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ASC-particle-induced Peritonitis
ASC颗粒诱导腹膜炎模型的构建

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Abstract

In response to pathogen infection and tissue damage, inflammasome sensors such as NLRP3 and AIM2 are activated, which triggers PYRIN domain (PYD)-mediated ASC nucleation, followed by self-perpetuating ASC polymerization, which ultimately culminates in caspase-1 activation, interleukin (IL)-1β and IL-18 processing and release and pyroptosis (Ratsimandresy et al., 2013; Cai et al., 2014). Inflammasomes release not only cytokines, but also the polymeric ASC danger particles (pASC) by pyroptosis, which perpetuate and propagate inflammasome responses to bystander cells to engage cell intrinsic ASC and caspase-1 (Baroja-Mazo et al., 2014; Franklin et al., 2014). In this protocol we describe intraperitoneal injection of polymeric ASC particles as a danger signal and measure neutrophil infiltration and levels of the pro-inflammatory cytokine IL-1β by ELISA in the peritoneal lavage (de Almeida et al., 2015).

Keywords: Inflammasome(炎性体), Danger signal(危险信号), Inflammation(炎症), Peritonitis(腹膜炎), NLRP3(NLRP3)

Materials and Reagents

  1. 5 ml syringes (BD, catalog number: 309646 )
  2. 25 G x1 ½ needles (BD, catalog number: 305127 )
  3. Conical tubes (15 ml) (Coring, Falcon®, catalog number: 352095 )
  4. C57BL/6 mice, typically of 8-12 weeks old (male or female)
  5. 1 x 105 pASC-GFP particles generated from stable or transiently expressing HEK293 cells and sorted by flow cytometry as described (Fernandes-Alnemri et al., 2007; Fernandes-Alnemri and Alnemri, 2008)
  6. 1x Dulbecco's phosphate-buffered saline (DPBS) (Corning, catalog number: 21-030-CV )
  7. HEPES
  8. KOH
  9. Magnesium chloride (MgCl2)
  10. EGTA
  11. CHAPS
  12. cOmplete protease inhibitor cocktail (Roche Diagnostics, catalog number: 11697498001)
  13. IL-1β ELISA kit (BD, catalog number: 559603 )
  14. Lysis buffer (see Recipes)

Equipment

  1. Centrifuge
  2. Fluorescence microscope
  3. Flow cytometry
  4. Surgical instruments such as tweezers and scissors
  5. Biosafety cabinet

Procedure

  1. Isolate pASC-GFP particles from 107 HEK293 cells stable or transiently transfected with ASC-GFP as described previously (Fernandes-Alnemri et al., 2007; Fernandes-Alnemri and Alnemri, 2008; Martín-Sánchez et al., 2015) or by flow cytometry.
  2. Remove media from cells, add DPBS and gently scrape cells. Spin down cells at 1,500 x g for 5 min. Discard supernatant.
  3. Prepare total cell lysates by hypotonic lysis of cell pellets in 20 mM HEPES-KOH, pH 7.5, 5 mM MgCl2, 0.5 mM EGTA, 0.1% CHAPS, supplemented with protease inhibitors. Use a syringe with 25 G needle to lyse the cells by syringing several times. Centrifuge cell lysates full speed to obtain cell lysates supernatant.
  4. Induce aggregation of ASC-GFP by incubation of cell lysates supernatant at 37 °C for 30 min as previously described (Fernandes-Alnemri et al., 2007; Fernandes-Alnemri and Alnemri, 2008). Typical amount of ASC-GFP particles recovered is between 2 x 105 and 4 x 105 per million cells.
  5. Add a small amount of supernatant to a microscope slide to confirm ASC polymerization into 1-3 μm aggregates by fluorescence microscopy using a standard GFP excitation/emission filter set (exitation: 484 nm; emission: 507 nm) (Figure 1).
  6. Sort ASC-GFP particles (1-2 μm) by flow cytometry by gating for small particles based on forward scatter versus side scatter. Next start by gating for FITC positive particles. Later confirm ASC-GFP particles by fluorescent microscopy (Figure 1). Particles can be stored at 4 °C for one year.
  7. Intraperitoneally inject mice with 1 x 105 pASC-GFP particles or the same volume DPBS for control mice (injection volume approximately 200 μl).
  8. 4 h later sacrifice mice, cut the skin and expose the peritoneal wall (Video 1).

