项目来源

河南省自然科学基金

项目主持人

宋波

项目受资助机构

郑州大学

项目编号

212300410017

立项年度

2021

立项时间

未公开

研究期限

未知 / 未知

项目级别

省级

受资助金额

未知

学科

医学科学

学科代码

未公开

基金类别

杰出青年科学基金

关键词

GSDMD ; 缺血性脑梗死 ; 中性粒细胞 ; CD4+T细胞 ; ischemic cerebral infarction ; CD11b+cells ; CD4+T cells

参与者

未公开

参与机构

郑州大学第一附属医院

项目标书摘要：摘要背景:缺血性脑梗死后的脑中的免疫环境由众多固有和适应性免疫细胞相互作用、相互调节形成。在缺血早期，动脉阻塞导致组织缺氧、血流动力学改变，炎症进程即刻从血管内开始。随着缺血级联反应进展，濒死和死亡细胞释放“危险信号”活化免疫系统，损伤相关分子模式是启动炎症介质上调的最主要信号，这些信号活化嘌呤受体和模式识别受体，启动启动先天性免疫和适应性免疫应答，外周固有免疫细胞(如中性粒细胞、单核—巨噬细胞)和适应性免疫细胞(如B细胞、T细胞)被大量募集至缺血半球。浸润缺血脑组织和损伤区域血管内的中性粒细胞吞噬功能下降，表达高水平活性氧，加重缺血性脑损伤。在缺血性脑梗死后小鼠缺血脑组织中T 细胞数量显著升高，与更差的功能预后相关。因此，免疫系统与缺血性脑梗死的病理机制密切相关。Gasdermin D(GSDMD)蛋白介导的细胞焦亡对众多免疫炎症疾病发生有着重要作用，最近研究发现细胞焦亡广泛参与缺血性脑梗死后免疫反应。细胞焦亡又称细胞炎性坏死，是一种依赖炎性caspases、并伴随着炎症反应的细胞程序性死亡，在形态学上与细胞凋亡、坏死存在明显差别，其特征性表现为细胞膜上形成众多孔道，导致细胞肿胀裂解，并伴随大量炎症细胞因子如白细胞介素-1β(interleukin-1β,IL-1β)、IL-18的释放。细胞焦亡可通过多种途径参与缺血性脑梗死的发展过程，抑制细胞焦亡可能成为一种可行的神经保护策略。焦亡相关炎症小体，如NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP1)、NLRP3 在神经胶质细胞及神经元中高表达，当脑缺血再灌注损伤时，这些活化的炎症小体会激活 caspase-1,诱导细胞发生焦亡，从而加重脑缺血再灌注损伤。GSDMD蛋白被认为是焦亡的执行蛋白，巨噬细胞、中性粒细胞、树突状细胞、角质形成细胞和肠上皮细胞均表达炎性小体成分，并可发生GSDMD依赖性焦亡。目前细胞焦亡在缺血性脑梗死中的研究均聚焦在中枢神经细胞中，针对外周免疫的抗炎作用机制研究尚不明确。抑制GSDMD靶点可以减少IL-1β等细胞因子的分泌,IL-1β是IL-1家族的重要成员之一。IL是指在白细胞或免疫细胞间相互作用的淋巴因子，活化T细胞、刺激B细胞增殖、活化中性粒细胞、诱导内皮促凝血活性、诱导内皮表面抗原是IL-1的主要生物学特性。