实验性感染<em>李斯特菌</em>作为研究主机干扰素γ响应模型
Summary
这个协议描述如何接种C57BL / 6J小鼠李斯特菌的EGD株（单增李斯特菌），并测量干扰素γ（IFN-γ）的反应由自然杀伤（NK）细胞，天然杀伤T（NKT）细胞，和自适应T淋巴细胞后感染。该协议还介绍了如何进行生存研究，小鼠感染后有修改的LD 50剂量的病原体。
Abstract
李斯特菌是革兰氏阳性细菌是食源性疾病的人类的原因。与此病原体的小鼠实验性感染一直对先天性和适应性免疫细胞和特定细胞因子的抗细胞内病原体宿主免疫中的作用非常翔实。 李斯特杆菌亚致死感染期间由先天细胞的IFN-γ生产是活化的巨噬细胞和病原体1-3的早期控制很重要。此外，IFN-γ的生产由自适应存储器淋巴细胞是用于在再感染4吸先天细胞活化重要的。 单增李斯特菌感染模型从而作为一个伟大的工具调查，旨在提高IFN-γ生产的新疗法是否在体内的IFN-γ反应产生影响，并有生产性生物效应，如增加细菌清除或改善生存鼠标从感染。这里描述的是用于如何进行C57BL / 6J小鼠用单增李斯特菌的EGD株腹膜内感染和流式细胞测量IFN-γ的生产由NK细胞，NKT细胞，和自适应淋巴细胞由流动的基本协议。另外，程序被描述为：（1）生长和用于接种制备的细菌，（2）测量在脾脏和肝脏，以及端点（3）测量动物存活的细菌负荷。还提供了有代表性的数据来说明这个感染模型如何可以用来测试特定剂对IFN-γ反应， 单增李斯特菌 ，并从该感染的小鼠的存活率的影响。
Introduction
IFN-γ是一种细胞因子是介导针对细胞内病原体的免疫力和用于控制肿瘤5生长至关重要。该细胞因子在细菌耐药性的重要性在于，与IFN-γ信号传导途径的突变是高度易感分枝杆菌和沙门氏菌6人类观察是显而易见的。同样地，小鼠的缺陷型中任一的IFN-γ或电阻的IFN-γ受体表现出缺陷分枝杆菌7-9和其他细胞内病原体包括单增李斯特菌 10,11， 硕大利什曼原虫 12， 鼠伤寒沙门氏菌 13，以及某些病毒11。除了杀灭病原体，IFN-γ在防止肿瘤14宿主防御了至关重要的作用。虽然较高的生产IFN-γ的是在感染或癌症的情况下是有利的，延长生产这种细胞因子的已链接吨Ø系统性自身免疫15-17的发展和类型的加速I型糖尿病中的非肥胖型糖尿病小鼠模型18。
IFN-γ的主要来源包括NK细胞，NKT细胞，γδT细胞，T辅助1（Th1细胞）细胞和细胞毒性T淋巴细胞（CTL）的5,19,20。 IFN-γ的提高都通过先天和适应性免疫：（1）上调主要组织相容性复合体（MHC）I类和II表达，（2）提高的共刺激分子的抗原呈递细胞上的表达，（3）增强的巨噬细胞吞噬作用和促炎细胞因子和杀微生物因素（ 例如 ，一氧化氮和活性氧），（4）促进幼稚CD4 + T细胞分化成Th1细胞的效应细胞，（5）促进抗体类别转换到免疫球蛋白（ Ig）的图2a和IgG3（在小鼠），（6）诱导产生趋化因子的招募免疫细胞为i的位点nfection，和（7）增强NK细胞和CTL应答5,19。定在宿主响应于病原体和肿瘤的IFN-γ的关键重要性，重组IFN-γ已经过测试作为各种感染和恶性肿瘤（在19中综述）的处理。但是，因为IFN-γ或Th1细胞促进细胞因子白细胞介素-12（IL-12）的全身给药与副作用相关联，并且与剂量相关毒性19,21，存在开发替代策略，以增加IFN-γ的生产的兴趣免疫细胞。的新的生物制剂和小分子的发展需要在体内筛选工具来测试这种试剂是否免疫应答期间增加的IFN-γ的生产，这是否转换成有意义的生物效应，如在动物存活增加。
