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: Os experimentos com ratos foram realizados de acordo com o animal institucional comitê de bem-estar (IACUC) orientações. 1. Reconstituição da Rag-1 - / - Os ratos com células I n Vitro Cultivadas Th17 (três dias antes da infecção) <ol><li> Para o estabelecimento de células Th17, cultura de células CD4 + CD44low CD62Lhigh CD25- células T ingénuas (3 x 104) de fundo em U em placas de 96 poços, sozinho ou co-cultura eles, juntamente com 3 x 104 células T CD4 + CD25 + Foxp3 + Tregs na presença de solúvel α-CD3 (1 ug / ml), α-CD28 (2 ng / ml) de anticorpos e células apresentadoras de antigénio, sob condições Th17 (polarizadas usando IL-6 (25 ng / mL), TGF-β (2 ng / ml) , α-IFN-γ (2 ug / ml) e α -IL-4 (2 ng / ml)) durante três a quatro dias. NOTA: células ingênuo e regs T foram classificados usando fluorescência de células activadas (FACS) umad procedimentos de isolamento de células magnético e cultivadas como descrito anteriormente 19-22. </li><li> No dia 4, depois de re-suspender as células Th17, utilizando a pipeta, recolher-los a partir de culturas, e centrifugar-las em condições estéreis. <ol><li> Tomar uma pequena alíquota destas células para examinar a sua viabilidade e para fenotipagem. Ressuspender-los em 500 ul de meio RPMI, adicionar iodeto de propídio (200 ng / ml) e avaliar a sua viabilidade imediatamente por citometria de fluxo. Execute fenotipagem por citometria de fluxo avaliação da produção de IL-17A após restimulação (ver secção 6.3). </li></ol></li><li> Centrifugar e lavar o grosso das células em PBS estéril a 480 xg durante 6 minutos a 4 ° C em todas as etapas, a menos que especificado de outra forma. </li><li> Use essas células para transferência adotiva em 6-8 semanas de idade CD45.2 RAG1 - / - ratos imunodeficientes. <ol><li> Ressuspender as células a 1 x 10 7 células / ml de densidade celular utilizando PBS estéril frio. </li><li> Injectar 100 ul de células por intraperitoneal injecção, utilizando agulhas 25 G em seringas de 1 ml de tuberculina, de tal modo que um ratinho receber 1 x 10 6 células. Alguns ratos receberão células Th17 PBS ou somente, e outros ratos receberão células Th17 que foram co-cultivados com células Treg. Observação: executar todos os passos de forma asséptica. </li></ol></li></ol> 2. Crescer C. albicans para a infecção (um dia antes da infecção) <ol><li> Antes da inoculação, dispersar cinco colônias do CAF2-1 C. albicans estirpe laboratorial em 100 ul de suspensão de PBS estéril e adicionar a suspensão ao caldo estéril. </li><li> Inocular 5 colônias de C. albicans em 50 ml de base de azoto de levedura (YNB sem aminoácidos) / peptona / meio de caldo de dextrose e incubar numa incubadora com agitação a 30 ° C durante 15-18 horas a 130 rpm. </li><li> Monitorar o caldo para a opacidade, uma vez que é uma indicação para o crescimento do fungo. </li></ol> 3. CandidaColheita e Counting Procedimento (No dia da infecção) <ol><li> Recolha de 10 ml de caldo de Candida em tubos de 15 ml. Para volumes maiores, coletar Candida em tubos de 50 ml. </li><li> Centrifugar os blastosporos a 1900 xg durante 5 min à temperatura ambiente em todas as etapas, a menos que especificado de outra forma. Agregar as blastosporos por centrifugação, seguida pela remoção do sobrenadante. </li><li> Se houver mais do que um tubo, reunir os blastosporos Candida a partir de múltiplos tubos, adicionando PBS estéril para os peletes e ressuspender-los em 10 ml de PBS para contagem. </li><li> Tomar 20 ul de blastosporos em tubos de microcentrífuga de 1,5 ml, adicionar 20 uL de paraformaldeído a 2X e incubar à temperatura ambiente durante 15-20 min. Contagem blastosporos fixos usando o hemocitômetro sob o microscópio. NOTA: O paraformaldeído é cancerígeno. Abrir a solução não diluída apenas na hotte. </li><li> No entretanto, repetir a lavagem dos blastosporos pelo menos duas vezes, por centrifugação them em PBS, e peletização blastosporos. Manter os peletes em tubos de 15 ml à temperatura ambiente, na capa ABSL1, prontos para a infecção. </li><li> Deixar alguns PBS estéril à temperatura ambiente durante ressuspensão dos blastosporos para a infecção. </li><li> Após a contagem, adicionar PBS estéril para o sedimento blastosporo para ajustar o número de células de levedura blastosporo a 2 x10 8 de levedura células / ml. Esta é a suspensão blastosporo que será utilizado para infectar os ratinhos. Observação: executar todos os passos de forma asséptica. </li></ol> 4. Ratos Infection (3-5 dias após a transferência celular) NOTA: O procedimento básico infecção é realizada como descrito anteriormente 19,23-25. <ol><li> Pesar o Rag-1 - / - ratos que receberam PBS ou as células três dias antes. </li><li> Calcula-se a dose de agentes anestésicos, utilizando o seu peso corporal. Anestesiar os ratos por administração de cetamina / xilazina mistura (16,1 mg / ml e 1,6 mg / ml), utilizando agulhas 25 G em seringas de 1 ml de tuberculina, por intrinjeção aperitoneal. <ol><li> Administrar 50 ul por 10 gramas de peso corporal. (Ou seja, 0,8 mg de cetamina e 0,08 mg de xilazina / 10 g de peso corporal ou 80 mg de cetamina e de xilazina 8 mg por kg de peso corporal, respectivamente). Observar o desenvolvimento de resposta toe pitada a cada 15 minutos após a anestesia. NOTA: Esta dosagem irá induzir 60-90 min da anestesia, o que é suficiente para o procedimento de infecção. </li><li> Aplicar a pomada lubrificante oftálmico aos olhos dos ratinhos para evitar que as córneas de secar. Como o lubrificante ocular pode secar, repita lubrificação oftálmica a cada 45 min. </li></ol></li><li> Injectar 1 mL de