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Infection of mouse macrophages with viable Mycobacterium leprae does not induce apoptosis
Ramanuj Lahiri1, Baljit Randhawa, James L Krahenbuhl
1Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA.
Abstract:
The role played by apoptosis in host response to Mycobacterium leprae is unclear. Here, we studied in vitro induction of apoptosis in mouse bone marrow-derived macrophages infected with live and irradiated M. leprae, as a function of multiplicity of infection under permissive (33 degrees C) and nonpermissive (37 degrees C) temperatures. The infected macrophages were scored for apoptosis by using DAPI (4',6-diamindino-2-phenylindole dihydrochloride) and Annexin V staining, along with activated Caspases 3 and 9 and TUNEL (terminal dUTP nick end labeling) assay. Our results show that, in contrast to uninfected cells, murine macrophages infected with live M. leprae demonstrated little, if any, apoptosis, even when macrophages had a heavy burden of live leprosy bacilli. In contrast, elevated levels of apoptosis were observed when macrophages were infected with irradiated M. leprae. The results strongly suggest that the viability and purity of the leprosy bacilli used for in vitro studies determines the extent of apoptosis observed in infected host cells.
Insights
Live Mycobacterium leprae did not induce apoptosis in macrophages, unlike irradiated bacteria. This suggests bacterial viability, not just presence, influences host cell death in leprosy research.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- The role of apoptosis in host response to Mycobacterium leprae remains poorly understood.
- Investigating programmed cell death in macrophages is crucial for understanding leprosy pathogenesis.
Purpose of the Study:
- To investigate the in vitro induction of apoptosis in mouse macrophages infected with Mycobacterium leprae.
- To determine the influence of bacterial viability and experimental conditions on apoptosis.
Main Methods:
- Murine bone marrow-derived macrophages were infected with live and irradiated M. leprae at varying multiplicities of infection and temperatures (33°C and 37°C).
- Apoptosis was assessed using DAPI and Annexin V staining, activated Caspase 3 and 9 assays, and the TUNEL assay.
Main Results:
- Macrophages infected with live M. leprae showed minimal apoptosis, irrespective of bacterial load.
- In contrast, infection with irradiated M. leprae led to significantly elevated levels of apoptosis.
- Apoptosis induction varied with temperature and multiplicity of infection.
Conclusions:
- Bacterial viability is a critical factor in M. leprae-induced apoptosis in macrophages.
- The preparation and viability of M. leprae are crucial for accurate in vitro studies on host cell response.
- These findings impact the interpretation of previous in vitro studies on leprosy and apoptosis.
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