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.

NOTE: The experiments using mice were performed in accordance to the institutional animal welfare committee (IACUC) guidelines.
1. Reconstituting the Rag-1-/- Mice with In Vitro Cultured Th17 cells (Three days prior to Infection)
<ol>
	<li>For establishing Th17 cells, culture CD4+&#160;CD44low&#160;CD62Lhigh&#160;CD25-&#160;na&#239;ve T cells (3 x 104) in U-bottom 96 well plates alone, or co-culture them along with 3 x 104&#160;CD4+CD25+Foxp3+Tregs&#160;in the presence of...

<ol>
	<li>Allam, J. P., 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=IL-23-producing+CD68(+)+macrophage-like+cells+predominate+within+an+IL-17-polarized+infiltrate+in+chronic+periodontitis+lesions.">IL-23-producing CD68(+) macrophage-like cells predominate within an IL-17-polarized infiltrate in chronic periodontitis lesions.</a> Journal of Clinical Periodontology. 38 (10), 879-886 (2011).</li><li>Loesche, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontal+disease:+link+to+cardiovascular+disease.">Periodontal disease: link to cardiovascular disease.</a> Compendium Of Continuing Education In Dentistry. 21 (6), 463-466 (2000).</li><li>Sfyroeras, G. S., Roussas, N., Saleptsis, V. G., Argyriou, C., Giannoukas, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+periodontal+disease+and+stroke.">Association between periodontal disease and stroke.</a> Journal Of Vascular Surgery. 55 (4), 1178-1184 (2012).</li><li>Huppler, A. R., Bishu, S., Gaffen, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucocutaneous+candidiasis:+the+IL-17+pathway+and+implications+for+targeted+immunotherapy.">Mucocutaneous candidiasis: the IL-17 pathway and implications for targeted immunotherapy.</a> Arthritis Research &amp; Therapy. 14 (4), 217 (2012).</li><li>Sitheeque, M. A., Samaranayake, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+hyperplastic+candidosis/candidiasis+(candidal+leukoplakia).+Critical+Reviews+In.">Chronic hyperplastic candidosis/candidiasis (candidal leukoplakia). Critical Reviews In.</a> Oral Biology And Medicine : An Official Publication Of The American Association Of Oral Biologists. 14 (4), 253-267 (2003).</li><li>Canabarro, 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=Association+of+subgingival+colonization+of+Candida+albicans+and+other+yeasts+with+severity+of+chronic+periodontitis.">Association of subgingival colonization of Candida albicans and other yeasts with severity of chronic periodontitis.</a> Journal Of Periodontal Research. 48 (4), 428-432 (2013).</li><li>Urzua, B., 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=Yeast+diversity+in+the+oral+microbiota+of+subjects+with+periodontitis:+Candida+albicans+and+Candida+dubliniensis+colonize+the+periodontal+pockets.">Yeast diversity in the oral microbiota of subjects with periodontitis: Candida albicans and Candida dubliniensis colonize the periodontal pockets.</a> Medical Mycology. 46 (8), 783-793 (2008).</li><li>Ergun, 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=Oral+status+and+Candida+colonization+in+patients+with+Sjogren's+Syndrome.">Oral status and Candida colonization in patients with Sjogren's Syndrome.</a> Medicina Oral, Patologia Oral Y Cirugia Bucal. 15 (2), e310-e315 (2010).</li><li>Hernandez, Y. L., Daniels, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+candidiasis+in+Sjogren's+syndrome:+prevalence,+clinical+correlations,+and+treatment.">Oral candidiasis in Sjogren's syndrome: prevalence, clinical correlations, and treatment.</a> Oral Surgery, Oral Medicine, And Oral Pathology. 68 (3), 324-329 (1989).</li><li>Gall, F., 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+spp.+in+oral+cancer+and+oral+precancerous+lesions.">Candida spp. in oral cancer and oral precancerous lesions.</a> The New Microbiologica. 36 (3), 283-288 (2013).</li><li>Grady, J. F., Reade, P. C. <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+as+a+promoter+of+oral+mucosal+neoplasia.">Candida albicans as a promoter of oral mucosal neoplasia.</a> Carcinogenesis. 13 (5), 783-786 (1992).</li><li>Dwivedi, P. P., Mallya, S., Dongari-Bagtzoglou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+immunocompetent+murine+model+for+Candida+albicans-promoted+oral+epithelial+dysplasia.">A novel immunocompetent murine model for Candida albicans-promoted oral epithelial dysplasia.</a> Medical Mycology. 47 (2), 157-167 (2009).