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1 대한안과학회지 2015 년제 56 권제 1 호 J Korean Ophthalmol Soc 2015;56(1): ISSN (Print) ISSN (Online) Original Article 실험적자가면역포도막염마우스모델에서 Nod 양수용체전사체의안내발현양상분석 Transcriptional Analysis of Nod-Like Receptors in a Mouse Model of Experimental Autoimmune Uveitis 김형민 1 김태완 2 정현정 3 류진숙 3 김미금 1,3 위원량 1,3 오주연 1,3 Hyeong Min Kim, MD 1, Tae Wan Kim, MD, PhD 2, Hyun Jeong Jeong 3, Jin Suk Ryu 3, Mee Kum Kim, MD, PhD 1,3, Won Ryang Wee, MD, PhD 1,3, Joo Youn Oh, MD, PhD 1,3 서울대학교의과대학서울대학교병원안과학교실 1, 서울특별시보라매병원안과 2, 서울대학교병원의생명연구원인공안구센터안면역각막재생연구실 3 Department of Ophthalmology, Seoul National University Hospital, Seoul National University College of Medicine 1, Seoul, Korea Department of Ophthalmology, Seoul Metropolitan Government Seoul National University Boramae Medical Center 2, Seoul, Korea Laboratory of Ocular Regenerative Medicine and Immunology, Seoul Artificial Eye Center, Seoul National University Hospital Biomedical Research Institute 3, Seoul, Korea Purpose: To evaluate the transcription pattern of Nod-like receptors (NLRs), the intracellular sensors, to detect danger signals in murine eyes with experimental autoimmune uveitis (EAU). Methods: EAU was induced in B6 (C57BL/6) mice by subcutaneous injection of human interphotoreceptor retinoid binding protein and intraperitoneal injection of pertussis toxin. At 1, 2, and 3 weeks post-immunization, the eyeballs were extracted and subjected to histological and molecular assays using real-time reverse transcription polymerase chain reaction. Results: The levels of nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain 1 (NLRP1), NLRP3, nucleotide-binding oligomerization domain-containing protein 1 (NOD1), and NOD2 transcripts were increased at 2 weeks and gradually reduced thereafter. Notably, NLRP3 showed the highest expression in the eyes with EAU. Similarly, the transcript level of pro-inflammatory cytokine, interleukin-1β, increased and reached a peak at 2 weeks post-immunization. The retinal structure was severely damaged by inflammation at 3 weeks post-immunization. Conclusions: Among NLRs, NLRP3 may induce inflammation in eyes after EAU immunization. J Korean Ophthalmol Soc 2015;56(1): Key Words: Experimental autoimmune uveitis, NLRP3, Nod-like receptors 포도막염은안과외래에서흔히보는질환으로전체실 Received: Revised: Accepted: Address reprint requests to Joo Youn Oh, MD, PhD Department of Ophthalmology, Seoul National University Hospital, #101 Daehak-ro, Jongno-gu, Seoul , Korea Tel: , Fax: jooyounoh77@gmail.com * This work was supported by the SNUH research fund ( ). 명원인의약 10% 정도를차지한다고알려졌다. 1 포도막염을일으키는원인에는결핵, 헤르페스, 매독, 톡소플라스마와같은감염성원인부터사르코이드증, 당뇨, 베체트병, 척추관절병증등전신적인염증질환이있으며, 내인성포도막염은대개발생기전이확실히밝혀지지는않았으나자가면역기전에의한것으로생각되고있다. 따라서자가면역포도막염의일차적인치료로스테로이드제제가사용되고있으나이러한면역억제제는비특이적으로면역반응을억제하므로장기간투여시전신적인부작용을초래할수있 c2015 The Korean Ophthalmological Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 99

