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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Il protocollo generale richiede 2 giorni, inclusi il primo giorno di induzione di sepsi e l'isolamento del liquido di BAL dall'animale e il secondo giorno per l'isolamento di esosomi dal mouse BALF. Tutte le procedure sono state esaminate e approvate dal comitato di uso Atlanta VA medical center e istituzionali Animal Care.
1. Mouse polmone settica acuta lesioni modello
<ol><li>Utilizzare 6-8 settimane - topi di C57BL/6J wild-type maschi vecchi (peso g 20-22) per l'animale modello ed eseguir...

<ol>
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Modelli murini di malattie sono comunemente utilizzati per valutare la funzione fisiologica dei geni specifici e per ridurre il costo della sperimentazione2. La ferita di polmone acuta settica descritta qui imita risposta infiammatoria visto in esseri umani con ARDS. Questo modello è rilevante per studiare la patogenesi molecolare, sviluppo di biomarcatori e per testare potenziali nuove terapie5.
Sistemi di co-coltura sono rilevanti per gli stud...

ALI e ARDS sono le forme pericolose di insufficienza respiratoria con ipossiemia severa causata da edema polmonare non cardiogeno che colpisce circa 1 milione di persone nel mondo ogni anno1. L'eziologia di ARDS include ferita diretta ai polmoni da infezioni o aspirazione e una varietà di insulti indiretti. Nell'ultimo decennio c'è stata una maggiore comprensione della patogenesi molecolare di ARDS, tuttavia, trattamenti specifici mirati per ARDS sono ancora essere sviluppato2,3.
Sono stati sviluppati diversi modelli animali di danno polmonare acuto che forniscono un ponte per la traduzione di terapie sperimentali per studi umani4,5. I modelli comunemente usati includono installazione locale di acido oleico, batteri, LPS e bleomicina. Altri approcci includono ischemia-riperfusione, puntura cecal legatura, lesione elasticizzato indotta da ventilazione meccanica, l'iperossia o somministrazione sistemica di batteri e LPS5. Questi modelli forniscono un sistema biologico utile per testare le ipotesi cliniche e per lo sviluppo di potenziali terapie. Per simulare umano ARDS, modelli animali dovrebbero riprodurre l'infiammazione e lesione acuta alle cellule epiteliali ed endoteliali con difetti nella funzione di barriera nei polmoni.
Gli esosomi sono vescicole di membrana con 20-200 nanometro di diametro, il cui contenuto molecolare contiene proteine, DNA, RNA e lipidi, e facilitano la comunicazione intercellulare nel microambiente del tessuto tramite trasferimento composizione molecolare. Gli esosomi sono secreti da più tipi di cellule, quali cellule endoteliali, cellule epiteliali, cellule muscolari lisce e cellule del tumore e presenti nel fluido del corpo umano. Studi indicano che gli esosomi regolano cross-talk tra le cellule immunitarie e le cellule stromali durante malattie infiammatorie, infettive e sterile, e loro rilascio anormale sembra essere regolata da vari stimoli naturali e sperimentali durante fisiologico e processi patologici6. Tale rete di comunicazione può svolgere un ruolo importante nella patogenesi di malattie polmonari e può influenzare la progressione fisiopatologico7,8. Come 18-22 nucleotidi RNA non codificanti, Mirna esiste nel siero sia dei tessuti e fluidi corporei, al plasma e modula l'espressione di mRNA a livello post-traduzionale9,10.
Mirna confezionati in esosomi influenzano la differenziazione e la funzione di più tipi di cellule, e livelli eccessivi sono associati con una varietà di malattie, compreso il cancro, malattie polmonari, obesità, diabete e malattia cardiovascolare11, 12,13,14,15,16. Entrata in cellule del destinatario e la spola di Mirna exosomal facilitare comunicazioni intercellulari modificando l'emostasi del microambiente17,18. Ferita di polmone acuta è un processo complesso, che prevedono più tipi di cellule con vasta comunicazione intercellulare attraverso esosomi8. miR-155 e miR-146a condividere il comune meccanismo di regolazione trascrizionale e contribuire alla risposta infiammatoria e la tolleranza immunitaria19,20. Gli studi recenti indicano che entrambi modulano la risposta infiammatoria via exosomal Mirna spola tra cellule immunitarie21. Tuttavia, i meccanismi molecolari sottostanti effetti modulatory di Mirna exosomal alveolare risposta all'endotossina rimangono poco chiari, senza dubbio la potenziale rilevanza clinica e traslazionale implicazione merito ulteriore indagine.
Modelli di co-coltura sono impiegati per definire l'interazione dei tipi cellulari specifici nell'ambiente complesso, quali l'infiammazione e cancro22,23. Queste piattaforme forniscono una strategia alternativa per interrogare i cross-talk tra tipi cellulari in particolare per le cellule immuni e strutturali.
Endotracheale, amministrazione aerosolizzato, intraperitoneale o sistemica di LPS sono ampiamente usati per indurre sperimentale del polmone lesioni24,25,26e hanno dimostrato di indurre permeabilità epiteliale ed endoteliale difetti. Qui usiamo LPS intraperitoneale per indurre un modello settico della ferita acuta del polmone in topi. Entro 24 h dalla somministrazione intraperitoneale di LPS, difetti di permeabilità sono indotte nei polmoni con il reclutamento di cellule infiammatorie. Inoltre, mostriamo che esosomi da BAL contengono miRNA-155 e miR-146a, ed esosomi da liquido di BAL inducono l'espressione di citochine pro-infiammatorie in cellule epiteliali destinatario, compreso IL-6 e TNF-α. Questi dati sono il primo a mostrare che i miRNA exosomal vengono secreti nel BAL in questo modello di danno polmonare acuto settico.

