Potential conflict of interest: Nothing to report.
Supported by grant DK075635 (to G. S.). The core resources supported by the University of Massachusetts Diabetes Endocrinology Research Center grant DK32520 were also used (G. S. is a member of the Diabetes Endocrinology Research Center).
Mitochondrial dysfunction is a pathogenic feature of nonalcoholic steatohepatitis (NASH). NASH complicates hepatotropic viral disease. The mitochondrial antiviral signaling protein (MAVS) is the adapter of helicase receptors involved in sensing double-stranded RNA (dsRNA). We hypothesized that impaired MAVS function may contribute to insufficient antiviral response and liver damage in steatohepatitis. We identified reduced MAVS protein levels and increased MAVS association with the proteasome subunit alpha type 7 (PSMA7) in livers from mice given a methionine–choline-deficient (MCD) diet. Decreased association of MAVS with mitochondria and increased cytosolic cytochrome c indicated mitochondrial damage in steatohepatitis. In vivo administration of the synthetic dsRNA polyinosinic:polycytidylic acid [poly(I:C)], but not lipopolysaccharide or cytidine–phosphate–guanosine-rich DNA, resulted in impaired induction of type I interferons (IFNs) and proinflammatory cytokines in steatohepatitis. Consistent with a defect in helicase receptor-induced signaling, there was loss of poly(I:C)-induced translocation of MAVS to the cytosol and decreased IFN regulatory factor 3 phosphorylation. Caspases 1 and 8, both of which cleave MAVS, were increased in MCD diet–fed mice. At baseline, steatohepatitis was associated with increased serum alanine aminotransferase (ALT), apoptosis and caspase 3 activation compared with controls. In contrast to apoptosis in controls, necrosis was induced by poly(I:C) stimulation in steatohepatitis. Hepatocyte necrosis was indicated by elevated serum high-mobility group box protein-1 and ALT and was correlated with increased expression of receptor-interacting protein 3 (RIP3), a master regulator of necrosis. Increased expression of MAVS, PSMA7, and RIP3 messenger RNA was also present in human NASH livers. Conclusion: Our novel findings suggest that mitochondrial damage in steatohepatitis extends to MAVS, an adapter of helicase receptors, resulting in inefficient type I IFN and inflammatory cytokine response but increased hepatocyte necrosis and RIP3 induction in response to a dsRNA viral challenge. These mechanisms may contribute to progressive liver damage and impaired viral clearance in NASH. (HEPATOLOGY 2011;)
Nonalcoholic fatty liver disease is the most rapidly increasing cause of liver disease in the western world.1 The spectrum of nonalcoholic fatty liver disease spans from steatosis to nonalcoholic steatohepatitis (NASH), which can lead to cirrhosis and hepatocellular cancer.1 Although the factors determining progression of NASH are yet to be fully defined, the clinical importance of increased susceptibility of the fatty liver to ischemia,2 bacterial lipopolysaccharide (LPS),3 viral infections,1 and drug-induced liver damage4 is emerging.
Comorbidity of NASH with viral infections caused by RNA viruses, such as hepatitis C and human immunodeficiency virus (HIV) remains a clinical challenge.1 Hepatitis C virus (HCV)-infected patients with significant steatosis or superimposed NASH have rapid progression of liver disease, increased rate of fibrosis, and a decreased likelihood of sustained virological response to standard antiviral therapy.5 In HIV infection, highly active antiretroviral therapy induces extensive alterations to liver lipid metabolism, including liver damage and even liver failure.6 Fatty liver also complicates viral infections, such as hepatitis A virus, cytomegalovirus, or Epstein-Barr virus, resulting in acute on chronic liver failure.
HCV, a single-stranded RNA virus, undergoes a double-stranded RNA (dsRNA) phase during viral replication.7 The innate immune response to dsRNA is triggered through Toll-like receptor (TLR) 3 and the helicase receptors retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5) and induces type I interferon (IFN) and proinflammatory cytokine production. The adapter mitochondrial antiviral signaling protein (MAVS), also called virus-induced signaling adaptor or IFNβ promoter stimulator protein-1, is associated with the outer membrane of mitochondria and is critical in both IFN and inflammatory cytokine induction after engagement of helicases by dsRNA.8, 9 Multiple hits have been proposed in the pathogenesis of NASH, including hepatocyte death as a result of toxic lipid metabolites and pathogen-derived factors that contribute to inflammation and liver damage.10 Indeed, necroinflammation is considered an indicator of NASH progression.1 There is evidence that mitochondrial damage contributes to apoptotic/necrotic cellular damage in NASH,11 but the role of MAVS in NASH and in response to pathogenic RNA has yet to be evaluated.
