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: Gli esperimenti con i topi sono stati eseguiti in conformità al comitato benessere degli animali istituzionale (IACUC) le linee guida. 1. Ricostituzione della Rag-1 - / - I topi con cellule I n Vitro Colta Th17 (Tre giorni prima di infezione) <ol><li> Per stabilire le cellule Th17, cultura CD4 + CD44low CD62Lhigh CD25- cellule T naive (3 x 104) in U-bottom 96 pozzetti da soli, o co-coltura con 3 x 104 CD4 + CD25 + Foxp3 + Tregs in presenza di solubile α-CD3 (1 mg / ml), α-CD28 (2 mg / ml) anticorpi e cellule presentanti l&#39;antigene, in condizioni Th17 (polarizzati utilizzando IL-6 (25 ng / ml), β TGF- (2 ng / ml) , α-IFN-γ (2 mg / ml) e α -IL-4 (2 mg / ml)) per tre o quattro giorni. NOTA: le cellule T reg ingenuo e sono stati ordinati con la fluorescenza delle cellule attivate (FACS) unad procedure di isolamento delle cellule magnetiche e coltivate come descritto in precedenza 19-22. </li><li> Il giorno 4, dopo risospendere le cellule Th17 con la pipetta, raccogliendoli da culture, e li centrifugare in condizioni sterili. <ol><li> Prendete una piccola aliquota di queste cellule per esaminare la loro vitalità e per fenotipizzazione. Li Risospendere in 500 ml di RPMI media, aggiungere ioduro di propidio (200 ng / ml) e valutare immediatamente la loro redditività mediante citometria a flusso. Eseguire fenotipizzazione mediante citometria di flusso valutazione della produzione di IL-17A dopo restimolazione (vedere paragrafo 6.3). </li></ol></li><li> Centrifugare e lavare la parte più consistente delle cellule in PBS sterile a 480 xg per 6 minuti a 4 ° C in tutte le fasi se non diversamente specificato. </li><li> Utilizzare queste cellule per il trasferimento adottivo in 6-8 settimane di età CD45.2 Rag1 - / - topi immunodeficienti. <ol><li> Risospendere le cellule a densità cellulare 1 x 10 7 cellule / ml usando freddo sterile PBS. </li><li> Iniettare 100 ml di cellule di intrapiniezione eritoneal, con 25 aghi G su 1 ml siringhe tubercolina, tale che un mouse riceve 1 x 10 6 cellule. Alcuni topi riceveranno cellule Th17 o PBS solo, e altri topi riceveranno cellule Th17 che erano co-coltura con cellule Treg. NOTA: Eseguire tutte le operazioni in modo asettico. </li></ol></li></ol> 2. Crescere C. albicans per infezione (un giorno prima infezione) <ol><li> Prima di inoculazione, disperdere cinque colonie della CAF2-1 C. albicans ceppo di laboratorio in 100 ml di sospensione sterile PBS, e aggiungere la sospensione al brodo sterile. </li><li> Seminare 5 colonie di C. albicans in 50 ml della base di azoto di lievito (YNB senza amminoacidi) / Peptone / Destrosio brodo e incubare in un incubatore shaker a 30 ° C per 15-18 ore a 130 rpm. </li><li> Monitorare il brodo per nuvolosità, dato che è un&#39;indicazione per la crescita del fungo. </li></ol> 3. CandidaRaccolta e procedura di conteggio (Nel giorno di infezione) <ol><li> Raccogliere 10 ml di brodo di Candida in provette da 15 ml. Per volumi maggiori, raccogliere Candida in 50 ml provette. </li><li> Centrifugare le blastospores a 1900 xg per 5 minuti a temperatura ambiente in tutte le fasi, se non diversamente specificato. Agglomerare le blastospores mediante centrifugazione, seguita dalla rimozione del supernatante. </li><li> Se vi è più di un tubo, in comune i blastospores Candida da più tubi, aggiungendo PBS sterile al pellet e risospendere in 10 ml di PBS per il conteggio. </li><li> Prendere 20 ml di blastospores in provette da 1,5 ml microcentrifuga, aggiungere 20 ml di paraformaldeide 2X e incubare a temperatura ambiente per 15-20 minuti. Contare