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.

Alle Experimente wurden in Übereinstimmung mit den Richtlinien der Institutional Animal Care und Use Committee (IACUC) von Baylor Research Institute durchgeführt. 1. Anzucht nicht typisierbaren Haemophilus influenzae (NTHi) und Vorbereiten des Inokulums <ol><li> Platte NTHi auf einem Schokolade - Agar - Platte und halten die Platte den Kopf in einem befeuchteten CO 2 Inkubator über Nacht bei 37 ° C und 5% CO 2. Am folgenden Tag Kultur der Bakterien in Gehirn-Herz-Infusionsbrühe. Dann fügen Sie 1 ml der Brühe in eine sterile 1,5 ml-Tube. </li><li> Zentrifuge bei 4.000 xg für 5 min bei Raumtemperatur. Dann wird der Überstand verworfen und erneut zu suspendieren, das Pellet in 1 ml sterile 1x PBS. Wiederholen Sie einmal diesen Schritt. </li><li> Transfer von 100 &amp; mgr; l der resuspendierten Lösung in 900 ul 1x PBS und Messen der Extinktion der verdünnten Kultur bei 600 nm Wellenlänge in einem Spektrophotometer. </li><li> Bestimmen Sie die Lebensfähigkeit und die Kolonie bildende Einheits (CFUs) des Inokulums durch Züchten 01.10 Reihenverdünnungen auf Schokolade - Agarplatten und über Nacht bei 37 ° C und 5% CO 2 inkubiert. </li><li> Bezogen auf die Lebensfähigkeit und die CFUs aus dem vorherigen Schritt bestimmt wird , das Inokulum auf eine Endkonzentration von 20 x 10 6 CFU / ml einzustellen. </li></ol> 2. intratracheale (it) Einträufeln <ol><li> Injizieren Sie die Maus intraperitoneal (ip) mit 0,1 ml / 10 g Körpergewicht der Maus mit einer 150 mg / ml Ketamin + 20 mg / ml Xylazin Lösung. Das Verfahren sollte mit einer Lichtquelle in der Haube getan werden, um das Tier warm zu halten. </li><li> Drücken Sie den Zehenzwischenraum der Maus, um zu überprüfen, dass die Maus ausreichend betäubt. </li><li> Platzieren Sie die Maus auf dem Rücken. Shave den ventralen Bereich des Halses und desinfizieren mit Chlorhexidin und 70% Ethylalkohol. </li><li> Heben Sie die Haut des oberen ventralen Teil des Halses unter Verwendung von Rattenzahnzange. Machen Sie einen kleinen Schnitt (0,5-1,0 cm) über dem Thymus mit einem Skalpellund sorgfältig den Muskel sezieren, um die Luftröhre aus. <ol><li> Unter Verwendung einer 1-ml-Spritze mit einer 27 G-Nadel, die Injektion sollte langsam 0,05 ml Luft, gefolgt von 0,05 ml NTHi erhalten von Abschnitt 1 oder Salzlösung als Kontrolle, gefolgt von einem weiteren 0,05 ml Luft. Halten Sie Tier vertikal für ca. 1 min. </li></ol></li><li> Schließen Sie den Schnitt mit chirurgischen Klebstoff. Halten Sie die Maus seitlich liegend und warm für ca. 10 min. </li></ol> 3. BALF Sammlung <ol><li> Opfere die Maus durch Genickbruch 24 Stunden nach der Infektion. </li><li> Öffnen Sie die Bauchhöhle der Maus mit anatomischen Schere und schneiden Sie die Bauchaorta zu bluten. Setzen Sie das ventrale Teil der Lunge. </li><li> Setzen Sie die Luftröhre und intubieren es mit einem 20-Gauge-Katheter. Instill 0,8 ml BALF Puffer (1x PBS, 0,1% Dextrose, 10 U / ml Heparin) und die Spülung durch leichtes Ansaugen sammeln. Wiederholen Sie diesen Vorgang dreimal. </li><li> Zählen der Gesamtzahl der Zellen in 100 &amp; mgr; l BALF Suspension unter Verwendung eines hemocytoMeter unter 10-facher Vergrößerung mit Trypan-Blau-Färbung. </li><li> Bereiten Sie Folien für modifizierte Giemsa. Aliquot 100 ul BALF in die entsprechenden Vertiefungen der Cytospin und Zentrifuge bei 500 xg für 5 min. Trocknen der Objektträger für 10 min und färben die Zellen modifiziert Giemsa-Färbung unter Verwendung von nach Protokollen des Herstellers. </li><li> Verwenden , um die verbleibenden Zellen für eine nachfolgende Analyse, wie beispielsweise Fluoreszenz - aktivierte Zellsortierung (FACS), die konfokale Mikroskopie, die Genexpression und die Western - Blot - 11-13. </li></ol> 4. Histopathologie Vorbereitung <ol><li> Für histopathologische Analyse, infizieren Mäuse mit NTHi wie in Schritt 2.4 beschrieben. Nach 24 Stunden der Infektion opfern, um die Tiere durch Genickbruch. Schneiden Sie die Brusthöhle mit anatomischen Schere öffnen die Lunge und die Luftröhre freizulegen, wie bereits erwähnt. Dann legen Sie die 20-Gauge-Katheter in die Trachea. </li><li> Füllen Sie die Lunge mit 2% warme Agarose-Lösung in 