Role of Autophagy in Regulating Cytokine-Induced Macrophage Cell Death and Systemic Inflammatory Responses

项目总结 这项指导临床科学家研究职业发展奖(K08)的目标是提供为期5年的 安东尼·奥维达尔，医学博士，成为一名独立调查员的培训途径。Orvedahl博士获得了 德克萨斯大学西南医学中心医学科学家培训项目的医学博士学位 他研究了自噬在先天抗病毒免疫中的作用。在接受儿科和传染病方面的培训后 在圣路易斯儿童医院/华盛顿大学，他加入了赫伯特·维珍的实验室，医学博士， 是一位著名的抗病毒免疫专家。加里·西尔弗曼，医学博士，将担任主要联合导师， 是一位新生儿学的执业内科科学家，在细胞死亡机制方面拥有公认的专业知识。在他的 通过全基因组CRISPR筛选的初步工作，Orvedahl博士确定了自噬的重要作用 调控干扰素γ诱导细胞死亡的基因(包括ATG5)。ATG5缺陷的抑制子CRISPR屏幕 研究背景表明，肿瘤坏死因子途径是细胞死亡超敏反应的媒介。然而，肿瘤坏死因子是 它本身不足以触发细胞死亡，这表明一个或多个额外的干扰素γ诱导因素起作用 肿瘤坏死因子诱导的死亡。初步工作表明，其中一个因素是干扰素γ诱导的线粒体。 免疫调节基因1(IRG1)。重要的是，携带髓系细胞特异性自噬基因的小鼠- 在肿瘤坏死因子诱导的休克模型中，缺乏表现出明显的存活率下降。但是，具体的类型 干扰素γ联合肿瘤坏死因子诱导细胞死亡的机制及其酶产物 衣康酸，以及这些因子在肿瘤坏死因子诱导休克中的体内作用尚不清楚。建议进行的研究 要在K08中解决这些问题，需要深入分析细胞死亡和线粒体动态平衡 使用多种显微镜、流式细胞术和生化技术。华盛顿大学学院 医学为执行本提案中概述的培训计划提供了理想的环境。美国商务部 儿科在培训独立的内科科学家方面有着长期的承诺和记录。Dr。 Orvedahl与该项目建立了成功的合作关系，并与 儿科和病理学/免疫科的研究人员。已经成立了一个监督委员会 其成员包括免疫新陈代谢、肠道炎症自噬、蛋白质等方面的专家 生物化学和巨噬细胞免疫反应。培训计划还包括技术研讨会。 作为关于格兰特精神的正式课程，这将有助于向独立过渡到 获奖期。导师、机构资源和有重点的培训活动的结合提供了 无与伦比的机会，以实现这项建议的目标。本文所述的近期研究目标 目的：1)探讨自噬对细胞因子诱导的巨噬细胞死亡的保护作用机制； 2)评估将线粒体与细胞死亡机制联系起来的新因子IRG1的作用；以及3)应用 这些发现在体内建立了致命性肿瘤坏死因子诱导休克的模型。

PROJECT SUMMARY The goal of this Mentored Clinical Scientist Research Career Development Award (K08) is to provide a 5-year training pathway for Anthony Orvedahl, MD, PhD, to become an independent investigator. Dr. Orvedahl obtained the MD, PhD degree in the Medical Scientist Training Program at the UT Southwestern Medical Center, where he studied the role of autophagy in innate antiviral immunity. After training in Pediatrics and Infectious Diseases at St. Louis Children's Hospital/ Washington University, he joined the lab of Herbert “Skip” Virgin, MD, PhD, who is a renowned expert in antiviral immunity. Gary Silverman, MD, PhD, who will serve as the primary Co-Mentor, is a practicing physician-scientist in Neonatology with recognized expertise in mechanisms of cell death. In his preliminary work using genome-wide CRISPR screening, Dr. Orvedahl identified an important role for autophagy genes (including Atg5) in regulating IFNγ-induced cell death. A suppressor CRISPR screen on an Atg5-deficient background revealed the TNF pathway as a mediator of the hypersensitivity to cell death. However, TNF was insufficient on its own to trigger cell death, which indicated one or more additional IFNγ-induced factors contribute to TNF-induced death. Preliminary work suggests that one of these factors is the IFNγ-induced mitochondrial enzyme, immune regulated gene 1 (IRG1). Importantly, mice with myeloid cell-specific autophagy gene- deficiency exhibited markedly decreased survival in a TNF-induced model of shock. However, the specific type of cell death induced by IFNγ in combination with TNF, the mechanism of IRG1 and its enzymatic product itaconate, and the in vivo role of these factors during TNF-induced shock remain unclear. The proposed studies to address these questions in this K08 will require in-depth analyses of cell death and mitochondrial homeostasis using multiple microscopic, flow cytometric, and biochemical techniques. Washington University School of Medicine provides an ideal environment to pursue the training plan outlined in this proposal. The Department of Pediatrics has a longstanding commitment and track record of training independent physician-scientists. Dr. Orvedahl has established successful collaborations related to this project, and interacts frequently with investigators in the departments of Pediatrics and Pathology/ Immunology. An oversight committee has been formed that includes advisors with expertise in immunometabolism, autophagy in intestinal inflammation, protein biochemistry, and macrophage immune responses. The training plan incorporates technical workshops, as well as formal coursework on grantsmanship, which will facilitate transition to independence towards the end of the award period. The combination of mentorship, institutional resources, and focused training activities offer an unparalleled opportunity to achieve the goals of this proposal. The immediate research goals described herein are to: 1) investigate the mechanism of autophagy in protection against cytokine-induced macrophage cell death; 2) evaluate the role of a novel factor, IRG1, that links mitochondria to the mechanism of cell death; and 3) apply these findings in vivo to a model of fatal TNF-induced shock.