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

项目摘要 这个指导临床科学家研究职业发展奖（K 08）的目标是提供一个为期5年的 Anthony Orvedahl，MD，PhD的培训途径，成为独立研究者。奥维达尔博士获得了 UT西南医学中心医学科学家培训计划的MD，PhD学位，其中 他研究了自噬在先天性抗病毒免疫中的作用。经过儿科和传染病培训后 在圣路易斯儿童医院/华盛顿大学，他加入了赫伯特“跳过”维珍，医学博士，博士，谁的实验室 是著名的抗病毒免疫专家加里西尔弗曼，医学博士，博士，谁将担任主要的共同导师， 是一个执业医师，科学家在新生儿与公认的专业知识，在细胞死亡的机制。在他 Orvedahl博士利用全基因组CRISPR筛选的初步工作，确定了自噬的重要作用 基因（包括Atg 5）在调节IFNγ诱导的细胞死亡中的作用。在Atg 5缺陷型细胞上的抑制CRISPR筛选 背景揭示了TNF途径作为对细胞死亡的超敏反应的介质。然而，TNF 其本身不足以触发细胞死亡，这表明一种或多种额外的IFNγ诱导因子起作用 TNF诱导的死亡初步研究表明，这些因素之一是IFNγ诱导的线粒体 免疫调节基因1（ImmuneRegulatedGene 1，IRG 1）重要的是，具有骨髓细胞特异性自噬基因的小鼠- 在TNF诱导的休克模型中，缺乏TNF的小鼠表现出明显降低的存活率。然而，具体类型 IFNγ联合TNF诱导细胞死亡的机制、IRG 1及其酶促产物的作用机制 衣康酸，和在TNF诱导的休克过程中，这些因素在体内的作用仍然不清楚。拟议的研究 要解决K 08中的这些问题，需要对细胞死亡和线粒体稳态进行深入分析 使用多种显微镜、流式细胞术和生物化学技术。华盛顿大学 医学提供了一个理想的环境，以追求本提案中概述的培训计划。部 儿科长期以来一直致力于培养独立的医生科学家。博士 Orvedahl已经建立了与该项目相关的成功合作，并经常与 儿科和病理学/免疫学部门的研究人员。一个监督委员会 形成，包括具有免疫代谢，肠道炎症自噬，蛋白质 生物化学和巨噬细胞免疫反应。培训计划还包括技术讲习班， 作为正式的granitarian课程，这将有助于在2010年年底向独立过渡。 奖励期。导师制、机构资源和重点培训活动相结合， 这是实现这一目标的最佳机会。本文所述的近期研究目标 目的：1）探讨自噬对苦参碱诱导的巨噬细胞死亡的保护作用机制; 2)评估一种新的因子IRG 1的作用，它将线粒体与细胞死亡机制联系起来; 3）应用 这些发现在体内致死性TNF诱导的休克模型。

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.