4% Formalin präpariert. Sobald derLungen sind voll aufgeblasen, statt Eis auf der Oberseite der Lunge die Agarose-Lösung zu verfestigen. </li><li> Dann entfernen Sie vorsichtig die Brust Stecker und steckte es in eine 50 ml-Lösung von 4% Formalin in 1x PBS, bis sie für Hämatoxylin und Eosin (H &amp; E) Abschnitte verarbeitet wird. </li></ol> 5. FACS Zellen in der BALF Anfärben <ol><li> Stellen 1 x 10 5 Zellen von der BALF Fluid in einer 96-Well - V-Boden Mikrotiterplatte. </li><li> Wasche die Zellen mit 300 ul kaltem PBS 1x und Schleudern bei 4.000 × g für 3 min bei 4 ° C. </li><li> Stain die Zellen mit Zellfarblösung in 100 ul 1x PBS (1: 1000 Verdünnung) und halten bei Raumtemperatur für 15 min. </li><li> Wasche die Zellen mit 300 ul kaltem PBS 1x bei 4.000 × g für 3 min bei 4 ° C. </li><li> Halten Zellen in 100 &amp; mgr; l FACS-Waschpuffer [Waschpuffer (1x PBS, 2% FBS, 1 mM EDTA, 0,1% Natriumazid], das 1: 100 verdünnt Fc-Block-für 15 min bei 4 ° C. </li><li> Centrifuge Zellen bei 4.000 × g für 3 min und entsorgen supernatant. </li><li> Stain Zellen mit 100 &amp; mgr; l Oberflächenfärbung Cocktail (1: 100 Verdünnung) in FACS-Waschpuffer für 30 min bei 4 ° C. </li><li> Waschen Zellen dreimal mit 300 &amp; mgr; l FACS-Waschpuffer bei 4.000 × g für 3 min bei 4 ° C. </li><li> Fix-Zellen in 100 &amp; mgr; l von 4% Paraformaldehyd für 15 min bei Raumtemperatur. </li><li> Wasche die Zellen zweimal mit 300 ul 1x PBS bei 400 × g für 3 min bei 4 ° C und resuspendieren in 300 &amp; mgr; l FACS-Waschpuffer. </li><li> Machen Sie einfarbige Färbung Kontrollen unter Verwendung von Durchflusszytometrie Perlen. <ol><li> Geben Sie einen Tropfen Perlen in den 96-Well-V-Boden-Mikrotiterplatte und waschen mit 200 ul 1x PBS. </li><li> 1 &amp; mgr; l-Färbung Antikörper auf 100 ul 1x PBS und Perlen und Inkubation für 5 min bei Raumtemperatur. </li><li> Waschen Sie Perlen zweimal mit 300 ul 1x PBS. </li></ol></li><li> Analysieren Sie einzelne gebeiztem Kontrollen und Zellen mittels Durchflusszytometrie 14. </li></ol> 6. Homogenisieren von Lungengewebe RNA zu isolieren <ol><li> Nach der Infektion über Nacht ein kleines Stück Lunge (20-40 mg) und halten Sie sie in RNA-stabilisierende Reagenz bei 4 ° C zu sammeln. Dann speichern Sie es bei -80 ° C bis zum nächsten Schritt. </li><li> Waschen Sie die Lungengewebe in kaltem 1x PBS loswerden der RNA-Stabilisierung Reagenz zu erhalten. </li><li> Platzieren das Gewebe in einem 1,5 ml Zentrifugenröhrchen, das Lyse-Puffer (1 ml Lyse-Puffer + 10 &amp; mgr; l β-Mercaptoethanol). </li><li> Hacken Sie das Gewebe in kleine Stücke spitzen Schere und homogenisieren für 20-40 sec ein schnurloses Motor Pellet zerstoßen. </li><li> Halten Sie das Lysat auf Eis für 5 min und fahren Sie mit Schritt 7.1. </li></ol> 7. RNA-Isolierung aus der Lunge <ol><li> Zentrifugieren Sie das Lysat bei 18.000 × g für 3 min. Entfernen Sie den Überstand durch Pipettieren und vorsichtig Transfer in ein neues 1,5-ml-Röhrchen. </li><li> 1 Volumen von 70% Ethanol zu dem Lysat. Mischen Sie sofort durch Pipettieren und warten 2-4 min. </li><li> Übertragen Sie bis zu 700 &amp; mgr; l Lysat auf eine Spin-Säule platziertin einem 2 ml Sammelröhrchen. </li><li> Zentrifuge bei 9.600 × g für 15 Sekunden und den Durchlauf verwerfen. </li><li> In 350 ul strengen Waschpuffer und Zentrifuge bei 9.600 × g für 15 Sekunden. Verwerfen Sie den Durchfluss durch. </li><li> In 80 ul DNase I direkt auf die Spinsäule Membran und halten Sie sie für 15 Minuten bei Raumtemperatur. </li><li> In 350 ul strengen Waschpuffer auf die Spin-Säule und Zentrifuge bei 9.600 × g für 15 Sekunden. Verwerfen Sie den Durchfluss durch. </li><li> In 500 ul RNA Waschpuffer und Zentrifuge bei 9.600 × g für 15 Sekunden. Verwerfen Sie den Durchfluss durch. </li><li> In 500 ul RNA Waschpuffer und Schleudern bei 9.600 × g für 2 min. Verwerfen Sie den Durchfluss durch. </li><li> Legen Sie die Spin-Säule in ein neues 2 ml Sammelröhrchen und Zentrifuge bei 9.600 × g für 1 min. Verwerfen Sie den Durchfluss durch. </li><li> Hinzufügen 30-50 &amp; mgr; l RNase-freiem Wasser direkt auf die spin-Säule zentrifugieren bei 9.600 × g für 1 min. Store RNA bei -80 ° C. </li><li> Führen RNA-Seq - Analyse 14. </li></ol>

