Fig 1.
Augmented IL-1β expression during viral FH.
(A) Cultured peritoneal exudative macrophages (PEMs) and RAW264.7 cells were infected with MHV-3 (MOI = 1) in vitro, and IL-1β p17 protein levels were detected in the indicated time points by western-blotting. C57BL/6 WT mice were infected with MHV-3 (100PFU), (B) PEMs and livers were isolated and the transcription of proIL-1β mRNA was measured by qPCR. *p<0.05, NS: no significant difference. (C) The expression of proIL-1β and IL-1β p17 in virus-infected liver tissues was detected by western-blotting. Three representative samples per group are shown. (D) The proIL-1β protein in PEMs isolated from MHV-3 infected mice at the indicated time points was detected by flow cytometry. The up panels are gate strategies and number indicates the percentage of positive cells in the gate. One representative sample from five mice per group is showed. (E) Serum IL-1β and (F) IL-1α levels in virus-infected mice were detected by ELISA. N = 5~10 per group,*p<0.05, NS: no significant difference.
Fig 2.
IL-1R1 deficiency attenuates MHV-3-induced hepatitis.
IL-1R1-/- mice and their C57BL/6 wild-type (WT) littermates were infected with MHV-3 (100 PFU), (A) The survival rate was monitored for a total of 20 days. One representative of three experiments with similar results is shown. *p<0.05. (B) The liver architecture was analyzed by H&E staining (left), and serum ALT/AST levels were determined with an AU5400 automatic biochemistry analyzer (right). Scale bar 20 μm, n = 5 per group, *p<0.05 and **p<0.001, NS: no significant difference. (C) The expression of Bgp1 in MHV-3-infected livers was compared by western-blotting. Three representative samples per group are shown. (D) The virus titers in livers at 72h post-infection were analyzed by plaque assay (up), and results were compared by statistical analysis (down). *p<0.05. (E) Peritoneal exudative macrophages (PEMs) were isolated from virus infected mice at 24h and the virions were detected by electron microscopy. Arrows indicate spherical virions. (F) The expression of Bgp1 in PEMs and RAW264.7 cells that treated with IL-1β (20 ng/ml) at the indicated time points was detected by western-blotting (up panel). RAW264.7 cells were treated with IL-1β (20 ng/ml) and PBS for a total 48h firstly, cells were further infected with MHV-3 and virus titers were detected by plaque assay at the indicated time points (down panel). *p<0.05 compared to PBS-treated counterparts.
Fig 3.
MHV-3 fails to induce FGL2 production and neutrophil infiltration in the livers of IL-1R1-/- mice.
IL-1R1-/- mice and their C57BL/6 WT littermates were infected with MHV-3 (100 PFU). (A) Peritoneal exudative macrophages (PEMs) were isolated and the expression of FGL2 was detected by western-blotting. (B) The expression of FGL2 in liver at 48h and 72h post-infection was analyzed by western-blotting. Four representative samples per group are shown. (C) Serum FGL2 levels in virus infected mice were measured by ELISA.*p<0.05 and **p<0.0001, NS: no significant difference, n = 5 per group. (D) The liver fibrinogen deposition post-infection was analyzed by immunohistochemistry. Scale bar 20 μm, n = 6~8 per group. (E) Liver recruitment of CD45+Gr-1high neutrophils after MHV-3 infection was measured by flow cytometry. The left panels are gate strategies, and number indicates the percentage of positive cells in the gate. One representative sample from five mice per group is showed. (F) Statistical analysis of liver CD45+Gr-1high neutrophil infiltration. *p<0.05 compared to WT littermates in each group, n = 5 per group.
Fig 4.
IL-1β synergistically acts with TNF-α to promote FGL2 overexpression through activation of NF-κB signaling.
(A) The expression of IL-1β-p17 and FGL2 was detected by immunofluorensent double staining. Arrows indicate positive cells, blue color indicates nuclear staining with 4',6-diamidino-2-phenylindole (DAPI), scale bar 20 μm. RAW264.7 cells were treated with IL-1β (20 ng/ml), TNF-α (100 ng/ml) alone or in combination, (B) The transcription of fgl2 at 24h post-cytokine treatment was detected by qPCR. *p<0.05, NS: no significant difference, one of three experiments with similar results is shown. (C) Immunoblot analysis of phosphorylated (p-) and total signaling proteins in whole-cell lysates of RAW264.7 cells stimulated for various times with IL-1β, TNF-α alone or in the combination. (D) RAW264.7 cells were treated with IL-1β/TNF-α for a total of 24h, and cells were further incubated with inhibitors against NF-κB (PDTC), ERK (PD98059) and p38-MAPK (SB203580) in the last 6h, the expression of FGL2 was detected by western-blotting. One of three experiments with similar results is shown.
Fig 5.
NLRP3 inflammasome involves in regulating the pathogenesis of MHV-3-mediated hepatitis.
