Name: th17 inflammation model of oropharyngeal candidiasis in immunodeficient mice
Uploaded: 2015-02-18T15:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice at JoVE.com

We thank Dr. Helene Bernstein for providing us with access to her flow cytometer. We also thank CFAR flow cytometry facility for the flow cytometry services. This project was in part supported by CTSC core utilization funding and STERIS corporation/University Hospitals-Division of Infectious diseases grant to PP.

NOTA: Los experimentos con ratones se realizaron de acuerdo con el comité de bienestar de los animales institucional (IACUC) directrices. 1. Reconstitución de la RAG-1 - / - ratones con células I n Vitro Cultivadas Th17 (Tres días antes de la infección) <ol><li> Para el establecimiento de las células Th17, cultura CD4 + CD25 CD44low CD62Lhigh células T naïve (3 x 104) en placas de 96 pocillos de fondo en U solo, o co-cultivo ellos junto con 3 x 104 células CD4 + CD25 + Foxp3 + Treg en presencia de solubles α-CD3 (1 mg / ml), α-CD28 (2 g / ml) anticuerpos y células presentadoras de antígeno, en condiciones Th17 (polarizados usando IL-6 (25 ng / ml), TGF-β (2 ng / ml) , α-IFN-γ (2 mg / ml) y α -IL-4 (2 mg / ml)) durante tres a cuatro días. NOTA: Las células ingenuo y T regs fueron ordenados usando células activadas por fluorescencia (FACS) und procedimientos de aislamiento celular magnética y se cultivaron como se ha descrito previamente 19-22. </li><li> El día 4, después de resuspensión de las células Th17 utilizando la pipeta, recogerlos a partir de cultivos, y centrifúguelas en condiciones estériles. <ol><li> Tomar una pequeña alícuota de estas células para examinar su viabilidad y por el fenotipo. Resuspender en 500 l de medio RPMI, añadir yoduro de propidio (200 ng / ml) y evaluar su viabilidad inmediatamente por citometría de flujo. Realizar fenotipificación por citometría de flujo de evaluación de la producción de IL-17A después de la re-estimulación (ver sección 6.3). </li></ol></li><li> Centrifugar y lavar el grueso de las células en PBS estéril a 480 xg durante 6 min a 4 ° C en todos los pasos a menos que se especifique lo contrario. </li><li> Utilice estas células para la transferencia adoptiva en 6-8 semanas de edad CD45.2 Rag1 - / - ratones inmunodeficientes. <ol><li> Resuspender las células a la densidad celular 1 x 10 7 células / ml usando PBS estéril frío. </li><li> Inyectar 100 l de las células por intrapinyección eritoneal, utilizando 25 G agujas en 1 ml jeringas de tuberculina, de tal manera que un ratón recibe 1 x 10 6 células. Algunos ratones recibirán células PBS o Th17 solamente, y otros ratones recibirán las células Th17 que fueron co-cultivadas con células Treg. NOTA: Realice todos los pasos de forma aséptica. </li></ol></li></ol> 2. Crecimiento C. albicans para la infección (un día antes de la infección) <ol><li> Antes de la inoculación, dispersar cinco colonias de la CAF2-1 C. cepa albicans laboratorio en 100 l de suspensión de PBS estéril, y añadir la suspensión al caldo estéril. </li><li> Inocular 5 colonias de la C. albicans en 50 ml de la base de nitrógeno de levadura (YNB sin aminoácidos) / Peptona medio / caldo de dextrosa y se incuba en un incubador agitador a 30 ° C durante 15-18 horas a 130 rpm. </li><li> Monitorear el caldo de nubosidad, ya que es una indicación para el crecimiento del hongo. </li></ol> 3. CandidaLa cosecha y el procedimiento de conteo (En el Día de la infección) <ol><li> Recoger 10 ml de caldo de Candida en tubos de 15 ml. Para volúmenes mayores, recoger Candida en tubos de 50 ml. </li><li> Centrifugar las blastosporas en 1900 xg durante 5 min a RT en todos los pasos a menos que se especifique lo contrario. Sedimentar las blastosporas por centrifugación, seguido por la eliminación del sobrenadante. </li><li> Si hay más de un tubo, agrupar los blastosporas de Candida a partir de múltiples