The authors acknowledge the Microscopy core facility at the Faculty of Health Sciences, Link&#246;ping University for providing access to advanced imaging systems; Karl-Eric Magnusson (Emeritus Scientist) at the Dept. of Clinical and Experimental Medicine, Link&#246;ping University for providing access to Imaris 3D/4D image processing software (Bitplane, Switzerland); and S. Braian for his help with the lung model cartoon. This work was supported by funds from the Swedish Research Council (Alternatives to animal research, 2012-1951) and Swedish Research Council (2012-3349) to M.L. and Swedish Foundation for Strategic Research to S.B. S.B. receive grants from the Karolinska Institutet, Swedish Research Council, the Swedish International Development Cooperation Agency (Sida) and the Swedish Civil Contingencies Agency (MSB), and the Swedish Heart and Lung Foundation (HLF). M.S. received grants from the Karolinska Institutet and Stockholm County Council.

<ol>
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L., 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=CFTR+expression+and+chloride+secretion+in+polarized+immortal+human+bronchial+epithelial+cells.">CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.</a> Am J Respir Cell Mol Biol. 10, 38-47 (1994).</li><li>Brighenti, S., Andersson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+immune+responses+in+human+tuberculosis:+learning+from+the+site+of+infection.">Local immune responses in human tuberculosis: learning from the site of infection.</a> J Infect Dis. 205, S316-S324 (2012).</li><li>Davis, J. 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The authors declare no competing financial interests.

The ability to recruit and form organized cell clusters at the site of infection is the hallmark of human TB 11. These dynamic structures known as tubercle granulomas primarily consist of immune cells (macrophages, monocytes, T-cells and B-cells) and multi-nucleated giant cells surrounding M. tuberculosis. The role of the granuloma has long been considered to wall off the infection, preventing local spread of bacteria. However, more recent studies show that granuloma formation is critical for early bacterial survival, growth and dissemination 12. A strategy of new studies is to identify molecules or pathways that could efficiently be targeted to inhibit the cellular migration in granuloma formation and/or TB dissemination. 
A caveat for novel studies on TB is the lack of models that recapitulate human TB. The most widely used experimental animals do not form true granuloma upon M. tuberculosis infection, and are therefore not appropriate choices for studies of TB 13-16. Non-human primates have the closest resemblance to human TB 17, but are not the preferred choice owing to high operational costs and ethical issues. Human TB is a complex immunological process and is difficult to model in vitro. Cell cultures of monolayers or co-cultures lack the 3D environment and tissue responses. Therefore, we have developed an innovative lung tissue model based on human primary immune cells and human lung-specific cell lines 8,9. The model displays characteristic features of human lung tissue, including epithelia with evenly integrated macrophages, formation of extracellular matrix, stratified epithelia and mucus secretion 9. 
The 3D human lung tissue model has several benefits over the in vitro single or co-cultures seeded on tissue culture plates or transwell inserts. First, the human lung-specific cells (fibroblasts and epithelial cells) are not commonly included in the in vitro single or co-cultures. Second, the immune cells and lung-specific cells are embedded in a 3D physiological context (collagen rich extra-cellular matrix products). The response of cells to a stimulus/infection and the migratory behaviour of cells, for instance formation of a granuloma, differ significantly between a 2D and 3D environment. Furthermore, the described method enables the 3D visualization and robust 3D quantitative analysis that provides pivotal information on spatial distribution and intricate cellular interactions. 
Experimental infection in the model tissue with M. tuberculosis resulted in clustering of macrophages at the site of infection, reminiscent of early TB granuloma (Figure 2 and 3). We have recently demonstrated that mutant strains defective in the ability to secrete the virulence factor ESAT-6 or Mycobacterium bovis BCG that lacks ESAT-6 did not induce the clustering of monocytes (no early granuloma), in contrast to the virulent M. tuberculosis 9. These data are consistent with the observations made from Mycobacterium marinum-infected zebrafish embryos, whose transparency allows for elegant live imaging of granuloma formation 12. As there is no gold-standard model for TB, we took advantage of the surgically resected tissue biopsies from TB patients for validation of the method 9. Our in vitro tissue model shares several characteristics with the lung and lymph node biopsies from TB patients, including the aggregation of macrophages in granuloma, the presence of both intra- and extracellular bacteria 18 and induction of necrosis 11. 
Although the described model has physiological relevance to human TB and has several advantages over other in vitro models, it has some limitations. For instance, out of more than 20 collagen proteins identified in humans, only type I is included to the model to mimic the extra-cellular matrix. However, type I collagen is a complex mixture of extra-cellular matrix products and is the most abundant collagen in the human body. Further, we have demonstrated the presence of collagen IV and several extra-cellular matrix proteins such as tropoelastin, vimentin and laminin, which are produced by the epithelial cells and fibroblasts in the tissue model, indicating the synthesis of new collagen 8. Presently, the lung tissue model only has monocytes and macrophages, besides lung-specific cells. It lacks neutrophils and lymphocytes that are also known to be present in the granuloma. Remarkably the model is not limited to the introduction of additional immune cells and is of interest to explore how they contribute to the complex cellular interactions in human TB. Implantation of primary alveolar macrophages, skin-specific cells and lung carcinoma cells has already been tested in the model. Since our objective was to use a model that closely resembles human TB, introduction of mouse cells have not been attempted.
In summary, the lung tissue model has implications for both basic mechanistic and applied studies. Potential applications of the lung model include the study of innate immunity, investigating mechanistic aspects of host defences such as phagosomal maturation, autophagy, production of cytokines, chemokines and anti-microbial peptides, and functional characterization of individual cell types. Strikingly, the in vitro tissue model allows manipulation of one or more cells types and provides a relevant tissue micro-environment, not only for studies on TB, but for a variety of infectious and non-infectious diseases that affect the lungs.

