Microbiome-derived Extracellular Vesicles Targeting Neutrophilic Asthma
- 주제(키워드) Asthma , Bacteria , Extracellular vesicle , Lactobacillus paracasei , Microbiome , Micrococcus luteus , Neutrophil
- 주제(DDC) 570
- 발행기관 아주대학교 일반대학원
- 지도교수 예영민
- 발행년도 2024
- 학위수여년월 2024. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000033447
- 본문언어 한국어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Bacterial extracellular vesicles (EVs) have been shown to regulate various diseases, but their function in asthma remains unclear. Therefore, this study aimed to evaluate the clinical significance of bacterial EVs in specific asthma phenotypes, to identify key molecules in EVs, and to investigate a potential therapeutic benefit of EVs for controlling airway inflammation. To measure relative abundance of EVs derived from Micrococcus luteus (MlEV) and Lactobacillus paracasei (LpEV) using ELISA for their specific IgG4 antibodies in serum, 65 asthmatic patients and 50 healthy controls (HCs) were enrolled. As a result, lower levels of MlEV- and LpEV-specific IgG4 were noted in asthmatic patients than in HCs, as well as in patients with neutrophilic asthma (NA) than in those with eosinophilic asthma (EA). Moreover, the serum levels IgG4 specific to MlEV and LpEV were positively correlated with baseline FEV1 (%). In asthmatic C57BL/6 mice induced by LPS, an improvement in neutrophilic inflammation in the airway by MlEV and LpEV was noted. Especially, MlEV reduced IL-1β and IL-17 production in BALF as well as the number of group 3 innate lymphoid cells (ILC3s) in the lungs. Moreover, LpEV decreased not only CXCL1 and IL-17 concentration in BALF but the proportion of Th17 cells in the lungs. In airway epithelial cells (AECs), MlEV and LpEV suppressed the release of IL-8, a chemoattract for neutrophils, by inhibiting NFκB and JNK pathway, respectively. In particular, MlEV were found to regulate miRNA composition in AECs. Among them, hsa-miR-4517 reduced IL-1β secretion via inhibiting NLRP3 inflammasome in monocytes, resulting in the inactivation of IL-17-producing ILC3s. Furthermore, metabolites present in LpEV were also found to decrease IL-8 released from AECs by suppressing JNK phosphorylation. In summary, MlEV and LpEV could suppress neutrophil inflammation in the airway by reducing neutrophil chemoattractant and various pro-inflammatory cytokines. Especially, their suppressive effect on airway inflammation might be related to pathophysiology of AECs by regulating expression of miRNAs or activation of signaling pathways. Therefore, MlEV and LpEV could be novel therapeutic agents, providing an insight into asthma treatment by using microbial EVs.
more목차
Ⅰ. Introduction 1
Ⅱ. Materials and Methods 3
A. Bacterial EV isolation 3
B. Metabolomic analysis 3
C. Patients recruitment 4
D. ELISA for EV-specific IgG antibodies 5
E. Dot blot analysis for EV-specific IgG antibodies 5
F. Mouse experiment 6
G. Flow cytometric analysis 7
H. EV fluorescence imaging 7
I. Metagenomic analysis 8
J. Airway epithelial cell stimulation 8
K. Isolation of mammalian EVs from airway epithelial cells 9
L. Microarray analysis 9
M. Monocyte isolation and activation 11
N. ILC isolation and activation 11
O. Neutrophil activation and migration 11
P. miRNA transfection 12
Q. Quantification of miRNA expression 12
R. Statistical analysis 13
Ⅲ. Results 14
A. Micrococcus luteus-derived extracellular vesicles: Therapeutic target for neutrophilic asthma by regulating miRNA in airway epithelial cells 14
1. Characterization of MlEV 14
2. Clinical characteristics of the study subjects 15
3. Lower prevalence of MlEV-specific IgG4 in asthmatic patients 17
4. Suppressive effect of MlEV on neutrophilic airway inflammation in mice 19
5. Effect of MlEV on pathophysiological conditions in AECs 22
6. Effect of miRNAs on IL-1β-producing monocytes 24
7. Clinical significance of hsa-miR-4517 in asthmatic patients 26
B. Lactobacillus paracasei-derived extracellular vesicles: Therapeutic strategy for neutrophilic asthma by inhibiting the JNK pathway in airway epithelial cells 28
1. Altered composition of gut microbial EVs in neutrophilic asthmatic mice 28
2. Characterization of LpEV 30
3. Low abundance of LpEV-specific IgG in mice with NA 31
4. Clinical characteristics of the study subjects 32
5. Lower prevalence of LpEV-specific IgG4 in asthmatic patients 34
6. Suppressive effect of LpEV on airway inflammation in mice with NA 36
7. Inhibition of the JNK pathway by LpEV metabolites in the airway 39
Ⅳ. Discussion 43
Ⅴ. Conclusion 45
Reference 46
국문요약 51
