激活和NLRP3炎性活性的人单核细胞来源的树突状细胞用IL-1β测量
Summary
树突状细胞（DC）分泌IL-1β响应于TLR8识别合成嘌呤，R848，接着NLRP3炎性体激活与尼日利亚菌素，因此，IL-1β可以用来测量NLRP3炎性活性。细胞内细胞因子染色法，免疫印迹和ELISA法用于准确地测量通过IL-1β表达NLRP3炎性引发和活化。
Abstract
从白细胞介素的分泌素（IL）产生的炎症过程-1家族细胞因子由免疫细胞引起局部或全身炎症反应，组织重塑和修复，以及病毒学控制1，2。白细胞介素-1β是先天免疫反应的基本要素，并有助于消除侵入的病原体，同时防止建立持续性感染1-5中。
Inflammasomes是关键信令平台白细胞介素1转化酶（ICE或胱天蛋白酶-1）的激活。在NLRP3炎性需要在DC中的至少两个信号，使IL-1β分泌6。亲IL-1β蛋白表达在静息细胞限制;因此，一个起动信号所必需的IL-1β的转录和蛋白表达的影响。第二信号中的多蛋白NLRP3炎性体的形成感测由NLRP3结果。树突状细胞，以respon的能力d，来所需的IL-1β分泌的信号可以使用合成嘌呤，R848，这是由TLR8在树突状细胞（moDCs），以素细胞人单核细胞检测到，随后激活NLRP3炎性体与细菌毒素进行试验和钾离子载体，尼日利亚。
单核细胞来源的DC很容易产生文化，并提供比纯化的人骨髓DC显著更多的细胞。这里介绍的方法不同于其他炎性测定法，它使用在体外人，而不是小鼠衍生的DC因此允许在人类疾病和感染的炎性体的研究。
Introduction
先天免疫系统的活化是必需的感染，疾病，和疫苗接种7中引导适应性免疫应答。树突状细胞是最有效的抗原呈递的先天免疫系统的细胞;它们是专门用于摄取抗原，迁移到淋巴结，并激活幼稚CD4 +和细胞毒性CD8 + T细胞8-10。启用快速病原体检测的先天免疫系统利用众多种系编码的模式识别受体（PRR），识别保守的病原体衍生的基序或细胞应激和损伤的宿主衍生的标记。 Toll样受体（TLRs）是膜结合的模式识别受体识别某些胞吞噬病原体相关的分子模式（PAMP）和相关联的危险性分子模式（D-AMPS）。通过样受体（最接近现代种）对比点头是细胞质和的PAMP和阻尼的多元化作出回应。点头样受体重新目前抵御病原体逃避细胞表面和细胞内吞猪蓝耳病的第二行。病原体衍生的，或“危险”的相互作用有关，与TLR和NLR配体因素导致DC成熟而导致与其他免疫细胞和促进T细胞和自然杀伤细胞活化11增加直流互动的状态。
白细胞介素-1β是对感染的宿主防御的重要组成部分。在识别微生物，高促炎性细胞因子，IL-1β，分泌和功能的化学引诱剂和先天免疫和适应性免疫细胞激活剂， 在体内的IL-1β主要是负责对急性期反应包括发热和炎症细胞因子合成12。
最最接近现代种含有被认为在配体感应，中央核苷酸结合域（NACHT）是IMPO运作一个C端富含亮氨酸重复结构域rtant为NLRP3低聚和N末端效应器结构域（PYD在NLRP3），通过蛋白蛋白相互作用介导的信号转导至下游目标。在NLRP3蛋白质定义了最强烈的研究炎性复杂。这种蛋白质是NLR家族的成员，并具有以形成多分子蛋白复合物NLRP3，适配器蛋白PYCARD（也称为ASC）和ICE组成的能力。当炎性激活PYCARD结合NLRP3 N端结构域，并通过caspase活化和募集结构域（CARD）域新兵冰。白细胞介素-1转换酶最初产生为包含CARD的动机在其N-末端的酶原。炎性形成的结果使二ICE分子充分接近诱导其催化活性。炎性复合物是必要的激活ICE从而允许它来转换细胞质亲IL-1β成熟的细胞因子。
白细胞介素的分泌成功-1β在区议会需要检测两个不同的独立危险信号。首先，TLR传感的PAMP，DAMPS，或细胞因子信号传导（TNFα或IL-1β）的导致细胞质亲IL-1β蛋白表达的上调。第二，往往是不同的，信号所必需的炎性复合物的形成ICE成熟的上游。一些炎性刺激信号包括细菌膜孔形成毒素（如尼日利亚菌素），溶酶体破坏晶体（如单钠尿酸盐结晶，密歇根州立大学），和细胞外的ATP。上游机制领先了这些不同的激活剂，以NLRP3炎性体的激活目前还不清楚。调查研究炎性体形成的信令上游建议，细胞内的事件，如感应低钾血症或活性氧（ROS）的间接激活炎性13-28。
当中的NLRP3炎性体的不同的病毒活化是流感，它提供了bOTH所需的IL-1β分泌3，29-33初级和次级信号。使用鼠标NLRP3基因敲除模型发现，IL-1β分泌的DC是NLRP3依赖32。此外，NLRP3基因敲除小鼠引起的白细胞减少感染的部位和有经验的死亡率较高2，5。最近的两篇论文提出了一种机制，在流感病毒感染NLRP3炎性体的激活;首先，通过识别病毒RNA由TLR7或TLR8（取决于响应细胞的TLR表达）或通过传感共生细菌被其他的TLR诱导细胞质亲IL-1β的表达，随后的第二信号，激活NLRP3的吸反式高尔基网络33，34上形成炎性由病毒离子通道蛋白M2。在后者的步骤，触发NLRP3炎性体是由细胞内离子的干扰来实现<em>的环境导致ROS的产生，这是简单地说，通过NLPR3感觉到作为一个信号，以形成炎性。不过，ICE活性流感感染过程中炎性激活上游的确切机制仍不清楚。
这个工作描述了一种技术，用于研究NLRP3炎性体在人类moDCs可以用作用于通过一个很好的途径相关的DC基于响应于TLR8结扎IL-1β分泌与R848然后激活炎性体的进一步研究的基础有价值已知的激活NLRP3的，尼日利亚。这种方法的变体可以与其它细胞类型，包括可以使用，但不限于：单核细胞，巨噬细胞，以及其他DC亚群，和上皮细胞。
Protocol
Representative Results
Discussion