En los seres humanos, las respuestas a la infección, la inflamación del tejido, el reclutamiento celular, la remodelación tisular y la regulación de la homeostasis del tejido son eventos complejos que implican diferentes tipos de células. Por lo tanto, estos procesos son los más estudiados en el entorno del tejido local. Anteriormente, esto ha sido principalmente posible utilizando modelos animales experimentales. Sin embargo, los animales experimentales utilizados tienen muchos límites ya que a menudo responden a los patógenos de una manera diferente que los humanos y también muestran un curso diferente de la enfermedad 1. Un humano en el modelo de tejido pulmonar vitro tiene las posibilidades para estudiar las respuestas inmunes específicas en el pulmón humano. Infección de tuberculosis humana (TB) es una enfermedad que afecta principalmente a los pulmones. Mycobacterium tuberculosis (M. tuberculosis), el agente causante de la TB, alcanza el pulmón a través de gotitas de aerosol que son transportados al espacio alveolar, donde las bacterias son engullidas por dendri pulmonarcélulas de tics y macrófagos alveolares como parte de la respuesta inmune innata a la infección 2,3. La fagocitosis del patógeno conduce a la compartimentación del error dentro de un fagosoma e idealmente como resultado la neutralización y el asesinato del agente patógeno por el fagocito. Hasta 50% de los individuos expuestos a M. la tuberculosis se cree que son capaces de eliminar la infección a través de la respuesta inmune innata 4. Otros resultados de la infección son un despeje de la adaptación del sistema inmunológico en una etapa posterior, la infección latente o en el peor de los casos la enfermedad activa crónica 5. Anteriormente no se han presentado los modelos de tejidos in vitro para el estudio de la tuberculosis humana. Cultivos de células individuales de los macrófagos humanos u otras células de sangre periférica a menudo se han utilizado 6,7. La desventaja de este enfoque es que no pueden reflejar la dinámica de los diferentes tipos de células que operan juntos en un tejido pulmonar expuesto a M. tuberculosis </em&gt;. Por lo tanto, hay una necesidad de un modelo in vitro para ser capaz de realizar estudios funcionales y mecanicistas sobre la tuberculosis. El modelo en el tejido pulmonar humano in vitro descrito aquí basada en células fue establecida originalmente por nuestro grupo de estudios sobre las funciones celulares dendríticas 8. Hemos adaptado este método para el estudio de la tuberculosis. El modelo de tejido pulmonar humano que aquí se presenta se compone de células epiteliales y fibroblastos 8 específicos de tejido. Estas células se cultivan en una matriz de colágeno en la parte superior de una membrana porosa en un inserto de transwell y las estructuras de forma que se asemeje tejido pulmonar humano normal (Figura 1). Cuando se expone al aire las células comienzan a secretar moco en la parte apical 8. Mediante la implantación de los macrófagos primarios humanos infectados con M. tuberculosis al modelo, hemos observado cómo las células inmunes migran en el tejido y forman primeras etapas de granulomas TB 9. Este es el primer modelo de tejido descr humanaibed para la tuberculosis y plantea una herramienta prometedora para el estudio de la respuesta inmune innata a la tuberculosis y otras enfermedades del pulmón. Hasta la fecha, hemos utilizado sólo monocitos y macrófagos como células inmunes en el modelo, pero el nivel de complejidad se puede aumentar mediante la inclusión de tipos de células relevantes adicionales. <img alt="Figura 1" src="/files/ftp_upload/53084/53084fig1.jpg" /> Figura 1. Esquema Esquema del modelo de tejido pulmonar. (A) El modelo se compone de células epiteliales pulmonares-específica humanos, M. tuberculosis infectados con macrófagos primarios y tinte rojo monocitos marcados sembró en colágeno incrustado fibroblastos preparados en un filtro transwell. La exposición de la modelo de tejido al aire inicia la producción de proteínas de la matriz extra-celular, la secreción de moco y la estratificación por el epitelio. El modelo de tejido 3D así desarrollada es una herramienta útil para estudiar M. infección de tuberculosis en un entorno que closely se asemeja a un pulmón humano. (B) imágenes microscópicas representativas de los diferentes pasos en la preparación del modelo de tejido. (C) estructura completa de la sección de tejido modelo de pulmón. Escala -. 100 m <a href="https://www.jove.com/files/ftp_upload/53084/53084fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cell culture inserts</td>
			<td>BD Falcon</td>
			<td>353092</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>MRC-5 cells, lung fibroblasts</td>
			<td></td>
			<td></td>
			<td>ATCC#CCL-171</td>
		</tr>
		<tr>
			<td>16HBE cells, lung epithelial cells</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. Dieter Gruenert, Mt. Zion Cancer Center, University of California, San Fransisco, USA&#160;</td>
		</tr>
		<tr>
			<td>5 x Dulbecco&#8217;s modified Eagle&#8217;s medium (5 x DMEM)</td>
			<td>Gibco</td>
			<td>12800-082</td>
			<td>Made from powder but add 5 times less water. Adjust pH to 7.3 and filter it using a 0.2 &#181;m filter.</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium with glucose (DMEM) 1x</td>
			<td>Gibco</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM) 1x with Earle&#8217;s salts</td>
			<td>Sigma</td>
			<td>M4655</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids Solution, 100x</td>
			<td>Life Technologies</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 200 mM (100x)</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-039</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3 (71.2 mg/ml)</td>
			<td></td>
			<td></td>
			<td>Prepared in house</td>
		</tr>
		<tr>
			<td>Heat inactivated Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Heat inactivated for 30 min, 56 &#176;C</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/ml)</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes buffer solution 1M</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (Pen Strep)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Axis-Shield</td>
			<td>7801</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure 0.5 M EDTA</td>
			<td>Gibco</td>
			<td>15575</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Collagen PA treated (500 ml)</td>
			<td>Organogenesis</td>
			<td>200-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure col purified Bovine Collagen solution (100 ml)</td>
			<td>Advanced biomatrix</td>
			<td>5005-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Extracellular matrix protein, Fibronectin (1 mg)</td>
			<td>BD</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human monocytes/macrophages</td>
			<td></td>
			<td></td>
			<td>Isolated from human whole blood or buffy coats.</td>
		</tr>
		<tr>
			<td>PKH26 Red fluorescent cell linker</td>
			<td>Sigma</td>
			<td>MINI26</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis H37Rv expressing green fluorescent protein</td>
			<td></td>
			<td></td>
			<td>M. tuberculosis H37Rv wild type was transformed with the pFPV2 plasmid constitutively expressing GFP.</td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 medium&#160;</td>
			<td>Difco</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Middlebrook ADC Enrichment</td>
			<td>BBL</td>
			<td>211887</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin B sulfate (20 &#181;g/ml)</td>
			<td>Sigma</td>
			<td>B5264</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold anti=-fade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin -EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iMaris Scientific 3D/4D image processing software, version 7.6.8</td>
			<td>Bitplane AG</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a 3d human lung tissue model for functional studies on mycobacterium tuberculosis infection

