炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化
Summary
该协议描述了从人血液样本中获取单核细胞来源巨噬细胞（MDM）的工作流程，一种在不损害细胞活力和行为的情况下将炎症半胱天冬酶双分子荧光互补（BiFC）报告基因有效引入人MDM的简单方法，以及一种基于成像的方法来测量活细胞中的炎症半胱天冬酶活化。
Abstract
炎症性半胱天冬酶包括半胱天冬酶-1、-4、-5、-11 和 -12，属于引发者半胱天冬酶的亚组。半胱天冬酶-1是确保炎症信号传导正确调节所必需的，并且在募集到炎症小体后通过接近诱导的二聚化激活。半胱天冬酶-1在单核细胞谱系中含量丰富，并诱导促炎细胞因子白细胞介素（IL）-1β和IL-18向活性分泌分子成熟。其他炎症性半胱天冬酶，半胱天冬酶-4和-5（及其小鼠同源半胱天冬酶-11）通过诱导焦磷酸化来促进IL-1β的释放。半胱天冬酶双分子荧光互补（BiFC）是一种用于测量炎症性半胱天冬酶诱导的接近度作为半胱天冬酶活化的读数的工具。半胱天冬酶-1，-4或-5原结构域，其中包含与炎症小体结合的区域，与黄色荧光蛋白Venus（Venus-N [VN]或Venus-C [VC]）的非荧光片段融合，当半胱天冬酶发生诱导接近时，这些片段与荧光金星复合物结合以重整荧光金星复合物。该协议描述了如何使用细胞核感染将这些报告基因引入原代人单核细胞来源的巨噬细胞（MDM），处理细胞以诱导炎症性半胱天冬酶活化，并使用荧光和共聚焦显微镜测量半胱天冬酶活化。这种方法的优点是它可用于鉴定活细胞中炎症性半胱天冬酶活化复合物的组分，要求和定位。然而，需要考虑仔细的控制，以避免损害细胞活力和行为。该技术是一种强大的工具，用于分析炎症小体水平的动态半胱天冬酶相互作用，以及询问活MDM和来自人类血液样本的单核细胞中的炎症信号级联。
Introduction
半胱天冬酶是半胱氨酸天冬氨酸蛋白酶的一个家族，可以分为引发子半胱天冬酶和刽子手半胱天冬酶。刽子手半挂酶包括半胱天冬酶-3、-6和-7。它们以二聚体的形式天然存在于细胞中，并被引发剂半胱天冬酶切割以执行细胞凋亡1。引发剂半胱天冬酶包括人半胱天冬酶-1、-2、-4、-5、-8、-9、-10和-12。它们被发现为无活性酶原（前半胱天冬酶），通过接近诱导的二聚化激活，并通过自体蛋白水解裂解2，3稳定。炎症性半胱天冬酶是引发子半胱天冬酶2 的一个子集，包括人类中的半胱天冬酶-1、-4、-5 和 -12，以及小鼠4、5 中的半胱天冬酶-1、-11 和 -12。它们不是凋亡作用，而是在炎症中起核心作用。它们介导蛋白水解处理和促白细胞介素（IL）-1β和亲IL-186，7的分泌，这是响应致病性入侵者释放的第一批细胞因子8，9。半叶酶-1在其激活平台募集时被激活;称为炎症小体的大分子量蛋白质复合物（图1A）10。半胱天冬酶-4、-5 和 -11 的二聚化通过非规范的炎性小体途径11，12 独立于这些平台发生。
典型炎性粒体是胞质多聚体蛋白复合物，由炎性粒体传感器蛋白、适应蛋白ASC（含有CARD的凋亡相关斑点样蛋白）和效应蛋白半胱天冬酶-110组成。研究最充分的规范炎小体是含有pyrin结构域（NLRP），NLRP1和NLRP3的NOD样受体家族，含有CARD（NLRC），NLRC4的NLR家族以及黑色素瘤2（AIM2）中不存在的。它们各自包含一个pyrin域，一个CARD或两个域。CARD域介导含CARD的半链酶与其上游激活剂之间的相互作用。因此，由N端pyrin结构域（PYD）和C端CARD基序13，14组成的支架分子ASC是将半胱天冬酶-1募集到NLRP110，NLRP315和AIM216 炎性小体所必需的。
每个炎症小体都以其独特的传感器蛋白命名，该传感器蛋白可识别不同的促炎刺激（图1B）。该途径的激活剂被称为规范刺激。炎症小体充当微生物成分和组织应激的传感器，并通过激活炎症半胱天冬酶17来组装以触发强大的炎症反应。炎症小体组装启动半胱天冬酶-1活化以介导其主要底物pro-IL-1β和pro-IL-18的成熟和分泌。此过程通过两步机制进行。首先，启动刺激通过激活NF-κB途径上调某些炎症小体蛋白和pro-IL-1β的表达。其次，细胞内（规范）刺激诱导炎症小体组装和前卡星酶-16，7的募集。
半胱天冬酶-4和半胱天冬酶-5是小鼠半胱天冬酶-11 11的人类同源物。它们通过细胞内脂多糖（LPS）以炎症小体独立的方式被激活，LPS是在革兰氏阴性细菌18，19，20的外膜中发现的分子，以及细胞外血红素，红细胞溶血21的产物。已经提出LPS直接与这些蛋白质的CARD基序结合并诱导其寡聚化20。半胱天冬酶-4或半胱天冬酶-5的活化通过裂解形成孔隙蛋白gasdermin D（GSDMD）18，19诱导称为焦磷酸化的炎症形式的细胞死亡来促进IL-1β的释放。此外，由半胱天冬酶-4和GSDMD介导的热融性死亡引起的钾离子外排诱导NLRP3炎症小体的活化和随后的半胱天冬酶-122，23的活化。因此，半胱天冬酶-4、-5 和 -11 被认为是 LPS 的细胞内传感器，它们能够诱导焦磷酸化和半胱天冬酶-1 激活以响应特定的刺激11，24。
图1：炎症半胱天冬酶和半胱天冬酶 – 双分子荧光互补（BiFC）测定。 （A）显示半胱天冬酶-BiFC系统的图表，其中与金星的每个非荧光片段（Venus-C或Venus-N）相连的两个半胱天冬酶-1蛋白结构域（C1-pro）被招募到NLRP3激活平台，迫使金星重新折叠并发出荧光。该复合物在显微镜下显示为绿点，并用作炎症性半胱天冬酶诱导的接近的读数，这是引发半胱天冬酶激活的第一步。（B）显示炎症小体成分和炎症性半胱天冬酶的结构域组织的示意图。 请点击此处查看此图的大图。
