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.

참고 : 마우스를 사용하여 실험 기관 동물 복지위원회 (IACUC) 가이드 라인에 따라 실시 하였다. (감염에 세 가지 일 이전) I N 체외 배양 된 Th17 세포와 마우스 - / - 1. 헝겊-1을 재구성 <ol><li> 수용성의 존재에 혼자 U-바닥 96 웰 플레이트, 또는 3 × 104 CD4 + CD25 + Foxp3의 + Tregs과 함께 공동 문화를에서 Th17 세포, 문화 CD4 + CD44low CD62Lhigh CD25- 나이브 T 세포 (× 104 3) 수립 α-CD3 (1 ㎍ / ㎖) α-CD28 (2 ㎍ / ㎖) Th17 조건 하에서 항체 및 항원 제시 세포 (IL-6 (25 NG / ㎖), TGF-의 β를 이용하여 편광 (이 NG / ㎖) , α-IFN-γ (2 ㎍ / ml) 및 α -IL-4 (2 ㎍ / ㎖)) 3~4일 대. 참고 : 나이브 세포와 T의, 규정 정렬 형광 활성화 된 셀을 사용하여 정렬 된 (FACS)자기 세포 분리 절차 D 배양 앞서 설명한 바와 같이 19-22. </li><li> 4 일에, 피펫을 사용하여 Th17 세포를 재 부유 한 후, 문화에서 그들을 수집하고, 멸균 조건을 원심 분리기. <ol><li> 자신의 생존과 표현형에 대한 검사 이러한 세포의 작은 나누어지는 가져 가라. 요오드화 프로피 듐 (중에는 200ng / ㎖)를 첨가하고, 유세포 분류법으로 즉시 평가 생존, R​​PMI 배지 500 μL에이를 재현 탁. restimulation 후 IL-17A 생산의 평가 유동 세포 계측법에 의해 표현형을 수행합니다 (6.3 절 참조). </li></ol></li><li> 달리 명시하지 않는 한 모든 원심 분리 단계에서 39 ° C에서 6 분 동안 480 XG에 멸균 PBS에서 세포 주 벌크 씻는다. </li><li> 6~8주 된 CD45.2 Rag1에 입양 전송을 위해 이러한 세포를 사용하여 - / - 면역 결핍 쥐. <ol><li> 찬 멸균 PBS를 사용하여 1 × 10 7 세포 / ㎖의 세포 밀도로 세포를 재현 탁. </li><li> intrap에 의한 세포의 100 μl를 주입eritoneal 주사 한 마우스가 1 × 10 6 세포를 수신하도록, 1 ㎖의 투베르쿨린 주사기에 25 G 바늘을 사용. 일부 마우스는 PBS 또는 Th17 세포를 받게됩니다, 다른 마우스는 TREG 세포와 공동 배양 Th17 세포를 받게됩니다. 참고 : 무균 모든 단계를 수행합니다. </li></ol></li></ol> 2. C. 성장 감염 알비 칸스 (일일 감염 이전에) <ol><li> 접종 전에 CAF2-1 C. 다섯 식민지를 분산 알비 칸스 실험실 멸균 PBS 현탁액 100 ㎕의 변형 및 멸균 국물에 서스펜션을 추가합니다. </li><li> C. 5 식민지를 접종 효모 질소 염기의 50 ㎖에 칸디다 알비 칸스 (아미노산없이 YNB) / 펩톤 / 덱 스트로스 브로 쓰 배지 및 130 rpm에서 15 내지 18 시간 동안 30 ℃에서 진탕 배양기에서 배양한다. </li><li> 즉, 균류의 성장을 표시 한, 백탁 국물 용 모니터. </li></ol> 3. 칸디다수확과 (감염의 날) 계수 절차 <ol><li> 15 ml의 튜브에 칸디다 균 배지 10 ㎖를 수집합니다. 큰 볼륨의 경우, 50 ㎖ 튜브에 칸디다를 수집합니다. </li><li> 달리 명시하지 않는 한 모든 단계에서 RT에서 5 분 동안 1,900 XG에 blastospores 원심 분리기. 