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

摘要 脓毒症的特征在于免疫细胞功能障碍，其与疾病的病理生理学相关， 随后的器官损伤和增加的相关死亡率。在小鼠实验性脓毒症中，调节 白细胞功能减少器官损伤并减轻感染的严重性。评估外周 血液白细胞的活化和功能可以作为预后的临床生物标志物， 用于管理人类脓毒症和炎性疾病的治疗策略。解决 炎症是具有抗炎和促消退机制的主动过程。有几 消退介质家族，称为“特化促消退介质（SPMs）"，作为激动剂 在白细胞和实验模型中， 这些SPM降低了脓毒症的严重程度和持续时间。最近，在微升数量的 外周血中，我们确定白细胞反应的功能表征更能提供信息 比白细胞计数更适合临床评估危重病的发展轨迹。针对NIGMS NOT-GM-19-054“探索现有或新的脓毒症人类生物标本采集的科学价值”， 提出的概念验证和规模扩大研究旨在确定我们的科学价值， 现有和正在进行的人脓毒症生物标本的危重疾病生物储存库登记， 患者健康记录数据。这些生物标本在就诊时、住院第3天和住院第7天收集， 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 用于脓毒症内定型/分层。在这里，我们建议使用等介电分离作为当代 尖端技术在这些生物标本的分析，以揭示白细胞活化过程中， 患者免疫反应的上升和消退将检验假设：脓毒症超 炎症反应是由有缺陷的内源性消退机制引起的，并且这些机制 在外周血白细胞活化和对SPM的功能反应中缺陷是明显的。 为了解决这一假设，我们计划了四个具体目标。在R21阶段，（1）证明可重现 从毛细血管血中分离和功能分析嗜中性粒细胞，和（2）证明获得深层 来自少量中性粒细胞的蛋白质组学和磷酸蛋白质组学图谱。在R33阶段，（3）纵向 分析脓毒症消退期间SPM途径活化，和（4）推断和模型生成， 用离体测定进行测试。本应用程序的目标是提高对 解决缺陷，释放无限制的脓毒症炎症，长期目标是新的治疗方法 疾病进展的目标和预后指标。该应用的重要产品包括 提供关于收集、利用和分析人体生物标本的最佳实践的指南， 最大限度地发挥其对整个脓毒症研究界的价值。

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.