的气管内管理<em>流感嗜血杆菌</em>在小鼠模型来研究气道炎症
Summary
We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.
Abstract
在这里，我们有效地描述的详细步骤和直接递送流感嗜血杆菌 到小鼠的下呼吸道。我们演示程序编制H.接种流感 ，H的内气管滴入流感进入肺，支气管肺泡灌洗液（BALF），BALF中的免疫细胞的分析，和RNA隔离差异基因表达分析的汇编。此过程可用于研究的任何细菌，病毒或真菌的肺部炎症反应。直接气管滴入被过度鼻内或气溶胶吸入程序大多优选的，因为它更有效地以较少的歧义开细菌接种到下呼吸道。
Introduction
炎症是针对传染剂的防御的基本免疫机制。它促进了消灭病原体和受损组织的修复。它还有助于恢复到正常的健康状态1。然而，失调炎症常常导致慢性炎症性疾病2。气道炎症是对于不同的肺部疾病，如慢性阻塞性肺疾病（COPD），哮喘和肺纤维化3的初始触发。
的不可分型（未包囊） 流感嗜血杆菌 （NTHi的）与慢性上下肺部炎性疾病4,5相关联。它是儿童和成人的下呼吸道分离慢性阻塞性肺疾病4,6,7的主导品种。的NTHi感染后的炎症反应的特征在于促炎性细胞因子（如TNF和IL-1β）的上调，并且它介导bý丝裂原活化蛋白激酶（MAPK）和核因子-κB（NF-κB）通过toll样受体（TLR）8。
小鼠模型是用于分析的，因为不同的基因缺陷线的可用性肺部炎性疾病的潜在病理非常有用的工具。几种方法已被用于接种活的/减毒的细菌和细菌产物，包括鼻内滴注和雾化吸入9,10。在这里，我们表现出内气管滴入。虽然不经常使用的，这种方法是更有效的和高重现性的，因为到下呼吸道的直接递送接种物的。
Protocol
Representative Results
Discussion
在此，我们描述了一个独特和微创手术用细菌肺病原体接种的小鼠的肺。我们证明，这个过程可用于研究是否可使用那些在炎症信号传导途径的基因缺陷小鼠不同基因的功能。这个过程也可以用来研究病毒和真菌的肺部感染的炎症反应。此过程在其他方法，例如鼻内或气溶胶吸入的优点是：（1）在此过程中，致病性接种物被直接滴注至下呼吸道; （2）来控制接种物大小的能力;和（3）减少细菌暴?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Carson Harrod for critical reading of the manuscript. We also thank Mr. Minghui Zeng and Drs. Mahesh Kathania and Prashant Khare for their contributions. This work was supported by grants from the American Cancer Society (Research Scholar grant, 122713-RSG-12-260-01-LIB) and the Sammons Cancer Center (Pilot Project grant) to K. Venuprasad.
Materials
| Chocolate agar plate | Fisher Scientific | CAS50-99-7 | |
| Dextrose Anhydrous | Themo Scientific | R01300 | |
| Heparin | Hospira,Inc | RL-3010 | |
| Deft quick solution | Sigma | GS500-500ML | |
| Syringe needle 20/26G | BD | (REF305115/175) | |
| Iml syringe | BD | REF 309602 | |
| Catheter 20GA | BD | REF 381433 | |
| Dissecting Scissors, straight, 10 cm long | kentscientific | INS600393 | |
| Iris Forceps, serrated, 10cm long | kentscientific | INS650915 | |
| Tweezer #5 Stainless steel, 11cm long | kentscientific | INS600095 | |
| 10% Formalin | Fisher Scientific | CAS 67-56-1 | |
| Agarose | peqlab | 35-1020 | |
| 5ml polystyrene round-bottom tubes | BD | REF 352058 | |
| 1.5 ml Microcentrifuge tubes | Light Labs | A-7001-R | |
| Reasy Mini kit | Qiagen | 74104 | |
| Pellet pestile motor (Tissue homoginizer) | Sigma | Z359971-1EA | |
| 96 well microtiter plates V bottom | Thermo | 2605 | |
| 1X PBS | Gibco | 10010-023 | |
| OneComp eBeads | eBioscience | 01-1111-42 | |
| CD45.2-APC | eBioscience | 17-0454-81 | Working dilution 1:100 |
| Ly-6G-eFlor 450 | eBioscience | 48-5931-82 | Working dilution 1:100 |
| BSA | HyClone | SH30574.03 | |
| RBC Lysis Buffer (10X) | Biolegend | 420301 | |
| Live/Dead fixable aqua dead cell stain kit | Invitrogen | L-34957 | |
| EDTA (0.5M) | lifetechnologies | 15575-020 | |
| CD16/CD32 FcBlock | BD | 553142 | |
| Facs tubes polystyrene round bottom tube | BD | 352052 | |
| Formaldehyde | Polyscience | 4018 |
References
- Medzhitov, R. Origin and physiological roles of inflammation. Nature. 454, 428-435 (2008).