tubos, la adición de PBS estéril para los pellets y resuspender en 10 ml de PBS para el conteo. </li><li> Tomar 20 l de blastosporas en 1,5 ml tubos de microcentrífuga, añadir 20 l de la paraformaldehído 2X e incubar a temperatura ambiente durante 15 a 20 min. Contar las blastosporas fijos utilizando el hemocitómetro bajo el microscopio. NOTA: El paraformaldehido es cancerígeno. Abra la solución sin diluir sólo en la campana de humos. </li><li> Mientras tanto, repetir el lavado de las blastosporas al menos dos veces, por centrifugación tdobladillo en PBS, y la granulación las blastosporas. Mantenga las pastillas en tubos de 15 ml a temperatura ambiente, en la campana ABSL1, listas para la infección. </li><li> Deje algo de PBS estéril a temperatura ambiente durante la resuspensión de las blastosporas de infección. </li><li> Después de contar, añadir PBS estéril al sedimento blastospore para ajustar el número de células de levadura blastosporo a 2 x10 8 de levadura células / ml. Esta es la suspensión blastospore que se utilizó para infectar los ratones. NOTA: Realice todos los pasos de forma aséptica. </li></ol> 4. Los ratones Infección (3-5 días después de la transferencia de la célula) NOTA: El procedimiento básico infección se realizó como se describió anteriormente 19,23-25. <ol><li> Pese la RAG-1 - / - ratones que recibieron PBS o las células tres días antes. </li><li> Calcular la dosis de los agentes anestésicos que utilizan su peso corporal. Anestesiar a los ratones mediante la administración de ketamina / mezcla de xilazina (16,1 mg / ml y 1,6 mg / ml), utilizando 25 g de agujas en 1 ml jeringas de tuberculina, por intrinyección aperitoneal. <ol><li> Administrar 50 l por 10 gramos de peso corporal. (Es decir, 0,8 mg de ketamina y 0,08 mg de xilazina / 10 g de peso corporal o 80 mg de ketamina y 8 mg de xilazina por kg de peso corporal, respectivamente). Observe la respuesta pizca dedo cada 15 minutos después de la anestesia. NOTA: Esta dosis inducirá 60-90 min de la anestesia, lo cual es suficiente para el procedimiento de infección. </li><li> Aplique el ungüento lubricante oftálmica a los ojos de los ratones para evitar las córneas se sequen. A medida que el lubricante ocular podría secarse y repetir lubricación oftálmica cada 45 min. </li></ol></li><li> Inyectar 1 ml de solución salina (0,9% NaCl) por vía subcutánea en la parte posterior adyacente a la extremidad anterior, para ayudar a la rehidratación de los ratones durante la anestesia. </li><li> Obtener una nueva, jaula limpia. Siga la infección procedimiento de un ratón a la vez, la colocación de cada ratón en la nueva jaula una vez infectados. Realice la infección PBS / farsa primero, y luego proceder a las gro de infección por Candidaups. </li><li> Coge el ratón anestesiado y abrir la boca para revelar la base de la lengua. <ol><li> Coloque una bola de algodón diámetro 3 mm saturado con 50 l de PBS o blastospore suspensión, por vía sublingual en la cavidad oral durante 90 min. Da la vuelta al ratón todos los frentes 15 min y la espalda para evitar la congestión pulmonar, lo que garantiza que las bolas de algodón no se mueven. </li></ol></li><li> Establecer un temporizador para cada ratón para asegurar 90 min de Sham o Candida inoculación en la cavidad oral. </li><li> Manténgalos en una jaula bajo la lámpara de calor (4 metros de distancia), y cada ratón sobre almohadillas de gel de calor. </li><li> Asegúrese de que la lengua se retira dentro y fuera de los dientes, para evitar los dientes lacerantes la lengua. </li><li> Al final de 90 minutos, retire la bola de algodón de la boca del ratón. Esté atento a cualquier ratón que pueden recuperarse de la anestesia antes de 75 minutos. En tal caso, anestesiar a ellos de nuevo con ketamina (40 mg / kg) solamente. </li></ol> 5. Publicar Procedure Monitoreo (durante los 90 minutos de anestesia) <ol><li> Use almohadillas