In this study, we hypothesized that steatohepatitis results in decreased antiviral immunity and increased susceptibility to liver damage in response to a viral challenge. Using the diet-induced NASH model in mice and polyinosinic:polycytidylic acid [poly(I:C)], a synthetic dsRNA sensed by the helicase receptors, we show that fatty livers fail to mount an efficient antiviral IFN response due to dissociation of MAVS from the mitochondria. We also found increased liver damage in steatohepatitis after poly(I:C) challenge that was associated with receptor-interacting protein 3 (RIP3) overexpression and necrosis. Our data offer novel mechanistic insights into the decreased antiviral immune defense in steatohepatitis and indicate that impaired response to virus-derived factors may promote RNA virus-induced liver injury in NASH.
Six- to 8-week-old female C57Bl/6 wild-type mice were fed a diet deficient in methionine and choline for 5 weeks. Control animals received a diet supplemented with methionine (3 g/kg) and choline bitartrate (2 g/kg) (Dyets Inc., Bethlehem, PA). Poly(I:C) (InvivoGen, San Diego, CA), a synthetic dsRNA (5 mg/kg), or cytidine–phosphate–guanosine-rich DNA (CpG)-ODN (InvivoGen, San Diego, CA; 5 mg/kg) or LPS (Sigma-Aldrich Co., St. Louis, MO; 0.5 mg/kg) were injected intraperitoneally for 2 or 6 hours. The study was approved by the Institutional Animal Use and Care Committee at the University of Massachusetts.
Biochemical Analysis and Cytokine Measurements.
Serum alanine aminotransferase (ALT) levels were determined using a kinetic method (D-TEK, Bensalem, PA), and liver triglyceride levels were assessed using an L-Type Triglyceride H kit (Wako Chemicals USA Inc., Richmond, VA). Serum cytokine levels were determined by way of BD Cytometric Bead Array (BD Biosciences, Sparks, MD). Liver thiobarbituric acid reactive substances (TBARS) were assayed using whole liver homogenates and an Oxi-TEK TBARS assay kit (ZeptoMetrix Corp., Buffalo, NY). Serum high-mobility group box protein-1 (HMGB1) protein levels were measured by enzyme-linked immunosorbent assay (IBL Transatlantic, Toronto, Ontario, Canada).
Sections of formalin-fixed livers were stained with hematoxylin and eosin. All slides were analyzed by way of microscopy.
RNA was purified using an RNeasy kit (Qiagen Sciences, Germantown, MD) and on-column DNA digestion. Complementary DNA was transcribed with the Reverse Transcription System (Promega Corp., Madison, WI). Real-time quantitative polymerase chain reaction was performed using the iCycler (Bio-Rad Laboratories Inc., Hercules, CA) as described12; primer sequences are shown in Table 1.
Table 1. Polymerase Chain Reaction Primer Sequences
5′-GTA ACC CGT TGA ACC CCA TT-3′
5′-CCA TCC AAT CGG TAG TAG CG-3′
5′-AGC TCC AAG AAA GGA CGA ACA T-3′
5′-GCC CTG TAG GTG AGG GTT GAT CT-3′
5′-TCA AGC CAT CCT TGT GCT AA-3′
5′-CTG ATG GAG GTC ATT GCA GA-3′
5′-CAG AAG CAC ACA TTG AAG AA-3′
5′-TGT AAG TAG CCA GAG GAA GG-3′
5′-CAG GAC GGT CTT ACC CTT TCC-3′
5′-AGG CTC GCT GCA GTT CTG TAC-3′
5′-GCT GCT AAA GAC GGA AAT CG-3′
5′-TCT TGT CGC TGT CAT TGA GG-3′
5′-AAA GAC GGT TCA CCG CAT AC-3′
5′-TCA ACC ACT CGA ATG TCA GC-3′
5′-GTG AGA TAC AAC GTA GCT GAC TG-3′
5′-TCC TGC ATC CAA GAT AGC AAG T-3′
5′-ACA GCG CCA TCA AAA TTA CC-3′
5′-ATT TGG TCC AGT CTG GTT GC-3′
5′-GCC AGC CAC CTA GAG ATC AG-3′
5′CCT CTG GAA CGC TAA TTT CG-3′
5′-AAC GTC TGT ATG GCC TTT GC-3′
5′-TAG GGC CGA GAT ACC AAA TG-3′
5′-GAA GTT CCC AAA TGG CCT CC-3′
5′-GTG AGG GTC TGG GCC ATA GA-3′
5′-ACA ACC ACG GCC TTC CCT ACT T-3′
5′-CACGAT TTC CCA GAG AAC ATG TG-3′
5′-TCT TTG AAG TTG ACG GAC CC-3′
5′-TGA GTG ATA CTG CCT GCC TG-3′
5′-GGG ACC TCA AGC CCT CTA AC-3′
5′-CTG GGT CCA AGT ACG CTA GG-3′
5′-GCT GCA GTT CAA GCA ACC AA-3′
5′-CCA CGA AGC ACT TCA CTT CA-3′
5′-GGG TAG GAT CCT TGG ATG GT-3′
5′-TCA GAA ACC TGC CTG GAA TC-3′
5′-ATG CCT CCC ATG CAC TTA TC-3′
5′-TAG CAT TGT GGA TGG AGC TG-3′
5′-CCC TGC TTG CAG AAG AG-3′
5′-GGC CTA AGG TCT TTC CAT CC-3′
5′-TCC GTC TGG TAT GGA GAA GG-3′
5′-ACA TCG ACA CAG TGC AGA GC-3′
5′-TCT TTT CAG GCA ACC ATT GC-3′
5′-ACT TGG AGC ATG GGA TGA AG-3′
5′-GTG GGT TTC GAC TTT GAT GG-3′
5′-TTG ATC TCG CCT CAT GAC AG-3′
5′-CTC TCT GCG AAA GGA CCA AG-3′
5′-TCG TAG CCC CAC TTC CTA TG-3′
Isolation of mitochondrial and cytosolic fraction from fresh liver tissue was based on the principle of differential centrifugation using a Mitochondrial Extraction kit (Imgenex Co., San Diego, CA).