近期的研究显示在神经免疫疾病中，抑制NLRP3的表达或减少IL-1β的分泌可以抑制致病型T细胞的形成，从而对疾病的发生发展起到抑制作用，但GSDMD在缺血性脑梗死中对T细胞反应中的作用尚不清楚。目的:1.通过野生型小鼠MCAO模型，利用药物抑制实验和蛋白免疫印迹分析、免疫荧光、流式细胞分析及病理染色等技术，研究GSDMD蛋白介导的细胞焦亡与缺血性脑梗死外周免疫的相关性。2.通过系统性Gsdmd敲除小鼠和条件性髓系细胞Gsdmd敲除小鼠MCAO模型，利用细胞耗竭实验和流式细胞分析等技术，研究GSDMD对缺血性脑梗死后中性粒细胞的影响及相关机制。3.通过系统性Gsdmd基因敲除小鼠、条件性髓系细胞或CD4+T细胞Gsdmd敲除小鼠，利用骨髓移植实验和流式细胞分析等技术，研究研究缺血性脑梗死后GSDMD蛋白对CD4+T细胞的影响及相关机制。方法:1.检测WT小鼠脑、脾脏组织中GSDMD、caspase-1/11、IL-1β和IL-18蛋白的表达。2.检测WT小鼠MCAO后各器官中的GSDMD蛋白表达水平和脑中单核/巨噬细胞焦亡水平，观察脑中焦亡细胞的规律。3.双硫仑药物干预GSDMD蛋白后观察小鼠MCAO后的行为学及病理改变。4.比较双硫仑注射小鼠与对照小鼠MCAO后脾脏中外周免疫细胞的水平。5.评估中性粒细胞水平对AIS患者3个月预后的预测价值。6.观察Gsdmd-/-小鼠MCAO模型中脑组织的病理变化，检测炎症水平。7.观察WT小鼠MCAO后1-5天脑、脾脏、血液及骨髓中中性粒细胞及单核细胞的变化规律。比较Gsdmd-/-小鼠与WT小鼠后脑、脾脏、血液及骨髓中中性粒细胞及单核细胞的水平。8.在腹腔注射抗Ly6G抗体小鼠及条件性髓系细胞 Gsdmd 基因敲除小鼠的MCAO模型中观察血液及骨髓中的中性粒细胞、单核细胞及骨髓祖细胞(BM、CMP、GMP)的水平。9.比较Gsdmd-/-小鼠与WT小鼠MCAO后脑和脾脏中CD4+T细胞(包括初始T细胞、效应T细胞、Th1和Th17细胞)的水平。10.比较Gsdmd-/-小鼠与WT小鼠MCAO后脾脏中单核/巨噬细胞及树突状细胞中MCH II分子复合物阳性细胞水平。11.RNA-Seq分析MCAO后Gsdmd-/-和WT小鼠脾脏中CD11b+细胞和CD4+T细胞的差异基因。qRT-PCR验证差异基因的表达。12.利用骨髓移植实验及条件性CD4+细胞敲除Gsdmd小鼠，比较骨髓细胞或CD4+细胞不表达GSDMD小鼠与对照小鼠MCAO后的神经功能、病理改变和脑中炎症水平。13.比较条件性髓系细胞 Gsdmd 基因敲除小鼠和同窝对照小鼠生理状态及MCAO后脾脏中CD4+T细胞及各亚型的水平。结果:1.TTC染色及激光散斑结果均显示成功构建小鼠MCAO模型。2.免疫印迹分析显示MCAO后第3天脑和脾脏中GSDMD、caspase-1/11和IL-1β/18的表达水平升高(P<0.05),细胞焦亡信号被激活。