JoVE Journal > Immunology and Infection
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Immunology and Infection

评估半胱天冬酶活化以评估先天免疫细胞死亡

Published: January 20, 2023
doi:
10.3791/64308
, ,
1Department of Immunology,St. Jude Children’s Research Hospital

Summary

该协议描述了一种评估半胱天冬酶活化（半胱天冬酶-1，半胱天冬酶-3，半胱天冬酶-7，半胱天冬酶-8，半胱天冬酶-9和半胱天冬酶-11）的综合方法，以响应 体外 和 体内 （小鼠）感染，无菌侮辱和癌症模型，以确定细胞死亡途径的启动，例如焦亡，细胞凋亡，坏死性凋亡和全细胞凋亡。

Abstract

先天免疫是应对病原体和无菌侮辱的关键第一道防线。这种反应的一个关键机制组成部分是启动先天免疫程序性细胞死亡（PCD），以消除感染或受损的细胞并传播免疫反应。然而，过量的PCD与炎症和病理有关。因此，了解PCD的激活和调节是表征先天免疫反应和确定整个疾病谱的新治疗靶点的核心方面。

该协议提供了通过监测半胱天冬酶来表征先天免疫PCD激活的方法，半胱天冬酶是半胱氨酸依赖性蛋白酶家族，通常与多种PCD途径相关，包括细胞凋亡，焦亡，坏死性凋亡和PANoptosis。最初的报告将半胱天冬酶-2、半胱天冬酶-8、半胱天冬酶-9和半胱天冬酶-10描述为引发剂半胱天冬酶，半胱天冬酶-3、半胱天冬酶-6和半胱天冬酶-7作为细胞凋亡中的效应半胱天冬酶，而后来的研究发现炎症性半胱天冬酶，半胱天冬酶-1，半胱天冬酶-4，半胱天冬酶-5和半胱天冬酶-11驱动焦亡。现在已知半胱天冬酶与其他先天免疫和细胞死亡分子在先前定义的PCD途径中存在广泛的串扰，确定了先天免疫和PCD的机制理解中的关键知识差距，并导致了PANoptosis的特征。PANoptosis是一种独特的先天免疫炎症PCD途径，由PANoptosome复合物调节，其整合了来自其他细胞死亡途径的成分，包括半胱天冬酶。

在这里，提供了评估半胱天冬酶响应各种刺激的激活的方法。这些方法允许 在体外 和 体内表征PCD途径，因为活化的半胱天冬酶经历蛋白水解切割，可以使用最佳抗体和印迹条件通过蛋白质印迹可视化。已经建立了方案和蛋白质印迹工作流程，可以评估来自同一细胞群的多个半胱天冬酶的活化，从而提供PCD过程的全面表征。该方法可以应用于发育，稳态，感染，炎症和癌症的研究领域，以评估健康和疾病中整个细胞过程中的PCD途径。

Introduction

先天免疫系统在感染期间和响应无菌刺激（例如组织损伤和体内平衡改变）时充当第一道防线。细胞表面和细胞质中的先天免疫传感器对病原体或损伤相关的分子模式（分别为PAMP或DAMP）做出反应，以触发炎症信号通路和细胞反应。先天免疫反应的关键过程之一是诱导细胞死亡以去除受感染或受损的细胞，并进一步驱动先天性和适应性免疫反应。程序性细胞死亡（PCD）是跨物种的高度保守的过程，突出了其作为先天免疫机制的进化重要性。

