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oai:repository.ajou.ac.kr:201003/85842024-10-11T02:21:19Zcom_201003_14372com_201003_14368col_201003_14374

The role of T cell Immunoglobulin Mucin domain in Herpes Simplex Virus-induced Behçet’s Disease mouse model 단순포진 바이러스로 유도한 베체트병 마우스 모델에서 T cell immunoglobulin mucin domain(Tim) 의 역할 심, 주아 손, 성향 대학원 의생명과학과 200724296 심, 주아 Tim-1 Tim-3 Tim-4 Galectin-9 Herpes simplex virus-induced systemic inflammation Behçet’s Disease mouse model 베체트병 마우스 모델 단순포진바이러스 The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. Individual TIM family members may serve as susceptibility markers for asthma, allergies and autoimmune diseases, as well as potential cell surface markers for T helper type (Th)1 and Th2 T cells. TIM-1 plays an important role in the regulation of immune responses and the development of autoimmune diseases. TIM-4 is a natural ligand of TIM-1, and interaction of TIM-1 and TIM-4 is involved in the regulation of Th cell responses and the modulation of the Th1/Th2 cytokines balance. TIM-4 expression was increased in patients with systemic lupus erythematosus (SLE). It has been also reported that TIM-3 expression was higher in patients with rheumatoid arthritis compared to controls. Further, Galectin-9 (Gal-9) has been identified as a TIM-3 ligand (L) and the TIM-3-TIM-3L interaction serves as a specific down-regulator of the Th1 immune response. Behçet’s disease (BD) is a chronic, multisystemic inflammatory disorder with arthritic, gastrointestinal, mucocutaneous, ocular, vascular, and central nervous system involvement. In herpes simplex virus induced BD mouse model, the expression of Tim-1 and Gal-9 was lower levels compared to asymptomatic BD normal (BDN) mice. The expression of Tim-3 and Tim-4 was higher in BD mice than BDN mice. In addition, Tim-1 vector injected BD mice showed changes of BD-like symptoms and decreased the severity score. Again, treatment with Tim-4 siRNA also improved the BD-like symptoms and decreased the severity score accompanied with up-regulation of regulatory T cells (Treg). Furthermore, administration of Gal-9 improved the BD-like symptoms, decreased the severity score, and increased Treg cells. In addition, Gal-9 induced improvement was associated with down-regulation of pro-inflammatory cytokines and induction of apoptosis. In the present study, we showed that the regulation of Tim-1 or Tim-4 affected the BD-like symptoms and Tim-3-Tim-3L interaction improved the inflammatory symptoms in BD mice. T cell immunoglobulin mucin domain (TIM) 단백질은 T 세포의 활성과 tolerance를 조절한다. TIM family는 천식, 알레르기, 자가면역질환에 대한 감수성이 높은 표식자로 알려져 있다. TIM-1은 면역반응 조절과 자가면역질환을 일으키는데 중요한 역할을 하며, TIM-4는 TIM-1의 ligand로 알려져 있다. Tim-1과 Tim-4의 상호작용은 Th 세포 반응의 조절과 Th1/Th2 사이토카인의 균형을 조절한다고 보고되었다. TIM-3와 TIM-3 ligand 인 galectin-9 (Gal-9) 의 상호작용은 Th1 면역반응을 특이적으로 저해한다고 알려져 있다. 베체트병은 만성 염증질환으로 재발성 구강궤양, 외음부궤양, 피부 및 안증상 등이 특징인 질병이다. 베체트병은 여러증상들이 동시에 발병하거나 초기 피부 점막 증상이 호전과 악화를 거듭하다 관절 증상, 안 증상, 신경계 증상까지 발전할 수 있다. 특히 안 증상은 대표적인 베체트병 주 증상으로 실명이라는 심각한 후유증을 초래할 수 있다. 이러한 증상을 조절할 수 있는 방법으로, 본 연구에서는 단순포진 바이러스로 유도한 베체트병 마우스 모델을 이용하여 Tim과의 연관성을 알아보고자 하였다. 우선 Tim-3 ligand인 Gal-9은 베체트병 마우스모델의 증상 군이 무증상 군 (바이러스 접종 후 증상이 나타나지 않은 군) 에 비하여 낮게 발현되었고, 반대로 Tim-3와 Tim-4는 높게 발현되었다. 그 발현의 차이가 증상 변화에 연관이 있는지 알아보기 위해 베체트병 마우스 모델에 Tim-1 vector를 투여한 결과 대조군과 비교하여 증상이 호전되었고 severity score는 감소하였다. Tim-4 siRNA를 투여한 후 증상이 완화되는 것을 관찰하였고 severity score 역시 감소하였으며 조절 T 세포는 증가하였다. Gal-9 투여는 증상호전과 severity score의 감소를 보였으며, pro-inflammatory 사이토카인을 감소시키고 세포자살을 유도하였다. 본 연구에서는, Tim-1과 Tim-4 발현 수준을 조절하여 베체트병 마우스 모델의 증상을 호전시키고, Tim-3L 증가에 의한 Tim-3-Tim-3L 상호작용이 베체트병 마우스모델의 염증 증상의 호전에도 영향을 주는 것을 확인하였다. TABLE OF CONTENTS ABSTRACT ⅰ TABLE OF CONTENTS ⅲ LIST OF FIGURES ⅴ LIST OF TABLES ⅶ Ⅰ. INTRODUCTION 1 Ⅱ. MATERIALS AND METHODS 4 A. Antibodies and reagents 4 B. Animal experiments 4 C. BD-like symptoms 5 D. Tim-1 DNA constructs 5 E. Preparation of Tim-4 small interfering RNA (siRNA) 6 F. Tim-1 vector and Tim-4 siRNA administration to BD mice 6 G. Gal-9 administration to BD mice 6 H. Flow cytometry 6 I. Enzyme-linked immunosorbent assay (ELISA) 7 J. Transmission electron microscopy (TEM) 7 K. Statistical analysis 8 Ⅲ. RESULTS 9 A. The frequencies of Tim-1 and Tim-4 expressing cells in normal healthy, BDN and BD mice 9 B. The expression of Tim-3 and Gal-9 in BD mice 14 C. Administration of Tim-1 vector up-regulates the frequency of Tim-1(+) cells in vivo lymph nodes 16 D. Administration of Tim-1 vector affected the BD-like symptoms 18 E. Tim-1 vector administration affected the regulatory cellular phenotypes 24 F. Pro-inflammatory cytokines were down-regulated by Tim-1 vector administration in BD mice 26 G. Tim-4 siRNA treatment down-regulated the expression of Tim-4 in normal healthy mice 28 H. Administration of siTim-4 changed BD-like symptoms 30 I. Treg cells were up-regulated in siTim-4 treated BD mice 35 J. Treatment with siTim-4 decreased the serum level of IL-17 in BD mice 37 K. Gal-9 treatment up-regulated the expression of Gal-9 in vitro and in vivo 39 L. Gal-9 administration improved BD-like symptoms 41 M. Gal-9 induced the expression of cell death-related molecules in BD mice 43 N. Gal-9 modulated the cell population in BD mice 47 O. Gal-9 regulated cytokine expression in BD mice 49 Ⅳ. DISCUSSION 51 Ⅴ. CONCLUSION 57 REFERENCES 58 국문요약 70 Doctor 2013-12-12T06:14:23Z 2013-12-12T06:14:23Z 2013 2013 Thesis Theses http://repository.ajou.ac.kr/handle/201003/8584 http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000013529 000000013529 en