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.

注:使用小鼠实验是按照该机构的动物福利委员会（IACUC）的指导方针进行。 1.重构的RAG-1 - / -小鼠I N 体外培养Th17细胞（前三天感染） <ol><li>在U型底96孔板单独或联合培养它们沿着用3×104 CD4 + CD25 +的Foxp3 + Treg细胞在可溶性存在建立Th17细胞，培养的CD4 + CD44low CD62Lhigh CD25 - 幼稚T细胞（3×104） α-CD3（1微克/毫升），α-CD28（2微克/毫升）的抗体和抗原呈递细胞，Th17的条件下（使用的IL-6（25毫微克/毫升），TGF-β极化（2纳克/毫升） α-IFN-γ（2微克/毫升）和α-IL-4（2微克/毫升））为三到四天。 注:使用荧光激活细胞分选的幼稚细胞和T 暂存器被排序（FACS）的ð磁性细胞分离程序和培养如前面描述19-22。 </li><li>在第4天，再悬浮用吸管Th17细胞后，从培养物收集它们，并且离心它们在无菌条件下。 <ol><li>取一小等分这些细胞，研究其可行性和表型。重悬他们在500μlRPMI培养基中，加入碘化丙啶（200毫微克/毫升），并通过流式细胞立即评估其可行性术。通过流式细胞仪再刺激评估IL-17A生产进行分型（见6.3）。 </li></ol></li><li>离心机，在4℃下在所有的步骤洗涤细胞的主要散装在无菌PBS以480×g离心6分钟，除非另有规定。 </li><li>使用这些细胞过继转移到6-8周龄CD45.2 RAG1 - / - 免疫缺陷小鼠。 <ol><li>使用冷无菌PBS悬浮细胞以1×10 7个细胞/ ml的细胞密度。 </li><li>注入由intrap 100微升的细胞eritoneal注射，使用上1毫升结核菌素注射器地下25针，使得一只小鼠接收1×10 6个细胞。一些小鼠只接受PBS或Th17细胞，和其它的小鼠将收到的Th17细胞共培养与Treg细胞。 注:执行所有步骤无菌。 </li></ol></li></ol> 2.成长C.白色假丝酵母感染（前一天感染） <ol><li>接种前，驱散CAF2-1 C.五殖民地白色念珠菌实验室菌株在100微升无菌PBS悬浮，并将悬浮液添加到无菌肉汤。 </li><li>接种C. 5殖民地白色念珠菌在50毫升的酵母氮碱（YNB无氨基酸）/蛋白胨/右旋糖肉汤培养基，孵育在摇床培养箱中在30℃下15-18小时，在130转。 </li><li>监视肉汤为混浊，因为这是一个指示用于对真菌的生长。 </li></ol> 3. 念珠菌收获和计数过程（感染的日） <ol><li>收集10毫升念珠菌肉汤的15ml管中。对于较大的卷，收集念珠菌在50毫升管。 </li><li>离心芽生孢子在1900×g离心5分钟，在RT中的所有步骤，除非另有规定。沉淀芽生孢子通过离心，然后除去上清液。 </li><li>如果有一个以上的管，从多个管凝聚念珠菌芽生孢子，加入无菌PBS的颗粒和重悬它们在10ml PBS中用于计数。 </li><li>采取20微升的芽生孢子在1.5ml微量离心管中，加入20微升2倍多聚甲醛和在室温下孵育15-20分钟。计数采用显微镜下血球固定芽生孢子。 注意:多聚甲醛是致癌物质。打开仅在通风橱的原液。 </li><li>在此期间，重复洗涤该芽生孢子的至少两倍，通过离心吨下摆的PBS，造粒的芽生孢子。保持在15ml管中的颗粒在室温，在ABSL1罩，准备感染。 </li><li>留下一些无菌PBS在室温下重悬芽生孢子感染。 </li><li>计数后，添加无菌PBS到芽生粒料以调节酵母细胞芽生号码的酵母细胞/ ml 2×10 8。这是芽生孢子悬浮液，将被用于感染的小鼠。 注:执行所有步骤无菌。 </li></ol> 4.感染小鼠（细胞转移后3-5天） 注:基本感染过程如前面19,23-25 ​​所述进行。 <ol><li>权衡RAG-1 - / - 小鼠接受PBS或细胞三天前。 </li><li>计算的使用他们的体重麻醉剂的剂量。麻醉小鼠通过施用氯胺酮/甲苯​​噻嗪混合物（16.1毫克/毫升和1.6毫克/毫升），利用地下25针上1毫升结核菌素注射器，由INTRaperitoneal注入。 <ol><li>管理每10克的机身重量50微升。 （ 即，分别为0.8毫克氯胺酮和0.08毫克的甲苯噻嗪/ 10克体重或80毫克氯胺酮和每公斤体重甲苯噻嗪的8毫克）。