有几种先天免疫PCD途径可以在所有细胞类型中激活。半胱天冬酶是高度保守、细胞内、半胱氨酸依赖性蛋白酶的关键家族，在许多 PCD 途径中至关重要，包括传统的非炎症性细胞凋亡途径，以及炎性 PCD 途径，如焦亡、坏死性凋亡和 PANoptosis1，2，3，4，5.有11种人半胱天冬酶和10种鼠半胱天冬酶是明确的，以及可能具有功能的假半胱天冬酶，并且大多数组成型表达为需要切割才能激活的无活性单体或二聚体前半胱天冬酶6，7。半胱天冬酶还包含用于募集和形成多蛋白复合物的重要结构域。这些包括半胱天冬酶活化和募集结构域（CARD），可以在半胱天冬酶-1，半胱天冬酶-2，半胱天冬酶-4，半胱天冬酶-5，半胱天冬酶-9和半胱天冬酶-11中找到，或死亡效应域（DED），存在于半胱天冬酶-8和半胱天冬酶-10中。通过其蛋白水解活性和形成多蛋白复合物的能力，半胱天冬酶是先天免疫PCD的关键驱动因素。

半胱天冬酶在先天免疫PCD中的作用首先在细胞凋亡中被鉴定，其中引发剂半胱天冬酶半胱天冬酶-2，半胱天冬酶-8，半胱天冬酶-9和半胱天冬酶-10激活刽子手半胱天冬酶，半胱天冬酶-3，半胱天冬酶-6和半胱天冬酶-7，以驱动细胞死亡8，9，10，11，12。引发半胱天冬酶可以通过不同的信号级联激活;外在途径通过细胞外配体诱导的死亡受体信号激活半胱天冬酶-8，内在途径通过破坏线粒体完整性激活半胱天冬酶-913。活化的引发剂半胱天冬酶切割接头，分离刽子手半胱天冬酶的大小催化亚基以产生其活性形式。然后，刽子手半胱天冬酶切割其底物以分解细胞，导致DNA降解，膜起泡，核碎裂和凋亡体的释放14，15。该过程通常以非裂解和非炎症形式的细胞死亡结束，同时通过风化细胞作用立即清除垂死细胞16。然而，胞吐作用缺陷或吞噬细胞缺乏可导致凋亡细胞积聚，然后发生溶解和炎症细胞死亡17，18。

炎症性半胱天冬酶，包括半胱天冬酶-1（人和小鼠），半胱天冬酶-4和半胱天冬酶-5（人）和半胱天冬酶-11（小鼠），已被发现在一种称为焦亡的炎症先天免疫PCD（III-PCD）形式中被激活。半胱天冬酶-1 活化与炎性小体的形成有关，炎性小体是含有胞质先天免疫传感器、衔接分子（含有 CARD [ASC] 的细胞凋亡相关斑点样蛋白）和半胱天冬酶-1 的多蛋白复合物。该复合物的形成允许半胱天冬酶-1进行邻近介导的自蛋白水解以释放其活性形式，其可以切割靶底物，包括促炎细胞因子白细胞介素（IL）-1β和IL-18以及造孔分子gasdermin D（GSDMD）19，20，21，22，23.半胱天冬酶-11、半胱天冬酶-4和半胱天冬酶-5在感应脂多糖（LPS）等PAMP后，也可以激活GSDMD，而不会上游形成炎症小体19，20。这些半胱天冬酶在与胞质LPS结合后经历二聚化，然后进行寡聚化和自切割以激活，这导致非规范炎症小体活化24，25，26和半胱天冬酶-1以细胞内在方式活化以诱导IL-1β和IL-18成熟20。这些促炎细胞因子的成熟和释放将这些半胱天冬酶描述为“炎症”。此外，已发现凋亡半胱天冬酶-8定位于炎性小体，在凋亡和焦亡过程之间提供了联系。研究发现，凋亡半胱天冬酶-8对于调节另一种形式的PCD也至关重要，称为坏死性凋亡。半胱天冬酶-8 的缺失导致自发受体相互作用丝氨酸-苏氨酸激酶 3 （RIPK3） 介导的混合谱系激酶结构域样假激酶 （MLKL） 激活，以驱动坏死性凋亡的 III-PCD 途径27，28，29，30，31，32，33，34，35。