de gel de calor durante la anestesia 90 min. <ol><li> Para mantener la temperatura del cuerpo y para evitar la asfixia, no permita que los ratones que se recuperan en la ropa de cama regular de ratón mazorca de maíz. </li></ol></li><li> Después de la recuperación de la anestesia, administrar un adicional de 1 ml de solución estéril de NaCl al 0,9% por vía subcutánea en dos lugares en la parte posterior. </li><li> Infórmate de los cinco PBS (simulado) los ratones inoculados por jaula. Casa sólo 1-2 ratones infectados por jaula. </li><li> Llene la tarjeta de procedimiento con las iniciales todos los días después del procedimiento para anotar los cambios en el peso corporal, hábitos de aseo y la salud en general. </li></ol> 6. Evaluación de la inflamación 6.1) La pérdida de peso <ol><li> Pesar los ratones cada día durante la infección, comenzando en el día antes del procedimiento infección. NOTA: Por lo general, los ratones inmunodeficientes inyectados con células Th17 y sin T regs sucumben a 20Pérdida de peso -30% debido al aumento de la carga de hongos 19 y exacerba la inflamación. </li></ol> 6.2) Histología de puntuación de la lengua <ol><li> Sacrificar los ratones por asfixia de CO 2 seguido por dislocación cervical. </li><li> Abra las mordazas y diseccionar la lengua con tijeras y pinzas. El uso de fórceps romos para sostener la lengua, y el uso de las tijeras llegan a la parte posterior de la boca para incidir el extremo posterior de la lengua. </li><li> Para hematoxilina inmuno y eosina (H &amp; E) tinción de los tejidos de la lengua, enjuague los tejidos de la lengua con helado de PBS. Añadir 5 ml de formalina al 10% durante 2-3 lenguas y fijarlos O / N en tubos de 15 ml. <ol><li> Al día siguiente, eliminar el formol y sumergir los tejidos en 5 ml de etanol al 70% para evitar la hiper-fijación. Enviarlos a un servicio comercial para continuar con la inclusión en parafina, corte y tinción de secciones de parafina. NOTA: El servicio de histología comercial se utiliza para realizar estos pasos. </li&gt; </ol></li><li> Una vez que los portaobjetos están de vuelta desde el servicio de histología comercial, el grado de la inflamación mediante la observación de las secciones de tejido bajo un microscopio de luz. <ol><li> Puntuación de 0 a 5, siendo 0 ausencia de inflamación, y 5 la inflamación más severa. Partitura la inflamación usando la Tabla 1. </li></ol></li></ol> 6.3) La producción de citocinas por las células Th17 NOTA: la producción de TNF-α excesiva es una de las lecturas para inmunopatología excesivas. Cuando los ratones se transfieren adoptivamente con células Th17 sólo, hace que la inmunopatología que está asociada con la producción excesiva de TNF-α en células Th17. <ol><li> Recoger una suspensión de células de bazo, los ganglios linfáticos cervicales, ganglios linfáticos axilares, y la lengua, usando métodos descritos previamente 19,26. </li><li> Reestimular las células con miristato acetato de forbol (PMA) (50 ng / ml) e ionomicina (500 ng / ml) durante 4 h con Brefeldina A (10 mg / ml) añadido en el last 2 hr. Lavar las células con PBS y fijarlos utilizando un kit de fijación / permeabilización comercial según las instrucciones del fabricante. </li><li> Realizar tinción de citoquinas intra celular utilizando el anti-TNF-α conjugados con fluorocromo, anti-IL-17A y anticuerpos ROR-γt como se describió anteriormente 19. </li><li> Brevemente, resuspender las células en el tampón de permeabilización 1X con el cóctel de anticuerpos anti-TNF-α, los anticuerpos ROR-γt anti-IL-17A y, cada anticuerpo a 2-3 mg / ml de concentración final. </li><li> Se incuban las células durante 1 hora a RT. </li><li> Después de la incubación, se lavan las células con tampón de permeabilización 1X. </li><li> Resuspender el sedimento celular en 500 l de PBS con 0,5% de albúmina de suero bovino para citometría de flujo análisis. </li></ol>