观察的脚趾捏响应麻醉后，每15分钟。 注意:此剂量将诱发60-90分钟麻醉的，这是不够的感染过程。 </li><li>适用的眼用润滑剂软膏在小鼠的眼中，以防止角膜干燥。由于眼睛的润滑剂可以干出，重复眼科润滑每隔45分钟。 </li></ol></li><li>注入1毫升盐水（0.9％NaCl）中皮下注射在背部邻接到前肢，帮助麻醉期间小鼠的补液。 </li><li>获得一个新的，干净的笼子。按照感染的程序一个鼠标的时间，将每只小鼠成一旦感染新的笼子。首先执行PBS /假感染，然后进行念珠菌感染GROups。 </li><li>拿起麻醉鼠标，打开口宽透露舌根。 <ol><li>放置用50μlPBS或芽生孢子悬浮液的饱和3毫米直径棉球，舌下在口腔中90分钟。翻转小鼠每隔15分钟正面和背面，以防止肺充血，确保棉球不移动。 </li></ol></li><li>设置一个定时器，每鼠，以确保在口腔中90分钟的深水或念珠菌接种。 </li><li>让他们下的热灯（4英尺远）一个笼子，每只小鼠对热凝胶垫。 </li><li>确保该舌片从牙齿内部和远离撤出，以避免齿划破舌头。 </li><li>在90分钟结束时，从鼠的口中除去棉花球。留意任何鼠标可能从麻醉中恢复早于75分钟。在这样的情况下，只用氯胺酮（40毫克/千克）再次麻醉它们。 </li></ol> 5.后ProcedurE监测（90分钟麻醉过程中） <ol><li>在90分钟的麻醉过程中使用的热凝胶垫。 <ol><li>为了保持体温，防止窒息，不要让老鼠恢复的常规玉米芯鼠标床上用品。 </li></ol></li><li>从麻醉中恢复后，施用额外的1毫升无菌的0.9％NaCl皮下的，背面的两个位置。 </li><li>跟上五PBS（假）每笼接种小鼠。房子只有1-2元笼感染小鼠。 </li><li>每天在手术后填写过程卡缩写记下的变化的体重，疏导习惯和总体健康状况。 </li></ol> 6.评估炎症 6.1）减肥<ol><li>每天在感染期间称重小鼠，开始在感染前过程的那一天。 注:通常情况下，注射Th17细胞和不要选丁字暂存器的免疫缺陷小鼠死于20-30％的重量损失是由于增加的真菌负担19和加剧炎症。 </li></ol> 6.2）组织学舌得分<ol><li>牺牲小鼠用CO 2窒息之后颈脱位。 </li><li>打开颌骨和解剖用剪刀和镊子的舌头。使用钝钳保持舌，以及使用该剪刀接触到口腔的后部，以切开舌的后端。 </li><li>对于免疫细胞化学苏木精和曙红舌组织（H＆E）染色，冲洗舌组织用冰冷的PBS。加入5毫升10％的福尔马林2-3舌头和解决这些问题O / N在15毫升管。 <ol><li>第二天，除去福尔马林浸泡组织在5毫升70％的乙醇，以防止超固定。送他们到商业服务，继续用石蜡包埋，切片和石蜡切片染色。 注:商业组织学服务用来执行这些步骤。 </LI&gt; </ol></li><li>一旦载玻片从商业组织学服务回，等级炎症通过观察在光学显微镜下的组织切片。 <ol><li>得分从0至5，其中0是无炎症，和5是最严重的炎症。评分使用表1中的炎症。 </li></ol></li></ol> 6.3）细胞因子产量Th17细胞注意:过量TNF-α生产的读数过度免疫病理之一。当小鼠过继与Th17细胞转移只，它导致了与过度的TNF-α产生中Th17细胞相关的免疫病理学。 <ol><li>收集来自脾脏，颈部淋巴结，腋窝淋巴结和舌单细胞悬浮液，使用先前描述的方法19,26。 </li><li>重新刺激细胞与佛波醇肉豆蔻乙酸酯（PMA）（50纳克/毫升）和离子霉素（500毫微克/毫升）4小时用布雷菲德菌素A（10微克/毫升），在升加入AST 2小时。用PBS洗涤细胞，并使用根据制造商的说明商用固定/透化试剂盒解决这些问题。 </li><li>使用荧光染料缀合的抗TNF-α，抗IL-17A和ROR-γT抗体如先前19描述的执行帧内蜂窝细胞因子染色。 </li><li>简言之，将悬浮在与抗TNF-α，抗IL-17A和ROR-γT抗体，每种抗体在2的鸡尾酒的1X透化缓冲液中的细胞 - 3微克/毫升的最终浓度。 </li><li>孵育细胞1小时，在室温。 </li><li>培养后，洗涤细胞用1X透化缓冲液。 </li><li>重悬在500μlPBS中的0.5％牛血清白蛋白进行流式细胞术分析细胞沉淀。 </li></ol>

