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Brown adipose tissue and GRIM-19 attenuate autoimmune diseases via inhibition of Th17 cells : Th17 세포 억제를 통한 갈색지방과 GRIM-19의 자가면역질환 완화효과
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 노상호 | - |
| dc.contributor.author | 문정현 | - |
| dc.date.accessioned | 2021-11-30T05:02:38Z | - |
| dc.date.available | 2021-11-30T05:02:38Z | - |
| dc.date.issued | 2021-02 | - |
| dc.identifier.other | 000000163828 | - |
| dc.identifier.uri | https://hdl.handle.net/10371/176176 | - |
| dc.identifier.uri | https://dcollection.snu.ac.kr/common/orgView/000000163828 | ko_KR |
| dc.description | 학위논문 (박사) -- 서울대학교 대학원 : 치의학대학원 치의과학과, 2021. 2. 노상호. | - |
| dc.description.abstract | Abstract
Brown adipose tissue and GRIM-19 attenuate autoimmune diseases via inhibition of Th17 cells Jeonghyeon Moon Program in Cancer and Developmental Biology The Graduate School Seoul National University Background: Brown adipose tissue (BAT) is known to secrete several factors that differ from those in white adipose tissue and has a large number of mitochondria. These reasons suggest that BAT might have potential positive advantages in the physiology of autoimmune diseases. The protein encoded by the Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19) is located in the mitochondrial inner membrane and is homologous to the NADH dehydrogenase 1-alpha subcomplex subunit 13 of the electron transport chain. It plays a critical role in mitochondrial functions. Multiple sclerosis (MS) is a demyelinating disease that damages the brain and spinal cord. Although both the cause and mechanism of MS progression remain unclear, it is accepted that an immune disorder is involved. Coenzyme Q10 (CoQ10), also known as ubiquinone, is a fat-soluble antioxidant. Although CoQ10 has not been approved as medication by the Food and Drug Administration, it is widely used in dietary supplements. It is known to increase mitochondrial functions and conditions. Taken together, this study focused on the therapeutic effect of mitochondrial regulation using BAT transplantation, GRIM-19 overexpression and CoQ10. Experiment 1: This data compared the functions of collagen-induced arthritis mice-derived BAT (CIA BAT) with normal mice-derived BAT (Nor BAT). DBA/1 J mice (6-7 weeks of age) were immunized by intradermal injection at the base of the tail with 100 μg of bovine type II collagen (CII) emulsified in complete Freunds adjuvant and incomplete Freunds adjuvant for boosting. CIA BAT and normal BAT were transplanted into CIA recipient mice. After transplantation, the author measured the anti-inflammatory functions of CIA BAT and normal BAT in mice. Normal BAT-transplanted mice showed significantly lower scores of bone damage, inflammation, and cartilage damage than sham control mice and CIA-transplanted mice. The proinflammatory cytokines in normal BAT-transplanted mice, such as IL-12, IL-17, IL-6, and tumor necrosis factor-α (TNF-α), tended to decrease than control mice and CIA-transplanted mice. Microarray analysis showed that the PI3K-AKT signaling pathway and IL-17 levels of CIA BAT tissues were significantly higher than those of normal BAT tissues. Experiment 2: To explore whether GRIM-19 ameliorated MS by increasing the levels of inflammatory cytokines and immune cells; this study used a mouse model of experimental