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The Association of CD83, Noise Stress, and Gut Microbiota in Systemic Inflammation of Herpes Simplex Virus-1 Induced Behçet’s Disease Mouse Model
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | Sohn Seonghyang | - |
| dc.contributor.author | S M SHAMSUL ISLAM | - |
| dc.date.accessioned | 2022-11-29T03:01:31Z | - |
| dc.date.available | 2022-11-29T03:01:31Z | - |
| dc.date.issued | 2022-08 | - |
| dc.identifier.other | 31922 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/21254 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2022. 8 | - |
| dc.description.abstract | PART I 단순 헤르페스 바이러스-1으로 유도된 베체트병 쥐에서 CD83 억제로 베체트병 증상의 개선 베체트병 (Behcet's disease, BD) 은 구강 궤양, 생식기 궤양, 피부 염증 및 포도막염의 재발을 특징으로 하는 만성 전신 염증성 질환이다. 장, 혈관 및 중추신경계에 잠재적으로 생명을 위협할 수 있는 다른 병변도 발생할 수 있다. BD 표현형의 이러한 이질성은 표적 치료 전략의 발전을 방해한다. BD의 발병기전은 완전히 이해되지는 않았지만 본 연구의 목적은 BD에서 공동 자극 인자의 연관성을 확인하는 것이다. ICR 마우스에 Herpes simplex virus type1 (HSV-1) 을 접종하면 BD 유사 증상이 발생한다. 공동 자극 분자의 세포 빈도는 유세포 분석으로 분석하였다. CD83+ 세포를 억제하기 위한 CD83 siRNA 형질주입은 BD 증상이 있는 마우스로 수행되었다. 수지상 세포의 형태적 변화는 투과전자현미경으로 조사하였고 혈장 사이토카인 수치는 효소결합면역흡착법으로 측정하였다. CD83+ 세포의 빈도는 무증상 BD 마우스와 비교하여 BD 마우스의 말초 혈액 백혈구 (PBL)에서 유의하게 증가하였다. CD83+ 세포의 빈도는 건강한 대조군과 비교하여 활성 BD 환자에서 유의하게 증가했다. BD 증상이 있는 마우스에서 CD83 siRNA의 복강 내 주사는 PBL 및 복막 대식세포에서 CD83+ 세포의 빈도를 유의하게 감소시켰고 BD 유사 증상을 개선했다. CD83 siRNA에 의한 CD83+ 세포의 억제는 수지상세포 성숙을 저해하였다. CD83 siRNA주입 중단은 생쥐에서 BD의 증상을 악화시켰고, CD83 siRNA의 재투여는 증상을 개선시켰다. 이러한 결과는 BD의 염증 증상에서 CD83+ 세포의 관련성을 분명히 보여준다. PART II 환경, 스트레스 및 단순 포진 바이러스-1과 베체트병 마우스 모델의 연관성 베체트병 (Behcet's disease, BD) 은 재발성 피부 점막 병변, 생식기 아프타 병변, 안구 염증, 중추신경계 침범 및 혈관염을 유발하는 원인이 알려지지 않은 전신 혈관염이며 만성, 재발 완화 과정을 갖는 경향이 있다. 본 연구의 목적은 Herpes simplex virus type-1 (HSV-1)에 의해 유도된 BD 마우스 모델에서 BD의 유도에 영향을 미치는 스트레스 및 환경적 요인의 영향을 조사하는 것이다. ICR 마우스는 HSV-1을 접종한 후 BD와 유사한 증상이 나타난다. 환경적 요인이 BD 발병에 미치는 영향을 조사하기 위해 HSV-1 접종 마우스를 conventional 및 SPF 시설에서 유지하였다. BD의 발생률과 면역 조절을 조사하기 위해 conventional 시설에 있는 마우스에 여러 스트레스 요인을 추가로 유도했다. 전사 인자 T-bet, Gata-3, Foxp3 및 Ror-gt mRNA 수준은 qRT-PCR로 확인하였다. 장내 미생물군에 대한 연구는 미생물군 구성의 차이를 확인하기 위해 마우스의 대변에서 16S rRNA 시퀀싱으로 수행되었다. 공동 자극 인자의 BD발생 빈도는 유세포 분석으로 분석되었다. Conventional 조건에서 HSV-1 접종 마우스에 소음 스트레스 유도는 HSV-1 접종 마우스 (15%) 보다 BD 발생률 (38.1%) 이 더 높았다. 또한 소음 스트레스는 CD83+ 세포의 빈도를 증가시켰고 전사 인자 T-bet 및 Ror-gt도 상향 조절했다. CD83 siRNA의 복강내 주사에 의한 CD83+ 세포의 억제는 소음 유발된 BD 증상이 있는 마우스에서 BD 유사 증상을 개선했다. 장내 미생물 조성의 차이는 SPF 및 conventional 조건에서 유지되는 정상 마우스에서도 관찰되었다. 이 결과는 HSV에 의해 유발된 BD의 발병률이 환경과 스트레스 요인에 의해 영향을 받는다는 것을 보여준다. PART III Eubacterium rectale은 CD83을 조절하여 의 HSV-1 유도된 베체트병 마우스의 전신 염증을 호전시킨다 이 연구의 목적은 미생물 Eubacterium rectale (E. rectale) 의 투여가 공동 자극 분자를 조절하고 Herpes simplex virus-1 (HSV-1) 유도된 베체트병 (Behçet’s disease, BD) 에서 전신 염증을 완화할 수 있는지 여부를 결정하는 것이다. BD는 원인을 알 수 없는 만성 다발성 염증 질환이다. 환경 요인, 면역 기능 장애 및 HSV 감염이 BD를 유발할 수 있다. E. rectale의 효과를 조사하기 위해 ICR 마우스에 HSV-1을 접종하여 BD 유사 증상이 발생하도록 하였다. 부티레이트 분비 세균인 E. rectale을 BD 증상이 있는 마우스에 경구 투여하여 임상 증상을 관찰하였다. 혈장 사이토카인 수준은 효소 결합 면역흡착 분석법으로 측정했으며, 장내 미생물총에 대한 연구는 마우스의 대변에서 16S rRNA 시퀀싱으로 수행하여 정상 마우스와 BD 마우스 간의 미생물 집단 구성의 차이를 확인하였다. 공동 자극 분자 및 조절 T 세포의 빈도는 유세포 분석으로 분석하였다. 부티레이트의 투여는 BD 마우스에서 CD83+ 세포의 빈도를 감소시켰다. 부티레이트를 분비하는E. rectale을 BD 마우스에 투여하면 CD83+ 세포의 빈도가 감소하고 BD 증상이 개선되면서 NK1.1+ 세포의 빈도가 크게 증가했다. CD83+ 세포의 빈도는 BD 마우스에 콜히친과 대장균을 병용 투여한 후에도 감소했다. 정상 및 BD 증상 마우스에서 장내 미생물 조성의 차이가 관찰되었다. 이러한 결과는 E. rectale이 BD 관리의 보충제로 사용될 수 있음을 시사한다. | - |