<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>

The authors declare that they have no competing financial interests.

该模型是基于对诱导口服C.白色念珠菌感染相关的Th17细胞炎症。因为没有T 暂存器的，所述的Th17细胞诱导的炎症是无节制，并导致解决不良免疫病理学。 体外衍生的极化作为Th17细胞被用于过继转移幼稚的CD4细胞。 40 - 培养的CD4 +细胞的50％表现出约3天可检测的IL-17A的表达（Th17细胞），并因此被用于注射的小鼠。模型的主要优点是，Th17细胞引起，可有效地改善与调节性T注射感染特定免疫病理学。因此该模型可用于测试和T 章注射免疫调节策略作为阳性对照。炎症是容易评估的基础上的重量损失，病理组织学得分和促炎性细胞因子产生的感染小鼠的Th17细胞。 吨后，他收养Th17细胞的转移，他们迁移到二级淋巴器官和各种组织，包括舌，感染的原发部位。感染后，其他炎性细胞也招募到舌头清除感染。然而，如果没有调节性T介导的免疫调节，Th17细胞本身并没有解决感染，但引起高度免疫病理学。共转移了T 暂存器彻底解决了炎症。有趣的是，小鼠在没有T 暂存器加剧炎症，也显示增加的真菌负担。我们之前的报告显示，这是由于减少了IL-17A生产的小鼠，比起那些以T 暂存器 。我们目前的实验支持这样的想法，这也可能是由于增加的TNF-α是恶化的炎症，可能通过增加上皮细胞凋亡和增加的真菌负担28。考虑的IL-17A的在粘膜免疫保护作用我们认为，炎性病理学，我们观察到与增加的真菌负担是不是由于由Th17细胞或缺乏宿主抗性的产生的IL-17A。它是由于TNF-α和由注入Th17细胞产生的可能的其它促炎细胞因子引起的。 - / -小鼠缺乏Th17细胞仅显示最小的炎症和随后的感染的分辨率，只有那些接受Th17细胞屈服于严重的体重减轻（ 图1），这可以通过该碎布观测进行验证。一致的这一数据，它也已表明以前碎布- / -小鼠清除主OPC感染没有太多的免疫病理学27。在这些条件下，依赖于IL-17A产生先天免疫细胞代偿机制已显示以清除感染的碎布- / -小鼠17,29,30。综合来看，虽然IL-17A由Th17细胞和中性粒细胞招聘生产的演奏感染的最初阶段起保护作用，EXCessive生产TNF-α和其他细胞因子的Th17细胞和嗜中性粒细胞的原因的继续存在的加剧免疫病理学。除了由Th17细胞产生的TNF-α促炎性细胞因子的作用保持在免疫病理学的上下文中进行调查。 该模型的主要限制是问题是否是生理有关人类的OPC。虽然Th17细胞被证明是病理介质在几个口腔疾病，Th17细胞的慢性活化是否是致病OPC中仍进行调查。与最近的发现暗示C的作用在炎症和癌症白色念珠菌 ，Th17细胞免疫病理学在口腔中是调查31的一个活跃的领域。从而此处描述的模型可以用于更仔细研究Th17细胞的具体作用和它们与先天免疫细胞如何相互作用，从而在口腔免疫疾病和口腔癌详细的见解。在该技术的关键步骤是合适的招聘和扩张的过继转移Th17细胞。这是高度依赖于生存力和IL-17A的生产移交前的体外极化Th17细胞。最好是测试他们的生存能力和细胞因子的产生，流式细胞仪分析19。妥善地执行的Th17细胞注射和小鼠感染会导致感染相关的Th17细胞免疫病理。该模型可用于测试策略，以挫败Th17细胞免疫病理学和口腔炎症的其他感染的上下文中也是如此。此外，使用该模型，可以执行二次再感染和研究在慢性感染的上下文中加剧免疫病理学Th17细胞的作用。作为该模型涉及到免疫缺陷的小鼠，它可能是非常相关的口腔生态失调见于艾滋病和PID的患者，谁重演慢性念珠菌感染患。

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.

