Questa ricerca è stata sostenuta in parte dal NIH premi 5T32AI07506, 5R01AI033774, 5R37AI033142, 5R01AI052733.

Tutto il lavoro animale è stato fatto in conformità con le normative e le linee guida dell&#39;Istituto di studi sugli animali con Albert Einstein College of Medicine. 1. Crescita Condizioni di C. neoformans H99 (sierotipo A) <ol><li> Grow individuale C. neoformans (Cn) colonie su piastre di agar Sabouraud. </li><li> Un giorno prima dell&#39;esperimento, selezionare una colonia e inoculare 10 ml di brodo Sabouraud. </li><li> Lasciare cultura a crescere durante la notte a 37 ° C agitando a velocità di 250 rpm. </li></ol> 2 Isolamento e preparazione di primaria Bone macrofagi da C57 / BL6 Mice <ol><li> Euthanize topi mettendo animale in una camera di CO 2 fino a non respirare più. Come seconda misura, cervicale dislocare il collo prima di iniziare la dissezione. Sterilizzare tutto il corpo con il 70% di alcol etilico. </li><li> Rimuovere entrambi i femori e tibia e posto le ossa in DMEM sterile. </li><li> Rimuovere il tessuto in eccesso e muscolare con delicate asciuga fino ossa sono completamente puliti. </li><li> Posizionare le ossa intatte solo in una capsula di Petri contenente il 70% di alcol etilico e incubare per 3 minuti per uccidere i microrganismi associati. Togliere le ossa e posto in capsula di Petri con DMEM sterile. </li><li> Tagliare con cautela estremità delle ossa. Tenere osso su un tubo conico vuoto 50 ml posti su ghiaccio e lavare accuratamente 10 ml di DMEM freddo utilizzando un ago da 25 G con ogni osso. NOTA: Ogni topo produrrà 4 ossa (2 femori, tibie 2). Pertanto un mouse dovrebbe produrre circa 40 ml di sospensione cellulare. </li><li> Sospensione cellulare Centrifugare a temperatura ambiente per 10 min a 650 x g. </li><li> Durante questo centrifugazione, preparare e filtro sterilizzare midollo osseo macrofagi alimentazione dei media che consiste di DMEM supplementato con il 20% L929, siero di vitello fetale 10%, 10% NCTC-109, 1% Pen-Strep, 1% HEPES, 1% L-glutammina , 1% di aminoacidi non essenziali, 0,1% 2-mercaptoetanolo. </li><li> Risospendere il pellet risultante con 10 ml di alimentazione dei supporti. Passare suspens cellulariion attraverso un filtro 70 micron cella di interrompere grumi di cellule e rimuovere i detriti di grandi dimensioni. </li><li> Tavola 1 ml di sospensione cellulare in 10 ml di media alimentare in capsule di Petri che vengono specificati per l&#39;uso coltura tissutale (per un totale di 10 piatti). </li><li> Consentono alle cellule di aderire a 37 ° C con 10% di CO 2. Aggiungere 5 ml di supporti di alimentazione fresca il giorno 3 Il giorno 7, completamente sostituire il supporto di alimentazione e macrofagi sono pronti per essere utilizzati. </li><li> Il giorno prima dell&#39;esperimento, staccare macrofagi di targa togliendo mezzi di alimentazione e aggiungere 5 ml di una cellula dolce strippaggio reagente alla piastra. </li><li> Incubare per 5-10 min a 37 ° C e rimuovere macrofagi con dolce pipettaggio. </li><li> Cellule pellet per centrifugazione a 650 xg per 10 min. </li><li> Risospendere il pellet in 1 ml di alimentazione dei supporti. Usando una diluizione 1:20, calcolare la concentrazione di macrofagi pipettando 10 ml di sospensione cellulare su un emocitometro. </li><li> Utilizzo di 14 millimetri con fondo trasparente piastre di Petri, un piattoconcentrazione di 1 x 10 5 macrofagi direttamente sul pozzo interno che contiene un massimo di 200 ml, quindi prendere precauzioni per non superare questo volume, come mostrato in Figura 4. </li><li> Consentire alle cellule di aderire alla piastra di Petri di vetro mettendo i piatti in un incubatore a 37 ° C per 1 ora. </li><li> Aggiungere 1-2 ml di alimentazione supporti integrati con LPS a 1 mg / ml e IFNg a 500 U / ml e permettono alle cellule di incubare una notte. </li></ol> 3 co-incubazione dei macrofagi murini primari e C. neoformans <ol><li> Il giorno dell&#39;esperimento, rimuovere 1 ml di cellule di coltura e lievito pellet pernottamento inoculate mediante centrifugazione per 5 min a 420 x g. </li><li> Lavare le cellule 3 volte con PBS sterile per rimuovere il supporto e prodotti criptococco extracellulari come la glucuronoxylomannan polisaccaride (GXM) alla velocità e il tempo di cui sopra. </li><li> Risospendere il pellet di cellule in 1 ml di PBS sterile e fare una diluizione 1: 100. Pipettare 10 ml di tegli diluizione su un emocitometro e contare le cellule. Aggiungi cellule di lievito in una MOI di 1: 5. Ad esempio, se ci sono 1 x 10 5 macrofagi / bene, fare una sospensione cellulare di 5 x 10 6 / ml di Cn e aggiungere 100 ml di questa soluzione per un importo complessivo di 5 x 10 5 Cn. </li><li> Registrare il volume calcolato di sospensione cellulare criptococcica a 1 ml di mezzi di alimentazione con l&#39;anticorpo monoclonale 18B7 ad una concentrazione di 10 mg / ml. Incubare sospensione cellulare a temperatura ambiente per 5 min. </li><li> Rimuovere i supporti di alimentazione dal fondo di vetro piatto di Petri e lavare 1 volta con PBS sterile. Aggiungere 100 ml di opsonizzati Cn direttamente al bene. </li><li> Consentono alle cellule di incubare per 2 ore a 37 ° C con il 10% di CO 2. </li><li> Dopo aver co-incubazione, controllare da microscopio che i macrofagi hanno interiorizzato Cn. Per essere considerati interiorizzato, le cellule criptococco dovrebbero chiaramente essere visto entro i confini del macrofago. </li><li> Lavare 3x Petri piatto con PBS sterile per rimuovere ogni e qualsiasi extracellular Cn. </li><li> Aggiungere 1-2 ml di alimentazione dei supporti, senza l&#39;aggiunta di mAb 18B7, e impostare microscopio. </li></ol> 4. Visualizing non-litica Esocitosi in tempo reale Utilizzando Luce Microscopia Digitale NOTA: Per eseguire questi esperimenti, un microscopio che viene fornito con una camera di incubazione allegato che include CO 2 la consegna è richiesta una unità di riscaldamento. <ol><li> Prima dell&#39;esperimento, pre-riscaldare la camera di incubazione per almeno 30 min a 37 ° C. Assicurarsi che l&#39;unità di CO 2 legge 5% all&#39;inizio dell&#39;esperimento. </li><li> Luogo fondo di vetro piatto di Petri su piattaforma microscopio, coprire con CO 2 coperchio, e chiudere tutte le porte per garantire l&#39;assenza di fughe di calore. </li><li> Utilizzando l&#39;obiettivo 10X con il microscopio a contrasto di fase, concentrarsi su una chiara campo di macrofagi infettati. Sulla base della sperimentazione, determinare l&#39;obiettivo utilizzato da quanti macrofagi necessario studiare. </li><li> Impostare il software microscopio a take un&#39;immagine ogni 4 minuti per un periodo di 24 ore. </li><li> Dopo 24 ore, l&#39;esperimento avrà raggiunto il completamento e sarà stato raccolto un totale di 361 immagini. Esportare queste immagini come .jpegs a compressione 5%. Utilizzando software Image J, visualizzare le immagini come una pila di Visual a 5 fotogrammi al sec. Se il filmato deve essere visto per la pubblicazione o una presentazione, salvarla sia come avi o mov di file a seconda di quale formato funziona meglio per lo spettatore. </li></ol>