| dc.description.tableofcontents | PARTI 1 1. INTRODUCTION 2 2. MATERIALS AND METHODS 5 2.1. Animals 5 2.2. BD symptomatic mice induced by HSV-1 5 2.3. Severity score of BD mouse 6 2.4. Patients clinical characteristics 7 2.5. Preparation and treatment of CD83 siRNA to mice 8 2.6. Generation of mouse bone-marrow derived DCs and transfection of CD83 siRNA 9 2.7. Flow cytometry analysis 9 2.8. Morphological observation under transmission electron microscopy 10 2.9. Measurement of cytokines concentration by ELISA 11 2.10. Statistical analysis 11 3. RESULTS 12 3.1. Frequencies of CD40, CD83, CD80, and CD86 expressing cells in normal, HSV-1 infected, BD normal (BDN), and BD mice 12 3.2. Frequency of CD83+ cells in healthy control, active BD, and inactive BD patients 15 3.3. Clinical characteristics and therapeutic history of Behçet’s disease patients 18 3.4. CD83 siRNA suppresses CD83+ cells in normal mice 21 3.5. CD83 siRNA treatment affects BD symptoms and decreases disease severity score in mice 24 3.6. CD83 siRNA suppresses CD83+ cell expression in bone marrow cells and modulates the morphology of DCs 29 3.7. Plasma IL-17 levels in BD mice treated with CD83 siRNA 32 4. DISCUSSION 34 5. CONCLUSION 37 PARTII 38 1. INTRODUCTION 39 2. MATERIALS AND METHODS 42 2.1. Animals 42 2.2. Induction of BD symptomatic mice by HSV-1 42 2.3. Severity score of BD mouse 43 2.4. Induction of stressors to normal and BD symptomatic mice 44 2.5. Analysis of microbiota in mouse feces by 16S rRNA sequencing 45 2.6. Taxonomy profiling, OTUs, alpha-diversity, and beta-diversity 46 2.7. Flow cytometry analysis 46 2.8. RNA extraction and quantitative RT-PCR 47 2.9. Preparation and treatment of CD83 siRNA to mice 50 2.10. Statistical analysis 51 3. RESULTS 52 3.1. Effects of stress and environment on the incidence of BD 52 3.2. Altered gut microbial compositions in normal mice under conventional and SPF conditions 54 3.3. Identification of several stressors that influence the expression of co-stimulatory molecules in mice 59 3.4. Noise stress upregulates co-stimulatory molecules in HSV-1 infected state 63 3.5. Noise stress modulates co-stimulatory molecules in BD symptomatic mice 65 3.6. Inhibition of CD83 expression ameliorates BD symptoms even after exposure to noise stress 68 4. DISCUSSION 71 5. CONCLUSION 75 PARTIII 76 1. INTRODUCTION 77 2. MATERIALS AND METHODS 80 2.1. Animals 80 2.2. BD symptomatic mice induced by HSV-1 80 2.3. Severity score of BD mouse 81 2.4. Analysis of microbiota in normal and BD mice feces by 16S rRNA sequencing 82 2.5. Taxonomy profiling, OTUs, alpha-diversity, and beta-diversity 83 2.6. Bacteria culture and preparation of lyophilized E. rectale 84 2.7. Experimental scheme and administration of E. rectale to mice 83 2.8. Flow cytometry analysis 85 2.9. Measurement of plasma cytokine IL-17 concentration by ELISA 86 2.10. Statistical Analysis 86 3. RESULTS 87 3.1. Altered gut microbial composition in HSV-1-induced BD symptomatic mice 87 3.2. Probiotic application and In vivo study design 94 3.3. Butyrate treatment reduces the frequency of CD83+ cells and improves symptoms in BD mice by increasing Treg cells 95 3.4. Dose dependence of E. rectale on the frequencies of co-stimulatory molecule expressing cells in normal and BD mice 100 3.5. Frequencies of Treg cells in E. rectale treated BD mice 105 3.6. E. rectale treatment increase NK