在这个模型中，无论是念珠菌 -感染的小鼠和未感染的小鼠中RAG-1 - / -背景的免疫缺陷过继与Th17细胞3-5天前，感染传送。总共10-12只小鼠用于实验，用2只小鼠中每一深水感染组，并且在每个所述念珠菌感染的群体的4-5。幼稚细胞来自同类Thy1.1的或CD45.1小鼠，使注入的Th17细胞被跟踪中使用Thy1.1的或CD45.1染色分别（ 图1A） 的体内 。联合转移的T 暂存器来源于CD45.2小鼠从CD45.1 Th17细胞区分开来。对于一些实验，CB-17 SCID Thy1.2受体小鼠中，使用从Thy1.1系Balb / C小鼠，并从同类Thy1.2小鼠获得Ť 暂存器获得的Th17供体细胞。只有念珠菌感染的小鼠，但不是未受感染的小鼠表现出的招聘和扩大重组CD45.1的+ </s向上&gt; Th17细胞在CLN，引流淋巴结。这一结果表明，真菌Th17细胞特异性应答是在舌引流淋巴结（ 图1A）启动的。结果表示来自一只小鼠的每个组中的数据。 仅在被共转移以T 暂存器的小鼠，在CLN检测FOXP3 + CD45.1阴性CD4 +细胞。这表明，所注入的Ť 暂存器还招募到感染部位（ 图1B）。结果表示来自一只小鼠的每个组中的数据。而感染RAG-1 - / -减肥和4天后感染的痊愈，可的松免疫抑制小鼠没有恢复，但进一步丢失的重量，如前面27所示。两者的小鼠的组接受Th17细胞在T 暂存器的存在或不存在初始体重减轻。但是，接收由体重减轻回收Ť 寄存器和小鼠解决了感染。 2米冰被深水感染组中的每个念珠菌感染组中使用，和4。有3只小鼠中可的松组。接收Th17细胞的小鼠，体重减轻逐步（ 图2）19。一些小鼠在该组中的不得不被安乐死。这些结果表明，所有的小鼠在该组中的权值的平均值。虽然中性粒细胞所需的感染的初始间隙，其连续存在和招聘的组织是未解决的炎症标志。因此中性粒细胞浸润进行了检查，在受感染的舌，通过确定在稍后的时间点，如天-7感染后嗜中性粒细胞标记物的Gr-1的表达，。该接收到的Th17细胞的小鼠只，有较高的炎性浸润（ 图3A），甚至包括天-4-的Gr-1 +嗜中性粒细胞在感染后，相对于注射以T 暂存器 （ 图3B）的小鼠。这些结果表明，在presencË了T 暂存器，小鼠解决炎症更有效地从感染中康复。 在感染后7天，脾（SPLN），CLN和舌组织中分离，单细胞悬液的流式细胞仪分析发了言。 CLN透露在接受仅Th17细胞的小鼠增加的CD45.1 Th17细胞（ 图1）和提高的TNF-α产生（ 图4A，4B）。另一方面，T 章收件人表现由Th17细胞（ 图1及图4A，4B）Th17细胞的频率和降低的TNF-α生成减少。在这些实验中，示出从CLN一个鼠标的，和舌组织数据采集并汇集来自2只小鼠。虽然在早期的时间点T中的IL-17A的产量为更高章收件人19，在两个组中，IL-17A的生产由Th17细胞是最小的（数据未示出）后面的时间点。碘酸雪夫氏染色舌显示增加真菌菌丝在于只收到Th17细胞小鼠，而接收Ť 暂存器小鼠以及清零天-5和天7真菌感染（ 图5）之后。此外，舌炎症组织病理学评分也显示降低的炎症分数接受Ť 暂存器小鼠相比，仅接受Th17细胞（ 图6）的小鼠。得分炎症是基于舌组织病理学参数，如，乳头的存在，表面上皮细胞层，可见酵母或菌丝，侵入菌丝指示非解决感染的完整性，基底层的增厚（指示正在进行组织修复），和浸润免疫细胞（嗜中性粒细胞和Th17细胞）的存在。这些结果表明，注射Th17细胞没有直接减少感染但增加所造成的初级C.真菌负担和免疫病理学白色念珠菌 infec化，而 ​​共转移了T 暂存器改善免疫病理学。 <img alt="图1" src="/files/ftp_upload/52538/52538fig1highres.jpg" /> 图1.注射Th17细胞和调节性T细胞的招募和扩大C的引流淋巴结（CLN） 白色念珠菌感染小鼠（A）RAG-1 - / - CD45.2小鼠重组与Th17细胞或Th17细胞+ 调节性T Th17的条件下极化5天的细胞。 Th17细胞被从CD45.2小鼠获得CD45.1同类系小鼠和T reg细胞获得。一些小鼠没有接受任何细胞（深水或念珠菌+ PBS）中。受体小鼠各组感染了假性对照或C.白色念珠菌 。在感染后5天，CLN是isolat编使单细胞悬浮液，并掌握其注入CD45.1表达Th17细胞通过流式细胞术（选通在FSC，SSC分散所有白细胞）（B）的 RAG-1 - / - CD45.2小鼠重构与Th17细胞或Th17细胞+ T reg细胞，如"A"和感染下进行 。 白色念珠菌。CLN中分离，使单细胞悬浮液，并测量CD45.1并通过流式细胞术的细胞内Foxp3的表达（门上的所有的CD4细胞）。在情节的调节性T门显示CD45.1负，FOXP3 + CD4 +细胞。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52538/52538fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52538/52538fig2highres.jpg" /> 图2.注入Th17细胞导致体重减轻用C<html> 白色念珠菌感染的小鼠对T 寄存器和共同给药改善从重量损失恢复。SCID CB-17小鼠重构与Th17细胞或Th17细胞+ T reg细胞如在图1A中。 3天后，将受体小鼠被感染C.白色念珠菌，每组中的一些小鼠感染与假控制。