用革兰氏阳性菌李斯特菌的小鼠实验性感染一直器乐模型破译IFN-γ在宿主免疫对细胞内病原体1,22的作用。与病原体静脉内或腹膜内（IP）的小鼠的感染导致细菌的脾脏和肝脏，在那里他们成为驻地巨噬细胞和肝细胞中的脾3和天后4之间存在的峰细菌负载内化的快速传播感染1,3,22。 NK细胞的IFN-γ生产是巨噬细胞活化和抗病原菌3初期电阻重要;然而在高感染剂量，产生IFN-γ也可能是有害的病原体间隙23。 NKT细胞也可在脾脏和肝脏的IFN-γ的病原体2,24早期控制期间的源极和该生产已被证明是放大由其它细胞类型，包括NK细胞2 IFN-γ的生产。另一方面，后作用自适应T淋巴细胞，CD8 + T细胞在杂色丘拉尔，是调解病原体的清除和提供抗再感染1,4,22保护非常重要。
这种感染模型一直研究人员有许多原因（1综述）具有吸引力。首先，感染病原体是高度可再现的和诱导强Th1和细胞免疫应答。其次，亚致死感染期间，细菌负荷集中在其中可以容易地测量在肝和脾。第三，病原体可以安全地在生物安全等级2（BSL2）条件下处理。第四，有机物和免疫反应，它产生已经被广泛表征。最后，各种突变体和转基因品系已开发了可用于使用。
这里描述的是用于与L的EGD株C57BL / 6J小鼠的接种基本协议菌等 25和用于测量γ –干扰素重新由NK，NKT和自适应淋巴细胞感染后sponses。还描述了如何测量脾细菌负荷和肝脏亚致死感染后，用改性的LD 50剂量的病原体的感染后进行存活研究。最后，有代表性数据示的这个协议如何用于从李斯特菌感染筛选的新的治疗药物上的IFN-γ反应和小鼠存活率的影响。
Protocol
安全声明本协议描述与现场单增李斯特菌感染小鼠。病原菌是谁不免疫功能低下训练有素的人员BSL2条件下安全处理。免疫力低下的人群包括孕妇，老人和个人谁是艾滋病毒感染或有需要使用免疫抑制疗法治疗慢性疾病。在处理感染的样本的人员应穿上防护白大褂或礼服，手套，口罩和护目镜。这里所描述的工作是由大学健康网络发行（UHN）生物安全办公室的证书（＃3287…
Representative Results
图3显示在24小时后的感染细胞计数的IFN-γ染色一些典型流脾NK和NKT细胞与病原体的10 5 CFU。该图还示出了表2中记载的染色面板的选通策略。 图4示出在一个实验中，其中雄性小鼠用PPARα拮抗剂IS001或媒介物对照处理得到的一些有代表性的数据，感染10 5 CFU 单增李斯特菌，然后24小时后分析在NK和NKT细胞的IFN-γ 。该…
Discussion
这里我们介绍与L的EGD株雄性或雌性C57BL / 6J小鼠25 单核细胞增生如何进行基本的实验性感染的协议。该协议被建立用于研究体内 31上的IFN-γ的生产的新的小分子IS001的由先天性和适应性淋巴细胞的影响的目的。通过监测细菌清除和生存感染后，被见解将获得的这些变化中的IFN-γ如何影响主机的控制感染的能力。
议定书中考虑的关键因素
<p c…Divulgations
The authors have nothing to disclose.
Acknowledgements
Development of this protocol was supported by an operating grant from CIHR (MOP97807) to SED.
