Endoplasmic reticulum stress-induced hepatocellular death pathways mediate liver injury and fibrosis via Stimulator of Interferon Genes

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon Regulatory Factor 3 (IRF3) regulates hepatocyte apoptosis and production of Type-I interferons (IFNs). In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the ER adapter, Stimulator of Interferon Genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically-induced liver fibrogenesis. To test this, we performed acute or chronic carbon-tetrachloride (CCl 4 ) administration to WT, IRF3-, TRAM-, TRIF-,

upstream of IRF3, were not protected from hepatocyte death and/or fibrosis suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis.Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3.Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis, and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.
Fibrosis represents a late-stage of liver disease that is common to all chronic liver diseases including viral hepatitis, alcoholic and nonalcoholic fatty liver disease, biliary liver diseases, and some genetic liver disease.In spite of their specific etiologies, common denominators of fibrosis are shared among all these liver diseases, including liver inflammation and hepatocyte death.Fibrosis results from chronic unresolved liver inflammation and may progress from fibrotic scarring to cirrhosis that ultimately leads to liver failure.Inflammation triggers liver fibrosis via a signaling event in which liver resident macrophages (Kupffer cells, KC) activate hepatic stellate cells (HSC) to deposit collagen (1,2) .Liver dysfunction results from the fibrotic tissue distorting the liver parenchyma.This process involves a dynamic and complex series of multicellular events, involving inflammation and HSC activation.The hepatocyte is primarily responsible for metabolism and detoxification, and as such, it is often exposed to damage due to toxic metabolites and reactive oxygen species.However, the role of hepatocyte death in liver fibrosis has been only partially elucidated.Data available up to date demonstrate that hepatocyte apoptosis activates HSC via paracrine mechanisms.Alternatively, HSC can be directly activated by apoptotic bodies (3) .Apoptosis in hepatocytes results from displacement or downregulation of the Bcl-2 family of anti-apoptotic proteins and subsequent activation of proapoptotic BH3-only proteins such as Bax and Bak (4) .These events lead to oligomerization of Bax and Bak, resulting in permeabilization of the outer mitochondrial membrane and execution of the intrinsic apoptotic process (5) .Recently, IRF3, a transcription factor that induces interferon-β (IFNβ), has been shown to play a major role in cell death by way of its newly characterized BH3-only domain.IRF3 associated with the pro-apoptotic adaptor Bax and triggered apoptosis in murine embryonic fibroblasts and murine hepatocytes (6)(7)(8) .However, it is not known whether other key signal transduction events in hepatocytes are required for the pathogenesis of fibrosis.For this reason, we sought to understand the nature of profibrogenic hepatocyte damage and specifically assess the potential role of hepatocyte apoptosis in determining the onset of fibrosis.In this study, we investigated the role of IRF3 in hepatocyte death and liver fibrosis.We used carbon-tetrachloride (CCl 4 ), a chemical that, in the short term, induces hepatocyte apoptosis (9) which is followed by secondary necrosis (10,11) .When administered repetitively, CCl 4 induces liver fibrosis (12) .Using two models of CCl 4 -induced liver injury, we investigated the complex multi-cellular events associated with fibrosis in a chronic model, as well as the tightly-controlled signal transduction events associated with early disruption of homeostasis and cell death in the acute model.We report that hepatocyte-specific endoplasmic reticulum (ER) stress and activation of IRF3 occurs and is mediated by the ER adaptor, STING.Activated IRF3 associates with the pro-apoptotic adaptor Bax and induces hepatocyte death.We initially hypothesized that IRF3 will utilize established pathways of antiviral response, including Type-I IFNs, to induce hepatocyte apoptosis.However, our results demonstrate that the pro-apoptotic role of IRF3 is independent of its function to induce Type-I IFNs such as IFN-β.Taken together, our data demonstrate that hepatocyte death is indispensable in liver fibrosis and that the pathogenic role of IRF3 in liver fibrosis is mediated via its pro-apoptotic effect in hepatocytes.

