FICZ Exposure and Viral Infection in Mice

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Nature Immunology
May 2016


The aryl hydrocarbon receptor (AHR) is known as a sensor for dioxins that mediates their toxicity, and also has important biophysiological roles such as circadian rhythms, cell differentiation and immune responses. 6-formylindolo(3,2-b)carbazole (FICZ), which is derived through the metabolism of L-tryptophan by ultraviolet B irradiation, is one of putative physiological ligands for AHR (Smirnova et al., 2016). It has recently been shown that endogenously-activated AHR signaling modulates innate immune response during viral infection (Yamada et al., 2016). This section describes how to treat mice with FICZ and to infect them with virus.

Keywords: Aryl hydrocarbon receptor (芳烃受体), 6-formylindolo(3,2-b)carbazole (6-甲酰基吲哚并(3,2-b)咔唑), Viral infection (病毒感染), Innate immunity (先天免疫), Interferon response (干扰素反应)


The role of AHR had so far been investigated mainly on the basis of experiments with dioxin treatment. On the other hand, it has been shown that AHR-mediated signaling is activated by endogenous tryptophan metabolites (FICZ, Kynurenine, etc.), heme metabolites (bilirubin etc.), and eicosanoids (Lipoxin A etc.). In particular, it was demonstrated that FICZ is a physiological high affinity ligand for AHR, and many accumulating reports have shown that FICZ is involved in various basic biological processes including adaptive responses to ultraviolet light, immune responses, genomic instability, and homeostasis of stem cells. Recently, Yamada et al. (2016) demonstrated its role in innate immune response: FICZ treatment in vivo suppresses type I interferon (IFN) production in response to viral infection and promotes levels of viral titer in sera of mice.

Materials and Reagents

  1. 0.1-10 μl pipet tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: QSP#TF104 )
  2. 1-200 μl pipet tips (Corning, catalog number: 4845 )
  3. 100-1,000 μl pipet tips (Corning, catalog number: 4846 )
  4. 1.5 ml microcentrifuge tubes (Corning, Axygen®, catalog number: MCT-150-A )
  5. 1 ml syringe (NIPRO, catalog number: 4987458080104 )
  6. 27 G hypodermic needle (TERUMO, catalog number: NN-2719S )
  7. Feather disposable scalpel (Sigma-Aldrich, catalog number: Z692395 )
  8. Falcon 6-well tissue culture plate (Corning, Falcon®, catalog number: 353046 )
  9. C57BL/6NJcl mice (male, 6 weeks of age, purchasable from CLEA Japan, model: C57BL/6NJcl mice )
  10. Vero cells (purchasable from ATCC, catalog number: CCL-81 )
  11. 6-formylindolo(3,2-b)carbazole (FICZ) (Enzo Life Sciences, catalog number: BML-GR206-0100 )
  12. Corn oil (stored at room temperature) (Sigma-Aldrich, catalog number: C8267 )
  13. Vesicular stomatitis virus (VSV) (Indiana strain: Kuroda et al., 2016)
  14. Phosphate-buffered saline (PBS) (pH 7.4) (NISSUI PHARMACEUTICAL, catalog number: 05913 )
  15. VeriKine-HSTM mouse IFN beta serum ELISA kit (Pestka Biomedical Laboratories, catalog number: 42410 )
  16. Dulbecco’s modified Eagle’s medium (DMEM) (‘Nissui’ ②) (NISSUI PHARMACEUTICAL, catalog number: 05919 )
  17. Methyl cellulose, 4,000 cps (Sigma-Aldrich, catalog number: 19-2930 )
  18. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437-028 )
  19. Acetic acids (KANTO CHEMICAL, catalog number: 01021-70 )
  20. Methanol (NACALAI TESQUE, catalog number: 21915-93 )
  21. Crystal violet (Sigma-Aldrich, catalog number: C6158 )
  22. Formaldehyde (Wako Pure Chemical Industries, catalog number: 063-04815 )
  23. FICZ solution (see Recipes)
  24. VSV stock solution (see Recipes)
  25. Fixation solution (see Recipes)
  26. Crystal violet staining solution (see Recipes)