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F., Kollias, G., Becker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+functions+of+TNF-alpha+in+the+regulation+of+the+intestinal+epithelial+response+to+inflammation.">Pleiotropic functions of TNF-alpha in the regulation of the intestinal epithelial response to inflammation.</a> International Immunology. 26 (9), 509-515 (2014).</li><li>Bishu, 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=The+adaptor+CARD9+is+required+for+adaptive+but+not+innate+immunity+to+oral+mucosal+Candida+albicans+infections.">The adaptor CARD9 is required for adaptive but not innate immunity to oral mucosal Candida albicans infections.</a> Infection and Immunity. 82 (3), 1173-1180 (2014).</li><li>Quintin, J., 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=Differential+role+of+NK+cells+against+Candida+albicans+infection+in+immunocompetent+or+immunocompromised+mice.">Differential role of NK cells against Candida albicans infection in immunocompetent or immunocompromised mice.</a> Eur J Immunol. 44 (8), 2405-2414 (2014).</li><li>Ramirez-Garcia, A., 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=Candida+albicans+and+cancer:+Can+this+yeast+induce+cancer+development+or+progression.">Candida albicans and cancer: Can this yeast induce cancer development or progression.</a> Critical Reviews In Microbiology. , 1-13 (2014).</li></ol>

The authors declare that they have no competing financial interests.

Este modelo se basa en la inducción de C. Oral infección albicans inflamación dependiente Th17. Debido a la ausencia de T regs, la inflamación de células Th17 inducida es desenfrenada y conduce a la inmunopatología mal resuelto. In vitro derivan las células CD4 naive polarizados ya que se utilizaron células Th17 para la transferencia adoptiva. 40 - 50% de las células CD4 + cultivadas muestran detectable la expresión de IL-17A alrededor de 3 días (células Th17), y por lo ...

Oral infections and inflammation have been related to cancer and cardiovascular diseases, and have dramatic impact on overall human health2,3. Opportunistic infections and inflammation caused by C. albicans are associated with primary immunodeficiencies (PID)4,5, inflammatory disorders such as periodontitis 6,7, Sjogren&#8217;s syndrome, and salivary gland disease8,9, as well as oral squamous cell carcinoma 10-12. C. albicans is a dimorphic commensal fungus that colonizes the mouths of 60% of healthy humans asymptomatically, yet it is the most common fungal pathogen causing infections when the host defense is weakened13-15. It causes recurring and chronic infections and inflammation in patients with AIDS and PID, and also in other immunocompromised individuals. As a commensal, its colonization load is associated with the change in the diversity of the overall oral microbiome16. As a pathogen it causes several forms of oropharyngeal candidiasis such as acute pseudomembranous, acute atrophic, chronic atrophic, chronic hypertrophic/hyperplastic, and angular cheilitis. 
Protection against C. albicans is determined not only by host immune resistance, but also by the ability to appropriately control Candida-induced immunopathology. Although commensals such as C. albicans contribute to modulation and exacerbation of other oral inflammatory conditions, the mechanisms by which dysbiosis occur during opportunistic infections are unclear. Besides the known role of adaptive Th17 cells in memory response to C. albicans17, their role in initiation and perpetuation of inflammation pathology during chronic infections remain unclear. Furthermore, oral inflammatory diseases such as Sjogren&#8217;s syndrome and periodontitis are associated with Th17 mediated pathology. Interestingly, these diseases are also strongly associated with frequent OPC. However, the interactions among Th17 cells, oral immunopathology of OPC and other oral inflammatory diseases are unstudied. 
Although mouse models of primary and secondary infection of oral candidiasis are available, a mouse model to study Candida infection associated Th17 inflammation, especially in the context of immunodeficiency is unavailable. This study presents a method for establishing oral Th17 inflammation associated with oral Candida infection in mice. Candida infection in mice is characterized by fungal lesions, inflammation in the tongue, decreased food intake, weight loss and eventually a moribund state. Oral pathology resembles chronic candidal infection lesions, as well as epithelial dysplasia in mouse oral cancer models12,18.

