Analyzing Caspase-8-Dependent GSDMD Cleavage in Response to Yersinia Infection

Area of Science:

• Immunology
• Cell Biology
• Microbiology

Background:

• Caspase-8 traditionally induces apoptosis, a non-inflammatory cell death.
• Pathogen-induced innate immune signaling inhibition can redirect caspase-8 to form a death-inducing complex with RIPK1 and FADD.
• This complex promotes pyroptosis, a lytic cell death, via gasdermin D (GSDMD) cleavage.

Purpose of the Study:

• To provide a detailed protocol for activating caspase-8-dependent GSDMD cleavage in murine bone marrow-derived macrophages (BMDMs).
• To investigate the mechanism of pyroptosis induction during Yersinia pseudotuberculosis infection in macrophages.
• To establish a reproducible method for studying caspase-8's role in pro-inflammatory cell death.

Main Methods:

• Murine bone marrow-derived macrophage (BMDM) harvesting and plating.
• Preparation of Yersinia pseudotuberculosis strains that induce the type 3 secretion system.
• Macrophage infection, followed by lactate dehydrogenase (LDH) release assay and Western blot analysis to assess cell death and GSDMD cleavage.

Main Results:

• Successful activation of caspase-8-dependent GSDMD cleavage was achieved in BMDMs upon Yersinia infection.
• The protocol allows for the quantification of pyroptosis through LDH release.
• Western blot analysis confirmed caspase-8 mediated GSDMD cleavage.

Conclusions:

• The described protocol effectively induces and measures caspase-8-driven pyroptosis in macrophages during Yersinia infection.
• This method facilitates further research into the role of caspase-8 in pathogen-mediated inflammatory cell death.
• Understanding this pathway is crucial for deciphering innate immune evasion strategies employed by bacteria like Yersinia.