La tuberculosis (TB) sigue teniendo una gran amenaza para la salud de las personas en todo el mundo, y hay una necesidad de modelos rentables, pero fiables para ayudarnos a entender los mecanismos de la enfermedad y promover los descubrimientos de nuevas opciones de tratamiento. Cultivos celulares in vitro de las monocapas o co-cultivos carecen de la tridimensional medio ambiente (3D) y las respuestas de los tejidos. En este documento, se describe un innovador modelo in vitro de un tejido pulmonar humano, que promete ser una herramienta eficaz para el estudio de los complejos acontecimientos que ocurren durante la infección por Mycobacterium tuberculosis (M. tuberculosis). El modelo de tejido 3D consiste en células epiteliales y fibroblastos específicos de tejido, que son cultivadas en una matriz de colágeno en la parte superior de una membrana porosa. Tras la exposición de aire, las células epiteliales estratificar y secretar moco en la parte apical. Mediante la introducción de los macrófagos primarios humanos infectados con M. tuberculosis al modo de tejidol, hemos demostrado que las células inmunitarias migran al tejido infectado y forman primeras etapas de granuloma TB. Estas estructuras recapitulan la característica distintiva de la tuberculosis humana, el granuloma, que es fundamentalmente diferente o no se observa comúnmente en modelos animales experimentales utilizados. Este método de cultivo organotípico permite la visualización en 3D y el análisis cuantitativo robusto que proporciona información fundamental sobre las características espaciales y temporales de las interacciones célula-patógeno de acogida. En conjunto, el modelo de tejido pulmonar proporciona un tejido microambiente fisiológicamente relevante para los estudios sobre la tuberculosis. Por lo tanto, el modelo de tejido pulmonar tiene implicaciones potenciales para ambos estudios mecanicistas y aplicados básicos. Es importante destacar que el modelo permite la adición o manipulación de tipos de células individuales, que de este modo amplía su uso para modelar una variedad de enfermedades infecciosas que afectan a los pulmones.