2 - 대한안과학회지 2015 년제 56 권제 1 호 - 다. 따라서내인성포도막염에서자가면역반응을조절할수있는여러방법들이모색되고있는데, 성공적인치료법개발을위해서는포도막염의발병기전에대한분자유전학적인이해가선행되어야한다. 2 그중에 Nod양수용체 (NOD-like receptor, NLR) 는최근세포내병원체를감지하는체계로발견되었는데, toll-like receptor (TLR) 단백질과구조적으로, 기능적으로유사하며선천성면역반응에관여하는것으로알려졌다. 3 또한 Nod 양수용체는 Interleukin (IL)-1β와같은염증성사이토카인을세포밖으로분비하여내인성위험신호에대해염증반응을유발하는역할을하는것으로알려졌다. 4 하지만자가면역질환에서이들수용체의발현양상과그역할에대해 서는알려진바가없다. 이에본연구에서는인간의내인성포도막염모델인실험적자가면역포도막염마우스모델에서 Nod양수용체전사체의시간에따른안구내발현을알아보고자하였다. 대상과방법 실험적자가면역포도막염의유발 6주령된무병원 (pathogen-free) B6 mouse (C57BL/6J) (Orient Bio Inc., Seongnam, Korea) 마우스를실험에사용하였다. 실험동물의사육은서울대학교병원임상의학연구소전임상실험실의무균동물실 (SPF free) 을이용하였고실 A B C D Figure 1. Assay for Nod-like receptors in the eyes after experimental autoimmune uveitis (EAU) immunization. Real-time RT-PCR analysis showed that the levels of NLRP1, NLRP3, NOD1, and NOD2 transcripts increased in the eyes after EAU immunization with a peak at the post-immunization 2 weeks, and gradually decreased until 3 weeks. (A) Time course of NLRP1 expression in the eye. (B) Time course of NLRP3 expression in the eye. (C) Time course of NOD1 expression in the eye. (D) Time course of NOD2 expression in the eye. Data are presented in mean ± SEM. N = 5 in each group. RT-PCR = reverse transcription polymerase chain reaction; NLRP = nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain; NOD = nucleotide-binding oligomerization domain-containing protein; SEM = standard error of the mean. * p < 0.01; p <

3 - 김형민외 : 실험적포도막염모델에서 Nod 양수용체의발현 - 험프로토콜은서울대학교병원임상의학연구소전임상실험실의동물실험윤리위원회의허가를받았다. 실험적자가면역포도막염을유발하기위해 250 µg의사람광수용체간레티노이드결합단백질 (human IRBP) 펩타이드 1-20, GPTHLFQPSLVLDMAKVLLD (20 mg/ml; Peptron, Daejeon, Korea) 를마우스발바닥에피하내주입하였다. Human IRBP는 Mycobacterium tuberculosis (2.5 mg/ml; BD Difco TM, Franklin Lakes, NJ) 가포함된 complete Freund's adjuvant (Sigma, Saint Louis, MO) 에 emulsion된것을사용 Figure 2. Assay for the pro-inflammatory cytokine interleukin (IL)-1β in the eyes after experimental autoimmune uveitis (EAU) immunization. Real-time RT-PCR analysis showed that the level of IL-1β transcript were gradually increased and reached at peak at the post-immunization 2 weeks after EAU immunization. Data are presented in mean ± SEM. N = 5 in each group. RT-PCR = reverse transcription polymerase chain reaction; SEM = standard error of the mean. * p < 0.05; p < 하였다. 동시에 0.7 µg의백일해독소 (Pertussis toxin; 300 µl, Sigma) 를복강내에주입하였다. 5 조직학적분석포도막염유발후 1, 2, 3주째에쥐를안락사하여안구를적출한후 10% 포름알데히드와파라핀으로고정을하였다. 4 µm 두께의냉동절편을만들고 hematoxylin과 eosion으로염색을시행하였다. 실시간역전사연쇄중합반응포도막염유발후 1, 2, 3주째에적출한안구를 RNA isolation reagent (RNA Bee, Tel-Test Inc., Frienswood, TX) 로용해하였고 sonicator (Ultrasonic Processor, Cole Parmer Instruments, Vernon Hills, IL) 로균질화하였다. RNA 는 RNeasy Mini Kit (Qiagen, Valencia, CA) 를이용하여추출하였고역전사효소 (SuperScript III, Invitrogen, Carlsbad, CA) 로이중가닥 cdna 를합성하였다. 