cGAS-mediated autophagy protects the liver from ischemia-reperfusion injury independently of STING

Abstract

Liver ischemia-reperfusion (I/R) injury occurs through induction of oxidative stress and release of damage-associated molecular patterns (DAMPs), including cytosolic DNA released from dysfunctional mitochondria or from the nucleus. Cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) is a cytosolic DNA sensor known to trigger stimulator of interferon genes (STING) and downstream type 1 interferon (IFN-I) pathways, which are pivotal innate immune system responses to pathogen. However, little is known about the role of cGAS/STING in liver I/R injury. We subjected C57BL/6 (WT), cGAS knockout (cGAS^−/−), and STING-deficient (STING^gt/gt) mice to warm liver I/R injury and that found cGAS^−/− mice had significantly increased liver injury compared with WT or STING^gt/gt mice, suggesting a protective effect of cGAS independent of STING. Liver I/R upregulated cGAS in vivo and also in vitro in hepatocytes subjected to anoxia/reoxygenation (A/R). We confirmed a previously published finding that hepatocytes do not express STING under normoxic conditions or after A/R. Hepatocytes and liver from cGAS^−/− mice had increased cell death and reduced induction of autophagy under hypoxic conditions as well as increased apoptosis. Protection could be restored in cGAS^−/− hepatocytes by overexpression of cGAS or by pretreatment of mice with autophagy inducer rapamycin. Our findings indicate a novel protective role for cGAS in the regulation of autophagy during liver I/R injury that occurs independently of STING.

NEW & NOTEWORTHY Our studies are the first to document the important role of cGAS in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that cGAS protects liver from I/R injury in a STING-independent manner.

INTRODUCTION

Liver ischemia-reperfusion (I/R) injury occurs clinically after hepatic resection surgery, liver transplantation, and hemorrhagic shock. Importantly, liver I/R injury is a major risk factor for the development of acute liver failure (4). I/R injury occurs primarily following return of blood flow and oxygen to hypoxic liver during reperfusion. In this process, massive amounts of reactive oxygen species (ROS) are produced and contribute to the pathogenesis of I/R injury. During oxidative stress, endogenous nucleic acids, such as mitochondrial DNA and nuclear DNA, are released into cytosol as well as the circulation. Previously, we have shown that DNA sensing pathways such as the Toll-like receptor 9 (TLR9) signaling pathway (22) and the absent in melanoma 2 (AIM2) inflammation pathway (39) are important for the pathogenesis of liver injury under oxidative stress. TLR9 activation through histone-DNA complexes leads to enhanced inflammatory signaling and greater damage, while activation of AIM2 signaling in response to high-mobility group protein B1 (HMGB1)-DNA complexes activates mitophagy and protection from cell death during hemorrhagic shock. We have also shown that RNA sensing pathways become activated in the absence of adequate adenosine deaminase acting on RNA 1 (ADAR1) expression and can exacerbate retinoic acid inducible gene I (RIG-I) dependent inflammation and liver injury following I/R (47). These studies indicate that the sensing of endogenous nucleic acids is one of the important mechanisms of oxidative stress-induced liver injury or protection.

Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) is a cytosolic DNA sensor. cGAS can directly bind to DNA and thus induce its conformational change in the active site, which catalyzes cGAMP production from ATP and GTP (37, 53). cGAMP then serves as a second messenger to activate stimulator of interferon genes (STING) and subsequently induce the production of type 1 interferon (IFN-I) and other inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1β, and IL-6 (23, 37, 51). A recent report demonstrated that transcription factor A, mitochondrial (TFAM) and HMGB1 can both orient DNA for more efficient DNA binding (2). The cGAS/STING DNA sensing pathway is known to be important for innate immune response to the detection of intracellular DNA from invading viral and bacterial pathogens. There is also evidence that endogenous DNA can be sensed by cGAS during pathological processes including autoimmune diseases and cancer (5). Previous findings from our laboratory group demonstrated that double-stranded DNA (dsDNA) is released into the cytosol following liver I/R (39). However, whether the cGAS pathway is activated during liver I/R is unknown.

In this study, we aimed to determine the role of the cGAS-STING pathway in liver I/R injury. We found that cGAS protects liver from I/R injury independently of STING. Deletion of cGAS, but not STING, suppressed hypoxia-induced autophagy in hepatocytes and thus led to apoptotic cell death during liver I/R. Collectively, our findings reveal a novel role for cGAS in protection of liver from I/R injury. This previously unrecognized mechanism may be useful in designing strategies to prevent liver reperfusion injury.

MATERIALS AND METHODS

Animals.

Male C57BL/6 wild-type (WT) and STING golden-ticket (STING^gt/gt) mice and cGAS knockout (cGAS^−/−) mice on a C57BL/6 background were purchased from Jackson Laboratory, and were bred in our animal facility. Commercial mice and laboratory-bred mice were cohoused for 2 wk before experimentation to mitigate effects of microbiome differences. Experimental mice were used between the ages of 8 and 12 wk with weights ranging from 20 to 30 g. Animal protocols were approved by the Animal Care and Use Committee of the University of Pittsburgh, and the experiments were performed in adherence to National Institutes of Health guidelines for the use of laboratory animals.

