Este trabajo fue apoyado por una beca de Ciencias Naturales e Ingeniería de Investigación de Canadá (NSERC, conceder ninguna 298.515-2011). AL es el destinatario de un Ph.D. beca de Ciencias Naturales e Ingeniería de Investigación de Canadá (NSERC) y MS fue apoyado por una subvención de los Institutos Canadienses de Investigación en Salud (CIHR, subvención no. MOP123246).

Todos los procedimientos se realizaron de acuerdo con el Consejo Canadiense de Cuidado de Animales directrices después de la aprobación por el Comité institucional Cuidado de Animales del Centre de recherche du Centre Hospitalier de la Universidad de Montreal (CRCHUM). 1. El aislamiento, identificación, y Cultura de Murino macrófagos peritoneales <ol><li> Preparar medio tioglicolato 3,8% de cerveza. Para ello, suspender 38 g de tioglicolato medio en 1000 ml de agua destilada. Llevar a hervir solución para disolver completamente el medio. Esterilizar en autoclave a 121 ° C durante 15 minutos. Almacene hasta 3 meses en la oscuridad, a temperatura ambiente 18. NOTA: Desechar si la turbidez se desarrolla lo que indica una contaminación bacteriana. La solución puede conservarse durante un máximo de un año en la oscuridad si se mantiene estéril. </li><li> Uso de 1 ml jeringas unidas a agujas 23 G, inyectar 1 ml de 3,8% de tioglicolato de Brewer medio en la cavidad peritoneal de cada mouse y esperar durante 3 días. Use nueva jeringa y la aguja para cada ratón. </li><li> Anestesiar los ratones por inyección intraperitoneal de pentobarbital sódico (100 mg / kg) y la eutanasia a los ratones por dislocación cervical. Confirmar anestesia adecuada marcando la frecuencia respiratoria. Por lo general, las respiraciones rápidas indican que el ratón no está profundamente anestesiado. </li><li> Lavar el abdomen de cada ratón con etanol al 70%. Usando una tijera, realizar una incisión lateral a lo largo de la línea media inferior de la peritoneo. </li><li> Usando unas pinzas, tire hacia atrás la piel del abdomen para exponer la piel peritoneal transparente. Uso de 5 ml jeringas unidas a agujas 20 G, inyectar 5 ml de DPBS frío en la cavidad peritoneal de cada ratón. </li><li> Realizar un masaje suave en la cavidad peritoneal y luego aspirar el líquido cuidadosamente sin perforar cualquier órgano. Retire la aguja y dispensar el líquido peritoneal en 50 ml tubos de centrífuga cónicos. </li><li> Centrifugar durante 10 min a 400 xg en una centrífuga refrigerada.Las células deben mantenerse frío durante todo el procedimiento. Desechar el sobrenadante y resuspender el sedimento de células en medio RPMI 1640. </li><li> Utilizando un hemocitómetro, contar las células y adaptarse a la densidad de células a 1 x 10 6 células / ml. </li><li> Caracterizar el fenotipo de células aisladas por citometría de flujo utilizando 1 x 10 6 células por ratón y antibodie contra F4 / 80 (un antígeno de superficie expresada en macrófagos). </li></ol> 2. Tratamientos Celulares <ol><li> Directamente, después del aislamiento, agregue 1 x 10 6 células en cada pocillo. Deje los macrófagos peritoneales murinos para adherirse a las placas de 6 pocillos cultivándolas durante 1 a 2 horas a 37 ° C. Retire las células no adherentes lavando suavemente 3 veces con PBS caliente. </li><li> Posteriormente, las células de cultivo de 900 l DMEM libre de suero durante 24 horas en la presencia de los siguientes ligandos TLR: (Pam3CSK4-TLR1 / 2 (0,5 mg / ml); Poly (I: C) -TLR3 (10 mg / ml) ; LPS-TLR4 (100 ng / ml); flagelina-TLR5 (100 ng / ml); FSL1-TLR6 / 2 (100 ng / ml); ssRNA40-TLR7 (1 mg / ml); ODN1826-TLR9 (1 M). NOTA: Prepararse para cada ligando una solución 10x. Añadir 100 l de este último a cada pocillo, realizando así una dilución de 10 veces. </li></ol> 3. Aislamiento de ARN <ol><li> Eliminar todas las células medianas y lisan directamente en las placas de seis pocillos mediante la adición de 1 ml de TRIzol a cada pocillo y el lisado celular que pasa varias veces a través de una pipeta. Incubar las muestras homogeneizadas durante 5 a 10 min a TA, para permitir la disociación completa de los complejos de nucleoproteína. </li><li> Traslado lisado en 1,5 ml RNasa y tubos de microcentrífuga-DNasa libre. Añadir 0,2 ml de cloroformo por TRIzol 1 ml. Agitar los tubos vigorosamente a mano durante 15 segundos e incubar ellos a TA durante 5 a 10 min. Centrifugar a 12000 xg durante 15 min a 4 ° C. Observar la mezcla se separa en una fase roja (fenol-cloroformo) inferior, una interfase y una fase acuosa superior incoloro. ARN permanece exclusivamente en la fase acuosa. </li><li> TranSfer cuidadosamente la fase acuosa superior a un tubo nuevo sin perturbar la interfase. Precipitar el RNA de ella por la mezcla con 0,5 ml de alcohol isopropílico. Incubar las muestras a temperatura ambiente durante 10 min y se centrifuga a 12.000 xg durante 10 min a 4 ° C. El ARN precipitados de la formación de un sedimento blanco en el lado y la parte inferior del tubo. </li><li> Eliminar el sobrenadante. Lavar el pellet de ARN una vez con 1 ml de etanol al 75%. Mezclar las muestras mediante vórtex y centrifugar a 7500 xg durante 5 min a 4 ° C. Brevemente secar el ARN (secar al aire durante 10 min). Disolver ARN en 0,1 ml de DEPC (RNasa libre de agua) y se incuba durante 10 minutos a 55 ° C. </li><li> Cuantificar ARN usando un espectrofotómetro. Para ello, diluir las muestras 100 veces en agua DEPC y leer en longitudes de onda de 260 nm. La concentración de ARN será entonces: OD 260 x 40 / ul x factor de dilución ng. </li><li> Igualar las concentraciones de ARN y síntesis de ADNc según las instrucciones del fabricante utilizando RT-PCR System para Kit primer capítulo de síntesis de cDNA. Comosegún las instrucciones del fabricante, medir los niveles de ARNm de genes por PCR en tiempo real (45 ciclos) en un sistema de detección de ADN en tiempo real. Normalizar los niveles de expresión de genes con genes de dos de limpieza, por ejemplo, β-actina y GAPDH. NOTA: Normalizar la concentración de ARN a 500 mg / ml y 0,5 mg usar para la síntesis de ADNc. </li></ol>

