C-Type Lectins and Immune Surveillance in ALD

项目总结 候选人是一名博士后研究员，致力于发展一个专注于 应用生物信息学了解酒精相关性肝病的病理生理学。他的前任 目前的博士后工作给了他回答涉及基因的问题的独特技能 在免疫系统中的表达/调节。职业发展计划描述了两年的指导 候选人将发展临床免疫学技能的研究，产生概述的测序数据 在提案中，学习领导技能，过渡到独立。在接下来的3年里，在获得了 独立教员职位，将致力于开发新的数据分析管道和建立细胞 生物和生物信息学工具，以了解先天免疫系统中的基因调节，这将使 未来建立ALD免疫学项目的候选人。研究计划：ALD是一种疾病谱系 这影响了全球越来越多的人。酒精性肝炎是一种严重的炎症性疾病， 会增加ALD疾病的发病率和严重程度。在急性胰腺炎中，先天免疫系统对 微生物副产品。饮酒导致肠道屏障破坏，导致肠道微生物渗入 门脉循环。肝脏免疫系统能够通过模式识别检测到这些微生物 受体，包括Toll样受体(TLRs)和C型凝集素受体(CTL)。而TLR一直是 CTL在提高天然免疫细胞对微生物产物的敏感度方面的作用得到了充分的研究，直到最近 与ALD的小鼠模型有关。CTL是PRR的一个大家族，它可以感知各种不同的 微生物，包括细菌、真菌和病毒。我们的工作发现，CTL基因家族的许多成员 在AH患者的肝脏和外周血单个核细胞中表达上调。CTL也是 外周血单核细胞对内毒素/TLR4信号的反应上调。我们预测CTL会因肠道反应而上调- 以增加对血液中可能存在的其他微生物的感知，使这一点 第二条先天免疫监视途径。在本提案中，我们将在三个阶段测试这一假设 旨在阐明这一免疫监视途径的功能作用和调节，以及通过 这在急性呼吸综合征中加剧了。首先，我们将使用从AH患者分离的PBMCs来测量增加的 对CTL激动剂的敏感性以及微生物/真菌生物群测序以确定哪些微生物副产物 在病人体内循环。其次，我们将使用单细胞RNA测序(scRNA-seq)来剖析 不同的单核细胞亚类，CTL表达的差异，以及不同类型的细胞对CTL的反应 激动剂。第三，由于CTL基因聚集在基因组中，我们将使用scRNA-seq和atac-seq(Assay 对于转座酶可访问的染色质)，以了解基因组中邻近基因的共同调节。总而言之， 这一建议将进一步加深我们对CTL在宿主/微生物相互作用中介导的免疫监视的理解 在急性肝炎疾病进展过程中，并有可能确定新的治疗靶点以减少炎症。

PROJECT SUMMARY The Candidate is a postdoctoral fellow committed to developing an academic research group focused on applying bioinformatics to understand the pathophysiology of alcohol-related liver diseases (ALD). His previous and current postdoctoral work has given him the unique skillset to answer questions involving gene expression/regulation in the immune system. The Career Development Plan describes 2 years of mentored research wherein the candidate will develop skills in clinical immunology, generate the sequencing data outlined in the proposal, and learn leadership skills to transition into independence. The next 3 years, after obtaining an independent faculty position, will be dedicated to developing new data analysis pipelines and establishing cell biological and bioinformatics tools to understand gene regulation in the innate immune system, which will allow the candidate to establish future projects in ALD immunology. Research Plan: ALD is a spectrum of disorders that affect a growing number of people worldwide. Alcoholic Hepatitis (AH) is a severe inflammatory disease that can increase the morbidity and severity of ALD disorders. In AH, the innate immune system is hypersensitive to microbial byproducts. Alcohol consumption causes gut-barrier disruption, leading to leakage of gut microbes into the portal circulation. The liver immune system is able to detect these microbes through pattern recognition receptors, including the Toll-like receptors (TLRs) and C-type Lectin receptors (CTLs). While TLRs have been well studied for their role in sensitizing the innate immune cells to microbial products, the CTLs have only recently been implicated in mouse models of ALD. CTLs are a large family of PRRs that sense a vast diversity of microbes, including bacteria, fungi, and viruses. Our work has found that many members of the CTL gene family are upregulated in the liver and peripheral blood mononuclear cells PBMCs of AH patients. CTLs were also upregulated in PBMCs in response to LPS/TLR4 signaling. We predict CTLs are upregulated in response to gut- derived LPS in order to increase sensing for other microbes that may be present in the blood, making this pathway a secondary innate immune surveillance pathway. In this proposal, we will test this hypothesis in three aims to address the functional role and regulation of this immune surveillance pathway, and the mechanism by which it is exacerbated in AH. First, we will use PBMCs isolated from AH patients to measure increased sensitivity to CTL agonists, as well as micro-/mycobiome sequencing to determine what microbial byproducts were in circulation in patients. Second, we will use single-cell RNA sequencing (scRNA-seq) to dissect the different monocyte subclasses, variation in CTL expression, and how different cell types respond to CTL agonists. Third, because CTL genes are clustered in the genome, we will use scRNA-seq and ATAC-seq (Assay for Transposase-Accessible Chromatin) to understand co-regulation of nearby genes in the genome. Altogether, this proposal will further our understanding of CTL mediated immune surveillance in host/microbial interactions during AH disease progression and potentially identify new therapeutic targets to decrease inflammation.