- Grivennikov, S. I., Greten, F. R., Karin, M. Immunity, inflammation, and cancer. Cell. 140, 883-899 (2010).
- Barnes, P. J. Immunology of asthma and chronic obstructive pulmonary disease. Nat Rev Immunol. 8, 183-192 (2008).
- Sethi, S., Murphy, T. F. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med. 359, 2355-2365 (2008).
- King, P. T., Sharma, R. The Lung Immune Response to Nontypeable Haemophilus influenzae (Lung Immunity to NTHi). J Immunol Res. 2015, 706376 (2015).
- Kapur, N., Grimwood, K., Masters, I. B., Morris, P. S., Chang, A. B. Lower airway microbiology and cellularity in children with newly diagnosed non-CF bronchiectasis. Pediatr Pulmonol. 47, 300-307 (2012).
- King, P. T., Holdsworth, S. R., Freezer, N. J., Villanueva, E., Holmes, P. W. Microbiologic follow-up study in adult bronchiectasis. Respir Med. 101, 1633-1638 (2007).
- Shuto, T., et al. Activation of NF-kappa B by nontypeable Hemophilus influenzae is mediated by toll-like receptor 2-TAK1-dependent NIK-IKK alpha /beta-I kappa B alpha and MKK3/6-p38 MAP kinase signaling pathways in epithelial cells. Proc Natl Acad Sci U S A. 98, 8774-8779 (2001).
- Moghaddam, S. J., et al. Haemophilus influenzae lysate induces aspects of the chronic obstructive pulmonary disease phenotype. Am J Respir Cell Mol Biol. 38, 629-638 (2008).
- Rajagopalan, G., et al. Intranasal exposure to bacterial superantigens induces airway inflammation in HLA class II transgenic mice. Infect Immun. 74, 1284-1296 (2006).
- Ganesan, S., et al. Elastase/LPS-exposed mice exhibit impaired innate immune responses to bacterial challenge: role of scavenger receptor A. Am J Pathol. 180, 61-72 (2012).
- Hogner, K., et al. Macrophage-expressed IFN-beta contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia. PLoS Pathog. 9, e1003188 (2013).
- Karisola, P., et al. Invariant Natural Killer T Cells Play a Role in Chemotaxis, Complement Activation and Mucus Production in a Mouse Model of Airway Hyperreactivity and Inflammation. PloS one. 10, e0129446 (2015).
- Theivanthiran, B., et al. The E3 ubiquitin ligase Itch inhibits p38alpha signaling and skin inflammation through the ubiquitylation of Tab1. Sci Signal. 8, 22 (2015).
- Venuprasad, K., Zeng, M., Baughan, S. L., Massoumi, R. Multifaceted role of the ubiquitin ligase Itch in immune regulation. Immunol Cell Biol. , (2015).
- Decramer, M., Janssens, W., Miravitlles, M. Chronic obstructive pulmonary disease. Lancet. 379, 1341-1351 (2012).
