Esta pesquisa foi apoiada em parte pelo NIH prêmios 5T32AI07506, 5R01AI033774, 5R37AI033142, 5R01AI052733.

Todo o trabalho animal foi feito de acordo com as normas e diretrizes do Instituto de Estudos de Animais no Albert Einstein College of Medicine. 1. Crescimento Condições de C. neoformans H99 (sorotipo A) <ol><li> Crescer indivíduo C. neoformans (Cn) colônias em placas de ágar Sabouraud. </li><li> Um dia antes do experimento, selecione uma colônia e inocular 10 ml de caldo Sabouraud. </li><li> Permitir que a cultura a crescer durante a noite a 37 ° C com agitação a uma velocidade de 250 rpm. </li></ol> 2 Isolamento e Preparação de primário macrófagos Osso de C57 / Bl6 Ratos <ol><li> Eutanásia ratos colocando animal numa câmara de CO 2 até não mais respiração. Como segunda medida, cervicalmente deslocar o pescoço antes de começar a dissecção. Esterilizar todo o corpo com álcool etílico a 70%. </li><li> Remova os dois fêmures e tíbia e os ossos no lugar estéril DMEM. </li><li> Retire o excesso de tecido e músculo usando delicate limpa até os ossos estão completamente limpos. </li><li> Coloque os ossos intactos apenas em uma placa de Petri contendo álcool etílico a 70% e incubar por 3 min para matar microorganismos associados. Retirar os ossos e lugar em placa de Petri com DMEM estéril. </li><li> Corte cuidadosamente extremidades dos ossos. Segurar osso sobre uma vazios tubo de 50 ml colocados em gelo e cuidadosamente descarga 10 ml de DMEM frio usando uma agulha G 25 através de cada osso. NOTA: Cada rato irá produzir quatro ossos (2 fêmures, duas tíbias). Portanto, um rato deve produzir aproximadamente 40 ml de suspensão de células. </li><li> Centrifugar a suspensão de células à temperatura ambiente durante 10 min a 650 x g. </li><li> Durante esta centrifugação, preparar e esterilizar filtro de macrófagos da medula óssea que consiste em meios de DMEM suplementado com 20% de L929, 10% de soro fetal de vitelo, 10% de NCTC-109, 1% de Pen-Strep, 1% de HEPES, 1% de L-Glutamina alimentação , 1% de aminoácidos não essenciais, 0,1% de 2-mercaptoetanol. </li><li> Ressuspender resultando pellet com 10 ml de alimentação de mídia. Passe suspens celularesião positivo através de um filtro de 70 um da célula para interromper aglomerações de células e remover os detritos grandes. </li><li> Placa 1 ml de suspensão de células para 10 ml de meio de alimentação em placas de Petri, que são especificadas para uso de cultura de tecidos (para um total de 10 placas). </li><li> Permitir que as células aderir a 37 ° C com 10% de CO 2. Adicionar 5 ml de meio de alimentação fresca no dia 3 No dia 7, substituir completamente a mídia de alimentação e macrófagos está pronto para ser usado. </li><li> O dia antes do experimento, retire macrófagos de placa removendo mídia alimentação e adicionar 5 ml de uma célula suave descascar reagente ao prato. </li><li> Incuba-se durante 5-10 minutos a 37 ° C e remover macrófagos com pipetagem suave. </li><li> Células de pelotas por centrifugação a 650 xg durante 10 min. </li><li> Ressuspender o sedimento em 1 ml de alimentação de mídia. Usando uma diluição de 1:20, o cálculo da concentração de macrófagos por pipetagem de 10 ul de suspensão de células para um hemocitómetro. </li><li> Usando 14 milímetros com fundo de vidro placas de Petri, placa Aconcentração de 1 x 10 5 macrófagos diretamente sobre o bem interior, que tem um máximo de 200 l, por isso tome cuidado para não exceder este volume como mostrado