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， 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，医院死亡和费用每年超过240亿美元。没有特定的治疗方法可以预防或 治疗与脓毒症和脓毒症-ALI相关的细胞功能障碍。虽然脓毒症的复杂机制 脓毒症和脓毒症-ALI的新发病机制涉及中性粒细胞和 巨噬细胞死亡，通过释放瓜氨酸化组蛋白3引起炎症和器官损伤 CitH3（CitH3）。肽基精氨酸脱亚胺酶（PAD）2和4催化CitH 3的形成。PAD 2和CitH 3 与脓毒症有关，发现脓毒症患者中PAD 2和CitH 3的血清水平升高。鼠标 模型进一步证明，Pad 2的敲除和泛PAD抑制剂的利用改善了 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，博士。 Alam和Li博士对脓毒症中的PAD-CitH 3通路进行了广泛的研究。该提案与 NIH R 01提案（1 R 01 HL 155116 - 01 A1）“PAD 2和CitH 3在脓毒症诱导的ALI发病机制中的作用”， Alam和Li博士特别关注阐明PAD的机制及其生理影响， 脓毒症中巨噬细胞的调节。在目标1中，我们将使用Pad 2-/-、Pad 4-/-和 Pad 2-/-Pad 4-/-小鼠来研究PAD 2和PAD 4在脓毒症和脓毒症-ALI结果中的相互作用。在 目的2，我们将使用腺相关病毒（AAV）介导的Cas9/gRNA递送来进行靶向治疗。 敲除和修复巨噬细胞中的Pad 2。脓毒症小鼠及其巨噬细胞的转归 将比较体细胞消融和靶向RNA敲除的活性， 体细胞消融小鼠的变化。对于目标3，我们将使用RNA测序和蛋白质组学来识别 与Pad 2消融补偿相关的基因。从这个项目中获得的知识将有一个 这对我们理解脓毒症中的PAD-CitH 3通路产生了重大影响。这将大大有助于 对脓毒症、脓毒症-ALI和脓毒症引起的多器官损伤的发病机制和潜在治疗的认识 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.