STING signaling activation inhibits HBV replication and attenuates the severity of liver injury and HBV-induced fibrosis - PubMed (original) (raw)

STING signaling activation inhibits HBV replication and attenuates the severity of liver injury and HBV-induced fibrosis

Yuqi Li et al. Cell Mol Immunol. 2022 Jan.

Abstract

The covalently closed circular DNA (cccDNA) of HBV plays a crucial role in viral persistence and is also a risk factor for developing HBV-induced diseases, including liver fibrosis. Stimulator of interferon genes (STING), a master regulator of DNA-mediated innate immune activation, is a potential therapeutic target for viral infection and virus-related diseases. In this study, agonist-induced STING signaling activation in macrophages was revealed to inhibit cccDNA-mediated transcription and HBV replication via epigenetic modification in hepatocytes. Notably, STING activation could efficiently attenuate the severity of liver injury and fibrosis in a chronic recombinant cccDNA (rcccDNA) mouse model, which is a proven suitable research platform for HBV-induced fibrosis. Mechanistically, STING-activated autophagic flux could suppress macrophage inflammasome activation, leading to the amelioration of liver injury and HBV-induced fibrosis. Overall, the activation of STING signaling could inhibit HBV replication through epigenetic suppression of cccDNA and alleviate HBV-induced liver fibrosis through the suppression of macrophage inflammasome activation by activating autophagic flux in a chronic HBV mouse model. This study suggests that targeting the STING signaling pathway may be an important therapeutic strategy to protect against persistent HBV replication and HBV-induced fibrosis.

Keywords: Autophagic flux; Epigenetic suppression of HBV cccDNA; HBV-induced liver fibrosis; Inflammasome activation; STING activation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

STING signaling pathway activation inhibited HBV gene expression and replication in an rcccDNA cell model. a Schematic illustration of the direct effect of DMXAA on prcccDNA/Cre-transfected AML12 cells. Briefly, AML12 cells were transfected with prcccDNA and pCMV-Cre plasmids. Forty-eight hours posttransfection, the cells were then treated with increasing doses of DMXAA (20 and 40 μg/mL) for another 48 h. b Cells were treated as shown in a, and the levels of HBsAg in the culture supernatants, pgRNA in the transfected cells, and intracellular HBV DNA were analyzed by ELISA, qRT–PCR and qPCR, respectively. c Cells were treated as shown in a, and the levels of intracellular HBV DNA were analyzed by Southern blotting. RC, relaxed circular DNA; RI, replicative intermediate. d Cells were treated as shown in a, rcccDNA was extracted by Hirt’s procedure as described in the Materials and Methods and measured by qPCR. e Schematic illustration of the indirect effect of DMXAA on prcccDNA/Cre-transfected AML12 cells. Briefly, RAW264.7 cells were treated with increasing doses of DMXAA (10 and 20 μg/mL). Twelve hours later, the culture medium of DMXAA-treated RAW264.7 cells was collected and used to treat the prcccDNA/Cre-transfected AML12 cells for another 48 h. Cells were treated as shown in e, and the levels of HBsAg, pgRNA, and HBV-DNA (f, g) and rcccDNA (h) were determined as shown in b–d. i, j The effect of the STING agonist DMXAA on cccDNA-driven transcription and HBV replication in a linear HBV monomer-transfected cccDNA cell model. k AML12 or RAW264.7 cells were treated with different doses of DMXAA (20 and 40 μg/mL for AML12 cells; 10 and 20 μg/mL for RAW264.7 cells). 4 h later, cell lysates were collected and subjected to Western blotting using antibodies against phosphorylated IRF3 (p-IRF3), total IRF3, phosphorylated TBK1 (p-TBK1), total TBK1, IκBα, phosphorylated p65 (p-p65), total p65 and GAPDH. l, m AML12 or RAW264.7 cells were treated as shown in k, and the mRNA levels of IFN-β (l) and TNF-α (m) were quantified by qRT–PCR (normalized to GAPDH). n The protein levels of STING in RAW264.7, AML12 or prcccDNA/Cre-transfected AML12 cells were determined by Western blotting using antibodies against STING and GAPDH. o–q The effect of STING knockdown in RAW264.7 cells on DMXAA-mediated innate immune activation and inhibition of rcccDNA transcription and HBV replication. Primary human hepatocytes (PHHs) or human KCs (hKCs) were treated with the STING agonist cGAMP (5 μg/mL) for 6 h, the levels of STING and the phosphorylation of IRF3 and p65 were determined by Western blotting (r), and the induction of IFN-β and TNF-α was determined by qRT–PCR (normalized to GAPDH) (s, t). u hKCs were treated with 2 μg/mL cGAMP for 12 h, and the culture supernatants of hKCs were then collected and used to treat HBV-infected PHHs. Forty-eight hours posttreatment, the levels of secreted HBsAg, pgRNA, intracellular HBV DNA and cccDNA were determined by ELISA, qRT–PCR and qPCR, respectively. The data are shown as the means ± SD and were compared by unpaired Student’s t test or one-way analysis of variance (ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001; NS, no significance

