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Enterococcus faecalis induces caspase-1 activation and IL-1β production in macrophages : Enterococcus faecalis에 의해 대식세포에서 유도되는 caspase-1 활성화와 IL-1β 생성
DC Field | Value | Language |
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dc.contributor.advisor | 최봉규 | - |
dc.contributor.author | 양하힘 | - |
dc.date.accessioned | 2017-07-19T08:24:13Z | - |
dc.date.available | 2017-07-19T08:24:13Z | - |
dc.date.issued | 2015-02 | - |
dc.identifier.other | 000000025657 | - |
dc.identifier.uri | https://hdl.handle.net/10371/130934 | - |
dc.description | 학위논문 (석사)-- 서울대학교 대학원 : 치의생명과학과, 2015. 2. 최봉규. | - |
dc.description.abstract | Objectives
Enterococcus faecalis is a Gram-positive bacterium and causes various diseases using its virulence factors. Inflammasome is a component of the innate immune system. Recent studies of inflammasome activation have focused on the pathogenesis of diverse inflammatory and autoimmune diseases. Inflammasome activation results in caspase-1 activation, which is required for the processing of pro-interleukin-1 beta (pro-IL-1β) to its secreted form (IL-1β) as well as pyroptosis, a form of pro-inflammatory cell death. The purpose of this study was to investigate whether endodontic infection associated with E. faecalis induces inflammasome activation. Methods THP-1 macrophages were treated with live E. faecalis. Caspase-1 activation, pro–IL-1β expression and IL-1β secretion were detected by immunoblotting, real-time reverse-transcription polymerase chain reaction (real-time RT-PCR), and enzyme-linked immunosorbent assay (ELISA), respectively. Pyroptosis was measured by lactate dehydrogenase (LDH) release and propidium iodide (PI) staining. Secreted IL-1β and pyroptosis were detected in the presence of caspase-1 inhibitors. Adenosine triphosphate (ATP) release was measured by an ATP bioluminescence assay kit. E. faecalis-induced inflammasome activation was measured by immunoblotting in the presence of the ATP receptor antagonist, oxATP. To determine whether the Nod-like receptor family protein 3 (NLRP3) inflammasome was associated with E. faecalis-induced caspase-1 activation and IL-1β production, knockdown of NLRP3 was conducted using siRNA. To verify whether E. faecalis internalization is a prerequisite for inflammasome activation, CFSE-labelled E. faecalis was detected by flow cytometry and confocal laser scanning microscopy in the presence or absence of cytochalasin D. Caspase-1 activation, pro-IL-1β expression and IL-1β production were detected by immunoblotting in the presence of cytochalasin D. To determine which signaling pathway contributes to the E. faecalis-induced pro-IL-1β expression, NF-κB pathway activation and MAP kinase pathway activation were measured by immunoblotting in the presence or absence of signaling inhibitors. Results E. faecalis induced caspase-1 activation and pro–IL-1β expression in macrophages, which resulted in IL-1β secretion. E. faecalis significantly induced ATP release, a mechanism of NLRP3 inflammasome activation, and oxATP treatment inhibited E. faecalis–induced caspase-1 activation. E. faecalis significantly increased LDH release and PI uptake, which are characteristics of pyroptosis. E. faecalis-induced caspase-1 activation and IL-1β secretion were decreased by the knockdown of NLRP3. E. faecalis was internalized into THP-1 macrophages, but blocking E. faecalis internalization by treatment with cytochalasin D did not affect E. faecalis-induced caspase-1 activation, pro-IL-1β expression and IL-1β production. The E. faecalis-induced pro-IL-1β expression was mediated via NF-κB and MAP kinase activation. These results suggest that E. faecalis may contribute to the progression of pulpal inflammation by stimulating excessive secretion of IL-1β and cell death. | - |
dc.description.tableofcontents | Abstract
1. Introduction 1 2. Materials and Methods 4 2.1. Chemicals and antibodies 4 2.2. Bacterial strain and growth conditions 5 2.3. Cell culture and treatment 5 2.4. Immunoblotting 7 2.5. Real-time RT-PCR 8 2.6. Enzyme-linked immunosorbent assay (ELISA) 9 2.7. Measurement of ATP release 9 2.8. RNA interference assay 10 2.9. Fluorescence labelling of bacteria and cells 11 2.10. Bacterial binding and internalization assay 12 2.11. Cell death assay 13 2.12. NF-κB and MAP kinase activation assay 14 2.13. Statistical analysis 15 3. Results 16 3.1. E. faecalis activates caspase-1 in THP-1 macrophages 16 3.2. E. faecalis induces pyroptosis 20 3.3. E. faecalis induces ATP release, resulting in caspase-1 activation 22 3.4. NLRP3 knockdown decreases caspase-1 activation and IL-1β secretion induced by E. faecalis 24 3.5. E. faecalis adheres to and is internalized into THP-1 cells 27 3.6. Inflammasome activation is not dependent on internalization of E. faecalis 33 3.7. E. faecalis activates NF-κB and MAP kinase pathway in THP-1 cells 35 4. Discussion 38 5. Conclusion 43 6. References 44 국문초록 | - |
dc.format | application/pdf | - |
dc.format.extent | 1680348 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | caspase-1 | - |
dc.subject | Enterococcus faecalis | - |
dc.subject | inflammasome | - |
dc.subject | interleukin-1 beta | - |
dc.subject | pyroptosis | - |
dc.subject.ddc | 617 | - |
dc.title | Enterococcus faecalis induces caspase-1 activation and IL-1β production in macrophages | - |
dc.title.alternative | Enterococcus faecalis에 의해 대식세포에서 유도되는 caspase-1 활성화와 IL-1β 생성 | - |
dc.type | Thesis | - |
dc.description.degree | Master | - |
dc.citation.pages | 55 | - |
dc.contributor.affiliation | 치의학대학원 치의생명과학과 | - |
dc.date.awarded | 2015-02 | - |
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