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.

所有实验均按照贝勒研究院的机构动物护理和使用委员会（IACUC）的指导方针进行。 1.培养不可分型流感嗜血杆菌 （NTHi的），并准备接种<ol><li>在巧克力琼脂平板板的NTHi和在37℃和5％CO 2在加湿的 CO 2培养箱中保持板倒置过夜。翌日，培养在脑心脏浸液肉汤的细菌。然后，加入1毫升肉汤进入无菌1.5毫升管。 </li><li>离心以4,000 xg离心在室温下5分钟。然后，弃去上清液并在1ml无菌1×PBS中重新悬浮沉淀。重复此步骤一次。 </li><li>在分光光度计转移100微升的再悬浮溶液注入900微升1×PBS中的与测量稀释的培养的吸光度在600nm处的波长。 </li><li>确定的生存能力和集落形成单位秒（CFU的）接种的通过培养在巧克力琼脂平板上1点10系列稀释，并在37℃下温育过夜，5％ 的 CO 2。 </li><li>基于从前面的步骤确定的生存能力和的CFUs，调整接种物，以20×10 6 CFU / ml的终浓度。 </li></ol> 2.气管内（IT）滴注<ol><li>用一个150毫克/毫升氯胺酮+ 20毫克/毫升甲苯噻嗪液取0.1毫升/ 10克体重小鼠注入小鼠腹腔内（IP）。该过程应在通风橱中进行与光源保持动物保暖。 </li><li>捏住鼠标的叉指空间，以验证该鼠标充分麻醉。 </li><li>将在它的后面鼠标。剃颈部腹侧区域，并与氯己定和70％的乙醇消毒。 </li><li>解除用鼠齿钳的颈部的上部腹侧部分的皮肤上。做一个小切口（0.5〜1.0厘米）胸腺上面用手术刀然后小心地解剖肌肉以暴露气管。 <ol><li>用1ml注射器用27克针，缓缓注入0.05毫升空气，接着从第1或盐水作为对照获得0.05毫升的NTHi，接着另一0.05毫升空气。保持动物的垂直约1分钟。 </li></ol></li><li>关闭与手术胶水切口。按住鼠标横向躺着和温暖约10分钟。 </li></ol> 3.收集肺泡灌洗液<ol><li>牺牲颈椎脱位24小时小鼠感染后。 </li><li>打开解剖剪鼠标的腹腔和切腹主动脉流血。揭露肺的腹一部分。 </li><li>暴露气管并用20号的导管插管它。灌输0.8毫升BALF缓冲液（1×PBS中，0.1％葡萄糖，10 U / ml肝素），并收集轻轻吸出灌洗。重复此步骤三次。 </li><li>算在100μlBALF悬浮液用血细胞的细胞的总数在放大10倍与台盼蓝染色米。 </li><li>准备修改姬姆萨染色幻灯片。等份加入100μlBALF进入细胞离心涂片和离心机在500×g离心5分钟的适当的孔中。干燥10分钟的幻灯片，并根据制造商的协议使用改性Giemsa染色细胞染色。 </li><li>使用剩余的细胞用于随后的分析，例如荧光激活细胞分选（FACS），共焦显微镜，基因表达和蛋白质印迹11-13。 </li></ol> 4.组织病理学准备<ol><li>对于组织病理学分析，感染与NTHi的小鼠如在步骤2.4中描述。感染后的24小时，通过颈脱位牺牲动物。切断与解剖剪刀以暴露肺和如前所述气管打开胸腔。然后，将20号导管插入气管。 </li><li>填充在4％福尔马林制备2％温暖琼脂糖溶液的肺部。一旦肺完全膨胀，地方冰对肺部的顶部，以巩固琼脂糖溶液。 </li><li>然后，小心地取出胸插头，把它在1X PBS 4％福尔马林50毫升的解决方案，直到它被用于苏木精伊红（H＆E）的部分进行处理。 </li></ol> 5. FACS BALF中染色的细胞<ol><li>把从BALF流体1×10 5个细胞在96孔V形底微量滴定板。 </li><li>在4℃下洗涤细胞用冷的1×PBS中的300微升和自旋在4000 xg离心3分钟。 </li><li>染色与细胞染色液细胞在100μl1×PBS中（1:1000稀释度），并保持在室温下15分钟。 </li><li>在4℃下洗涤细胞用300μl冷1×PBS中的在4000 xg离心3分钟。 </li><li>保持细胞在100μl含有1 FACS洗涤缓冲液[洗涤缓冲液（1×PBS，2％FBS，1mM EDTA中，0.1％叠氮化钠]的:100稀释的Fc块在4℃下15分钟。 </li><li>离心细胞以4000×g离心3分钟，并弃去SUPernatant。 </li><li>染色的细胞用100μl表面染色鸡尾酒（1:100稀释）在FACS洗涤缓冲液在4℃，30分钟。 </li><li>在4℃以4000 xg离心用300μlFACS洗涤缓冲液中洗涤细胞三次3分钟。 </li><li>固定的细胞在100μl的4％多聚甲醛在室温下15分钟。 </li><li>在4℃下用300μl1×PBS中洗涤细胞两次，在400×g离心3分钟，并在300微升的FACS洗涤缓冲液中重新悬浮。 </li><li>请使用流式细胞仪珠单色染色控制。 <ol><li>添加珠粒一滴到96孔V形底微量滴定板并用200μl1×PBS洗涤。 </li><li>加入1μl染色抗体与100μl的1×PBS中并珠和在室温下孵育5分钟。 </li><li>用300μl1X PBS洗两次珠。 </li></ol></li><li>通过流式细胞仪分析14单染色控制和细胞。 </li></ol> 6.均质肺组织来隔离RNA <ol><li>感染后，收集一小块肺（20-40毫克），并保持它的RNA在4℃稳定试剂过夜。然后，在-80℃储存，直到下一个步骤。 </li><li>用冷1X PBS肺组织摆脱RNA稳定试剂。 </li><li>放置组织到1.5毫升离心管中含有裂解缓冲液（1毫升的裂解缓冲液+10μl的β巯基乙醇）。 </li><li>使用尖剪刀切碎的组织切成小块并采用无绳马达粒料杵20-40秒均质。 </li><li>保持在冰上裂解5分钟，并与步骤7.1进行。 </li></ol> 7.从肺RNA分离<ol><li>离心溶胞产物18000 xg离心3分钟。通过移液除去上清液，并仔细转移到一个新的1.5毫升管。 </li><li> 1体积的70％乙醇加入到裂解物。吹打立即混匀，等待2-4分钟。 </li><li>转移到700微升裂解物以置于旋转柱在2 ml收集管。 </li><li>离心机以9,600×g离心15秒，弃去流过。 </li><li>加入350微升严格的洗涤液，离心9,600 XG为15秒。丢弃的流量通过。 </li><li> 80微升DNA酶添加我直接向旋转柱膜并保持在室温下15分钟。 </li><li>添加350微升严格洗涤缓冲液到旋转柱和离心机以9,600×g离心15秒。丢弃的流量通过。 </li><li>以9,600 XG加入500μlRNA洗涤液，离心15秒。丢弃的流量通过。 </li><li>以9,600 XG加入500μlRNA洗涤液和旋转2分钟。丢弃的流量通过。 </li><li>放置在新的2ml收集管中，离心旋转柱以9,600 xg离心1分钟。丢弃的流量通过。 </li><li>直接以9,600 xg离心添加30-50微升不含RNA酶的水，以旋转柱，离心1分钟。在-80°C储存RNA。 </li><li>执行RNA测序分析14。 </li></ol>