le blastospores fissi utilizzando il emocitometro sotto il microscopio. NOTA: Paraformaldeide è cancerogeno. Aprire la soluzione diluita solo nella cappa. </li><li> Nel frattempo, ripetere lavare i blastospores almeno due volte, per centrifugazione them in PBS, e pellet le blastospores. Tenere il pellet in 15 ml provette a RT, nella cappa ABSL1, pronto per l&#39;infezione. </li><li> Lascia un po &#39;sterile PBS a temperatura ambiente per risospendere le blastospores di infezione. </li><li> Dopo conteggio, aggiungere PBS sterile al pellet Blastospora per regolare il numero di cellule di lievito Blastospora a 2 x10 8 del lievito cellule / ml. Questa è la sospensione Blastospora che verrà utilizzato per infettare i topi. NOTA: Eseguire tutte le operazioni in modo asettico. </li></ol> 4. Topi Infezione (3-5 giorni dopo il trasferimento Cell) NOTA: La procedura di infezione di base viene eseguita come descritto in precedenza 19,23-25. <ol><li> Pesare il Rag-1 - / - topi che hanno ricevuto PBS o le cellule tre giorni prima. </li><li> Calcolare la dose di agenti anestetici che utilizzano il loro peso corporeo. Anestetizzare topi somministrando ketamina / xilazina miscela (16,1 mg / ml e 1,6 mg / ml), con 25 aghi G su 1 ml siringhe tubercolina, da intriniezione aperitoneal. <ol><li> Somministrare 50 ml per 10 g di peso corporeo. (Cioè, 0,8 mg di ketamina e 0,08 mg di xilazina / 10 g di peso corporeo o 80 mg di ketamina e 8 mg di xilazina per kg di peso corporeo, rispettivamente). Osservare per la risposta pizzico punta ogni 15 minuti dopo l&#39;anestesia. NOTA: Questo dosaggio indurrà 60-90 min di anestesia, che è sufficiente per la procedura di infezione. </li><li> Applicare il lubrificante pomata oftalmica negli occhi dei topi per evitare le cornee si secchi. Come il lubrificante occhio potrebbe seccarsi, ripetere la lubrificazione oftalmica ogni 45 min. </li></ol></li><li> Iniettare 1 ml di soluzione fisiologica (NaCl 0,9%) per via sottocutanea sul dorso adiacente alla zampa anteriore, per aiutare reidratazione dei topi durante l&#39;anestesia. </li><li> Ottenere un nuovo, gabbia pulita. Seguire la procedura di infezione un mouse alla volta, posizionando ciascun topo nella nuova gabbia volta infettati. Eseguire il / infezione farsa PBS prima, e poi passare alle gro infezione da Candidaups. </li><li> Prendete il mouse anestetizzato e aprire la bocca per rivelare la base della lingua. <ol><li> Inserire una sfera di diametro 3 millimetri di cotone saturato con 50 ml di PBS o Blastospora sospensione, sublinguale nella cavità orale per 90 min. Capovolgere il mouse ogni 15 minuti parte anteriore e posteriore per evitare congestione polmonare, assicurando che i batuffoli di cotone non si muovono. </li></ol></li><li> Impostare un timer per ogni topo per garantire 90 min di Sham o Candida inoculazione nella cavità orale. </li><li> Tenerli in una gabbia sotto la lampada di calore (4 metri di distanza), e ogni mouse su cuscinetti di gel di calore. </li><li> Assicurarsi che la lingua è ritirata all&#39;interno e lontano dai denti, per evitare i denti lacerano lingua. </li><li> Alla fine di 90 min, rimuovere il batuffolo di cotone dalla bocca del mouse. Guarda per qualsiasi mouse che può recuperare da anestesia prima di 75 minuti. In tal caso, le anestetizzare nuovo con ketamina (40 mg / kg) soltanto. </li></ol> 5. Posta immissioe Monitoraggio (durante la 90 min Anesthesia) <ol><li> Usare tamponi di gel di calore durante la 90 min anestesia. <ol><li> Per mantenere la temperatura corporea e per prevenire il soffocamento, non permettere topi per recuperare il