Liver I/R.

A nonlethal model of segmental (70%) hepatic warm ischemia and reperfusion was used as described previously (45). Briefly, all structures in the portal triad (hepatic artery, portal vein, bile duct) to the left and median liver lobes were occluded with a microvascular clamp for 60 min, and reperfusion was initiated by clamp removal. The temperature of the mice during ischemia was maintained at 34°C using a warming incubator chamber. Sham animals underwent anesthesia, laparotomy, and exposure of the portal triad without hepatic ischemia. Animals were euthanized at predetermined time points (6 and 24 h) after reperfusion to obtain serum and liver samples.

Hepatocyte and nonparenchymal cell isolation.

Hepatocytes were isolated from mice by an in situ collagenase (type VI, Worthington) perfusion technique, as described previously (36). Hepatocytes were separated from nonparenchymal cells by two cycles of differential centrifugation (50 g for 2 min) and further purified over a 30% Percoll gradient. Hepatocyte purity exceeded 98%, as assessed by light microscopy, and viability typically was above 95%, as determined by trypan blue exclusion assay. Nonparenchymal cells (NPCs) were isolated from mice as described (44). The NPCs did not contain hepatocytes as detected by light microscopy.

Cell culture and treatment.

Hepatocytes (4 × 10⁵ cells/plate for 6-well plates, 5 × 10⁶ cells/plate for 10-cm plates) were plated on gelatin-coated (42) culture plates in Williams medium E with 10% calf serum, 15 mM HEPES, 10⁻⁶ M insulin, 2 mM l-glutamine, and 100 U/ml penicillin-streptomycin. NPCs (2.5 × 10⁷cells/plate) were plated on 10-cm plates in Williams medium E containing 5% calf serum, 15 mM HEPES, 10⁻⁶ M insulin, 2 mM l-glutamine, and 100 U/ml penicillin-streptomycin. Cells were allowed to attach to plates for at least 4 h before treatment. To simulate ischemia, hepatocytes were incubated in modular incubator chamber (MIC-101, patent no. 5352414), which was flushed with the anoxic gas mixture (95% Nnitrogen and 5% carbon dioxide) with a rate of 20 l/min in 15 min. To simulate reperfusion, hepatocytes were returned to 21% normoxic conditions in the chamber.

Liver damage assessment.

Alanine aminotransferase (ALT) levels were measured using the DRI-CHEM 4000 Chemistry Analyzer System (Heska). The ALT values were expressed as international units per liter. Liver samples were embedded in paraffin and stained with hematoxylin and eosin (H&E) for histopathology assessment. Necrotic area was measured with ImageJ software.

H&E staining and scoring method.

Mice were euthanized after designated treatment. The right upper lung lobe and the left lateral lobe of the liver were perfused with PBS and fixed in 2% paraformaldehyde (PFA). Tissues were then placed in 2% PFA for an additional 2 h and then switched to 30% sucrose in distilled water solution for 24 h. The tissue was then slowly frozen in liquid nitrogen-cooled 2-methylbutane according to a standardized protocol for cryopreservation. Cryostat sections of the tissues (6 µm) were stained with H&E to evaluate histopathological cumulative changes among treatment groups. Images of five randomly selected fields were acquired using an Olympus Provis light microscope (Malvern, NY) with ×100 magnification. Samples were scored by three independent members of the Center for Biologic Imaging according to Suzuki’s score, on a scale of 0–4, for characterizing liver damage I/R (3).

Reagents.

N-acetyl-l-cysteine (NAC, Sigma-Aldrich), 10 min before A/R stimulation, was added to each well with a dose of 0.5 or 5 mM. Rapamycin was purchased from LC Laboratories, and 8 mg/kg (24, 35) was injected into mice 1 h before ischemia using intraperitoneal injection; 100 nM (54) was added to hepatocytes of each well 10 min before A/R stimulation. Dimethyl sulfoxide (DMSO) was purchase from ATCC. Antibodies for Western blot analysis were as follows: anti-cGAS (1:1,000, no. 31659), anti-STING (1:1,000, no. 13647), anti-caspase3 (1:500, no. 9665), anti-cleaved caspase3 (1:500, no. 9661), anti-SQSTM1/p62 (1:1,000, no. 5114), anti-p70 S6 kinase (1:1,000, no. 2708), anti-phospho-p70 S6 kinase (1:1,000, no. 9234), anti-COX IV (1:1,000, no. 4850), and anti-GAPDH (1:1,000, no. 97166) from Cell Signaling Technology. Anti-LC3B (1:1,000, NB100-2220) was from Novus, anti-cytochrome c (1:1,000, ab133504) and anti-β-actin (1:5,000, ab8226) were from Abcam; anti-Beclin1 (1:1,000, sc-48341) was from Santa Cruz Biotechnology. Secondary antibodies (1:10,000, no. 31460, no. 31430) were from Thermo Fisher Scientific. For Western blot analysis, cell lysis buffer (1:10, Cell Signaling Technology, no. 9803) was used for whole cell lysis and tissue lysis together with protease inhibitors. The procedure of Western blot analysis was as previously described (8). Western gel images were quantified by densitometry analysis using ImageJ software and presented as a ratio of loading controls.

