Molecular tools and model systems for studying inflammasome activation
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Upon cellular stress or invasion by pathogens such as Mycobacterium tuberculosis (Mtb), inflammasomes are activated as an innate immune response, an important host-defense mechanism to resist pathogens. Activation of NLRP3 recruits ASC and caspase-1 for oligomerization and eventually to IL-1β secretion and pyroptosis through gasdermin D. In the first part of this study, we knocked out NLRP3 and GSDMD genes with CRISPR/Cas9 technology and confirmed their essential roles in canonical inflammasome pathway by using different genetic and molecular tools. We have compared the efficiency of different guide RNAs and verified the functional roles of inflammasome proteins. Additionally, we attempted to knock out secretory autophagy related proteins FIP200 and SEC22B for they were found to play additional roles in IL-1β secretion, and ESCRT machinery proteins ALIX and ALG-2 for they play major role in balancing cell death and viability and IL-1β release in inflammasome activation. We also wished to explore other recent model systems, BlaER1 cells, for (human) inflammasome research that might show different responses. In the last part of this study, we performed molecular cloning to insert epitope and fluorescent tags within GSDMD in order to visualize intracellular localization and GSDMD cleavage during pyroptotic cell death. Overall, this project established genetic and molecular tools to study inflammasome activation, and have demonstrated the importance of different genes in inflammasome regulation and pyroptotic cell death.