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In vivo Analysis of Neutrophil Infiltration during LPS-induced Peritonitis
LPS诱导腹膜炎过程中嗜中性粒细胞免疫浸润分析   

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Abstract

Bacterial lipopolysaccharide (LPS) is present in the outer membrane of Gram-negative bacteria and functions as pathogen-associated molecular pattern (PAMP) (Whitfield and Trent, 2014). LPS therefore is a potent activator of inflammatory responses leading to cytokine release and neutrophils recruitment. The lipid A moiety of LPS activates the complex consisting of the LPS binding protein (LBP), CD14, MD-2 and Toll-like receptor 4 (TLR4) and the non-canonical inflammasome-linked caspases-4, 5 and 11, which in turn activate the canonical NLRP3 inflammasome (Shi et al., 2014; Hagar et al., 2013; Kayagaki et al., 2013; Hoshino et al., 1999; Poltorak, 1998; Nagai et al., 2002; Park et al., 2009; Ratsimandresy et al., 2013). In particular, the cytokine interleukin (IL)-1β produced in response to inflammasome activation has a crucial role in neutrophil recruitment through promoting neutrophil adhesion and migration (McDonald et al., 2010).This protocol allows studying of inflammatory response induced by LPS that affect neutrophil infiltration by tracking myeloperoxidase (MPO) activity in vivo (de Almeida et al., 2015).

Keywords: Inflammation(炎症), Peritonitis(腹膜炎), Sepsis(脓毒症), in vivo imaging(活体成像), Myeloperoxidase(髓过氧化物酶)

Materials and Reagents

  1. Insulin syringes (Thermo Fisher Scientific, catalog number: 14-841-31 )
  2. 0.22 μm filters
  3. C57BL/6 mice, typically of 8-12 weeks old mice (male or female)
  4. LPS E.coli 0111:B4 (Sigma-Aldrich, catalog number: L2630-100MG )
  5. Dulbecco's phosphate-buffered saline (DPBS) (Corning, catalog number: 21-030-CV )
  6. XenoLight RediJect inflammation probe (PerkinElmer, catalog number: 760535 )
  7. Luminol sodium salt (Sigma-Aldrich, catalog number: A4685 )
  8. Isofluorane (Henry Schein, IsothesiaTM, catalog number: 10014450 )
  9. 5 mg/ml LPS (see Recipes)
  10. 20 mg/ml luminol sodium salt stock solution (see Recipes)

Equipment

  1. Anesthesia machine (VetEquip, model: 901808 ) or similar anesthesia equipment
  2. Rechargeable trimmer (Braintree Scientific, catalog number: VLP-323 75 )
  3. Scale (Kent Scientific, catalog number: SCL66110 )
  4. Biosafety cabinet
  5. IVIS spectrum (PerkinElmer, model: 124262 ) or a comparable luminescence imaging equipment

Software

  1. Living Image software (PerkinElmer)

Procedure

  1. Two days before the LPS intraperitoneal injection, place mice in the anesthesia machine and once the mice are anesthetized shave abdominal area with a trimmer, as fur quenches the luminescence signal (Figure 1).


    Figure 1. A representative picture of mice under anesthesia getting their abdominal area shaved with a trimmer

  2. Weigh mice.
  3. In the day of the experiment dilute LPS in DPBS and prepare syringes for injection.
  4. Intraperitoneally inject mice with 2.5 mg/kg of LPS or the same volume DPBS for the control group (injection volume approximately 200 μl).
  5. 3 h later intraperitoneally inject mice with 200 mg/kg of XenoLight Rediject inflammation probe or 200 mg/kg luminol sodium salt (injection volume approximately 200 μl).
  6. Place mice in the IMPAC6 anesthesia chamber attached to the IVIS spectrum.
  7. Transfer mice to the IVIS spectrum and place mice abdomen facing up into the chamber and position each nose inside the cone that delivers the isofluorane (Figure 2).
  8. Start imaging anesthetized mice 10 min post XenoLight Rediject inflammation probe injection with a 5 min exposure capturing in vivo bioluminescence generated by the activity of MPO as a marker for infiltration of neutrophils. In order to imagine 5 mice select field of view D and select 1.5 cm subject height (Figures 2 and 3) (Gross et al., 2009; Tseng and Kung, 2012).


