Fig 1.
Macrophages treated with HBV displayed high OXPHOS and low glycolytic activities.
(A) Analysis of OCR (left panel) and ECAR (right panel) of THP-1-derived macrophages (THP-1(M0)) that were treated with LPS+ IFN-γ (THP-1(LPS+IFN-γ)), IL-4+IL-13 (THP-1(IL-4+13)), HCV (THP-1(M0)+HCV) or HBV (THP-1(M0)+HBV). THP-1(M0) that was not treated was used as the negative control. (B) Analysis of OCR (left panel) and ECAR (right panel) of human M1-like macrophages (M(GM-CSF)), M2-like macrophages (M(CSF-1)), and M(GM-CSF) treated with HCV (M(GM-CSF)+HCV) or HBV (M(GM-CSF)+HBV).
Fig 2.
HBeAg induces OXPHOS and suppresses glycolysis induced by HBV both in vitro and in vivo.
(A) Analysis of OCR (left panel) and ECAR (right panel) of THP-1 macrophages that were treated with recombinant 1 μg/mL HSA, HBeAg, HBsAg or HBcAg for 48 hours. (B) Analysis of OCR (left panel) and ECAR (right panel) of THP-1 macrophages with the incubation medium harvested from Huh7 cells transfected with pUC19, the 1.3mer wild-type HBV genomic DNA or the 1.3mer HBV genomic DNA that was incapable of expressing only HBeAg (HBV-e-Mt). (C) Analysis of OCR (left panel) and ECAR (right panel) of Kupffer cells (KC) isolated from mice without (Mock) or with the hydrodynamic injection of pUC19, 1.3mer wild-type HBV DNA or 1.3mer HBeAg-null HBV DNA.
Fig 3.
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.
Fig 4.
HBeAg induces GLS and increases glutamate to promote OXPHOS.
(A) THP-1 M0 macrophages with various treatments were subjected to metabolomic analysis. The volcano plot displays changes of metabolites induced by the treatment of LPS+IFN-γ, IL-4+IL-13, pUC19, HBV or HCV. The boxed area denotes glutamate that was increased by different doses of HBV. In contrast, HCV reduced glutamate levels. (B) The levels of glutamate in THP-1 macrophages with different treatments as shown in (A) were compared. In both (A) and (B), 20, 60 and 120 indicated the volume (in μL) of the incubation media used to treat THP-1 macrophages. M1, THP-1 macrophages treated with LPS+IFN-γ; M2, THP-1 macrophages treated with IL-4+IL-13. (C) The GLS1 RNA of THP-1 macrophages with or without the treatment of HBV were quantified by RT-qPCR. (D) The GLS1 RNA in THP-1 macrophages without or with the treatment of 1 μg/mL HSA, HBsAg, HBcAg or HBeAg were quantified by RT-qPCR. (E) THP-1 macrophages treated with 1 μg/mL HSA, LPS, HBsAg, HBcAg or HBeAg were lysed for immunoblot analysis of GLS1 and GLS2. GAPDH served as the loading control. (F) Kupffer cells isolated from mice that had been injected with pUC19 or the 1.3mer HBV genomic DNA without or with mutations that abolished the expression of HBsAg, HBcAg or HBeAg were analyzed for the levels of GLS1 RNA by RT-qPCR. Mice not injected with DNA (mock) was used as the control. (G) THP-1 macrophages (MΦ) with or without the treatment of 20 mM glutamate HSA or HBeAg were subjected to Seahorse analysis for their OCRs. (H) THP-1 macrophages were treated with HBeAg in the presence or absence of GLS inhibitors Telaglenastat (1 μM) and compound 968 (5 μM) and then subjected to Seahorse analysis for their OCR. (I) THP-1 macrophages without (-) or with (+) the treatment of 20 mM glutamate were not treated or treated with 1 μg/mL HSA, HBsAg, HBcAg or HBeAg for 48 hours. The incubation media were then harvested for the measurement of IL-β by ELISA. In (B-D), (F) and (I), N.S., not significant; **, p<0.01; ***, p<0.001.
Fig 5.
HBeAg differentially induces apoptosis of macrophages.
(A) THP-1 macrophages stimulated with HSA, HBsAg, HBcAg, HBeAg together with control IgG or HBeAg in the presence of anti-HBeAg for three days were analyzed for their viability using the CCK8 assay. (B) THP-1 macrophages were stimulated with the incubation medium of Huh7 cells that had been transfected with pUC19 (mock), the 1.3mer wild-type HBV genomic DNA, or the 1.3mer HBV genomic DNA with mutations that abolished the expression of HBsAg, HBcAg, the X protein or HBeAg and then analyzed for their viability using the CCK8 assay. (C) THP-1 M0, M1-like and M2-like macrophages without or with HBeAg treatment were stained for annexin V and with propidium iodide and analyzed by flow cytometry. The statistical analysis of annexin V-positive cells is shown in the bar chart. (D) Kupffer cells were isolated from mice four days after the injection of pUC19 or the HBV genomic DNA and stained for CD11b and F4/80, two markers of Kupffer cells. The staining using the isotype antibody control was also shown. The statistical analysis is shown in the bar chart. In (A-D), N.S., not significant; *, p<0.05; **, p<0.01; ***, p<0.001.