normale assestamento del mouse corn-cob. </li></ol></li><li> Dopo il recupero dall&#39;anestesia, amministrare un ulteriore 1 ml di sterile 0,9% NaCl per via sottocutanea in due posizioni sul retro. </li><li> Tieniti cinque PBS (sham) topo inoculato per gabbia. Solo 1-2 Casa topi infettati per gabbia. </li><li> Compilare la scheda di procedura con le iniziali ogni giorno dopo la procedura di annotare i cambiamenti nel peso corporeo, governare le abitudini e la salute generale. </li></ol> 6. Valutare l&#39;infiammazione 6.1) La perdita di peso <ol><li> Pesare i topi ogni giorno durante l&#39;infezione, a partire dal giorno precedente procedura di infezione. NOTA: in genere, i topi immunodeficienti iniettati con le cellule Th17 e senza regs T soccombono 20-30% Di perdita di peso a causa di maggiore onere fungina 19 e aggravata infiammazione. </li></ol> 6.2) Istologia punteggio della lingua <ol><li> Sacrifica i topi da CO 2 asfissia seguita da dislocazione cervicale. </li><li> Aprire le ganasce e sezionare fuori la lingua con le forbici e pinze. Utilizzare pinze smussato per tenere la lingua, e utilizzando le forbici protendono verso il retro della bocca di incidere all&#39;estremità posteriore della lingua. </li><li> Per ematossilina immunocitochimica e eosina (H &amp; E) colorazione dei tessuti lingua, lavare i tessuti lingua con ghiaccio freddo PBS. Aggiungere 5 ml di formalina al 10% per 2-3 lingue e fissarli O / N in provette da 15 ml. <ol><li> Il giorno successivo, togliere formalina e immergere i tessuti in 5 ml di etanolo al 70% per evitare iper-fissazione. Send them out al servizio commerciale per continuare con paraffina, sezionamento e colorazione delle sezioni di paraffina. NOTA: istologia servizio commerciale viene utilizzato per eseguire questa procedura. </li&gt; </ol></li><li> Una volta che le diapositive sono tornati dal servizio istologia commerciale, di grado l&#39;infiammazione osservando le sezioni di tessuto al microscopio ottico. <ol><li> Punteggio da 0 a 5, con 0 essendo senza infiammazione, e 5 è l&#39;infiammazione più grave. Nota l&#39;infiammazione utilizzando Tabella 1. </li></ol></li></ol> 6.3) la produzione di citochine da parte delle cellule Th17 NOTA: L&#39;eccessiva produzione di TNF-α è una delle letture per eccessiva immunopatologia. Quando i topi sono adoptively trasferiti con cellule Th17 solo, provoca immunopatologia associato eccessiva produzione di TNF-α nelle cellule Th17. <ol><li> Raccogliere una sospensione singola cellula da milza, linfonodi cervicali, linfonodi ascellari, e la lingua, usando i metodi descritti in precedenza 19,26. </li><li> Restimolare le cellule con acetato di forbolo miristato (PMA) (50 ng / ml) e Ionomicina (500 ng / ml) per 4 ore con Brefeldina A (10 mg / ml) aggiunto nella last 2 hr. Lavare le cellule con PBS e fissare utilizzando un kit di fissaggio / permeabilizzazione commerciale secondo le istruzioni del produttore. </li><li> Eseguire intra citochine cellulari colorazione utilizzando il fluorocromo coniugato anti-TNF-α, anti-IL-17A e anticorpi ROR-γt come descritto in precedenza 19. </li><li> Brevemente, risospendere le cellule nel buffer permeabilizzazione 1X con il cocktail anti-TNF-α, anti-IL-17A e anticorpi ROR-γt, ciascun anticorpo a 2 - 3 g / ml concentrazione finale. </li><li> Incubare le cellule per 1 ora a RT. </li><li> Dopo l&#39;incubazione, lavare le cellule con tampone permeabilizzazione 1X. </li><li> Risospendere il pellet cellulare in 500 ml di PBS con 0.5% albumina sierica bovina per citometria a flusso. </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.