<ol>
	<li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+physiological+roles+of+inflammation.">Origin and physiological roles of inflammation.</a> Nature. 454, 428-435 (2008).</li><li>Grivennikov, S. I., Greten, F. R., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity,+inflammation,+and+cancer.">Immunity, inflammation, and cancer.</a> Cell. 140, 883-899 (2010).</li><li>Barnes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+asthma+and+chronic+obstructive+pulmonary+disease.">Immunology of asthma and chronic obstructive pulmonary disease.</a> Nat Rev Immunol. 8, 183-192 (2008).</li><li>Sethi, S., Murphy, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+in+the+pathogenesis+and+course+of+chronic+obstructive+pulmonary+disease.">Infection in the pathogenesis and course of chronic obstructive pulmonary disease.</a> N Engl J Med. 359, 2355-2365 (2008).</li><li>King, P. T., Sharma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lung+Immune+Response+to+Nontypeable+Haemophilus+influenzae+(Lung+Immunity+to+NTHi).">The Lung Immune Response to Nontypeable Haemophilus influenzae (Lung Immunity to NTHi).</a> J Immunol Res. 2015, 706376 (2015).</li><li>Kapur, N., Grimwood, K., Masters, I. B., Morris, P. S., Chang, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+airway+microbiology+and+cellularity+in+children+with+newly+diagnosed+non-CF+bronchiectasis.">Lower airway microbiology and cellularity in children with newly diagnosed non-CF bronchiectasis.</a> Pediatr Pulmonol. 47, 300-307 (2012).</li><li>King, P. T., Holdsworth, S. R., Freezer, N. J., Villanueva, E., Holmes, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiologic+follow-up+study+in+adult+bronchiectasis.">Microbiologic follow-up study in adult bronchiectasis.</a> Respir Med. 101, 1633-1638 (2007).</li><li>Shuto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Proc Natl Acad Sci U S A. 98, 8774-8779 (2001).</li><li>Moghaddam, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemophilus+influenzae+lysate+induces+aspects+of+the+chronic+obstructive+pulmonary+disease+phenotype.">Haemophilus influenzae lysate induces aspects of the chronic obstructive pulmonary disease phenotype.</a> Am J Respir Cell Mol Biol. 38, 629-638 (2008).</li><li>Rajagopalan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+exposure+to+bacterial+superantigens+induces+airway+inflammation+in+HLA+class+II+transgenic+mice.">Intranasal exposure to bacterial superantigens induces airway inflammation in HLA class II transgenic mice.</a> Infect Immun. 74, 1284-1296 (2006).</li><li>Ganesan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastase/LPS-exposed+mice+exhibit+impaired+innate+immune+responses+to+bacterial+challenge:+role+of+scavenger+receptor+A.">Elastase/LPS-exposed mice exhibit impaired innate immune responses to bacterial challenge: role of scavenger receptor A.</a> Am J Pathol. 180, 61-72 (2012).</li><li>Hogner, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage-expressed+IFN-beta+contributes+to+apoptotic+alveolar+epithelial+cell+injury+in+severe+influenza+virus+pneumonia.">Macrophage-expressed IFN-beta contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia.</a> PLoS Pathog. 9, e1003188 (2013).</li><li>Karisola, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invariant+Natural+Killer+T+Cells+Play+a+Role+in+Chemotaxis,+Complement+Activation+and+Mucus+Production+in+a+Mouse+Model+of+Airway+Hyperreactivity+and+Inflammation.">Invariant Natural Killer T Cells Play a Role in Chemotaxis, Complement Activation and Mucus Production in a Mouse Model of Airway Hyperreactivity and Inflammation.</a> PloS one. 10, e0129446 (2015).</li><li>Theivanthiran, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+E3+ubiquitin+ligase+Itch+inhibits+p38alpha+signaling+and+skin+inflammation+through+the+ubiquitylation+of+Tab1.">The E3 ubiquitin ligase Itch inhibits p38alpha signaling and skin inflammation through the ubiquitylation of Tab1.</a> Sci Signal. 8, 22 (2015).</li><li>Venuprasad, K., Zeng, M., Baughan, S. L., Massoumi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+role+of+the+ubiquitin+ligase+Itch+in+immune+regulation.">Multifaceted role of the ubiquitin ligase Itch in immune regulation.</a> Immunol Cell Biol. , (2015).</li><li>Decramer, M., Janssens, W., Miravitlles, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+obstructive+pulmonary+disease.">Chronic obstructive pulmonary disease.</a> Lancet. 379, 1341-1351 (2012).</li></ol>