Fig 6.
HBeAg induces DR5 to promote apoptosis but not pyroptosis of macrophages.
(A) THP-1 macrophages were incubated with the incubation medium of Huh7 cells that had been transfected with pUC19, the 1.3mer HBV genomic DNA or the HBV genomic DNA with mutations that abolished the expression HBsAg, HBcAg or HBeAg followed by RT-qPCR analysis of DR4 and DR5 RNAs. (B) THP-1 macrophages were treated with HSA, HBsAg, HBcAg, HBeAg+control IgG or HBeAg+anti-HBeAg and then lysed for analysis of DR4, DR5 RNAs by RT-qPCR. (C) THP-1 macrophages treated the same way as in (B) were lysed for immunoblot analysis. (D) Kupffer cells isolated from control mice and TGD mice were treated with HSA, LPS or HBeAg and then lysed for immunoblot analysis. (E) The M1-like M(GM-CSF) and the M2-like M(CSF-1) human MDMs were treated with HSA, LPS, or HBeAg and then lysed for immunoblot analysis. (F) THP-1 macrophages transduced with a lentiviral vector that expressed the nonspecific shRNA (shNS) or the DR5 shRNA (shDR5) were stimulated with HSA, LPS, HBsAg or HBeAg and then lysed for immunoblot analysis. (G) THP-1 macrophages without (shNS) or with DR5 silencing (shDR5) were not treated (mock) or treated with HSA, LPS, HBsAg or HBeAg and then analyzed for their viability using the CCK8 assay. In (A), (B) and (G), N.S., not significant; **, p<0.01; ***, p<0.001.
Fig 7.
TLR4 mediates the effects of HBeAg on macrophages.
(A) THP-1 macrophages transduced by a lentiviral vector that expressed nonspecific shRNA (shNS), TLR2 shRNA (shTLR2) or TLR4 shRNA (shTLR4) were not treated (mock) or treated with LPS, HBsAg or HBeAg. The incubation media were then harvested for the measurement of IL-1β levels by ELISA. (B) Kupffer cells isolated from control mice were treated with nonspecific siRNA (siNS), TLR2 siRNA (siTLR2) or TLR4 siRNA (siTLR4) followed by the treatment with LPS, HBsAg or HBeAg. The incubation media were then harvested for the quantification of mouse IL-1β (mIL-1β) by ELISA. (C) Human MDMs treated with siNS, siTLR2 or siTLR4 were treated with LPS, HBsAg or HBeAg. The incubation media were then harvested for quantification of IL-1β by ELISA. (D) THP-1 macrophages without or with TLR2 or TLR4 silencing were not treated (mock) or treated with HSA, LPS, HBsAg or HBeAg for three days. The viability of cells was then analyzed by the CCK8 assay. (E) THP-1 macrophages without (shNS) or with TLR4 silencing (shTLR4) were not treated (mock) or treated with HBeAg. Cells were then stained with propidium iodide and with the anti-annexin V antibody for the analysis of apoptosis by flow cytometry. (F) THP-1 macrophages without (shNS) or with TLR4 silencing (shTLR4) were treated with HSA, LPS, HBsAg or HBeAg and then lysed for immunoblot analysis. (G) THP-1 macrophages without (shNS) or with TLR4 silencing (shTLR4) were treated with HSA, LPS, HBsAg or HBeAg and then lysed for immunoblot analysis. (H) THP-1 macrophages without (shNS) or with TLR4 silencing (shTLR4) were treated with HSA or HBeAg and then subjected to Seahorse assay for the OCR. (I) HEK293 cells were transfected with the expression plasmid of Flag-tagged TLR4, lysed and treated with GST-HBeAg, and then incubated with glutathione beads for the pull-down experiment. The protein sample was then analyzed by immunoblot using the anti-Flag, anti-GST and anti-TLR4 antibodies. Input represents the total cell lysates prior to the GST-pulldown experiment. GAPDH was used as the loading control. (J) THP-1 macrophages with the treatment of either GST, HBeAg-GST or HBcAg were subjected to the proximity ligation assay (PLA) for the co-localization analysis. Red dots represent positive colocalization signals. DAPI was used to stain the nuclei (blue color). The chart to the right is the average number of PLA signals per cell after a total of 50 cells were analyzed. In (A-E), N.S., not significant; ***, p<0.001.