<ol>
	<li>Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+macrophages+in+development+and+disease.">Trophic macrophages in development and disease.</a> Nat Rev Immunol. 9 (4), 259-270 (2009).</li><li>Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+activation+of+macrophages.">Alternative activation of macrophages.</a> Nat Rev Immunol. 3 (1), 23-35 (2003).</li><li>Koury, M. J., Ponka, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+erythropoiesis:+the+roles+of+folate,+vitamin+B12,+and+iron.">New insights into erythropoiesis: the roles of folate, vitamin B12, and iron.</a> Annu Rev Nutr. 24, 105-131 (2004).</li><li>Sheftel, A. D., Kim, S. F., Ponka, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-heme+induction+of+heme+oxygenase-1+does+not+alter+cellular+iron+metabolism.">Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism.</a> J Biol Chem. 282 (14), 10480-10486 (2007).</li><li>Hershko, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Storage+iron+regulation.">Storage iron regulation.</a> Prog Hematol. 10, 105-148 (1977).</li><li>Collins, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Withholding+iron+as+a+cellular+defence+mechanism--friend+or+foe.">Withholding iron as a cellular defence mechanism--friend or foe.</a> Eur J Immunol. 38 (7), 1803-1806 (2008).</li><li>Noyes, W. D., Bothwell, T. H., Finch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+reticulo-endothelial+cell+in+iron+metabolism.">The role of the reticulo-endothelial cell in iron metabolism.</a> Br J Haematol. 6, 43-55 (1960).</li><li>Layoun, A., Huang, H., Calve, A., Santos, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+signal+adaptor+protein+MyD88+is+required+for+sustained+endotoxin-induced+acute+hypoferremic+response+in+mice.">Toll-like receptor signal adaptor protein MyD88 is required for sustained endotoxin-induced acute hypoferremic response in mice.</a> Am J Pathol. 180 (6), 2340-2350 (2012).</li><li>Zumerle, S., 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=Targeted+disruption+of+hepcidin+in+the+liver+recapitulates+the+hemochromatotic+phenotype.">Targeted disruption of hepcidin in the liver recapitulates the hemochromatotic phenotype.</a> Blood. 123 (23), 3646-3650 (2014).</li><li>Nguyen, N. B., Callaghan, K. D., Ghio, A. J., Haile, D. J., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepcidin+expression+and+iron+transport+in+alveolar+macrophages.">Hepcidin expression and iron transport in alveolar macrophages.</a> Am J Physiol Lung Cell Mol Physiol. 291 (3), L417-L425 (2006).</li><li>Nemeth, E., 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=Hepcidin,+a+putative+mediator+of+anemia+of+inflammation,+is+a+type+II+acute-phase+protein.">Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.</a> Blood. 101 (7), 2461-2463 (2003).</li><li>Nemeth, E., 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=Hepcidin+regulates+cellular+iron+efflux+by+binding+to+ferroportin+and+inducing+its+internalization.">Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.</a> Science. 306 (5704), 2090-2093 (2004).</li><li>Constante, 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=Distinct+requirements+for+Hfe+in+basal+and+induced+hepcidin+levels+in+iron+overload+and+inflammation.">Distinct requirements for Hfe in basal and induced hepcidin levels in iron overload and inflammation.</a> Am J Physiol Gastrointest Liver Physiol. 291 (2), G229-G237 (2006).</li><li>Zhang, X., Goncalves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+characterization+of+murine+macrophages.">The isolation and characterization of murine macrophages.</a> Curr Protoc Immunol. Chapter 14 (Unit 14 11), (2008).</li><li>Ray, A., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+peritoneal+cavity+cells.">Isolation of mouse peritoneal cavity cells.</a> J Vis Exp. (35), (2010).