<table><tbody><tr><td>CAF-2</td><td>University of Pittsburgh (Sarah Gaffen)</td><td>-</td><td>Candida culture</td></tr><tr><td>U-bottom 96 well plates&amp;nbsp;</td><td>Fisher</td><td>055588</td><td>Used for cell culture</td></tr><tr><td>a-CD3&amp;nbsp;</td><td>eBiosciences</td><td>16-0031-85</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>a-CD28&amp;nbsp;</td><td>eBiosciences</td><td>16-0281-85</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>m-IL-6</td><td>Bio Basic</td><td>RC232</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>h-TGF-b&amp;nbsp;</td><td>R&amp;amp;D</td><td>240-B</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>a-IFN-g&amp;nbsp;</td><td>eBiosciences</td><td>16-7311-85</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>a-IL-4&amp;nbsp;</td><td>eBiosciences</td><td>16-7041-85</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>&amp;alpha;-IL17A ef660</td><td>eBiosciences</td><td>50-7177-82</td><td>Used for cell culture - 1:50</td></tr><tr><td>&amp;alpha;-TNFa-PE-Cy7</td><td>eBiosciences</td><td>25-7423-41</td><td>Used for cell culture - 1:100</td></tr><tr><td>&amp;alpha;-RORgt PE&amp;nbsp;</td><td>eBiosciences</td><td>12-6981-82</td><td>Used for cell culture - 1:50</td></tr><tr><td>YNB w/o amino acids)/Peptone/Dextrose broth medium&amp;nbsp;</td><td>Bio Basic</td><td>S507.SIZE</td><td>Mediium for candida growth</td></tr><tr><td>Shaker incubator&amp;nbsp;</td><td>New Brunswick Scientific</td><td>Innova 4300</td><td>Incubation growth for candida</td></tr><tr><td>15 ml tubes</td><td>Bio Basic</td><td>BT888-SY</td><td>Used for cell culture and candida growth</td></tr><tr><td>50 ml tubes</td><td>Bio Basic</td><td>CT 788-YS</td><td>Used for cell culture and candida growth</td></tr><tr><td>Table top Centrifuge</td><td>VWR International LLC</td><td>82017-654</td><td>For pelleting candida -900 g</td></tr><tr><td>Allegra Centrifuge</td><td>Beckman Coulter</td><td>392302</td><td>Cell culture - 480 g</td></tr><tr><td>1.5 ml eppendorf tubes</td><td>Bio Basic</td><td>BT620-NS</td><td>Preparing final concentration of candida</td></tr><tr><td>2x paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Fixing candida for count</td></tr><tr><td>Hemocytometer</td><td>VWR International LLC</td><td>15170-172</td><td>Counting cells</td></tr><tr><td>PBS - Phosphate Buffered Saline</td><td>Bio Basic</td><td>PD8117</td><td>Preparation of buffers</td></tr><tr><td>Ketamine/xylazine&amp;nbsp;</td><td>Case Western Reserve University - Animal Resource Center</td><td>-</td><td>Obtained from ARC approved protocol for anesthesia</td></tr><tr><td>Ophthalmic lubricant ointment&amp;nbsp;</td><td>Allergan</td><td>-</td><td>Eye ointment for animals to prevent dryness</td></tr><tr><td>Saline (0.9% NaCl)</td><td>G Biosciences</td><td>786-561</td><td>Administerd to animals to prevent dehydration</td></tr><tr><td>3-mm-diameter cotton wool ball&amp;nbsp;</td><td>VWR International LLC</td><td>BP7603</td><td>3 mm balls for candida infection</td></tr><tr><td>Heat gel pads</td><td>Case Western Reserve University - Animal Resource Center</td><td>-</td><td>for maintaining the body temperaturre and fast recovery</td></tr><tr><td>Hematoxylin and Eosin staining</td><td>Histoserv - MD</td><td>-</td><td>Tissue histology</td></tr><tr><td>10% formalin&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>JC1111/MC</td><td>Tissue histology</td></tr><tr><td>70% ethanol&amp;nbsp;</td><td>VWR International LLC</td><td>97064-490</td><td>Sterilization purpose</td></tr><tr><td>PMA - Phorbol 12-myristate 13-acetate</td><td>Sigma-Aldrich</td><td>P1585-1MG</td><td>Restimulation of cells</td></tr><tr><td>Ionomycin</td><td>Life Technologies</td><td>124222 1mg</td><td>Restimulation of cells</td></tr><tr><td>Fixation permeabilization kit</td><td>eBiosciences</td><td>E16913-106</td><td>Fixing cells for Flow cytometry</td></tr><tr><td>Tuberculin syringes</td><td>BD Biosciences</td><td>309659</td><td>Mice injection</td></tr><tr><td>25 G x 3/8 needles</td><td>BD Biosciences</td><td>309626</td><td>Mice injection</td></tr><tr><td>Rag1 -/- mice</td><td>Jackson laboratories</td><td>Stock no: 002216</td><td>Recipient mice</td></tr><tr><td>CD45.1 congenic mice</td><td>Jackson laboratories</td><td>Stock no:002014&amp;nbsp;</td><td>Donor Th17 cells</td></tr><tr><td>CB17-SCID mice</td><td>Jackson laboratories</td><td>Stock no: 001303</td><td>Recipient mice</td></tr><tr><td>Balb/c mice</td><td>Jackson laboratories</td><td>Stock no: 000651</td><td>Donor Th17 cells</td></tr></tbody></table>