<ol>
	<li>Voelz, K., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcal+Interactions+With+the+Host+Immune+System.">Cryptococcal Interactions With the Host Immune System.</a> Eukaryotic cell. 9 (6), 835-846 (2010).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+interactions+with+macrophages:+evasion+and+manipulation+of+the+phagosome+by+a+fungal+pathogen.">Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen.</a> Cellular microbiology. 15 (3), 403-411 (2012).</li><li>Sabiiti, W., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+infection+by+the+human+fungal+pathogen+Cryptococcus+neoformans.">Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans.</a> Future microbiology. 7 (11), 1297-1313 (2012).</li><li>Bliska, J. B., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pathogenic+bacteria+and+fungi+&#8212;+a+case+of+convergent+evolution.">Intracellular pathogenic bacteria and fungi &#8212; a case of convergent evolution.</a> Nature Reviews Microbiology. 7, 165-171 (2008).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+spread+and+massive+vacuole+formation+after+Cryptococcus+neoformans+infection+of+murine+macrophages.">Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages.</a> BMC immunology. 8 (1), 16 (2007).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cell-to-cell+spread+of+a+pathogenic+yeast.">Direct cell-to-cell spread of a pathogenic yeast.</a> BMC immunology. 8, 15 (2007).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+extrusion+and+host-cell+survival+after+Cryptococcus+neoformans+phagocytosis+by+macrophages.">Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages.</a> Curr Biol. 16 (21), 2161-2165 (2006).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expulsion+of+live+pathogenic+yeast+by+macrophages.">Expulsion of live pathogenic yeast by macrophages.</a> Curr Biol. 16 (21), 2156-2160 (2006).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+fungal+pathogen+Cryptococcus+neoformans+escapes+macrophages+by+a+phagosome+emptying+mechanism+that+is+inhibited+by+Arp2/3+complex-mediated+actin+polymerisation.">The human fungal pathogen Cryptococcus neoformans escapes macrophages by a phagosome emptying mechanism that is inhibited by Arp2/3 complex-mediated actin polymerisation.</a> PLoS pathogens. 6 (8), (2010).</li><li>Carnell, M., 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=Actin+polymerization+driven+by+WASH+causes+V-ATPase+retrieval+and+vesicle+neutralization+before+exocytosis.">Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.</a> The Journal of cell biology. 193 (5), 831-839 (2011).</li><li>Voelz, K., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+regulates+the+outcome+of+intracellular+macrophage+parasitism+by+Cryptococcus+neoformans.">Cytokine signaling regulates the outcome of intracellular macrophage parasitism by Cryptococcus neoformans.</a> Infection and immunity. 77 (8), 3450-3457 (2009).</li><li>Bain, M. J., Lewis, E. L., Okai, B., Quinn, J., Gow, A. R. N., Erwig, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-lytic+expulsion/exocytosis+of+Candida+albicans+from+macrophages.">Non-lytic expulsion/exocytosis of Candida albicans from macrophages.</a> Fungal genetics and biology. 49 (9), 677-678 (2012).</li></ol>

