The AIM2 inflammasome is involved in both autoimmune diseases and the host defense against pathogens,and the study of its activation mechanism will be very meaningful.To establish an activation system for the AIM2 inflammasome in vitro,the gene fragments of AIM2,ASC,pro-Caspase-1,and pro-IL-1β from human monocytic cells(THP-1 cells)were amplified by using PCR.Subsequently,the recombinant plasmids including p3×Flag-AIM2,p3×Flag-ASC,p3×Flag-pro-Caspase-1,and p3×Flag-pro-IL-1β were constructed.These obtaining plasmids were transfected into HEK293T cells,and their expressions were confirmed by western blot analysis.The results were showed that the recombinant proteins of AIM2,ASC,Caspase-1,and IL-1β were well-expressed tagged with Flag in the form of fusion expression in HEK293T cells,and the specific bands were shown at approximately 41.9ku,34.1ku,48.5ku,and 24.6ku,respectively.Notably,endogenous AIM2 protein was also detected at 39ku in HEK293T cells,indicating endogenous AIM2 was expressed in HEK293T cells.Co-transfection of plasmids with different concentrations into HEK293T cells,inluding or excluding poly(dA:dT)transfection,was performed,and IL-1β secretion levels were assessed by indirect ELISA.The results demonstrated that IL-1β was undetectable in the supernatants of the control group,the group transfected with p3×Flag-pro-IL-1β alone,or the group co-transfected with p3×Flag-ASC together with p3×Flag-pro-IL-1β.However,high concentrations of IL-1β were detectable in the supernatants of the experimental groups co-transfected with p3×Flag-ASC,p3×Flag-pro-Caspase-1,p3×Flag-pro-IL-1β,and co-transfected with p3×Flag-AIM2,p3×Flag-ASC,p3×Flag-pro-Caspase-1,p3×Flag-pro-IL-1β,especially for p3×Flag-AIM2 co-transfection group exhibiting higher IL-1β secretion levels in HEK293T cells.Additionally,after poly(dA:dT)stimulation,a significant increase in IL-1β secretion levels was observed compared to the control group(P<0.001),confirming successful construction of the AIM2 inflammasome activation system in vitro.Furthermore,an indirect ELISA was used to test the IL-1β secretion levels in HEK293T cells,where the different concentrations of p3×Flag-AIM2 were co-transfected with other plasmids,followed by poly(dA:dT)transfection.The results were showed that the IL-1β secretion levels were increased significantly with the increasing doses of p3×Flag-AIM2(P<0.001,P<0.01,P<0.05,P<0.001),whether or not the poly(dA:dT)was transfected,in HEK293T cells.It is indicating that there was a positive correlation between AIM2 protein expression level and AIM2 inflammasome activity.Additionally,Listeria monocytogenes,a known activator of the AIM2 inflammasome,were employed to infect the activated inflammasome system,and the alive bacterial would be tested.The results indicated that the intracellular bacterial quantity was significantly lower than the control group at 2 hours and 6 hours post-infection(P<0.001,P<0.01),suggesting that the AIM2 inflammasome activation system plays a role in resisting microbial infections.In conclusion,this study successfully established an AIM2 inflammasome activation system and provided initial evidence of its anti-infection function.These findings lay the foundation for further elucidating the specific mechanisms underlying this activation system's function.
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