Cytokine measurement.

Plasma and liver tissue lysis samples were analyzed with ELISA kits specific for IL-6, IL-1β, (C-X-C motif L1 (CXCL1), and monocyte chemoattractant protein-1 (MCP-1) (R&D Systems) according to the manufacturer’s instructions to assess their concentrations.

Real-time RT-PCR.

Two-step real-time RT-PCR was performed as previously described (9). The forward and reverse primer pairs were specific for cGAS (Invitrogen) as follows: forward: 5′-ACGAGAGCCGTTTTATCTCGTACCC-3′; reverse: 3′-TGTCCGGAAGATTCACAGCATGTTT-5′. All samples were assayed in triplicate, and data were normalized to actin mRNA abundance.

Immunofluorescence and confocal microscopy.

Liver tissue samples were prepared as described previously (38). Cell death was measured by incubation with In Situ Cell Death Detection TMR Red (Roche no. 12156792910) according to the manufacturer’s protocol for 30 min at 37°C followed by a 1-h incubation at room temperature with 2 μg/ml goat anti-rabbit IgG conjugated with Cy3 (Jackson Immunoresearch) in PBB (Thermo Fisher) to stain for actin on both staining sets. Hoechst nuclear stain (Sigma, B-2883) was applied at room temperature for 30 s followed by a single rinse of PBS to remove excess dye. Imaging conditions were maintained at identical settings, with original gating performed using the negative control (no primary antibody). Images were taken from six random fields/section with a Nikon A1 confocal microscope (purchased with 1S10OD019973-01 awarded to Dr. Simon C. Watkins). Quantification was performed using NIS Elements (Nikon).

Cellular ROS detection.

Cellular ROS detection was performed using a fluorescence intensity-based method with a DCFDA-Cellular Reactive Oxygen Species Detection Assay Kit (Abcam). Hepatocytes (2.5 × 10⁴ cells/plate) were plated on a 96-well microplate 45 min before completion of A/R stimulation, and 100 μl of DCFDA (25 µM) was added to each well. DCFDA fluorescence was measured by microplate reader at wave length at 485 nm/535 nm.

Mouse cGAS plasmid construction and transfection.

Mouse cGAS cDNA (pUNO1-cGAS) was purchased from Invivogen. High-fidelity PCR was performed using Phusion Polymerase (NEB) from the vector, using the following primers: forward: 5′-CCATGGAAGATCCGCTAGAA-3′, reverse: 5′-ATCTTATCAGATCTGGCCAGCT-3′. The reverse primer creates a BglII restriction endonuclease 3′ of the stop codon. The purified PCR amplimer was digested with BglII, gel purified, and then ligated in-frame to the 3 × FLAG moiety of pCmv3 × FLAG (Addgene). The plasmid was prepared by digesting with EcoRI and blunting and then digesting with BamHI followed by gel purifying. Clones were verified by DNA sequencing. The cGAS plasmid DNA was transfected into hepatocytes with GeneJammer Transfection Reagent (Agilent, no. 204130) for 3:1 (reagent-to-DNA ratio) according to the protocol. A/R was performed after incubation of cells for 24 h.

Measurement of cytochrome-c release.

Cytosolic fractions of liver tissue from WT and cGAS^−/− mice were prepared as described (20). Briefly, livers were homogenized and centrifuged to pellet the mitochondria. The supernatant was collected, and protein concentration was determined by BCA assay (Thermo Scientific), followed by detection of cytochrome c by Western blot analysis.

Measurement of autophagic flux.

Autophagic flux was assessed by increase in GFP-LC3 puncta or Western blot analysis of LC3I:II conversion in hepatocytes after treatment with bafilomycin (50 nM, Sigma) for 1 h, or transfecting hepatocytes with GFP-LC3 plasmid (38). GFP-LC3 transfection of cells was imaged with a ZeissLSM510 laser-scanning confocal microscope. The numbers of GFP-positive puncta were counted for each cell. The number of LC3 puncta was obtained by counting the number of cells with at least five green dots per cell from at least 30 cells per treatment. The average transfection efficiency was assessed using immunofluorescence staining. Transfection efficiency can vary but was typically around 50% for primary hepatocytes with Genejammer after 24-h transfection (data not shown).

Statistical analysis.