</li><li>Leijh, P. C., van Zwet, T. L., ter Kuile, M. N., van Furth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+thioglycolate+on+phagocytic+and+microbicidal+activities+of+peritoneal+macrophages.">Effect of thioglycolate on phagocytic and microbicidal activities of peritoneal macrophages.</a> Infect Immun. 46 (2), 448-452 (1984).</li><li>Dy, M., Debray-Sachs, M., Kamoun, P., Hamburger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+thioglycollate+on+macrophage+lysosomal+enzymes.">Effect of thioglycollate on macrophage lysosomal enzymes.</a> Biomedicine. 29 (5), 167-170 (1978).</li><li>Li, Y. M., Baviello, G., Vlassara, H., Mitsuhashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycation+products+in+aged+thioglycollate+medium+enhance+the+elicitation+of+peritoneal+macrophages.">Glycation products in aged thioglycollate medium enhance the elicitation of peritoneal macrophages.</a> J Immunol Methods. 201 (2), 183-188 (1997).</li><li>Layoun, A., Santos, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+cell+wall+constituents+induce+hepcidin+expression+in+macrophages+through+MyD88+signaling.">Bacterial cell wall constituents induce hepcidin expression in macrophages through MyD88 signaling.</a> Inflammation. 35 (4), 1500-1506 (2012).</li><li>Oppenheim, J. J., Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alarmins:+chemotactic+activators+of+immune+responses.">Alarmins: chemotactic activators of immune responses.</a> Curr Opin Immunol. 17 (4), 359-365 (2005).</li><li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+recognition+and+innate+immunity.">Pathogen recognition and innate immunity.</a> Cell. 124 (4), 783-801 (2006).</li><li>Lang, R., Patel, D., Morris, J. J., Rutschman, R. L., Murray, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+gene+expression+in+activated+and+resting+primary+macrophages+by+IL-10.">Shaping gene expression in activated and resting primary macrophages by IL-10.</a> J Immunol. 169 (5), 2253-2263 (2002).</li><li>Wang, C., 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=Characterization+of+murine+macrophages+from+bone+marrow,+spleen+and+peritoneum.">Characterization of murine macrophages from bone marrow, spleen and peritoneum.</a> BMC Immunol. 14 (6), (2013).</li><li>Austin, P. E., McCulloch, E. A., Till, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+factor+in+L-cell+conditioned+medium+capable+of+stimulating+colony+formation+by+mouse+marrow+cells+in+culture.">Characterization of the factor in L-cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture.</a> J Cell Physiol. 77 (2), 121-134 (1971).</li><li>Cannon, G. J., Swanson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macrophage+capacity+for+phagocytosis.">The macrophage capacity for phagocytosis.</a> J Cell Sci. 101 (Pt 4), 907-913 (1992).</li><li>Wang, Y., Harris, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+in+renal+disease.">Macrophages in renal disease.</a> J Am Soc Nephrol. 22 (1), 21-27 (2011).</li><li>Edwards, J. P., Zhang, X., Frauwirth, K. A., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+functional+characterization+of+three+activated+macrophage+populations.">Biochemical and functional characterization of three activated macrophage populations.</a> J Leukoc Biol. 80 (6), 1298-1307 (2006).</li><li>Hoover, D. L., Nacy, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+to+kill+Leishmania+tropica:+defective+intracellular+killing+of+amastigotes+by+macrophages+elicited+with+sterile+inflammatory+agents.">Macrophage activation to kill Leishmania tropica: defective intracellular killing of amastigotes by macrophages elicited with sterile inflammatory agents.</a> J Immunol. 132 (3), 1487-1493 (1984).</li><li>Schleicher, U., Bogdan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+culture+and+flow-cytometric+characterization+of+primary+mouse+macrophages.">Generation culture and flow-cytometric characterization of primary mouse macrophages.</a> Methods Mol Biol. 531, 203-224 (2009).</li></ol>