na Figura 4. </li><li> Permitir que as células adiram ao prato de Petri de vidro, colocando os pratos em uma incubadora a 37 ° C durante 1 hora. </li><li> Adicionar 1-2 ml de alimentação de meio suplementado com LPS a 1 mg / ml e IFNg em 500 u / ml, e permitir que as células a incubar durante a noite. </li></ol> 3 Co-incubação de macrófagos murino primário e C. neoformans <ol><li> No dia da experiência, remover um ml de células e de cultura de sedimento de levedura durante a noite inoculados por centrifugação durante 5 min a 420 x g. </li><li> Lave as células três vezes com PBS estéril para remover a mídia e produtos criptocócicas extracelulares como o glucuronoxylomannan polissacarídeo (GXM) na velocidade e tempo mencionado acima. </li><li> Ressuspender o sedimento de células em 1 ml de PBS estéril e fazer uma diluição a 1: 100. Pipeta de 10 mL de tele diluição para um hemocitómetro e contar as células. Adicionar células de levedura a uma MOI de 1: 5. Por exemplo, se houver um x 10 5 macrófagos / bem, faça uma suspensão de células de 5 x 10 6 / ml de Cn e adicione 100 ml desta solução para um montante total de 5 x 10 5 Cn. </li><li> Adicionar o volume calculado de suspensão de células criptocócica a 1 ml de meios de alimentação com o anticorpo mAb 18B7 a uma concentração de 10 ug / ml. Incubar suspensão de células à temperatura ambiente durante 5 min. </li><li> Remova a mídia de alimentação de vidro de fundo placa de Petri e lavar 1x com PBS estéril. Adicionar 100 ul de opsonizado Cn directamente a bem. </li><li> Permitir que as células a incubar durante 2 horas a 37 ° C com 10% de CO 2. </li><li> Depois de co-incubação, verifique com o microscópio que os macrófagos internalizaram Cn. Para ser considerado internalizada, as células criptocócicas deve ser visto claramente dentro dos limites do macrófago. </li><li> Lavar 3x prato de Petri com PBS estéril para remover todo e qualquer extracellular Cn. </li><li> Adicionar 1-2 ml de alimentação de mídia sem a adição de mAb 18B7 e configurar microscópio. </li></ol> Exocitose 4 Visualizando Non-lítica em tempo real Digital Utilizando Microscopia de Luz NOTA: Para realizar estes experimentos, um microscópio que vem com uma câmara de incubação anexo que inclui CO 2 entrega e uma unidade de aquecimento é necessário. <ol><li> Antes da experiência, pré-aquecer a câmara de incubação de, pelo menos, 30 minutos a 37 ° C. Certifique-se de que a unidade de CO 2 lê 5% no início da experiência. </li><li> Lugar com fundo de vidro placa de Petri na plataforma de microscópio, cubra com CO 2 tampa e feche todas as portas para garantir que nenhum calor escapa. </li><li> Usando a objetiva de 10X com o microscópio em contraste de fase, o foco em um campo claro de macrófagos infectados. Com base na experiência, determinar o objectivo usado por quantos macrófagos precisam de ser estudados. </li><li> Configurar o seu software microscópio para take uma imagem a cada 4 minutos, por um período de 24 horas. </li><li> Após 24 horas, o experimento terá atingido a conclusão e um total de 361 imagens terão sido recolhidas. Exportar estas imagens como .jpegs a uma compressão de 5%. Usando o software Image J, ver imagens como uma pilha Visual em 5 quadros por segundo. Se o filme tem que ser visto para publicação ou uma apresentação, salvá-lo como qualquer um avi ou mov arquivo dependendo do formato que funciona melhor para o telespectador. </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>