</li><li>Cheng, S. C., Joosten, L. A., Kullberg, B. J., Netea, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+Candida+albicans+and+the+mammalian+innate+host+defense.">Interplay between Candida albicans and the mammalian innate host defense.</a> Infection and Immunity. 80 (4), 1304-1313 (2012).</li><li>Marodi, L., Johnston, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+Candida+species+disease+in+infants+and+children:+occurrence,+risk+factors,+management,+and+innate+host+defense+mechanisms.">Invasive Candida species disease in infants and children: occurrence, risk factors, management, and innate host defense mechanisms.</a> Current Opinion In Pediatrics. 19 (6), 693-697 (2007).</li><li>Kumamoto, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+gastrointestinal+Candida+colonization.+Current+Opinion+In.">Inflammation and gastrointestinal Candida colonization. Current Opinion In.</a> Microbiology. 14 (4), 386-391 (2011).</li><li>Kraneveld, E. 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=The+relation+between+oral+Candida+load+and+bacterial+microbiome+profiles+in+Dutch+older+adults.">The relation between oral Candida load and bacterial microbiome profiles in Dutch older adults.</a> PloS One. 7 (8), e42770 (2012).</li><li>Hernandez-Santos, N., 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=Th17+cells+confer+long-term+adaptive+immunity+to+oral+mucosal+Candida+albicans+infections.">Th17 cells confer long-term adaptive immunity to oral mucosal Candida albicans infections.</a> Mucosal Immunology. 6 (5), 900-910 (2013).</li><li>Hasina, R., 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=ABT-510+is+an+effective+chemopreventive+agent+in+the+mouse+4-nitroquinoline+1-oxide+model+of+oral+carcinogenesis.">ABT-510 is an effective chemopreventive agent in the mouse 4-nitroquinoline 1-oxide model of oral carcinogenesis.</a> Cancer Prevention Research. 2 (4), 385-393 (2009).</li><li>Pandiyan, P., 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=CD4(+)CD25(+)Foxp3(+)+regulatory+T+cells+promote+Th17+cells+in+vitro+and+enhance+host+resistance+in+mouse+Candida+albicans+Th17+cell+infection+model.">CD4(+)CD25(+)Foxp3(+) regulatory T cells promote Th17 cells in vitro and enhance host resistance in mouse Candida albicans Th17 cell infection model.</a> Immunity. 34 (3), 422-434 (2011).</li><li>Pandiyan, P., 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+role+of+IL-15+in+activating+STAT5+and+fine-tuning+IL-17A+production+in+CD4+T+lymphocytes.">The role of IL-15 in activating STAT5 and fine-tuning IL-17A production in CD4 T lymphocytes.</a> J Immunol. 189 (9), 4237-4246 (2012).</li><li>Pandiyan, P., Lenardo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+CD4+CD25+Foxp3++regulatory+T+cell+survival.">The control of CD4+CD25+Foxp3+ regulatory T cell survival.</a> Biology Direct. 3 (6), (2008).</li><li>Pandiyan, P., Zheng, L., Ishihara, S., Reed, J., Lenardo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4(+)CD25(+)Foxp3(+)+regulatory+T+cells+induce+cytokine+deprivation-mediated+apoptosis+of+effector+CD4(+)+T+cells.">CD4(+)CD25(+)Foxp3(+) regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4(+) T cells.</a> Nat Immunol. 8 (12), 1353-1362 (2007).</li><li>Conti, H. R., Huppler, A. R., Whibley, N., Gaffen, S. L., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+candidiasis.">Animal models for candidiasis.</a> Current Protocols In Immunology. 105, 11-19 (2014).</li><li>Conti, H. R., 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=Th17+cells+and+IL-17+receptor+signaling+are+essential+for+mucosal+host+defense+against+oral+candidiasis.">Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis.</a> J Exp Med. 206 (2), 299-311 (2009).</li><li>Kamai, Y., 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=New+model+of+oropharyngeal+candidiasis+in+mice.">New model of oropharyngeal candidiasis in mice.</a> Antimicrobial Agents And Chemotherapy. 45 (11), 3195-3197 (2001).</li><li>Pandiyan, P., Bhaskaran, N., Zhang, Y., Weinberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+T+cells+from+mouse+oral+tissues.">Isolation of T cells from mouse oral tissues.</a> Biological Procedures Online. 16 (1), 4 (2014).</li><li>Gladiator, A., Wangler, N., Trautwein-Weidner, K., LeibundGut-Landmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+IL-17-secreting+innate+lymphoid+cells+are+essential+for+host+defense+against+fungal+infection.">Cutting edge: IL-17-secreting innate lymphoid cells are essential for host defense against fungal infection.</a> J Immunol. 190 (2), 521-525 (2013).</li><li>Leppkes, M., Roulis, M., Neurath, M. 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>

