Mechanistically defining the role for each NFkB family member in pro-inflammatory macrophage activation

项目总结 世界卫生组织(WHO)估计，每年约有600万人死于败血症，其中 中低收入国家感受到的负担最大。根据世界卫生组织的数据，在过去的十年中，每十人中就有一人死亡 怀孕是产妇败血症的结果，其中95%以上的病例发生在发展中国家。最后， 疾病控制和预防中心估计，所有新冠肺炎患者中有3%-17%将发展为 急性呼吸窘迫综合征(ARDS)，这是该患者群体死亡的主要原因。脓毒症 和ARDS，代表感染期间免疫系统过度激活和调节失调的严重后果。 尽管这种病理的负担已经被认识到，但这种临床恶化的分子基础 人们对此仍然知之甚少。NFkB是一种普遍存在的转录因子，在病毒和细菌感染时被激活 感染控制先天免疫细胞的行为，因此很可能是免疫失调的关键因素。 NFkB是一个包含5个亚基(cRel、relA、p50、p52和RelB)的转录因子家族，它们可以组合地相互作用 形成15个可能的二聚体物种来调节基因表达。虽然，NFkB已经被很好地研究了，但仍然有一个 缺乏对单个NFkB二聚体在免疫细胞激活中作用的研究。初步 分析支持中心假设，即NFkB的每个二聚体都具有独特的生物学功能。理解 单个NFkB二聚体的作用可能为促炎基因的精确调控开辟道路 表情。此外，我的数据支持这样一种假设，即cRel同源二聚体对于选择性 调节一小群基因，包括与共调节蛋白相关的Il12b和p50同源二聚体 激活包括IL6、IL1b和Lcn2在内的关键促炎基因。这项研究使用了一种骨髓 衍生巨噬细胞模型系统结合原代腹膜巨噬细胞的提取 在体外用大肠杆菌刺激，或用盲肠结扎和脓毒症穿孔模型体内刺激，以确定二聚体 NFkB在巨噬细胞活化中的特殊作用及二聚体特异基因的分子机制 监管。这项建议将探索cRel同源二聚体在巨噬细胞激活中的机制作用；以及 Ii)研究巨噬细胞活化过程中p50同源二聚体的特异性基因调控。这项研究建议将重点放在 关于cRel和p50同源二聚体，因为它们在病毒和细菌的PRR下游具有强大的活性，以及它们的 在免疫细胞激活中的高度特异性作用。完成后，这项研究将为 NFkB通过揭示使二聚体具有特定功能的潜在机制而采用的调节逻辑 有可能。此外，它还可以发现免疫系统调节的特定治疗靶点，以帮助减缓或阻止 在脓毒症和ARDS中可见免疫细胞过度激活。

PROJECT SUMMARY The World Health Organization (WHO) estimates that ~6 million people die from sepsis each year, with the greatest burden felt in low to middle income countries. According to the WHO, one out of ten deaths during pregnancy is a result of maternal sepsis with over 95% of these cases arising in developing countries. Finally, the Centers for Disease Control and Prevention, estimates that 3-17% of all patients with COVID-19 will develop acute respiratory distress syndrome (ARDS), the primary cause of mortality in this patient population. Sepsis and ARDS, represent severe consequences of immune system overactivation and dysregulation during infection. Although the burden of this pathology is recognized, the molecular underpinnings of this clinical deterioration remain poorly understood. NFkB is a ubiquitous transcription factor that is activated during viral and bacterial infection to control innate immune cell behavior and is thus likely to be a key player in immune dysregulation. NFkB is a family of TFs containing 5 subunits (cRel, RelA, p50, p52, and RelB) that combinatorically interact to form 15 possible dimeric species to regulate gene expression. Although, NFkB is well studied, there remains a paucity of research investigating the function of individual NFkB dimers in immune cell activation. Preliminary analysis supports the central hypothesis that each dimer of NFkB has a unique biological function. Understanding the role of individual NFkB dimers could open avenues for the precise modulation of pro-inflammatory gene expression. Further, my data supports the hypothesis that cRel homodimers are necessary for the selective regulation of a small group of genes including Il12b; and p50 homodimers associate with a co-regulatory protein to activate key pro-inflammatory genes including Il6, Il1b and Lcn2. This research employs a bone marrow derived macrophage model system in combination with the extraction of primary peritoneal macrophages stimulated ex vivo with E. coli or in vivo with a cecal ligation and puncture model of sepsis, to define dimer specific roles of NFkB in macrophage activation and the molecular mechanisms underlying dimer specific gene regulation. This proposal will i) explore the mechanistic role of cRel homodimers in macrophage activation; and ii) investigate p50 homodimer specific gene regulation in macrophage activation. This research proposal focuses on cRel and p50 homodimers due to their potent activation downstream of viral and bacterial PRRs and their highly specific roles in immune cell activation. Upon completion, this research will provide novel insights into the regulatory logic employed by NFkB by revealing the underlying mechanisms that make dimer specific functions possible. Further, it could uncover specific therapeutic targets for immune system modulation to help slow or halt exaggerated immune cell activation seen in sepsis and ARDS.