炎性细胞因子是一体的操纵先天和适应性免疫反应对抗病毒感染。分泌IL-1β已经证明流感病毒感染3，43，44中增加。由这些细胞因子响应于识别病毒在人树突状细胞加工的精确机制尚不完全清楚。骨髓DC分离试剂盒是昂贵和费时的。分离试剂盒和足总杯的排序可能会无意间应力或激活细胞。此外，还有经常分离的细胞用于实验的量不足。 在体外培养 45，46人原髓细胞DC的还好…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢奥利维尔曼彻斯，博士，达沃尔Frleta博士和梅根奥布莱恩博士的支持和反馈。这项研究是由过敏和传染病国家研究所的支持，并完成了与美国国立卫生研究院授予露丝属Kirschstein国家研究服务奖个人博士前奖学金（F31），以促进多样性与健康有关的研究（AI089030）和RO1（AI081848资金）。
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| IL-4 | R&D | ||
| GM-CSF | Genzyme | NDC 58468-0180-2 | We acquire this item through our local pharmacy with a prescription |
| RPMI 1640 with L-glutamine | Cellgro | 10-040-CV | |
| Peripheral blood mononuclear cells | New York Blood Center | PBMCs were isolated from the blood of healthy donors | |
| 12-well tissue culture plates | Sigma-Aldrich | 3516 | |
| 96-well round bottom tissue culture plates | Sigma-Aldrich | 3799 | |
| α-IL-1β-FITC | R&D | IC201F | |
| FITC isotype control | Miltenyi Biotec | 130-092-213 | |
| α-β-Tubulin | Santa Cruz | SC-9014 | |
| α-IL-1β | R&D | mab201 | |
| PVDF Immobilon-FL membrane | Millipore | IPFL00010 | |
| gradient 4-12 % polycrylamide gel | Bio Rad | 161-1159 | |
| laemmli sample buffer | Bio Rad | 161-0737 | |
| BSA | Equitech Bio Inc | 30% solution sterile/filtered | |
| PFA | Electron Microscopy Sciences | 15710 | 16% solution |
| human inflammatory cytokine bead array kit | BD | 551811 | |
| nigericin | Invivogen | tlrl-nig | |
| R848 | 3M Corp. | ||
| α-CD14 | BD | 340436 | |
| α-CD11c | BD | 555392 | |
| β-mercaptoethanol | Sigma-Aldrich | M6250-10ML | |
| TBS | On site stock room | ||
| Tween-20 | Sigma-Aldrich | P2287-100mL | |
| Nunc EasYFlask 225cm2, Filter Cap, 70mL working volume, 30/Cs | Thermo Scientific | 159933 | |
| 20 μM Sterile Disposable Filter Units | Thermo Scientific | 569-0020 | |
| Gentamicin | Invitrogen | 15750060 | |
| Hepes | Invitrogen | 15630080 | |
| goat α-mouse IRDye 800CW | Licor | 926-32210 | |
| donkey α-rabbit IRDye 680RD | Licor | 926-68073 | |
| Spectra multicolor broad range protein ladder | Thermo Scientific | 26634 | |
| Tris Glycine SDS 10x | Bio Rad | 1610732 | |
| Tris Glycine 10x | Bio Rad | 161-0734 | |
| Methanol – 4L | Fisher Scientific | A433P-4 | |
| Prolong Gold antifade Reagent with DAPI | Life Technologies | P-36931 | |
| 8 chamber polystyrene vessel tissue culture treated glass slide | BD Falcon | 354108 | |
| Poly-L-Lysine | Sigma | P4707 |
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