Un modelo de tejido pulmonar 3D para la tuberculosis humana puede ser utilizado eficazmente para estudiar las interacciones huésped-patógeno en M. infección por tuberculosis. Los pasos básicos de este método, las imágenes microscópicas representativas de diferentes pasos y una estructura microscópica general de una sección de tejido se indica en la Figura 1. El modelo tiene varios componentes del tejido pulmonar humano, incluyendo fibroblastos de pulmón, células epiteliales bronquiales y monocitos primarios / macrófagos incrustado en el entorno del tejido 3D. Además de la incorporación de componentes de tejido de pulmón humano, el modelo se asemeja a condiciones fisiológicas a saber, la estratificación de los epitelios y la secreción de moco. Un ejemplo para el uso del modelo de tejido pulmonar en el seguimiento de una infección de TB se presenta en la Figura 2. Para visualizar la M. tuberculosis migración celular -immune e interacción, que introdujeron los macrófagos infectados con M. tuberculosis que expresan GFP(verde) junto con los monocitos PKH26 marcado recién aisladas (rojo) en el modelo de tejido (azul, manchado DAPI para los núcleos). En el día 7 después de la adición de M. tuberculosis células infectadas en el modelo de tejido, microscopía confocal revela la agrupación de los monocitos de color rojo en el sitio de infección (verde) (Figura 2), que imita las lesiones característicos de la TB humana 9. Una serie de imágenes representativas para la visualización 3D de M. tuberculosis infectados con modelo de tejido y la cuantificación de los grupos de células se muestra en la Figura 3. La visualización en 3D da la flexibilidad para el usuario para interactuar, examinar y cuantificar varias características en una imagen en 3D. La disposición espacial de las bacterias verdes y rojos racimos de monocitos se puede ver desde la vista apical, rotacional y lateral como se ilustra en la figura 3B, lo que revela la agrupación de los monocitos en el sitio de M. Los racimos de tuberculosis. no se observido en los tejidos no infectados (Figura 3A). Hemos cuantificado el tamaño y el número de grupos de células de monocitos y encontramos que el tamaño (volumen) de grupos de células se ha mejorado (p &lt;0,001), mientras que el número de monocitos individuales disminuyó (p &lt;0,01) en M. tuberculosis tejidos infectados en comparación con los modelos de tejido no infectadas (Figura 3C y 3D). Estos datos valida nuestra anterior conclusión de la formación de granulomas a principios de M. infección tuberculosa observa en los modelos de tejido pulmonar analizados en secciones de tejido 2D 9. Nuestros datos sugieren que el modelo de tejido proporciona un hábitat natural 3D para investigar el complejo de acogida por células M. red de comunicación tuberculosis. También se encontró que la visualización en 3D y el análisis cuantitativo son mejores herramientas para el estudio de las características del modelo de tejido (Figura 3). La cuantificación de un grupo de células (granuloma por ejemplo) a menudo stretch<html> hes a varias capas de células y pueden ser capturados por completo por un análisis cuantitativo 3D. Además, la visualización de las características espaciales y temporales exactas de las células individuales o bacterias en el modelo permite estudios de imagen en vivo, de migración y de seguimiento en un laboratorio designado. <img alt="Figura 2" src="/files/ftp_upload/53084/53084fig2.jpg" /> Figura 2. monocitos en el clúster modelo tejido alrededor virulenta M. tuberculosis. confocal de imágenes representativas de los no infectados y M. se presenta la tuberculosis modelo de tejido infectado. Paneles de verde (M. tuberculosis- GFP), rojo (monocitos PKH26 marcado), azul (núcleos teñidos con DAPI) y canales fusionadas muestran el reclutamiento de monocitos en el tejido infectado en comparación con los tejidos infectados. Scale - 100 micras.large.jpg &quot;style =&quot; font-size: 14px; line-height: 28px; &quot;target =&quot; _ blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta figura. <img alt="Figura 3" src="/files/ftp_upload/53084/53084fig3.jpg" /> Figura 3. Visualización 3D y el análisis cuantitativo de modelo de tejido proporcionan información útil. Imágenes representativas de visualización en 3D de todo el modelo de tejido (A) del tejido infectado, (B) infectados con M. tuberculosis, a través de seccionamiento óptico Zeiss LSM700 usando microscopio confocal y análisis cuantitativo por el software de procesamiento de imágenes Imaris (versión 7.6.8). Estas imágenes fueron adquiridas a 20X aumentos, 14 z-pilas que cubren un espesor de tejido de 19,5 m con 1,5 micras intervalo, permitiendo la visualización de la posición horizontal apical, de rotación, vista vertical y lateral de rotación (A y B). (C) Quaanálisis ntitative de grupos de células de monocitos revelan mejorada (p &lt;0,0001) tamaño de los clusters granuloma primeros después M. infección de tuberculosis cuando se compara con la ausencia de infección. (D) Cuantificación de número de monocitos mostró una disminución (p &lt;0,01) en el tejido infectado en comparación con el tejido no infectado, reiterando más grupos en el tejido infectado. Verde - M. GFP tuberculosis, Red - monocitos marcados PKH26, Azul - núcleos celulares, Escala -. 100 m <a href="https://www.jove.com/files/ftp_upload/53084/53084fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection at JoVE.com