虽然半胱天冬酶历来根据它们引发的细胞死亡类型被归类为“凋亡”或“炎症性”，但越来越多的证据表明，通过半胱天冬酶的先天免疫PCD途径之间存在广泛的串扰3，4。例如，来自炎性小体的炎性半胱天冬酶-1在其典型激活位点34处切割凋亡半胱天冬酶-7。半胱天冬酶-1活化也可导致凋亡底物的切割，例如聚（ADP-核糖）聚合酶1（PARP1）36。在缺乏GSDMD的细胞中，半胱天冬酶-1也可以裂解半胱天冬酶-337，38。此外，典型的凋亡半胱天冬酶-3可以裂解加斯皮明E（GSDME）以诱导PCD17，18，并将GSDMD加工成无活性形式40。此外，已经观察到半胱天冬酶-8募集到炎性小体复合物中39，40，41，42，43，44，45，并且半胱天冬酶-8是典型和非典型炎症小体活化的关键调节因子39。在许多炎症条件下，半胱天冬酶-8和半胱天冬酶-1也存在重叠和冗余的作用，并且以火光凋亡，凋亡和坏死性凋亡成分的激活为特征的先天免疫PCD发生在疾病谱39，46，47，48，49，50中。

基于炎症和凋亡半胱天冬酶之间的这种串扰，确定了先天免疫和PCD的机制理解中的一个关键差距，导致了PANoptosis的发现。PANoptosis是III-PCD的一种独特形式，其响应病原体，PAMP，DAMP和体内平衡的改变而被激活，并由PANoptosomes调节 – 多方面的大分子复合物，整合了来自其他细胞死亡途径的成分44，50，51，52，53，54，55.PAN凋亡中生物学效应的全部不能单独通过焦亡，细胞凋亡或坏死性凋亡来单独解释3，4，35，36，39，46，47，48，因为PAN凋亡的特征是激活多种半胱天冬酶，包括半胱天冬酶-1，半胱天冬酶-11，半胱天冬酶-8，半胱天冬酶-9，半胱天冬酶-3和/或半胱天冬酶-7，取决于上下文 44，48，49，50，51，52，53，54，56，57，58，59，60，61，62. PANoptosis越来越多地与传染病和炎症性疾病以及癌症和癌症治疗有关3，4，35，36，39，44，46，47，48，49，50，51，52，53，54，56
，57，58，59，60，61，62，63，64，65，66.

鉴于半胱天冬酶在细胞死亡途径中的重要作用，包括在细胞凋亡、焦亡、坏死性凋亡和全细胞凋亡中，开发表征其激活并了解PCD途径的全部复杂性的技术非常重要。这里的协议详细介绍了一种刺激细胞并测量半胱天冬酶后续活化的方法（图1）。该方法利用半胱天冬酶的蛋白水解裂解，这通常是其激活所必需的，作为研究它们的手段。通过蛋白质印迹，可以确定蛋白质大小，从而可以清晰地观察和区分无活性的半胱天冬酶原及其活化的裂解形式。

该协议的主要优点是1）它能够评估来自单个内源性细胞群的多个半胱天冬酶的激活，以更准确地确定PCD激活和2）使用相对简单的实验室技术，不需要广泛的培训或昂贵的设备。以前的方案使用蛋白质印迹、荧光报告基因或抗体染色来监测培养上清液、细胞和组织裂解物、通过显微镜和体内67，68，69，70，71的全细胞中的半胱天冬酶活化，但这些技术通常只监测样品中的一种或两种半胱天冬酶。此外，虽然含有半胱天冬酶切割位点的合成肽底物在切割时发出荧光，已被用于监测细胞或组织裂解物69中的半胱天冬酶活化，但这些底物通常可以被一种以上的半胱天冬酶切割，使得难以确定该系统中单个半胱天冬酶的特异性激活。此外，使用蛋白质印迹而不是使用荧光报告基因或其他基于标签的方法允许研究人员使用内源性细胞，而不是使用报告基因创建特定的细胞系。使用内源性细胞有多种优点，包括许多永生化细胞系缺乏关键细胞死亡分子72，73，这可能会影响结果。此外，使用内源性细胞可以评估不同的细胞类型，例如巨噬细胞，上皮细胞和内皮细胞，而不是单个谱系。蛋白质印迹也是一种相对简单且具有成本效益的技术，可以在世界各地的实验室中进行，而无需大型昂贵的设备或复杂的设置。

