The Mechanism Of The Interaction Between HCMV And AIM2 Inflammasome In THP-1 Derived Macrophages

BackgroundAIM2 is a cytosolic dsDNA sensor.Once senseing the dsDNA,AIM2 recruits pro-caspase-1 through ASC and assembles the inflammasome to activate caspase-1,which cleaves pro-IL-1? and pro-IL 18 to there active forms,IL-1? and IL18,as well as induces pyroptosis.AIM2 inflammasome has a pivotal role in response to multiple pathogens with double-stranded DNA,including MCMV;however,its role in HCMV infections remains unclear.HCMV,as a dsDNA virus,can trigger the maturity and release of the inflammasome associated pro-inflammatory cytokines,IL-1? and IL-18.indicating the activation of inflammasome.While TLR-9 and ZBP1,the known HCMV DNA sensors,cannot assemble inflammasomes.Thus,we speculate the dsDNA sensor AIM2,which can assemble inflammasome,may be involved in the immune response to HCMV.In addition,HCMV has developed a variety of strategies to evade host immunity.The tegument protein pUL83 is an important immune evasion protein,several studies have reported that pUL83 binds to specific cellular proteins,such as IFI16,to affect their functions.As AIM2 and IFI16 belong to HIN200 family and have similar structural domains,we hypothesize that pUL83 can affect AIM2 inflammasome through interacting with AIM2 protein to evade immune response.Objectives1.To clarify that HCMV can be sensed by AIM2 and triggers the activation of AIM2 inflammasome;2.Detecting the impact of HCMV infection on AIM2-mediated molecular events by siRNA approach to determine whether HCMV induced activation of inflammasome depends on AIM2.3.To investigate the interaction between pUL83 and AIM2;4.To study the impact of pUL83-AIM2 interaction on AIM2 inflammasome.MethodsHCMV triggers the activation of AIM2 inflammasome in THP-1 derived macrophages1.THP-1 cells were stimulated by PMA(100 ng/ml)for 24 to induce cellular differentiation;2.HCMV AD 169 strain was used to infect THP-1 derived macrophages(MOI=1)for 1 h,3 h,6 h,12 h,24 h,48 h,and 96 h.Regular DMEM was used as mock infection control.poly(dA:dT)was transfected into macrophages(1 ?g/ml)for 24 hours as a positive control.3.Supernatants were collected for ELISA to detect mature IL-1? and IL-18,and for detection of LDH to assess cell death.4.Cells infected with HCMV for 1 h,6 h,and 24 h,as well as mock infected cells and poly(dA:dT)transfected cells were harvested for co-immunoprecipitation and immunoblotting to detect the interaction between AIM2 and ASC.The effect of AIM2 gene silencing on HCMV induced inflammasome activation and viral gene transcription1.AIM2 specific siRNA or non-specific siRNA(Stealth negative control LO GC)was transfected into THP-1 derived macrophages for 72 h to obtain the AIM2-defient macrophage(S+)or mock silenced macrophages(S-).Both S+ and S-were infected(S+/V+ and S-/V+)or mock infected(S+/V and S-/V-)with HCMV;2.Cells infected/mock infected for 1 h,6 h,and 24 h were collected to detect the protein levels of AIM2,pro-caspase-1,p10,pro-IL-1?,and IL-1?;3.Cells were infected for 1 h,3 h,6 h,12 h,and 24 h,and then cell supernatants were collected for ELISA to detect mature IL-1? and IL-18,and for detection of LDH to assess cell death;4.Cells infected for 1 h,6 h,12 h,18 h,24 h,48 h,and 72 h were collected to abstract total RNA for RT-qPCR assay to detect the transcription of immediate early gene UL122,early gene UL54,and late gene UL83;HCMV tegument protein pUL83 interacts with human AIM2 protein1.Specific primers were designed and synthesized based on HCMV AD 169 strain UL83 gene and human AIM2 gene sequences picked from NCBI Genbank website for amplifying this two desired genes;2.The desired genes UL83 and AIM2 were inserted into pM and pVP16 respectively by in-fusion method.Theoretically,the recombinant vectors pM-UL83 and pVP-AIM2 express BD-pUL83 and AD-AIM2 respectively;3.pM-UL83 and pVP-AIM2 were transfected into HEK293T cells by Calcium transfection reagent for 72 h.Then the cells were collected for western-blot assay to detect the expression of recombinant proteins;4.pM-UL83,pVP-AIM2 and Secretion alkaline phosphatase(SEAP)reporter vector were co-transfected into HEK293T cells by Calcium transfection reagent for 72 h.Then the cell supernatants were collected for detection of SEAP using chemiluminescence assay,and the cells were harvested for detection of pUL83-AIM2 interaction using co-immunoprecipitation and western-blot;5.THP-1 derived macrophages were infected with HCMV AD 169 strain(MOI=1)for 6 h,12 h,and 24 h.Regular DMEM was used as mock infection control.poly(dA:dT)was transfected into macrophages(1 pg/ml)for 24 hours as a positive control.Co-immunoprecipitation and western-blot assays were used to detect the interaction between pUL83 and AIM2;Immunofluorescence assay was used to detect the co-localization of pUL83 and AIM2.pUL83 down-regulates AIM2 inflammasome through binding to AIM2 protein1.Specific primers were designed and synthesized based on