Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA; Department of Pediatrics, Harvard Medical School, Boston, Massachusetts.
Department of Hematology, Lausanne University Hospital, Switzerland.
Am J Hematol. 2018 Feb;93(2):269-276. doi: 10.1002/ajh.24970. Epub 2017 Dec 6.
NET formation in mice (NETosis) is supported by reactive oxygen species (ROS) production by NADPH oxidase and histone hypercitrullination by peptidylarginine deiminase 4 (PAD4). Rac1 and Rac2, expressed in polymorphonuclear neutrophils (PMNs), regulate the cytoskeleton, cell shape, adhesion, and migration and are also essential components of the NADPH oxidase complex. We aimed to explore the role of the Rac signaling pathway including the upstream guanosine exchange factor (GEF) activator, Vav, and a downstream effector, the p21-activated kinase, Pak, on NETosis in PMNs using a previously described flow-cytometry-based assay. Rac2 PMNs showed reduced levels of citrullinated histone H3 (H3Cit)-positive cells and defective NETosis. Rac1 ; Rac2 PMNs demonstrated a further reduction in PMA-induced H3Cit levels and a more profound impairment of NETosis than deletion of Rac2 alone, suggesting an overlapping role of these two highly related proteins. Genetic knockouts of Vav1, or Vav2, did not impair H3Cit response to phorbol myristate ester (PMA) or NETosis. Combined, Vav1 and Vav3 deletions decreased H3Cit response and caused a modest but significant impairment of NETosis. Pharmacologic inhibition of Pak by two inhibitors with distinct mechanisms of action, led to reduced H3Cit levels after PMA stimulation, as well as significant inhibition of NETosis. We validated the importance of Pak using Pak2 PMNs, which demonstrated significantly impaired histone H3 citrullination and NETosis. These data confirm and more comprehensively define the key role of the Rac signaling pathway in PMN NETosis. The Rac signaling cascade may represent a valuable target for inhibition of NETosis and related pathological processes.
NET 形成在老鼠中（NETosis）是由 NADPH 氧化酶产生的活性氧（ROS）和组蛋白瓜氨酸化酶 4（PAD4）支持的。Rac1 和 Rac2 在多形核中性粒细胞（PMN）中表达，调节细胞骨架、细胞形状、黏附和迁移，也是 NADPH 氧化酶复合物的重要组成部分。我们旨在使用以前描述的基于流式细胞术的测定法，探索 Rac 信号通路（包括上游鸟嘌呤核苷酸交换因子（GEF）激活剂 Vav 和下游效应物 p21 激活激酶 Pak）在 PMN 中 NETosis 中的作用。Rac2 PMN 显示出降低的瓜氨酸化组蛋白 H3（H3Cit）阳性细胞水平和 NETosis 缺陷。Rac1; Rac2 PMN 显示出 PMA 诱导的 H3Cit 水平进一步降低，并且比单独缺失 Rac2 更严重地损害 NETosis，表明这两种高度相关的蛋白质具有重叠作用。Vav1 或 Vav2 的基因敲除不会损害对佛波酯（PMA）或 NETosis 的 H3Cit 反应。Vav1 和 Vav3 的联合缺失减少了 H3Cit 对 PMA 的反应，并导致 NETosis 适度但显著受损。通过两种具有不同作用机制的抑制剂对 Pak 的药理学抑制导致 PMA 刺激后 H3Cit 水平降低，并且 NETosis 受到显著抑制。我们使用 Pak2 PMN 验证了 Pak 的重要性，其显示出明显受损的组蛋白 H3 瓜氨酸化和 NETosis。这些数据证实并更全面地定义了 Rac 信号通路在 PMN NETosis 中的关键作用。Rac 信号级联可能代表抑制 NETosis 和相关病理过程的有价值的靶标。