autoimmune encephalomyelitis (EAE) to this end. Six-to-eight-week-old male C57BL/6, IFNγ-knockout (KO), and GRIM-19 transgenic mice were used; EAE was induced in all strains. A GRIM-19 overexpression vector was electrophoretically injected intravenously. IL-17A expression decreased and IFNγ expression increased in EAE mice that received injections of the GRIM-19-overexpression vector. GRIM-19 transgenic mice expressed more IFNγ than did wild-type mice; this inhibited EAE development. However, the effect of GRIM-19 overexpression on the EAE of IFNγ-KO mice did not differ from that of the empty vector. Experiment 3: Some studies have shown that CoQ10 has anti-inflammatory effects on various autoimmune disorders through enhancement of mitochondrial functions. This study investigated the anti-inflammatory effects of CoQ10. CoQ10 was administered orally to CIA mice for 10 weeks. This data showed that pro-inflammatory cytokines were significantly decreased in CoQ10 injected mice. Th17 cell and phosphorylated STAT3-expressed cell populations were also decreased in CoQ10 injected mice. When human peripheral blood mononuclear cells (PBMCs) were treated with CoQ10, the IL-17 expression of PBMCs in the CoQ10-treated group was significantly reduced. Conclusion: These results suggest that the transplantation of normal brown fat, GRIM-19 overexpression and injection of CoQ10 have a therapeutic effect in autoimmune animal models. It also may have potential therapeutic functions in autoimmune disease patients. Keywords: Brown adipose tissue, Rheumatoid arthritis, Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19), Multiple sclerosis (MS), helper T17 (Th17) cell, Interleukin-17, Coenzyme Q10 Student Number: 2015-22085 (These data were published in Scientific Reports. 2020 Jul 23;10(1):12374. and Immune Network. 2020 Oct 19;20(5):e40.) | - |
| dc.description.abstract | Th17 세포 억제를 통한 갈색지방과 GRIM-19의 자가면역질환 완화효과
문정현 종양및발달생물학 전공 치의과학과 서울대학교 대학원 (지도교수: 노상호, D.V.M., Ph.D.) 배경: 갈색지방조직은 백색지방조직과는 다른 여러 요인을 분비하는 것으로 알려져 있으며 많은 수의 미토콘드리아를 가지고 있다. 이는 갈색지방이 자가면역질환의 생리학적 관점에서 잠재적인 긍정적 이점을 가질 수 있음을 시사한다. Retinoid-interferon-induced mortality-19 (GRIM-19) 과 관련된 유전자에 의해 암호화된 단백질은 미토콘드리아 내막에 위치하며 전자 수송 사슬의 NADH dehydrogenase 1-alpha subcomplex subunit 13 과 함께 위치하며, 이는 미토콘드리아 기능에 중요한 역할을 한다. 다발성경화증(MS)은 뇌와 척수를 손상시키는 탈수 초성 질환이다. 다발성경화증과 류마티스 관절염 진행의 원인과 기전은 모두 불분명하지만, 면역 질환이 관련되어 있는 것으로 여겨진다. 유비퀴논으로도 알려진 코엔자임 Q10 (CoQ10)은 지용성 항산화제이다. CoQ10은 FDA에서 의약품으로 승인되지 않았 지만식이 보조제에 널리 사용된다. 이는 미토콘드리아 기능과 상태를 증가시키는 것으로 알려져 있다. 종합적으로 이 연구는 갈색지방이식, GRIM-19 과발현 및 CoQ10을 사용한 미토콘드리아 조절의 치료 효과에 초점을 맞추었다. 실험1: 이 연구는 콜라겐유도 관절염 마우스 유래의 갈색지방 (CIA BAT)의 기능을 정상 마우스 유래 갈색지방 (Nor BAT) 과 비교하였다. 6-7주령의 DBA/1J 마우스는 complete Freunds adjuvant와 추가 면역반응을 위한 incomplete Freunds adjuvant에 유화된 100μg의 제 Ⅱ형 콜라겐 (CⅡ)을 꼬리 기저의 피부 내 주사를 통해 면역반응을 유도하였다. 이 후에 CIA BAT 과 Nor BAT 은 각각 콜라겐유도 관절염 마우스에 이식되었다. 이식 후 생쥐에서 CIA BAT 과 Nor BAT의 항 염증기능을 측정하였다. Nor BAT을 이식한 마우스는 대조군과 CIA BAT을 이식한 마우스보다 뼈 손상, 염증 및 연골 손상 임상 점수가 현저히 낮게 측정되었다. Nor BAT을 이식한 마우스에서는 대조군과 CIA BAT을 이식한 마우스에 비해서 IL-12, IL-17, IL-6 및 종양괴사인자-α (TNF- α) 와 같은 염증성 사이토카인이 감소하는 것을 확인하였다. 마이크로어레이 분석은 PI3K-AKT 신호 전달 경로와 IL-17 생성경로의 발현 수준이 CIA BAT에서 Nor BAT 보다 유의미하게 높았다. 실험2: GRIM-19이 염증성 사이토카인 및 면역세포를 조절하여 MS를 개선했는지 여부를 조사하였다. 이를 위해 실험적자가면역 뇌척수염 (EAE)의 마우스 모델을 사용하였다. 