    Figure 2. A representative example of in vivo imaging in 8 wk of age male C57BL/6 mice after i.p. injection of PBS (left) or LPS (2.5 mg/kg body weight) (right)


    Figure 3. Screen capture image of the IVIS acquisition control panel

  9. Quantify the MPO signal using the Living Image software. First select region of interest (ROI) using ROI tools and choose to automatically draw measurement ROIs and perform ROI analyses to measure photon radiance. Also measure background ROI and subtract from your ROI measurement. Use average radiance to plot your graph.

Recipes

  1. 5 mg/ml LPS
    Dilute LPS in DPBS.
    Filter sterillize with a 0.22 μm filter and aliquot stock solution at -80 °C.
  2. 20 mg/ml luminol sodium salt stock solution
    Prepare 20 mg/ml luminol sodium salt stock solution in DPBS.
    Note: Luminol sodium salt for injection has to be prepared fresh each time in DPBS (20 mg/ml), filter sterillize with a 0.22 μm filter and protected from light until use.

Acknowledgments

This protocol was adapted from a previously published study (de Almeida et al., 2015). This work was supported by grants from the National Institutes of Health (AI099009 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. 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.
  2. Gross, S., Gammon, S. T., Moss, B. L., Rauch, D., Harding, J., Heinecke, J. W., Ratner, L. and Piwnica-Worms, D. (2009). Bioluminescence imaging of myeloperoxidase activity in vivo. Nat Med 15(4): 455-461.
  3. Hagar, J. A., Powell, D. A., Aachoui, Y., Ernst, R. K. and Miao, E. A. (2013). Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock. Science 341(6151): 1250-1253.
  4. Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K. and Akira, S. (1999). Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 162(7): 3749-3752.
  5. Kayagaki, N., Wong, M. T., Stowe, I. B., Ramani, S. R., Gonzalez, L. C., Akashi-Takamura, S., Miyake, K., Zhang, J., Lee, W. P., Muszynski, A., Forsberg, L. S., Carlson, R. W. and Dixit, V. M. (2013). Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341(6151): 1246-1249.
  6. McDonald, B., Pittman, K., Menezes, G. B., Hirota, S. A., Slaba, I., Waterhouse, C. C., Beck, P. L., Muruve, D. A. and Kubes, P. (2010). Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 330(6002): 362-366.
  7. Nagai, Y., Akashi, S., Nagafuku, M., Ogata, M., Iwakura, Y., Akira, S., Kitamura, T., Kosugi, A., Kimoto, M. and Miyake, K. (2002). Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol 3(7): 667-672.
  8. Park, B. S., Song, D. H., Kim, H. M., Choi, B. S., Lee, H. and Lee, J. O. (2009). The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature 458(7242): 1191-1195.
  9. Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B. and Beutler, B. (1998). Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282(5396): 2085-2088.
  10. 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.
  11. Shi, J., Zhao, Y., Wang, Y., Gao, W., Ding, J., Li, P., Hu, L. and Shao, F. (2014). Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514(7521): 187-192.
  12. Tseng, J. C. and Kung, A. L. (2012). In vivo imaging of inflammatory phagocytes. Chem Biol 19(9): 1199-1209.
  13. Whitfield, C. and Trent, M. S. (2014). Biosynthesis and export of bacterial lipopolysaccharides. Annu Rev Biochem 83: 99-128.

