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Updated: Dec 9, 2025
Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection
Published on: September 2, 2014
[Enterovirus 71 can induce autophagy and apoptosis of THP-1 macrophages]
Wenying Luo1, Lawei Yang2, Qingjun Pan3
1Department of Clinical Laboratory, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, China.
Objective:
To investigate enterovirus 71 (EV71)-induced of autophagy, apoptosis and the related signaling pathways in THP-1 macrophages.
Methods:
THP-1 macrophages were infected with EV71 at the multiplicity of infection (MOI) of 0.1 for 2, 8 or 16 h, and the cell proliferation and toxicity were analyzed using CCK-8 kit. The intracellular viral nucleic acid in THP-1 macrophages were detected by fluorescence quantitative PCR, and the ultrastructural changes of the cells were observed using transmission electron microscopy. Cell apoptosis induced by EV71 infection was detected using Hoechst 33342 staining and AnnexinV/PI double staining. Western blotting was performed for analysis of changes in autophagy and apoptosis of the cells and in the expressions of the related proteins. The effect of EV71 infection on apoptosis of THP-1 macrophages incubated with 3-MA and Ac-DEVD-CHO inhibitor for 2 h was assessed using Western blotting.
Results:
EV71 infection significantly lowered the cell survival rate of THP-1 macrophages at 2, 8 h and 16 h after the infection (P < 0.05). The total copy number of viral nucleic acid in THP-1 macrophages incubated with EV71 increased significantly and progressively over time (P < 0.01). Intracellular autophagosomes and virions could be seen in EV71-infected THP-1 macrophages. The total apoptotic rate of the infected cell also increased significantly over time (P < 0.01). EV71 infection significantly increased LC3 conversion (LC3-Ⅱ/ LC3-I) and the expression of cleaved caspase 3 protein and decreased the protein expressions of p62, Bcl-2 and caspase-3 (P < 0.01) without causing obvious changes in cleaved caspase-8 (P>0.05). 3-MA significantly inhibited the EV71-induced autophagy of THP-1 macrophages and reduced LC3 conversion (LC3-Ⅱ/LC3-I) and p62 protein expression at 8 h after EV71 infection (P < 0.01). Compared with DMSO, Ac-DEVD-CHO significantly inhibited EV71-induced apoptosis of THP-1 macrophages (15.5% vs 7.7%, P < 0.01).
Conclusions:
EV71 not only can infect and replicate in THP-1 macrophages, but also induces autophagy and cell apoptosis possibly by activating LC3/p62 autophagy pathway and caspase apoptosis pathway.
Insights
Enterovirus 71 (EV71) infects THP-1 macrophages, triggering autophagy and apoptosis. This study reveals EV71 activates the LC3/p62 autophagy and caspase apoptosis pathways, impacting macrophage survival.
Area of Science:
- Virology
- Immunology
- Cell Biology
Background:
- Enterovirus 71 (EV71) is a significant human pathogen.
- Macrophages play a crucial role in the immune response to viral infections.
- Understanding EV71's interaction with macrophages is vital for developing antiviral strategies.
Purpose of the Study:
- To investigate the effects of EV71 infection on THP-1 macrophages.
- To elucidate the mechanisms of autophagy and apoptosis induced by EV71.
- To identify the key signaling pathways involved in EV71-mediated macrophage response.
Main Methods:
- THP-1 macrophages were infected with EV71.
- Cell viability, viral load, and ultrastructural changes were assessed.
- Apoptosis was measured using Hoechst 33342 and AnnexinV/PI staining.
- Western blotting analyzed autophagy and apoptosis-related protein expression.
- Inhibitors of autophagy (3-MA) and apoptosis (Ac-DEVD-CHO) were used to confirm pathway involvement.
Main Results:
- EV71 infection reduced THP-1 macrophage viability and increased viral replication.
- Autophagosomes and virions were observed in infected cells, indicating active infection.
- EV71 induced significant apoptosis and altered the expression of key proteins in autophagy (LC3, p62) and apoptosis (caspase-3) pathways.
- Autophagy inhibition by 3-MA and apoptosis inhibition by Ac-DEVD-CHO reduced EV71-induced cellular damage.
Conclusions:
- EV71 effectively infects and replicates within THP-1 macrophages.
- The virus induces both autophagy and apoptosis in these cells.
- These cellular responses are mediated through the activation of the LC3/p62 autophagy and caspase apoptosis signaling pathways.
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