상등액을 제거하여 원심 분리를 blastospores 펠렛. </li><li> 하나 이상의 튜브가있는 경우, 펠릿 멸균 PBS를 첨가 다중 튜브의 칸디다 blastospores 수영장 카운팅 및 PBS 10 ㎖로 재현 탁 그들을. </li><li> 1.5 ml의 마이크로 원심 튜브에 blastospores의 20 μl를 타고 배 파라 포름 알데히드의 20 μl를 추가하고 15 ~ 20 분 동안 실온에서 품어. 현미경으로 혈구를 사용하여 고정 blastospores를 계산합니다. 참고 : 파라 포름 알데히드는 발암입니다. 만 흄 후드의 원액을 엽니 다. </li><li> 한편, t를 원심 분리하여, 두 번 이상 반복 세척 blastosporesPBS에서 밑단하고 blastospores을 펠렛. 감염에 대한 준비, ABSL1 후드, RT에서 15 ml의 튜브에 펠릿을 유지합니다. </li><li> 감염 blastospores을 재 부유에 대한 RT에서 일부 멸균 PBS를 남겨주세요. </li><li> 카운팅 한 후, 효모 세포 / ml의 X10이 8 blastospore 효모의 세포 수를 조정할 blastospore 펠렛을 멸균 PBS를 추가한다. 이는 쥐를 감염 사용될 blastospore 현탁액이다. 참고 : 무균 모든 단계를 수행합니다. </li></ol> 4. 마우스 감염 (셀 전송 후 3 ~ 5 일) 주 : 앞서 설명 된 바와 같이 19,23-25 ​​기본적인 감염 과정이 수행된다. <ol><li> - / - PBS 3 일 이전에 세포를받은 쥐 헝겊-1의 무게. </li><li> 자신의 체중을 이용하여 마취제의 용량을 계산합니다. INTR에 의해, 1 ㎖의 투베르쿨린 주사기에 25 G 바늘을 사용하여, 케타민 / 자일 라진 (xylazine)의 혼합물 (16.1 ㎎ / ㎖ 및 1.6 ㎎ / ㎖)을 투여하여 쥐를 마취aperitoneal 주입. <ol><li> 체중 10g 당 50 μL를 관리. (즉, 케타민 0.8 mg의 체중의 자일 라진 / 10g 또는 0.08 mg의 케타민과 80 mg의 체중 kg 당 자일 라진 8 mg을 각각). 발가락 핀치 응답을 마취 후 매 15 분을 준수하십시오. 참고 :이 투여 량은 감염 과정에 대한 충분한 마취의 60 ~ 90 분을 유도 할 것이다. </li><li> 건조로부터 각막을 방지하기 위해 마우스의 눈 안과 윤활유 연고를 적용합니다. 눈의 윤활유가 건조 수있는 바와 같이, 안과 윤활마다 45 분을 반복합니다. </li></ol></li><li> 마취 중 마우스의 재수 화를 돕기 위해 앞다리 피하 후면에 인접한 식염수 (0.9 %의 NaCl) 1ml를 주입한다. </li><li> 새로운, 깨끗한 케이지를 얻습니다. 일단 감염 새로운 케이지에 각 마우스를 배치, 한 번에 감염 절차를 하나의 마우스를 따르십시오. 제 PBS / 가짜 감염을 수행 한 다음 칸디다 감염 촉진제로 진행업. </li><li> 마취 마우스를 들고 혀의 기지를 공개하는 넓은 입을 엽니 다. <ol><li> 설하 90 분 동안 구강, PBS 또는 blastospore 현탁액 50 μL로 포화 3mm 직경 목화 공을 놓습니다. 코튼 볼이 움직이지 않는 것을 보장, 폐 울혈을 방지하기 위해 다시 쥐에게 15 분마다 전면 플립합니다. </li></ol></li><li> 구강에 가짜 또는 칸디다 균 접종 90 분을 보장하기 위해 모든 마우스에 대한 타이머를 설정합니다. </li><li> 가열 램프 (4피트 거리)에서 케이지, 열 젤 패드에 각 마우스에 보관하십시오. </li><li> 혀는 혀를 lacerating 치아를 방지하기 위해 치아 내부와 멀리 철회되어 있는지 확인합니다. </li><li> 90 분의 끝에서 마우스의 입에서 면봉을 제거한다. 75분보다 빨리 마​​취에서 회복 수있는 마우스 시계. 이러한 경우에, 단지 케타민 (40 ㎎ / kg)으로 다시 마취. </li></ol> 5. 