RESULTS
IRF3 deficiency attenuates chronic CCl 4 -mediated liver injury and fibrosis-Repetitive episodes of liver injury, such as those induced by administration of CCl 4 (a chemical inducer of hepatocyte death), result in the development of liver fibrosis, which is a hallmark of advanced and often irreversible liver disease in humans.We previously showed that IRF3-deficient mice were protected from alcohol-induced liver disease and that IRF3 deficiency in hepatocytes prevented fibrogenesis in the liver in alcoholic liver disease (8).We evaluated this hypothesis using a mouse model of liver injury and fibrosis using CCl 4 or vehicle, in a six-week regimen of bi-weekly intraperitoneal injections.Deficiency of IRF3 significantly attenuated liver damage, as demonstrated by significantly lower levels of serum ALT and substantially improved histological findings compared to CCl 4 -treated wild-type controls (Fig. 1A,B)., IRF3-deficient mice had decreased TUNEL positive staining in the liver compared to WT mice, suggestive of decreased apoptosis (Fig. 1C).Interestingly, TUNEL positive staining was predominantly present in hepatocytes.IRF3-deficient mice showed significant protection from liver fibrosis, indicated by reduced alpha-smooth muscle actin (α-SMA) immunohistochemistry and Sirius-red staining compared to WT mice (Fig. 1D,E).The protection from liver fibrosis in IRF3-deficient mice was accompanied by significantly lower expression of Acta2 and Col1a2 (Fig. 1F), encoding α-SMA and Type-I collagen, and by decreased expression of Ifnb and Isg15, two commonly used markers of IRF3-mediated transcriptional response (Fig. 1G).
Acute CCl 4 induces ER stress and phosphorylation of IRF3 in hepatocytes-Disruption of intracellular homeostasis can lead to an accumulation of misfolded proteins associated with the unfolded protein response (UPR) in the ER, also known as ER stress.Excessive ER stress is associated with pro-apoptotic signaling and can be pathogenic (13,14) .To test if CCl 4 -induced acute hepatocyte injury induces an early ER stress response, we analyzed livers of mice at various time points after a single administration of CCl 4 .We found an early upregulation of Xbp1 mRNA splicing (sXbp1), a marker of early ER stress (15,16) at one and two hours after administration of CCl 4 (Fig. 2A).Analysis of cell populations from murine livers showed that CCl 4 selectively upregulated Xbp1 mRNA splicing in hepatocytes but not in liver mononuclear cells (LMNCs) (Fig. 2B).
Previous reports have shown that ER stress results in IRF3 activation in mouse embryonic fibroblast, and that Xbp1 splicing enhances the IFN-β response in immune cells (17,18) .In our study, acute administration of CCl 4 led to an early activation of IRF3, a pro-apoptotic BH3-only protein, as seen by the phosphorylation in whole cell liver lysates (Fig. 2C), at the same time as splicing of Xbp1 mRNA (Fig. 2A).The phosphorylation of IRF3 had a functional effect, as liver Ifnb1 mRNA and serum IFN-β, transcriptional targets of IRF3, showed an early upregulation (Fig. 2D).To test if direct induction of ER stress can lead to IRF3 phosphorylation, we treated primary hepatocytes with thapsigargin in vitro and found an early increase in phosphorylated IRF3 (Fig. 2E).Furthermore, inhibition of Tank-binding kinase 1 (TBK1), a kinase responsible for IRF3 phosphorylation, with BX795 showed a dose-dependent reduction in IRF3 phosphorylation in thapsigargin-treated hepatocytes (Fig. 2E).Building upon previous findings, our data indicate that acute administration of CCl 4 results in hepatocytespecific ER stress and phosphorylation of IRF3 in vivo, and that in vitro inhibition of a kinase upstream of IRF3 inhibited phosphorylation of IRF3 induced by ER stress.Our data support the hypothesis that CCl 4 activates IRF3 in hepatocytes via ER stress.
Acute CCl 4 -induced liver injury is ameliorated by the deficiency of IRF3 independent of TRIF or TRAM, or Type-I IFN signaling-Next, we asked if IRF3 plays a role in liver injury after acute CCl 4 administration.We found that IRF3-KO mice were partially protected from liver injury after acute CCl 4 administration, as indicated by the significant attenuation in serum ALT compared to WT controls (Fig. 3A) and Caspase-3 cleavage in the liver (Fig. 3B).To test if the pathogenic phenotype of IRF3 involvement was mediated by its function as a transcription factor, we tested mice deficient in Type-I IFN receptor (IFNAR1) that lack Type-I IFN signaling and found that IFNAR1-KO mice had no protection from CCl 4mediated liver injury, phosphorylation of IRF3 or apoptotic signaling in the liver compared to WT mice (Fig. 3C,D).This data suggested that the pathogenic role of IRF3 may be independent of its transcriptional role.
