用：我们将比较关键的作用MYXV 诱导的人（例如 THP-1）或鼠（例如 RAW426.7）的抗肿瘤反应和清除中的炎性体和 NF-κB 成分（即 ASC-1、caspase-1、NLRP3 和 NF-κB1）分别在 NSG 免疫功能低下小鼠和基于 C57BL/6 的免疫功能正常小鼠模型中研究 M013 变异在选择性中的作用；使用 M013KO 与野生型 MYXV 以及仅选择性抑制炎性体或 NF-κB 通路的表达 M013 变体的病毒进行敲除小鼠。