Name: assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy
Uploaded: 2014-07-09T21:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

Questo lavoro è stato sostenuto dal National Institute of Allergy and Infectious Disease di Grant R01AI079253 (a BHS), e dal National Cancer Institute Cancer Support Center Concessione CA016056 al Roswell Park Cancer Institute.

Tutte le procedure eseguite su animali in questo studio sono stati approvati dal Comitato cura degli animali ed uso al Roswell Park Cancer Institute e rispettate tutte le statali, federali e istituti nazionali di regolamentazione di salute. 1. In vivo macrofagi Labeling Nota: PHK26 è un colorante lipofilo che sarà ripreso da fagociti sia in circolazione e nei tessuti quando somministrato per via endovenosa (iv). PKH26 è stato utilizzato per l&#39;etichettatura in vivo per minimizzare la quantità di manipolazione post-raccolta del macrofago. PKH-26 ha il vantaggio di accumulare stabilmente nei macrofagi alveolari per periodi prolungati. Attendere 10 giorni dopo la somministrazione permette per la liquidazione di tutte le cellule marcate dalla circolazione lasciando solo alveolare macrofagi stabile etichettati con PKH26 8, 9. PKH26 è un fluorocromo rosso che ha eccitazione (551 nm) ed emissioni (567 nm) caratteristiche compatibilicon i sistemi di rodamina o di rilevazione ficoeritrina. Qualsiasi procedura di etichettatura può introdurre artefatti, inclusa la perdita di vitalità. Tuttavia, dato che utilizziamo lo stesso approccio per l&#39;etichettatura dei macrofagi in diversi genotipi del mouse (ad esempio, WT vs NADPH ossidasi-carenti), gli effetti di questi manufatti sarebbero uniforme. <ol><li> Diluire soluzione madre di PKH26 (1 x 10 -3 M in etanolo) 1:5 in diluente C fornito dal produttore. </li><li> Carico: 100 ml di diluito PKH26 in una siringa da 1/2 ml insulina (29 G x 1/2 &quot;) per ogni mouse. </li><li> Somministrare a topi mediante iniezione ev (sia vena della coda o retro orbitale) almeno 10 giorni prima del raccolto. </li></ol> 2. Raccolta alveolari macrofagi di lavaggio broncoalveolare (BAL) Il numero di topi da utilizzare per un esperimento può variare. Una resa tipica di un mouse non stimolato è nell&#39;intervallo di 3-5 x 10 5 macrofagi alveolari, ma questo numero può variare notevolmente basEd da fattori quali la dimensione del mouse, esperienza della persona che effettua il lavaggio, e il numero di instillazioni di liquido di lavaggio e il volume prelevato dai polmoni. In questo protocollo lavaggio polmonare viene eseguita con una cavità toracica chiuso con ripetuti lavaggi 1 ml contribuisce ad aumentare la quantità di cellule. <ol><li> Euthanize topo somministrando una dose letale di anestetico avertin (12,5 mg) per via intraperitoneale (ip) utilizzando condizioni asettiche per conservare la soluzione sterile. </li><li> Spray topo accuratamente con etanolo al 70%. </li><li> Rimuovere pelle dall&#39;estremità craniale animale esporre il torace (Figura 1A). <ol><li> Fare un piccolo taglio nella pelle appena sotto il diaframma. </li><li> Inserire dito nel taglio e tirare pelle dall&#39;estremità craniale dell&#39;animale (Figura 1A). </li><li> Premere leggermente sulla testa, estendendo la parte ventrale del collo (Figura 1A). </li></ol></li><li> Tagliare un piccolo foro attraverso la gabbia toracicanel lato destro del torace per sgonfiare i polmoni e anche permettere la visualizzazione dei polmoni durante le seguenti fasi. </li><li> Individuare la trachea, e con attenzione sezionare il tessuto circostante. <ol><li> Rimuovere le ghiandole salivari, muscoli sterno-ioideo e laringe, ecc con pinze curve. </li><li> Sollevare delicatamente con una pinza curve e tagliare con le forbici il tessuto sottile che copre la trachea (avventizia) rivelando la struttura cartilaginea anelli sottostante. </li></ol></li><li> Inserire