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Reconstruction of the Mouse Inflammasome System in HEK293T Cells
HEK293T 细胞中炎症小体系统的重建   

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Abstract

The NLRP3 (NLR family, Pyrin domain containing 3) inflammasome is a multiprotein complex comprised of NLRP3, pro-caspase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and the protein kinase NIMA related kinase 7 (NEK7) (Shi et al., 2016; He et al., 2016; Schmid-Burgk et al., 2016). When cells are exposed to microbes and/or danger signals, the inflammasome assembles and serves as a platform for the activation of caspase-1. Caspase-1 activation promotes the processing and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β), IL-18, and IL-33 as well as pyroptosis induction (Gross et al., 2011; Arend et al., 2008), which elicit inflammatory responses. Here, we describe how to co-transfect the NLRP3 inflammasome components into HEK293T cells, which enables inflammasome activation and the production of IL-1β upon stimulation with nigericin.

Background

Inflammasomes are multiprotein complexes that control inflammatory responses and coordinate immune responses against invading microbes. Reconstitution of the NLRP3 inflammasome in vitro provides an easy and efficient way to study the regulation of inflammasome activation. In this protocol, NEK7 was introduced into the in vitro NLRP3 inflammasome system and the ratio among the NLRP3 inflammasome components was optimized, making the reconstituted NLRP3 inflammasome more similar to the physiological inflammasome in vivo. Nigericin was used to activate the inflammasome as we have observed that it induces a rapid rate of IL-1β secretion compared to other inflammasome activators. Using this protocol, the levels of IL-1β can be assayed to determine NLRP3 inflammasome function under physiological conditions as well as after gene knockdown or overexpression.

Materials and Reagents

  1. Costar 24 well clear TC-treated multiple well plate (Corning, Costar®, catalog number: 3527 )
  2. 1.5 ml Eppendorf tubes
  3. HEK293T cells (ATCC, catalog number: CRL-11268 )
  4. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 10569-044 )
  5. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
  6. Penicillin-streptomycin (10,000 U/ml; 10,000 μg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-163 )
  7. Pro-IL-1β-Flag, NLRP3-Flag, ASC1-Flag, pro-caspase-1-Flag and NEK7-HA plasmids
    Note: Pro-IL-1β, NLRP3, ASC1, pro-caspase-1, and NEK7 were amplified by standard PCR techniques and were subsequently inserted into mammalian expression vectors using the In-Fusion® HD cloning kit per manufacturer’s instructions (click here for a detailed protocol and Shi et al., 2016). All plasmids were submitted to Addgene.
    pCMV-pro-IL1β-C-Flag (Addgene, catalog number: 75131 )
    pcDNA3-N-Flag-NLRP3 (Addgene, catalog number: 75127 )
    pcDNA3-N-Flag-ASC1 (Addgene, catalog number: 75134 )
    pcDNA3-N-Flag-Caspase-1 (Addgene, catalog number: 75128 )
    pcDNA3-N-HA-NEK7 (Addgene, catalog number: 75142 )
  8. IL-1β ELISA Kit (affymetrix ,eBioscience, catalog number: 88-7013-76 )
  9. Plasmid Plus Midi Kit (QIAGEN, catalog number: 12943 )
  10. LB medium (MP Bio, catalog number: 3002-31 )
  11. Opti-MEM® (Thermo Fisher Scientific, GibcoTM, catalog number: 51985-034 )
  12. Lipofectamine® 2000 transfection reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668-019 )
  13. Nigericin (Sigma-Aldrich, catalog number: N7143-10MG )
  14. Ethanol
  15. SuperSignalTM West Pico chemiluminescent substrate (Thermo Fisher Scientific)
  16. DMEM cell culture medium (see Recipes)
  17. Nigericin stock solution (see Recipes)

Equipment

  1. 37 °C, 5% CO2 forced-air incubator (Thermo Fisher Scientific, Fisher Scientific, model: Sanyo Incubator Panasonic MCO-19AIC(UV) CO2 )
  2. Shaker incubator (Eppendorf, model: New Brunswick I24 )
  3. Pipettes (Mettler-Toledo, ShopRainin)
  4. Microcentrifuge (Eppendorf, model: Centrifuge 5424 )
  5. ELISA plate reader (Bio Tek Instruments, model: Synergy Neo2 Multi-Mode Reader )
  6. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDrop 2000c Spectrophotometer )

