Role of MLKL in Alcohol-associated Liver Disease

项目总结 这位候选人是一名年轻的调查员，致力于发展专注于理解的学术生涯 乙醇破坏免疫细胞生存和死亡途径之间平衡的机制。使用一个 在肝脏疾病和相关的先天免疫方面有很强的背景，应聘者有特别的 在使用体内和细胞培养模型进行拟议研究方面的专业知识。候选人目前的情况 工作为她提供了制定自己的研究计划并开始过渡到 独立。职业发展计划概述了为期两年的技术技能指导培训和 旨在促进成功过渡到独立的职业发展活动。为期3年的课程 在成功招聘助理教授职位后，独立的科学和职业发展 并对其进行了概述。候选人的导师拥有卓越的科学生产力和良好的业绩记录 成功的指导，并可以为候选人提供一个坚实的研究环境在她的实验室。研究 计划：酒精性肝病(ALD)仍然是一个主要的社会经济负担，发病率和 死亡率。先天免疫的激活不仅有助于ALD的进展，而且对解决ALD也是至关重要的 受伤的原因。对促生存和促死亡通路的适当调节对先天免疫的能力至关重要。 在乙醇诱导的损伤中，细胞快速反应以维持肝脏的动态平衡。MLKL介导的坏死性下垂 在组织损伤/修复中起双重作用。MLKL介导的免疫细胞坏死性下垂是有益的还是 在乙醇的背景下是有害的是未知的。在初步工作中，髓系细胞中MLKL缺乏加剧了 乙醇诱导的损伤，与乙醇诱导的肝巨噬细胞和 中性粒细胞。重要的是，我们发现高脂增加了肝脏F4/80细胞的坏死/坏死性下垂。 提示MLKL可能通过促进巨噬细胞死亡和分解来保护乙醇所致的损伤。 发炎的症状。综上所述，这些数据让我们假设髓系细胞特异性的MLKL限制了乙醇- 通过调节肝脏免疫细胞的存活和死亡来诱导损伤。为了验证这一假设，肝脏免疫细胞 从MLKL BM肝脏分离的NPC的流式细胞术分析将表征种群和死亡 高狂欢之后的嵌合体。然后，我们将挑战特定于细胞的敲除(LysM、Clec4f和MRP8 Cre交叉 与MLKL1/f1)结合，以区分MLKL对MALD的细胞特异性贡献。此外，磷酸蛋白质组学 结合有针对性的机械方法将被用来确定乙醇介导的激活 是RIP3依赖和/或RIP3非依赖的。此外，由于中性粒细胞和中性粒细胞胞外 陷阱(Net)在ALD中很重要，该项目将进一步探索中性粒细胞特异性MLKL限制 乙醇通过调节Net诱导损伤。综上所述，这项建议将为未来奠定坚实的基础 酒精性损伤和炎症的机制研究和临床干预。

Project summary The Candidate is a young investigator dedicated to developing an academic career focused on understanding the mechanisms by which ethanol disrupts balance between immune cell survival and death pathways. With a strong background in liver diseases and related innate immunity, the candidate has developed particular expertise in the use of in vivo and cell culture models to conduct the proposed studies. The Candidate’s current work has provided her with the opportunity to develop her own research program and begin her transition to independence. The Career Development Plan outlines 2-years of mentored training on technical skills and career development activities designed to promote the successful transition to independence. A 3-year program of independent scientific and career development after successful recruitment as an Assistant Professor position is also outlined. The Candidate’s Mentor has a proven track-record of excellent scientific productivity and successful mentorship and can provide the Candidate with a solid research environment in her lab. Research plan: Alcohol-associated liver disease (ALD) remains a major socioeconomic burden with high morbidity and mortality. Activation of innate immunity not only contributes to progression of ALD, but is also critical for resolution of injury. Appropriate regulation of pro-survival and pro-death pathways is critical to the ability of innate immune cells to rapidly respond to maintain liver homeostasis in ethanol-induced injury. MLKL-mediated necroptosis plays a dual role in tissue injury/repair. Whether MLKL-mediated necroptosis of immune cells is beneficial or detrimental in the context of ethanol is unknown. In preliminary work, Mlkl deficiency in myeloid cells exacerbated ethanol-induced injury, associated with increased ethanol-induced accumulation of hepatic macrophages and neutrophils. Importantly, we found that Gao-binge increased necrosis/necroptosis of hepatic F4/80+ cells, suggesting that MLKL may protect from ethanol-induced injury by promoting macrophage death and resolution of inflammation. Together, these data led us to hypothesize that myeloid cell-specific MLKL restricts ethanol- induced injury by regulating hepatic immune cell survival and death. To test this hypothesis, hepatic immune cell populations and death will be characterized by flow cytometric analysis of isolated NPCs from livers of Mlkl BM chimeras after Gao-binge. We will then challenge cell-specific knock-outs (LysM, Clec4f and MRP8 CRE crossed with Mlklfl/fl) to ethanol, to distinguish cell-specific contributions of MLKL to mALD. Further, a phospho-proteomics paired with targeted mechanistic approaches will be utilized to determine whether ethanol-mediated activation of MLKL is RIP3-dependent and/or RIP3-independent. Additionally, since neutrophils and neutrophil extracellular traps (NETs) are important in ALD, the project will further explore whether neutrophil-specific MLKL limits ethanol-induced injury by regulating NETs. Altogether, this proposal will provide a solid foundation for future mechanistic studies and clinical interventions for ethanol-induced injury and inflammation.