Name: bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury
Uploaded: 2018-05-21T15:00:00
Description: Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

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Het totale protocol vereist 2 dagen, met inbegrip van de eerste dag van sepsis inductie en isolatie van BAL vloeistof uit het dier, en de tweede dag voor exosomes isolatie van de muis BALF. Alle procedures zijn herzien en goedgekeurd door het institutionele Animal Care en gebruik Comité aan Atlanta VA medical center.
1. Acute septische Lung Injury muismodel
<ol><li>Gebruik 6-8 weken - oude mannelijke wild-type C57BL/6J muizen (gewicht 20-22 g) voor het dier model, en uitvoeren van alle proc...

<ol>
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Muismodellen van ziekten worden vaak gebruikt om te evalueren van de fysiologische functie van specifieke genen en om de kosten van experimenten2. De acute septische Long schade beschreven hier bootst ontstekingsreactie gezien bij mensen met ARDS. Dit model is relevante onderzoeken de Moleculaire Pathogenese, ontwikkeling van biomarkers en het testen van mogelijke nieuwe therapieën5.
Co cultuur systemen zijn relevant voor in vivo onderz...

ALI en ARDS zijn de levensbedreigende vormen van respiratoire insufficiëntie met ernstige hypoxemia veroorzaakt door niet-cardiogeen longoedeem die ongeveer 1 miljoen mensen wereldwijd treft jaarlijks1. De etiologie van ARDS omvat directe schade aan de longen van infecties of aspiratie en een scala aan indirecte beledigingen. De afgelopen tien jaar is er een toegenomen begrip van de moleculaire pathogenese van ARDS, specifieke gerichte behandelingen voor ARDS zijn echter nog worden ontwikkeld2,3.
Verschillende dierlijke modellen van acute long letsel hebben ontwikkeld die voorzien in een brug vertalen van experimentele therapieën op menselijke studies4,5. Gebruikte modellen omvat lokale installatie van oliezuur, bacteriën, LPS en bleomycine. Andere benaderingen omvatten ischemie-reperfusie, cecal afbinding punctie, mechanische ventilatie-geïnduceerde stretch letsel, hyperoxia of systemische toediening van bacteriën en LPS5. Deze modellen bieden een nuttige biologisch systeem om te testen van klinische hypothesen en voor de ontwikkeling van mogelijke therapieën. Als u wilt nabootsen menselijke ARDS, moeten dierlijke modellen reproduceren ontsteking en acute schade aan de epitheliale en endotheliale cellen met afwijkingen in de functie van de barrière in de longen.
Exosomes zijn membraan blaasjes met 20-200 nm in diameter, waarvan de moleculaire inhoud bevat eiwitten, DNA, RNA en lipiden, en vergemakkelijken van Inter cellulaire communicatie in weefsel communicatie via moleculaire samenstelling overdracht. Exosomes worden uitgescheiden door meerdere soorten cellen, zoals de endotheliale cellen, epitheliaale cellen, zachte spiercellen en tumorcellen, en bestaan in menselijke lichaamsvloeistof. Studies tonen aan dat exosomes cross-talk tussen immuuncellen en stromale cellen tijdens de bestrijding van infectiekiemen en steriele ontstekingsziekten reguleren, en hun abnormale vrijlating lijkt te worden geregeld door verschillende natuurlijke en experimentele prikkels tijdens fysiologische en ziekteprocessen6. Dergelijke communicatie-netwerk kan een belangrijke rol spelen in de pathogenese van longziekten en pathofysiologische progressie7,8kan beïnvloeden. Zo 18-22 nucleotide niet-coderende RNAs, miRNAs bestaan in zowel weefsels en lichaamsvloeistoffen, plasma, sera en moduleren van mRNA uitdrukking bij posttranslationele niveau9,10.
Verpakte miRNAs in exosomes differentiatie en functie van meerdere soorten cellen beïnvloeden, en buitensporige niveaus worden geassocieerd met een verscheidenheid van ziekten, waaronder kanker, longziekten, obesitas, diabetes en hart-en vaatziekten11, 12,13,14,15,16. Inwerkingtreding van de ontvangende cellen en pendelen van exosomal miRNAs vergemakkelijken intercellulaire communicatie wijzigen de hemostase communicatie17,18. Acute Long-blessure is een complexe processen, waarbij meerdere celtypen met uitgebreide intercellulaire communicatie via exosomes8. miR-155 en miR-146a delen van de gemeenschappelijke transcriptionele reguleringsmechanisme en bijdragen aan de ontstekingsreactie en de immuun tolerantie19,20. Recente studies tonen aan dat zowel ontstekingsreactie moduleren via exosomal miRNAs pendelen tussen immuuncellen21. Echter, de moleculaire mechanismen die ten grondslag liggen aan f effecten van exosomal miRNAs op de alveolaire reactie op endotoxine onduidelijk blijven, ongetwijfeld de potentiële klinische relevantie en translationeel implicatie verdienste verder onderzoek.
Co cultuur modellen gebruikt om te definiëren van de wisselwerking tussen de specifieke celtypes in de complexe omgeving, zoals de ontsteking en kanker22,23. Deze platforms bieden een alternatieve strategie te ondervragen cross talk tussen celtypen, met name voor immuun en structurele cellen.
Intra-tracheale, aërosol, intraperitoneaal of systemische toediening van LPS worden veel gebruikt voor het opwekken van experimentele long letsel24,25,26, en voor het opwekken van epitheliale en endotheel permeabiliteit is gebleken defecten. We gebruiken hier intraperitoneaal LPS ertoe een septische model van acute lung injury in muizen. Binnen de 24u intraperitoneale toediening van LPS, permeabiliteit defecten veroorzaakt in de longen met de aanwerving van ontstekingscellen. Verder laten we zien dat de exosomes van de BAL miRNA-155 en miR-146a bevatten, en exosomes aan de BAL vloeistof opwekken van pro-ontsteking cytokinen expressie in ontvangende epitheliale cellen, met inbegrip van IL-6 en TNF-α. Deze gegevens zijn de eerste om te laten zien dat exosomal miRNAs bal in dit model van acute septische long letsel worden uitgescheiden.

