Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
J Neuroinflammation. 2020 Feb 4;17(1):49. doi: 10.1186/s12974-020-1730-y.
Multiple sclerosis (MS) is a chronic debilitating immune-mediated disease of the central nervous system (CNS) driven by demyelination and gray matter neurodegeneration. We previously reported an experimental autoimmune encephalomyelitis (EAE) MS mouse model with elevated serum CXCL1 that developed severe and prolonged neuron damage. Our findings suggested that CXCR2 signaling may be important in neuronal damage, thus implicating neutrophils, which express CXCR2 in abundance, as a potential cell type involved. The goals of this study were to determine if CXCR2 signaling in neutrophils mediate neuronal damage and to identify potential mechanisms of damage.
EAE was induced in wild-type control and neutrophil-specific Cxcr2 knockout (Cxcr2 cKO) mice by repeated high-dose injections of heat-killed Mycobacterium tuberculosis and MOG peptide. Mice were examined daily for motor deficit. Serum CXCL1 level was determined at different time points throughout disease development. Neuronal morphology in Golgi-Cox stained lumbar spinal cord ventral horn was assessed using recently developed confocal reflection super-resolution technique. Immune cells from CNS and lymphoid organs were quantified by flow cytometry. CNS-derived neutrophils were co-cultured with neuronal crest cells and neuronal cell death was measured. Neutrophils isolated from lymphoid organs were examined for expression of reactive oxygen species (ROS) and ROS-related genes. Thioglycolate-activated neutrophils were isolated, treated with recombinant CXCL1, and measured for ROS production.
Cxcr2 cKO mice had less severe disease symptoms at peak and late phase when compared to control mice with similar levels of CNS-infiltrating neutrophils and other immune cells despite high levels of circulating CXCL1. Additionally, Cxcr2 cKO mice had significantly reduced CNS neuronal damage in the ventral horn of the spinal cord. Neutrophils isolated from control EAE mice induced vast neuronal cell death in vitro when compared with neutrophils isolated from Cxcr2 cKO EAE mice. Neutrophils isolated from control EAE mice, but not Cxcr2 cKO mice, exhibited elevated ROS generation, in addition to heightened Ncf1 and Il1b transcription. Furthermore, recombinant CXCL1 was sufficient to significantly increase neutrophils ROS production.
CXCR2 signal in neutrophils is critical in triggering CNS neuronal damage via ROS generation, which leads to prolonged EAE disease. These findings emphasize that CXCR2 signaling in neutrophils may be a viable target for therapeutic intervention against CNS neuronal damage.
多发性硬化症（MS）是一种由脱髓鞘和灰质神经退行性变驱动的中枢神经系统（CNS）慢性衰弱性免疫介导疾病。我们之前报道了一种实验性自身免疫性脑脊髓炎（EAE）MS 小鼠模型，其血清 CXCL1 升高，导致严重和持久的神经元损伤。我们的研究结果表明，CXCR2 信号可能在神经元损伤中很重要，因此丰富表达 CXCR2 的中性粒细胞可能是潜在的参与细胞类型。本研究的目的是确定中性粒细胞中的 CXCR2 信号是否介导神经元损伤，并确定潜在的损伤机制。
通过重复给予热灭活结核分枝杆菌和 MOG 肽，在野生型对照和中性粒细胞特异性 Cxcr2 敲除（Cxcr2 cKO）小鼠中诱导 EAE。每天检查小鼠的运动功能障碍。在疾病发展的不同时间点测定血清 CXCL1 水平。使用最近开发的共聚焦反射超分辨率技术评估高尔基-考克斯染色腰椎脊髓腹角的神经元形态。通过流式细胞术定量 CNS 和淋巴器官中的免疫细胞。从淋巴器官分离中性粒细胞，并检测其活性氧物种（ROS）表达和 ROS 相关基因。分离硫代乙醇酸盐激活的中性粒细胞，用重组 CXCL1 处理，并测量 ROS 产生。
与对照小鼠相比，Cxcr2 cKO 小鼠在疾病高峰期和晚期的症状较轻，尽管循环 CXCL1 水平较高，但中枢神经系统浸润的中性粒细胞和其他免疫细胞水平相似。此外，Cxcr2 cKO 小鼠的脊髓腹角中枢神经系统神经元损伤明显减少。与 Cxcr2 cKO EAE 小鼠分离的中性粒细胞相比，来自对照 EAE 小鼠的中性粒细胞在体外诱导了大量神经元细胞死亡。来自对照 EAE 小鼠的中性粒细胞，但不是 Cxcr2 cKO 小鼠的中性粒细胞，表现出升高的 ROS 生成，以及 Ncf1 和 Il1b 转录的升高。此外，重组 CXCL1 足以显著增加中性粒细胞的 ROS 生成。
中性粒细胞中的 CXCR2 信号通过 ROS 生成在触发中枢神经系统神经元损伤中起关键作用，从而导致 EAE 疾病的延长。这些发现强调，中性粒细胞中的 CXCR2 信号可能是针对中枢神经系统神经元损伤的治疗干预的一个可行靶点。