The authors have no competing financial interests.

Los macrófagos son cruciales para la supervivencia y proporcionan un objetivo tentador para manipular el anfitrión de objetivos inmunológicos. El descubrimiento de TLRs y otras moléculas de reconocimiento han llevado a cabo los macrófagos para el centro de debate inmunológica. Los macrófagos responden a una variedad de estímulos, incluyendo citoquinas, moléculas de daño asociado moleculares patrón (20) amortigua y moléculas asociadas con grupos de patógenos (PAMPs) 21. Estas respuestas...

El sistema reticuloendotelial fagocítica se compone de células en diversos tejidos y órganos tales como la médula ósea, la sangre, el hígado y el bazo. Los macrófagos se distribuyen ampliamente en todo el cuerpo, donde principalmente participan en innata y adaptativa la respuesta inmune para controlar las infecciones y claros. Además de su papel en la defensa del huésped, los macrófagos también juegan un papel importante en la cicatrización de heridas y en el mantenimiento de la homeostasis del tejido 1,2. Además, los macrófagos no sólo son importantes para la función inmune, pero también participan activamente en la homeostasis del hierro 3. En el cuerpo, aproximadamente 80% de hierro está presente en la hemoglobina dentro de los eritrocitos, que cuando senescentes son fagocitadas por los macrófagos 4. Todos los días, estos macrófagos reciclan 25 mg de hierro eritrocitos derivados y proporcionar su transporte en el plasma 5. Por otra parte, durante la infección y la inflamación, los macrófagos pro-inflamatorias secuestran hierro sérico para reducir DISPONIBIL hierrodad a los patógenos, tanto a nivel sistémico y local 6-8. Además, los estudios han demostrado que los macrófagos y principalmente los hepatocitos producen un péptido antimicrobiano llamado hepcidina que es considerado el maestro regulador del metabolismo del hierro 9, 10. La hepcidina se incrementa principalmente por estímulos inflamatorios y es parcialmente responsable de secuestro de hierro en los macrófagos en inflamación crónica 11-13. Como expresión de hepcidina en los macrófagos no se entiende muy bien, hemos estudiado el posible papel de los receptores tipo Toll (TLR) en este reglamento. Los TLRs se encuentran principalmente en los macrófagos y juegan un papel central en su activación. Además, LPS expresión de hepcidina inducida en el hígado es dependiente de TLR4 13. Por lo tanto, para ejecutar nuestro estudio, hemos utilizado un método basado en el aislamiento de macrófagos peritoneales murinos. Líneas celulares de macrófagos se utilizan ampliamente en espárrago macrófagoss; no obstante, la cultura extendida puede provocar la pérdida de genes y la disminución de las funciones inmunes en estas líneas celulares. Por lo tanto, el aislamiento de los macrófagos de la cavidad peritoneal es crucial. La cavidad peritoneal de ratón presenta un sitio ideal para cosechar los macrófagos 13-15. Macrófagos peritoneales murinos aislados son convenientes para varios estudios con respecto a su función inmunológica. Sin embargo, el número de macrófagos en el peritoneo es insuficiente para estudios extensos y se estima alrededor de 1 x 10 6 macrófagos por ratón. Por lo tanto, para aumentar la producción de macrófagos, un agente de provocar estéril tal como tioglicolato se inyectó en la cavidad peritoneal anterior a la cosecha de células. Después de la inyección de tioglicolato, el rendimiento de los macrófagos por ratón se incrementó en 10 veces. A pesar del aumento en los macrófagos rendimiento, tioglicolato actos medianas de cerveza como un irritante que induce una respuesta inflamatoria, lo que resulta en el reclutamiento de macrófagos, que may pero no es necesario afectar a la expresión génica. Por lo tanto, un grupo de control que consiste en macrófagos no tratados debe ser incluido en cada experimento. En nuestras manos, la expresión de hepcidina que está muy estimulada por la inflamación no se detectó en tioglicolato no tratado provocada macrófagos peritoneales. Por otra parte, los estudios han demostrado que tioglicolato de Brewer recluta numerosos macrófagos, pero no activa ellos 16. Por otro lado, tioglicolato de Brewer suscitó macrófagos mostraron un aumento en la enzima lisosomal, pero una disminución en matar microorganismos ingeridos 17. Sin embargo, la capacidad fagocítica no se vio afectada en comparación con los macrófagos no-suscitó 16. Una vez cultivadas en platos, los macrófagos peritoneales se convierten adherente, por lo tanto permitiendo su separación de otro tipo de células aisladas de la cavidad peritoneal. Posteriormente, los macrófagos aislados fueron desafiados con diferentes agonistas de TLR.Finalmente, el ARNm se extrajo de las células cultivadas y la expresión génica se analizó mediante reacción en cadena de la polimerasa con transcriptasa inversa cuantitativa (QRT-PCR).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Charles River Laboratories, Inc. (Wilmington, MA, USA)</td>