The authors have nothing to disclose.

Hier beschreiben wir eine einzigartige und minimal-invasive Verfahren, um die Lungen von Mäusen, die mit einer bakteriellen Lungen Pathogen zu beimpfen. Wir zeigen, dass dieses Verfahren verwendet werden kann, um die Funktion der verschiedenen Gene zu untersuchen, die in Mäusen unter Verwendung von Genen von Entzündungssignalwege defizient sind. Dieses Verfahren kann auch die Entzündungsreaktionen gegen virale und fungale Infektionen der Lunge zu untersuchen verwendet werden. Die Vorteile dieses Verfahrens gegenüber anderen Verfahren, wie intranasale oder Aerosolinhalation sind (1) in diesem Verfahren wird das pathogene Inokulum direkt an die unteren Atemwege instilliert; (2) die Fähigkeit, das Inokulum Größe zu steuern; und (3) ein geringeres Risiko einer bakteriellen Exposition gegenüber dem Handler. Die Exposition der Trachea chirurgisch für Bakterien Einträufeln ist ein entscheidender Schritt, wo Pflege Schäden an umliegenden Gewebe zu vermeiden, getroffen werden müssen, verursachen, vor allem die Blutgefäße. Instillation von Bakterien in die Trachea should sorgfältig durch Erstickung die Möglichkeit des Todes der Mäuse zu vermeiden, durchgeführt werden. Es wird vorgeschlagen, dass das Inokulum (etwa 50 ul) langsam freigesetzt werden und die Luft vor und nach dem Inoculum injiziert werden. nach dem Einträufeln der Maus vertikal zu halten für eine Weile und halten sie seitlich erleichtert Liegerad schnellere Genesung. Der Hauptnachteil dieses Verfahrens besteht darin, dass Mehrfachbelichtungen innerhalb kurzer Zeit nicht möglich sind aufgrund des Erfordernisses der Chirurgie. Ein mögliches Problem ist, dass, wenn der Katheter zu tief in die Bronchien platziert ist, wird der BALF-Zellzahl niedriger sein. Dieses Problem kann durch Ziehen des Rohres leicht nach oben gelöst werden. Da die Inzidenz von Atemwegsentzündungserkrankungen sind weltweit 3,16 zu erhöhen, das Verständnis der Pathophysiologie dieser Krankheiten ist von zentraler Bedeutung. Die Technologien, die in dieser Studie beschriebenen könnte dazu beitragen, wichtige regulatorische Wege zu erkennen und könnten die Entwicklung erleichternneuer Behandlungsmethoden.