测量特异性引发剂半胱天冬酶活化是困难的，并且通过成像方法没有多少方法可以做到这一点。半胱天冬酶双分子荧光互补（BiFC）可用于直接在活细胞中可视化炎症半胱天冬酶活化（图1A）25。该技术最近已被改编用于人单核细胞来源的巨噬细胞（MDM）21。半胱天冬酶BiFC测量炎症性半胱天冬酶活化的第一步，诱导接近以促进二聚化。使用编码含CARD的半胱天冬酶pro结构域的质粒的表达，这些质粒融合到光稳定黄色荧光蛋白Venus（Venus-C [VC]）和Venus-N [VN]）的非荧光片段中。当两个半胱天冬酶前跨域被招募到它们的激活平台或经历诱导的接近时，金星的两半被靠近并被迫重新折叠和发出荧光（见 图1A，B）。这提供了特定炎症性半胱天冬酶激活的实时读数。
人类MDM大量表达炎症小体基因和模式识别受体，识别危险信号和病原体产物。这为炎症半胱天冬酶途径的询问提供了理想的细胞类型。此外，它们可以来自外周血，甚至来自患者样本，以评估特定疾病状态下的炎症性半胱天冬酶激活。该协议描述了如何使用核感染，一种基于电穿孔的转染方法将BiFC半胱天冬酶报告基因引入MDM，如何处理细胞以诱导炎症性半胱天冬酶活化，以及如何使用显微镜方法可视化活性半胱天冬酶复合物。此外，该方法可以适于确定这些复合物的分子组成，亚细胞定位，动力学和这些高度有序结构的大小25，26，27。
Protocol
Representative Results
Discussion
该协议描述了从从人血液样本中分离的单核细胞中获取巨噬细胞的工作流程，以及一种在不影响细胞活力和行为的情况下将炎症性半胱天冬酶BiFC报告基因有效地引入人MDM的方法。
该协议利用BiFC技术35 用分裂荧光蛋白Venus的非荧光片段标记半胱天冬酶募集结构域（CARD）处的炎症半胱天冬酶。炎症性半胱天冬酶编码由大（p20）和小（p10）亚基组成的催化结构域…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢LBH实验室过去和现在的成员，他们为这项技术的发展做出了贡献。该实验室由NIH / NIDDK T32DK060445（BEB），NIH / NIDDK F32DK121479（BEB），NIH / NIGMS R01GM121389（LBH）提供支持。图2是使用Biorender软件绘制的。
Materials
| 48 well tissue culture2:34 plates | Genesee Scientific | 25-108 | |
| 10 cm Tissue Culture Dishes | VWR | 25382-166 | |
| 2 Mercaptoethanol 1000x | Thermo Fisher Scientific | 21985023 | |
| 8 well chambered coverglass with 1.5 HP coverglass | Cellvis | c8-1.5H-N | |
| AutoMACS columns | Miltenyi (Biotec) | 130-021-101 | For automated separation using AutoMACS Pro Separator only |
| AutoMACS Pro Separator | Miltenyi (Biotec) | 130-092-545 | For automated separation using AutoMACS Pro Separator only |
| AutoMACS Pro Washing Solution | Miltenyi (Biotec) | 130-092-987 | For automated separation using AutoMACS Pro Separator only |
| AutoMACS Rinsing Solution | Miltenyi (Biotec) | 130-091-222 | For automated separation using AutoMACS Pro Separator only |
| AutoMacs running buffer | Miltenyi (Biotec) | 130-091-221 | For manual or automated separation using QuadroMACS or AutoMACS pro Separator |
| Axio Observer Z1 motorized inverted microscope equipped with a CSU-X1A 5000 spinning disk unit | Zeiss | Any confocal microscope equipped with a laser module fitted with laser lines of 568 nm (RFP) and 488 or 512 nm (GFP or YFP) wavelengths can be used | |
| AxioObserver A1, Research Grade Inverted Microscope | Zeiss | Any epifluorescence microscope with fluorescence filters capable of exciting 568 nm (RFP) and 488 or 512 nm (GFP or YFP) wavelengths can be used | |