脑组织免疫组化显示，在MCAO后脑中GSDMD 免疫阳性细胞较假手术组增多。3.MCAO后第3天WT小鼠的肝脏、脾脏和肾脏的GSDMD蛋白表达水平明显高于脑干及脑皮层。CD31+内皮细胞周围有大量GSDMD免疫+细胞或caspase-1+细胞聚集。MCAO后第3天脑中CD11b+Ly6C+单核/巨噬细胞焦亡水平明显升高(P<0.05)。4.蛋白印迹分析显示，双硫仑显著降低GSDMD-N 32 kDa的表达(P<0.05)。与对照组小鼠相比，双硫仑在治疗5天后降低了MCAO小鼠的神经行为损伤程度(P<0.05),减小的梗死体积(P<0.05),改善梗死区周围皮层区神经元的无序分布，以及尼氏小体和细胞水肿情况。5.注射双硫仑小鼠脾脏中中性粒细胞、巨噬细胞、经典树突状细胞以及表达MHC II类分子复合物的CD11b+和CD11c+较未给药小鼠减少(P<0.05),浆细胞样树突状细胞、B细胞、NK细胞及CD4+、CD8+细胞无明显改变。6.多因素logistic回归分析显示，中性粒细胞与AIS患者3个月不良结局显著相关(P<0.05),单核细胞无显著相关性。ROC曲线显示，最佳鉴别变量为中性粒细胞。7.成功构建Gsdmd基因敲除小鼠,HE染色显示，与野生型小鼠相比,Gsdmd-/-小鼠的神经细胞丢失和细胞间隙更小，炎症性细胞更少。ELISA结果显示,Gsdmd基因的敲除导致MCAO后第3天外周血中IL-1β、IL-6和TNF-α的浓度下降(P<0.05)。8.WT小鼠脑中中性粒细胞和单核细胞在第1天增加，并在第3天达到峰值(P<0.05),Ly6Chi促炎单核细胞数量在MCAO后1-5天持续升高，而抗炎Ly6Clo单核细胞在MCAO后5天内没有明显变化。Gsdmd-/-小鼠脑中中性粒细胞和炎性Ly6Chi单核细胞减少(P<0.05),抗炎 Ly6Clo单核细胞有上升的趋势。9.WT小鼠MCAO后1-5天血液和脾脏中中性粒细胞和单核细胞在第1天水平最高(P<0.05)。与WT小鼠相比,MCAO后第1天Gsdmd-/-小鼠血液和脾脏中中性粒细胞降低。10.WT小鼠MCAO后第1-5天骨髓中中性粒细胞先降低后升高,Ly6C+单核细胞持续减少,Ly6Clo抗炎单核细胞短暂减少后增加。Gsdmd-/-小鼠在MCAO后第1天仅抑制Ly6C+单核细胞水平(P<0.05)。MCAO后第3天GSDMD缺失对中性粒细胞无影响。MCAO后第5天,Gsdmd-/-小鼠的中性粒细胞比例显著降低(P<0.05)。11.腹腔注射抗Ly6G抗体可有效降低外周血中中性粒细胞(P<0.05),对单核细胞无影响。与同型对照小鼠相比,MCAO后中性粒细胞耗竭小鼠血液及骨髓中的中性粒细胞减少(P<0.05),而CD11b+细胞和Ly6C+单核细胞改变无统计学差异。12.成功构建条件性髓系细胞Gsdmd敲除(Gsdmdfl/flLyz2Cre)小鼠，与对照组小鼠相比,MCAO后3天,Gsdmdfl/flLyz2Cre小鼠较对照小鼠骨髓中中性粒细胞减少(P<0.05),而单核细胞无统计学差异。