cells in BD mice 108 3.7. E. rectale treatment reduces disease severity scores and improves symptoms in BD mice 110 3.8. Co-administration of colchicine and E. rectale downregulates CD83 in BD mice 112 3.9. Lyophilized E. rectale reduces the frequency of CD83+ cells in BD mice 116 3.10. Plasma IL-17 levels in BD mice treated with E. rectale 118 4. DISCUSSION 120 5. CONCLUSION 124 REFERENCES 125 ABSTRACT IN KOREAN LANGUAGE 137 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | The Association of CD83, Noise Stress, and Gut Microbiota in Systemic Inflammation of Herpes Simplex Virus-1 Induced Behçet’s Disease Mouse Model | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2022. 8 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 1254163 | - |
| dc.identifier.uci | I804:41038-000000031922 | - |
| dc.identifier.url | https://dcoll.ajou.ac.kr/dcollection/common/orgView/000000031922 | - |
| dc.subject.keyword | Behçet’s disease | - |
| dc.subject.keyword | CD83 | - |
| dc.subject.keyword | Eubacterium rectale | - |
| dc.subject.keyword | Inflammation | - |
| dc.subject.keyword | Microbiota | - |
| dc.subject.keyword | Mouse model | - |
| dc.subject.keyword | Noise stress | - |
| dc.description.alternativeAbstract | Part I CD83 Inhibition Ameliorates Behçet’s Disease-Like Symptoms in Herpes Simplex Virus-1 Induced Behçet’s Disease Mice Behçet’s disease (BD) is a chronic systemic inflammatory disease characterized by relapses of mouth ulcers, genital ulcers, skin inflammation, and uveitis. Other potentially life-threatening lesions in the intestine, blood vessels, and central nervous system may also develop. This heterogeneity of the BD phenotype hinders the advancement of targeted therapeutic strategies. The purpose of the study was to identify the association of co-stimulatory molecules with severity of symptoms of BD. BD-like symptoms were induced in ICR mice by inoculation with herpes simplex virus (HSV-1). The frequencies of co-stimulatory molecules were analyzed by flow cytometry. CD83 siRNA transfections to BD symptomatic mice were carried out to inhibit CD83 expression. Morphological changes of dendritic cells (DCs) were studied by transmission electron microscopy and blood plasma cytokine levels were measured by enzyme-linked immunosorbent assay. The frequency of CD83+ cells was significantly increased in peripheral blood leukocytes (PBL) of BD mice, compared with asymptomatic BD normal mice. The frequency of CD83+ cells was also significantly elevated in patients with active BD compared with healthy controls. In BD symptomatic mice, intraperitoneal injection of CD83 siRNA significantly reduced the frequency of CD83+ cells in PBL and in peritoneal macrophages, injection of CD83 siRNA improved BD-like symptoms. Transfection of CD83 siRNA into bone marrow monocytes interrupt DCs maturation but discontinuation of CD83 siRNA injection exacerbated the BD-like symptoms whereas readministration of CD83 siRNA improved the symptoms. These results clearly show the involvement of CD83 expression in the inflammatory symptoms of BD. Part II Association of Environment and Stress with the Incidence of Behçet’s Disease in Herpes Simplex Virus-1 Infected Mouse Model Behçet’s disease (BD) is a systemic vasculitis of unknown etiology that causes recurrent mucocutaneous lesions, genital aphthous lesions, ocular inflammation and central nervous system