CCl 4 -induced liver fibrosis activates hepatic stellate cells via TLR4 (19,20), enhanced by signal transduction emanating from the TLR4 complex induced by gut bacterial products and LPS (1) .To investigate if activation of IRF3 in acute CCl 4 -liver injury was mediated by the intracellular MyD88-independent TLR4 adaptors TIR-domain-containing adapter-inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM), or the kinase TBK1, we first inhibited activity of their downstream kinase, TBK1, by the chemical inhibitor BX795 in vivo.Pre-treatment of mice with BX795 significantly attenuated the extent of liver injury and apoptotic signaling in the liver after administration of CCl 4 (Fig. 3E,F).The decrease in phosphorylated IRF3 (Fig. 3F) with BX795 pre-treatment further suggests that IRF3 requires activation and phosphorylation of TBK1 in order to exert a pathogenic effect in CCl 4 -induced liver injury.In contrast, TRAM-and TRIF-KO mice were each equally as susceptible to acute CCl 4 damage and apoptosis in the liver as WT mice (Fig. 3G,H).Importantly, TRAM-and TRIF-KO mice had similar levels of phosphorylated IRF3 compared to WT mice (Fig. 3H), suggesting that IRF3 activation by acute CCl 4 occurs independent of the TLR4 receptor complex and its adaptors.Together this data suggested that TBK1-mediated activation of IRF3 is pathogenic, leading to increased liver injury.Furthermore, Type-I IFNs do not have a significant role in acute liver injury after CCl 4 administration in spite of being activated by IRF3.Lastly, activation of IRF3 by acute CCl 4 is independent of the canonical MyD88-independent TLR4 adaptor-mediated receptor signaling.Therefore, we hypothesized that activation of IRF3 in the liver by acute CCl 4 likely involves different signaling pathways.
CCl 4 induces early association of IRF3 with STING in the ER and association of IRF3 with Bax in the mitochondria in the liver-Given the robust protection from acute or chronic CCl 4induced liver injury in mice deficient in IRF3, we next investigated subcellular localization and mechanisms of early IRF3 activation.First, we evaluated the kinetics of IRF3 activation in WT mice upon acute CCl 4 -mediated injury and found an early phosphorylation of IRF3 at two and four hours in whole cell liver lysates (Fig. 4A).Next, we analyzed the intracellular distribution of IRF3.Analysis of subcellular liver fractions revealed that administration of CCl 4 resulted in localization of phosphorylation of IRF3 not only in the cytoplasmic (Fig. 4B) and nuclear extracts (Fig. 4C), but also in the ER (Fig. 4D) and mitochondrial fractions (Fig. 4E).The finding of phosphorylated IRF3 in cytoplasmic and nuclear extracts (Fig. 4A,B) was consistent with the transcriptional role of IRF3 in induction of Ifnb.However, the finding of phosphorylated IRF3 in the ER (Fig. 4D) and mitochondria (Fig. 4E) was unexpected and prompted us to search for ER-and mitochondria-specific binding partners of IRF3.
It has been reported that during viral infections, IRF3 associates with ER via binding to Stimulator of IFN Gene (STING, alias Tmem173 or MPYS), an adaptor protein residing in the ER membrane.We found that a single administration of CCl 4 in vivo resulted in a significant increase in STING in whole cell liver lysates (Fig. 4F) and in ER extracts (Fig. 4H) after a pull-down with IRF3 antibody, suggesting an association between IRF3 and STING in the liver of mice exposed to CCl 4 .
Based on the recent report that IRF3 plays a key role in viral-mediated apoptosis mediated by Bax (6) , and on our previous report data that IRF3 is required for hepatocyte apoptosis triggered by FasL (8) , we hypothesized that IRF3 would associate with pro-apoptotic proteins involved in the intrinsic, mitochondrial pathway of hepatocytes apoptosis.We found an increased association between IRF3 and Bax at two hours after administration of CCl 4 (Fig. 4I), followed by Casp-8 activation and apoptosis (Fig. 4J,K).The timing of this association correlates with the increased presence of phosphorylated IRF3 in the mitochondrial fraction, suggesting that only active and phosphorylated IRF3 has a pro-apoptotic role when found in the mitochondria.Moreover, the presence of phosphorylated IRF3 in the mitochondria and its association with BAX coincided with activation and cleavage of the initiator Caspase-8 and the executioner Caspase-3 (Fig. 4J).Our data suggest that ER stress and phosphorylation of IRF3 (Fig. 2A,C), which occurred within one hour, was followed by proapoptotic signaling and hepatocyte damage (Fig. 4I,J,K), which were established within two hours.Taken together, our data indicated that following acute administration of CCl 4 , IRF3 interacts with the ER-associated protein, STING, and with the pro-apoptotic molecule, Bax.Importantly, the association of IRF3 with STING or phospho-TBK1 in the ER occurred within the first two hours after CCl 4 administration (Fig. 4F,H,G), concurrent with phosphorylation of IRF3 and upregulation of Ifnb in the liver (Fig. 2C,D); all of these events occurred prior to the development of significant liver injury (Fig. 3).