    Video 1. Peritoneal lavage. The video shows how to wash peritoneal cavity after injection of pASC-GFP particles to measure IL-1β by ELISA.

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  9. Inject 4 ml of DPBS into the peritoneal cavity.
  10. Shake the mouse to wash the peritoneal cavity.
  11. Use the syringe to recover DPBS injected by gradually pulling out the plunge.
  12. Remove the needle and add content to a conical tube.
  13. Spin down the cells at 1,200 x g for 5 min.
  14. Transfer supernatant to fresh conical tubes and measure IL-1β by ELISA. Typical IL-1β concentrations are up to 2 ng/ml and a representative result is shown in our previous publication (de Almeida et al., 2015).  

Representative data



Figure 1. Total cell lysates from stable ASC-GFP-expressing HEK293 cells were GFP-sorted by flow cytometry after inducing ASC polymerization and control (Ctrl) and ASC-GFP (pASC)-containing fractions analyzed by fluorescence microscopy. HEK293 cell lysates were used as a negative control.

Notes

  1. ASC-GFP particles should be protected from light until use.

Recipes

  1. Lysis buffer
    20 mM HEPES-KOH, pH 7.5
    5 mM MgCl2
    0.5 mM EGTA
    0.1% CHAPS
    Protease inhibitors

Acknowledgments

This protocol was adapted from a previously published study (de Almeida et al., 2015). This work was supported by grants the National Institutes of Health (AI099009, AI120625, AI120618 and AR064349 to C.S., AR066739 to A.D., AI120625 and AI120618 to C.S. and A.D., T32AR007611 to L.d.A. and the American Heart Association 13GRNT17110117 to C.S.).

References

  1. Baroja-Mazo, A., Martin-Sanchez, F., Gomez, A. I., Martinez, C. M., Amores-Iniesta, J., Compan, V., Barbera-Cremades, M., Yague, J., Ruiz-Ortiz, E., Anton, J., Bujan, S., Couillin, I., Brough, D., Arostegui, J. I. and Pelegrin, P. (2014). The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol 15(8): 738-748.
  2. Bondar, V. S. and Puzyr, A. P. (2000). Use of nanodiamond particles for rapid isolation of recombinant apoobelin from Escherichia coli. Dokl Biochem 373(1-6): 129-131.
  3. Cai, X., Chen, J., Xu, H., Liu, S., Jiang, Q. X., Halfmann, R. and Chen, Z. J. (2014). Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell 156(6): 1207-1222.
  4. de Almeida, L., Khare, S., Misharin, A. V., Patel, R., Ratsimandresy, R. A., Wallin, M. C., Perlman, H., Greaves, D. R., Hoffman, H. M., Dorfleutner, A. and Stehlik, C. (2015). The PYRIN domain-only protein POP1 inhibits inflammasome assembly and ameliorates inflammatory disease. Immunity 43(2): 264-276.
  5. Fernandes-Alnemri, T., Alnemri, E. S. (2008). Chapter thirteen assembly, purification, and assay of the activity of the ASC pyroptosome. Methods Enzymol 442: 251-270.
  6. Fernandes-Alnemri, T., Wu, J., Yu, J. W., Datta, P., Miller, B., Jankowski, W., Rosenberg, S., Zhang, J. and Alnemri, E. S. (2007). The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ 14(9): 1590-1604.
  7. Franklin, B. S., Bossaller, L., De Nardo, D., Ratter, J. M., Stutz, A., Engels, G., Brenker, C., Nordhoff, M., Mirandola, S. R., Al-Amoudi, A., Mangan, M. S., Zimmer, S., Monks, B. G., Fricke, M., Schmidt, R. E., Espevik, T., Jones, B., Jarnicki, A. G., Hansbro, P. M., Busto, P., Marshak-Rothstein, A., Hornemann, S., Aguzzi, A., Kastenmuller, W. and Latz, E. (2014). The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation. Nat Immunol 15(8): 727-737.
  8. Martín-Sánchez, F., Gómez, A. I. and Pelegrín, P. (2015). Isolation of particles of recombinant ASC and NLRP3. Bio-protocol 5(10): e1480.
  9. Ratsimandresy, R. A., Dorfleutner, A. and Stehlik, C. (2013). An update on PYRIN domain-containing pattern recognition receptors: from immunity to pathology. Front Immunol 4: 440.