该协议广泛应用于生物学，以了解半胱天冬酶的细胞死亡依赖性和细胞死亡依赖性功能，包括它们的支架作用和在其他炎症信号通路中的功能74。应用这种方法允许在研究先天免疫PCD途径和跨疾病和病症的炎症信号传导中采用统一的方法，并且该协议可用于识别关键的调节过程和机制连接，这将为未来治疗策略的发展提供信息。

Protocol

动物的使用和程序得到了圣裘德儿童研究医院动物使用和护理委员会的批准。 1. 准备解决方案 准备L929条件培养基。板1×106 个L929细胞（参见 材料表）在含有50mLL929培养基的182cm2 组织培养瓶中（参见 表1 培养基的制备）。 在37°C的加湿培养箱中用5%CO2培养细胞生长细胞。 7天后，收集?…

Representative Results

已经观察到PANoptosis对许多细菌，病毒和真菌感染以及其他炎症刺激以及癌细胞的反应44，48，49，50，51，52，53，54，56，57，58，60，61</su…

Discussion

监测半胱天冬酶裂解和活化提供了先天免疫PCD活化作为先天免疫反应一部分的最全面图片之一。这里描述的方案展示了一种监测半胱天冬酶活化以响应IAV，HSV1和F. novicida感染以及无菌触发LPS + ATP的策略，但许多其他刺激可以诱导PCD，并且可以用于该方法，如几个出版物所示44，48，49，50，51，52，<sup cl…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢Kanneganti实验室成员的意见和建议，并感谢J. Gullett博士的科学编辑支持。我们实验室的工作得到了美国国立卫生研究院（NIH）拨款AI101935，AI124346，AI160179，AR056296和CA253095（给T.-D.K.）和美国黎巴嫩叙利亚相关慈善机构（给T.-D.K.）的支持。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。