Die Entzündung ist eine grundlegende Immunabwehrmechanismus gegen infektiöse Erreger. Es fördert die Erreger Beseitigung und Reparatur von beschädigtem Gewebe. Es erleichtert auch die Erholung zu einem normalen , gesunden Zustand 1. Jedoch führt dysregulated Entzündung oft zu chronischen entzündlichen Erkrankungen 2. Entzündung der Atemwege ist ein erster Auslöser für verschiedene Lungenerkrankungen wie chronische obstruktive Lungenerkrankung (COPD), Asthma und Lungenfibrose 3. Die nicht-typisierbaren (nicht eingekapselten) Haemophilus influenzae (NTHi) mit chronischen oberen und unteren Lungenentzündungserkrankungen 4,5 verbunden. Es ist die dominante Spezies aus den unteren Atemwege von Kindern und Erwachsenen mit chronisch - obstruktiver Lungenerkrankung 4,6,7 isoliert. Die Entzündungsantwort nach der NTHi-Infektion wird durch die Hochregulation von proinflammatorischen Zytokinen (wie TNF und IL-1β) gekennzeichnet, und es wird vermittelt by Mitogen - aktivierte Proteinkinase (MAPK) und nuclear factor-kappaB (NF-kappaB) über Toll-like Rezeptoren (TLR) 8. Mausmodelle sind sehr nützliche Werkzeuge zur Analyse der zugrundeliegenden Pathologie der Lungenentzündungskrankheit aufgrund der Verfügbarkeit der verschiedenen Gen-defizienten Linien. Live / attenuierten Bakterien und bakterielle Produkte zu inokulieren, einschließlich intranasale Instillation und aerosolized Inhalations 9,10 mehrere Verfahren verwendet worden. Hier zeigen wir, intratracheale Instillation. Obwohl weniger häufig verwendet wird, ist dieser Ansatz effizienter und hoch reproduzierbare wegen der direkten Abgabe des Inokulums auf den unteren Atemwege.

<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

Hier beschreiben wir ein detailliertes Verfahren , um effizient und direkt Haemophilus influenzae liefern in die unteren Atemwege von Mäusen. Wir zeigen das Verfahren zur Herstellung von H. influenzae Inokulum, intratracheale Instillation von H. influenzae in die Lunge, Sammlung von broncho-alveolären Lavage (BALF), Analyse von Immunzellen in der BALF und RNA - Isolation für Differentialgenexpressionsanalyse. Dieses Verfahren kann verwendet werden, um die Lungenentzündungsreaktion auf alle Bakterien, Viren oder Pilze zu untersuchen. Direkt tracheale Instillation wird meist über intranasale oder Aerosolinhalationsverfahren bevorzugt, da es effizienter das bakterielle Inokulum in die unteren Atemwege mit geringer Mehrdeutigkeit liefert.