Fig. 2

STING signaling pathway activation inhibited HBV gene expression and replication in a chronic rcccDNA mouse model. a Schematic illustration of the effect of STING activation on HBV transcription and replication in a chronic rcccDNA mouse model. Briefly, Alb-Cre Tg mice were injected with Ad-rcccDNA (1.5 × 109 PFU) by the tail vein. On Day 45 postinjection, rcccDNA mice were treated with 10 mg/kg DMXAA or vehicle by intraperitoneal injection for the indicated times. On Day 15 after the initial DMXAA treatment, the effect of STING activation on HBV replication and the innate immune response was examined. b, c Serum levels of HBsAg and HBV DNA in DMXAA-treated or untreated mice were determined by ELISA and qPCR, respectively (n = 10/group). d The levels of HBcAg in the liver tissues of DMXAA-treated or untreated mice were determined by immunohistochemistry. e The levels of pgRNA in the liver tissues of DMXAA-treated or untreated mice were analyzed by qRT–PCR (n = 10/group). f Intracellular HBV DNA in the liver tissues of DMXAA-treated or untreated mice was determined by Southern blotting. g The level of rcccDNA in the liver tissues of DMXAA-treated or untreated mice was determined by qPCR (n = 6/group). h, i Serum levels of IFN-β or TNF-α were determined by ELISA (n = 6/group). j, k The mRNA levels of IFN-β or TNF-α in the livers of DMXAA-treated or untreated mice were determined by qRT–PCR (normalized to GAPDH) (n = 6/group). l PMHs or KCs were isolated from DMXAA-treated or untreated mice and analyzed by Western blotting using antibodies against p-IRF3, IRF3, p-TBK1, TBK1, IκBα, p-p65, p65, STING, and GAPDH. m, n The mRNA levels of IFN-β or TNF-α in PMHs or KCs were quantified by qRT–PCR (normalized to GAPDH) (n = 6/group). The data are shown as the means ± SD and were compared by unpaired Student’s t test. ***P < 0.001; ****P < 0.0001; NS, no significance

Fig. 3

The STING signaling pathway participates in the epigenetic regulation of HBV cccDNA. RAW264.7 cells were treated with increasing doses of DMXAA (10 and 20 μg/mL). Twelve hours later, the culture medium of DMXAA-treated RAW264.7 cells was used to treat prcccDNA/Cre-transfected AML12 cells (a) or linear HBV monomer-transfected AML12 cells (c) for another 48 hours. AML12 cells were then harvested and used for ChIP assays with the indicated antibodies. RAW264.7 cells were treated with increasing doses of cGAMP (5 and 10 μg/mL). Twelve hours later, the culture medium of cGAMP-treated RAW264.7 cells was used to treat prcccDNA/Cre-transfected AML12 cells (b) or linear HBV monomer-transfected AML12 cells (d) for another 48 h. AML12 cells were then harvested and used for ChIP assays with the indicated antibodies. e rcccDNA mice were treated with or without DMXAA, and the livers were harvested and used for ChIP assays with the indicated antibodies (n = 3/group). The data are shown as the means ± SD and were compared by unpaired Student’s t test or one-way analysis of variance (ANOVA). *P < 0.05, **P < 0.01; NS, no significance