il catetere nella trachea. <ol><li> Utilizzando pinze curve, posizionare una sutura dietro (dorsale a) la trachea e tirare una lunghezza 10 cm attraverso l&#39;apertura. </li><li> Avvio sopra la cricoide (l&#39;anello ispessito della cartilagine) guida con attenzione un catetere G 22 lungo la trachea fermandosi dove la trachea scompare dietro lo sterno. </li><li> Fermamente legare sutura intorno trachea e il catetere per una perfetta aderenza. Rimuovere l&#39;ago dal catetere. </li></ol></li><li> Montare a 4 vie rubinetto. <ol><li> Riempireuno 6 ml siringa con DPBS, 1% FBS e attribuiscono al 4-way rubinetto come indicato nella figura 1B. </li><li> Collegare un vuoto 6 ml siringa nella posizione indicata sul 4-way rubinetto (Figura 1B). </li><li> Collegare la porta aperta sul 4-way rubinetto per catetere (Figura 1B). Fare attenzione a non rimuovere il catetere dalla trachea. </li></ol></li><li> Raccogliere liquido di lavaggio dai polmoni. Nota: Gli investigatori possono scegliere di utilizzare minori volumi di liquido di lavaggio per ridurre al minimo la possibilità di danni al tessuto. È importante che lo stesso approccio venga applicato coerentemente in tutti i gruppi di topo. <ol><li> Girare la maniglia sul rubinetto in modo che la siringa vuota è chiuso e iniettare lentamente ~ 1 ml DPBS, 1% FBS per gonfiare i polmoni. Osservare l&#39;inflazione polmone attraverso il taglio foro nella fase 2.4. </li><li> Fare attenzione a NON gonfiare eccessivamente i polmoni. </li><li> Girare la maniglia del rubinetto in modo che l&#39;intero siringa è chiuso, e prelevare con cautela l&#39;influenza<html> id dai polmoni. </li><li> Osservare i polmoni si sgonfiano attraverso il foro praticato nel punto 1.4. Il catetere può essere necessario spostare leggermente in avanti o indietro per ottenere un buon pareggio. Prendere precauzioni speciali per mantenere il catetere all&#39;interno della trachea. </li><li> Ripetere i punti 2.8.1-2.8.4 5-6x finché tutto il DPBS, 1% FBS è stata immessa e prelevata dai polmoni. Nota: il recupero del fluido polmonare non sarà 100% del fluido iniettato. </li></ol></li><li> Siringa vuota con il liquido di lavaggio in una provetta da 50 ml. Mantenere le cellule a temperatura ambiente. </li><li> Agglomerare cellule per centrifugazione a 300 xg per 5 min a temperatura ambiente e versare il surnatante. Questo può essere salvata per l&#39;analisi di citochine, se lo desideri. </li><li> Risospendere le cellule in 1-5 ml di RPMI 1640 con 10% di FBS e contare su un emocitometro. Nel topo non stimolato,&gt; 95% di cellule raccolte da BAL sono ritenute macrofagi, che può essere confermata mediante citologia. </li></ol> 3. Coltura e raccolta funghi &quot;&gt; L&#39;Aspergillus fumigatus utilizzato in questo protocollo esprime GFP in tutte le fasi del suo ciclo di vita. Spore, germlings e ife di questo ceppo fungino sono state fornite dal Dott. Margo Moore, Simon Fraser University, Burnaby, BC, Canada. <ol><li> Piatto conidi di un ceppo di Aspergillus fumigatus esprimere GFP su Sabouraud cuore cervello infusione (BHI) slant agar con cloramfenicolo (0,05 g / L) e Gentamicina (0,5 g / L). </li><li> Incubare al buio liberamente ricoperto per 7-10 giorni a temperatura ambiente. </li><li> Harvest conidi mediante lavaggio slant con 10 ml di 0,01% di Tween 20 in DPBS. Leggermente raschiare il fungo con il stripette per allentare. Sciacquare l&#39;inclinazione più volte per aumentare il rendimento. </li><li> Passare sospensione conidi attraverso un colino cella 100 micron in una conica da 50 ml. </li><li> Pellet conidi per centrifugazione a 400 xg per 10 min. </li><li> Risospendere conidi in 50 ml DPBS e contare con un emocitometro. </li><li> Lavare due volte con DPBS e diluire a concentrazione desideratistrazione. </li></ol> 4. Fungina Challenge e Microscopia confocale Per studiare il ruolo dei macrofagi