포스트 Procedur전자 (90 분 마취시) 모니터링 <ol><li> 90 분 마취 중 열 젤 패드를 사용합니다. <ol><li> 체온을 유지하고 질식을 방지하기 위하여, 생쥐는 보통 옥수수 냉이 마우스 침구에 복구 할 수 없다. </li></ol></li><li> 마취에서 회복 한 후, 뒷면에 두 개의 위치에 멸균 0.9 % 염화나트륨 피하의 추가 1 ml에 관리 할 수​​ 있습니다. </li><li> 다섯 PBS (가짜) 케이지 당 접종 한 마우스에 계속. 케이지 당 하우스 만 1-2 감염된 쥐. </li><li> 습관과 전반적인 건강을 정리, 체중의 변화를주의하는 것이 시술 후 매일 이니셜 절차 카드를 작성합니다. </li></ol> 6. 염증 평가 6.1) 체중 감소 <ol><li> 감염 절차 이전에 하루에 시작, 감염 동안 매일 마우스의 무게를. 참고 : 일반적으로, Th17 세포없이 T의, 규정에 주입 된 면역 결핍 마우스 (20)에 굴복인해 -30 %의 중량 손실은 19 진균 부담 증가 및 염증을 악화. </li></ol> 혀의 6.2) 조직학 득점 <ol><li> 자궁 경부 전위 다음 CO 2 질식하여 쥐를 희생. </li><li> 턱을 열고 가위와 집게로 혀를 해부하다. 혀를 잡아 무딘 집게를 사용하고, 가위 입의 뒤쪽으로 다가 사용하는 혀의 뒤쪽 끝을 절개. </li><li> 면역 세포 화학 헤 마톡 실린 및 에오신 혀 조직의 (H &amp; E) 염색의 경우, 얼음 차가운 PBS와 혀의 조직을 씻어. 15 ml의 튜브 / N을 2-3 방언을 10 % 포르말린 5 mL를 추가하고 O를 수정. <ol><li> 다음날, 포르말린을 제거하고, 하이퍼 고정을 방지하기 위해 70 % 에탄올 5 ml의 조직 몰입. 단면 및 파라핀 섹션의 염색, 파라핀 삽입을 계속 상용 서비스로 보내십시오. 참고 : 상업 조직학 서비스는 다음 단계를 수행하는 데 사용됩니다. </리&gt; </ol></li><li> 슬라이드 다시 상업 조직학 서비스에서 일단, 학년 광학 현미경 조직 절편을 관찰하여 염증. <ol><li> 0은 염증 되 않으며, 5 가장 심한 염증 인 상태 0-5 점수. 표 1을 사용하여 염증 점수. </li></ol></li></ol> Th17 세포에 의해 6.3) 사이토킨 생성 참고 : 과도한 TNF-α 생산은 과도한 면역 병리의 판독 중 하나입니다. 마우스를 적응 적 Th17 세포 만 전송되면, 그것은 Th17 세포에서 과도한 TNF-α의 생산과 관련된 면역 병리 발생된다. <ol><li> 전술 한 방법을 사용하여 비장 19,26, 경부 림프절, 겨드랑 lymphnodes, 혀에서 단일 세포 현탁액을 수집. </li><li> Brefeldin A (10 ㎍ / ㎖)으로 4 시간 동안 포르 볼 미리 스테이트 아세테이트 (PMA) (50 NG / ml) 및 이오 노마 이신 (500 NG / ㎖)와 함께 세포를 Restimulate는 L 첨가대서양 표준시 2 시간. PBS로 세포를 세척하고 제조업체의 지침에 따라 상용 고정 / 투과성으로 키트를 사용하여 고정한다. </li><li> 앞서 설명한 바와 같이 19를 형광 접합 된 항 TNF-α, IL-항 17a 및 ROR - γt 항체를 이용하여 세포 내 사이토 카인 염색법을 수행한다. </li><li> / ml의 최종 농도 3 μg의 - 간단히, 항 TNF-α, IL-항 17a 및 ROR - γt 항체, 각각의 AT (2) 항체의 칵테일 (1X)의 투과성으로 버퍼에 세포를 재현 탁. </li><li> RT에서 1 시간 동안 세포를 배양한다. </li><li> 배양 후, 1X의 투과성으로 버퍼로 세포를 씻어. </li><li> 유세포 분석을 0.5 % 소 혈청 알부민 500 μl의 PBS에서 세포 펠렛을 재현 탁. </li></ol>