Deficiency in STING, but not in Type-I IFN signaling, attenuates liver fibrosis-Acute
CCl 4 -induced liver injury resulted in ER stress and IRF3 activation, as well as early localization of IRF3 in the ER in association with STING, an ERadaptor protein.Furthermore, IRF3 demonstrated a pathogenic role, independent of the TLR4adaptor complex, in models of acute or chronic CCl 4 -induced liver injuries, we then hypothesized that activation of IRF3 was through STING after chronic CCl 4 administration in mice.In support of this hypothesis, we observed that Tmem173 Gt mice, which lack the STING protein (21) had significantly attenuated liver injury induced by repetitive injections of CCl 4 (Fig. 5A,B) to a similar extent to that observed in IRF3-deficient mice (Fig. 1A).Further, STING-deficient mice (Tmem173 Gt ) showed protection from liver fibrosis (Fig. 5C) as well as a decrease in deposition α-SMA and Type1-Collagen protein in the liver (Fig. 5D,E) and expression of profibrogenic markers (Fig. 5F).
IRF3 is the transcription factor responsible for Type-I IFN production, a pro-inflammatory cytokine important in creating an antiviral state.In addition to IRF3, IRF7 is another major regulator of Type-I IFN production (22) .Administration of CCl 4 resulted in significantly upregulated Ifnb1 and the IFN inducible gene Isg15 in the liver in WT mice, while IRF3-deficient mice lacked induction of Type-I IFN genes (Fig. 1F).However, IRF7 or Type I IFNs did not mediate the pathogenic effect of IRF3 in the liver as demonstrated by lack of protection from liver injury (Fig. 5H,I) and fibrosis (Fig. 5J,K) in IRF7or IFNAR1-KO mice, respectively.The lack of Type-I IFN induction or signaling in INFAR1-KO mice resulted in aggravated liver fibrosis, as demonstrated by increased Sirius-red area (Fig. 5J) and augmented expression of Acta2 and Col1a2 (Fig. 5K) compared to WT mice, which is consistent with the previously reported antifibrotic role of Type-I IFNs (23,24) .Taken together, this data implied that IRF3, but not IRF7 or Type-I IFN signaling, play a pathogenic role in chronic liver injury and fibrosis induced by chronic CCl 4 administration.In addition, the data demonstrate the pathogenic role of STING, a binding partner of IRF3 (Fig. 4 and ( 25) ), as a determinant in the development of liver injury and fibrosis in this model.

STING mediates pro-apoptotic activation of IRF3 in a chronic CCl 4 -induced model of fibrosis-
To further characterize the role of STING in mediating the CCl 4 -induced liver injury in the chronic CCl 4 model, we evaluated phosphorylation of TBK1 and IRF3.After six weeks of exposure to CCl 4 , STING-deficient mice had decreased phosphorylation of TBK1 (Fig. 6A) as well as decreased phosphorylation of IRF3 (Fig. 6B) compared to WT mice.Previous reports have studied the pro-apoptotic association of IRF3 with Bax.To test if STING-mediated activation of IRF3 results in pro-apoptotic signaling, we tested Caspase activation in STING-deficient mice using the chronic CCl 4 -mediated liver injury model.We found that STING-deficient mice (Tmem173 Gt ) given a six-week regimen of CCl 4 exhibited a significant reduction in the cleavage and activation of the apoptosis initiator Caspase-8 (Fig. 6C) and the apoptosis executioner Caspase-3 (Fig. 6D) compared to WT mice.
This data was consistent with the association between IRF3, STING and Bax that we found in the liver following a single exposure to CCl 4 (Fig. 4H,I) and indicated the possibility that ER stress may be a source of IRF3 activation in this model.In addition, this data also supported the possibility that in the chronic model of CCl 4induced liver injury and fibrosis, IRF3 represents an activator of pro-apoptotic signaling in the liver.