Materials
| Brain Heart Infusion Broth, Modified | BD | 299070 | any brand should be appropriate |
| Agar | BD | 214010 | any brand should be appropriate |
| Triton X-100 | Sigma-Aldrich | X100 | any brand should be appropriate |
| 1xPBS | Sigma | D8537 | any brand should be appropriate |
| TissueLyser II | Qiagen | 85300 | any brand should be appropriate |
| Ammonium Chloride (NH3Cl) | any brand should be appropriate | ||
| KHCO3 | any brand should be appropriate | ||
| Na2EDTA | any brand should be appropriate | ||
| RPMI 1640 | Gibco | 22400089 | any brand should be appropriate |
| Fetal Bovine Serum | Gibco | 12483 | Before use, heat-inactivate at 56 °C for 30 min |
| L-glutamine | Gibco | 25030 | any brand should be appropriate |
| Non-essential amino acids | Gibco | 11140 | any brand should be appropriate |
| Penicillin/Streptomycin | Gibco | 15140 | any brand should be appropriate |
| GolgiStop Protein Transport Inhibitor (containing Monensin) | BD | 554724 | Use 4 μl in 6 ml cell culture |
| 16% Paraformaledehye | Electron Microscopy Sciences | 15710 | Dilute to 4% PFA in ddH20 or 1xPBS |
| 10 x Perm/Wash buffer | BD | 554723 | Dilute 10x in ddH20 |
| Fc block, Anti-Mouse CD16/CD32 Purified | eBioscience | 14-0161 | Dilute 1:50 |
| Fixable Viability Dye eFluor 506 | eBioscience | 65-0866 | Dilute 1:1000 (we have also used viability dyes from Molecular Probes) |
| anti-Mouse CD4-PE-Cy5 (GK1.5) | eBioscience | 15-0041 | Manufacturer recommends a certain test size; however this should be titrated before use. |
| anti-Mouse CD8-FITC (53-6.7) | eBioscience | 11-0081 | Manufacturer recommends a certain test size; however this should be titrated before use. |
| PBS57/mCD1d tetramer-APC | NIH Tetramer Core Facility | N/A | Obtained as a gift from the facility |
| anti-Mouse TCRβ-PE-Cy7 (H56-597) | eBioscience | 25-5961 | Manufacturer recommends a certain test size; however this should be titrated before use. |
| anti-Mouse NKp46-APC-eFluor780 (29A1.4) | eBioscience | 47-3351 | Manufacturer recommends a certain test size; however this should be titrated before use. |
| anti-Mouse CD45 PE-Cyanine7 (30-F11) | eBioscience | 25-0451 | Manufacturer recommends a certain test size; however this should be titrated before use. |
| anti-Mouse IFN gamma-PE (XMG1.2) | eBioscience | 12-7311 | Manufacturer recommends a certain test size; however this should be titrated before use. |
| OneComp eBeads | eBioscience | 01-1111 | Manufacturer recommends a certain test size; however this should be titrated before use. |
| Mouse IFN gamma ELISA kit | eBioscience | 88-7314 | Used for measuring the interferon gamma in the culture supernatant |
| 50 mL vented tubes for culture | Used for culturing the bacteria, any brand should be appropriate | ||
| 1.5 ml microcentrifuge tubes | any brand should be appropriate | ||
| bacterial petri dishes | any brand should be appropriate | ||
| 2 ml cyrovials | any brand should be appropriate | ||
| UV spectrometer | any brand should be appropriate | ||
| safety engineered needles | any brand should be appropriate | ||
| C57BL6/J | Jackson laboratories | Stock#000664 | Order for arrival at 7 wks |
| Bleach | For decontamination | ||
| 70% Ethanol | For decontamination | ||
| Glass beads | any brand should be appropriate | ||
| Centrifuge | rotor, buckets, bucket covers. | ||
| Microcentrifuge | any brand should be appropriate | ||
| Sterile Glycerol | any brand should be appropriate | ||
| Pipette Tips | any brand should be appropriate | ||
| Pipette | any brand should be appropriate | ||
| Surgical instruments | any brand should be appropriate | ||
| 70 micron strainers | any brand should be appropriate | ||
| 3 ml syringe | any brand should be appropriate | ||
| Pipette gun | any brand should be appropriate | ||
| Filtration Units | any brand should be appropriate | ||
| Trypan Blue | Dilute 1 to 9 in ddH20, any brand should be appropriate | ||
| Hemocytometer | any brand should be appropriate | ||
| Round bottomed plates | any brand should be appropriate | ||
| FACs tubes | BD | ||
| BD LSR II | BD | Any flow cytometer could be used for acquisition that has an appropriate laser configuration and filter set to discriminate the fluorochormes | |
| Flowjo software | Treestar | Used for data analysis. Other types of data analysis software will also be appropriate | |
| Multichannel pipettor (0-300 µl) | Eppendorf | Used for washing cells and adding antibodies during flow cytometry staining | |
| Acetic Acid | Used for washing glass beads, any brand should be appropriate | ||
| Microbank Bacterial Preservation System | Pro-lab Diagnositics | Used as an alternative to glycerol stocks for long-term storage of bacteria |
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Citer Cet Article
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