NADPH ossidasi in difesa ospite, la capacità dei macrofagi alveolari isolati da topi di tre genotipi per controllare la crescita delle spore di Aspergillus fagocitati è stata valutata. I genotipi utilizzati in questo studio includono; 1) topi wild-type, 2) Ncf1 * / * Mn-topi con una mutazione naturale Ncf1 (Ncf1 codifica per p47 phox, una componente necessaria del fagociti ossidasi NADPH) lasciando loro NADPH ossidasi carente, e 3) Ncf1 * / * Mn + topi transgenici (MN denota il transgene ospitare tipo selvatico Ncf1 sotto il controllo del promotore di CD68 umano) dove l&#39;attività NADPH ossidasi è stato ricostituito nella popolazione di monociti / macrofagi 10, 11. Microscopia confocale È più adatto per questo test per la presenzae la crescita di conidi non fagocitato sugli scivoli. Il microscopio confocale permetterà la visualizzazione di piani individuali all&#39;interno l&#39;asse Z, e consentire la differenziazione tra fungo cresce intorno piuttosto che all&#39;interno dei macrofagi. Tutte le immagini confocale vengono acquisite con un obiettivo a immersione in olio 63X. Z-stack sono acquisiti con un fattore di zoom elettronico di 2.5. Lo spessore di un Z-stack è determinato utilizzando DAPI e immagini in campo chiaro per individuare la parte superiore e inferiore del campo. Z-stack variava da 14 a 24 micron, con singole sezioni ~ 1 micron di spessore. Per la quantificazione dei macrofagi, singole scansioni di zoom elettronico 1X sono stati eseguiti su 10 campi per genotipo. Macrofagi intatti sono stati identificati sulla base PKH26 e colorazione DAPI della membrana e nucleo rispettivamente. Conidi sono stati identificati in base all&#39;immagine campo chiaro espressione FITC e. <ol><li> Preparare vetrini sterili e piastre <ol><li> Sotto una cappa di sicurezza biologica, immergere 22 x 22 mm coprioggetto di vetro nel 70% ethanol per 5-10 min. </li><li> Sciacquare coprioggetto in PBS sterile. </li><li> Utilizzando pinze sterili, posizionare delicatamente un singolo vetrino sul fondo di ciascun pozzetto di una singolarmente confezionati 6-pozzetti sterile. </li></ol></li><li> Seed ~ 1 x 10 6 raccolto PKH26 etichettato macrofagi alveolari sospesi in 300 ml RPMI 1640, 10% FBS su ogni vetrino. Il numero esatto può variare a seconda del rendimento del raccolto. </li><li> Lasciare macrofagi di aderire incubando a 37 ° C con 5% di CO 2 durante la notte. </li><li> La mattina seguente, aggiungere ~ 1 x 10 7-GFP che esprimono A. fumigatus conidi sospeso in 100 ml di pre-riscaldato (37 ° C) RPMI 1640, 10% FBS. </li><li> Piastra Rientro a 37 ° C incubatore con il 5% di CO 2. </li><li> Le cellule devono essere fissati per un minimo di 2 ore a temperatura ambiente o per una notte a 4 ° C. A 3, 7, e 14 hr cellule fix aggiungendo un volume uguale di 2% di formaldeide in ogni pozzetto (concentrazione finale 1% di formaldeide). Piastre posto per l&#39;punti temporali presto a 4 ° C durante la notte. Per il punto finale di tempo (14 ore), fissare le cellule a temperatura ambiente per 2 ore. </li><li> Dopo la fissazione, rimuovere delicatamente coprioggetto con pinze e sciacquare in DPBS. </li><li> Rimuovere il liquido in eccesso ponendo il bordo del vetrino su un Kimwipe piegato. </li><li> Applicare 6 mezzo di montaggio microlitri fluorescenza con DAPI ad un vetrino da microscopio in vetro. </li><li> Posare un bordo del vetrino sul vetrino e rilasciare delicatamente con lato della cella giù nel mezzo di montaggio. Mezzo di montaggio si diffonderà e formare uno strato anche sotto il vetrino. Non c&#39;è bisogno di premere sul vetrino. </li><li> Sigillare i bordi del coprioggetto con smalto trasparente e lasciare asciugare all&#39;aria. </li><li> Conservare preparati vetrini in una scatola slitta (protetto dalla luce) a temperatura ambiente fino al momento di analizzare mediante microscopia confocale. </li></ol>