Early pro-apoptotic activation of IRF3 by CCl 4 is hepatocyte-specific and mediated by STING-To further explore the mechanism of cell death and liver injury and to test whether induction of cell death in the acute CCl 4 liver injury model was mediated by STING, we evaluated activation of Caspase-8 and Caspase-3 in mouse livers.We found that Caspase-8 and -3 were both activated at 8 and 12 hours after CCl 4 injection in WT mice (Fig. 7A,B), while the STING-deficient mice (Tmem173 Gt ) had complete absence of cleaved caspases (Fig. 7C,D).
To better understand the underlying mechanism behind the protection observed in mice deficient in either IRF3 or STING in a chronic CCl 4 injury, we tested the dynamics of hepatocyte damage by comparing WT mice with Tmem173 Gt mice during an acute CCl 4 liver injury.We found protection from liver injury in STING-deficient mice (Tmem173 Gt ) after CCl 4 administration as seen by serum ALT and H&E histology (Fig. 7E,F).WT mice exhibited extensive necrosis at 12 hours, peaking at 24 hours, as seen by irregular histological morphology, while the STING-deficient mice did not (Fig. 7F).Surprisingly, we found that one dose of CCl 4 was sufficient to elicit a pro-fibrogenic response in WT mice as early as 24 hours after injection (Fig. 7G,H).In comparison to WT mice, the STING-deficient mice (Tmem173 Gt ) were protected from this early pro-fibrogenic response in the liver.
Our data established that CCl 4 -mediated ER stress occurred primarily in hepatocytes and that phospho-IRF3 after ER stress occurs in primary hepatocytes (Fig. 2B,C).To assess which liver cell type is responsible for IRF3 phosphorylation and caspase activation after CCl 4 administration in vivo, we isolated liver mononuclear cells (LMNCs), hepatic stellate cells (HSCs) and primary hepatocytes from mouse livers 9 hours post CCl 4 injection.We found that phosphorylation of IRF3 occurred predominantly in hepatocytes, and not in LMNCs or HSCs (Fig. 7I).In assessing cell type-specific contribution to apoptotic signaling, we found increased Casp-8 cleavage in hepatocytes but not in HSCs or LMNCs.Similarly, there was an increase in Caspase-3 cleavage predominantly in hepatocytes, albeit some cleaved Caspase-3 was detected in HSCs (Fig. 7I).The phosphorylation of IRF3 and Caspase-8 cleavage found exclusively in the hepatocyte population was consistent with our previous findings of TUNEL positive staining in hepatocytes (Fig. 1C) and in IRF3 association with Caspase-8 and BAX in total liver lysates (Fig. 4I).
Taken together, this data demonstrated that activation of IRF3 in acute or chronic liver damage is triggered in hepatocytes via the adaptor STING.Deficiency in either STING or IRF3 resulted in significant decreased hepatocyte death and fibrosis.
DISCUSSION Previous studies described the role of STING activation in viral infections in immune cells (26)(27)(28)(29)(30) .In hepatocytes, we demonstrated earlier that STING and IRF3 mediate the pathogenesis in alcoholic liver disease, a role independent of microbial components (8) .In the present study, our data demonstrate a hepatocytespecific role of STING and IRF3 in mediating hepatic apoptosis as an early event in the development of liver fibrosis.
Hepatocytes are responsible for metabolism and for detoxifying portal blood, which can be rich in bacterial products.For this reason, innate immunity plays an important role in the liver; even though this organ is rich in immune cells, parenchymal cells have an abundance of receptors and are capable of initiating immune signaling (31-34) .In our study, TBK1-mediated ER stress was detected early after CCl 4administration only in hepatocytes and not in LMNCs.Similarly, phosphorylated IRF3 was detected in hepatocytes exposed to CCl 4 in vivo but not in LMNCs or HSCs.Furthermore, in support of our hypothesis of the pro-apoptotic role of IRF3, CCl 4 -mediated Caspase-8 cleavage was detected only in hepatocytes and was absent from LMNCs and HSCs.Lastly, compared to hepatocytes exposed to CCl 4 , Caspase-3 cleavage was reduced in HSCs and absent in LMNCs.The decreased level of Caspase-3 cleavage in HSCs compared to hepatocytes is likely due to the absence of concurrent phosphorylated IRF3 and Caspase-8 cleavage, suggesting of an alternate mechanism of toxicity and cell death signaling independent of IRF3 in HSCs.It is possible that the mechanisms that tightly regulate apoptotic signaling may also be responsible for hepatic stellate cell activation, leading to fibrogenesis.