Software

  1. GraphPad Prism 6 software (http://www.graphpad.com/scientific-software/prism/)

Procedure

  1. Plate HEK293T cells in 24-well microplates at a density of 2 x 105 cells per well in 0.5 ml complete DMEM cell culture medium and incubate at 37 °C in a forced-air incubator overnight.
  2. Extract the pro-IL-1β-Flag, NLRP3, ASC, pro-caspase-1 and NEK7 plasmids from their respective bacterial cultures (35 ml) by using a QIAGEN Plasmid Plus Midi Kit according to the manufacturer’s instructions.
    Note: Click here for a detailed protocol on how to prepare a liquid culture of the Addgene plasmid. Briefly, add 35 ml of LB medium (with the appropriate antibiotic) to a tube or flask for each Addgene plasmid. Inoculate the LB medium using a sterile pipette tip and incubate 12-18 h at 37 °C in a shaking incubator.
  3. In 1.5 ml Eppendorf tubes, dilute 1.5 μl of Lipofectamine® 2000 in 100 μl Opti-MEM per well.
  4. In 1.5 ml Eppendorf tubes, dilute the pro-IL-1β-Flag (200 ng/well), ASC (20 ng/well), pro-caspase-1 (100 ng/well), NLRP3 (200 ng/well) and NEK7 (200 ng/well) plasmids in 100 μl Opti-MEM.
    Note: Avoid transfecting more than the amount indicated for the ASC plasmid. Excess ASC promotes self-aggregation and induces NLRP3-independent caspase-1 activation.
  5. Add the diluted DNA to the diluted Lipofectamine® 2000 (1:1 ratio), incubate for 5 min at room temperature, and then add the DNA-lipid complex to the HEK293T cells in 0.5 ml DMEM cell culture medium.
  6. 24-36 h after transfection, replace the medium with 250 μl DMEM cell culture medium.
  7. Supernatants are collected 6-12 h after media change.
  8. Optional: To stimulate the NLRP3 inflammasome, 10 μg/ml nigericin can be directly added to the 250 μl DMEM media one hour before supernatant collection (step 7).
  9. ELISA (or Western blot) to assay the amount of mature IL-1β in the supernatants. Perform ELISA according to the manufacturer’s instructions.
    Note: When using Western blot to detect the mature IL-1β, the supernatants do not need to be concentrated. Mature IL-1β in 15 μl of supernatant could be detected by SuperSignalTM West Pico chemiluminescent substrate (Thermo Fisher Scientific).

Data analysis

ELISA data should be collected from a minimum of two independent experiments with at least 3 replicates per treatment. Figure 1 shows representative ELISA data after transfection of the indicated plasmids into HEK293T cells. The statistical significance of the differences between samples can be determined with GraphPad Prism 6 software and Student’s t-test (unpaired, two-tailed). A P value of < 0.05 is considered statistically significant.



Figure 1. Levels of secreted IL-1β from reconstituted HEK293T cells. Transfected components in each sample are indicated below the X-axis. 50 μl supernatants of each HEK293T cell culture sample were used for the IL-1β ELISA. Representative data were graphed using GraphPad Prism 6 software.

Recipes

  1. DMEM cell culture medium
    Mix 500 ml DMEM with 50 ml FBS and 5 ml penicillin-streptomycin (10,000 U/ml; 10,000 μg/ml)
    Store at 4 °C until use
  2. Nigericin stock solution
    Dissolve 10 mg nigericin in 1 ml pure ethanol and mix thoroughly 
    Store at -80 °C until use

Acknowledgments

This protocol was adapted from the previously published study, Lu et al. (2012) and was performed by Shi et al. (2016). This work was supported by the US National Institutes of Health (U19 AI100627).