All data were analyzed using GraphPad Prism software (GraphPad, San Diego, CA). Statistical analysis was performed using the two-tailed Student’s t-test for calculating the statistical significance of two experimental groups. One-way analysis of variance for calculating the statistical significant differences between the means of three or more independent groups. A P value of <0.05 was considered statistically significant. All values are presented as means ± SE.

RESULTS

Deletion of cGAS aggravates liver I/R injury.

To determine the role of cGAS in an in vivo model of liver I/R, we subjected WT and cGAS^−/−mice to warm I/R, with 60 min of liver ischemia followed by 6 or 24 h of reperfusion. Liver injury was assessed by measuring serum ALT levels and area of necrosis in liver sections stained with H&E. ALT levels in cGAS^−/− mice subjected to I/R were significantly higher than those of WT mice at both 6 and 24 h after reperfusion (Fig. 1, A and B), with levels highest at 6 h. The Suzuki histological scores and extent of necrosis identified by H&E staining of liver sections were consistent with ALT levels, with significantly more liver damage in cGAS^−/− mice than in WT mice at both 6 and 24 h of reperfusion (Fig. 1C). We confirmed that cGAS was upregulated in the liver of WT mice subjected to I/R and that cGAS was not expressed in cGAS^−/− mice before or after I/R (Fig. 1D). cGAS protein levels were higher at 6 h than at 24 h (Fig. 1E).

Fig. 1.

Depletion of cyclic GMP-AMP (cGAMP) synthase (cGAS) aggravates liver ischemia-reperfusion (I/R) injury. A: plasma alanine aminotransferase (ALT) levels in wild-type (WT) and cGAS knokout (cGAS^−/−) mice after sham surgery (Sham) or 1-h ischemia and 6-h reperfusion (I/R); n = 5 in each of the sham groups, n = 16 in each of the liver I/R groups. B: plasma ALT levels in WT and cGAS^−/− mice after sham surgery or 1-h ischemia and 24-h reperfusion; n = 5 in each of the sham groups, n = 6 in WT I/R group, n = 7 in cGAS^−/− I/R group. Each data point represents a single mouse. C: representative liver H&E (original magnification ×10) of WT and cGAS^−/− mice after sham surgery or 1-h ischemia and 6- or 24-h reperfusion. Liver damage evaluated by Suzuki’s histological score, quantified in bar graph. Dotted lines indicate measured areas of necrosis, quantified in bar graph; n = 3 for sham; n = 5 for 1-h ischemia and 6- or 24-h reperfusion groups, respectively. D: Western blot for cGAS in livers from WT and cGAS^−/− mice in sham surgery and 1-h ischemia and 6-h reperfusion groups. Densitometry of cGAS bands relative to β-actin loading control quantified by ImageJ software and presented in bar graph. E: Western blot for cGAS in whole liver lysates from WT mice after sham surgery or 1-h ischemia and 6- or 24-h reperfusion. Densitometry of cGAS bands relative to β-actin loading control quantified by ImageJ software and presented in bar graph. One mouse liver per lane. Images are representative of data from multiple mice per experimental group. Data ae presented as means ± SE.*P < 0.05, **P < 0.01.

STING is not protective in liver after I/R injury.

To determine whether STING also influences liver injury after I/R, WT and STING^gt/gt mice were subjected to warm liver I/R as described above. Surprisingly, STING deficiency did not phenocopy cGAS^−/−mice. There was no significant difference in ALT levels (Fig. 2A) or liver necrosis area (Fig. 2B) between WT and STING^gt/gt mice at 6 h.

Fig. 2.

Stimulator of interferon genes (STING) is not protective in liver I/R injury. A: plasma ALT levels in WT and STING-deficient (STING^gt/gt) mice after sham surgery or 1-h ischemia and 6-h reperfusion. Each data point represents a single mouse; n = 6 in each of the sham groups, n = 9 in each of the liver I/R groups. B: representative liver H&E (original magnification ×10) of WT and STING^gt/gt mice after sham surgery or 1-h ischemia and 6-h reperfusion. Dotted lines indicate measured areas of necrosis, quantified in bar graph; n = 3 for sham and n = 5 for 1-h ischemia and 6-h reperfusion. C: Western blot for STING expression in primary isolated hepatocytes or nonparenchymal cells (NPCs) under normoxia (Ctrl) and after 10-h anoxia/12-h reoxygenation (A/R). Densitometry of STING bands relative to β-actin loading control quantified by ImageJ software and presented in bar graph. Images are representative of data from multiple mice per experimental group or ≥3 independent in vitro experiments. Data presented as means ± SE. **P < 0.01; ^#P < 0.05 (NPC vs. hepatocyte); ns, not significant.