Regulated IRE1-dependent decay of mRNA (RIDD), a key component of the unfolded protein response, results in degradation of viral mRNA transcripts and spliced Xbp1 (13,35) .However, there are multiple lines of evidence suggesting that IRF3 activation can result from other forms of noninfectious cell damage that involve Xbp1 splicing (17,18,36) .
Here we show that STING activation may provide the link between induction of ER stress and IRF3.Moreover, the rapid nature of the phosphorylation of IFR3 and Xbp1 splicing in our experiments due to an acute CCl 4 -induced liver injury underscores the importance of STING and its location in the ER.
Viral infections as well as noninfectious injuries are associated with induction of cell death signals, emphasizing the importance of the BH3domain within IRF3 as responsible for its proapoptotic properties in the mitochondria.It is also possible that CCl 4 -induced cell death signaling from STING activation of IRF3 and BAX may trigger ER membrane permeability (37) in addition to the effects on the mitochondrial membrane integrity.
A previous study by Knockaert et al. (38) of CCl 4 in mouse livers interestingly found no apoptosis or pro-apoptotic caspase activity at three or 24 hours, the only two time-points they tested.It is important to highlight that our data showed Caspase-8 and -3 cleavage peaking between eight and twelve hours in WT mice, while apoptotic signaling had resolved by 24 hours (Fig. 7A,B).Importantly, the STING-deficient mice had no detectable Caspase-8 or -3 cleavage (Fig. 7C,D), indicating protection from apoptosis and secondary cell death.
Moreover, the Knockaert et al. study similarly found CCl 4 -mediated necrosis at 24 hours (Fig. 4K,7F), further supporting our hypothesis that early, and extensive pro-apoptotic signaling leads to necrosis, or secondary necrosis, which have overlapping components (39) .In support of the study by Roychowdhury et al. (40), our data indicate that an absence of early apoptosis is also associated with an absence in early fibrogenesis and detectable necrosis at the level of liver histology in STING-deficient mice.
Pro-apoptotic signaling may play a beneficial role in the resolution of inflammation after liver injury, specifically when it is targeted to infiltrating immune cells such as neutrophils (41).Uncontrolled parenchymal apoptosis, however, although typically controlled and tightly regulated, may be the tipping point responsible for subsequent necrosis.
As summarized in Fig. 7J, our data suggest that overwhelming levels of IRF3-mediated apoptosis may eventually lead to a form of secondary necrosis.Taken together, our data provides strong evidence that inhibition of parenchymal apoptotic signaling is sufficient to reduce necrosis and amplified liver damage.
In conclusion, our study builds on the previous paradigm of fibrogenesis.The CCl 4induced liver damage, ER stress and death of hepatocytes is mediated by the engagement of the ER adaptor protein, STING, with phosphorylated IRF3, resulting in activation of the mitochondrial pathway of hepatocyte death.As such, fibrosis is more than a pathology strictly driven by inflammation.Rather, hepatocyte death appears to be a separate key component in the process of liver fibrosis.
Other mice received a six-week regiment of CCl 4 injections, given twice per week.All animals received proper care in agreement with animal protocols approved by the Institutional Animal Use and Care Committee of the University of Massachusetts Medical School.