简介

响应病原体感染和组织损伤,激活炎症小体传感器如NLRP3和AIM2,其触发PYRIN结构域(PYD)介导的ASC成核,随后是自我永存的ASC聚合,其最终终止于胱天蛋白酶-1激活,白细胞介素 IL)-1β和IL-18加工和释放和pyroptosis(Ratsimandresy等人,2013; Cai等人,2014)。 Inflammasomes不仅释放细胞因子,而且还通过pyroptosis释放聚合的ASC危险颗粒(pASC),其使得对旁观者细胞的炎症反应永久化并增殖,从而使细胞内在ASC和胱天蛋白酶-1参与(Baroja-Mazo等人 。,2014; Franklin 。。,2014)。 在该方案中,我们描述了腹膜内注射聚合物ASC颗粒作为危险信号,并通过ELISA在腹膜灌洗中测量嗜中性粒细胞浸润和促炎细胞因子IL-1β的水平(de Almeida等人, 2015)。

关键字:炎性体, 危险信号, 炎症, 腹膜炎, NLRP3

材料和试剂

  1. 5ml注射器(BD,目录号:309646)
  2. 25 G x1 ½针(BD,目录号:305127)
  3. 锥形管(15ml)(Coring,Falcon ,目录号:352095)
  4. C57BL/6小鼠,通常为8-12周龄(雄性或雌性)
  5. 1×10 5 pASC-GFP颗粒,其由稳定或瞬时表达HEK293细胞产生并通过流式细胞术分选(Fernandes-Alnemri等人,2007; Fernandes-Alnemri 和Alnemri,2008)
  6. 1×Dulbecco磷酸盐缓冲盐水(DPBS)(Corning,目录号:21-030-CV)
  7. HEPES
  8. KOH
  9. 氯化镁(MgCl 2)
  10. EGTA
  11. CHAPS
  12. cOmplete蛋白酶抑制剂混合物(Roche Diagnostics,目录号:11697498001)
  13. IL-1βELISA试剂盒(BD,目录号:559603)
  14. 裂解缓冲液(见配方)

设备

  1. 离心机
  2. 荧光显微镜
  3. 流式细胞术
  4. 外科器械如镊子和剪刀
  5. 生物安全柜

程序

  1. 如先前所述,分离用ASC-GFP稳定或瞬时转染的10 7个HEK293细胞的pASC-GFP颗粒(Fernandes-Alnemri等人,2007; Fernandes-Alnemri和Alnemri ,2008;Martín-Sánchez等人,2015)或流式细胞术。
  2. 从细胞中取出培养基,添加DPBS并轻轻刮除细胞。在1,500×g下旋转细胞5分钟。弃去上清液。
  3. 通过在补充有蛋白酶抑制剂的20mM HEPES-KOH,pH 7.5,5mM MgCl 2,0.5mM EGTA,0.1%CHAPS中的细胞沉淀的低渗裂解制备总细胞裂解物。使用带有25 G针头的注射器通过注射几次裂解细胞。离心细胞裂解物全速以获得细胞裂解物上清液
  4. 如前所述(Fernandes-Alnemri等人,2007; Fernandes-Alnemri和Alnemri,2008)通过在37℃下孵育细胞裂解物上清液诱导ASC-GFP的聚集30分钟。回收的ASC-GFP颗粒的典型量为2×10 5至4×10 5个/百万细胞。
  5. 加入少量上清液到显微镜载玻片,以确认ASC聚合成1-3微米聚集体通过荧光显微镜使用标准的GFP激发/发射滤光片集(激发:484纳米;发射:507纳米)(图1)。 />
  6. 基于前向散射与侧向散射通过对小颗粒进行门控,通过流式细胞术分选ASC-GFP颗粒(1-2μm)。接下来通过门控FITC阳性颗粒开始。随后通过荧光显微镜确认ASC-GFP颗粒(图1)。颗粒可以在4℃保存一年。
  7. 腹膜内注射1×10 5 pASC-GFP颗粒或对照小鼠相同体积的DPBS(注射体积约200μl)。
  8. 4小时后处死小鼠,切开皮肤并暴露腹膜壁(视频1)
    <! - flashid1944v100开始 - >
    视频1.腹膜灌洗。 视频显示如何在注射pASC-GFP颗粒后通过ELISA测量IL-1β来清洗腹膜腔。
    <! - [if!IE]> - <! - <![endif] - >