Gsdmdfl/flLyz2Cre小鼠骨髓中GMP水平降低(P<0.05),MP和CMP无统计学差异。13.MCAO后第3天Gsdmd-/-小鼠大脑中的CD4+T细胞较WT小鼠减少(P<0.05),而脾脏中CD4+T细胞并无统计学差异。Gsdmd-/-小鼠脾脏中初始T细胞增多(P<0.05),效应T细胞、Th1细胞、Th17减少(P<0.05)。14.MCAO后小鼠敲除Gsdmd后脾脏中单核/巨噬细胞及树突状细胞中MCH II分子复合物阳性细胞水平较野生型小鼠降低。15.MCAO后脾脏中Gsdmd敲除的CD11b+细胞中显著下调的前30个基因与白细胞迁移、细胞迁移、调节细胞因子的产生、细胞粘附分子结合活性、活性自由基和髓系细胞生成相关。qRT-PCR的结果显示Gsdmd敲除的CD11b+细胞中Slfn4、Il1b、H2-T24、Ccl2、Ccr2的表达水平显著下调(P<0.05)。Th1/Th2细胞分化、TNF信号通路、RIG-I样受体信号通路、NOD样受体信号通路、造血细胞谱系和抗原处理和呈递是Gsdmd-/-CD11b+细胞中前30条差异通路。Gsdmd敲除的CD4+T细胞中表达显着降低的前30个基因与适应性免疫反应、过氧化物酶活性、炎症和抗原处理、免疫球蛋白产生以及B细胞增殖的调节等相关,RIG-I样受体信号通路、原发性免疫缺陷、自然杀伤细胞介导的细胞毒性、NF-kappa B信号通路、B细胞受体信号通路、抗原处理和呈递是Gsdmd-/-CD4+T细胞前30条差异通路。16.Gsdmdfl/flCD4Cre与其对照Gsdmdfl/fl小鼠相比在MCAO后第3天的梗死体积及脑中CD4+T、CD8+T和CD11b+细胞的水平无差异。骨髓移植实验结果显示,MCAO后用GSDMD蛋白缺陷型骨髓重建的小鼠在mNSS评分和圆筒试验中比用WT骨髓重建的小鼠表现更好，具有较小的梗死体积、更低的脑中继发性死亡细胞及炎症细胞水平较低(P<0.05)。17.Gsdmdfl/flLyz2Cre小鼠较对照小鼠MCAO后第3天脾脏中CD4+T细胞水平无统计学差异，但初始T细胞增多，效应T细胞、Th1及Th17细胞减少(P<0.05),不影响B细胞及NK细胞的水平。生理状态下，髓系细胞中GSDMD蛋白对MCAO后CD4+T细胞的启动及分化无影响。结论1.缺血性脑梗死后GSDMD蛋白介导的细胞焦亡信号在早期被激活,GSDMD蛋白介导的细胞焦亡通过调节外周免疫改善缺血性脑梗死后脑损伤。2.GSDMD蛋白的表达缺失可降低MCAO后大脑整体炎症程度和中性粒细胞比例。早期(缺血性脑梗死后第1天)炎性白细胞的产生和补充依赖于GSDMD蛋白。中性粒细胞固有GSDMD对骨髓和血液中中性粒细胞的产生和供应至关重要，可能通过调节骨髓造血在MCAO后的病理过程中发挥作用。3.髓系细胞中GSDMD蛋白的缺失减弱细胞因子和趋化因子的释放，抑制CD4+T细胞在二级淋巴器官的启动及向Th1、Th17细胞的分化，从而缓解T细胞介导的神经炎症。