6-8 주령 수컷 C57BL/6, IFNƳ 넉아웃 (KO) 및 GRIM-19 과발현 형질 전환 마우스를 사용하였고 EAE는 모든 군에서 유도되었다. GRIM-19 과발현 벡터를 정맥주사 한 직후 전기충격을 통해 마우스 체내에 주입하였다. IL-17A 및 IFNƳ 발현 수준은 ELISA 및 정량적 PCR을 통해 평가되었다. GRIM-19 과발현 벡터를 주사 한 EAE 마우스에서 IL-17A 발현이 감소하고 IFNƳ 발현이 증가하였다. GRIM-19 과발현 형질 전환 마우스는 야생형 마우스보다 더 많은 IFNƳ를 발현하였고 EAE의 발달이 억제되는 것을 확인하였다. 그러나, IFNƳ KO 마우스에 대한 GRIM-19 과발현의 효과는 대조군 벡터의 효과와 유의미한 차이점을 가지지 않았다. GRIM-19 발현은 IFNƳ 발현을 증가시키고 Th17/Treg 세포의 균형을 조절하였다. 실험3: 일부 연구에서 CoQ10은 미토콘드리아 기능의 향상을 통해 다양한 자가면역질환에 항 염증 효과가 있음을 증명하였다. 이 연구는 CoQ10의 RA 동물모델에서 항 염증 효과를 조사했다. CoQ10은 CIA 마우스에게 10 주 동안 경구 투여되었다. 이는 CoQ10 투여 마우스에서 염증성 사이토카인이 현저하게 감소했음을 보여주었다. Th17 세포 및 인산화 된 STAT3 발현 세포 집단도 CoQ10이 투여된 마우스에서 감소되었다. 인간 말초 혈액 단핵 세포 (PBMC)에 CoQ10을 처리했을 때 CoQ10 처리 군에서 PBMC의 IL-17 발현이 현저하게 감소했다. 결론: 이 연구는 정상 갈색지방 이식, GRIM-19 과발현 및 CoQ10 주입이 자가면역 동물모델에서 치료효과가 있음을 시사한다. 또한 자가면역질환 환자에서 잠재적인 치료 기능을 가질 수 있음을 확인하였다. 주요어: 갈색지방조직, 류마티스관절염, Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19), 다발성경화증, 헬퍼T세포17 (Th17), 인터루킨-17, 코엔자임 Q10 학번: 2015-22085 | - |
| dc.description.tableofcontents | Table of Contents
Abstract ⅰ Table of Contents ⅴ List of Tables ⅷ List of Figures ⅸ Chapter 1. Literature Review 1 1.1 Characteristic of adipose tissue and Retinoid-Interferon-Induced Mortality-19 (GRIM-19)………………………………..2 1.1.1 Characteristic of each fat tissue type 1.1.2 GRIM-19 and mitochondria 1.1.3 Coenzyme Q10 1.2 Inflammatory disease and T helper 17 cell………………………5 1.2.1 Rheumatoid arthritis (RA) 1.2.2 Multiple sclerosis (MS) 1.2.3 T helper 17 cell (Th17) and interleukin 17 (IL-17) 1.3 Objective of the study…………………………………………………8 Chapter 2. Brown adipose tissue ameliorates autoimmune arthritis via inhibition of Th17 cells ….9 2.1 Introduction……………………………………………………………..10 2.2 Materials and methods……………………………………………….13 2.2.1 Animals 2.2.2 Type II collagen (CII) immunization and induction of CIA 2.2.3 Assessment of arthritis 2.2.4 Immunohistochemistry 2.2.5 Histological assessment of arthritis 2.2.6 Confocal microscopy 2.2.7 Flow cytometry 2.2.8 Murine T-cell isolation and differentiation 2.2.9 Co-culture of Th17 cells with adipocytes 2.2.10 Enzyme-linked immunosorbent assay (ELISA) 2.2.11 Western blotting 2.2.12 Transplantation of BAT 2.2.13 Microarray analysis 2.2.14 Statistical analysis 2.3 Results…………………………………………………………………...21 2.3.1 Experimental RA requires normal BAT activity for therapy 2.3.2 Transplantation of normal brown adipose tissue ameliorates proinflammatory cytokines and impacts anti-inflammatory cytokine secretion 2.3.3 Normal BAT ameliorates balance of Th17/Treg cells 2.3.4 Brown adipose tissue inhibits IL-17 secretion and strongly induces IL-10 expression 2.3.5 Comparison of gene expression between normal BAT with CIA BAT in murine 2.3.6 PI3K-AKT signaling-associated genes and IL-17 signaling-associated genes were highly expressed in CIA BAT, compared to normal BAT 2.4 Discussion………………………………………………………………48 Chapter 3. GRIM-19 ameliorates multiple sclerosis in a mouse model of experimental autoimmune encephalomyelitis with reciprocal regulation of IFNγ/Th1 and IL-17A/Th17 cells 52 3.1 Introduction……………………………………………………………53 3.2 Materials and methods………………………………………………56 3.2.1 Animals 3.2.2 Experimental autoimmune encephalomyelitis (EAE) model 3.2.3 Injection of GRIM-19 3.2.4 Immunohistochemistry 3.2.5 Immunostaining for confocal microscopy 3.2.6 Western blotting 3.2.7 Quantitative polymerase chain reaction (qPCR) analysis 