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The authors have nothing to disclose.

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）减少细菌暴露于处理程序的风险。 手术灌输细菌气管暴露是至关重要的一步，其中必须小心，以免造成损伤周围组织，尤其是血管。 菌的滴注进气管的sh乌尔德仔细进行，以避免因窒息老鼠的死亡的可能性。有人建议，该接种物（约50微升）缓慢释放和前和接种后的空气被注入。按住鼠标垂直滴入了一段时间之后，保证它们的横向躺着有利于更快的恢复。这个过程的主要缺点是，很短的时间周期内多次曝光是不可能的，因为该手术的要求。一个潜在的问题是，如果在导管放置太深在支气管中，BALF细胞计数会更低。这个问题可以通过拉动管略微向上来解决。 由于气道炎症性疾病的发病率在世界范围内不断增加3.16，了解这些疾病的病理生理机制是最重要的。在这项研究中所描述的技术，可以帮助确定主要调控途径，并能够促进发展新的治疗模式。

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。

<table border="0" cellpadding="0" cellspacing="0" width="705">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Chocolate agar plate</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">CAS50-99-7</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Dextrose Anhydrous</td>
			<td style="width:127px;">Themo Scientific</td>
			<td style="width:119px;">R01300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Heparin</td>
			<td style="width:127px;">Hospira,Inc</td>
			<td style="width:119px;">RL-3010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Deft quick solution</td>
			<td style="width:127px;">Sigma</td>
			<td>GS500-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:293px;">Syringe needle 20/26G&#160;</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">(REF305115/175)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Iml syringe</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">REF 309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Catheter 20GA</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">REF 381433</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Scissors, straight, 10 cm long</td>
			<td>kentscientific</td>
			<td>INS600393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iris Forceps, serrated, 10cm long</td>
			<td>kentscientific</td>
			<td>INS650915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tweezer #5 Stainless steel, 11cm long</td>
			<td>kentscientific</td>
			<td>INS600095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">10% Formalin</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">CAS 67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Agarose</td>
			<td style="width:127px;">peqlab</td>
			<td style="width:119px;">35-1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">5ml polystyrene round-bottom tubes</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">REF 352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">1.5 ml Microcentrifuge tubes</td>
			<td style="width:127px;">Light Labs</td>
			<td style="width:119px;">A-7001-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Reasy Mini&#160; kit&#160;</td>
			<td style="width:127px;">Qiagen</td>
			<td align="right" style="width:119px;">74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Pellet pestile motor (Tissue homoginizer)</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">Z359971-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">96 well microtiter plates V bottom</td>
			<td style="width:127px;">Thermo</td>
			<td align="right" style="width:119px;">2605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">1X PBS</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">OneComp eBeads</td>
			<td style="width:127px;">eBioscience</td>
			<td style="width:119px;">01-1111-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">CD45.2-APC</td>
			<td style="width:127px;">eBioscience</td>
			<td style="width:119px;">17-0454-81</td>
			<td>Working dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Ly-6G-eFlor 450</td>
			<td style="width:127px;">eBioscience</td>
			<td style="width:119px;">48-5931-82</td>
			<td>Working dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">BSA</td>
			<td style="width:127px;">HyClone</td>
			<td style="width:119px;">SH30574.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">RBC Lysis Buffer (10X)</td>
			<td style="width:127px;">Biolegend</td>
			<td style="width:119px;">420301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Live/Dead fixable aqua dead cell stain kit</td>
			<td style="width:127px;">Invitrogen</td>
			<td style="width:119px;">L-34957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">EDTA (0.5M)</td>
			<td style="width:127px;">lifetechnologies</td>
			<td style="width:119px;">15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">CD16/CD32 FcBlock</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">553142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Facs tubes polystyrene round bottom tube</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">352052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Formaldehyde</td>
			<td style="width:127px;">Polyscience</td>
			<td style="width:119px;">4018</td>
			<td></td>
		</tr>
	</tbody>
</table>

intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation

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。

气管内滴注导致BALF中（ 图1A，左面板）比安装用盐水一个白血球数显着增加。白细胞的分类计数分析清楚地显示增加的中性粒细胞浸润（ 图1，右图）。在BALF中的细胞的FACS分析进一步证实嗜中性粒细胞数量的增加（ 图1B）。肺组织的H＆E染色的切片的组织学分析显示增加的气道炎症（ 图1C）。这些数据共同表明，气管内滴注在小鼠中诱导的气道炎症。支持使用这种方法的用于研究调节气道病信号途径，我们使用是缺陷的E3泛素连接酶痒的小鼠。痒已知参与调节炎症信号传导途径15。我们接种年龄和性别匹配的野生型C57BL / 6控制和痒 - / - 小鼠的NTHi。我们分离的RNA从控制WT和瘙痒的​​肺组织 - / - 小鼠和进行全转录组测序，以确定差异表达的炎症基因。 如图2中所示，痒不足导致的几个基因的差异表达。这表明，气管内滴注可用于调查肺部炎症，基因表达谱和信号通路。 <img alt="图1" src="/files/ftp_upload/53964/53964fig1.jpg" /> 图1:的NTHi的气管内接种在小鼠中诱导的肺部炎症小鼠用的NTHi（10 6 CFU /鼠）或盐水对照接种。在接种后24小时，处死小鼠，收集（A）的BALF。总白细胞，单核细胞和嗜中性粒细胞数进行了测定。数据表示平均值±SEM。 N = 4只/组。 *表示相对于盐水处理的小鼠P &lt;0.05。 （B）在BALF中的细胞用抗Gr1的抗体染色，并通过FACS分析。 （C）H＆E染色的肺切片显示白细胞浸润和炎症。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53964/53964fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53964/53964fig2.jpg" /> 图2:在WT和瘙痒的肺部基因表达差异- / -小鼠以下气管内的NTHi接种的NTHi接种24小时后，RNA的从两个WT的肺中分离（1,2）和两个痒- / - 。 （1，2）的小鼠。 （A）的RNA的质量用生物分析仪进行分析。 （B）热图显示Z值（从解释为SD衡量远平均）为每百万日志2计数（日志172痒痒使用磨边机鉴定差异表达基因2 CPM） - / -相比于WT小鼠<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53964/53964fig2large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

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.

的气管内管理<em&gt;流感嗜血杆菌</em&gt;在小鼠模型来研究气道炎症

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

10.1K 次观看.  Baylor Research Institute. 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. 

视频: Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

在体内成像方法区分急性和慢性炎症

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

增加了恢复时间，并减少LPS管理研究迷走神经刺激机制的有限炎症反应

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

精密切割的小鼠肺切片可视化现场肺树突状细胞

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

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