NLRP3 inflammasome is a multiprotein complex of innate immunity that is used to activate caspase 1, which proteolytically activates IL-1β, IL-18 and gasdermin D. After the activation of NLRP3 the interleukins are released through gasdermin D pores or by necrosis and trigger inflammation. Inflammasome assembly is triggered by many PAMPs and DAMPs, which act via the canonical, non-canonical or alternative NLRP3 activation pathway. NLRP3 inflammasome activity is also connected with inflammaging and accompanies geriatric diseases such as type 2 diabetes, Alzheimer's disease and atherosclerosis. It is not yet clear which of the activation pathways is involved in this effect. The regulation of NLRP3 activity is also poorly understood. The purpose of our work was to systematically evaluate the effect of lifespan-enhancing compounds on canonical, non-canonical and alternative NLRP3 inflammasome activation to obtain information on the regulation of NLRP3 activity in aging. We selected compound that extended the lifespan of model organisms through activation of AMPK or autophagy. On iBMDM cells, we successfully established a protocol for non-canonical NLRP3 activation by LPS transfection and a protocol for alternative NLRP3 activation by peptidoglycan. We have shown that rapamycin, spermidine, spermine, resveratrol, curcumin, metformin, AICAR and PT-1 inhibited IL-1β maturation by the non-canonical inflammasome. The majority of tested compounds also inhibited IL-1β release by the alternative inflammasome, however, particularly spermine and spermidine exposed some cytotoxic activity at prolonged incubation. Metformin, spermidine and spermine were also tested in canonical NLRP3 activation setup, where spermine and spermidine inhibited IL-1β release, yet some cytotoxicity was observed at the same time. Interestingly, the indirect AMPK activator metformin even increased the cellular response to canonical activation of NLRP3. The second aim of our work was to prepare an iBMDM cell line that inducibly expresses constitutively active AMPK to study the effect of AMPK on NLRP3 response. By molecular cloning and retroviral transduction, we have successfully prepared CA/iBMDM and nCA/iBMDM cell lines, each expressing a variant of constitutively active AMPKα1 (1-312) T183D. We confirmed by western blot that doxycycline induces the synthesis of CA-AMPK and nCA-AMPK enzymes in CA/iBMDM and nCA/iBMDM cell lines, respectively. We have not yet uniformly confirmed the physiological activity of the enzymes, but both enzymes have indicated an effect on ACC phosphorylation after doxycycline induction. Our work provided good candidates for further research on NLRP3 inhibition and its regulation in aging. In addition, we prepared two cell lines where we could study the effect of AMPK on NLRP3 inflammasome activity after we optimize the induction.
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