(A) Peritoneal exudative macrophages (PEMs) and RAW264.7 cells were infected with MHV-3 (MOI = 1) in vitro, and the expression of NLRP3 inflammasome complex components in the indicated time points was detected by western-blotting. (B) The transcription of NLRP3 and proCaspase-1 mRNA in liver tissues isolated from MHV-3 infected C57BL/6 WT mice was detected by qPCR (up penal), and the protein levels were measured by western-blotting (down penal). *p<0.05, NS: no significant difference. (C) The expression of IL-1β-p17, FGL2 and Bgp1 in NLRP3-/-, Caspase-1-/- and C57BL/6 WT livers at 0h and 72h post-infection was measured by western-blotting. (D) Liver fibrinogen deposition was analyzed by immunohistochemistry, the architecture was analyzed by H&E staining and cellular apoptosis was analyzed using TUNEL staining (left). Arrow indicates positive cells, blue color indicates nuclear staining with DAPI, scale bar 20 μm, n = 5 per group. Serum ALT and AST activities were determined with an AU5400 automatic biochemistry analyzer (right).*p<0.05, ** p<0.001, n = 5 per group, NS: no significant difference. (E) The virus titers in livers at 72h post-infection were analyzed by plaque assay (left), and their levels were compared by statistical analysis (right). *p<0.05, n = 5 per group. (F) The survival rate of virus infected mice was monitored for a total of 20 days. One representative of four experiments with similar results is shown. p<0.05 was considered significant different.
Fig 6.
IL-18-/- mice are susceptible to MHV-3-mediated hepatitis.
IL-18-/- mice and their C57BL/6 WT littermates were treated with MHV-3 (100PFU). (A) Peritoneal exudative macrophages (PEMs) and liver tissues were isolated from C57BL/6 WT mice, and the transcription of proIL-18 mRNA was measured by qPCR. *p < 0.05. (B) Serum IL-18 levels in virus-infected WT mice at the indicated time points were measured by ELISA. **p < 0.001, n = 5~8 per group, NS: no significant difference. (C) RAW264.7 cells and SVE-10 endothelial cells were treated with IFN-γ (50 ng/ml), TNF-α (100 ng/ml) and IL-18 (20 ng/ml) alone or in combination, and fgl2 mRNA transcription was detected by qPCR at 24h. *p < 0.05, NS: no significant difference. (D) The expression of FGL2 in MHV-3 infected livers was compared by western-blotting. Three representative samples per group are shown. (E) Liver fibrinogen deposition was analyzed by immunohistochemistry, architecture was detected by H&E staining, and cell apoptosis by TUNEL staining. Scale bar 20 μm, arrows indicates TUNEL-positive cells, blue color indicates nuclear staining with 4',6-diamidino-2-phenylindole (DAPI), n = 5 per group. (F) Liver expression of Bgp1 was detected by western-blot (up) and the virus titers in livers at 72h post-infection were analyzed by plaque assay, and their levels were compared by statistical analysis (down). *p<0.05, n = 5 per group. (G) The survival rate of virus-infected mice was monitored for 20 days. One of three experiments with similar results is shown. NS: no significant difference.
Fig 7.
Enhanced ROS in macrophages following MHV-3 infection.
PEMs and RAW264.7 cells were infected with MHV-3 (MOI = 1) in vitro, the NOX-derived ROS (DCFH), mitochondrial damage (stained with MitoTracker Red FM/MitoTracker Green FM) and the secretion of mitochondrial ROS (MitoSOX) were detected by flow cytometry (A). The gate strategies were similar to Fig 1D, and number indicates the percentage of positive cells in the gate. One of three experiments with similar results was shown. (B) The expression of NADPH oxidase subunits including gp91phox, p47phox and p67phox and NOX-4 in virus infected Raw264.7 cells was measured by western-blot. (C) Transmission electron microscopy analysis of mitochondrial morphology in virus infected cells. Arrows indicate the damaged mitochondria, whereas arrow heads indicate the normal mitochondria. (D) RAW264.7 cells were infected with MHV-3 for a total 24h, and cells were incubated with different doses of DPI in the last 4h. The secretion of DCFH and MitoSOX was measured by flow cytometry. Data were normalized to the increase in fluorescence of the MHV-3 infected alone sample without DPI treatment for each experiment (n = 3 independent experiments). (E) The expression of Caspase-1 p20 and IL-1β p17 in DPI treated RAW264.7 cells as well as PEMs was detected by western-blotting. One representative of three experiments with similar results is shown.
Fig 8.
p47phox deficiency limited NLRP3 inflammasome activation and attenuated MHV-3 mediated hepatitis.
The p47phox-/- mice and their C57BL/6 WT littermates were infected with MHV-3 (100PFU). (A) The peritoneal macrophages (PEMs) were isolated at 24h post-infection and NOX-derived ROS (DCFH) secretion was detected by flow cytometry. One representative of three experiments with similar results is shown. (B) The survival rate of virus infected mice was monitored. *p<0.05. One of three experiments with similar results is shown. (C) The expression of Casp-1 p20, IL-1β p17, Bgp1 and FGL2 at 72h post-MHV-3 infection was compared by western-blotting. (D) Serum IL-1β and FGL2 levels were measured by ELISA. N = 5 per group, *p<0.05 and **p<0.001, NS: no significant difference. (E) Liver fibrinogen deposition was analyzed by immunohistochemistry, the architecture was analyzed by H&E-staining and cellular apoptosis was analyzed using TUNEL staining (left). Scale bar 20 μm; arrow indicates positive cells; blue color indicates nuclear staining with DAPI, n = 5 per group. Serum ALT and AST activities were determined with an AU5400 automatic biochemistry analyzer (right). *p<0.05 and **p<0.001, n = 5 per group. (F) Liver virus titers at 72h of infection were analyzed by plaque assay (left), and their levels were compared by statistical analysis (right). *p<0.05. (G) MHV-3 infected p47phox-/- mice were treated with mouse recombinant IL-1β protein (100 ng/day/mouse) and the survival rate was monitored. One of three experiments with similar results is shown. *p<0.05 compared top47phox-/- +PBS+MHV-3 group.