  1. Pipettes (PIPETMAN P2) (Gilson, catalog number: F144801 )
  2. Pipettes (PIPETMAN P20) (Gilson, catalog number: F123600 )
  3. Pipettes (PIPETMAN P1000) (Gilson, catalog number: F123602 )
  4. Centrifuge
  5. Deep freezer
  6. Biosafety hood in a biosafety level 3 (BSL3) facility (HITACHI, model: SCV-1303 ECII-AG )
  7. Labnet VX100 vortex (Labnet International, catalog number: 13111-LV2 )


  1. In vivo treatment with FICZ
    1. FICZ is dissolved at 10 mg/ml in corn oil (see Recipes).
    2. Mice are intraperitoneally injected with 200 μl of corn oil (as a negative control) or 10 mg/ml FICZ solution (2 mg per mouse).
  2. In vivo infection with VSV
    1. An aliquot of VSV stock is suspended in PBS to 1.5 x 109 PFU/ml.
    2. At 12 h after treatment with FICZ, mice were intraperitoneally infected with 200 μl of PBS (as an uninfected control) or this virus solution (3 x 108 PFU per mouse).
  3. Measurement of serum interferon-beta levels and viral titers
    1. At 12 h after infection with VSV, collect blood samples from mouse tail veins by making a cut around 5 mm from the tip of the mouse tail using scalpel blade.
    2. Allow the whole blood to clot by leaving it undisturbed at room temperature for 30 min.
    3. Remove the clot by centrifuging at 1,000 x g for 10 min at 4 °C.
    4. For measurement of serum interferon-beta levels, use VeriKine-HSTM mouse IFN beta serum ELISA kit. ELISA is performed according to the manufacturer’s protocol.
    5. For measurement of serum viral titer, plaque-forming assay is performed. In this assay, 5 x 106 Vero cells on 6-well plate is infected with virus of serum samples for 1 h and overlaid with 2 ml of DMEM containing 2.4% methylcellulose and 5% heat inactivated FBS for 24 h. Plaques (i.e., focus of dead cells) can be more visible by staining live cells through the following method: Cells are fixed for 15 min with 1 ml of fixation solution (see Recipes), and then remove the above-mentioned, overlaid media with running water. Plaques are counterstained with 1 ml of crystal violet solution for 15 min, and washed with running water. A representative image of counterstained plaques is shown in Figure 1.

      Figure 1. An image of plaques. This is the result of VSV infection in Vero cells. One representative well of the 6-well plate is shown. Live cells are stained with crystal violet, while dead cells following viral infection, so-called ‘plaques’, are not stained.

Data analysis

Statistical significance between two samples is determined by Student’s t-test. Each sample is prepared in septuplicate (cf. Figure 2d of Yamada et al. [2016]).


  1. FICZ solution
    Dissolve 10 mg FICZ in 1 ml corn oil at room temperature. Vortexing is required to completely dissolve.
    Note: In this experiment, freshly prepared solution of FICZ is always used without filtration. 
  2. VSV stock solution
    VSV is suspended with PBS to 2.0 x 109 PFU/ml and stored at -80 °C in deep freezer
    1. When you thaw a viral stock, you should thaw it on ice.  
    2. It is also important not to use it again, because of its possible degradation of virus titers by freeze and thaw action.
  3. Fixation solution
    Fixation solution is composed of 30% acetic acid and 70% methanol
  4. Crystal violet staining solution
    Crystal violet staining solution is prepared with PBS containing 0.0005% crystal violet (w/v) and 4% formaldehyde (v/v) 


This protocol was adapted from Yamada et al. (2016). This was supported by a grant from the Ministry of Health, Labour and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant-in-aid for scientific research (A) [25253030] and grant-in-aid for scientific research on innovative areas [25115502, 23112701]), the Kato Memorial Bioscience Foundation, the Yasuda Medical Foundation, the Takeda Science Foundation and the Waksman Foundation of Japan to A.T.