solução salina (NaCl a 0,9%) por via subcutânea no lado de trás do membro anterior, para ajudar a re-hidratação dos ratos durante a anestesia. </li><li> Obter uma nova gaiola, limpo. Siga o procedimento de uma infecção do mouse em um momento, colocando cada rato na gaiola nova, uma vez infectado. Realize a infecção PBS / sham em primeiro lugar, e depois prosseguir para as gro infecção por Candidaups. </li><li> Pegue o rato anestesiado e abra a boca para revelar a base da língua. <ol><li> Coloque uma bola de algodão 3 mm de diâmetro saturado com 50 ul de PBS ou blastosporo suspensão, sublingualmente na cavidade oral durante 90 min. Virar os ratos a cada 15 minutos frente e de trás para evitar congestão pulmonar, garantindo que as bolas de algodão não se movem. </li></ol></li><li> Configurar um temporizador para cada rato para garantir 90 min de Sham ou Candida inoculação na cavidade oral. </li><li> Mantê-los em uma gaiola sob a lâmpada de calor (4 metros de distância), e cada mouse sobre almofadas de gel de calor. </li><li> Certifique-se de que a língua é retirado dentro e longe dos dentes, para evitar dentes dilacerante da língua. </li><li> No final de 90 minutos, retire a bola de algodão da boca do mouse. Preste atenção para qualquer rato que pode recuperar da anestesia mais cedo do que 75 minutos. Em tal caso, anestesiar-los novamente com cetamina (40 mg / kg) apenas. </li></ol> 5. Publicar Procedure Monitoramento (durante a anestesia 90 min) <ol><li> Use almofadas de gel de calor durante a anestesia 90 min. <ol><li> Para manter a temperatura do corpo e para evitar sufocamento, não permitem que os ratos se recuperar no mouse cama sabugo de milho regular. </li></ol></li><li> Após a recuperação da anestesia, administrar um adicional de 1 ml de NaCl a 0,9% esterilizado subcutaneamente em dois locais no dorso. </li><li> Mantenha-se cinco PBS (sham) camundongos inoculados por gaiola. Casa apenas 1-2 camundongos infectados por gaiola. </li><li> Preencha o cartão de procedimento com as iniciais todos os dias após o procedimento para anotar as mudanças no peso corporal, higiene hábitos e saúde em geral. </li></ol> 6. Avaliação da Inflamação 6.1) A perda de peso <ol><li> Pesar os ratinhos todos os dias durante a infecção, começando no dia anterior ao processo de infecção. NOTA: Normalmente, os ratos imunodeficientes injetados com células Th17 e sem regs T sucumbir a 20-30% De perda de peso devido ao aumento da carga fúngica 19 e exacerbado inflamação. </li></ol> 6.2) pontuação Histologia da língua <ol><li> Sacrifício dos ratinhos por asfixia com CO 2, seguida por deslocação cervical. </li><li> Abra as mandíbulas e dissecar a língua com tesouras e pinças. Use uma pinça rombo para manter a língua, e usando a tesoura chegar à parte de trás da boca para entalhar a extremidade traseira da língua. </li><li> Para hematoxilina immunocytochemical e eosina (H &amp; E) a coloração dos tecidos da língua, lavar os tecidos língua com PBS gelado. Adicionar 5 ml de formol a 10% por 2-3 línguas e corrigi-los O / N em tubos de 15 ml. <ol><li> No dia seguinte, remover a formalina e mergulhar os tecidos em 5 ml de etanol a 70% para evitar a hiper-fixação. Enviá-los para o serviço comercial para continuar com parafina, corte e coloração de cortes de parafina. NOTA: serviço histologia comercial é usado para executar estes passos. </li&gt; </ol></li><li> Uma vez que as lâminas estão de volta a partir do serviço histologia comercial, grau de inflamação, observando as secções de tecido sob um microscópio de luz. <ol><li> Pontuação de 0 a 5, sendo 0 sem inflamação, e 5 sendo o mais grave inflamação. Pontuação da inflamação usando Tabela 1. </li></ol></li></ol> 6.3) A produção de citocinas pelas células Th17 NOTA: A produção excessiva de TNF-α é uma das leituras de imunopatologia excessiva. Quando os ratos são transferidos adoptivamente com células Th17 só, faz com que a imunopatologia que está associado com a produção excessiva de TNF-α em células Th17. <ol><li> Coletar uma suspensão de células individuais do baço, linfonodos cervicais, linfonodos axilares, e língua, utilizando métodos previamente descritos 19,26. </li><li> Reestimular as células com miristato acetato de forbol (PMA) (50 ng / ml) e ionomicina (500 ng / ml) durante 4 h com Brefeldina A (10 ug / ml) adicionada no last 2 h. Lavam-se as células com PBS e corrigi-los utilizando um kit de fixação / permeabilização comercial de acordo com as instruções do fabricante. </li><li> Realização da coloração de citocinas celular intra utilizando o anti-TNF-α conjugado com fluorocromo, anti-IL-17A e anticorpos ROR-γt como anteriormente descrito 19. </li><li> Resumidamente, ressuspender as células em tampão de permeabilização 1X com o cocktail de anticorpos anti-TNF-α, anti-IL-17A e anticorpos ROR-γt, cada anticorpo a 2-3 ug / ml de concentração final. </li><li> Incubam-se as células durante 1 h à TA. </li><li> Após a incubação, lavar as células com tampão de permeabilização 1X. </li><li> Ressuspender o sedimento celular em 500 ul de PBS com 0,5% de albumina de soro bovino para análise de citometria de fluxo. </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 é baseado na indução C. bucal infecção albicans dependente inflamação Th17. Devido à ausência de regs T, a inflamação induzida por células Th17 desprendido e leva a imunopatologia mal resolvidas. In vitro derivados de células T CD4 + naive polarizadas como células Th17 foram utilizados para a transferência adoptiva. 40 - 50% das células CD4 + cultivadas células mostram detectável expressão de IL-17A por volta do dia 3 (células Th17), e por isso foram ...