To further explore the STING-independent role of cGAS, isolated primary hepatocytes and NPCs from WT and knockout mice were subjected to A/R, recapitulating the effects of the in vivo warm I/R model. Western blot analysis confirmed a recently published finding that hepatocytes do not express STING under normoxic conditions (43). Importantly the expression of STING in hepatocytes was also not induced by A/R. However, liver NPCs (Kupffer, sinusoidal endothelial, and stellate cells) from WT and cGAS^−/− mice did express STING (Fig. 2C), although levels were reduced in cGAS^−/− compared with WT and were not upregulated by A/R in either WT or cGAS^−/− cells. STING^gt/gt mice were also confirmed to not express measurable levels of STING in either cell type under normoxic conditions or after A/R.

Systemic inflammation was assessed by levels of IL-6, IL-1β, CXCL-1, and MCP-1. The levels of these inflammatory cytokines were elevated after I/R; however, there was no significant difference between WT and cGAS^−/− mice (data not shown). Taken together, our results suggest that cGAS is protective through a mechanism that is independent of STING in the liver and in response to I/R.

Oxidative stress mediates cGAS upregulation in liver.

Oxidative stress can induce DNA oxidation, and this was previously shown to upregulate activation of the cGAS-STING pathway after ultraviolet irradiation in immune cells (17). To test whether cGAS expression itself was regulated by oxidative stress in liver, we first confirmed oxidative stress injury by detecting the generation of ROS after anoxia and reoxygenation in hepatocytes. Anoxia and reoxygenation of cultured hepatocytes resulted in increased ROS production (Fig. 3A). A/R lead to an increase in cGAS expression at both the mRNA and protein levels (Fig. 3, B and C), and that upregulation was markedly suppressed by N-acetylcysteine (NAC), a scavenger of ROS. Low levels of cGAS expression were observed in hepatocytes cultured under normoxia, and this was also blocked by NAC suggesting that plating hepatocytes lead to low-level ROS generation.

Fig. 3.

Oxidative stress mediates cGAS upregulation in vivo and in vitro. A: measurement of ROS by DCDF fluorescence in WT hepatocytes subjected to 10 h of anoxia and 2, 6, or 8 h of reoxygenation (A/R). B: expression of cGAS mRNA in WT and cGAS^−/− hepatocytes after normoxia (Ctrl) or 10-h anoxia with either 6- or 8-h reoxygenation and with or without N-acetylcysteine (NAC) treatment, NAC was added 10 min before A/R stimulation. C: Western blot for cGAS in whole cell lysates of WT hepatocytes after normoxia (Ctrl) or 10-h anoxia and 2- or 12-h reoxygenation with or without treatment with NAC. Densitometry of cGAS bands relative to β-actin loading control quantified by ImageJ software and presented in bar graph. Images are representative of data from ≥3 independent in vitro experiments. Data presented as means ± SE. *P < 0.05, **P < 0.01.

cGAS protects hepatocytes from cell death in vivo and in vitro.

To further explore the protective role of cGAS in hepatocytes, we assessed levels of apoptosis in liver and hepatocytes from WT and cGAS^−/− mice. More TMR-positive cells were observed in the liver of cGAS^−/− mice after I/R than were seen in liver of WT mice (Fig. 4A). We also assessed the release of cytochrome c into the cytosol, a measure of the intrinsic apoptosis signaling pathway (11, 32). As shown in Fig. 4B, cytosolic levels of cytochrome c in the livers of cGAS^−/− mice after I/R were higher than the levels in livers from WT mice. In parallel with the increased of cytochrome c in the cytosol, we also saw markedly decreased levels of cytochrome c in the mitochondria of livers from cGAS^−/− mice after I/R, suggesting increased apoptosis in cGAS^−/− mice after I/R. Similarly, there was less procaspase-3 and significantly more cleaved (activated) caspase-3 in the livers of cGAS^−/− mice compared with WT after I/R (Fig. 4C). Consistent with the in vivo results, cGAS deficiency resulted in higher levels of hepatocyte apoptosis compared with WT hepatocytes after A/R (10 h anoxia/6, 8, or 12 h reoxygenation), as determined by lower procaspase-3 and increased cleaved caspase-3 levels (Fig. 4D). Importantly, the greater levels of apoptosis in cGAS^−/− hepatocytes after A/R (10/12 h) was completely abrogated by transfection with a plasmid expressing murine cGAS (Fig. 4E). These findings suggest that cGAS deficiency resulted in higher levels of liver and hepatocyte apoptosis, possibly driven by increased mitochondrial dysfunction and subsequent increased levels of oxidative stress injury.