This model is based on inducing oral C. albicans infection dependent Th17 inflammation. Because of the absence of Tregs, the Th17 cell induced inflammation is unrestrained and leads to poorly resolved immunopathology. In vitro derived na&#239;ve CD4 cells polarized as Th17 cells were used for the adoptive transfer. 40 - 50% of the cultured CD4+ cells show detectable IL-17A expression around day 3 (Th17 cells), and therefore were used for the injection in mice. The major advantage of the model...

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 border="0" cellpadding="0" cellspacing="0" width="1289">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">CAF-2</td>
			<td style="width:411px;">University of Pittsburgh (Sarah Gaffen)</td>
			<td style="width:119px;">-</td>
			<td style="width:380px;">Candida culture</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">U-bottom 96 well plates&#160;</td>
			<td style="width:411px;">Fisher</td>
			<td style="width:119px;">055588</td>
			<td>Used for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-CD3&#160;</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">16-0031-85</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-CD28&#160;</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">16-0281-85</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">m-IL-6</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">RC232</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">h-TGF-b&#160;</td>
			<td style="width:411px;">R&#38;D</td>
			<td style="width:119px;">240-B</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-IFN-g&#160;</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">16-7311-85</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-IL-4&#160;</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">16-7041-85</td>
			<td>Polarization of cells to Th17 conditions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">&#945;-IL17A ef660</td>
			<td style="width:411px;">eBiosciences</td>
			<td>50-7177-82</td>
			<td>Used for cell culture - 1:50</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">&#945;-TNFa-PE-Cy7</td>
			<td>eBiosciences</td>
			<td>25-7423-41</td>
			<td>Used for cell culture - 1:100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">&#945;-RORgt PE&#160;</td>
			<td>eBiosciences</td>
			<td>12-6981-82</td>
			<td>Used for cell culture - 1:50</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">YNB w/o amino acids)/Peptone/Dextrose broth medium&#160;</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">S507.SIZE</td>
			<td>Mediium for candida growth</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Shaker incubator&#160;</td>
			<td style="width:411px;">New Brunswick Scientific</td>
			<td style="width:119px;">Innova 4300</td>
			<td>Incubation growth for candida</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">15 ml tubes</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">BT888-SY</td>
			<td>Used for cell culture and candida growth</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">50 ml tubes</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">CT 788-YS</td>
			<td>Used for cell culture and candida growth</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table top Centrifuge</td>
			<td style="width:411px;">VWR International LLC</td>
			<td style="width:119px;">82017-654</td>
			<td>For pelleting candida -900 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Allegra Centrifuge</td>
			<td style="width:411px;">Beckman Coulter</td>
			<td style="width:119px;">392302</td>
			<td>Cell culture - 480 g</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1.5 ml eppendorf tubes</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">BT620-NS</td>
			<td>Preparing final concentration of candida</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">2x paraformaldehyde</td>
			<td style="width:411px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td>Fixing candida for count</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemocytometer</td>
			<td style="width:411px;">VWR International LLC</td>
			<td style="width:119px;">15170-172</td>