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Un modelo de tejido 3D Pulmón humano de Estudios funcionales en<em&gt; Mycobacterium tuberculosis</em&gt; Infección

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

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Research

JoVE Journal

Immunology and Infection

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

18.8K vistas.  Linköping University. Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Video: A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Preparación de la muestra de<em&gt; Mycobacterium tuberculosis</em&gt; Extractos de Estudios de Resonancia Magnética Nuclear metabolómica

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Investigación

Inmunología e Infección

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Crecimiento de Mycobacterium tuberculosis Las biopelículas

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Citometría de flujo El aislamiento de células primarias murino tipo II alveolares epiteliales de Estudios funcionales y moleculares

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Un modelo experimental para estudiar la tuberculosis-Malaria Coinfección sobre Transmisión Natural de Mycobacterium tuberculosis Y Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Demostración de un resistente a múltiples fármacos<em&gt; Mycobacterium tuberculosis</em&gt; Microarray Amplificación

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Un nuevo enfoque para la recuperación Microdissection<em&gt; Mycobacterium tuberculosis</em&gt; Las transcripciones específicas de formalina parafina fijo Embedded pulmón granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Sistema para la eficacia y la citotoxicidad de detección de inhibidores de focalización intracelular<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Análisis de 18FDG PET/CT la proyección de imagen como una herramienta para estudiar la tuberculosis del Mycobacterium infección y tratamiento en Primates no humanos

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Proyección de imagen de tuberculosis del Mycobacterium en ratones con reportero enzima fluorescencia

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modelado de la Tuberculosis de Mycobacterium marinum infección pez cebra adulto

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...