Role of MLKL in Alcohol-associated Liver Disease

项目概要 候选人是一位年轻的研究者，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。与一个 候选人在肝脏疾病和相关先天免疫方面有很强的背景，已经发展出特殊的能力 使用体内和细胞培养模型进行拟议研究的专业知识。候选人目前的 工作为她提供了发展自己的研究计划并开始过渡的机会 独立性。职业发展计划概述了为期 2 年的技术技能和指导培训 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 成功聘任助理教授职位后的独立科学和职业发展 还概述了。候选人的导师拥有出色的科学生产力和良好的记录 成功的指导，可以为候选人的实验室提供坚实的研究环境。研究 计划：酒精相关性肝病（ALD）仍然是一个主要的社会经济负担，发病率高， 死亡率。先天免疫的激活不仅有助于 ALD 的进展，而且对于解决 ALD 也至关重要 的伤害。促生存和促死亡途径的适当调节对于先天免疫的能力至关重要 细胞在乙醇引起的损伤中快速做出反应以维持肝脏稳态。 MLKL介导的坏死性凋亡 在组织损伤/修复中发挥双重作用。 MLKL 介导的免疫细胞坏死性凋亡是否有益或 对于乙醇的危害尚不清楚。前期工作中，骨髓细胞 Mlkl 缺乏加剧 乙醇引起的损伤，与乙醇引起的肝巨噬细胞积累增加有关 中性粒细胞。重要的是，我们发现高暴饮会增加肝 F4/80 细胞的坏死/坏死性凋亡， 表明 MLKL 可能通过促进巨噬细胞死亡和消退来防止乙醇引起的损伤 炎症。总之，这些数据使我们推测骨髓细胞特异性 MLKL 限制乙醇- 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 通过对 Mlkl BM 肝脏中分离的 NPC 进行流式细胞术分析来表征种群数量和死亡情况 高狂欢之后的嵌合体。然后我们将挑战细胞特异性敲除（LysM、Clec4f 和 MRP8 CRE 交叉 与 Mlklfl/fl) 到乙醇，以区分 MLKL 对 mALD 的细胞特异性贡献。此外，磷酸化蛋白质组学 与靶向机制方法相结合将用于确定乙醇介导的激活是否 MLKL 是 RIP3 依赖的和/或 RIP3 独立的。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，该项目将进一步探讨中性粒细胞特异性 MLKL 是否存在限制 乙醇通过调节 NET 引起的损伤。总而言之，该提案将为未来的发展奠定坚实的基础。 乙醇引起的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.