简介

细菌脂多糖(LPS)存在于革兰氏阴性细菌的外膜中,并且作为病原体相关分子模式(PAMP)(Whitfield和Trent,2014)。因此LPS是炎症反应的有效活化剂,导致细胞因子释放和嗜中性粒细胞募集。 LPS的脂质A部分激活由LPS结合蛋白(LBP),CD14,MD-2和Toll样受体4(TLR4)组成的复合物和非规范的炎症小体连接的半胱天冬酶-4,5和11反过来激活典型的NLRP3炎症小体(Shi等人,2014; Hagar等人,2013; Kayagaki等人,2013; Hoshino等人,1999; Poltorak,1998; Nagai等人,2002; Park等人,2009; Ratsimandresy等人, et al 。,2013)。特别地,响应于炎症小体激活产生的细胞因子白细胞介素(IL)-1β在通过促进嗜中性粒细胞粘附和迁移的中性粒细胞募集中具有关键作用(McDonald等人,2010)。该方案允许研究的由LPS诱导的炎症反应,其通过跟踪体内髓过氧化物酶(MPO)活性来影响嗜中性粒细胞浸润(de Almeida等人,2015)。

关键字:炎症, 腹膜炎, 脓毒症, 活体成像, 髓过氧化物酶

材料和试剂

  1. 胰岛素注射器(Thermo Fisher Scientific,目录号:14-841-31)
  2. 0.22μm过滤器
  3. C57BL/6小鼠,通常为8-12周龄小鼠(雄性或雌性)
  4. LPS大肠杆菌0111:B4(Sigma-Aldrich,目录号:L2630-100MG)
  5. Dulbecco磷酸盐缓冲盐水(DPBS)(Corning,目录号:21-030-CV)
  6. XenoLight RediJect炎症探针(PerkinElmer,目录号:760535)
  7. 鲁米诺钠盐(Sigma-Aldrich,目录号:A4685)
  8. 异氟烷(Henry Schein,Isothesia TM ,目录号:10014450)
  9. 5mg/ml LPS(参见配方)
  10. 20 mg/ml鲁米诺钠盐储备溶液(见配方)

设备

  1. 麻醉机(VetEquip,型号:901808)或类似的麻醉设备
  2. 可充电微调器(Braintree Scientific,目录号:VLP-323 75)
  3. Scale(Kent Scientific,目录号:SCL66110)
  4. 生物安全柜
  5. IVIS光谱(PerkinElmer,型号:124262)或类似的发光成像设备

软件

  1. 生活图像软件(PerkinElmer)

程序

  1. 在LPS腹膜内注射前两天,麻醉机中的小鼠和一旦小鼠麻醉,用修剪器刮剃腹部区域,作为毛发淬灭发光信号(图1)。


    图1.在麻醉下用微调器剃除其腹部区域的小鼠的代表性图片

  2. 称重小鼠。
  3. 在实验当天,在DPBS中稀释LPS,并制备注射用注射器
  4. 腹膜内注射2.5mg/kg LPS或相同体积DPBS的小鼠用于对照组(注射体积约200μl)。
  5. 3小时后,用200mg/kg的XenoLight Rediject炎症探针或200mg/kg鲁米诺钠盐(注射体积约200μl)腹膜内注射小鼠。
  6. 将小鼠在IMPAC6麻醉室连接到IVIS光谱。
  7. 将小鼠转移到IVIS光谱,将小鼠腹部朝上放入小室,并将每个鼻子放在提供异氟烷的锥体内(图2)。
  8. 开始成像麻醉的小鼠XenoLight Rediject炎症探针注射后10分钟,5分钟暴露捕获由MPO活性产生的体内生物发光作为嗜中性粒细胞浸润的标志物。为了想象5只小鼠选择视野D并选择1.5cm对象高度(图2和3)(Gross等人,2009; Tseng和Kung,2012)。


    图2. i.p.后8周龄雄性C57BL/6小鼠体内成像的代表性实例。注射PBS(左)或LPS(2.5mg/kg体重)(右)


    图3. IVIS采集控制面板的屏幕截图

  9. 使用Living Image软件对MPO信号进行量化。首先使用ROI工具选择感兴趣区域(ROI),并选择自动绘制测量ROI,并执行ROI分析以测量光子辐射。还测量背景ROI,并从您的ROI测量中减去。使用平均辐射率绘制图表。

食谱

  1. 5mg/ml LPS
    在DPBS中稀释LPS。
    用0.22μm过滤器和等分试样储液在-80℃下过滤灭菌
  2. 20mg/ml鲁米诺钠盐储备液
    在DPBS中制备20mg/ml鲁米诺钠盐储备溶液 注意:注射的鲁米诺钠盐必须每次在DPBS(20mg/ml)中新鲜制备,用0.22μm过滤器过滤除菌,并避光使用。