			<td>Counting cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS - Phosphate Buffered Saline</td>
			<td style="width:411px;">Bio Basic</td>
			<td style="width:119px;">PD8117</td>
			<td>Preparation of buffers</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Ketamine/xylazine&#160;</td>
			<td style="width:411px;">Case Western Reserve University - Animal Resource Center</td>
			<td style="width:119px;">-</td>
			<td>Obtained from ARC approved protocol for anesthesia</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmic lubricant ointment&#160;</td>
			<td style="width:411px;">Allergan</td>
			<td style="width:119px;">-</td>
			<td>Eye ointment for animals to prevent dryness</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Saline (0.9% NaCl)</td>
			<td style="width:411px;">G Biosciences</td>
			<td style="width:119px;">786-561</td>
			<td>Administerd to animals to prevent dehydration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-mm-diameter cotton wool ball&#160;</td>
			<td style="width:411px;">VWR International LLC</td>
			<td style="width:119px;">BP7603</td>
			<td>3 mm balls for candida infection</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Heat gel pads</td>
			<td style="width:411px;">Case Western Reserve University - Animal Resource Center</td>
			<td style="width:119px;">-</td>
			<td>for maintaining the body temperaturre and fast recovery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin and Eosin staining</td>
			<td style="width:411px;">Histoserv - MD</td>
			<td style="width:119px;">-</td>
			<td>Tissue histology</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10% formalin&#160;</td>
			<td style="width:411px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">JC1111/MC</td>
			<td>Tissue histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">70% ethanol&#160;</td>
			<td style="width:411px;">VWR International LLC</td>
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			<td>Sterilization purpose</td>
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		<tr height="20">
			<td height="20" style="height:20px;">PMA - Phorbol 12-myristate 13-acetate</td>
			<td style="width:411px;">Sigma-Aldrich</td>
			<td style="width:119px;">P1585-1MG</td>
			<td>Restimulation of cells</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Ionomycin</td>
			<td style="width:411px;">Life Technologies</td>
			<td style="width:119px;">124222 1mg</td>
			<td>Restimulation of cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fixation permeabilization kit</td>
			<td style="width:411px;">eBiosciences</td>
			<td style="width:119px;">E16913-106</td>
			<td>Fixing cells for Flow cytometry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">Tuberculin syringes</td>
			<td>BD Biosciences</td>
			<td align="right">309659</td>
			<td>Mice injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G x 3/8 needles</td>
			<td>BD Biosciences</td>
			<td align="right">309626</td>
			<td>Mice injection</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Rag1 -/- mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no: 002216</td>
			<td>Recipient mice</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD45.1 congenic mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no:002014&#160;</td>
			<td>Donor Th17 cells</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:380px;">CB17-SCID mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no: 001303</td>
			<td>Recipient mice</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:380px;">Balb/c mice</td>
			<td style="width:411px;">Jackson laboratories</td>
			<td style="width:119px;">Stock no: 000651</td>
			<td>Donor Th17 cells</td>
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	</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 this model, both the Candida-infected mice and uninfected mice in Rag-1-/- immunodeficient background were adoptively transferred with Th17 cells 3-5 days prior to the infection. A total of 10-12 mice were used for the experiments, with 2 mice in each Sham infected groups, and 4-5 in each of the Candida infected groups. Naive cells were derived from congenic Thy1.1 or CD45.1 mice, so that injected Th17 cells were tracked in vivo using Thy1.1 or CD45.1 staining respectively (<stron...

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.

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 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. 