Fig. 4

STING signaling activation alleviated liver injury and fibrosis in rcccDNA mice. a Schematic illustration of the effect of STING activation on HBV-induced fibrosis in a chronic rcccDNA mouse model. Briefly, rcccDNA mice were treated with 10 mg/kg DMXAA or vehicle by intraperitoneal injection twice per week for 4 weeks. On the day after the last DMXAA treatment, the effect of STING activation on liver injury and HBV-induced fibrosis was examined (n = 6/group). b, c The levels of ALT or AST in mouse sera were determined by ALT or AST ELISA kits. d The levels of α-SMA in liver sections were determined by immunohistochemical staining, and liver collagen deposition was assessed by Sirius red staining and Masson’s trichrome staining. Right panel: The percentages of α-SMA-, Sirius red- and Masson-positive areas were measured in 5 random fields from each animal. 10 mg/kg data are represented as the means ± SEM and were compared by unpaired Student’s t test. *P < 0.05, **P < 0.01. Scale bar: 100 μm. e–h The levels of α-SMA, Col 1a1, MMP2, and TIMP1 in livers were determined by qRT–PCR (normalized to GAPDH). Except for the data in d, all data are shown as the means ± SD and were compared by unpaired Student’s t test. *P < 0.05, **P < 0.01

Fig. 5

STING signaling pathway activation suppressed inflammasome activation in macrophages. a Serum levels of IL-1β from Ad-control or Ad-rcccDNA-transduced mice were determined by ELISA at the indicated time points (n = 6/group). b The levels of caspase-1 p20 in the liver tissues of Ad-control or Ad-rcccDNA-transduced mice were determined by Western blotting. Right panel: The relative level of caspase-1 p20 was determined by densitometric analysis, and the value of Ad-control mice was set as 1.0 (n = 6/group). Anti-IL-1β or isotype control antibodies (25 mg/mL) were intraperitoneally administered twice per week for 4 weeks to the rcccDNA chronic mouse model (n = 5/group): c Serum levels IL-1β were measured by ELISA. d, e The levels of ALT or AST in mouse sera were determined by ALT or AST ELISA kits. f Liver fibrosis was evaluated by determining the α-SMA-, Sirius red- and Masson-positive areas. Right panel: The percentages of α-SMA-, Sirius red- and Masson-positive areas were measured in 5 random fields from each animal. The data are represented as the means ± SEM and were compared by unpaired Student’s t test. **P < 0.01. Scale bar: 100 μm. Serum levels IL-1β (g) and IL-18 (h) in rcccDNA mice treated with or without DMXAA were determined by ELISA (n = 6/group). i The levels of caspase-1-p20 in the liver tissues of rcccDNA mice treated with or without DMXAA were determined by Western blotting and quantified as shown in b (n = 6/group). j The levels of caspase-1-p20 in PMHs and KCs from rcccDNA mice treated with or without DMXAA were determined by Western blotting and quantified as shown in b (n = 3/group). k The levels of IL-1β secreted by PMHs and KCs from rcccDNA mice treated with or without DMXAA were determined by ELISA (n = 3/group). l Schematic illustration of the effect of siRNA-mediated STING knockdown on DMXAA-mediated inhibition of inflammasome activation. m, n Cells were treated as shown in l, and the protein levels of IL-1β in culture supernatants and the levels of caspase-1 p20 in cell lysates were determined by ELISA and Western blotting, respectively. Except for the data in f, all data are shown as the means ± SD and were compared by unpaired Student’s t test or one-way analysis of variance (ANOVA). *P < 0.05, **P < 0.01