Fig. 4.

cGAS protects hepatocytes and liver from apoptosis in vivo and in vitro. A: confocal images from WT and cGAS^−/− liver stained with TMR red (red) and Hoescht nuclear stain (blue) after sham surgery or 1-h ischemia/6-h reperfusion (original magnification ×40). Percentage of TMR-positive cells was quantified using ImageJ software and represented in bar graph. B: Western blot for cytosolic cytochrome c (cyto c) and Cox IV in WT and cGAS^−/− liver cytosolic extract after sham surgery or I/R. Densitometry of cytosolic cyto c bands relative to β-actin loading control quantified by ImageJ software and presented in bar graph. Western blot for mitochondrial cyto c in WT and cGAS^−/− liver mitochondrial extract after sham surgery or I/R. Densitometry of cytosolic cyto c bands relative to Cox IV loading control quantified by ImageJ software and presented in bar graph. C: Western blot for procaspase-3 and cleaved caspase-3 in whole cell lysates from WT and cGAS^−/− liver after sham surgery or I/R. Densitometry of procaspase-3 and cleaved caspase-3 bands relative to β-actin loading control quantified by ImageJ software and presented in bar graphs. D: Western blot for procaspase-3 and cleaved caspase-3 in whole cell lysates from WT and cGAS^−/− hepatocytes subjected to normoxia (Ctrl) or 10-h anoxia with 6-, 8-, or 12-h reoxygenation. Densitometry of procaspase-3 and cleaved caspase-3 relative to β-actin loading control quantified by ImageJ software and presented in bar graph. E: Western blot for cGAS, procaspase-3, and cleaved caspase-3 in whole cell lysates from WT and cGAS^−/− hepatocytes under normoxia (Ctrl) or after 10-h anoxia and 12-h reoxygenation with or without overexpression of cGAS. Densitometry of cGAS, procaspase-3 and cleaved caspase-3 bands relative to β-actin loading control quantified by ImageJ software and presented in bar graphs. Images are representative of data from multiple mice per experimental group or ≥3 independent in vitro experiments. Data presented as means ± SE. *P < 0.05, **P < 0.01.

Deficiency in cGAS suppresses hepatic autophagy after oxidative stress injury.

Redox stress increased by hypoxia can lead to increased autophagic flux as a protective mechanism. If this response is inadequate, cells can undergo apoptosis (6, 28, 38). A recent study suggested that cGAS is required for dsDNA or herpes simplex virus-1 stimulation-induced autophagy (30). Given this finding, we hypothesized that cGAS-mediated protection in I/R occurs via regulation of hepatic autophagy during oxidative stress injury. To test this hypothesis, we determined levels of expression of LC3-II [a marker for autophagosomes (33)] in WT and cGAS^−/− livers. Levels of LC3-II increased in WT liver after I/R but not in cGAS^−/− livers. In addition, levels of p62 in livers of cGAS^−/− mice, which typically drop during increased autophagy, instead increased compared with WT mice following I/R (Fig. 5A). In vitro, we also found lower levels of LC3-II in hepatocytes isolated from cGAS^−/− mice compared with WT hepatocytes following A/R with 8 or 12 h of reoxygenation. The p62 expression in hepatocytes of cGAS^−/− mice significantly increased compared with WT mice following A/R (Fig. 5B). These data suggest an important role for cGAS in the regulation of protective autophagy in hepatocytes during I/R injury. Interestingly, we did not find a significant difference in beclin-1 expression after liver I/R injury (data not shown).

Fig. 5.

Deficiency of cGAS suppresses hepatic autophagy after oxidative stress. A: Western blot for LC3-II (a marker for autophagosomes) and p62 in whole liver lysates from WT and cGAS^−/− mice after sham surgery or 1-h ischemia/6-h reperfusion. Densitometry of LC3-II and p62 bands measured by ImageJ software and represented in bar graphs relative to β-actin loading control. B: Western blot for LC3-II and p62 in whole cell lysates from WT and cGAS^−/− hepatocytes under normoxia (Ctrl) or after 10-h anoxia and 8- or 12-h reoxygenation. Densitometry of LC3-II and p62 bands relative to β-actin loading control quantified by ImageJ software and presented in bar graph. Images are representative of data from multiple mice per experimental group or ≥3 independent in vitro experiments. Data presented as means ± SE. *P < 0.05, **P < 0.01.

Because the process of autophagy is a dynamic pathway, autophagosome accumulation may represent either increased autophagosome formation or blockade of its degradation (33). To address these possibilities, we performed autophagic flux assays in cultured hepatocytes, where we inhibited autophagolysosomal fusion and degradation with bafilomycin treatment. GFP-LC3-II was transfected into hepatocytes, and the accumulation of GFP-LC3-II puncta was detected by confocal microscopy and LC3-II levels by Western blot. As expected, we found that the number of GFP-LC3-II-positive autophagosomes in WT hepatocytes increased with bafilomycin treatment both under normoxic conditions and after A/R (10/8 h); however there was no significant increase in GFP-LC3-II puncta in cGAS^−/− hepatocytes (Fig. 6A). Similarly, bafilomycin treatment did not significantly increase levels of LC3-II in cGAS^−/− hepatocytes compared with WT cells after normoxia or A/R (Fig. 6B). Taken together, our results suggest that cGAS is an important inducer of autophagosome formation and regulates induction of hepatic autophagy.