References

  1. Arend, W. P., Palmer, G. and Gabay, C. (2008). IL-1, IL-18, and IL-33 families of cytokines. Immunol Rev 223: 20-38.
  2. Gross, O., Thomas, C. J., Guarda, G. and Tschopp, J. (2011). The inflammasome: an integrated view. Immunol Rev 243(1): 136-151.
  3. He, Y., Zeng, M. Y., Yang, D., Motro, B. and Núñez, G. (2016). NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature 530(7590): 354-357.
  4. Lu, B., Nakamura, T., Inouye, K., Li, J., Tang, Y., Lundback, P., Valdes-Ferrer, S. I., Olofsson, P. S., Kalb, T., Roth, J., Zou, Y., Erlandsson-Harris, H., Yang, H., Ting, J. P., Wang, H., Andersson, U., Antoine, D. J., Chavan, S. S., Hotamisligil, G. S. and Tracey, K. J. (2012). Novel role of PKR in inflammasome activation and HMGB1 release. Nature 488(7413): 670-674.
  5. Schmid-Burgk, J. L., Chauhan, D., Schmidt, T., Ebert, T. S., Reinhardt, J., Endl, E. and Hornung, V. (2016). A genome-wide CRISPR (clustered regularly interspaced short palindromic repeats) screen identifies NEK7 as an essential component of NLRP3 inflammasome activation. J Biol Chem 291(1): 103-109.
  6. Shi, H., Wang, Y., Li, X., Zhan, X., Tang, M., Fina, M., Su, L., Pratt, D., Bu, C. H., Hildebrand, S., Lyon, S., Scott, L., Quan, J., Sun, Q., Russell, J., Arnett, S., Jurek, P., Chen, D., Kravchenko, V. V., Mathison, J. C., Moresco, E. M., Monson, N. L., Ulevitch, R. J. and Beutler, B. (2016). NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component. Nat Immunol 17(3): 250-258.

简介

核糖体足迹或Ribo-seq,彻底改变了翻译研究。它最初是为培养中的酵母和哺乳动物细胞开发的(Ingolia等人,2009)。本文中,我们描述了来自先前公开的多核糖体分离程序的植物优化的亲手核糖体印迹方案(Ingolia等人,2009; Mustroph等人,2009 )和核糖体印迹(Ingolia等人,2009; Ingolia等人,2013)。使用该协议,我们能够成功地分离和分析来自体外生长的拟南芥植物(黑暗生长的幼苗)的不同阶段的高质量核糖体印迹[Merchante < ,以及在液体培养物中生长的板和植物中的13天龄小植物[未发表的结果])。

[背景] 翻译在调节基因活性中的中心作用早已被公认,但是在响应于特定刺激的全基因组翻译定量变化的系统探索最近才变得技术上可行。最初为培养中的酵母和哺乳动物细胞开发的核糖体印迹技术(通常称为Ribo-seq)已经彻底改变了翻译调节和基因表达的研究,因为其允许确定核糖体在基因组 - 大规模和单密码子分辨率(Ingolia等人,2009)。在开发Ribo-seq之前,用于研究植物中翻译调节的最常见方法是通过蔗糖梯度离心或翻译核糖体亲和纯化(TRAP)分离多核糖体RNA,然后进行Northern印迹,qRT-PCR,微阵列或RNA-seq。称为多核糖体分析的第一种方法依赖于通过超速离心在蔗糖梯度上分离不同的多聚体部分(Branco-Price等人,2008; Missra和vonArnim,2014; Li等人al 。,2015)。通过比较不同的植物生长条件或突变体,可以推断翻译速率的变化从观察多态性部分之间mRNAs分布的变化。例如,如果转录物在单体部分中变得更丰富,伴随着更高级多核糖体的减少,则认为该mRNA的翻译被下调。然而,这种技术的关键限制是其低分辨率的高阶多核糖体(和因此翻译的温和的定量变化经常错过)和无法区分多糖体RNAs经历主动翻译与装载被拘留的核糖体(为例如,那些"卡在"转录物的上游开放阅读框中)。第二多核糖RNA分离技术TRAP基于表位标记的核糖体蛋白的稳定表达,随后是整个核糖体及其相关mRNA的免疫沉淀(Zanneti等人,2005; Reynoso 等。,2015)。虽然后一种方法适应于翻译的全局和组织特异性研究(通过以普遍存在与组织特异性方式驱动标记的核糖体蛋白的表达来实现),但是其使用限于可以产生转基因品系的可转化品种。此外,TRAP样品本身的转录组分析不提供翻译的定量测量(除非与Ribo-seq [Juntawong等人,2014]一起),作为任何具有与其结合的一个或多个核糖体的mRNA将通过TRAP纯化。此外,由于TRAP依赖于表位标记,并且标签可能干扰核糖体的功能,所以TRAP转基因株系中某些mRNA的翻译调节可能被破坏,例如,由于降低标记的核糖体在翻译的某些阶段与特定蛋白质缔合的能力。 TRAP的另一个限制是其通常使用核糖体蛋白的特定的冗余同工型用于标记,例如RPL18,因此可能仅纯化携带该RPL18变体的核糖体的子集。鉴于植物基因组中存在多个RPL18样蛋白,使用一种特定核糖体蛋白同种型标记缺失利用替代RPL18同种型的核糖体。
  我们的研究选择的方法Ribo-seq不涉及转基因系生成或亲和纯化,因此避免了上述早期技术的许多限制。最重要的是,核糖体足迹技术的单一密码子决议允许研究人员映射mRNAs上的核糖体,从而清楚地区分拥有生产性核糖体翻译主要基因开放阅读框架的转录本与非生产性核糖体相关的转录本在5'UTR。这个方法不仅关闭...

