HBeAg induces DAP3 and mitochondrial proteins to promote OXPHOS of macrophage both in vitro and in vivo.

(A) THP-1 M0 macrophages treated with the incubation medium of Huh7 cells that had been transfected with pUC19, the 1.3mer wild-type HBV genomic DNA or the 1.3mer HBeAg-null HBV genomic DNA were analyzed by RT-qPCR for the RNA levels of mitochondrial proteins, DAP3 and IL-1β. (B) THP-1 M0 macrophages treated with HSA, LPS+IFN-γ (i.e., M1 macrophages), IL4+IL13 (i.e., M2 macrophages), 1 μg/mL HBeAg, HBsAg or HBcAg for 48 hours were analyzed by RT-qPCR for the expression of mitochondrial genes, DAP3 and IL-1β. (C) THP-1 macrophages treated with 1 μg/mL HSA, HBsAg, HBcAg or HBeAg for 48 hours were lysed for immunoblot analysis. β-actin served as a loading control. (D) Human MDMs with (HBV+) and without (HBV-) treatment and Kupffer cells that were isolated from mice that had been injected with pUC19 (HBV-) or the 1.3mer HBV genomic DNA (HBV+) were analyzed for DAP3 and IL-1β RNAs by RT-qPCR. (E) Kupffer cells isolated from mice three days after the injection of pUC19, 1.3mer wild-type HBV genomic DNA or the HBV genomic DNA mutant that was incapable of expressing HBsAg, HBcAg or HBeAg were lysed for immuoblot analysis. (F) THP-1 macrophages were transduced with a lentiviral vector that expressed a nonspecific shRNA (shNS) or the DAP3 shRNA (shDAP3) and then treated with the incubation medium of Huh7 cells that were transfected with pUC19 (HBV-) or the 1.3mer HBV genomic DNA (HBV+). The OCR of THP-1 macrophages was then measured by Seahorse assay. (G) THP-1 macrophages with and without DAP3 silencing as in (F) were treated with 1 μg/mL HSA, HBsAg, HBcAg or HBeAg for 48 hours and then subjected to Seahorse analysis of OCR. (H) THP-1 macrophages were transduced with a control lentiviral vector or a lentiviral vector that expressed DAP3, stimulated with 100 ng/mL LPS for 24 hours and then subjected to Seahorse analysis of OCR. In (A), (B) and (D), N.S., statistically not significant; *, p<0.05; **, p<0.01; ***, p<0.001.