human ASC?caspase-1 and IL-1? gene sequences picked from NCBI Genbank website for amplifying these three desired genes.These desired genes(retaining the termination codons)were severally inserted into pDsRed-N1 vectors.Theoretically,the recombinant vectors express ASC,pro-caspase-1,and pro-IL-1? respectively;2.HEK293T cells transfected with pVP-AIM2,ASC,caspase-1,and IL-1? recombinants were named as rHEK293T cells;72 hours later.rHEK293T cells were harvested to detect AIM2,ASC,pro-caspase-1,and pro-IL-1? proteins by western-blot.3.rHEK293T cells were transfected with poly(dA:dT)for 6 h and 24 h and then harvested to abstract total protein;rHEK293T cells were transfected with pM-UL83 and poly(dA:dT)successively and then harvested to abstract total protein,protein levels of AIM2,ASC,pro-caspase-1,p10,pro-IL-1?,and IL-1(3 were determined by western-blot assay;4.HEK293T cells transfected with ASC,caspase-1,and IL-1? recombinants were named as rHEK293T(AIM2-)cells,which were used for assays described in step 3.ResultsHCMV triggers the activation of AIM2 inflammasome in THP-1 derived macrophages1.The concentrations of IL-1? and IL-18 increased over the course of HCMV infection.statistic differences showed after 12 h on IL-1? level and 48 h on IL-18 level respectively between HCMV infection and mock infection groups;Compared with mock infection,HCMV infection cells showed significantly higher cell death rate at 3 h,48 h,and 96 h;2.AIM2 combined to ASC in THP-1 derived macrophages 1-24 h post HCMV infectionThe effect of AIM2 gene silencing on HCMV induced inflammasome activation and viral gene transcription1.The expression of AIM2 protein increased in S-cells 6 h post HCMV infection compare with that in mock infected S-cells.There was almost no AIM2 in S+ cells.2.pro-caspase-1 constitutively expressed in THP-1-derived cells;There was little p10 in mock-infected cells;p10 was weakly detected at 1 h post infection in S-cells,and increased 6-24 h post-infection.While there was no p10 detected in S+ cells 1 h and 6 h post infection,it was detected in lower amount,compared to S-cells,24 h post infection3.Both S+ and S-cells exhibited high expression of pro-IL-1? as early as 1 h post infection.This enhancement continued to 6 hr.However,this enhancement reduced either dramatically in S-cells or slightly in S+ cells at 24 h post infection.While the level of mature IL-1? increased over the infection time in S-cells,there was no apparent increase in IL-1? in infected S+ cells compared to uninfected cells at 1 h and 6 h post infection.Notably,the mature IL-1(3 increased in infected S+ cells,but still lower than that in S-cells at 24 h post infection;4.The concentrations of IL-1? and IL-18,as well as the cell death rate in the supernatants of HCMV infected S+ cells were lower than those in infected S-cells.5.The transcription of HCMV early gene UL54 and late gene UL83 were higher in infected S+ cells compared with those in infected S-cells.HCMV tegument protein pUL83 interacts with human AIM2 protein1.pM-UL83 and pVP-AIM2 were successfully recombined and could express the desired proteins in HEK293T cells;2.increased SEAP were detected in HEK293T cells co-transfected with pM-UL83,pVP-AIM2 and SEAP reporter vector through two-hybrid and chemiluminescence assay;3.pM-UL83 and pVP-AIM2 expressed DNA BD-pUL83 and AD-AIM2 proteins were detected to interact with each other in HEK293T cells through Co-immunoprecipitation and western-blot assays;4.pUL83 and AIM2 were detected to interact with each other and co-localized in cytoplasm 6?12 h post HCMV infection through co-immunoprecipitation,western-blot and immunofluorescence assays.pUL83 down-regulates AIM2 inflammasome through binding to AIM2 protein1.ASC?caspase-1 and IL-1? expression vectors were successfully recombined and could express the desired proteins in HEK293T cells;2.After transfecting with poly(dA:dT),rHEK293T cells expressed more pro-IL-1? and expressed p10 and IL-1? proteins;rHEK293T cells transfected with pM-UL83 and poly(dA:dT)successively expressed high level of pUL83,while the level of AIM2,pro-caspase-1,pro-IL-1? p10 and IL-1? were reduced compared with those in rHEK293T cells transfected with poly(dA:dT)alone;3.The protein levels of ASC,pro-caspase-1,and pro-IL-1? in poly(dA:dT)transfected remained rHEK293T(AIM2-)cells remained unchanged compared with un-transfected rHEK293T(AIM2-)cells regardless whether pUL83 existed or not.there was no detectable p10 and IL-1?.Conclusions1.HCMV infection induces the assembly of AIM2 inflammasome and the maturation of IL-1? and IL-18 as well as cell death in THP-1 derived macrophages;2.AIM2 is indispensable in HCMV induced inflammasome activation in THP-1 derived macrophages;3.AIM2 inflammasome inhibits HCMV gene transcription to a certain degree;4.HCMV tegument protein pUL83 interacts with AIM2 in the cytoplasm during the early stages of HCMV infection;5.The pUL83/AIM2 interaction down-regulates the activation of AIM2 inflammasome.