S-Space College of Dentistry/School of Dentistry (치과대학/치의학대학원) Dept. of Dental Science(치의과학과) Theses (Master's Degree_치의과학과)
Enterococcus faecalis induces caspase-1 activation and IL-1β production in macrophages
Enterococcus faecalis에 의해 대식세포에서 유도되는 caspase-1 활성화와 IL-1β 생성
- 치의학대학원 치의생명과학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 : 치의생명과학과, 2015. 2. 최봉규.
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.
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.
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.