在合并臭氧和LPS诱导穆林急性肺损伤期间可视化肺细胞适应
Summary
臭氧和细菌内毒素暴露的小鼠的结合显示广泛传播的细胞死亡，包括嗜中性粒细胞死亡。我们观察到细胞适应，如细胞骨质跛脚膜的中断，复杂V ATP合成酶亚单位β和血管他汀在支气管-阿尔韦奥拉厕所的细胞表达增加，抑制肺免疫反应和延迟中性粒细胞招募。
Abstract
肺部不断面临无菌（颗粒或活性毒素）和传染性（细菌、病毒或真菌）炎症等直接和间接的侮辱。压倒性的宿主反应可能导致呼吸受损和急性肺损伤，其特点是肺中性粒细胞的招募，由于病理逻辑宿主免疫，凝固和组织重塑反应。敏感的微观方法，可视化和量化Murine肺细胞适应，以响应低剂量（0.05ppm）臭氧，一种强大的环境污染物，结合细菌脂聚糖，TLR4激动剂，是至关重要的，以了解宿主炎症和修复机制。我们描述了对各种肺和全身体室的全面荧光微观分析，即支气管-肺泡乳液、肺血管灌注液、左肺冷冻切除和骨骼骨髓灌注。我们显示肺巨噬细胞、嗜中性粒细胞、肺乳腺组织以及骨髓细胞的损伤，这些细胞与被分析隔间中以离散化学素梯度为标志的延迟（高达36-72 h）免疫反应相关。此外，我们呈现肺细胞外基质和细胞细胞细胞相互作用（行为素，图布林），线粒体和活性氧物种，抗凝血性质氨酸，其抗血管生成肽片段血管他汀，线粒体ATP合成酶复合V亚单位，α和β。这些代孕标记，当辅之以足够的 体外 细胞检测和 体内 动物成像技术，如静脉显微镜，可以提供重要信息，了解肺对新型免疫调节剂的反应。
Introduction
急性肺损伤 （ALI） 是肺部对传染性或其他有害刺激的重要病理反应，其特点是同时激活凝固性、纤维化和与生俱来的免疫系统1。嗜中性粒细胞通过收费状受体（TLR）系列2、3、4迅速感知微生物以及细胞内损伤模式。嗜中性粒细胞释放预先形成的细胞因子和细胞毒性颗粒含量，然后可能导致附带组织损伤。随之而来的藻类损伤与继发细胞死亡一起受损，导致腺苷三磷酸盐（ATP）5等分子的释放，从而引发免疫调节的恶性循环。
在理解ALI时，一个尚未解决的问题涉及如何在气孔膜内启动损伤的问题。电子运输复合物V，F1F0 ATP合成酶，是一种线粒体蛋白，已知在炎症期间在细胞（包括内皮、白细胞、上皮）血浆膜上随处可见。细胞细胞细胞，由作用素和图布林组成，分别具有许多细胞形状和功能调节以及线粒体蛋白质。我们最近表明，由内源性分子、血管他汀、沉默中性粒细胞招募、活化和脂糖（LPS）引起的肺部炎症6对ATP合成酶的阻断。因此，生化（ATP合成酶）和免疫（TLR4）机制都可能调节肺部炎症期间的肺气道屏障。
接触臭氧（O3），环境污染物，损害肺功能，增加肺部感染的易感性，而短期低水平的O3暴露增加那些有基础心肺疾病7，8，9，10，11，12，13，14的死亡率的风险。因此，接触与生理相关的O3浓度为ALI研究炎症7、8的基本机制提供了一个有意义的模型。我们的实验室最近建立了低剂量O3诱导ALI15的穆林模型。在对低O3浓度进行剂量和时间反应后，我们观察到，暴露在0.05ppm O3中2小时，诱发急性肺损伤，其特征是肺ATP合成酶复合V亚单位β（ATP®）和血管他汀表达，类似于LPS模型。静脉肺成像显示肺活体作用微细丝的杂乱无章，表明肺部损伤， 和消融的肺膜隔膜活性氧物种 （ROS） 水平 （表示基线细胞信号的废除） 和线粒体膜潜力 （表示急性细胞死亡） 后 2 小时暴露到 0.05 ppm O315，这与异质肺18FDG 保留16，中性粒体招募和细胞因子释放有关， 最明显的是IL-16和自卫队-1+。我们最近的研究传达的信息是，当O3暴露在浓度低于允许的0.063ppm（每天）超过8小时（每天）的允许限值时，会产生指数级的高毒性。重要的是，对于这些亚临床O3暴露能否调节TLR4介质机制，如细菌内毒素17，目前还不清楚。因此，我们研究了双击O3和LPS暴露模型，并观察了免疫和非免疫细胞适应。
我们描述了对各种肺部和全身体室的全面荧光微观分析，即支气管-肺泡熔岩液（即， BAL）对肺血管空间（即LVP）进行采样，在内皮屏障、左肺冷冻时，对肺血管和肺间膜进行采样，以观察熔岩肺组织中残留的常驻肺细胞和粘附白细胞，代表循环白细胞的外周血液和胸骨和股骨骨髓分别在炎症期间采样造血细胞动员的近部和离位位。
Protocol
Representative Results
Discussion
本研究中提出的方法突出了多个隔间分析对于研究肺部炎症期间的多个细胞事件的有用性。我们已在第 二表中总结了研究结果。我们和许多实验室广泛研究了对鼻内LPS灌输的穆林反应，其特点是肺中性粒细胞的快速招募，其峰值在6-24小时之间，随后，分辨率开始生效。最近，我们已表明，仅亚临床O3（ 在0.05ppm为2 h）就可诱发C57BL/6NJ亚株15的显著肺损伤，其?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究由国家核创新委员会主席的赠款以及西尔维亚·费多鲁克加拿大核创新中心的启动资金资助。西尔维亚·费多鲁克加拿大核创新中心由萨斯喀彻温省创新中心资助。荧光成像是在WCVM成像中心进行的，该中心由国家SERC资助。杰西卡·布罗科斯（理学硕士学生）和曼普雷特·考尔（硕士生）由西尔维亚·费多鲁克加拿大核创新中心的启动基金资助。