Materials

0.45 μm filter Millipore SCHVU05RE
10 mL syringe BD Biosciences 309604
12% polyacrylamide gel with 10 wells  Bio-Rad 4561043
12-well plate  Corning 07-200-82
18 G needle  BD Biosciences 305195
25 G needle  BD Biosciences 305122
50 mL tube  Fisher Scientific 50-809-218
70 μm cell strainer  Corning 431751
150 mm tissue culture dishes Corning 430597
182-cm2 tissue culture flask Genesee Scientific 25-211
Accessory white trans tray Cytiva 29-0834-18
Anti–caspase-1 antibody AdipoGen AG-20B-0042-C100
Anti–caspase-11 antibody Novus Biologicals NB120-10454
Anti–caspase-3 antibody Cell Signaling Technology 9662
Anti–caspase-7 antibody Cell Signaling Technology 9492
Anti–caspase-8 antibody Cell Signaling Technology 4927
Anti–caspase-9 antibody Cell Signaling Technology 9504
Anti–cleaved caspase-3 antibody  Cell Signaling Technology 9661
Anti–cleaved caspase-7 antibody  Cell Signaling Technology 9491
Anti–cleaved caspase-8 antibody  Cell Signaling Technology 8592
Anti-mouse HRP-conjugated secondary antibody  Jackson ImmunoResearch Laboratories 315-035-047
Anti-rabbit HRP-conjugated secondary antibody  Jackson ImmunoResearch Laboratories 111-035-047
Anti-rat HRP-conjugated secondary antibody  Jackson ImmunoResearch Laboratories 112-035-003
Anti–β-Actin antibody (C4) HRP Santa Cruz sc-47778 HRP
ATP InvivoGen tlrl-atpl
BBL Trypticase Soy Broth BD Biosciences 211768
Bead bath Chemglass Life Sciences CLS-4598-009
Biophotometer D30 Eppendorf 6133000010
BME Sigma M6250
Bromophenol blue  Sigma BO126
Cell scrapers CellTreat Scientific Products 229315
Chemiluminescence imager (Amersham 600)  Cytiva 29083461
CO2 chamber VetEquip 901703
Cuvettes Fisher Scientific 14-955-129
Dissecting scissors Thermo Fisher Scientific 221S
DMEM Thermo Fisher Scientific 11995-073
DTT Sigma 43815
Eelectrophoresis apparatus  Bio-Rad 1658004
Ethanol Pharmco 111000200
Fetal bovine serum  Biowest S1620
Filter paper Bio-Rad 1703965
Forceps Fisher Scientific 22-327379
Francisella novicida (U112 strain) BEI Resources NR-13
Gel releaser  Bio-Rad 1653320
Gentamycin Gibco 15750060
Glycerol Sigma G7893
Glycine Sigma G8898
HCl Sigma H9892
Heat block Fisher Scientific 23-043-160
Herpes simplex virus 1 (HF strain) ATCC VR-260
High glucose DMEM  Sigma D6171
Human anti–caspase-1 antibody R&D Systems MAB6215
Human anti–caspase-8 antibody Enzo ALX-804-242
Humidified incubator  Thermo Fisher Scientific 51026282
Image analysis software ImageJ v1.53a
IMDM Thermo Fisher Scientific 12440-053
Influenza A virus (A/Puerto Rico/8/34, H1N1 [PR8])  constructed per Hoffmann et al.
L929 cells ATCC CCL-1 cell line for creating L929-conditioned media
L-cysteine  Thermo Fisher Scientific BP376-100
Luminata Forte Western HRP substrate Millipore WBLUF0500 standard-sensitivity HRP substrate
MDCK cells ATCC CCL-34 cell line for determining IAV viral titer
Methanol Sigma 322415
Microcentrifuge Thermo Fisher Scientific 75002401
Non-essential amino acids  Gibco 11140050
Nonfat dried milk powder Kroger
NP-40 solution  Sigma 492016
PBS Thermo Fisher Scientific 10010023
Penicillin and streptomycin  Sigma P4333
Petri dish Fisher Scientific 07-202-011
PhosSTOP Roche PHOSS-RO
Power source  Bio-Rad 164-5052
Protease inhibitor tablet Sigma S8820
PVDF membrane  Millipore IPVH00010
Rocking shaker Labnet S2035-E
SDS Sigma L3771
Sodium chloride  Sigma S9888
Sodium deoxycholate Sigma 30970
Sodium hydroxide Sigma 72068
Sodium pyruvate  Gibco 11360-070
Square Petri dish Fisher Scientific FB0875711A
Stripping buffer Thermo Fisher Scientific 21059
Super Signal Femto HRP substrate Thermo Fisher Scientific 34580 high-sensitivity HRP substrate
Tabletop centrifuge Thermo Fisher Scientific 75004524
Trans-Blot semi-dry system  Bio-Rad 170-3940
Tris Sigma TRIS-RO
Tween 20  Sigma P1379
Ultrapure lipopolysaccharide (LPS) from E. coli 0111:B4 InvivoGen tlrl-3pelps
Vero cells ATCC CCL-81 cell line for determining HSV1 viral titer
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Caspase ActivationInnate Immune Cell DeathDisease MechanismsTherapeutic StrategiesCell Death ProcessesBone Marrow-derived Macrophages (BMDMs)Influenza A VirusProtein CollectionSDS-PAGECaspase Lysis Buffer
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Han, J., Tweedell, R. E., Kanneganti, T. Evaluation of Caspase Activation to Assess Innate Immune Cell Death. J. Vis. Exp. (191), e64308, doi:10.3791/64308 (2023).
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