Intratracheale Instillation führte zu einer deutlich erhöhten Anzahl von Leukozyten in der BALF (1A, linkes Feld) als Anlage mit Kochsalzlösung. Die Differentialzählung Analyse der Leukozyten zeigte deutlich erhöhte neutrophile Infiltration (Abbildung 1, rechts). Die FACS - Analyse der Zellen in der BALF bestätigt weiter die erhöhte Anzahl von Neutrophilen (1B). Histologische Analyse von H &amp; E-gefärbten Schnitten des Lungengewebes zeigten eine Entzündung der Atemwege (1C) erhöht. Diese Daten legen nahe, dass kollektiv intratracheale Instillation Atemwegsentzündung bei Mäusen induziert. Um die Verwendung dieses Ansatzes zur Untersuchung von Signalwegen zu unterstützen, die Atemwegs Pathogenese regulieren, haben wir Mäuse, die für die E3-Ubiquitin-Ligase Itch defizient sind. Krätze ist bekannt , bei der Regulierung der Entzündungssignalwege 15 mit einbezogen werden. Wir geimpftalters- und geschlechtsangepassten Wildtyp C57BL / 6-Steuerung und Itch - / - Mäuse mit NTHi. Wir isolierten RNA aus den Lungengeweben von Kontroll WT und Itch - / - Mäusen und ausgeführt ganze Transkriptom Sequenzierung der Entzündungs ​​Gene zu identifizieren, die differentiell exprimiert werden. In differenzielle Expression von mehreren Genen , wie in Abbildung 2, Mangel führte Itch gezeigt. Dies legt nahe, daß intratracheale Instillation verwendet werden kann, Lungenentzündung, Genexpressionsprofile und Signalwege zu untersuchen. <img alt="Abbildung 1" src="/files/ftp_upload/53964/53964fig1.jpg" /> Abbildung 1: intratrachealen Inokulation von NTHi induziert Lungenentzündung bei Mäusen Mäuse wurden mit NTHi geimpft (10 6 CFU / Maus) oder einer Salzkontrolle.. 24 Stunden nach der Inokulation wurden die Mäuse getötet (A) BALF wurde gesammelt. Die gesamten Leukozyten, Monozyten und Neutrophilen-Zahlen bestimmt. Die Daten sindals Mittelwert ± SEM. n = 4 Mäuse / Gruppe. * Zeigt p &lt;0,05 im Vergleich zu Kochsalzlösung behandelten Mäusen. (B) Die Zellen in der BALF wurden mit anti-Gr1 - Antikörper gefärbt und durch FACS analysiert. (C) H &amp; E-gefärbten Lungenschnitten Leukozyten - Infiltration und Entzündung zeigt. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53964/53964fig1large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53964/53964fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/53964/53964fig2.jpg" /> Abbildung 2: Differentielle Genexpression in der Lunge von WT und Itch - / - Mäusen nach der intratrachealen NTHi Inokulation 24 Stunden nach NTHi Inokulation RNA aus den Lungen von zwei WT isoliert wurde (1, 2) und zwei Itch - / -. (1, 2) Mäusen. (A) wurde die RNA Qualität analysiert , um eine Bioanalyzers verwenden. (B) Heatmap zeigt Z-Scores (als Maß für sd interpretiert weg vonder Mittelwert) für die Zählung log 2 pro Million (log 2 CPM) von 172 differentiell exprimierten Gene mit Edger im Itch identifiziert - / -. im Vergleich zu WT - Mäusen <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53964/53964fig2large.jpg" target="_blank">Bitte klicken Sie hier eine größere Version dieser Figur zu sehen.</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.

Intratracheale Verabreichung von<em&gt; Haemophilus influenzae</em&gt; In Mausmodelle zur Untersuchung Atemwegsentzündung

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 Aufrufe.  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. 

Serie: 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.

Co-Kultur Modelle Pseudomonas aeruginosa Biofilme auf lebenden menschlichen Zellen gezüchtet Airway

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

Forschung

Immunologie und Infektion

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.

Analyse der Lungen-Reifung von dendritischen Zellen und Migration während der allergischen Atemwegsentzündung

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.

Aufbau einer Liquid-bedeckten Kultur Polarized Menschliche Airway Epithelial Calu-3-Zellen, um Host-Zell-Antwort, um Atemwegspathogene Studie<em&gt; In-vitro-</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.

In-vivo-Imaging-Methode zu unterscheiden, akuten und chronischen Entzündungen

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.

Die intranasale Verabreichung von rekombinantem Influenza Vaccines in chimären Maus-Modelle zu Mucosale Immunität Studieren

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.

Co-Immunopräzipitation der Maus Mx1 Protein mit dem 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 ...

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.

Ein optimiertes Verfahren zur Isolierung und Ausbau unveränderlichen natürlichen Killer T-Zellen aus der Milz der Maus

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.

Erhöhte Erholzeit und abnehmend LPS-Verabreichung die Vagusnervstimulation Mechanisms in beschränkten Entzündungsreaktionen zu studieren

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.

Precision-cut Maus Lung Slices Live-Pulmonary Dendritische Zellen sichtbar zu machen

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.