From the Unidad de Investigación Neurovascular, Departamento de Farmacología y Toxicología, Facultad de Medicina, Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense Madrid, and Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain (A.G.-C., V.D.-L., C.P.-M., A.M., I.B., M.I.C., J.d.l.P., S.P.-T., M.A.M., I.L.).
Area of Cell and Developmental Biology, Fundación Centro Nacional Investigaciones Cardiovasculares (CNIC), Madrid, Spain, and Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximillians-Universität München, Germany (A.H.).
Stroke. 2019 Oct;50(10):2922-2932. doi: 10.1161/STROKEAHA.119.025085. Epub 2019 Aug 27.
Background and Purpose- After stroke, the population of infiltrated neutrophils in the brain is heterogeneous, including a population of alternative neutrophils (N2) that express M2 phenotype markers. We explored the role of TLR4 (toll-like receptor 4) on neutrophil infiltration and polarization in this setting. Methods- Focal cerebral ischemia was induced by occlusion of the middle cerebral artery occlusion in TLR4-KO and WT (wild type) mice. Infarct size was measured by Nissl staining and magnetic resonance imaging. Leukocyte infiltration was quantified 48 hours after middle cerebral artery occlusion by immunofluorescence and flow cytometry. To elucidate mechanisms underlying TLR4-mediated N2 phenotype, a cDNA microarray analysis was performed in neutrophils isolated from blood 48 hours after stroke in WT and TLR4-KO mice. Results- As demonstrated previously, TLR4-deficient mice presented lesser infarct volumes than WT mice. TLR4-deficient mice showed higher density of infiltrated neutrophils 48 hours after stroke compared with WT mice, concomitantly to neuroprotection. Furthermore, cytometric and stereological analyses revealed an increased number of N2 neutrophils (YM1 cells) into the ischemic core in TLR4-deficient mice, suggesting a protective effect of this neutrophil subset that was corroborated by depleting peripheral neutrophils or using mice with TLR4 genetically ablated in the myeloid lineage. Finally, cDNA microarray analysis in neutrophils, confirmed by quantitative polymerase chain reaction, showed that TLR4 modulates several pathways associated with ischemia-induced inflammation, migration of neutrophils into the parenchyma, and their functional priming, which might explain the opposite effect on outcome of the different neutrophil subsets. Conclusions- TLR4 deficiency increased the levels of alternative neutrophils (N2)-an effect associated with neuroprotection after stroke-supporting that modulation of neutrophil polarization is a major target of TLR4 and highlighting the crucial role of TLR4 at the peripheral level after stroke. Visual Overview- An online visual overview is available for this article.
背景与目的-中风后，大脑中浸润的中性粒细胞群体具有异质性，包括表达 M2 表型标志物的替代性中性粒细胞（N2）群体。我们探讨了 TLR4（ toll样受体 4）在这种情况下对中性粒细胞浸润和极化的作用。方法-通过阻断大脑中动脉闭塞来诱导 TLR4-KO 和 WT（野生型）小鼠的局灶性脑缺血。通过尼氏染色和磁共振成像测量梗死面积。通过免疫荧光和流式细胞术在大脑中动脉闭塞后 48 小时量化白细胞浸润。为了阐明 TLR4 介导的 N2 表型的机制，我们对中风后 48 小时从 WT 和 TLR4-KO 小鼠血液中分离的中性粒细胞进行了 cDNA 微阵列分析。结果-如前所述，TLR4 缺陷型小鼠的梗死体积小于 WT 小鼠。与 WT 小鼠相比，TLR4 缺陷型小鼠在中风后 48 小时浸润的中性粒细胞密度更高，同时具有神经保护作用。此外，细胞计数和立体学分析显示 TLR4 缺陷型小鼠缺血核心内 N2 中性粒细胞（YM1 细胞）数量增加，表明该中性粒细胞亚群具有保护作用，这一作用得到了外周中性粒细胞耗竭或使用骨髓中 TLR4 基因缺失的小鼠的证实。最后，对中性粒细胞的 cDNA 微阵列分析，通过定量聚合酶链反应进行验证，表明 TLR4 调节了与缺血诱导炎症、中性粒细胞向实质迁移及其功能启动相关的几个途径，这可能解释了不同中性粒细胞亚群对结果的相反影响。结论-TLR4 缺陷增加了替代性中性粒细胞（N2）的水平-这是中风后神经保护的一种作用-支持了对中性粒细胞极化的调节是 TLR4 的主要靶点，并突出了 TLR4 在中风后外周水平的关键作用。