材料和试剂

  1. Costar 24孔透明TC处理的多孔板(Corning,Costar ,目录号:3527)
  2. 1.5 ml Eppendorf管
  3. HEK293T细胞(ATCC,目录号:CRL-11268)
  4. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM ,目录号:10569-044)
  5. 胎牛血清(FBS)(Gemini Bio Products,目录号:100-106)
  6. 青霉素 - 链霉素(10,000U/ml;10,000μg/ml)(Thermo Fisher Scientific,Gibco TM ,目录号:15140-163)
  7. Pro-IL-1β-标志,NLRP3-Flag,ASC1-Flag,半胱天冬酶-1标志和NEK7-HA质粒。 注意:通过标准PCR技术扩增Pro-IL-1β,NLRP3,ASC1,半胱天冬酶-1和NEK7,随后使用In-Fusion?HD克隆试剂盒根据制造商的说明书将其插入哺乳动物表达载体中点击这里查看详细的协议,Shi等人,2016)。所有质粒都提交给Addgene。
    pCMV-pro-IL1β-C-Flag(Addgen,目录号:75131)
    pcDNA3-N-Flag-NLRP3(Addgen,目录号:7512??7) pcDNA3-N-Flag-ASC1(Addgen,目录号:75134)
    pcDNA3-N-Flag-Caspase-1(Addgen,目录号:7512??8) pcDNA3-N-HA-NEK7(Addgen,目录号:75142)
  8. IL-1βELISA试剂盒(affymetrix,eBioscience,目录号:88-7013-76)
  9. Plasmid Plus Midi Kit(QIAGEN,目录号:12943)
  10. LB培养基(MP Bio,目录号:3002-31)
  11. Opti-MEM (Thermo Fisher Scientific,Gibco TM ,目录号:51985-034)
  12. Lipofectamine 2000转染试剂(Thermo Fisher Scientific,Invitrogen TM ,目录号:11668-019)
  13. 尼日利亚霉素(Sigma-Aldrich,目录号:N7143-10MG)
  14. 乙醇
  15. SuperSignal TM West Pico化学发光底物(Thermo Fisher Scientific)
  16. DMEM细胞培养基(见配方)
  17. 尼日利亚储备液(见配方)

设备

  1. 37℃,5%CO 2强制空气培养箱(Thermo Fisher Scientific,Fisher Scientific,型号:Sanyo Incubator Panasonic MCO-19AIC(UV)CO 2) />
  2. 摇床培养箱(Eppendorf,型号:New Brunswick I24)
  3. 移液器(Mettler-Toledo,ShopRainin)
  4. 微量离心机(Eppendorf,型号:Centrifuge 5424)
  5. ELISA板读数器(Bio Tek Instruments,型号:Synergy Neo2多模式读数器)
  6. 分光光度计(Thermo Fisher Scientific,Thermo Scientific ,型号:NanoDrop 2000c分光光度计)

软件

  1. GraphPad Prism 6软件( http://www.graphpad.com/科学软件/棱镜/

程序

  1. 将24孔板中的HEK293T细胞以0.5x10 4个细胞/孔的密度在0.5ml完全DMEM细胞培养基中以2×10 5个细胞的密度铺板,并在37℃在强制空气培养箱中孵育过夜。 />
  2. 通过使用QIAGEN Plasmid Plus Midi Kit根据制造商的说明。 > 注意:点击此处  关于如何制备Addgene质粒的液体培养物的详细方案。简言之,向每个Addgene质粒的管或烧瓶中加入35ml LB培养基(具有合适的抗生素)。接种LB培养基使用无菌移液管尖端和培养在振荡培养箱中在37°C 12-18小时。
  3. 在1.5ml Eppendorf管中,在每孔100μlOpti-MEM中稀释1.5μlLipofectamine 2000。
  4. 在1.5ml Eppendorf管中,稀释前IL-1β-标志(200ng /孔),ASC(20ng /孔),胱天蛋白酶原-1(100ng /孔),NLRP3(200ng /孔) NEK7(200ng /孔)质粒在100μlOpti-MEM中 注意:避免转染超过ASC质粒指示的量。过量的ASC促进自身聚集并诱导NLRP3依赖性胱天蛋白酶-1激活。
  5. 将稀释的DNA加入到稀释的Lipofectamine 2000(1:1比例)中,在室温下孵育5分钟,然后将DNA-脂质复合物加入到0.5ml DMEM细胞培养物中的HEK293T细胞中中等
  6. 转染后24-36小时,用250μlDMEM细胞培养基更换培养基
  7. 培养基更换后6-12小时收集上清液
  8. 可选:为了刺激NLRP3炎症小体,在上清液收集(步骤7)之前1小时可以将10μg/ml尼日利亚菌素直接加入到250μlDMEM培养基中。
  9. ELISA(或Western印迹)来测定上清液中成熟IL-1β的量。根据制造商的说明进行ELISA。
    注意:当使用Western印迹检测成熟IL-1β时,上清液不需要浓缩。通过SuperSignalTM West Pico化学发光底物(Thermo Fisher Scientific)可以检测15μl上清液中的成熟IL-1β。

