Role of peptidylarginine deiminase in modulating macrophage functions in sepsis

项目摘要 脓毒症是一种危及生命的器官功能障碍，由多方面的失调的宿主反应引起 感染，包括脓毒症引起的急性肺损伤(脓毒症-ALI)。脓毒症是最常见的原因-- 在美国，每年医院死亡的费用超过240亿美元。没有特定的治疗方法来预防或 治疗与脓毒症和脓毒症-ALI相关的细胞功能障碍。而脓毒症的复杂机制 败血症和脓毒症的一种新的发病机制-ALI涉及中性粒细胞和 通过释放瓜氨酸组蛋白3引起炎症和器官损伤的巨噬细胞死亡 释放(CitH3)。精氨酸脱亚胺酶(PAD)2和4催化CitH3的形成。PAD2和CitH3 已发现败血症患者血清PAD2和CitH3水平升高与脓毒症有关。小白鼠 模型进一步表明，Pad2基因的敲除和PAN-PAD抑制剂的利用有所改善 在几种不同的脓毒症模型中存活并预防器官功能障碍。在过去的十年里，戴维斯博士。 Alam和Li博士在脓毒症中对Pad-CitH3途径进行了广泛的研究。该提案与以下内容相关联 NIH R01建议(1R01HL155116-01A1)“PAD2和CitH3在脓毒症诱导的ALI发病机制中的作用”。 Alam和Li博士重点阐述了PAD的机制及其生理影响和 巨噬细胞在脓毒症中的调节作用。在目标1中，我们将使用Pad 2-/-、Pad4-/-和 Pad2-/-Pad4-/-小鼠，研究PAD2和PAD4在脓毒症和脓毒症-ALI结局中的相互作用。在……里面 目的2，我们将使用腺相关病毒(AAV)介导的Cas9/gRNA递送进行靶向 巨噬细胞中Pad2的敲除和修复。脓毒症小鼠及其巨噬细胞的结局 将比较躯体消融和靶向RNA敲除的活动，以评估可能的代偿 小鼠躯体消融后的变化。对于目标3，我们将使用RNA测序和蛋白质组学来鉴定 与Pad2消融代偿相关的基因。从这个项目中获得的知识将有一个 对我们理解PAD-CitH3途径在脓毒症中的作用有重要影响。这将对未来的发展做出重大贡献 脓毒症、脓毒症-ALI及脓毒症所致多脏器感染的发病机制及治疗探讨 功能障碍。

Project Summary Sepsis is a life-threatening organ dysfunction caused by a multifaceted dysregulated host response to an infection, including sepsis induced acute lung injury (sepsis-ALI). Sepsis is the most common cause of in- hospital deaths and costs more than $24 billion annually in the US. No specific therapy exists to prevent or treat the cellular dysfunction associated with sepsis and sepsis-ALI. While the complex mechanisms of sepsis have remained poorly defined, an emerging pathogenesis of sepsis and sepsis-ALI involves neutrophil and macrophage cell death causing inflammation and organ damage through the release of citrullinated histone 3 release (CitH3). Petidylarginine deiminase (PAD) 2 and 4 catalyze the formation of CitH3. PAD2 and CitH3 have been linked to sepsis with findings of elevated serum levels of PAD2 and CitH3 in septic patients. Mouse models have further demonstrated that knockout of Pad2 and utilization of pan-PAD inhibitors improved survival and prevented organ dysfunction in several different sepsis models. Throughout the past decade, Dr. Alam and Dr. Li have conducted extensive work on the PAD-CitH3 pathway in sepsis. This proposal is linked to the NIH R01 proposal (1R01HL155116-01A1) “PAD2 and CitH3 in Pathogenesis of Sepsis-induced ALI” by Dr. Alam and Dr. Li with a specific focus on elucidating the mechanisms of PAD and their physiologic impacts and modulation of macrophages in sepsis. In Aim 1, we will conduct experiments using Pad 2-/-, Pad4-/-, and Pad2-/-Pad4-/- mice to investigate the interaction of PAD2 and PAD4 in sepsis and sepsis-ALI outcomes. In Aim 2, we will use adeno-associated virus (AAV)- mediated delivery of Cas9/gRNA to perform targeted knockout and restoration of Pad2 in macrophages. Outcomes of mice with sepsis and their macrophage activity with somatic ablation and targeted RNA knockout will be compared to assess possible compensatory changes in mice with somatic ablation. For Aim 3, we will use RNA sequencing and proteomics to identify genes associated with the compensation of Pad2 ablation. The knowledge gained from this project will have a significant impact on our understanding of the PAD-CitH3 pathway in sepsis. This will contribute significant insight in to the pathogenesis and potential treatment of sepsis, sepsis-ALI, and sepsis induced multi-organ dysfunction.