    要播放视频,您需要安装较新版本的Adobe Flash Player。

    获取Adobe Flash Player

    <! - [if!IE]> - >
    <! - <![endif] - >
    <! - flashid1944v100结束 - >
  9. 将4ml DPBS注入腹膜腔。
  10. 摇动鼠标以清洗腹膜腔。
  11. 使用注射器通过逐渐拔出插入物来恢复注射的DPBS
  12. 取出针头,向锥形管中加入内容物。
  13. 以1,200×g 旋转细胞5分钟。
  14. 将上清转移到新鲜的锥形管中,并通过ELISA测量IL-1β。 典型的IL-1β浓度高达2ng/ml,并且在我们先前的出版物(de Almeida等人,2015)中显示了代表性的结果。  

代表数据



来自稳定的表达ASC-GFP的HEK293细胞的总细胞裂解物在诱导ASC聚合后通过流式细胞术进行GFP分选,并且通过荧光显微镜分析对照(Ctrl)和含ASC-GFP(pASC)的级分。/strong> HEK293细胞裂解物用作阴性对照。

笔记

  1. ASC-GFP颗粒应避光使用。

食谱

  1. 裂解缓冲液
    20mM HEPES-KOH,pH 7.5 5mM MgCl 2/
    0.5mM EGTA 0.1%CHAPS
    蛋白酶抑制剂

致谢

该方案改编自以前发表的研究(de Almeida等人,2015)。 这项工作由国家卫生研究院(AI099009,AI120625,AI120618和AR064349到C.S.,AR066739到A.D.,AI120625和AI120618到C.S.和A.D.,T32AR007611到L.d.A.和美国心脏协会13GRNT17110117到C.S.)支持。

References

  1. Baroja-Mazo, A., Martin-Sanchez, F., Gomez, A. I., Martinez, C. M., Amores-Iniesta, J., Compan, V., Barbera-Cremades, M., Yague, J., Ruiz-Ortiz, E., Anton, J., Bujan, S., Couillin, I., Brough, D., Arostegui, J. I. and Pelegrin, P. (2014). The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol 15(8): 738-748.
  2. Bondar, V. S. and Puzyr, A. P. (2000). Use of nanodiamond particles for rapid isolation of recombinant apoobelin from Escherichia coli. Dokl Biochem 373(1-6): 129-131.
  3. Cai, X., Chen, J., Xu, H., Liu, S., Jiang, Q. X., Halfmann, R. and Chen, Z. J. (2014). Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell 156(6): 1207-1222.
  4. de Almeida,L.,Khare,S.,Misharin,AV,Patel,R.,Ratsimandresy,RA,Wallin,MC,Perlman,H.,Greaves,DR,Hoffman,HM,Dorfleutner,A.and Stehlik, (2015)。  PYRIN仅结构域蛋白POP1抑制炎症小体组装和改善炎性疾病。 免疫 43(2):264-276。
  5. Fernandes-Alnemri,T.,Alnemri,ES(2008)。  焦斑病:ASC二聚体的超分子组装体介导炎症细胞死亡胱天蛋白酶-1激活。 细胞死亡差异 14(9):1590-1604。
  6. Franklin,BS,Bossaller,L.,De Nardo,D.,Ratter,JM,Stutz,A.,Engels,G.,Brenker,C.,Nordhoff,M.,Mirandola,SR,Al- Amoudi, Mangan,MS,Zimmer,S.,Monks,BG,Fricke,M.,Schmidt,RE,Espevik,T.,Jones,B.,Jarnicki,AG,Hansbro,PM,Busto,P.,Marshak-Rothstein,A 。,Hornemann,S.,Aguzzi,A.,Kastenmuller,W. and Latz,E。(2014)。  衔接子ASC具有增殖炎症的细胞外和"朊病毒"活性。 Nat Immunol 15(8):727-737 。
  7. Martín-Sánchez,F.,Gómez,AI和Pelegrín,P.(2015)。  重组ASC和NLRP3的颗粒的分离。 生物协议 5(10):e1480。
  8. Ratsimandresy,RA,Dorfleutner,A.和Stehlik,C.(2013)。  含有PYRIN结构域的模式识别受体的更新:从免疫到病理。前免疫 4:440。
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引用:de Almeida, L., Dorfleutner, A. and Stehlik, C. (2016). ASC-particle-induced Peritonitis. Bio-protocol 6(19): e1944. DOI: 10.21769/BioProtoc.1944.
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