<table border="0" cellpadding="0" cellspacing="0" style="width:1276px;" width="1276">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:691px;">lipopolysaccharide</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:224px;">Escherichia coli 055:B5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS pH7.2(1X)</td>
			<td>Life technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mirVana miRNA isolation kit</td>
			<td>Thermo Fisher</td>
			<td align="right">449774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>Thermo Fisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mmu-miR-155 (002571) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">has-miR-146a (000468) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U6snRNA primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TNF-&#945;(Mm00443258_m1 ) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IL-6 (Mm00446190_m1&#160;&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZO-1 (Mm00493699_m1&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minimal essential medium (MEM)</td>
			<td>Thermo Fisher</td>
			<td>11095-072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trysin-EDTA solution(0.05%)</td>
			<td>Thermo Fisher</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome Spin Columns (MW3000)</td>
			<td>Thermo Fisher</td>
			<td align="right">4484449</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Beckman Coulter Optima L-100XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>L-100XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-clear centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td align="right">344058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Applied Biosystems 7500 Fast Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">transmission electron microscopes (TEM)</td>
			<td>JEOL Ltd</td>
			<td>120 kV TEM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olympus BX41 microscope</td>
			<td>Olympus</td>
			<td>BX41</td>
			<td></td>
		</tr>
	</tbody>
</table>

bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury

Acute long letsel (ALI) en acute respiratory distress syndrome (ARDS) vormen een heterogene groep van longziekten die nog steeds een hoge morbiditeit en mortaliteit. De moleculaire pathogenese van ALI wordt beter gedefinieerd; echter, vanwege de complexe aard van de ziekte moleculaire therapieën moeten nog worden ontwikkeld. Hier gebruiken we een lipopolysaccharide (LPS) geïnduceerde muismodel van acute septische long letsel af te bakenen de rol van exosomes in de inflammatoire respons. Met behulp van dit model, hebben wij laten zien dat muizen die zijn blootgesteld aan intraperitoneaal LPS afscheiden exosomes in Broncho-alveolaire lavage (BAL) vloeistof uit de longen die zijn ingepakt met de miRNA en cytokinen tot regeling van de ontstekingsreactie. Verder gebruikt een modelsysteem co cultuur, laten we zien dat exosomes verlost van macrofagen expressie van strakke junction eiwitten in bronchiale epitheliale cellen verstoren. Deze resultaten suggereren dat 1) cross talk tussen aangeboren immuun en structurele cellen via de exosomal pendelen bijdraagt aan het inflammatoire respons en verstoring van de structurele barrière en 2) gericht op deze miRNAs een nieuw platform bieden kan te behandelen ALI en ARDS.

Om te induceren septische long letsel, werden muizen behandeld met intraperitoneaal LPS (15 mg/kg). Binnen de 24u van LPS administratie, werd neutrofiele toestroom gezien in de longen, zoals weergegeven in figuur 1A. Muis BAL vloeistof werd getrokken, gevolgd door isolatie en zuivering van exosomes. Morfologie van BALF exosomes werd bevestigd door Transmissie Electronenmicroscopie (figuur 1B).
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Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Bronchoalveolar Lavage Exosomes Lipopolysaccharide-geïnduceerde septische long letsel

Muizen blootgesteld aan intraperitoneaal LPS afscheiden exosomes in Broncho-alveolaire lavage (BAL) vloeistof die zijn ingepakt met miRNAs. Met behulp van een systeem co cultuur tonen we dat exosomes vrijgegeven in de BAL vloeistof expressie van strakke junction eiwitten in bronchiale epitheliale cellen verstoren en verhogen van expressie van pro-ontsteking cytokinen die long letsel accentueren.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