免疫抑制小鼠接受醋酸可的松（皮质醇）注射。百分重量变化在复原用表示细胞和感染白色念珠菌小鼠，相对于D-1（前一天感染）被示出。数据标准化相对重量数据未感染（深水+ PBS）的小鼠。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52538/52538fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> "SRC ="/文件/ ftp_upload / 52538 / 52538fig3highres.jpg"/&gt; 图3.注入Th17细胞引起舌免疫病理学在C中。 白色念珠菌感染的小鼠和共同给药对T 暂存器降低 C的舌片的免疫病理组织学评估白色念珠菌感染小鼠。小鼠重组，指示细胞和病毒感染如在图1A中。感染后第5天，舌片是从小鼠中分离。（A）的舌片的矢状切片用H＆E以评估炎症和免疫细胞的浸润（IF）。 （PA）和（EP）表示乳头和舌分别上皮层。显微图像观看at50X倍率的幻灯片。（B）的组织学免疫染色，使用抗Gr-1抗体的Gr-1的中性粒细胞标记物 （CLONE:1A8-Ly6g）在第5天的舌片部感染后（褐色，表示为IF）。放大100倍观察显示的幻灯片显微图像。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52538/52538fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52538/52538fig4highres.jpg" /> 对T 暂存器图4.联合政府减少注射Th17细胞TNF-α。 （A）RAG-1 - / - CD45.2小鼠重组与Th17细胞或Th17细胞+ Treg 细胞和感染C.白色念珠菌 ， 如图1A。在感染后第5天，CLN和舌组织中分离，使单细胞悬浮液。将这些细胞再刺激用PMA和离子霉素的细胞内staini之前纳克和流式细胞术分析。流式细胞等高线图内的ROR-γT的Th17细胞和TNF-α的表达示（选通对CD4 +细胞）。（B）的TNFαpositive细胞从实验中的"A"进行的百分比的统计表示。数据代表4只未感染组，6只在每个感染组，并从两个独立的实验汇集。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52538/52538fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52538/52538fig5highres.jpg" /> 图5.增加免疫病理学还与C升高真菌的负担有关。 白色念珠菌感染小鼠。CB-17 SCID麦克风E的重组，BALB / c小鼠Th17细胞，Th17细胞或+ Treg 细胞在Figure1A，并感染了C.白色念珠菌 。示从感染小鼠舌的组织学评价。于感染后第7天，舌收获并用高碘酸席夫（PAS）染色，以评估真菌损害，炎症和浸润细胞（IF），并以检测真菌（CA）。幻灯片被视为at100X放大。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52538/52538fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52538/52538fig6highres.jpg" /> 图6.牛逼暂存器改善免疫病理学用C。白色念珠菌感染小鼠。 </s仲&gt; CB-17 SCID小鼠重构与BALB / c小鼠Th17细胞，或Th17细胞+ T reg细胞如在图1A中 ，与感染了C。白色念珠菌 。从评估， 如图5感染小鼠的舌头的平均组织学评分的统计表示。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52538/52538fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td>得分</td><td>特点</td></tr><tr><td> 0 </td><td> 0 - 没有可见的酵母或菌丝，在舌背表面完好表面上皮，乳头清楚本和完好的（可以是或可以不是浸润在组织内的单核细胞），无增稠基底层，没有浸润免疫细胞。 </td></tr><tr><td> 1 </td><td> 1 - 没有明显的酵母或菌丝E，衣衫褴褛或轻微受损表面上皮，乳头明显存在以最小的损伤和偶发浸润的免疫细胞。 </td></tr><tr><td> 2 </td><td> 2 - 乳头减少，但目前有损伤，基底层增厚和频繁浸润的免疫细胞。 </td></tr><tr><td> 3 </td><td> 3 - 可见酵母和菌丝侵入，乳头破损，受损上皮病变偶尔，基底层增厚和小簇浸润的免疫细胞的证据偶尔集群。 </td></tr><tr><td> 4 </td><td> 4 - 频繁的集群可见的酵母和菌丝侵入，乳头破损，受损的上皮细胞，免疫细胞浸润很小，基底层和频繁集群浸润的免疫细胞增厚频繁病变的证据。 </td></tr><tr><td>五</td><td> 5 - 入侵的菌丝，整个背表面广泛受损上皮细胞，大量渗入广泛的证据免疫细胞，很少或根本没有检测到乳头，基底层增厚和高频率和大集群浸润的免疫细胞。 </td></tr></tbody></table> 表1:组织学评分值