  1. Kuroda, M., Fujikura, D., Nanbo, A., Marzi, A., Noyori, O., Kajihara, M., Maruyama, J., Matsuno, K., Miyamoto, H., Yoshida, R., Feldmann, H. and Takada, A. (2015). Interaction between TIM-1 and NPC1 is important for cellular entry of Ebola virus. J Virol 89(12): 6481-6493.
  2. Smirnova, A., Wincent, E., Vikström Bergander, L., Alsberg, T., Bergman, J., Rannug, A. and Rannug, U. (2016). Evidence for new light-independent pathways for generation of the endogenous aryl hydrocarbon receptor agonist FICZ. Chem Res Tpxicol 29: 75-86.
  3. Yamada, T., Horimoto, H., Kameyama, T., Hayakawa, S., Yamato, H., Dazai, M., Takada, A., Kida, H., Bott, D., Zhou, A. C., Hutin, D., Watts, T. H., Asaka, M., Matthews, J. and Takaoka, A. (2016). Constitutive aryl hydrocarbon receptor signaling constrains type I interferon-mediated antiviral innate defense. Nat Immunol 17(6): 687-694.


芳烃受体(AHR)被称为二恶英介导其毒性的传感器,也具有重要的生物生理学作用,如昼夜节律,细胞分化和免疫应答。通过紫外线B照射通过L-色氨酸的代谢衍生的6-甲酰基吲哚(3,2-b)咔唑(FICZ)是AHR的推定生理配体之一(Smirnova等人)。 ,2016)。最近已经显示内源性激活的AHR信号调节病毒感染期间的先天免疫应答(Yamada等人,2016)。本节介绍如何用FICZ治疗小鼠并用病毒感染。

背景 迄今为止,AHR的作用主要是在二恶英治疗实验的基础上进行了调查。另一方面,已经显示AHR介导的信号传导是由内源色氨酸代谢物(FICZ,Kynurenine,等等),血红素代谢物(胆红素等)激活的。 ,和类二十烷酸(Lipoxin A等)。特别地,已经证明FICZ是AHR的生理高亲和力配体,许多积累的报道显示FICZ参与各种基本生物学过程,包括对紫外线的适应性反应,免疫应答,基因组不稳定性和干细胞的体内平衡。最近,Yamada等人。 (2016)证明其在先天免疫应答中的作用:体内FICZ治疗抑制响应于病毒感染的I型干扰素(IFN)产生并促进小鼠血清中病毒滴度的水平。

关键字:芳烃受体, 6-甲酰基吲哚并(3,2-b)咔唑, 病毒感染, 先天免疫, 干扰素反应


  1. 0.1-10μl移液管吸头(Thermo Fisher Scientific,Thermo Scientific TM,目录号:QSP#TF104)
  2. 1-200μl移液管提示(康宁,目录号:4845)
  3. 100-1,000μl移液管吸头(康宁,目录号:4846)
  4. 1.5ml微量离心管(Corning,Axygen ,目录号:MCT-150-A)
  5. 1 ml注射器(NIPRO,目录号:4987458080104)
  6. 27 G皮下注射针(TERUMO,目录号:NN-2719S)
  7. 羽毛一次性手术刀(Sigma-Aldrich,目录号:Z692395)
  8. Falcon 6孔组织培养板(Corning,Falcon ®,目录号:353046)
  9. C57BL/6NJcl小鼠(男性,6周龄,可从日本CLEA购买,型号:C57BL/6NJcl小鼠)
  10. Vero细胞(可从ATCC购买,目录号:CCL-81)
  11. 6-甲酰基吲哚(3,2-b)咔唑(FICZ)(Enzo Life Sciences,目录号:BML-GR206-0100)
  12. 玉米油(在室温下储存)(Sigma-Aldrich,目录号:C8267)
  13. 水泡性口炎病毒(VSV)(Indiana株:黑田等人,2016)
  14. 磷酸盐缓冲盐水(PBS)(pH 7.4)(NISSUI PHARMACEUTICAL,目录号:05913)
  15. VeriKine-HS TM小鼠IFNβ血清ELISA试剂盒(Pestka Biomedical Laboratories,目录号:42410)
  16. Dulbecco改良Eagle培养基(DMEM)("Nissui"②)(NISSUI PHARMACEUTICAL,目录号:05919)
  17. 甲基纤维素,4000cps(Sigma-Aldrich,目录号:19-2930)
  18. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437-028)
  19. 乙酸(KANTO CHEMICAL,目录号:01021-70)
  20. 甲醇(NACALAI TESQUE,目录号:21915-93)
  21. 结晶紫(Sigma-Aldrich,目录号:C6158)
  22. 甲醛(Wako Pure Chemical Industries,目录号:063-04815)
  23. FICZ解决方案(见配方)
  24. VSV储备溶液(见配方)
  25. 固定溶液(见配方)
  26. 结晶紫染色溶液(参见食谱)