Fig. 6.

cGAS regulates autophagic flux in hepatocytes. A: confocal microscopy images of WT and cGAS^−/− hepatocytes overexpressing green fluprescent protein (GFP)-LC3 (green) and Hoescht nuclear stain (blue) and subjected to normoxia or 10-h anoxia with 8-h reoxygenation and with/without 60 min bafilomycin (baf) treatment. GFP-LC3 puncta were counted per cell and represented in bar graph. Scale bar, 100 μm. B: Western blot for LC3-II in whole cell lysates from WT and cGAS^−/− (KO) hepatocytes subjected to normoxia (Ctrl) or A/R (10-h/8-h) with/without bafilomycin. Densitometry of LC3-II bands measured by ImageJ software and represented in bar graph relative to β-actin loading control. Images are representative of data from ≥3 independent in vitro experiments. Data presented as means ± SE. *P < 0.05, **P < 0.01, ns = not significant.

Induction of autophagy rescues cGAS^−/− mice from excessive liver I/R injury.

We next assessed whether cGAS^−/− mice were capable of protective autophagy during liver I/R injury. cGAS^−/− mice were injected with rapamycin (8 mg/kg ip) or vehicle (DMSO) 1 h before ischemia to induce autophagy, as previously described (24, 35). Mice were subjected to 60 min of warm hepatic ischemia followed by 6 h of reperfusion. We confirmed that rapamycin blocked mammalian target of rapamycin complex 1 (mTORC1) autophagy inhibitor by assessing total p70 S6 kinase and phosphorylation of p70 S6 kinase, a downstream target of mTORC1. No significant phospho-p70 S6 was determined by Western blot in rapamycin-treated mouse livers, confirming effective blockade of the inhibitory effects of mTORC1 (Fig. 7A). We also confirmed that autophagy was significantly induced in rapamycin-treated mice by assessing p62 degradation and beclin-1 levels (Fig. 7A). Importantly, pretreatment with rapamycin significantly reduced serum ALT levels, suggesting significant protection from liver I/R injury (Fig. 7B). To determine whether increased autophagy with rapamycin treatment resulted in reduced apoptosis and cell death, we assessed levels of cleaved caspase-3 and measured TMR-positive cells. Rapamycin-induced autophagy significantly reduced apoptosis in cGAS^−/− liver, shown by reduced TMR-positive cells and reduced cleaved caspase-3 (Fig. 7, C and D). In vitro rapamycin decreased p62 levels in cGAS^−/− hepatocytes in vitro to a greater extent than in WT mice (Fig. 7E), suggesting increased autophagosome formation for its removal. Thus, cGAS^−/− mice were capable of protective autophagy.

Fig. 7.

Induction of autophagy rescues cGAS^−/− mice from excessive liver injury. A: Western blot for total p70 S6 kinase (p70 S6K) and phospho-p70 S6K kinase (P-p70S6K), p62, beclin-1, in whole liver lysates from cGAS^−/− mice pretreated with rapamycin (Rapa, 8 mg/kg) or DMSO control and subjected to sham surgery or I/R. Densitometry of p70S6K, P-p70S6K, p62, and beclin-1 relative to GAPDH loading control, quantified by ImageJ software and presented in bar graphs. B: plasma ALT levels in cGAS^−/− mice pretreated with rapamycin or DMSO control and subjected to sham surgery or 1-h ischemia/6-h reperfusion; n = 5 in each of the sham groups, n = 5 in each of the liver I/R groups. Each data point represents one mouse. C: confocal microscopy images of TMR red (red) and Hoescht nuclear staining (blue) in liver sections from cGAS^−/− mice pretreated with rapamycin or DMSO control and subjected to sham surgery or I/R (original magnification ×20). Percentage of TMR-positive cells quantified and represented in bar graph. Images are representative of data from multiple mice per experimental group. D: Western blot for procaspase-3 and cleaved caspase-3 in whole liver lysates from cGAS^−/− mice pretreated with rapamycin or DMSO control and subjected to sham surgery or I/R. Densitometry of procaspase-3 and cleaved caspase-3 bands relative to GAPDH loading control quantified by ImageJ software and presented in bar graphs. E: Western blot for p62 in WT and cGAS^−/− hepatocytes pretreated with rapamycin (Rapa, 100 nM) or DMSO control and subjected to normoxia (Ctrl) or A/R (10-h/8-h). Densitometry of p62 bands relative to GAPDH loading control quantified by ImageJ software and presented in bar graph. Images are representative of data from multiple mice per experimental group or ≥3 independent in vitro experiments. Data presented as means ± SE. *P < 0.05, **P < 0.01.

DISCUSSION

The cGAS-STING pathway is critical for initiating innate immune responses to invading microbial pathogens such as DNA viruses and bacteria (16, 31, 49). This pathway can also contribute to autoimmune diseases and irradiation-mediated cell stress through the recognition of endogenous DNA within the cytosol (7, 14, 15, 19, 34). Most recently, one study indicated that the cGAS-STING pathway regulated senescence upon the recognition of cytosolic chromatin fragments (18). Our results are the first to document the critical role of cGAS in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that cGAS protects liver from I/R injury in a STING-independent manner.