Gli autori dichiarano che non abbiamo interessi finanziari concorrenti o altri conflitti di interesse.

Qui abbiamo descritto un metodo con cui le interazioni fungine-macrofagi possono essere registrati e analizzati in tempo reale per un periodo di 24 ore. Il nostro laboratorio ha utilizzato questo protocollo per studiare molti degli aspetti temporali della esocitosi non-litica e continuerà ad utilizzare la microscopia in tempo reale per accertare le componenti morfologiche che circondano questo processo. Per questa tecnica per produrre risultati ottimali, particolare attenzione deve essere d...

Le fasi di una infezione fungina tra le cellule criptococco e macrofagi sono ben documentati 1-3. Dopo cellule di lievito vengono ingeriti dai macrofagi, una varietà di interazioni possono verificarsi: il macrofago può lisare rilasciando il suo carico fungina nello spazio extracellulare, o potrebbe controllare l&#39;infezione mantenendo le cellule di lievito entro i confini della sua membrana cellulare 4. Tuttavia, alcuni anni fa un nuovo risultato è stato descritto indipendentemente da due gruppi: esocitosi non-litico, un processo in cui un macrofago cancellata alcune o tutte le celle criptococco nell&#39;ambiente circostante o una cellula vicina e sia host e patogeno rimangono vitali 5 -8. Diversi studi hanno cercato di comprendere i meccanismi molecolari alla base di questa interazione, ma abbiamo ancora non hanno una conoscenza completa su ciò che guida questo fenomeno. La capacità di catturare immagini in tempo reale e successivamente analizzare infettare piùEd macrofagi permette di rispondere a molte domande sulle proprietà fisiche circostanti esocitosi non-litica per quanto riguarda i tempi, la capacità spaziale, il cambiamento nella morfologia e anche lo stress di macrofagi nel corso di una infezione fungina. Permettendo alle cellule di essere ospitati in un ambiente che è paragonabile a quello di un incubatore, possiamo intravedere la natura dinamica delle interazioni cellulari di macrofagi-fungine. I ricercatori hanno usato questa tecnica per studiare vari elementi di questo processo. Il ruolo del phagosome in questo processo è stato reso evidente con colorazione fluorescente e actina agenti di blocco 9, mentre un altro gruppo ha usato questo metodo per dimostrare che esocitosi non-litica è stata bloccata in cellule che hanno avuto la WASH nucleazione domini proteici cancellato 10. L&#39;effetto di segnalazione delle citochine è stata anche determinata mediante microscopia in tempo reale 11. Questo processo non è limitato a C. neoformans come è stato osservato anche con Candida albicans, another patogeno fungino che ha la capacità di infettare i macrofagi 12. Questi risultati sono esempi di come questo metodo può fornire una serie di informazioni in un&#39;area che sappiamo così poco.