  1. 移液器(PIPETMAN P2)(Gilson,目录号:F144801)
  2. 移液器(PIPETMAN P20)(Gilson,目录号:F123600)
  3. 移液器(PIPETMAN P1000)(Gilson,目录号:F123602)
  4. 离心机
  5. 深冻柜
  6. 生物安全等级3(BSL3)设施(日立,型号:SCV-1303 ECII-AG)的生物安全罩
  7. Labnet VX100涡流(Labnet International,目录号:13111-LV2)


  1. 使用FICZ进行体内治疗
    1. FICZ以10毫克/毫升溶解在玉米油中(见食谱)。
    2. 腹腔注射200μl玉米油(作为阴性对照)或10mg/ml FICZ溶液(每只小鼠2mg)。
  2. VSV感染体内
    1. 将等分的VSV原液悬浮在PBS中至1.5×10 9 PFU/ml。
    2. 用FICZ处理12小时后,用200μlPBS(作为未感染对照)或该病毒溶液(每只小鼠3×10 8 PFU)腹膜内感染小鼠。
  3. 测量血清干扰素-β水平和病毒滴度
    1. 感染VSV后12小时,使用解剖刀刀片从小鼠尾部切开5mm左右,从小鼠尾静脉采集血液样品。
    2. 允许全血凝血,将其在室温下静置30分钟。
    3. 通过在4℃下以1,000×g离心10分钟去除凝块。
    4. 对于血清干扰素-β水平的测量,使用VeriKine-HS 小鼠IFNβ血清ELISA试剂盒。 ELISA根据制造商的方案进行。
    5. 为了测量血清病毒滴度,进行斑块形成测定。在该测定中,6孔板上的5×10 6个Vero细胞用血清样品的病毒感染1小时,并用2ml含有2.4%甲基纤维素和5%热灭活FBS的DMEM覆盖, 24小时。通过以下方法染色活细胞,斑块(,即死细胞的焦点)可以更明显:细胞用1ml固定溶液固定15分钟(参见食谱),然后除去上述的覆盖媒体与流水。将噬斑用1ml结晶紫溶液重新染色15分钟,并用流水洗涤。复染斑块的代表性图像如图1所示  





  1. FICZ解决方案
  2. VSV库存解决方案
    将VSV用PBS悬浮至2.0×10 9 PFU/ml,并储存在-80℃深冷冻箱中。 注意:

    1. em>再次使用它也是重要的,因为它可以通过冻结和解冻作用降低病毒滴度。
  3. 固定解决方案
  4. 结晶紫染色溶液




  1. Kuroda,M.,Fujikura,D.,Nanbo,A.,Marzi,A.,Noyori,O.,Kajihara,M.,Maruyama,J.,Matsuno,K.,Miyamoto,H.,Yoshida, Feldmann,H。和Takada,A.(2015)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/25855742"target ="_ blank" > TIM-1和NPC1的相互作用对于埃博拉病毒的细胞进入是重要的。 89(12):6481-6493。
  2. Smirnova,A.,Wincent,E.,VikströmBergander,L.,Alsberg,T.,Bergman,J.,Rannug,A.and Rannug,U.(2016)。< a class ="ke-insertfile" href ="https://www.ncbi.nlm.nih.gov/pubmed/26686552"target ="_ blank">用于产生内源芳烃受体激动剂FICZ的新的光依赖途径的证据。 > Chem Res Tpxicol 29:75-86。
  3. Yamada,T.,Horimoto,H.,Kameyama,T.,Hayakawa,S.,Yamato,H.,Dazai,M.,Takada,A.,Kida,H.,Bott,D.,Zhou,AC,Hutin ,D.,Watts,TH,Asaka,M.,Matthews,J.andTakaoka,A。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm。本发明的芳基烃受体信号传导限制I型干扰素介导的抗病毒先天性防御。 Nat Immunol 17(6):687-694 。
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引用:Yamada, T. and Takaoka, A. (2017). FICZ Exposure and Viral Infection in Mice. Bio-protocol 7(1): e2096. DOI: 10.21769/BioProtoc.2096.