致谢

该方案改编自以前发表的研究(de Almeida等人,2015)。这项工作得到国立卫生研究院(AI099009和AR064349至C.S.,AR066439至A.D.,AI120625和AI120618至C.S.和A.D.,T32AR007611至L.d.A.和美国心脏协会13GRNT17110117至C.S.)的赠款支持。

参考文献

  1. 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。
  2. Gross,S.,Gammon,ST,Moss,BL,Rauch,D.,Harding,J.,Heinecke,JW,Ratner,L.and Piwnica-Worms,D。(2009)。< a class ="ke -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19305414"target ="_ blank">髓过氧化物酶活性的体内生物发光成像 em> Nat Med 15(4):455-461。
  3. Hagar,JA,Powell,DA,Aachoui,Y.,Ernst,RK和Miao,EA(2013)。  细胞质LPS激活半胱天冬酶11:对TLR4非依赖性内毒素性休克的影响。科学 341(6151):1250-1253。
  4. Hoshino,K.,Takeuchi,O.,Kawai,T.,Sanjo,H.,Ogawa,T.,Takeda,Y.,Takeda,K.and Akira,S。(1999) ke-insert file"href ="http://www.ncbi.nlm.nih.gov/pubmed/10201887"target ="_ blank">切缘:Toll样受体4(TLR4)缺陷型小鼠对脂多糖低反应性: TLR4作为Lps基因产物的证据。 Immunol 162(7):3749-3752。
  5. Kayagaki,N.,Wong,MT,Stowe,IB,Ramani,SR,Gonzalez,LC,Akashi-Takamura,S.,Miyake,K.,Zhang,J.,Lee,WP,Muszynski,A.,Forsberg,LS ,Carlson,RW和Dixit,VM(2013)。  非经典炎症小体通过细胞内LPS独立于TLR4的激活。科学 341(6151):1246-1249。
  6. McDonald,B.,Pittman,K.,Menezes,GB,Hirota,SA,Slaba,I.,Waterhouse,CC,Beck,PL,Muruve,DA和Kubes,P。(2010)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/20947763"target ="_ blank">血管内危险信号将嗜中性粒细胞引导至无菌性炎症部位 Science 330(6002):362-366。
  7. Nagai,Y.,Akashi,S.,Nagafuku,M.,Ogata,M.,Iwakura,Y.,Akira,S.,Kitamura,T.,Kosugi,A.,Kimoto,M.and Miyake, 2002)。  MD-2在LPS应答中的重要作用和TLR4分布。 Immunol 3(7):667-672。
  8. Park,BS,Song,DH,Kim,HM,Choi,BS,Lee,H.and Lee,JO(2009)。  TLR4-MD-2复合物识别脂多糖的结构基础。 458(7242):1191- 1195。
  9. Poltorak,A.,He,X.,Smirnova,I.,Liu,MY,Van Huffel,C.,Du,X.,Birdwell,D.,Alejos,E.,Silva,M.,Galanos, Freudenberg,M.,Ricciardi-Castagnoli,P.,Layton,B.and Beutler,B。(1998)。  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.
  10. (a),(b),(a),(b),(c),(d),(d) ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25119034"target ="_ blank">炎性胱天蛋白酶是细胞内LPS的先天免疫受体。/em> 514(7521):187-192。
  11. Tseng,JC and Kung,AL(2012)。  < em> in vivo imaging of inflammatory phagocytes. Chem Biol 19(9):1199-1209。
  12. Whitfield,C.和Trent,MS(2014)。  细菌脂多糖的生物合成和输出。 Annu Rev Biochem 83:99-128。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:de Almeida, L., Dorfleutner, A. and Stehlik, C. (2016). In vivo Analysis of Neutrophil Infiltration during LPS-induced Peritonitis. Bio-protocol 6(19): e1945. DOI: 10.21769/BioProtoc.1945.
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