数据分析

ELISA数据应从最少两次独立实验收集,每次处理至少重复3次。图1显示了将所示质粒转染到HEK293T细胞中后的代表性ELISA数据。样品之间的差异的统计学显着性可以用GraphPad Prism 6软件和Student's t-检验(未配对,双尾)确定。 P 值< 0.05被认为具有统计显着性


图1.来自重建的HEK293T细胞的分泌的IL-1β的水平。每个样品中的转染组分在X轴下方指示。将每种HEK293T细胞培养物样品的50μl上清液用于IL-1βELISA。使用GraphPad Prism 6软件绘制代表性数据。

食谱

  1. DMEM细胞培养基
    将500ml DMEM与50ml FBS和5ml青霉素 - 链霉素(10,000U/ml;10,000μg/ml)混合
    在4°C储存,直到使用
  2. 尼日利亚储备液
    将10 mg尼日利亚菌素溶解在1 ml纯乙醇中,然后充分混匀
    储存于-80℃直至使用

致谢

该方案改编自先前公开的研究,Lu等人。 (2012),并由Shi等人进行。 (2016年)。这项工作得到了美国国家卫生研究院(U19 AI100627)的支持。

参考文献

  1. Arend,WP,Palmer,G。和Gabay,C.(2008)。  IL-1,IL-18和IL-33家族的细胞因子。 Immunol Rev 223:20-38。
  2. Gross,O.,Thomas,CJ,Guarda,G.and Tschopp,J.(2011)。  inflammasome:综合视图 Immunol Rev 243(1):136-151。
  3. He,Y.,Zeng,MY,Yang,D.,Motro,B.和Nú?ez,G。(2016)。  NEK7是NLRP3激活下游钾流出的必要介质。 530(7590):354-357。
  4. Lu,B.,Nakamura,T.,Inouye,K.,Li,J.,Tang,Y.,Lundback,P.,Valdes- Ferrer,SI,Olofsson,PS,Kalb,T.,Roth, Zou,Y.,Erlandsson-Harris,H.,Yang,H.,Ting,JP,Wang,H.,Andersson,U.,Antoine,DJ,Chavan,SS,Hotamisligil,GSand Tracey,KJ(2012)。   PKR在炎症小体激活和HMGB1释放中的新作用。/a> 自然 488(7413):670-674。
  5. Schmid-Burgk,JL,Chauhan,D.,Schmidt,T.,Ebert,TS,Reinhardt,J.,Endl,E。和Hornung,V。(2016)。  全基因组CRISPR(聚簇定期间隔短回文重复序列)筛选鉴定NEK7是NLRP3炎症小体激活的必要组分。  J Biol Chem   291(1):103-109。
  6. Shi,H.,Wang,Y.,Li,X.,Zhan,X.,Tang,M.,Fina,M.,Su,L.,Pratt,D.,Bu,CH,Hildebrand, ,S.,Scott,L.,Quan,J.,Sun,Q.,Russell,J.,Arnett,S.,Jurek,P.,Chen,D.,Kravchenko,VV,Mathison,JC,Moresco,EM ,Monson,NL,Ulevitch,RJ和Beutler,B。(2016)。  NLRP3激活和有丝分裂是由NEK7(一种新的炎症组件)协调的互斥事件。 Nat Immunol 17(3):250-258。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Shi, H., Murray, A. and Beutler, B. (2016). Reconstruction of the Mouse Inflammasome System in HEK293T Cells. Bio-protocol 6(21): e1986. DOI: 10.21769/BioProtoc.1986.
提问与回复

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当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。