Application Abstract: Backgroud:The immune environment in the brain after ischemic cerebral infarction is formed by the interaction and mutual regulation of many innate and adaptive immune cells.In the early stage of ischemia,arterial occlusion leads to tissue hypoxia and hemodynamic changes,and the inflammatory process immediately begins in the blood vessel.As the ischemic cascade progresses,dying and dying cells release"danger signals"to activate the immune system.Damage related molecular patterns are the most important signals to initiate the up-regulation of inflammatory mediators.These signals activate purine receptors and pattern recognition receptors to activate innate and adaptive immune responses.Peripheral innate immune cells(e.g.,neutrophils,mononuclear macrophages)and adaptive immune cells(e.g.,B cells,T cells)are recruited to the ischemic hemisphere in large numbers.The phagocytic function of neutrophils infiltrating ischemic brain tissue and blood vessels in the injured area decreased and expressed high levels of reactive oxygen species,which aggravated ischemic brain injury.The number of T cells in ischemic brain tissue was significantly increased after ischemic cerebral infarction in mice,and was associated with poorer functional prognosis.Therefore,the immune system is closely related to the pathological mechanism of ischemic cerebral infarction.Gasdermin D(GSDMD)protein-mediated pyroptosis plays an important role in the occurrence of many immune inflammatory diseases.Recent studies have found that pyroptosis is widely involved in the immune response after ischemic cerebral infarction.Pyroptosis,also known as inflammatory necrosis,is a kind of programmed cell death dependent on inflammatory caspases and accompanied by inflammatory reactions.It is significantly different from apoptosis and necrosis in morphology.It is characterized by the formation of numerous pores on the cell membrane,leading to cell swelling and lysis,and accompanied by the release of a large number of inflammatory cytokines such as interleukin-1β(IL-1β)and IL-18.Pyroptosis can be involved in the development of ischemic cerebral infarction through various ways.Inhibition of pyroptosis may be a feasible neuroprotective strategy.Pyroptosis related inflammatory bodies,such as(NOD-like receptor thermal protein domain associated protein 3,NLRP1)and NLRP3,are highly expressed in glial cells and neurons.When cerebral ischemia-reperfusion injury occurs,these activated inflammatory bodies activate caspase-1 and induce pyroptosis,thus aggravating cerebral ischemia-reperfusion injury.GSDMD protein is considered to be the executive protein of pyrosis.Macrophages,neutrophils,dendritic cells,keratinocytes and intestinal epithelial cells all express inflammatory body components and GSDMD-dependent pyrosis can occur.At present,studies on pyroptosis in ischemic cerebral infarction focus on central nerve cells,and the anti-inflammatory mechanism of peripheral immunity is still unclear.Inhibition of GSDMD targets can reduce the secretion of cytokines such as IL-1β,which is an important member of the IL-1 family.IL is a lymphoid factor that interacts with leukocytes or immune cells.Activation of T cells,stimulation of proliferation of B cells,activation of neutrophils,induction of endothelial procoagulant activity and induction of endothelial surface antigen are the main biological characteristics of IL-1.Recent studies have shown that inhibiting the expression of NLRP3 or reducing the secretion of IL-1β can inhibit the formation of pathogenic T cells and thus the development and progression of the disease in neuroimmune diseases.However,the role of GSDMD in T cell response in ischemic cerebral infarction remains unclear.Objective:1.The correlation between GSDMD protein-mediated pyroptosis and peripheral immunity in ischemic cerebral infarction was studied in wild-type mice MCAO model by drug inhibition assay,western blotting analysis,immunofluorescence,flow cytometry and pathological staining.2.The effect of GSDMD on neutrophils after ischemic cerebral infarction and its related mechanism were studied by cell depletion assay and flow cytometry in mice with Gsdmd knockout model and conditioned myeloid cell Gsdmd knockout model.3.The effect of GSDMD protein on CD4+T cells after ischemic cerebral infarction was studied by using bone marrow transplantation and flow cytometry in mice with systematic Gsdmd knockout,conditioned myeloid cells or CD4+T cell Gsdmd knockout.Methods:1.The expression of GSDMD,caspase-1/11,IL-1β and IL-18 in brain and spleen tissues of WT mice were detected.2.The expression level of GSDMD protein in various organs of WT mice after MCAO and the pyroptosis level of monocytes/macrophages in brain were detected,and the rule of pyroptosis cells in brain was observed.3.The