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천식기전에서 호중구 활성의 역할
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 박해심 | - |
| dc.contributor.author | PHAM, DUY LE | - |
| dc.date.accessioned | 2019-10-21T07:27:49Z | - |
| dc.date.available | 2019-10-21T07:27:49Z | - |
| dc.date.issued | 2016-08 | - |
| dc.identifier.other | 22763 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/18859 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2016. 8 | - |
| dc.description.tableofcontents | CHAPTER-I Neutrophilic autophagy and extracellular DNA trap in severe asthma 1 I. INTRODUCTION 2 II. MATERIALS AND METHODS 5 A. Study subjects recruitment 5 B. Antibodies and reagents 6 C. Sputum induction and blood collection 6 D. Assessment of sputum neutrophil count 7 E. SNP selection and genotyping 7 F. Preparation of ATG5 constructs for promoter assays 8 G. Cell culture 8 H. Endogenous mRNA expressions of ATG5 and ATG7 10 I. Transient transfection and dual luciferase promoter assay 10 J. Peripheral blood neutrophil (PBN) and eosinophil (PBE) isolation from asthmatic patients 11 K. Induction of autophagy and NET from PBNs 12 L. Evaluation of autophagy and protein expressions by Western blot 12 M. Evaluation of NET production by PicoGreen assay 12 N. Confocal microscopy analysis 13 O. PBN migration assay 13 P. NET induction and isolation for target cell treatment 14 Q. Evaluating NET effects on A549 cells, SAECs and PBEs isolated from asthmatic patients 14 R. Evaluation of cell death and cell detachment 15 S. Evaluation of IL-8, ECP and eosinophil derived neurotoxin (EDN) in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA) 15 T. Statistical analysis 15 III. RESULTS 17 A. Clinical characteristics of ATG genetic study subjects 17 B. Allele, genotype and haplotype frequency of ATG5 and ATG7 polymorphisms 18 C. Clinical characteristics according to ATG5 and ATG7 polymorphisms 23 D. Transcriptional activity of ATG5 promoter polymorphisms 23 E. Clinical characteristics of the study subjects in neutrophilic autophagy and NET study 26 F. Autophagy and NET production from PBNs of SA and NSA patients 27 G. NET levels was positively correlated with autophagy levels and airway obstruction 30 H. Autophagy inhibitors reduced neutrophil migration 31 I. Isolated NET- induced airway epithelial cell death and detachment 32 J. NET-induced degradation of tight-junction (TJ) proteins in AECs is mediated by neutrophilic enzyme, NE and MPO 35 K. NET induced IL-8 production from A549 cells and SAECs 38 L. NET induced eosinophil degranulation and primary lysis 38 IV. DISCUSSION 41 CHAPTER-II Serum S100A8 and S1009 enhance neutrophilic inflammation in baker’s asthma 48 I. INTRODUCTION 49 II. MATERIALS AND METHODS 51 A. Study subjects 51 B. Skin prick tests and measurement of serum specific antibodies to wheat flour 51 C. TLR4 gene genotyping 52 D. ELISA to measure serum levels of S100A8, S100A9, myeloperoxidase (MPO), tumor necrosis factor (TNF )-α, and IL 8 52 E. Statistical analysis 52 III. RESULTS 54 A. Characteristics of the baker’s asthma study subjects 54 B. Increased serum levels of S100A8 and S100A9 in the bakery workers 54 C. Associations of serum S100A8 and S100A9 with several serum cytokines 58 D. Associations of serum S100A8 with TLR4 gene polymorphisms 60 IV. DISCUSSION 62 V. CONCLUSIONS 66 REFERENCES 67 -국문요약- 76 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 천식기전에서 호중구 활성의 역할 | - |
| dc.title.alternative | Roles of Neutrophil Activation in Asthma | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | DUY LE PHAM | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2016. 8 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 758561 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000022763 | - |
| dc.subject.keyword | Asthma | - |
| dc.subject.keyword | autophagy | - |
| dc.subject.keyword | baker’s asthma | - |
| dc.subject.keyword | genetic polymorphism | - |
| dc.subject.keyword | neutrophil | - |
| dc.subject.keyword | S100 protein | - |
| dc.description.alternativeAbstract | Background: Neutrophilic inflammation has been implicated in allergic asthma as well as occupational asthma, which is usually associated with severe phenotype. Autophagy plays crucial roles in innate as well as adaptive immune responses by contributing to the proliferation, activation and biological function of various immune cells such as macrophages, neutrophils, mast cells, T and B cells. Autophagy could regulate neutrophil survival and formation of neutrophil extracellular DNA trap (NET), which is an extracellular release of decondensed chromatin from neutrophils triggered by various stimuli including cytokines [e.g. intetleukin(IL)-8 or tumor necrosis factor (TNF)-α], phorbol 12-myristate 13-acetate (PMA) or microbial proteins. In allergic asthma, autophagy and NET were detected in asthmatic airways and inflammatory cells; however, their roles in pathogenesis of asthma have not yet been identified. S100A8 and S100A9 are Ca2+- and Zn2+-binding proteins of the S100 family, which could be produced by lipopolysaccharide-stimulated granulocytes and provoke an innate