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Updated: Oct 2, 2026
Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages
Published on: May 21, 2018
Nitric oxide-mediated protection of A. actinomycetemcomitans-infected murine macrophages against apoptosis
Keisuke Nakashima1, Jun Tomioka, Satsuki Kato
1Department of Periodontology and Endodontology, Health Sciences University of Hokkaido, Hokkaido, 061-0293, Japan.
Abstract:
We investigated apoptotic cell death in murine macrophage cell line J774.1 following Actinobacillus actinomycetemcomitans infection. Infected macrophages generally kill bacteria within phagosomes with nitric oxide (NO). Our previous study demonstrated that DNA fragmentation in infected cells increased significantly on addition of S-Methylisothiourea (SMT), a selective inhibitor of inducible NO synthetase (iNOS). The purpose of the present study was to determine the mechanism via which NO affects apoptosis of infected macrophages. J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1. The infected cells were then cultured in the presence or absence of SMT (400 microM). Culture supernatant was removed 21 h after the infection to measure LDH activity. Additionally, cellular proteins were extracted from the infected cells and measured for histone-associated DNA fragmentation and caspase-1, -3, -5, -6, -8, -9 activities. LDH activity and DNA fragmentation were significantly elevated by the infection; moreover, levels increased further on addition of SMT. Caspase activity of infected cells, particularly caspase-3, was significantly higher than that of uninfected cells. Furthermore, caspase activity increased on addition of SMT. These findings indicate that NO protects infected J774.1 cells, at least in part, against apoptotic cell death via a decrease in caspase activity.
Insights
Nitric oxide (NO) protects macrophages from apoptosis during Actinobacillus actinomycetemcomitans infection. Inhibiting NO synthesis increased DNA fragmentation and caspase activity, indicating NO
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- Macrophages combat Actinobacillus actinomycetemcomitans infection using nitric oxide (NO).
- Previous research indicated increased DNA fragmentation with S-Methylisothiourea (SMT), an inducible NO synthase (iNOS) inhibitor, in infected cells.
- The role of NO in macrophage apoptosis during infection requires further elucidation.
Purpose of the Study:
- To investigate the mechanism by which NO influences apoptosis in macrophages infected with A. actinomycetemcomitans.
- To determine the impact of NO inhibition on cellular damage and apoptotic pathways.
Main Methods:
- Murine macrophage cell line J774.1 infected with A. actinomycetemcomitans Y4.
- Treatment with S-Methylisothiourea (SMT) to inhibit inducible NO synthase (iNOS).
- Measurement of lactate dehydrogenase (LDH) activity, DNA fragmentation, and caspase activities (caspase-1, -3, -5, -6, -8, -9).
Main Results:
- Infection elevated LDH activity and DNA fragmentation, with further increases upon SMT addition.
- Caspase activity, particularly caspase-3, was significantly higher in infected cells and increased further with SMT treatment.
- SMT treatment exacerbated infection-induced cellular damage and apoptotic markers.
Conclusions:
- Nitric oxide (NO) plays a protective role in preventing apoptosis of J774.1 macrophages during A. actinomycetemcomitans infection.
- NO appears to exert its protective effect by reducing caspase activity.
- Inhibition of NO synthesis enhances susceptibility to apoptosis in infected macrophages.