Fig. 6

STING signaling pathway activation suppressed inflammasome activation by inducing autophagic flux. a The levels of LC3 in the liver tissues of rcccDNA mice treated with or without DMXAA were examined by Western blotting. Lower panel: The relative level of LC3II was determined by densitometric analysis, and the value of Ad-control mice was set as 1.0. b The levels of LC3 and p62 in PMHs or KCs from rcccDNA mice treated with or without DMXAA were determined by Western blotting. Right panel: The relative levels of LC3II or p62 were determined by densitometric analysis, and the value of vehicle-treated cells was set as 1.0. c The levels of LC3 and p62 in KCs with or without STING knockdown were determined by Western blotting and quantified as shown in b. d Schematic illustration of the effect of blocking DMXAA-mediated autophagic flux using leupeptin (200 μg/mL) on inflammasome activation induced by LPS (500 ng/mL) plus ATP (5 nM) in KCs. e KCs were treated as shown in d, and the levels of LC3 and p62 were determined by Western blotting. The relative level of p62 was determined by densitometric analysis, and the value of control cells was set as 1.0. f, g KCs were treated as shown in d, and the levels of caspase 1-p20 and secreted IL-1β were determined by Western blotting and ELISA, respectively. h Human KCs (hKCs) were treated with the STING agonist cGAMP (5 μg/mL) for 6 h, and the levels of LC3 and p62 were determined by Western blotting. i LPS plus ATP-stimulated hKCs were treated with the STING agonist cGAMP in the presence or absence of leupeptin, and the levels of caspase 1-p20, LC3II, and p62 were examined by Western blotting. j Human KCs were treated as described in i, and secreted IL-1β was measured by ELISA. The data are shown as the means ± SD and were compared by unpaired Student’s t test. *P < 0.05,**P < 0.01

Fig. 7

STING activation attenuated HBV-induced liver fibrosis via autophagy-mediated suppression of inflammasomes. a Schematic illustration of the effect of blocking STING-mediated autophagic flux on inflammasome activation and liver fibrosis in rcccDNA mice. Briefly, rcccDNA mice were treated with DMXAA (10 mg/kg) with or without leupeptin (40 mg/kg) twice per week for 4 weeks. 5 h after the last DMXAA plus leupeptin treatment, the effect of DMXAA on autophagic flux, inflammasome activation, liver injury, and fibrosis was determined (n = 6/group). b The effect of DMXAA on the levels of LC3 and p62 in KCs in the presence or absence of leupeptin was determined by Western blotting. Right panel: The relative level of p62 was determined by densitometric analysis, and the value of control mice was set as 1.0. c The effect of DMXAA on the levels of caspase-1 p20 in KCs in the presence or absence of leupeptin was determined by Western blotting. Right panel: The relative level of caspase-1 p20 was determined by densitometric analysis, and the value of control mice was set as 1.0. d The effect of DMXAA on the serum levels IL-1β in rcccDNA mice in the presence or absence of leupeptin was determined by ELISA. e, f The effect of DMXAA on the levels of ALT and AST in rcccDNA mouse sera in the presence or absence of leupeptin. g The effect of DMXAA on liver fibrosis in rcccDNA mice in the presence or absence of leupeptin was evaluated by immunohistochemical staining for α-SMA, Sirius red staining, and Masson’s trichrome staining. Right panel: The percentages of α-SMA-, Sirius red- and Masson-positive areas were measured in five random fields from each animal. The data are represented as the means ± SEM and were compared by unpaired Student’s t test. **P < 0.01. Scale bar: 100 μm. h–k The effect of DMXAA on the mRNA expression of α-SMA, Col 1a1, MMP2, and TIMP1 in the liver tissue of rcccDNA mice in the presence or absence of leupeptin was determined by qRT–PCR (normalized to GAPDH). Except for the data in g, all data are shown as the means ± SD and were compared by unpaired Student’s t test. *P < 0.05, **P < 0.01

Fig. 8

Schematic illustration of the inhibitory effect of STING activation on HBV cccDNA and HBV-induced liver fibrosis. STING signaling activation in macrophages leads to the functional suppression of cccDNA in hepatocytes via epigenetic modification. In addition to inhibiting HBV replication, STING activation ameliorates HBV-induced liver fibrosis by suppressing the activation of macrophage inflammasomes by stimulating autophagic flux

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STING signaling activation inhibits HBV replication and attenuates the severity of liver injury and HBV-induced fibrosis - PubMed (original) (raw)