Monitoring pro-resolving leukocyte responses in peripheral blood predicts clinical severity during sepsis

摘要 脓毒症的特征是免疫细胞功能障碍，这与疾病的病理生理学有关 随之而来的器官损伤和相关死亡率增加。在小鼠实验性脓毒症中，调节 白细胞功能可减少器官损伤，减轻感染的严重程度。对外围设备的评估 血白细胞活化和功能可作为临床预后的生物标志物，并提示新的 人类败血症和炎症性疾病的治疗策略。解决方案 炎症是一个具有抗炎和促消炎机制的活跃过程。有几个 作为激动剂的决议调解人家族，被称为“专门的支持决议调解人(SPM)” AT同源受体在白细胞和实验模型上转导细胞类型特异性反应 这些SPM可降低脓毒症的严重程度和持续时间。最近，以微升为单位的 外周血中，我们确定白细胞反应的功能特征更具信息量 比白细胞计数用于临床评估危重疾病的轨迹。对NIGMS的回应 NOT-GM-19-054“探索现有或新的败血症人类生物标本的科学价值”， 拟议的概念验证和扩大研究旨在确定我们的 现有和正在进行的人类败血症生物检疫菌危重病生物信息库登记 患者健康记录数据。这些生物标本是在分娩时收集的，住院第3天和第7医院至 与患者的临床轨迹保持一致，与临床数据集相关联，并代表变革潜力 用于脓毒症内分型/分层。在这里，我们建议使用等电介质分离作为当代 在分析这些生物标本方面的尖端技术，以揭示在 患者免疫反应的上升和分辨率将检验这一假设：脓毒症高- 炎症反应是由有缺陷的内源性分解机制引起的，这些 外周血白细胞活化和对SPM的功能反应存在明显缺陷。 为了解决这一假设，我们计划了四个具体目标。在R21阶段，(1)演示可重复性 从毛细血管血液中分离中性粒细胞并进行功能分析，以及(2)证明有能力获得深部 来自少量中性粒细胞的蛋白质组和磷蛋白质组图谱。在R33阶段，(3)纵向 脓毒症缓解过程中SPM通路激活的分析，以及(4)推理和模型的生成 用体外试验进行检测。本应用程序的目标是更好地理解 释放脓毒症不受抑制的炎症的解决缺陷，并为新的治疗方法制定更长期的目标 疾病进展的目标和预后指标。此应用程序的重要产品包括 就收集、利用和分析人类生物检疫的最佳做法提供指导 最大限度地提高它们对整个脓毒症研究社区的价值。

Abstract Sepsis is characterized by immune cell dysfunction, which is linked to the pathophysiology of the disease with consequent organ injury and increased associated mortality. In experimental sepsis in mice, regulating leukocyte function decreases organ injury and lessens the severity of the infection. Assessment of peripheral blood leukocyte activation and function may serve as prognostic clinical biomarkers and inform new therapeutic strategies for the management of human sepsis and inflammatory diseases. Resolution of inflammation is an active process with anti-inflammatory and pro-resolving mechanisms. There are several families of resolution mediators, termed “specialized pro-resolving mediators (SPMs)”, that serve as agonists at cognate receptors to transduce cell-type specific responses on leukocytes and in experimental model systems these SPMs decrease the severity and duration of sepsis. Recently, in microliter quantities of peripheral blood, we determined that functional characterization of leukocyte responses was more informative than leukocyte counting for clinical assessment of the trajectory of critical illness. In response to the NIGMS NOT-GM-19-054 “Exploring the Scientific Value of Existing or New Sepsis Human Biospecimen Collections”, the proposed proof of concept and scale-up studies are designed to determine the scientific value of our existing and ongoing Registry of Critical Illness biorepository of human sepsis biospecimens with associated patient health record data. These biospecimens are collected at presentation, hospital day 3 and hospital 7 to align with the clinical trajectory of the patient, linked to clinical datasets and represent transformative potential for sepsis endotyping/stratification. Here, we propose the use of isodielectric separation as a contemporary cutting-edge technology in the analysis of these biospecimens to uncover leukocyte activation during the upslope and resolution of patient’s immune responses that will test the hypothesis: Sepsis hyper- inflammatory responses result from defective endogenous resolution mechanisms and that these defects are evident in peripheral blood leukocyte activation and functional responses to SPMs. To address this hypothesis, we plan four specific aims. In the R21 phase, (1) demonstrate reproducible isolation and functional analysis of neutrophils from capillary blood, and (2) demonstrate ability to obtain deep proteomic and phosphoproteomic profiles from small numbers of neutrophils. In the R33 phase, (3) longitudinal analysis of SPM pathway activation during sepsis resolution, and (4) inference and model generation and testing with ex vivo assays. The goals of this application are to develop an improved understanding of the resolution defects that unleash unrestrained inflammation of sepsis with a longer term goal for new therapeutic targets and prognostic indicators of disease progression. Important products of this application include provision of guidance on the best practices for collecting, utilizing, and analyzing human biospecimens to maximize their value for the entire sepsis research community.