immune-mediated airway inflammation. S100A9 protein is known to be involved in uncontrolled neutrophilic asthma. The role of S100A8 and S100A9 in baker’s asthma pathogenesis has not been investigated. Objectives: This thesis has investigated: 1) associations of neutrophilic inflammation in asthma with genetic variants of autophagy related gene (ATG) 5 and ATG7; 2) levels of autophagy and NET production from peripheral blood neutrophils (PBNs) of severe asthma (SA) and non-severe asthma (NSA) patients, as well as the roles of NET in airway inflammation; 3) involvement of serum S100A8 and S100A9 in neutrophilic inflammation of occupational asthma induced by wheat flour Methods: In the first study, we recruited 408 asthma patients and 201 normal controls (NC) to a genetic association study of ATG5 and ATG7 variants with neutrophilic airway inflammation. ATG5 (-769T>C, -335G>A, and 8830C>T) and ATG7 (-100A>G and 25108G>C) polymorphisms were genotyped using the SNaPshot method. The transcriptional activities of ATG5 -769T>C and -335G>A variants were investigated by luciferase reporter assays. In the second study, 68 asthma patients (including 30 SA and 38 NSA patients) were recruited to investigate the production levels of neutrophilic autophagy and NET from PBNs. Isolated PBNs were treated with interleukin (IL)-8 (100 ng/ml). Autophagy (LC3 expression) and NET production levels were evaluated by Western bloting, immunofluorescent microscopy and PicoGreen assay. Cell survival and tight junction protein (occludin and claudin-1) expression levels in airway epithelial cells (AECs) were evaluated by CCK8 assay and Western blot analysis. Peripheral blood eosinophils (PBEs) were isolated from peripheral blood of human donors using MACS system. Secretion levels of IL-8, eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) were measured by ELISA. In baker’s asthma cohort, we recruited 381 bakery workers and 100 unexposed NCs. Skin prick tests for bakery allergens were performed to determine atopic status of study subjects. Serum levels of S100A8, S100A9, myeloperoxidase (MPO), tumor necrosis factor (TNF)-α, and IL-8 were measured using ELISA. Predictive values of serum S100A8 and S100A9 in bakery workers were estimated by receiver-operating characteristic (ROC) curves. Polymorphisms of toll like receptor 4 gene (TLR4) -2027A>G and -1608T>C, were genotyped using the SnaPshot method. Results: In asthma patients, the GA/AA genotypes at ATG5 -335G>A SNP were significantly associated with higher neutrophil counts in sputum (P<0.05); the CC/TT genotype at ATG5 8830C>T was significantly associated with lower FEV1% predicted value (P<0.05). Promoter activity assay revealed that DNA fragments containing ATG5 -769T and -335G alleles exhibited significantly higher promoter activities compared to those with -769C and -335A in both human AECs (A549, P<0.01) and human mast cells (HMC-1, P<0.001). GG and CC genotype at ATG7 -100A>G and 25108G>C were significantly associated with high serum levels of IL-8 (P<0.001 and P<0.00, respectively). In the second study, naïve and IL-8 treated neutrophils of SA patients expressed higher autophagy and NET production levels than NSA patients (P<0.001 for both experiments). IL-8 increased autophagy and NET levels in the PBNs of SA, but not in the NSA group. NET levels correlated with autophagy levels in PBNs (P <0.001). Levels of IL-8 induced NET production negatively correlated with FEV1/FVC (r=-0.700, P=0.016). NET dose-dependently induced cell death, cell detatchment, degradation of tight-junction proteins and IL-8 production from AECs dose dependently. NET-induced ECP and EDN releases were higher in PBEs of SA patients compared to NSA patients. In baker’s asthma study, the bakery workers had higher serum levels of S100A8 and S100A9 compared to the NCs (P<0.001); however, no significant differences were noted according to work-related symptoms. The area under the ROC curve of serum S100A8 was 0.886 for occupational exposure (P< 0.001). The CC genotype at TLR4 -1608T>C was significantly associated with a higher serum S100A8 level (P=0.025). Serum S100A8 and S100A9 levels were correlated with serum levels of MPO (r=0.396 and 0.189, respectively), TNF-α (r=0.536 and 0.280, respectively), and IL-8 (r=0.540 and 0.205, respectively; P<0.001 for all). Conclusions: This thesis demonstrated the role of neutrophils in the airway inflammatory mechanism of adult asthma. Neutrophilic airway inflammation in asthma could be regulated by genetic polymorphisms of ATG5 and ATG7. Neutrophilic autophagy and NET could enhance asthma severity by damaging airway epithelium and triggering inflammatory responses of AECs and eosinophils. Modulating neutrophil autophagy and NET production may be a new therapeutic strategy for SA treatment. In baker’s asthma, S100A8 and S100A9 could be involved in neutrophilic inflammation of airway under the regulation of TLR4 polymorphisms. Additionally, serum S100A8 level could be a potential biomarker for predicting occupational exposure to wheat flour in bakery workers. | - |
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