Questo modello è basato su indurre orale C. infezione albicans dipendente infiammazione Th17. A causa della mancanza di T reg, l&#39;infiammazione delle cellule Th17 indotta è sfrenato e porta a immunopatologia mal risolto. In vitro derivato cellule CD4 naive polarizzate come le cellule Th17 sono stati utilizzati per il trasferimento adottivo. 40 - 50% della coltura CD4 + cellule mostrano rilevabile espressione di IL-17A intorno al giorno 3 (cellule Th17), e quindi sono stati util...

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.

In questo modello, sia i topi -infected Candida e topi non infetti in Rag-1 - / - sfondo immunodeficienti sono state adoptively trasferiti con le cellule Th17 3-5 giorni prima l&#39;infezione. Un totale di 10-12 topi sono stati utilizzati per gli esperimenti, con 2 topi in ciascun sham infettati gruppi, e 4-5 in ciascuno dei gruppi infetti Candida. Cellule naive sono stati ottenuti da congenici Thy1.1 o CD45.1 topi, in modo che le cellule Th17 iniettate sono stati monitorati in vivo...

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 Infiammazione Modello di orofaringea candidosi in topi immunodeficienti

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 Views.  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.

Protocolli per l&#39;inoculazione vaginale e Prelievo dei campioni nel modello murino sperimentale di Candida Vaginite

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

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Immunologia e infezioni

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 differenziazione di Th17 Naïve linfociti 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.

L&#39;utilizzo di orofaringea intratracheale PAMP Amministrazione e lavaggio broncoalveolare per valutare la risposta immunitaria nei topi Host

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.

Modellazione mucosa Candidosi in Zebrafish larvale da vescica natatoria 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.

Il modello di infezione del Waxworm di Galleria mellonella per candidosi disseminata

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 efficace con curcuminoidi per la candidosi orale nei topi

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.

Autore in primo piano: Esplorazione della terapia fotodinamica con la curcumina in un modello murino per la candidosi orale

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 fotodinamica antimicrobica mediata da curcuminoidi su un modello murino di candidosi orale

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

Stabilire un modello murino per la PJI correlata al biofilm di 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.

Autore in primo piano: Avanzamento della ricerca sulle infezioni articolari protesiche associate al biofilm della 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.

Un modello di infezione da Candida albicans articolare periprotesica nel topo

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

Th17 Infiammazione Modello di orofaringea candidosi in topi immunodeficienti

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Protocolli per l'inoculazione vaginale e Prelievo dei campioni nel modello murino sperimentale di Candida Vaginite

Isolamento e differenziazione di Th17 Naïve linfociti T CD4

L'utilizzo di orofaringea intratracheale PAMP Amministrazione e lavaggio broncoalveolare per valutare la risposta immunitaria nei topi Host

Modellazione mucosa Candidosi in Zebrafish larvale da vescica natatoria Injection

Il modello di infezione del Waxworm di Galleria mellonella per candidosi disseminata

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

Terapia fotodinamica antimicrobica mediata da curcuminoidi su un modello murino di candidosi orale

Terapia fotodinamica antimicrobica mediata da curcuminoidi su un modello murino di candidosi orale

Terapia fotodinamica antimicrobica mediata da curcuminoidi su un modello murino di candidosi orale

Un modello di infezione da Candida albicans articolare periprotesica nel topo