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.

口咽念珠菌病的免疫缺陷小鼠Th17细胞炎症模型

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

9.9K 次观看.  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. 

视频: 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.

实验小鼠模型阴道接种和样品采集的协议念珠菌阴道炎

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

科研

免疫与感染

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.

隔离和幼稚的CD4 T细胞的Th17细胞分化

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.

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

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.

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

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.

弥散性念珠菌感染模型

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

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

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

<meta charset="utf8"></head><body>皮肤树突状细胞差异亚群在皮下白色念珠菌感染中诱导 Th17 免疫的体内功能

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

体内 皮肤树突状细胞差异亚群在皮下白色 念珠菌 感染中诱导 Th17 免疫的功能

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

使用姜黄素类化合物治疗小鼠口腔念珠菌病的有效 aPDT

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.

作者聚焦:探索姜黄素在口腔念珠菌病小鼠模型中的光动力疗法

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.

口咽念珠菌病的免疫缺陷小鼠Th17细胞炎症模型

摘要

探索更多视频

实验小鼠模型阴道接种和样品采集的协议念珠菌阴道炎

隔离和幼稚的CD4 T细胞的Th17细胞分化

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

弥散性念珠菌感染模型

体内皮肤树突状细胞差异亚群在皮下白色念珠菌感染中诱导 Th17 免疫的功能

体内皮肤树突状细胞差异亚群在皮下白色念珠菌感染中诱导 Th17 免疫的功能

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

口咽念珠菌病的免疫缺陷小鼠Th17细胞炎症模型

摘要

探索更多视频

实验小鼠模型阴道接种和样品采集的协议念珠菌阴道炎

隔离和幼稚的CD4 T细胞的Th17细胞分化

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

弥散性念珠菌感染模型

体内 皮肤树突状细胞差异亚群在皮下白色 念珠菌 感染中诱导 Th17 免疫的功能

体内 皮肤树突状细胞差异亚群在皮下白色 念珠菌 感染中诱导 Th17 免疫的功能

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

体内皮肤树突状细胞差异亚群在皮下白色念珠菌感染中诱导 Th17 免疫的功能

体内皮肤树突状细胞差异亚群在皮下白色念珠菌感染中诱导 Th17 免疫的功能