			<td>475</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioglycollate</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>19032-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% sodium pentobarbital (Used for mice anesthesia (80 mg/kg, i.p.))</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate-buffered saline (DPBS), placed on ice and will serve to harvest macrophages</td>
			<td>WISENT INC Canada (QC)</td>
			<td>311-425-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 (Supplement with penicillin, streptomycin, L-glutamine, and 10 % fetal calf serum).</td>
			<td>WISENT INC Canada (QC)</td>
			<td>350-000-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>1 and 5 ml syringes</td>
			<td>BD USA (NJ)</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well plates</td>
			<td>Corning Incorporated (NY, USA)</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-pm25</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyionosine&#8211;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-PIC</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FLIC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoprotein synthetic FSL1 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FSL</td>
			<td></td>
		</tr>
		<tr>
			<td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &#956;g/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-LRNA-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Type B CpG oligonucleotide ODN1826 (1 &#956;M)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>11B16-MM</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIZOL</td>
			<td>Invitrogen, (Burlington, ON, Canada)</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr>
			<td>20g and 23g needles</td>
			<td>BD USA (NJ)</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes placed on ice</td>
			<td>Sarstedt (Newton, MA, USA)</td>
			<td>62.547.205</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cells Lysis Buffer</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>R7757-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80 antibody</td>
			<td>BIO-RAD ( CA, USA)</td>
			<td>MCA497APC</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16/CD32 antibodies</td>
			<td>Pharmingen, {Mississauga, ON, CA)</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>Coulter Epics Elite counter, Coulter, (Hialeah, FL,USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well plates</td>
			<td>Corning Incorporated (NY, USA)</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-pm25</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyionosine&#8211;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-PIC</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FLIC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoprotein synthetic FSL1 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FSL</td>
			<td></td>
		</tr>
		<tr>
			<td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &#956;g/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-LRNA-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Type B CpG oligonucleotide ODN1826 (1 &#956;M)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>11B16-MM</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIZOL</td>
			<td>Invitrogen, (Burlington, ON, Canada)</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf tubes</td>
			<td>Axygen Scietific (CA,USA)</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific (ON, Canada)</td>
			<td>UN1888</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>JT Baker (PA, USA)</td>
			<td>70566</td>
			<td></td>
		</tr>
		<tr>
			<td>75% ethanol (in DEPC treated water)</td>
			<td>Commercial Alchohols (QC, Canada)</td>
			<td>17394</td>
			<td></td>
		</tr>
		<tr>
			<td>0.01% diethyl pyrocarbonate (DEPC) treated water (let stand overnight and autoclave)</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>216.542.8</td>
			<td></td>
		</tr>
		<tr>
			<td>Omniscript RT-PCR system</td>
			<td>Qiagen, (Mississauga, ON, Canada)</td>
			<td>205113</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotor Gene 3000</td>
			<td>Montreal Biotech, (Kirkland, QC, Canada)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect SYBR Green I PCR kits</td>
			<td>Qiagen, (Mississauga, ON, Canada)</td>
			<td>204141</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation

Durante la infección y la inflamación, los monocitos circulantes abandonan el torrente sanguíneo y migran hacia los tejidos, donde se diferencian en macrófagos. Los macrófagos expresan receptores de superficie de tipo Toll (TLRs), que reconocen patrones moleculares conservados a través de la evolución en una amplia gama de microorganismos. TLRs jugar un papel central en la activación de macrófagos que generalmente se asocia con la alteración de la expresión génica. Los macrófagos son críticos en muchas enfermedades y se han convertido en objetivos atractivos para la terapia. En el siguiente protocolo, se describe un procedimiento para aislar los macrófagos peritoneales murinos utilizando medio de tioglicolato de Brewer. Este último será impulsar la migración de monocitos en el peritoneo, en consecuencia esto elevará el rendimiento de los macrófagos por 10 veces. Varios estudios se han llevado a cabo utilizando la médula ósea, el bazo o peritoneales macrófagos derivados. Sin embargo, los macrófagos peritoneales se mostró a ser más maduro tras el aislamiento y son más estables en su funcionaldad y fenotipo. Por lo tanto, los macrófagos aislados de la cavidad peritoneal murino presentan una importante población de células que pueden servir en diferentes estudios inmunológicos y metabólicos. Una vez aislado, los macrófagos fueron estimulados con diferentes ligandos de TLR y en consecuencia se evaluó la expresión génica.

En primer lugar, caracterizamos los macrófagos peritoneales murinos aislados por citometría de flujo. Para ello, hemos utilizado (F4 / 80) anticuerpos que reconocen específicamente marcadores única expresadas por los macrófagos. Se requiere esta caracterización para determinar el porcentaje de aislado de macrófagos y distinguirlos entre células obtenidas durante el proceso de aislamiento. Como se muestra en la (Figura 1), el porcentaje de células que expresan el antígeno F4 / 80 fue encontrado...

Watch this Scientific Journal Video about Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation at JoVE.com

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Aislamiento de macrófagos peritoneales murinos para llevar a cabo análisis de expresión génica Al Toll-like Receptores Estimulación

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

isolation-murine-peritoneal-macrophages-to-carry-out-gene-expression

Research

JoVE Journal

Immunology and Infection

36.1K vistas.  Institut du cancer de Montréal. We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Video: Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Aislamiento de células de ratón cavidad peritoneal

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Investigación

Inmunología e Infección

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

Detección de actividad sialidasa Neu1 en la regulación de Toll-like receptor de activación

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Imagen cuantitativa de linaje específico Toll-like receptor-mediada por la señalización en los monocitos y células dendríticas de pequeñas muestras de sangre humana

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

El aislamiento, tratamiento y análisis de células gingivales murinos

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

El uso de<em&gt; Flexneri Shigella</em&gt; Para estudiar las interacciones Autophagy Citoesqueleto

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Aislamiento y activación de los linfocitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila exterior vesículas de membrana: Aislamiento y análisis de su potencial pro-inflamatoria en los macrófagos

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Aislamiento de Salmonella typhimurium-que contienen fagosomas de los macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

Utilizando el análisis filogenético para investigar origen de genes eucariotas

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Evaluación de los efectos de drogas basados en anticuerpos en médula ósea murina y activación de los macrófagos peritoneales

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...