th17 inflammation model of oropharyngeal candidiasis in immunodeficient mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

En este modelo, tanto los ratones infectados con Candida y ratones no infectados en RAG-1 - / - de fondo inmunodeficiente se transfirieron de forma adoptiva con células Th17 3-5 días antes de la infección. Un total de 10-12 ratones fueron utilizados para los experimentos, con 2 ratones en cada Sham infectados grupos y 4-5 en cada uno de los grupos infectados Candida. Naive células se obtuvieron a partir Thy1.1 o CD45.1 congenic ratones, de modo que las células Th17 inyectados fueron ras...

Watch this Scientific Journal Video about Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice at JoVE.com

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

Th17 Inflamación Modelo de candidiasis orofaríngea en ratones inmunodeficientes

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

th17-inflammation-model-oropharyngeal-candidiasis-immunodeficient

Research

JoVE Journal

Medicine

9.9K vistas.  Case Western Reserve University. Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice. 

Video: Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

Protocolos para la inoculación dentro de la vagina y toma de muestras en el modelo experimental de ratón de Candida Vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

Investigación

Inmunología e Infección

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Aislamiento y diferenciación de Th17 Naïve linfocitos T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

La utilización de la orofaringe Intratraqueal PAMP Administración y lavado broncoalveolar para evaluar el anfitrión respuesta inmune en ratones

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Modelado de las mucosas Candidiasis en larvas de pez cebra por inyección de Swimbladder

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

El modelo de infección de Waxworm Galleria mellonella para la Candidiasis diseminada

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting cells. When the skin encounters invading pathogens, different cutaneous DCs initiate a distinct T cell immune response to protect the body. Among the invading pathogens, fungal infection specifically drives a protective interleukin-17-producing Th17 immune response. A protocol was developed to efficiently differentiate Th17 cells by intradermal Candida albicans infection to investigate a subset of cutaneous DCs responsible for inducing Th17 immunity. Flow cytometry and gene expression analyses revealed a prominent induction of Th17 immune response in skin-draining lymph nodes and infected skin. Using diphtheria toxin-induced DC subset-depleting mouse strains, CD301b+ dermal DCs were found to be responsible for mounting optimal Th17 differentiation in this model. Thus, this protocol provides a valuable method to study in vivo function of differential subsets of cutaneous DCs to determine Th17 immunity against deep skin fungal infection.

<meta charset="utf8"></head><body>Función in vivo de subconjuntos diferenciales de células dendríticas cutáneas para inducir inmunidad Th17 en la infección intradérmica por Candida albicans

Here, we demonstrate the in vivo function of cutaneous dendritic cell subsets in Th17 immunity of deep dermal Candida albicans infection.

In vivo Función de los subconjuntos diferenciales de células dendríticas cutáneas para inducir inmunidad Th17 en la infección intradérmica por Candida albicans

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting ...

APDT eficaz con curcuminoides para la candidiasis oral en ratones

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 &#181;M, 40 &#181;M, and 80 &#181;M) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm2; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch&#39;s ANOVA and Games-Howell tests (&#945; = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 &#181;M or 80 &#181;M curcuminoids. aPDT mediated by 80 &#181;M curcuminoids promoted a 2.47 log10 reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

Autor destacado: Explorando la terapia fotodinámica con curcumina en un modelo murino para la candidiasis oral

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

Th17 Inflamación Modelo de candidiasis orofaríngea en ratones inmunodeficientes

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