During liver I/R injury, the excessive generation of ROS promotes mitochondrial permeability transition (MPT) and cell death (25, 29). These events can initiate the release of endogenous DNA from mitochondria and the nucleus (21, 26, 38). Ample evidence indicates that nucleic acid sensing takes place early during liver I/R. Our previous studies indicated that histones can enhance DNA-mediated TLR9 pathway activation and contribute to liver I/R injury (22). Recently, we found that the sensing of HMGB1-DNA complexes by cytosolic AIM2 activates inflammasome-dependent mitophagy in hypoxic hepatocytes (39). Additionally, there are many other intracellular DNA sensors have been identified, including DNA-dependent activator of IFN-regulatory factors (DAI) IFI16 and IFIX (10, 41, 46). Notably, recent work provided evidence showing that cGAS and IFI16 cooperated in the sensing of intracellular DNA (1). Given this finding, the integrated functions of different DNA sensors in liver I/R injury may need to be investigated further. However, our current and previous results oint to important roles for cytosolic DNA sensing pathways in hepatocytes as pathways that regulate protective autophagy during oxidative stress.

Our data indicate that STING is not required for the cGAS- protective pathway in hepatocytes after liver I/R injury. This is established due to the fact that STING is not expressed in hepatocytes. In a murine hepatitis B virus (HBV) infection model, transfection of hepatocytes with STING restored antiviral action during HBV infection (43). This indicates that functional STING is capable of regulating hepatocyte IFN-1-inducible protein during antiviral defense. STING is not only a required factor for cytokine signaling but is also critical for the induction of autophagy in a cGAS-independent manner upon cytosolic pathogen DNA stimulation (49, 50). How cGAS regulates autophagy is not known and will require further analysis.

Autophagy plays a vital role in both normal and pathological conditions. Oxidative stress has been shown to induce autophagy in different types of cells (40), but the role of autophagy in cell survival during oxidative stress remains controversial. Our research group previously suggested that autophagy is a protective process during liver I/R injury (12). A number of studies indicate that autophagy may also be important in attenuating apoptosis via clearance of damaged mitochondria during liver I/R injury (13, 29, 48). In this study, we demonstrate that cGAS induces autophagy and protects hepatocytes lacking STING from apoptosis after oxidative stress. It has been previously shown that cGAS induces autophagy through direct interaction with beclin-1 during DNA virus infection (30). cGAS competitively binds beclin-1 and results in dissociation of the negative autophagy factor rubicon from the beclin1-phosphatidylinositol 3-kinase class III (PI3KC3) autophagy complex, leading to PI3KC3 activation and autophagy induction (30). As expected, our finding indicates that treatment of cGAS^−/− mice with rapamycin, an inducer of PI3KC3 activity (52), restores autophagy induction in knockout mice. Notably, our data indicate that beclin-1 expression did not change in either WT or cGAS^−/− mice. This may suggest that cGAS has an effect on beclin1-PI3KC3 complex to activate PI3KC3, and this effect is not dependent on beclin-1 expression. In addition, a recent study indicates that cGAMP generated by cGAS also induces the activation of Unc-51-like autophagy-activating kinase-1 (ULK1) autophagy kinase, suggesting that the cGAS-mediated autophagy may be involved in the ULK1 complex pathway (27). However, the mechanisms through which cGAS regulates autophagy are not well known and will require further analysis.

In summary, determining the cellular mechanisms leading to liver I/R injury are important and directly relevant to clinical situations, including surgical liver resection, liver transplantation, and hypoxia secondary to trauma and hemorrhagic shock. In this study, we have described a novel function for cGAS, a known sensor of cytosolic DNA and cell damage, in the regulation of protective autophagy responses to liver I/R. Importantly, this function of cGAS is independent of its usual downstream signaling partner STING. We showed that cGAS expression is increased by oxidative stress/ROS production and that cGAS is important in induction of autophagy and initial formation of autophagosomes. Understanding these pathways may help in the development of future treatments to prevent liver I/R injury and so help patients undergoing surgery and transplantation.

GRANTS

This work was supported by National Institute of General Medical Science Grants RO1 GM-044100 and RO1 GM-050441 (to T. R. Billiar) and R01 GM-102146 (to M. J. Scott).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Z.L., Z.Y., Q.S., R.A.S., T.L., P.A.L., and H.H. performed experiments; Z.L. and H.X. analyzed data; Z.L., M.D., M.J.S., F.H., and T.R.B. interpreted results of experiments; Z.L. prepared figures; Z.L. and M.J.S. drafted manuscript; M.D., F.H., and T.R.B. conceived and designed research; M.D., J.E.G., M.J.S., and T.R.B. edited and revised manuscript; F.H. and T.R.B. approved final version of manuscript.