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.

Neste modelo, tanto os camundongos infectados por Candida e camundongos não infectados em Rag-1 - / - fundo imunodeficientes foram adotivamente transferidos com células Th17 3-5 dias antes da infecção. Um total de 10-12 ratinhos foram utilizadas para as experiências, com dois ratos em cada Sham grupos infectados e 5/4 em cada um dos grupos infectados Candida. Células naive foram derivadas de Thy1.1 ou CD45.1 ratinhos congénicos, de modo que as células Th17 injectados foram rastreados...

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 Inflamação Modelo de orofaringe Candidíase em imunodeficientes 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 ...

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9.9K visualizações.  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. 

Vídeo: 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 inoculação vaginal e coleta de amostra no Modelo Experimental de Rato Candida Vaginite

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

Imunologia e Infecção

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.

Isolamento e Th17 Diferenciação de Linfócitos T CD4 Naïve

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.

A utilização de orofaringe Intratraqueal Administração PAMP e Lavagem Broncoalveolar para avaliar a resposta imune do hospedeiro em camundongos

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.

Modelagem da mucosa candidíase em larval Zebrafish por swimbladder Injection

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.

O modelo de infecção Waxworm Galleria mellonella para candidíase disseminada

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.

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

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

aPDT eficaz usando curcuminóides para candidíase oral em camundongos

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 em destaque: Explorando a terapia fotodinâmica com curcumina em um modelo murino para candidíase 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.

Terapia fotodinâmica antimicrobiana mediada por curcuminóides em um modelo murino de candidíase oral

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for ...

Estabelecendo um modelo de camundongo para PJI relacionada ao biofilme de Candida albicans

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

Autor em destaque: Avançando na pesquisa sobre infecções articulares protéticas associadas ao biofilme de Candida albicans 

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.

Modelo de Infecção por Candida albicans em Articulação Periprotética em Camundongos

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

Th17 Inflamação Modelo de orofaringe Candidíase em imunodeficientes Mice

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Isolamento e Th17 Diferenciação de Linfócitos T CD4 Naïve

A utilização de orofaringe Intratraqueal Administração PAMP e Lavagem Broncoalveolar para avaliar a resposta imune do hospedeiro em camundongos

Modelagem da mucosa candidíase em larval Zebrafish por swimbladder Injection

O modelo de infecção Waxworm Galleria mellonella para candidíase disseminada

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

Terapia fotodinâmica antimicrobiana mediada por curcuminóides em um modelo murino de candidíase oral

Terapia fotodinâmica antimicrobiana mediada por curcuminóides em um modelo murino de candidíase oral

Terapia fotodinâmica antimicrobiana mediada por curcuminóides em um modelo murino de candidíase oral

Modelo de Infecção por Candida albicans em Articulação Periprotética em Camundongos