The overall goal of this experiment is to delineate the role of exosomes in the inflammatory responses in an LPS induced, acute septic lung injury mouse model. So this method can help answer key questions related to cell cross talk in acute lung injury and acute respiratory distress syndrome. Especially intercellular communication within the alveolar-micro environment via exosome shuttling.The main advantage of this technique is that it allows the isolation and characterization of highly purified bronchoalveolar lavage exosomes derived from endotoxin treated septic mice with acute lung injury. Demonstrating the procedure is Dr.Zhihong Yuan and Dr.Chetna Bedi. To begin, load a one milliliter syringe equipped with a 27 gauge needle with 0.1 milliliters of LPS and PBS per mouse and manually restrain the first six to eight week old male C57 black 6J mouse.Deliver the LPS intra-peritoneally and return the animal to its cage. 24 hours later, make a five millimeter incision in the center of the neck of the first animal and use blunt forceps to gently separate the muscles to expose the trachea. Next, gently insert a 27 gauge needle attached to a sterile 10 milliliter syringe into the trachea and inject one milliliter of sterile PBS.Then immediately replace the 10 milliliter syringe with a three milliliter syringe equipped with a new 27 gauge needle. Aspirate the bronchoalveolar lavage fluid or BOLF and add the BOLF to an appropriately labeled 15 milliliter conical tube on ice. To isolate the exosomes, centrifuge the BOLF samples and transfer the supernatants to new 15 milliliter tubes.Centrifuge the samples again and transfer the supernatants to ultra-centrifuge tubes to remove the larger, cellular particles. Transfer the supernatants to new conical tubes and load the samples into 0.2 micron syringe filters to remove any remaining larger particles. Add the filtered samples to new ultra-centrifuge tubes to pellet the exosomes.Carefully discard the supernatants and re-suspend the pellets in 100 microliters of PBS per tube. Then transfer the exosome samples into individual 1.5 milliliter tubes for their minus 80 degree Celsius storage. To purify the exosomes, add 0.4 microliters of the appropriate exosome dye to one tube containing 100 microliters of suspension solution per sample and quickly add one thawed exosome sample to each tube of dye.Thoroughly mix the exosomes with the dye and incubate the samples at room temperature for five minutes, protected from light. At the end of the incubation, place one exosome spin column per sample into a 1.5 milliliter illusion tube and transfer the exosome solutions to the center of each spin column. Then centrifuge the samples, discard the columns, and store the alluded samples at minus 20 degrees Celsius.To measure exosome uptake by recipient cells, first seed one times 10 to the fourth mouse lung epithelial 12 cells in 0.5 milliliters of medium into each well of an eight well chamber slide for a 16 hour culture at 37 degrees Celsius in 5%carbon dioxide. After thawing, add 10 microliters of the purified fluorescent labeled exosomes to each well and return the cell cultures to the incubator for one hour. At the end of the incubation, wash the cells with PBS and fix each co-culture with 1%paraformaldehyde for 10 minutes at room temperature.Remove the medium and then mount the slide with medium supplemented with DAPI and image the samples by fluorescence microscopy at a 40 times magnification. To verify exosome interaction with recipient cells, seed two times 10 to the fifth mouse lung epithelial 12 cells in two milliliters of medium per well in a 12 well culture plate containing the appropriate culture medium for a two hour incubation at 37 degrees Celsius and 5%carbon dioxide. At the end of the incubation, add 20 micrograms of exosomes to three wells of epithelial cells per experimental group and return the cells to the incubator.After 24 hours, remove the medium, wash the cells with PBS, and add 600 microliters of lysis buffer to each well with gentle shaking. Then transfer the entire contents of each well into individual 1.5 milliliter tubes and analyze the samples according to the standard real time PCR protocols. Within 24 hours of LPS administration, neutrophilic influx is observed in the lungs of LPS treated animals.After their isolation and purification as just demonstrated, the presence of exosomes in the BOLF from septic lung injury animals can be confirmed by transmission electron microscopy. The expression of select pro-inflammatory micro RNA is significantly increased in bronchoalveolar fluid exosomes from LPS treated animals compared to controlled PBS treated mice. LPS treated exosomes are taken up by mouse bronchiole epithelial cells as evidenced by the co-localization of fluorescent dye labeled exosomes with DAPI labeled cells after one hour of co-culture.Pro-inflammatory mediator expression is significantly increased in epithelial cells co-cultured with exosomes from LPS treated animals compared to co-culture with exosomes from control PBS treated mice. Further, cells treated with exosomes from LPS treated mice demonstrate a significantly attenuated expression of zonula occludens one compared to controls indicating a less intact cell integrity after LPS treated exosome co-culture. The intra-peritoneal injection of LPS, the bronchoalveolar lavage extraction, and exosome delegation from the bronchoalveolar lavage all the steps can be completed in a relatively short amount of time.Following this procedure, other methods like exosome and epithelial cell co-culture can be performed to answer additional questions about the cross talk between innate and structural cells in the lung alveolar micro-environment. Cross talk between innate immune and structural cells via exosome shuttling contributes to the inflammatory response and to structural barrier disruption suggesting exosomal micro-RNA targeting may provide a normal platform for treating acute lung injury and acute respiratory distress syndrome. After watching this video you should have a good understanding of how to induce septic lung injury in mice, isolate bronchoalveolar lavage fluid from mice, isolate exosomes from the lavage fluid, and use a co-culture model for interrogating intracellular cross talk.Don't forget that working with LPS can be hazardous and that precautions such as wearing the appropriate personal protective equipment should always be taken while performing this procedure.

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