Data_Sheet_1_Temporal and Spatial Dynamics of Inflammasome Activation After Ischemic Stroke.docx

Background: The inflammasome represents a highly pro-inflammatory mechanism. It has been identified that inflammasome was activated after ischemic stroke. However, the impact of inflammasomes on stroke outcomes remains contradictory. The participating molecules and the functioning arena of post-stroke inflammasome activation are still elusive.
Methods: In the present study, blood samples from stroke patients were collected and analyzed with flow cytometry to evaluate the correlation of inflammasome activation and stroke outcomes. A stroke model was established using male C57/Bl6 mice with transient middle cerebral artery occlusion (tMCAO, 1 h). The dynamics of inflammasome components, cell type, and location of inflammasome activation and the therapeutic effects of inhibiting post-stroke inflammasome executors were evaluated.
Results: We found that a high level of inflammasome activation might indicate detrimental stroke outcomes in patients and mice models. Post-stroke inflammasome activation, especially NLRP3, cleaved Caspase-1, cleaved Caspase-11, IL-1β, IL-18, and GSDMD, peaked at 3–5 days and declined at 7 days with the participation of multiple components in mice. Macrophage that infiltrated into the ischemic lesion was the main arena for post-stroke inflammasome activation among myeloid cells according to the data of mice. Among all the members of the Caspase family, Caspase-1 and −11 served as the main executing enzymes. Inhibiting Caspase-1/−11 signaling efficiently suppressed DAMPs-induced macrophage inflammasome activation and displayed neuroprotection to stroke models including infarct size (Control: 48.05 ± 14.98; Cas1.i: 19.34 ± 12.21; Cas11.i: 21.43 ± 14.67, P < 0.001) and neurological deficit score (0 d-Control: 2.20 ± 0.63; 0 d-Cas1.i: 2.20 ± 0.63; 0 d-Cas11.i: 2.20 ± 0.63; 1 d-Control: 2.50 ± 0.53; 1 d-Cas1.i: 1.50 ± 0.71; 1 d-Cas11.i: 2.00 ± 0.67; 2 d-Control: 2.30 ± 0.48; 2 d-Cas1.i: 1.30 ± 0.48; 2 d-Cas11.i: 1.50 ± 0.53; 3 d-Control: 2.00 ± 0.67; 3 d-Cas1.i: 1.20 ± 0.42; 3 d-Cas11.i: 1.30 ± 0.48, P < 0.001).
Conclusions: Taken together, inflammasome activation played a detrimental role in stroke pathology. Targeting post-stroke inflammasome executing enzymes fitting in the dynamics of macrophages might obtain potential and efficient therapeutic effects.

**背景**：炎性体（inflammasome）是一类强效促炎机制。已有研究证实，缺血性脑卒中（ischemic stroke）后会发生炎性体激活，但炎性体对脑卒中预后的影响仍存在争议，卒中后炎性体激活所涉及的分子及作用位点尚不明确。
**方法**：本研究收集脑卒中患者的血液样本，采用流式细胞术（flow cytometry）进行分析，以评估炎性体激活与脑卒中预后的相关性。选用雄性C57/Bl6小鼠构建短暂性大脑中动脉闭塞（transient middle cerebral artery occlusion, tMCAO，1小时）脑卒中模型，检测炎性体组分的动态变化、炎性体激活的细胞类型与定位，并评估抑制卒中后炎性体执行因子的治疗效果。
**结果**：本研究发现，高水平的炎性体激活可能预示患者及小鼠模型的不良脑卒中预后。在小鼠体内，卒中后炎性体激活（尤其是NLRP3、活化半胱天冬酶-1（cleaved Caspase-1）、活化半胱天冬酶-11（cleaved Caspase-11）、白细胞介素1β（IL-1β）、白细胞介素18（IL-18）及细胞焦亡蛋白GSDMD）在造模后3~5天达到峰值，7天时逐渐下降，且该过程涉及多种炎性体组分。小鼠实验数据显示，在髓系细胞中，浸润至缺血病灶的巨噬细胞是卒中后炎性体激活的主要作用位点。在半胱天冬酶（Caspase）家族所有成员中，半胱天冬酶-1与半胱天冬酶-11是主要的执行性酶类。抑制半胱天冬酶-1/11信号通路可有效抑制损伤相关分子模式（damage-associated molecular patterns, DAMPs）诱导的巨噬细胞炎性体激活，并对脑卒中模型展现出神经保护作用，具体体现在脑梗死体积[对照组：48.05±14.98；Casp1.i组：19.34±12.21；Casp11.i组：21.43±14.67，P<0.001]与神经功能缺损评分[0天-对照组：2.20±0.63；0天-Casp1.i组：2.20±0.63；0天-Casp11.i组：2.20±0.63；1天-对照组：2.50±0.53；1天-Casp1.i组：1.50±0.71；1天-Casp11.i组：2.00±0.67；2天-对照组：2.30±0.48；2天-Casp1.i组：1.30±0.48；2天-Casp11.i组：1.50±0.53；3天-对照组：2.00±0.67；3天-Casp1.i组：1.20±0.42；3天-Casp11.i组：1.30±0.48，P<0.001]的改善上。
**结论**：综上，炎性体激活在脑卒中病理过程中发挥有害作用。针对卒中后炎性体执行性酶类、契合巨噬细胞激活动态特征的干预策略，或可获得具有潜力且高效的治疗效果。