This article details the interaction between influenza A viruses and bone marrow-derived macrophages (BMDMs), focusing on the activation of the PANoptosome complex. The study elucidates the roles of ZBP1 and various caspases in the immune response to viral infection.
Begin with a multi-well plate containing adhered bone marrow-derived macrophages.
Introduce influenza A viruses that fuse with macrophages, releasing ribonucleoproteins containing viral RNA.
The macrophages' Z-DNA binding protein 1, or ZBP1 interacts with viral ribonucleoproteins, triggering ZBP1 oligomerization, with kinases and caspase 8.
The oligomerized ZBP1 further recruits inflammasome sensor proteins, adaptor proteins, and procaspase-1, forming a multimeric complex — a PANoptosome.
Within the PANoptosome, initiator caspase-8 activates through proximity cleavage, further activating effector caspases, including caspase-3 and 7.
Additionally, the inflammasome sensor proteins within the PANoptosome activate a signaling cascade, converting pro-caspase-1 to its active form, caspase-1.
Concurrently, viral infection induces the release of cytochrome-c from the mitochondria, triggering apoptosome activation, and converting pro-caspase-9 to its active form.
Viral infection also triggers the non-canonical inflammasome pathway, activating caspase-11.
Introduce a lysis buffer to lyse the macrophages; collect the lysate, and heat it to inactivate proteases, preventing caspases' degradation.
Centrifuge and collect the supernatant containing multiple caspases for further analysis.
After isolating the bone marrow, and differentiating the BMDMs, infect the cells with the influenza A virus. Calculate the virus volume needed at a multiplicity of infection of 20 plaque-forming units. Remove the media from the BMDMs, and wash the cells once with 500 microliters of PBS. Add 450 microliters of influenza A virus in high glucose DMEM without heat-inactivated FBS to each well, and incubate the plates at 37 degrees Celsius for one hour in a humidified incubator to allow absorption.
At the end of the one-hour incubation, add 50 microliters of heat-inactivated FBS, and return the plates to the incubator at 37 degrees Celsius for a total of 12 hours. After 12 hours of incubation, remove the plate from the incubator. Aspirate 150 microliters of the supernatant, and discard or save this for supernatant analysis. Do not remove the remaining supernatant.
Now, create the protein collection solution by combining 50 microliters of caspase lysis buffer and 100 microliters of 4X SDS buffer per well. Then, add 150 microliters of the mix to each well.
For each well, pipette the mixture to collect the lysed cells and supernatant. While pipetting, scrape the bottom of the well with the pipette tip to disrupt the cells. After scraping and pipetting, collect the protein lysate into labeled 1.5-milliliter tubes. Using a heat block, heat all the tubes to 100 degrees Celsius for 12 minutes. Remove the tubes from the heat block and centrifuge at 14,500 g for 30 seconds at room temperature to pellet any insoluble components.
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