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Usando questo approccio ex vivo per analisi dell&#39;attività antifungina di macrofagi insieme in vivo sfida fungina, abbiamo precedentemente dimostrato che NADPH ossidasi in macrofagi svolge un ruolo importante nella difesa contro A. fumigatus 4. L&#39;uso di isolati macrofagi alveolari ci permette di concentrarsi sulla loro attività antifungina in assenza di altre cellule immunitarie (ad esempio, neutrofili reclutati) che difendersi Aspergillus in vivo. <p ...

Macrofagi alveolari sono i fagociti di prima linea che incontrano i microbi inalati. I macrofagi riconoscono motivi Aspergillus dai recettori di riconoscimento patogeni, ingerire e limitano la crescita delle spore per via inalatoria (conidi), e avviare le risposte infiammatorie. Il ossidasi dei fagociti NADPH converte l&#39;ossigeno molecolare per anione superossido e valle intermedi reattivi dell&#39;ossigeno (ROI). Malattia granulomatosa cronica (CGD) è una malattia ereditaria della NADPH ossidasi caratterizzata da infezioni batteriche e fungine gravi e le risposte infiammatorie eccessive. Anche se NADPH ossidasi è fondamentale per neutrofilo-mediato difesa ospite 1-3, l&#39;importanza della NADPH ossidasi nei macrofagi non è ben definito. Precedenti studi hanno dimostrato che i macrofagi alveolari ingerire ed uccidere le spore di Aspergillus, considerando che i neutrofili principalmente di mira il palco ife 5. Tuttavia, ci sono stati Resul contrastantits come il ruolo della NADPH ossidasi in macrofagi nel controllare la crescita di A. fumigatus spore 6, 7. L&#39;obiettivo di questo metodo è stato quello di delineare il ruolo specifico della NADPH ossidasi nei macrofagi nel mediare difesa dell&#39;ospite contro A. spore fumigatus. Abbiamo trovato che NADPH ossidasi in macrofagi alveolari controlla la crescita di fagocitato A. fumigatus spore 4. Per modellare più strettamente la risposta dell&#39;ospite ai funghi per via inalatoria, abbiamo usato macrofagi alveolari raccolte da lavaggio broncoalveolare da topi non stimolati subito dopo il sacrificio. L&#39;uso di isolati macrofagi alveolari ci ha consentito di concentrarsi sulla loro attività antifungina in assenza di altre cellule immunitarie (ad esempio, neutrofili reclutati) che difendersi Aspergillus in vivo. In questo metodo, macrofagi alveolari sono state colorate in vivo prima della raccolta in modo da minimizzare la quantità di manipolazione post-raccolta. &lt;/ P&gt; Un altro vantaggio di questo protocollo è l&#39;uso di un GFP che esprimono A. ceppo fumigatus. Questo fungo esprime GFP dalla fase conidi attraverso la fase di ife e non ha bisogno di ulteriori colorazione. Per ottenere immagini con maggiore dettaglio, abbiamo scelto microscopia confocale che permette la ricostruzione di strutture tridimensionali delle immagini ottenute. Ricostruzione tridimensionale dà la capacità di distinguere tra ife crescono intorno piuttosto che in o attraverso un macrofago. Conidi può anche essere precisamente distinto come intracellulare contro extracellulare.

