The Autophagic Changes Of THP-1 Macrophage Cells Induced By Human Cytomegalovirus And Virus Proliferation

Objectives1. To confirm the dynamic autophagic changes of THP-1 macrophages after human cytomegalovirus infection;2. To observe the temporal expression of AIM2 inflammasome downstream effector IL-1β in THP-1 macrophages after human cytomegalovirus infection;3. To determine the transcription of UL122 gene in HCMV infected THP-1 macrophages;4. To explore the role of innate immune cell autophagy and AIM2 inflammatory pathway in the anti HCMV mechanism.Methods1. The differentiation of THP-1 cells: THP-1 cells were cultured in vitro, and PMA(100ng/ml) was used to stimulate the differentiation of cells into THP-1 derived macrophages, the cells were used for next steps;2. The experiment groups:(1)HCMV AD169 standard strain infection group(HCMVgroup);(2)Heat inactivated HCMV infection group(Heat-HCMV group);(3)Mock group(M group);(4)Positive control group(autophagy induced by 400 nmol rapamycin, RAPA group). For the HCMV group, the cells were infected at a MOI of 1.3. Evaluation of THP-1 macrophage autophagy(1) We use western blot assay to detect the dynamic changes of beclin1 expression and LC3 transformation(LC3-II/LC3-I), which are both autophagy-related, 1h, 3h, 6h, 12 h,24h and 48 h after infection;(2) The cells were observed in the cytoplasm of the formation of autophagic vacuoles number by transmission electron microscope 6h, 12 h and 24 h after infection;4. Determination of IL-1β sequential expression in THP-1 derived macrophages using ELISA method: 1h, 3h, 6h, 12 h, 24 h and 48 h after infection, the cell culture supernatant were collected and the IL-1β concentration were determined by ELISA in HCMV, Heat-HCMV and M group; cells transfected with poly(d A:d T) for 24 h were set as positive control;5. The expression of HCMV UL122 gene: the temporal changes of UL122 gene transcription 1h, 3h, 6h, 12 h, 24 h and 48 h after infection were determination by real time RT-PCR method.Results1. The dynamic changes of beclin1 expression and LC3 transformation in THP-1 derived macrophages: for HCMV infected group, autophagy-related protein beclin1 increaed significantly 1h post infection; LC3 transformation increased dramatically 6h post infection; however, beclin1 expression and LC3 transformation decreased obviously compared to Heat-HCMV and RAPA group(p<0.05) when it came to 24 h and 48 h post infection. For Heat-HCMV group, beclin1 increaed significantly 1h post infection; LC3 transformation increased dramatically 6h post infection; and they stayed at high expression level 48 h post infection;2. The change of autophagic vacuoles numbers in THP-1 derived macrophages: the number of autophagic vacuoles increased 6h post HCMV infection, but decreased significantly24 h post infection; for Heat-HCMV group, the autophagic vacuoles number continued to increase after 48h;3. The changes of IL-1β expression in THP-1 derived macrophages: the IL-1βconcentration gradually increased(P>0.05) 1h post HCMV infection and reached the same level of postive control group 3h and 6h post HCMV infection; and were significantly higher than postive control group 12 h, 24 h and 48 h post HCMV infection(P <0.05). The IL-1β levels in Heat-HCMV group were significantly lower(P<0.05)than HCMV group 3h post treatment, but there was no obvious difference when compared to mock group(P>0.05);4. The HCMV gene UL122 transcription did not increase significantly until 24 h post infection, it was 2.12 times compared to 1h post infection(P<0.05); the transcription reached 3.43 times 48 h post infection(P<0.05).Conclusion1. In the early stage of infection HCMV promote autophagy in THP-1 derived macrophages,but tend to inhibit autophagy 24 h post infection; however, heat inactivated HCMV virons tend to stimulate autophagy constantly, which implies that HCMV can escape from being cleared by inhibiting the autophagy of innate immune cells;2. IL-1β levels stayed at high levels during HCMV infection, but inactivated virons had no such effect, which implies that the effect may be related to an AIM2 inflammasome pathway activaction in THP-1 derived macrophages;3. Compared with HEL, which is susceptible to HCMV infection, the immediate early gene expression of HCMV delayed significantly in THP-1 derived macrophages, this probably due to the autophagy and AIM2 inflammasome pathway activaction in innate immune cells at the early stage of infection.