In vitro experiments-Primary hepatocytes and liver mononuclear cells were isolated as described previously (42).Primary hepatocytes were cultured in Waymouth's medium supplemented with 10% fetal bovine serum and 1% insulin, transferrin, selenium solution.Primary hepatocytes were seeded in 6-well collagen-coated plates (Biocoat, Becton Dickinson, Bedford, MA).Before starting stimulation experiments, hepatocytes were rested for 4 hours.Subsequently culture media was replaced, and stimulation was performed as indicated in the figure legends.The BX795 (10µM or 100µM) and thapsigargin (1µM) were purchased from Sigma (St. Louis, MO).RNA Analysis-RNA analysis and PCR primers are described in Table 1.sXbp1 PCR was performed and products were separated on 3% agarose gel as described previously (8) .Protein quantification-Liver whole-cell lysates and nuclear preparations were extracted as described previously (8) .Mitochondrial and total ER fractions were prepared using specific extraction kits from Imgenex (San Diego, CA) as per manufacturer's protocol.Immunoblotting was performed as described in (43).Antibodies specific for phospho-IRF3, phospho-TBK1, STING, Casp-8, Casp-3, cleaved Casp-8, cleaved Casp-3 and Bax were from Cell Signaling (Danvers, MA).Antibodies against the total IRF3, GRP78 and KDEL, and control IgG were from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA).Beta-actin, beta-tubulin, porin and TATA-binding protein (TBP) antibodies were from Abcam (Cambridge, MA).The TrueBlot system from eBioscience (San Diego, CA) was used for immunoprecipitation assays and performed as per manufacturer's instructions.Statistical Analysis-Statistical significance was determined using two-sided t-test; Two-way ANOVA was used in Figures 3A-D

FIGURE 2. Acute CCl 4 induces ER stress and TBK1-mediated phosphorylation of IRF3 in hepatocytes.
WT mice received a single injection of corn oil or CCl 4. At indicated time points, mice were sacrificed, and splicing of Xbp1 mRNA (sXBP1) in the liver, indicating ER stress, was evaluated by PCR, and products were separated on 3% agarose gel (A).Two hours after CCl 4 administration, hepatocytes or LMNCs from WT mice were isolated and analyzed for sXBP1 by PCR (B).At indicated time points, phosphorylation of IRF3 was assessed by immunoblotting in liver (C) while Ifnb1 mRNA expression in liver and as serum IFN-β were measured by RT-PCR and ELISA, respectively (D).N= 4 mice per time point.Primary hepatocytes isolated from WT mice were treated with 1µM thapsigargin, and were pretreated with DMSO or BX795 (10µM or 100µM), and analyzed for phospho-IRF3 (E).

FIGURE 4. Acute CCl 4 induces early association of IRF3 with STING in the ER and association of IRF3
with Bax in the mitochondria in the liver.WT mice received a single injection of CCl 4 .At indicated time points, mice were sacrificed, and phosphorylation of IRF3 was assessed by immunoblotting in liver whole-cell lysate (A) or cytoplasmic (B), nuclear (C), ER (D), and mitochondrial (E) extracts.Using immunoprecipitation with anti-total IRF3 antibody for protein pull-down, we evaluated association between total IRF3 and phosphorylated TBK1 or STING in the whole-cell (F), mitochondrial (G), and ER (H) extracts.Similarly, we evaluated association between total IRF3 and cleaved Caspase-8 p43 fragment or BAX in liver whole-cell lysate (I), with representative IgG control shown (two hours after injection).At indicated time points, cleavage of Caspase-8 and Caspase-3 was assessed by immunoblotting in liver (J).Liver injury and apoptosis was assessed by H&E liver histology and TUNEL staining on paraffinembedded liver sections (K).).N= 4 mice per time point.

FIGURE 5. Deficiency in STING, but not in Type-I IFN signaling, attenuates liver fibrosis.
WT or STING-deficient mice (Tmem173 Gt ) were injected with oil or CCl 4 for six weeks and sacrificed 48 hours after final injection.Liver injury was assessed by serum ALT (A) and H&E staining (B).Liver fibrosis was evaluated by measuring Sirius-red staining and the percent-positive area stained was quantified using ImageJ (C).Liver fibrosis was also evaluated by immunoblot for alpha-smooth muscle actin (D) or Type-1 collagen (E).mRNA expression in liver for Acta2 and Col1a2 (F) as well as Ifnb1 and Isg15 (G) were measured by PCR to assess liver fibrosis and IRF3 activation, respectively.WT, IRF7-or IFNAR1-KO mice injected with oil or CCl 4 for six weeks and sacrificed sacrificed 48 hours after final injection.Liver injury was assessed by serum ALT (H) and H&E staining (I).Liver fibrosis was evaluated by Sirius-red staining and quantified using ImageJ (J).mRNA expression in liver for Acta2 and Col1a2 (K) as well as Ifnb1 and Isg15 (L) were measured by RT-PCR.N= 8-9 mice (CCl 4 -treated, per genotype); 3-4 mice (oiltreated, per genotype).WT mice received a single injection of CCl 4 or oil (baseline) and sacrificed nine hours later.Liver mononuclear cells (LMNC), hepatic stellate cells (HSC) or hepatocytes were isolated from livers and probed for phosphorylated IRF3, total IRF3, cleaved Caspase-8 and cleaved Caspase-3 by immunoblot (I).N=3 per condition (LMNC and HSC were pooled from 3 mice); N=1 (hepatocytes isolated from single mouse per condition); experiment was repeated three times (I).Schematic of pro-apoptotic STING and IRF3 activation from CCl 4 administration in mice (J).In CCl 4 -treated hepatocytes, ER stress results in phosphorylation of TBK1 via STING, followed by phosphorylation of IRF3.IRF3 associates with BAX in the mitochondria through its BH3-only domain, leading to pro-apoptotic Caspase-3 activation and hepatocyte apoptosis.After chronic CCl 4 administration, hepatocyte apoptosis is associated with secondary necrosis, which results in liver fibrosis.