3.2.8 Measurement of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) by immunofluorescence 3.2.9 Measurement of intracellular reactive oxygen species (ROS) and mitochondrial superoxide production 3.2.10 Flow cytometry 3.2.11 T-cell isolation, Th17 differentiation, and enzyme-linked immunosorbent assays (ELISAs) 3.2.12 Statistical analysis 3.3 Results…………………………………………………………………...65 3.3.1 The severity of EAE pathology was reduced by GRIM-19 overexpression via reduction of IL-17A levels 3.3.2 GRIM-19 overexpression was therapeutically efficacious 3.3.3 EAE pathology was inhibited in GRIM TG mice via elevation of the IFNγ level and Treg cell numbers 3.3.4 The mitochondrial function was improved in GRIM19 TG mice 3.3.5 EAE pathology was not inhibited in IFNγ-deficient mice injected with the GRIM-19 vector 3.3.6 GRIM-19 overexpression inhibited the progression of high fat-induced obesity 3.4 Discussion………………………………………………………………86 Chapter 4. CoenzymeQ10 suppressed rheumatoid arthritis via STAT3/Th17 targeting 90 4.1 Introduction……………………………………………………………91 4.2 Materials and methods………………………………………………93 4.2.1 Animals 4.2.2 Arthritis induction and treatment 4.2.3 Clinical assessment of arthritis 4.2.4 Histological analyses 4.2.5 Immunohistopathological analyses 4.2.6 Confocal microscopy of immunostaining 4.2.7 Cell culture 4.2.8 Statistical analyses 4.3 Results…………………………………………………………………...98 4.3.1 The pathogenesis of rheumatoid arthritis was suppressed by CoQ10 in CIA mice 4.3.2 Expression of pro-inflammatory cytokines was decreased in joints of CoQ10-treated mice 4.3.3 Expression of IL-17 and phosphorylated STAT3 was reduced by CoQ10 in spleen CD4+ T cells 4.3.4 IL-17 levels decreased in peripheral blood mononuclear cells following treatment with CoQ10 4.4 Discussion…………………………………………………………….106 Chapter 5. Conclusion 108 References 111 Abstract in Korean 123 List of Tables Table 2.1. The significant pathways in CIA BAT compared to nor BAT….35 Table 2.2. The gene list which has significant changes in CIA BAT compared to nor BAT……………………………………………………...37 Table 3.1. The PCR primers used………………………………………….61 List of Figures Figure 2.1. Comparison of phenotypes of by fat type……………………..22 Figure 2.2. Transplantation of normal brown adipose tissue reduces tissue damage, inflammation, and development of experimental rheumatoid arthritis…………………………………………………………..…………23 Figure 2.3. Transplantation of normal brown adipose tissue reduces secretion of proinflammatory cytokines…………………………………...25 Figure 2.4. Populations of Th17 and Treg cells were reduced by transplantation of normal BAT in CIA mice..……………………………...27 Figure 2.5. Splenic tissue from each group of mice was monitored by immunofluorescence staining.……………………………………………..28 Figure 2.6. BAT inhibits Th17 differentiation and induces IL-10 expression in vitro.……………………………………………………………………..30 Figure 2.7. Comparison of gene expression between normal BAT and CIA BAT in mice by microarray analysis..……………………………………...32 Figure 2.8. Comparison of genetic function between normal BAT and CIA BAT in mice by microarray analysis..…………………………….