<table><tbody><tr><td>Sabouraud Dextrose Agar</td><td>BD</td><td>DF0109-17-1</td><td></td></tr><tr><td>Sabouraud Dextrose Broth</td><td>BD</td><td>DF0382-17-9</td><td></td></tr><tr><td>Dubelcco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Corning Cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Atlanta Biologicals</td><td>S12450</td><td></td></tr><tr><td>NCTC 109</td><td>Invitrogen</td><td>21340-039</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>HEPES Buffer</td><td>Corning Cellgro</td><td>25-060-Cl</td><td></td></tr><tr><td>Glutamax</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Corning Cellgro</td><td>25-025-Cl</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Invitrogen</td><td>21985-023</td><td>Toxic</td></tr><tr><td>3003 Tissue Culture Petri Dishes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>08772E</td><td></td></tr><tr><td>CellStripper</td><td>Corning Cellgro</td><td>25-056-Cl</td><td></td></tr><tr><td>Mat-tek Glass Bottom Culture Dishes</td><td>MatTek</td><td>P35GC-1.5-14-C</td><td></td></tr><tr><td>LPS</td><td>Sigma</td><td>L3137</td><td></td></tr><tr><td>Mouse IFN-g</td><td>Roche</td><td>NC 9222016</td><td></td></tr><tr><td>mAb 18B7</td><td>Non-commerical antibody produced in our lab</td><td></td><td></td></tr><tr><td>Axiovert 200M Microscope with Incubating Chamber</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

visualizing non-lytic exocytosis of cryptococcus neoformans from macrophages using digital light microscopy

Molti aspetti l&#39;infezione di macrofagi da Cryptococcus neoformans sono stati ampiamente studiati e ben definito. Tuttavia, una particolare interazione che non è chiaramente compreso è esocitosi non-litico. In questo processo, le cellule di lievito vengono rilasciati nello spazio extracellulare con un meccanismo poco compreso che lascia entrambi i macrofagi e Cn praticabile. Qui, descriviamo come seguire un gran numero di macrofagi infettati singolarmente per un periodo di 24 ore l&#39;infezione mediante microscopia-tempo trascorso. Macrofagi infetti sono alloggiati in una camera di riscaldamento con un atmosfera di CO 2 collegato ad un microscopio che fornisce le stesse condizioni di un incubatore di coltura cellulare. Microscopio digitale diretta in grado di fornire informazioni sulle interazioni dinamiche tra un host e agente patogeno che non è disponibile da immagini statiche. Essere in grado di visualizzare ogni cellula infettata può fornire indizi su come i macrofagi gestire infezioni fungine, e viceversa. Questa tecnica isa potente strumento per studiare le dinamiche che stanno dietro un fenomeno complesso.

Le immagini che questa tecnica produce possono essere analizzati in una varietà di modi per raccogliere informazioni che circonda ciò che accade dopo che le cellule sono fagocitati C. neoformans. Come si vede nella figura 1, ci sono circa 100 i macrofagi che sono o infetto o non infetto. Ognuno di questi macrofagi dà allo spettatore l&#39;opportunità di studiare le interazioni ospite-patogeno su un livello molto microscopico. Come le figure 2 e 3 mostrano, ...

Watch this Scientific Journal Video about Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy at JoVE.com

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Descriviamo come visualizzare macrofagi C. neoformans (Cn) interazioni in tempo reale, con particolare enfasi sul processo di esocitosi non-litica mediante microscopia ottica digitale. Utilizzando questa tecnica individuale macrofagi infetti può essere studiato per accertare i vari aspetti di questo fenomeno.

Visualizzazione non-litica Esocitosi di<em&gt; Cryptococcus neoformans</em&gt; Dai macrofagi utilizzando la luce Microscopia Digitale

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

visualizing-non-lytic-exocytosis-cryptococcus-neoformans-from

Research

JoVE Journal

Immunology and Infection

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

11.7K Views.  Albert Einstein College of Medicine.  Descriviamo come visualizzare macrofagi C. neoformans (Cn) interazioni in tempo reale, con particolare enfasi sul processo di esocitosi non-litica mediante microscopia ottica digitale. Utilizzando questa tecnica individuale macrofagi infetti può essere studiato per accertare i vari aspetti di questo fenomeno. 

Video: Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

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 ...

Ricerca

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 ...

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

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

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 ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Macrofagi Colesterolo Depletion e il suo effetto sulla fagocitosi di<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Time-lapse Imaging 3D della fagocitosi dai macrofagi del topo

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

Uso complementare di tecniche microscopiche e lettura di fluorescenza nello studio delle interazioni Cryptococcus-Amoeba

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

Visualizzazione della morte delle cellule litiche macrofaschi durante l'infezione micobatterica negli embrioni di pesce zebra tramite la microscopia intravitale

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Visualizzazione della prossimità indotta da caspasi infiammatorie nei macrofagi derivati da monociti umani

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

Visualizzare le fasi iniziali della fagocitosi

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

Biologia

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

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