실시간역전사연쇄중합반응은 TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad, CA) 를이용하여시행하였으며, 18s rrna probe (TaqMan Gene Expression Assays ID, Hs _g1) 를이용하여유전자발현기준점을동일하게하였다. 사용한 PCR probe set는모두 Taqman Gene Expression Assay kit를 Applied Biosystems 에서구입하였다. 통계분석 SPSS software version 12.0 (SPSS Inc., Chicago, IL) 을이용하여통계분석을시행하였다. Student s t 방법을이용하여유의한차이가있는지분석하였으며, 통계적유의성은 p-value<0.05로정의하였다. 1 week 2 weeks 3 weeks A B C Figure 3. Histological findings of the eye. Hematoxylin-eosin staining of the eye showed that the retinal structure including the photoreceptor layer was severely disorganized with massive infiltration of inflammatory cells in the vitreous cavity and in the retina of BSS-treated experimental autoimmune uveitis mice on day 21 (Original magnification x100, Hematoxylin-eosin staining). (A) Hematoxylin-eosin staining of the eye at one week after EAU induction. (B) Hematoxylin-eosin staining of the eye at two weeks after EAU induction. (C) Hematoxylin-eosin staining of the eye at three weeks after EAU induction. BSS = balanced salt solution. 101

4 - 대한안과학회지 2015 년제 56 권제 1 호 - 결과 자가면역포도막염유발후안구내에서 Nod양수용체인 NLRP (nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain)1, NLRP3, NOD (nucleotidebinding oligomerization domain)1, NOD2 의전사체의양이증가하기시작하여 2주째에최대치를보인후 3주째에는감소하였다 (Fig. 1). 이중 NLRP3 전사체의양이가장많았다. 또한, 염증성사이토카인인 IL-1β의전사체의발현도포도막염유발후 2주째에가장높아서 Nod양수용체와비슷한발현양상을보였다 (Fig. 2). 하지만자가면역포도막염유발후망막의구조적파괴는 3주째에가장심하게관찰되었다 (Fig. 2). 포도막염유발후 1주째에는정상과다름없는망막의구조를보였지만 2 주째에는망막광수용체층의파괴가일부분에서관찰되기시작하였다. 3주째에는유리체강내염증세포가침착하고망막층의부종이일어났으며특히망막광수용체층의뒤틀림을동반한파괴가심하게관찰되었다 (Fig. 3). 고찰 포도막염은안구의중간층을이루고있는포도막에발생하는염증성질환으로미국에서 100,000명당 115명의환자가있으며, 특히만성포도막염의경우적절한치료가이루어지지않으면인접한안구조직즉공막, 각막, 망막조직에비가역적인손상을일으켜전체실명의약 10%, 전체실명원인의빈도중 4위를차지하는질환이다. 6,7 포도막염의치료는부신피질호르몬과면역억제제인데적어도 2-3년이상의장기치료가필요해눈과신체다른부위의심각한부작용을초래하여치료를지속하지못하는경우가종종발생하며스테로이드에반응을안하는포도막염도있다. 8 따라서안전하고효과가지속되는새로운약물의개발이요구되고있는상황이다. 염증성질환에대해새로운치료법을개발하기위해서는염증성질환의발병기전에대한분자유전학적새로운이해가필요하다. 최근급성감염성염증질환뿐아니라파킨슨씨병, 알츠하이머병, 심근경색, 비만, 당뇨, 뇌졸중등만성염증성질환에서도중요한발병원인으로염증조절복합체인인플라마좀의역할이새로이대두되고있다. 염증조절복합체는카스파제-1 (caspase-1) 을활성화시켜 IL-1β를성숙시키고분비하게하여염증반응을유도한다. 염증조절복합체에는 NOD양수용체로세포내신호를감지하는수용체와 caspase recruitment domain (CARD) 복합체를유도하는어댑터단백질 (adaptor protein) 이속해있다. 9 본연구에서는실험적자가면역포도막염에서염증조절복합체인 NOD양수용체중기존연구에서여러염증성질환및자가염증질환에관여하는것으로알려진 NLRP1, NLRP3, NOD1, NOD2의 4가지를선정하여분석하였다. 이중 NOD1은 caspase recruitment domain 4 유전자 (CARD4), NOD2는 CARD15 유전자에서발현되는것으로알려졌으며, CARD15 유전자의변이로인한 NOD2의선천성면역체계발현으로크론병이발병하는것으로유명하다. 10 NOD2 변이로나타나는 Blau syndrome 또한유명한데다발장기의염증을일으키며피부염, 관졀염과함께눈에서는포도막염을일으키는것으로도알려졌다. NLRP1, NLRP3은이들단백질이활성화되어선천성면역체계이상으로제2형당뇨병과같은대사성질환이나암, 류마티스관절염및자가면역질환을일으킨다는연구들이많다. 11,12 본연구에서유발된자가면역포도막염에서는위의 4가지 NOD양수용체의안구내발현이대표적인염증성싸이토카인인 IL-1β의발현양상과비슷하게 2주째에가장최고점을찍었으며그이후로점진적으로감소하는양상을보였다. 