………..33 Figure 2.9. The protein expression levels of normal BAT and CIA BAT…36 Figure 2.10. KEGG pathway of IL-17 production in the comparison of CIA BAT and nor BAT………………………………………………………….46 Figure 2.11. KEGG pathway of NF-κB in the comparison of CIA BAT and nor BAT……………………………………………………………………47 Figure 3.1. The IL-17A level decreased and that of IFNγ increased in mice with experimental autoimmune encephalomyelitis (EAE) that overexpressed GRIM-19…………………………………………………………………...66 Figure 3.2. After 5 weeks, the mice were sacrificed and the splenocytes of the mice were isolated and analyzed..……………………………………..67 Figure 3.3. The IL-17A, IL-10, and IFNγ levels (as revealed by ELISA) in the culture media of mouse splenocytes cultured under conditions triggering Th17 differentiation……………………………………………………..…68 Figure 3.4. After end of the experiment, immunofluorescent spleen images derived via confocal microscopy………………………………………..…70 Figure 3.5. GRIM-19 overexpression was therapeutic in EAE mice……..71 Figure 3.6. The H&E images showed the hippocampal CA1-CA2 region..72 Figure 3.7. The development of EAE pathology was inhibited in GRIM-19 transgenic (GRIM19 TG) mice..……………………………………….…..75 Figure 3.8. The population of proinflammatory cells was inhibited by GRIM-19 transgenic mice…………………………………………………76 Figure 3.9. The H&E staining monitored the hippocampal region……….77 Figure 3.10. GRIM-19 overexpression induces the mitochondrial functions…………………………………………………………………...79 Figure 3.11. The pathology of EAE did not differ significantly in IFNγ-deficient mice given GRIM-19 gene therapy, compared to those that were not………………………………………………………………………….81 Figure 3.12. The GRIM-19 has no immune regulation effect on IFNγ-deficient condition…………………………………………………………82 Figure 3.13. Expression of GRIM19 protects against diet-induced obesity……………………………………………………………………...84 Figure 3.14. Analysis of fat tissue weight in GRIM-19 TG and wild type mice………………………………………………………………………..85 Figure 4.1. Pathology scores of CoQ10-injected mice……………………99 Figure 4.2. Immunohistochemistry of joint tissues in vehicle- and CoQ10-injected mice……………………………………………………………...101 Figure 4.3. Immunofluorescence of spleen tissues………………………103 Figure 4.4. IL-17 levels were detected by ELISA in human peripheral blood mononuclear cells (PBMC) in anti-CD3 conditions……………………...105 | - |
| dc.format.extent | 138 | - |
| dc.language.iso | eng | - |
| dc.publisher | 서울대학교 대학원 | - |
| dc.subject | Brown adipose tissue | - |
| dc.subject | Rheumatoid arthritis | - |
| dc.subject | Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19) | - |
| dc.subject | Multiple sclerosis (MS) | - |
| dc.subject | helper T17 (Th17) cell | - |
| dc.subject | Interleukin-17 | - |
| dc.subject | Coenzyme Q10 | - |
| dc.subject | 갈색지방조직 | - |
| dc.subject | 류마티스관절염 | - |
| dc.subject | 다발 성경화증 | - |
| dc.subject | 헬퍼T세포17 (Th17) | - |
| dc.subject | 인터루킨-17 | - |
| dc.subject | 코엔자임 Q10 | - |
| dc.subject.ddc | 617.6 | - |
| dc.title | Brown adipose tissue and GRIM-19 attenuate autoimmune diseases via inhibition of Th17 cells | - |
| dc.title.alternative | Th17 세포 억제를 통한 갈색지방과 GRIM-19의 자가면역질환 완화효과 | - |
| dc.type | Thesis | - |
| dc.type | Dissertation | - |
| dc.contributor.AlternativeAuthor | Jeonghyeon Moon | - |
| dc.contributor.department | 치의학대학원 치의과학과 | - |
| dc.description.degree | Doctor | - |
| dc.date.awarded | 2021-02 | - |
| dc.contributor.major | 종양및발달생물학 | - |
| dc.identifier.uci | I804:11032-000000163828 | - |
| dc.identifier.holdings | 000000000044▲000000000050▲000000163828▲ | - |
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