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Measuring Innate Immune Responses to Bacterial Viability
Julien Moretti1,2, Nicolas Vabret3, J Magarian Blander4,5,6
1Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, New York, NY, 10021, USA.
Methods in Molecular Biology (Clifton, N.J.)
|November 28, 2017
Summary
The innate immune system detects bacterial viability using messenger RNA (mRNA) to activate inflammasome and type I interferon pathways, crucial for robust immune responses against Gram-negative bacteria.
Area of Science:
- Immunology
- Microbiology
- Molecular Biology
Background:
- The innate immune system utilizes viability-associated pathogen-associated molecular patterns (vita-PAMPs), like prokaryotic mRNA, to sense microbial viability.
- Detection of Gram-negative bacterial viability by phagocytes uniquely activates inflammasome and type I interferon pathways, driving potent innate and adaptive immune responses.
Purpose of the Study:
- To provide a detailed protocol for investigating inflammasome and type I interferon pathway activation.
- To examine these pathways in mouse bone marrow-derived macrophages stimulated with live or killed Gram-negative and Gram-positive bacteria.
Main Methods:
- Generation and handling of mouse bone marrow-derived macrophages.
- Bacterial culture, inactivation, and preparation of bacterial mRNA.
- Macrophage stimulation assays using live or killed bacteria.
- Measurement of interleukin-1β secretion (inflammasome hallmark) and TBK1/IRF3 activation and type I interferon secretion (type I interferon pathway hallmark).
Main Results:
- The protocol enables the study of differential immune pathway activation based on bacterial viability and type.
- Successful stimulation and measurement of key inflammasome and type I interferon pathway components are demonstrated.
Conclusions:
- This protocol offers a comprehensive method for dissecting the innate immune response to bacterial viability.
- It facilitates the understanding of how sensing bacterial mRNA contributes to inflammasome and type I interferon activation, impacting host defense strategies.