특히 NLRP3가가장많이발현됨을알수있었다. NLRP3 염증조절복합체는선천성면역체계에관여하여여러자가면역질환과관련이있음이알려졌지만, 13,14 안구내질환에서의작용은알려진바가없다. 본연구는 NOD양수용체가자가면역포도막염의안내에서유의하게증가하고이중 NLRP3의발현이가장특징적으로높음을확인하였다. 앞으로실제로안구내염증성질환에서 NLRP3를비롯한 NOD양수용체의역할을규명하기위한후속연구가필요하겠다. REFERENCES 1) Chang JH, Wakefield D. Uveitis: a global perspective. Ocul Immunol Inflamm 2002;10: ) Caspi RR. A look at autoimmunity and inflammation in the eye. J Clin Invest 2010;120: ) Martinon F, Tschopp J. NLRs join TLRs as innate sensors of pathogens. Trends Immunol 2005;26: ) Ogura Y, Sutterwala FS, Flavell RA. The inflammasome: first line of the immune response to cell stress. Cell 2006;126: ) Caspi RR. Experimental autoimmune uveoretinitis in the rat and mouse. Curr Protoc Immunol 2003;Chapter 15:Unit ) Gritz DC, Wong IG. Incidence and prevalence of uveitis in Northern California; the Northern California Epidemiology of Uveitis Study. Ophthalmology 2004;111: ; discussion ) LeHoang P. The gold standard of noninfectious uveitis: corticosteroids. Dev Ophthalmol 2012;51: ) Willermain F, Rosenbaum JT, Bodaghi B, et al. Interplay between innate and adaptive immunity in the development of non-infectious uveitis. Prog Retin Eye Res 2012;31: ) Martinon F, Mayor A, Tschopp J. The inflammasomes: guardians 102

5 - 김형민외 : 실험적포도막염모델에서 Nod 양수용체의발현 - of the body. Annu Rev Immunol 2009;27: ) Strober W, Murray PJ, Kitani A, Watanabe T. Signalling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol 2006;6: ) Vandanmagsar B, Youm YH, Ravussin A, et al. The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance. Nat Med 2011;17: ) Rosenzweig HL, Planck SR, Rosenbaum JT. NLRs in immune privileged sites. Curr Opin Pharmacol 2011;11: ) Franchi L, Warner N, Viani K, Nuñez G. Function of Nod-like receptors in microbial recognition and host defense. Immunol Rev 2009;227: ) Chen M, Wang H, Chen W, Meng G. Regulation of adaptive immunity by the NLRP3 inflammasome. Int Immunopharmacol 2011;11: = 국문초록 = 실험적자가면역포도막염마우스모델에서 Nod 양수용체전사체의안내발현양상분석 목적 : 내인성포도막염의실험모델인실험적자가면역포도막염마우스모델의눈에서세포내위험신호를감지하는역할을하는 Nod 양수용체전사체의시간에따른발현을알아보고자하였다. 대상과방법 : 마우스에사람광수용체간레티노이드결합단백질 (human IRBP) 의피하내주입및백일해독소 (Pertussis toxin) 의복강내주입을하여실험적자가면역포도막염을유발하고, 유발후 1, 2, 3 주째눈을적출하여실시간역전사연쇄중합반응 (real time RT-PCR) 으로분자유전학적인분석을시행하였고, 헤마톡실린 - 에오진염색으로조직학적분석을시행하였다. 결과 : 자가면역유발후 2 주째에 Nod 양수용체인 NLRP1, NLRP3, NOD1, NOD2 의전사체발현이가장증가하였고, 그뒤로점진적으로감소하였다. 이중 NLRP3 의발현이가장높았다. 염증성사이토카인인 Interleukin-1β 의발현도포도막염유발후 2 주째에가장높았다. 망막의구조적파괴는포도막염유발후 3 주째에가장심하게관찰되었다. 결론 : Nod 양수용체중 NLRP3 가실험적자가면역포도막염에서위험신호를감지하여염증반응을시작하는데중요한역할을할가능성이있다. < 대한안과학회지 2015;56(1):99-103> 103

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( )Kju269.hwp 만성세균성전립선염모델흰쥐에서 의항염효과 Anti-inflammatory Effect of Lycopene on Chronic Bacterial Prostatitis Rat Model Cho Hwan Yang, Dong Wan Sohn, Yong-Hyun Cho From the Department of Urology, The Catholic University