behavioral and pathological changes of mice after GSDMD protein intervention with disulfiram were observed.4.The levels of peripheral immune cells in spleen were compared between mice injected with disulfiram and control mice.5.To evaluate the predictive value of neutrophil levels for 3-month prognosis of AIS patients.6.The pathological changes of brain tissue in Gsdmd-/-mouse MCAO model were observed to detect the level of inflammation.7.The changes of neutrophils and monocytes in brain,spleen,blood and bone marrow of WT mice were observed 1-5 days after MCAO.The levels of neutrophils and monocytes in posterior brain,spleen,blood and bone marrow of Gsdmd-/-mice and WT mice were compared.8.The levels of neutrophils,monocytes and bone marrow progenitor cells(BM,CMP,GMP)in blood and bone marrow were observed in the MCAO model of mice injected with anti-Ly6G antibody and conditioned myeloid Gsdmd gene knockout mice.9.The levels of CD4+T cells(including primary T cells,effector T cells,Th1 cells and Th17 cells)in the brain and spleen of Gsdmd-/-and WT mice after MCAO were compared.10.The levels of MCH II molecular complex positive cells in mononuclear/macrophage and dendritic cells in spleen of Gsdmd-/-mice and WT mice after MCAO were compared.11.Differential genes of CD11b+cells and CD4+T cells in spleen of Gsdmd-/-and WT mice after MCAO were analyzed by RNA-Seq.The expression of differential genes was verified by qRT-PCR.12.Bone marrow transplantation experiment and conditional CD4+cell knockout of Gsdmd mice were used to compare the neurological function,pathological changes and levels of brain inflammation in mice without GSDMD expression in bone marrow cells or CD4+cells compared with control mice.13.The physiological status of Gsdmd knockout mice and littermates control mice were compared,as well as the levels of CD4+T cells and subtypes in spleen after MCAO.Results:1.TTC staining and laser speckle results showed that mouse MCAO model was successfully constructed.2.Western blotting analysis showed that the expression levels of GSDMD,caspase-1/11 and IL-1β/18 in brain and spleen on the 3 days after MCAO were increased(P<0.05),and the scorched-death signal was activated.Immunohistochemistry of brain tissue showed that GSDMD+cells were more in brain after MCAO than in sham operation group.3.On the 3 days after MCAO,the expression level of GSDMD protein in liver,spleen and kidney of WT mice was significantly higher than that in brain stem and cortex.A large number of GSDMD+cells or caspase-1+cells clustered around CD31+endothelial cells.CD11b+Ly6C+monocyte/macrophage pyrodeath levels in brain were significantly increased on the 3 days after MCAO(P<0.05).4.Western blot analysis showed that disulfide significantly decreased the expression of GSDMD-N 32 kDa(P<0.05).Compared with the control group,disulfiram reduced the degree of neurobehavioral damage(P<0.05)and infarct volume(P<0.05)in MCAO mice after 5 days of treatment,and improved the disordered distribution of neurons in the cortical area around the infarct area,as well as the condition of nissl bodies and cell edema.5.The number of neutrophil granulocytes,macrophages,classical dendritic cells and CD11b+and CD11c+expressing MHC Class II molecular complex in spleen of mice injected with disulfiram was decreased compared with that of mice not given disulfiram(P<0.05),while plasmacytoid dendritic cells,B cells,NK cells,CD4+and CD8+cells had no significant changes.6.Multivariate logistic regression analysis showed that neutrophils were significantly correlated with adverse outcomes in AIS patients at 3 months(P<0.05),while monocytes had no significant correlation.The ROC curve showed that the best discriminating variable was neutrophils.7.Gsdmd knockout mice were successfully constructed,and HE staining showed that Gsdmd-/-mice had smaller nerve cell loss,smaller cell space,and fewer inflammatory cells than wild-type mice.ELISA results showed that knockout of Gsdmd gene resulted in decreased concentrations of IL-1β,IL-6 and TNF-α in peripheral blood on day 3 after MCAO(P<0.05).8.Neutrophils and monocytes in the brain of WT mice increased on day 1 and reached a peak on day 3(P<0.05).The number of Ly6Chi pro-inflammatory monocytes continued to increase on day 1-5 after MCAO,while the number of anti-inflammatory Ly6Clo monocytes did not change significantly on day 5 after MCAO.In Gsdmd-/-mice,neutrophils and inflammatory Ly6Chi monocytes decreased(P<0.05),and anti-inflammatory Ly6Clo monocytes increased.9.Neutrophil and monocyte levels in blood and spleen of WT mice 1-5 days after MCAO were the highest on day 1(P<0.05).Compared with WT mice,neutrophils in blood and spleen of Gsdmd-/-mice were reduced on day 1 after MCAO.10.On day 1-5 after MCAO,neutrophils in bone marrow of WT mice decreased first and then