Lipoxin Receptor Agonist BML-111Negatively Modulates THP-1Macrophages Immune Response During Human Cytomegalovirus(HCMV) Infection

Objectives:①to investigate the immunomodulatory effect of BML-111on THP-1macrophages during HCMV infection;②to observe the influence of BML-111on HCMV replication in THP-1macrophages and human embryonic lung fibroblasts(HEL)Methods:①a cellular model of THP-1macrophages infected by HCMV was established: THP-1cells were converted into macrophages induced by100nmol/L PMA, and the group division in the experiment was as follows:mock infection,HCMV infection, HCMV+BML-111. HCMV MOI=0.5,the concentration of BML-111was100nmol/L;②the influence of BML-111on the protein expression of inflammatory cytokines was observed:the secreted ILl-β、TNF-α, IL-10and TGF-βin the supernatant of THP-1macrophages medium were Detected by ELISA at0、1h、2h、4h、12h、24h、48h and72h;③the influence of BML-111on the mRNA expression of inflammatory cytokines was observed:test the mRNA levels of ILl-β,TNF-aα, IL-10and TGF-βin the THP-1macrophages were tested by SYBR green real-time RT-PCR at0,1h、2h、4h、12h、24h、48h and72h;④the influence of BML-111on the translocation of NF-κB p65subunit was observed:NF-κB p65in the nucleus of THP-1macrophages Detectede by Western Blot at4h;⑤the influence of BML-111on the HCMV replication in THP-1macrophages was observed:the mRNA levels of IE86and pp65in the THP-1macrophages were tested by SYBR green real-time RT-PCR at0,1h、2h、4h、12h、24h、36h48h and72h;⑥the influence of BML-111on the HCMV replication in HELs was observed:the group division in this part was as follows:HCMV infection and HCMV+BML-111. the patho-morphous changes were observed under microscope,and the infective virus titer changes were examined by plaque assay.Results:①THP-1cells grew in suspension originally, then the cells adhered to the dish bottom and had the morphological characteristics of macrophages after48h of culture with100nmol/LPMA which had been converted into macrophages.②ELISA shows:compared with the group of mock infection, the levels of all the cytokines both in the HCMV infection group and in the HCMV+BML-111group increased significantly; compared with the group of HCMV infection, the levels of lLl-βand TNF-α in the HCMV+BML-111group decreased significantly, the level of TGF-β of the HCMV+BML-111group increased significantly, and there was no differences of the level of IL-10between them.③SYBR green real-time RT-PCR shows:compared with the group of mock infection, the mRNA levels of all the cytokines of the HCMV group and HCMV+BML-111group increased significantly; compared with the group of HCMV infection, the mRNA levels of all the cytokines of the HCMV+BML-111group decreased significantly.④Western Blot shows:compared with the group of mock infection, the level of NF-κB p65in the nucleus of the HCMV group and HCMV+BML-111group increased significantly; compared with the group of HCMV infection, NF-κB p65in the nucleus of the HCMV+BML-111group decreased significantly.⑤SYBR green real-time RT-PCR shows:compared with the group of mock infection, the mRNA levels of IE86and pp65of the HCMV group and HCMV+BML-111group increased significantly; compared with the group of HCMV infection, the mRNA levels of IE86and pp65of the HCMV+BML-111group increased significantly in the early stage after infection, but the mRNA level of pp65decreased significantly in the medium and late stages after infection.⑥Observation under microscope shows:the degree of the patho-morphous of the HCMV+BML-111group reached100%earlier than the group of HCMV infection; and plaque assay shows:the infective virus titer reached the peak earlier than the group of HCMV infection too.Conclusions:①BML-111negatively modulates THP-1macrophages immune response during human cytomegalovirus(HCMV) infection②The negatively modulation of BML-111were related with inhibiting nuclear translocation of NF-κB p65, decreasing the pro-inflammatory cytokines ILl-βand TNF-a,increasing the anti-inflammatory cytokine TGF-β,with no influence on the anti-inflammatory cytokine IL-10.③BML-111accelerates the replication of HCMV in the early stage of infection, but inhibits the expression of pp65gene in the late stage.④BML-111doesn’t increase the peak value of the infective HCMV titer in vitro.