involvement, and tends to have a chronic, relapsing-remitting course. The goal of this study was to investigate the effects of stress and environmental factors that affected the induction of BD in the herpes simplex virus-1 (HSV-1) induced BD mouse model. To investigate the effects of environmental factors on the incidence of BD, institute of cancer research mice after HSV-1 inoculation were maintained in conventional and specific pathogen free (SPF) facilities. In conventional facility, mice were subjected to induced stressors and the incidence of BD and immune regulations were investigated. Transcription factors T-bet, Gata-3, Foxp3, and Ror-gt mRNA levels were studied by qRT-PCR. Studies on gut microbiota were conducted by 16S rRNA sequencing in the feces of mice to determine the differences in the composition of the microbial populations. The frequencies of co-stimulatory molecules were analyzed by flow cytometry. Induction of noise stress to HSV-1 inoculated mice in conventional facility caused higher incidence of BD (38.1%) than only HSV-1 inoculation did (15%). Differences in gut microbial composition were also observed in normal mice maintained in SPF and conventional conditions. Additionally, noise stress increased the frequency of CD83+ cells and the mRNA expression of transcription factors T-bet as well as Ror-gt. Inhibition of CD83+ cells by intraperitoneal injection of CD83 siRNA improved BD-like symptoms in noise-induced BD symptomatic mice. Our results showed that the incidence of HSV-1 induced BD was influenced by the environment and stressors. Part III Eubacterium Rectale Modulates CD83 expression to Attenuate HSV-1 Induced Behçet’s disease-Like Systemic Inflammation in Mice The purpose of this study was to determine whether administration of the microorganism Eubacterium rectale (E. rectale) could modulate co-stimulatory molecules expression and alleviate systemic inflammation in herpes simplex virus type-1 (HSV-1) induced Behçet’s disease (BD). BD is a chronic multisystemic inflammatory disease of unknown cause. Environmental factors, immunological dysfunction, and HSV infection may trigger BD. To investigate the effects of E. rectale, ICR mice were inoculated with HSV-1 to development of BD-like symptoms. E. rectale, a butyrate secreting bacterium was orally administered to BD symptomatic mice, and clinical manifestations were observed. Plasma cytokine levels were measured by enzyme-linked immunosorbent assay, and studies on gut microbiota were conducted by 16S rRNA sequencing in the feces of mice. The frequencies of co-stimulatory molecules and regulatory T cells were analyzed by flow cytometry. Differences in gut microbial composition in normal and BD symptomatic mice were observed. Administration of butyrate decreased the frequency of CD83+ cells in BD mice. The administration of butyrate secreting E. rectale to BD mice reduced the frequency of CD83+ cells and significantly increased the frequency of NK1.1+ cells with an improvement of BD symptoms. The frequency of CD83+ cells was also decreased after the co-administration of colchicine and E. rectale to BD mice. Collectively, E. rectale administration reduces CD83 expression and inflammation in BD model mice, thus these findings suggest that E. rectale can be used as a supplement in the management of BD. | - |
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