STING mediates pro-apoptotic activation of IRF3 in a chronic
and Figure 7E to determine the global effect of BX795 on serum ALT.Numbers in the graphs indicate global P values.Data are shown as scatter plots of individual data plots ± S.D. to represent error and were considered statistically significant at P <

FIGURE 3 .
FIGURE 3. Acute CCl 4 -induced liver injury is ameliorated by the deficiency of IRF3 or inhibition of TBK1, independent of TRIM or TRAM , Type-I IFN signaling.WT or IRF3-KO mice received a single injection of CCl 4. Serum ALT levels were assessed (A) and apoptosis was assessed by measuring Caspase-3 cleavage in the liver by immunoblot (B).WT or IFNAR1-KO mice received a single injection of CCl 4. Serum ALT levels were assessed (C) and IRF3 activation and apoptosis were assessed by probing for phosphor-IRF3 and Caspase-3 cleavage in the liver by immunoblot (D).WT were pretreated with DMSO or BX795, a TBK1-inhibitor, two hours before receiving a single injection of corn oil (vehicle) or CCl 4. Serum ALT levels were assessed (E) and IRF3 activation and apoptosis were assessed by probing for phosphor-IRF3 and Caspase-3 cleavage in the liver by immunoblot (F).WT, TRIF-or TRAM-KO mice received a single injection of CCl 4 .Serum ALT levels were assessed (G) and IRF3 activation and apoptosis were assessed by probing for phosphor-IRF3 and Caspase-3 cleavage in the liver by immunoblot (H).Mice were bled serially at indicated time points.N= 8-9 mice (CCl 4 -treated, per genotype); 3-4 mice (oil-treated, per genotype) (A,C,G).N= 3-5 mice (CCl 4 -treated, pre-treated with BX795); 3 mice (oil-treated, pre-treated with DMSO) (E).

FIGURE 7 .
FIGURE 7. Early pro-apoptotic activation of IRF3 by CCl 4 is hepatocyte-specific and mediated by STING.WT or STING-deficient mice (Tmem173 Gt ) received a single injection of CCl 4 or oil (baseline) and sacrificed at indicated time-points.Whole cell liver lysates were probed for cleaved Caspase-8 (A,B), and cleaved Caspase-3 (C,D) by immunoblot.Liver injury was assessed by serum ALT (E) and H&E staining at indicated time points (F).Deposition of alpha-smooth muscle actin (α-SMA) 24hrs after injection was evaluated by immunoblots from whole cell liver lysate (G), while mRNA expression in liver for Acta2 and Col1a2 (H) was assessed by PCR.N= 5-8 mice per time point, per genotype (A-H).WT mice received a single injection of CCl 4 or oil (baseline) and sacrificed nine hours later.Liver mononuclear cells (LMNC), hepatic stellate cells (HSC) or hepatocytes were isolated from livers and probed for phosphorylated IRF3, total IRF3, cleaved Caspase-8 and cleaved Caspase-3 by immunoblot (I).N=3 per condition (LMNC and HSC were pooled from 3 mice); N=1 (hepatocytes isolated from single mouse per condition); experiment was repeated three times (I).Schematic of pro-apoptotic STING and IRF3 activation from CCl 4 administration in mice (J).In CCl 4 -treated hepatocytes, ER stress results in phosphorylation of TBK1 via STING, followed by phosphorylation of IRF3.IRF3 associates with BAX in the mitochondria through its BH3-only domain, leading to pro-apoptotic Caspase-3 activation and hepatocyte apoptosis.After chronic CCl 4 administration, hepatocyte apoptosis is associated with secondary necrosis, which results in liver fibrosis.