<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

Lesioni polmonari acute (ALI) e sindrome da distress respiratorio (ARDS) rappresentano un gruppo eterogeneo di malattie polmonari che continua ad avere un'alta morbosità e mortalità. Patogenesi molecolare delle ALI è essere meglio definiti; Tuttavia, a causa della natura complessa della malattia terapie molecolari devono ancora essere sviluppato. Qui usiamo un modello di topo indotta lipopolysaccharide (LPS) di danno polmonare acuto settica di delineare il ruolo degli esosomi nella risposta infiammatoria. Utilizzando questo modello, siamo stati in grado di dimostrare che i topi che sono esposti a LPS intraperitoneali secernono esosomi nel fluido di lavaggio (BAL) di bronco-alveolare dai polmoni che sono confezionati con miRNA e citochine che regolano la risposta infiammatoria. Ulteriormente utilizzando un sistema di modello di co-coltura, mostriamo che gli esosomi rilasciati dai macrofagi interrompono espressione delle proteine di giunzione stretta in cellule epiteliali bronchiali. Questi risultati indicano che 1) cross-talk tra cellule innate immune e strutturali attraverso la marcia exosomal contribuiscono alla risposta infiammatoria e rottura della barriera strutturale e 2) targeting questi miRNA può fornire una nuova piattaforma per trattare ALI e ARDS.

Per indurre il danno polmonare settico, i topi sono stati trattati con LPS intraperitoneali (15 mg/kg). Entro 24 h dalla amministrazione dei LPS, neutrophilic afflusso è stato visto nei polmoni, come mostrato in Figura 1A. Liquido di BAL del mouse è stato disegnato, seguita da isolamento e purificazione di esosomi. Morfologia di BALF esosomi è stata confermata da microscopia elettronica di trasmissione (Figura 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

Il lavaggio broncoalveolare esosomi in lesioni polmonari settici Lipopolysaccharide indotta

Topi esposti a LPS intraperitoneali secernono esosomi nel fluido di lavaggio (BAL) bronco-alveolare che sono confezionati con Mirna. Utilizzando un sistema di co-coltura, indichiamo che esosomi rilasciati nel liquido di BAL perturbare l'espressione delle proteine di giunzione stretta in cellule epiteliali bronchiali ed aumentano l'espressione delle citochine pro-infiammatorie che accentuano la ferita di polmone.

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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