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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.

이 모델은 경구 C. 유도에 기초 알비 칸스 (Candida albicans) 감염 따라 Th17 염증. 때문에 T의, 규정의 부재로, Th17 세포에 의한 염증은 무제한이며 제대로 해결 면역 병리로 연결됩니다. 시험관이 Th17 세포가 입양 전송에 사용했다 편광 순진한 CD4 세포를 유도. 40 - 배양 된 CD4 + 세포의 50 %가 약 3 일 검출 가능한 IL-17A 식 (Th17 세포)를 보여 주며, 따라서 마우스에서 주사에 사용 ...

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.

이 모델에서, 걸 레-1의 칸디다 -infected 마우스 및 감염되지 않은 쥐 모두 - / - 면역 결핍 배경이 적응 적 감염 3-5 일 전에 Th17 세포를 옮겼다. 10-12 마우스의 총 위장 감염이 각 그룹의 생쥐 및 칸디다 감염, 각 그룹에서 4-5으로, 실험에 사용 하였다. 주입 Th17 세포는 각각 (도 1a)을 Thy1.1 또는 CD45.1 염색법을 사용하여 생체 내에서 추적 된되도록 나이브 세포, CD4...

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.

면역 결핍 마우스의 구인두 칸디다증의 Th17 염증 모델

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 de Cartagena UDEC

Research

JoVE Journal

Medicine

9.9K 조회수.  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. 

비디오: 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.

의 실험 마우스 모델에서 질 접종 및 샘플 수집을위한 프로토콜 칸디다 질염

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

연구

면역학 및 감염병학

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.

분리 및 나이브 CD4 T 림프구의 TH17 분화

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.

마우스의 호스트 면역 반응을 평가하기 위해 구인두 기관 내 PAMP 관리 및 폐포의 활용

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.

Swimbladder 주입에 의해 애벌레 Zebrafish의 점막 칸디다증 모델링

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.

갤러리아 mellonella Waxworm 감염 모델 전파 칸디 다에 대 한

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>피내 칸디다 알비칸스 감염에서 Th17 면역을 유도하기 위한 피부 수지상 세포의 차등 부분 집합의 생체 내 기능

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

생체 내 피내 칸디다 알비칸스 감염에서 Th17 면역을 유도하기 위한 피부 수지상 세포의 차등 부분 집합의 기능

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

마우스의 구강 칸디다증에 대한 커큐미노이드를 사용한 효과적인 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.

저자 스포트라이트: 구강 칸디다증을 위한 쥐 모델에서 커큐민을 사용한 광역학 요법 탐색

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.

면역 결핍 마우스의 구인두 칸디다증의 Th17 염증 모델

요약

더 많은 비디오 탐색

의 실험 마우스 모델에서 질 접종 및 샘플 수집을위한 프로토콜 칸디다 질염

분리 및 나이브 CD4 T 림프구의 TH17 분화

마우스의 호스트 면역 반응을 평가하기 위해 구인두 기관 내 PAMP 관리 및 폐포의 활용

Swimbladder 주입에 의해 애벌레 Zebrafish의 점막 칸디다증 모델링

갤러리아 mellonella Waxworm 감염 모델 전파 칸디 다에 대 한

생체 내 피내 칸디다 알비칸스 감염에서 Th17 면역을 유도하기 위한 피부 수지상 세포의 차등 부분 집합의 기능

생체 내 피내 칸디다 알비칸스 감염에서 Th17 면역을 유도하기 위한 피부 수지상 세포의 차등 부분 집합의 기능

구강 칸디다증의 쥐 모델에 대한 커큐미노이드 매개 항균 광역학 요법

구강 칸디다증의 쥐 모델에 대한 커큐미노이드 매개 항균 광역학 요법

구강 칸디다증의 쥐 모델에 대한 커큐미노이드 매개 항균 광역학 요법