Os autores declaram que não temos concorrentes interesses financeiros ou outros conflitos de interesse.

Aqui, descrevemos um método através do qual as interacções macrófagos-fúngicas podem ser registados e analisados ​​em tempo real, ao longo de um período de 24 horas. Nosso laboratório tem usado esse protocolo para estudar muitos dos aspectos temporais da exocitose não-lítica e continuará a usar microscopia em tempo real para determinar os componentes morfológicos que cercam este processo. Para esta técnica para produzir resultados ideais, deve ser dada especial atenção a d...

As fases de uma infecção fúngica entre células criptocócica e macrófagos são bem documentados 1-3. Após as células de levedura são ingeridos pelos macrófagos, uma grande variedade de interacções podem ocorrer: o macrófago pode lisar a libertar a sua carga de fungos para o espaço extracelular, ou pode controlar a infecção, mantendo as células de levedura dentro dos limites da sua membrana celular 4. No entanto, há vários anos, um novo resultado foi descrito de forma independente por dois grupos: exocitose não-lítico, um processo em que um macrófago expurgado algumas ou todas as células criptocócica para o ambiente circundante ou uma célula vizinha e tanto o hospedeiro e patógeno permanecer viáveis ​​5 -8. Vários estudos têm tentado compreender os mecanismos moleculares por trás dessa interação, mas que ainda não têm uma compreensão completa sobre o que impulsiona este fenômeno. A capacidade de capturar imagens em tempo real e posteriormente analisar infectar múltiplased macrófagos permite responder a muitas perguntas sobre as propriedades físicas que cercam exocitose não-lítica em relação ao tempo, a capacidade espacial, mudança na morfologia e até mesmo estresse de macrófagos durante uma infecção fúngica. Ao permitir que as células a serem alojados num ambiente que seja comparável ao de uma incubadora, podemos entrever a natureza dinâmica das interacções celulares de macrófagos-fúngica. Os pesquisadores usaram esta técnica para estudar vários componentes deste processo. O papel do fagossoma neste processo foi feito utilizando a coloração fluorescente aparente e actina agentes de bloqueio 9, enquanto que um outro grupo utilizado este método para mostrar que a exocitose não-lítico foi bloqueada em células que têm tido a LAVAGEM nucleação domínios proteicos suprimido 10. O efeito de sinalização de citoquinas também foi verificado por microscopia em tempo real 11. Este processo não está limitado a C. neoformans como foi também observado com Candida albicans, diferentr patógeno, que tem a capacidade de infectar macrófagos 12. Estes achados são exemplos de como este método pode fornecer uma riqueza de informações para uma área que sabemos tão pouco.

<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

Muitos aspectos da infecção de macrófagos por Cryptococcus neoformans têm sido extensivamente estudada e bem definido. No entanto, uma interação especial, que não é claramente compreendido é exocitose não-lítico. Neste processo, as células de levedura são libertadas para o espaço extracelular através de um mecanismo mal entendido que deixa ambos os macrófagos e Cn viável. Aqui, nós descrevemos a seguir como um grande número de macrófagos infectados individualmente por um período de 24 horas de infecção por microscopia de tempo decorrido. Macrófagos infectados estão alojados numa câmara de aquecimento, com uma atmosfera de CO 2 ligado a um microscópio que fornece as mesmas condições que uma incubadora de cultura de células. Microscopia digital ao vivo pode fornecer informações sobre as interações dinâmicas entre um hospedeiro e patógeno que não está disponível a partir de imagens estáticas. Ser capaz de visualizar cada célula infectada pode fornecer pistas de como macrófagos lidar com infecções fúngicas, e vice-versa. Esta técnica isa ferramenta poderosa para estudar as dinâmicas que estão por trás de um fenômeno complexo.

As imagens que esta técnica produz podem ser analisados ​​em uma variedade de maneiras de coletar informações em torno do que acontece depois que as células tenham fagocitado C. neoformans. Como pode ser visto na Figura 1, há aproximadamente 100 macrófagos que estão infectados ou não infectados. Cada um desses macrófagos dá ao espectador a oportunidade de estudar interações patógeno-hospedeiro em um nível muito microscópico. Como as Figuras 2 e 3</str...

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

Nós descrevemos como visualizar macrófago C. neoformans (Cn) interações em tempo real, com especial ênfase para o processo de exocitose não-lítica usando microscopia de luz digital. Usando esta técnica individualmente macrófagos infectados podem ser estudados para determinar os vários aspectos deste fenómeno.

A visualização não-lítica exocitose de<em&gt; Cryptococcus neoformans</em&gt; De macrófagos Usando Digital Microscopia de Luz

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

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Research

JoVE Journal

Immunology and Infection

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

11.7K visualizações.  Albert Einstein College of Medicine.  Nós descrevemos como visualizar macrófago C. neoformans (Cn) interações em tempo real, com especial ênfase para o processo de exocitose não-lítica usando microscopia de luz digital. Usando esta técnica individualmente macrófagos infectados podem ser estudados para determinar os vários aspectos deste fenómeno. 

Vídeo: 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-célula Microscopia vídeo de Fagocitose patógeno fúngico

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

Imunologia e Infecção

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.

Uso de Citometria de imagem para quantificação de fungos patogênicos em associação com células do hospedeiro

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.

Avaliar a atividade anti-fúngica isolada de macrófagos alveolares por microscopia confocal

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.

Macrófago Colesterol Exaustão e seu efeito sobre a fagocitose de<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 ...

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.

Imagens ao vivo de antifúngicos Atividade por neutrófilos humanos primários e monócitos em resposta à<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 ...

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 complementar de técnicas microscópicas e leitura de fluorescência no estudo de Cryptococcus-Amoeba interações

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.

Visualização da morte de células ticas de macrófago durante infecção micobacteriana em embriões de zebrafish via microscopia intravital

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.

Visualização de Caspases Inflamatórias Induzidas proximidade em macrófagos derivados de monócitos humanos

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.

Visualizando os estágios iniciais da fagocitose

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 ...

A visualização não-lítica exocitose de

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