Department of Intensive Care Unit, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou 310014, Zhejiang, P.R. China.
Zhejiang University School of Medicine, Zhejiang University, Hangzhou 310029, Zhejiang, P.R. China.
Theranostics. 2021 Sep 13;11(19):9431-9451. doi: 10.7150/thno.61902. eCollection 2021.
The immunosuppressive, inflammatory microenvironment orchestrated by neutrophil extracellular traps (NETs) plays a principal role in pathogenesis of sepsis. Fibroblast growth factor-inducible molecule 14 (Fn14) has been established as a potential target for septic acute kidney injury (AKI), making further therapeutic benefits from combined NETs and Fn14 blockade possible. The concurrence of NETs and Fn14 in mice and patients with septic AKI were assessed by immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and studies. Survival, histopathological and biochemical analyses of wild-type and PAD4-deficient ; PAD4 mice with septic AKI were applied to evaluate the efficacy of either pharmacological or genetic NETs interruption in combination with Fn14 blockade. Molecular mechanisms underlying such effects were determined by CRISPR technology, fluorescence-activated cell sorter analysis (FACS), cycloheximide (CHX) pulse-chase, luciferase reporter and chromatin immunoprecipitation (ChIP) assay. NETs formation is concurred with Fn14 upregulation in murine AKI models of abdominal, endotoxemic, multidrug-resistant sepsis as well as in serum samples of patients with septic AKI. Pharmacological or genetic interruption of NETs formation synergizes with ITEM-2, a monoclonal antibody (mAb) of Fn14, to prolong mice survival and provide renal protection against abdominal sepsis, the effects that could be abrogated by elimination of macrophages. Interrupting NETs formation predominantly perpetuates infiltration and survival of efferocytic growth arrest-specific protein 6 (GAS6) macrophages in combination with ITEM-2 therapy and enhances transcription of tubular cell-intrinsic Fn14 in a DNA methyltransferase 3a (DNMT3a)-independent manner through dismantling the proteasomes-mediated turnover of homeobox protein Hox-A5 (HOXA5) upon abdominal sepsis challenge or LPS stimuli. Pharmacological NETs interruption potentiates the anti-septic AKI efficacy of ITEM-2 in murine models of endotoxemic and multidrug-resistant sepsis. Our preclinical data propose that interrupting NETs formation in combination with Fn14 mAb might be a feasible therapeutic strategy for septic AKI.
中性粒细胞胞外诱捕网（NETs）所构成的免疫抑制性炎症微环境在脓毒症发病机制中发挥主要作用。成纤维细胞生长因子诱导分子 14（Fn14）已被确定为脓毒症急性肾损伤（AKI）的潜在靶点，因此联合 NETs 和 Fn14 阻断可能带来进一步的治疗益处。通过免疫荧光、免疫组织化学、酶联免疫吸附试验（ELISA）和研究评估了脓毒症 AKI 小鼠和患者中 NETs 和 Fn14 的共存情况。应用野生型和 PAD4 缺陷型；PAD4 小鼠的脓毒症 AKI 的生存、组织病理学和生化分析来评估 NETs 形成的药理学或基因中断与 Fn14 阻断联合的疗效。通过 CRISPR 技术、荧光激活细胞分选分析（FACS）、环己酰亚胺（CHX）脉冲追踪、荧光素酶报告基因和染色质免疫沉淀（ChIP）测定确定了这些效应的潜在机制。NETs 的形成与 Fn14 的上调在腹部、内毒素、多药耐药性脓毒症的小鼠 AKI 模型以及脓毒症 AKI 患者的血清样本中同时发生。NETs 形成的药理学或基因中断与 ITEM-2（Fn14 的单克隆抗体（mAb））协同作用，延长了腹部脓毒症小鼠的生存时间，并提供了肾脏保护作用，这些作用可以通过消除巨噬细胞来消除。中断 NETs 的形成主要通过破坏空泡蛋白 A5（HOXA5）的蛋白酶体介导的周转，在与 ITEM-2 治疗结合时促进效应细胞吞噬生长停滞特异性蛋白 6（GAS6）巨噬细胞的浸润和存活，并以 DNA 甲基转移酶 3a（DNMT3a）非依赖性方式增强管细胞固有 Fn14 的转录，这是腹部脓毒症挑战或 LPS 刺激后发生的情况。药理学 NETs 中断增强了 ITEM-2 在内毒素和多药耐药性脓毒症的小鼠模型中的抗脓毒症 AKI 疗效。我们的临床前数据表明，NETs 形成的中断与 Fn14 mAb 的联合可能是脓毒症 AKI 的一种可行治疗策略。