| CD14+ MICROBEADS | Miltenyi (Biotec) | 130-050-201 | For manual or automated separation using QuadroMACS or AutoMACS pro Separator |
| DPBS without calcium chloride and magnesium chloride | Sigma | D8537-6x500ML | |
| DsRed mito plasmid | Clontech | 632421 | Similar plasmids that can be used as fluorescent reporters can be found on Addgene |
| Fetal Bovine Serum | Thermo Fisher Scientific | 10437028 | |
| Ficoll-Paque PLUS 6 x 100 mL | Sigma | GE17-1440-02 | |
| GlutaMAX Supplement (100x) | Thermo Fisher Scientific | 35050079 | |
| GM-CSF | Thermo Fisher Scientific | PHC2011 | |
| Hemin BioXtra, from Porcine, ≥96.0% (HPLC) | Sigma | 51280-1G | |
| HEPES | Thermo Fisher Scientific | 15630106 | |
| Inflammatory caspase BiFC plasmids | Available by request from LBH lab | ||
| LPS-EB Ultrapure | Invivogen | TLRL-3PELPS | |
| LS Columns | Miltenyi (Biotec) | 130-042-401 | For manual separation using QuadroMACS Separator only |
| MACS 15 mL Tube Rack | Miltenyi (Biotec) | 130-091-052 | For manual separation using QuadroMACS Separator only |
| MACS MultiStand | Miltenyi (Biotec) | 130-042-303 | For manual separation using QuadroMACS Separator only |
| mCherry plasmid | Yungpeng Wang Lab | Similar plasmids that can be used as fluorescent reporters can be found on Addgene | |
| Neon Transfection System | Thermo Fisher Scientific | MPK5000 | Includes Neon electroporation device, pipette and pipette station |
| Neon Transfection System 10 µL Kit | Thermo Fisher Scientific | MPK1096 | Includes resuspension buffer R, resuspension buffer T, electrolytic buffer E, 96 x 10 µL Neon tips and Neon electroporation tubes |
| Nigericin sodium salt, ready made solution | Sigma | SML1779-1ML | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140122 | |
| Poly-D-Lysine Hydrobromide | Sigma | P7280-5mg | |
| QuadroMACS Separator | Miltenyi (Biotec) | 130-090-976 | For manual separation using QuadroMACS Separator only |
| qVD-OPh | Fisher (ApexBio) | 50-101-3172 | |
| RPMI 1640 Medium | Thermo Fisher Scientific | 11875119 | |
| Trypsin-EDTA (0.25%), phenol red | Thermo Fisher Scientific | 25200072 | |
| UltraPure 0.5 M EDTA, pH 8.0 | Thermo Fisher Scientific | 15575020 | |
| Zeiss Zen 2.6 (blue edition) software | Zeiss | Any software used to operate the confocal microscope of choice |
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