The AIM2 Inflammasome Pathway Is Critical For Innate Immunocyte Against Human Cytomegalovirus Infection

Objectives:①To confirm AIM2 is a DNA sensor to detect HCMV;②To identify the influence of HCMV infection to the signaling molecules of AIM2 inflamasome pathway in THP-1-derived macrophages;③To confirm AIM2 inflammasome pathway involves in the pro-inflammatory cytokine response of innate immunity against HCMV infection.Methods:①Established a cell model of HCMV infected THP-1-derived macrophages:HCMV (AD 169 strain) inoculated with MOI= 1 to establish a cell model of HCMV infected THP-1-derived macrophages, while established a mock infected control group, a LPS+ ATP control group and a poly(dA:dT) control group;②Used real-time RT-PCR to detect the gene expression level of DNA sensors AIM2, polⅢ, DAI, and TLR9 in both HCMV infected group and mock infected control group;③Used real-time RT-PCR to detect the gene expression level of IL-1βin both HCMV infected group and mock infected control group;④Used western blot to detect the protein expression level of AIM2, ASC, Caspase-1 and IL-1βat 1 h,3 h,6 h,12 h and 24 h post infection in HCMV infected group, mock infected control group and LPS+ATP control group; And established a poly(dA:dT) control group at 24 h after the poly(dA:dT) transfected;⑤AIM2 siRNA and ASC siRNA were respectively transfected into THP-1-derived macrophages, then infected with HCMV AD 169 strain, while established a mock transfected control group. Used western blot to detect the protein expression level of AIM2, ASC, Caspase-1 and IL-1βin THP-1-derived macrophages at 6 h post infection, and compared with the mock infected control group;⑥Treated THP-1-derived macrophages with a specific inhibitor of Caspase-1 (Ac-YVAD-CHO), then infected with HCMV AD 169 strain, while established a mock treated control group; then used western blot to detect the protein expression level of AIM2, ASC, Caspase-1, pro-IL-1βand IL-1βat 6 h post infection, then compared with the mock infected control group.Results:①HCMV infected THP-1-derived macrophages that increased the gene expression level of DNA sensors AIM2, PolIII and DAI at 6 h post infection, however, that of TLR9 did not change significantly;②HCMV infected THP-1-derived macrophages that increased the gene expression level of pro-inflammatory cytokine IL-1βat 6 h post infection;③HCMV infected THP-1-derived macrophages that increased the protein expression level of AIM2 inflammasome related proteins AIM2, ASC, and Caspase-1 according to the time;④HCMV infected THP-1-derived macrophages increased the protein expression level of pro-inflammatory cytokines IL-1β;⑤AIM2 siRNA inhibited the protein expression level of AIM2 inflammasome related proteins AIM2, Caspase-1 and IL-1βinduced by HCMV infection, but had no effect on the protein expression of ASC;⑥ASC siRNA inhibited the protein expression levels of AIM2 inflammasome related proteins ASC, Caspase-1 and IL-1βinduced by HCMV infection, but had no effect on the protein expression of AIM2;⑦Ac-YVAD-CHO is a specific inhibitor of Caspase-1 which had no effect on the protein expression levels of AIM2 inflammasome related proteins AIM2, ASC, and Caspase-1 and pro-IL-1β, but inhibited the protein expression level of IL-1βinduced by HCMV infection.Conclusions:①AIM2 is a DNA sensor and localizes in cytoplasm to detect HCMV dsDNA;②HCMV infection can increase the protein expression level of AIM2 inflammasome pathway related proteins AIM2, ASC, and Caspase-1;③HCMV infection can increase the protein expression level of pro-inflammatory cytokines IL-1β;④AIM2 inflammasome pathway involves in the mature and activation of IL-1βof innate immune response against HCMV infection.