increased,Ly6C+monocytes continued to decrease,and Ly6Clo anti-inflammatory monocytes briefly decreased and then increased.Gsdmd-/-mice inhibited only Ly6C+monocyte level on day 1 after MCAO(P<0.05).The absence of GSDMD on day 3 after MCAO had no effect on neutrophils.On day 5 after MCAO,the proportion of neutrophil in Gsdmd-/-mice was significantly decreased(P<0.05).11.Intraperitoneal injection of anti-Ly6G antibody could effectively reduce neutrophils in peripheral blood(P<0.05),but had no effect on monocytes.Compared with the control mice,neutrophilia in blood and bone marrow of neutrophilic depletion mice after MCAO was decreased(P<0.05),but there was no statistical difference in the changes of CD11b+cells and Ly6C+monocytes.12.Conditional myeloid cell knockout(Gsdmdfl/flLyz2Cre)mice were successfully constructed.Compared with control mice,3 days after MCAO,Gsdmdfl/flLyz2Cre mice showed neutropenia in bone marrow compared with control mice(P<0.05),but there was no statistical difference in monocytes.The level of GMP in bone marrow of Gsdmdfl/flLyz2Cre mice was decreased(P<0.05),and there was no statistical difference between MP and CMP.13.On the 3 days after MCAO,CD4+T cells in the brain of Gsdmd-/-mice were less than those in WT mice(P<0.05),but there was no significant difference in CD4+T cells in the spleen.Naïve T cells increased in the spleen of Gsdmd-/-mice(P<0.05),while effective T cells,Th1 cells and Th17 decreased(P<0.05).14.The level of MCH II molecular complex positive cells in monocytes/macrophages and dendritic cells in spleen of MCAO mice after Gsdmd knockout was lower than that of wild-type mice.15.The first 30 genes significantly down-regulated in Gsdmd knockout CD11b+cells in spleen after MCAO were associated with leukocyte migration,cell migration,production of regulatory cytokines,binding activity of cell adhesion molecules,active free radicals,and myeloid cell generation.The results of RT-qPCR showed that the expression levels of Slfn4,Il1b,H2-T24,Ccl2 and Ccr2 in Gsdmd knockout CD11b+cells were significantly down-regulated(P<0.05).Th1/Th2 cell differentiation,TNF signaling pathway,RIG-I receptor signaling pathway,Nod-like receptor signaling pathway,hematopoietic cell lineage,and antigen processing and presentation are the top 30 differentiating pathways in Gsdmd-/-CD11b+cells.The top 30 genes with significantly reduced expression in CD4+T cells knockout by Gsdmd are associated with adaptive immune response,peroxidase activity,inflammation and antigen processing,immunoglobulin production,and regulation of B cell proliferation.RIG-I receptor signaling pathway,primary immune deficiency,natural killer cell-mediated cytotoxicity,NF-kappa B signaling pathway,B cell receptor signaling pathway,antigen processing,and presentation are the top 30 differential pathways in Gsdmd-/-CD4+T cells.16.Gsdmdfl/flCD4Cre showed no difference in infarct volume and levels of CD4+T,CD8+T and CD11b+cells on day 3 after MCAO compared with control Gsdmdfl/fl mice.The results of bone marrow transplantation showed that mice with GSDMD protein deficient bone marrow reconstruction after MCAO performed better in mNSS score and cylinder test than mice with WT bone marrow reconstruction,with smaller infarct volume,lower levels of secondary dead cells and inflammatory cells in the brain(P<0.05).17.There was no significant difference in the level of CD4+T cells in spleens of Gsdmdfl/flLyz2Cre mice compared with control mice on the 3 days after MCAO,but the level of B cells and NK cells was not affected by the increase of naïve T cells and the decrease of effective T cells,Th1 and Th17 cells(P<0.05).Under physiological conditions,GSDMD protein in myeloid cells had no effect on the initiation and differentiation of CD4+T cells after MCAO.Conclusion:1.After ischemic cerebral infarction,GSDMD protein mediated cell focal death signal is activated in the early stage,and the loss of GSDMD protein expression can improve the neurological injury and pathological process in ischemic cerebral infarction.2.The loss of GSDMD protein expression can reduce the degree of global brain inflammation and the proportion of neutrophils after MCAO.Early(day 1 after ischemic infarction)inflammatory leukocyte production and recruitment depend on GSDMD protein.Neutrophil intrinsic GSDMD is essential for the production and supply of neutrophils in bone marrow and blood and may play a role in the pathological process after MCAO by regulating bone marrow hematopoietic production.3.The loss of GSDMD protein in myeloid cells weakens the release of cytokines and chemokines,inhibits the initiation of CD4+T cells in secondary lymphoid organs and their differentiation into Th1 and Th17 cells,thus alleviating T-cell-mediated neuroinflammation.

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河南省