<table><tbody><tr><td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td><td>Sigma</td><td>PKH26GL-1KT</td><td></td></tr><tr><td>2,2,2 Tribromoethanol</td><td>Sigma</td><td>T48402</td><td>Used to make AvertinAnesthetic</td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma</td><td>A-1685</td><td>Used to make AvertinAnesthetic</td></tr><tr><td>Dulbecco&amp;#039;s Phosphate Buffered Saline (without calcium and magnesium)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma</td><td>A0171</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>P91444</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>22 G x 1.00 inch i.v. catheter</td><td>BD</td><td>381523</td><td>Insyte Autoguard Winged</td></tr><tr><td>Insulin syringes</td><td></td><td></td><td></td></tr><tr><td>6 ml Syringes</td><td></td><td></td><td></td></tr><tr><td>4-way stopcock</td><td>Fisher Scientific</td><td>50-700-077</td><td></td></tr><tr><td>Suture thread</td><td></td><td></td><td></td></tr><tr><td>50 ml conical centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>gauze sponges (4 inch square)</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 with L-glutamine</td><td>Corning</td><td>10-040-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30396.03</td><td>heat inactivated</td></tr><tr><td>Hema 3 Stain Set</td><td>Fisher Scientific</td><td>22-122-911</td><td></td></tr><tr><td>Cytoseal 60</td><td>Thermo Scientific</td><td>8310-4</td><td></td></tr><tr><td>Sabouraud Brain Heart Infusion Agar with Chloramphenicol and Gentamicin Slants</td><td>BD</td><td>297252</td><td></td></tr><tr><td>&lt;em&gt;Aspergillus fumigatous&lt;/em&gt; strain expressing green fluorescent protein</td><td></td><td></td><td>provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada</td></tr><tr><td>100 &amp;mu;m Cell strainers</td><td>BD Falcon</td><td>64753-00</td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Biological Safety Cabinet Class II</td><td></td><td></td><td></td></tr><tr><td>Sorval ST40 Centrifuge with swing bucket rotor</td><td></td><td></td><td></td></tr><tr><td>Leica DM1000 light microscope</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>Cytospin 2 centrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td></td><td></td><td>37 &amp;deg;C, 5%&amp;nbsp; CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>22 x 22 mm micro cover glass</td><td>VWR</td><td>48366-227</td><td>glass coverslips</td></tr><tr><td>6-well Cell culture plates</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>VWR</td><td>BDH0502-1LP</td><td></td></tr><tr><td>Vectashield mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Leica TCS SP2 system with a laser point scanner</td><td></td><td></td><td>Mounted on DMIRE2 fluorescence microscope</td></tr></tbody></table>

assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy

Il polmone è un&#39;interfaccia in cui cellule ospiti sono regolarmente esposti ai microbi e prodotti microbici. Macrofagi alveolari sono i fagociti di prima linea che incontrano i funghi per via inalatoria e altri microbi. Macrofagi e altre cellule immunitarie riconoscono motivi Aspergillus dai recettori di riconoscimento patogeno e avviare risposte infiammatorie a valle. Il ossidasi dei fagociti NADPH genera intermedi reattivi dell&#39;ossigeno (ROI) ed è fondamentale per la difesa ospite. Anche se NADPH ossidasi è fondamentale per neutrofilo-mediato difesa ospite 1-3, l&#39;importanza della NADPH ossidasi nei macrofagi non è ben definito. L&#39;obiettivo di questo studio è stato quello di delineare il ruolo specifico della NADPH ossidasi nei macrofagi nel mediare difesa dell&#39;ospite contro A. fumigatus. Abbiamo trovato che NADPH ossidasi in macrofagi alveolari controlla la crescita di fagocitato A. fumigatus spore 4. Qui, descriviamo un metodo per valutare la capacità di un topomacrofagi lveolar (AMS) per controllare la crescita delle spore di Aspergillus fagocitati (conidi). Macrofagi alveolari sono macchiati in vivo e dieci giorni più tardi isolati da topi da lavaggio broncoalveolare (BAL). I macrofagi sono placcati su vetrini, poi seminate con la proteina fluorescente verde (GFP) che esprimono A. spore fumigatus. A volte specificato, le cellule vengono fissate e il numero di macrofagi intatte con spore fagocitati è valutata mediante microscopia confocale.

<html> Raccolta dei macrofagi alveolari da BAL da topi non stimolati si tradurrà in una popolazione pura&gt; 95% in base citologia. Il 3 e 7 ore (Figura 2) punti temporali precedono la fase di ife di crescita fungina e consentono un confronto di efficienza fagocitaria dei conidi tra i genotipi. Il punto di tempo 14 hr è dove genotipi possono essere confrontati per quanto riguarda la capacità di inibire il passaggio di conidi fagocitato alla fase hyphal tessuto invasivo (Figura 3). Mac...

Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Un metodo per valutare la capacità di isolato macrofagi alveolari del mouse per controllare la crescita di spore di Aspergillus fagocitati mediante microscopia confocale.

Valutare l&#39;attività anti-fungina isolato Alveolar macrofagi dal Microscopia confocale

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

assessing-anti-fungal-activity-isolated-alveolar-macrophages-confocal

Research

JoVE Journal

Immunology and Infection

11.4K Views.  Roswell Park Cancer Institute.  Un metodo per valutare la capacità di isolato macrofagi alveolari del mouse per controllare la crescita di spore di Aspergillus fagocitati mediante microscopia confocale. 

Video: Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Raccolta murini macrofagi alveolari e Valutare l&#39;attivazione cellulare indotta da nanoparticelle polianidride

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Ricerca

Bioingegneria

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-cellula Microscopia Video di funghi patogeni fagocitosi

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Immunologia e infezioni

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

L&#39;uso di Citometria immagine per la quantificazione dei funghi patogeni in associazione con cellule ospiti

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Immagini dal vivo di antifungini attività da Human neutrofili e monociti primaria in risposta a<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualizzazione del macrofago trappole extracellulari mediante microscopia confocale

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

Studiare il ruolo dei macrofagi alveolari in Breast Cancer metastasi

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

Medicina

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

Valutazione In Vivo dell'efferocitosi del macrofago alveo dopo l'esposizione all'ozono

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Misurare la fagocitosi di Aspergillus fumigatus Conidia da parte dei Leucociti Umani utilizzando Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and immunological tolerance, while in immunocompromised patients, conidia can colonize airways and cause severe invasive respiratory disorders. Various cells in different airway compartments are involved in the immune response that prevents fungal invasion; however, the spatio-temporal aspects of pathogen elimination are still not completely understood. Three-dimensional (3D) imaging of optically cleared whole-mount organs, particularly the lungs of experimental mice, permits detection of fluorescently labeled pathogens in the airways at different time points after infection. In the present study, we describe an experimental setup to perform a quantitative analysis of A. fumigatus conidia distribution in the airways. Using fluorescent confocal laser scanning microscopy (CLSM), we traced the location of fluorescently labeled&#160;conidia in the bronchial branches and the alveolar compartment 6 hours after oropharyngeal application to mice. The approach described here was previously used for detection of the precise pathogen location and identification of the pathogen-interacting cells at different phases of the immune response. The experimental setup can be used to estimate the kinetics of the pathogen elimination in different pathological conditions.

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 &#181;m in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models.

Analisi quantitativa basata su microscopia a scansione laser confocale della distribuzione di conidi di Aspergillus fumigatus nel polmone di topo a montaggio intero otticamente cancellato

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

Efficienza della clearance fungina dei macrofagi

Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events involved in macrophage-pathogen interactions, as well as for the determination of the time of death of internalized conidia. The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The evasion of phagocytosis and escape of phagosomes are mechanisms of fungal virulence. Here, we report the methods that can be used for the analysis of the phagocytosis, clearance, and viability of T. stromaticum conidia, a fungus which is used as a biocontrol and biofertilizer agent and is capable of inducing human infections. The protocol consists of 1) Trichoderma culture, 2) washing to obtain conidia, 3) the isolation of peripheral blood mononuclear cells (PBMCs) using the polysucrose solution method and the differentiation of the PBMCs into macrophages, 4) an in vitro phagocytosis method using round glass coverslips and coloration, and 5) a clearance assay to assess the conidia viability after conidia phagocytosis. In summary, these techniques can be used to measure the&#160;fungal clearance efficiency of macrophages.

Autore in primo piano: Svelare l'interazione tra Trichoderma stromaticum e il sistema immunitario dei mammiferi

The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The purpose of this manuscript is to present a method for evaluating the phagocytosis and clearance abilities of human peripheral blood mononuclear-derived macrophages stimulated with Trichoderma stromaticum conidia.

Saggio di vitalità dei conidi di Trichoderma stromaticum all'interno di macrofagi di derivazione mononucleare del sangue periferico umano

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial ...

Valutare l'attività anti-fungina isolato Alveolar macrofagi dal Microscopia confocale

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Raccolta murini macrofagi alveolari e Valutare l'attivazione cellulare indotta da nanoparticelle polianidride