Materials
| 33-plex Bioplex chemokine panel | Biorad | 12002231 | |
| 63X oil (NA 1.4-0.6) Microscope objectives | Leica | HCX PL APO CS (11506188) | |
| Alexa 350 conjugated goat anti-mouse IgG (H+L) | Invitrogen | A11045 | |
| Alexa 488 conjugated goat anti-mouse IgG (H+L) | Invitrogen | A11002 | |
| Alexa 488 conjugated phalloidin | Invitrogen | A12370 | |
| Alexa 555 conjugated mouse anti-α tubulin clone DM1A | Millipore | 05-829X-555 | |
| Alexa 568 conjugated goat anti-hamster IgG (H+L) | Invitrogen | A21112 | |
| Alexa 568 conjugated goat anti-rat IgG (H+L) | Invitrogen | A11077 | |
| Alexa 633 conjugated goat anti-rabbit IgG (H+L) | Invitrogen | A21070 | |
| Armenian hamster anti-CD61 (clone 2C9.G2) IgG1 kappa | BD Pharmingen | 553343 | |
| C57BL/6 J Mice | Jackson Laboratories | 64 | |
| Confocal laser scanning microscope | Leica | Leica TCS SP5 | |
| DAPI (4′,6-diamidino-2-phenylindole) | Invitrogen | D1306 | aliquot in 2 µl stocks and store at -20°C |
| Inverted fluorescent wide field microscope | Olympus | Olympus IX83 | |
| Ketamine (Narketan) | Vetoquinol | 100 mg/ml | Dilute 10 times to make a 10 mg/ml stock |
| Live (calcein)/Dead (Ethidium homodimer-1) cytotoxicity kit | Invitrogen | L3224 | |
| Mouse anti-ATP5A1 IgG2b (clone 7H10BD4F9) | Invitrogen | 459240 | |
| Mouse anti-ATP5β IgG2b (clone 3D5AB1) | Invitrogen | A-21351 | |
| Mouse anti-NK1.1 IgG2a kappa (clone PK136) | Invitrogen | 16-5941-82 | |
| Pierce 660 nm protein assay | Thermoscientific | 22660 | |
| Rabbit anti-angiostatin (mouse aa 98-116) IgG | Abcam | ab2904 | |
| Rabbit anti-CX3CR1 IgG (RRID 467880) | Invitrogen | 14-6093-81 | |
| Rat anti-Ki-67 (clone SolA15) IgG2a kappa | Invitrogen | 14-5698-82 | |
| Rat anti-Ly6G IgG2a kappa (clone 1A8) | Invitrogen | 16-9668-82 | |
| Rat anti-Ly6G/Ly6C (Gr1) IgG2b kappa (clone RB6-8C5) | Invitrogen | 53-5931-82 | |
| Rat anti-mouse CD16/CD32 Fc block (clone 2.4G2) | BD Pharmingen | 553142 | |
| Reduced mitotracker orange | Invitrogen | M7511 | |
| Xylazine (Rompun) | Bayer | 20 mg/ml | Dilute 2 times to make a 10 mg/ml stock |
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