Whole liver lysates or mitochondrial fractions were extracted and Western blotting was performed as described.12 The following antibodies were employed: MAVS (Santa Cruz Biotechnology Inc., sc-6881), cytochrome c (Imgenex, IMG101-A), caspase 1 p10 (Santa Cruz Biotechnology Inc., sc-514), cleaved caspase 8 (Imgenex, IMG5703), RIP3 (Abcam, ab72106), β-actin (Abcam, ab6276), β-tubulin (Abcam, ab6046), and Tim23 (BD Biosciences, 611222).
Native Gel Electrophoresis.
A Native PAGE Novex Bis-Tris Gel System (Invitrogen Life Science, Carlsbad, CA) was used. Liver samples were lysed using 5% Digitonin as a mild detergent and separated on Native PAGE Novex 3-12% Bis-Tris gels. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane, fixed with 8% acetic acid, diluted in distilled water and identified with specific primary antibodies followed by horseradish peroxidase–labeled secondary antibodies and chemiluminescence assay.
Whole liver lysates were precleared with anti-rabbit immunoglobulin G beads followed by overnight incubation with 5 μg of the primary antibody (proteasome subunit alpha type 7 [PSMA7] or MAVS) and precipitated with immunoglobulin G beads. The immunprecipitates were lysed and denatured using β-mercaptoethanol containing buffer and heating. The proteins were separated on a polyacrylamid gel, transferred to a nitrocellulose membrane, and detected using specific antibodies (MAVS, PSMA7).
Human Liver Samples.
Human liver tissue was obtained from biopsies from clinically and biopsy-proven NASH patients without fibrosis and from patients with chronic hepatitis B. Liver samples were frozen immediately and kept in liquid nitrogen before RNA extraction. RNA was extracted as above. The study was approved by the Committee for the Protection of Human Subjects in Research at the University of Massachusetts. Human normal liver and liver tumor total RNA were purchased from OriGene Technologies (Rockville, MD).
Statistical significance was determined using the nonparametric Kruskal-Wallis test and Mann-Whitney tests. Data are shown as mean ± SE and were considered statistically significant at P < 0.05.
Type I IFN Induction Is Decreased in Steatohepatitis in Response to Poly(I:C) Stimulation.
Poly I:C, a synthetic dsRNA, is a surrogate for viral infection.13 dsRNA is recognized by TLR3 and helicase receptors and induces robust type I IFN response leading to anti-viral immunity.14 Antiviral responses to RNA are important in HCV and HIV infection.6, 7 We show for the first time that poly(I:C)-induced type I IFN production is significantly decreased in mice with steatohepatitis (Fig. 1). We found decreased serum protein (Fig. 1A) and liver messenger RNA (mRNA) levels of IFNβ (Fig. 1B) and IFNα4 (Fig. 1C) in mice fed a methionine–choline-deficient (MCD) diet compared with control mice fed a methionine–choline-supplemented (MCS) diet. Consistent with impaired type I IFN production after poly(I:C) stimulation, induction of IFN-inducible gene (ISG) 56 (Fig. 1D) and ISG15 (Fig. 1E) was also significantly decreased in MCD diet–induced steatohepatitis. These results suggest that steatohepatitis results in impaired type I IFN response to dsRNA viral challenge.
Impaired Type I IFN Induction in Steatohepatitis Is Restricted to the RIG-I/Mda5 Pathway.
To further evaluate the significance of impaired type I IFN induction in steatohepatitis, we employed stimulations that induce type I IFNs by way of receptor pathways different from dsRNA recognition by TLR3 and its adapter, TIR domain-containing adaptor inducing IFN-β (TRIF), or RIG-I/Mda5 and their adapter MAVS, respectively.14 LPS is recognized by TLR4 and uses the adapters TRIF and myeloid differentiation factor 88 (MyD88), whereas CpG DNA, a ligand for TLR9, uses solely the MyD88 adapter in type I IFN induction.14
We found increased TLR3, Mda5, and RIG-I, as well as their corresponding adapters, TRIF and MAVS, at the mRNA levels in fatty livers compared with livers of control mice (Fig. 2A). In contrast to poly(I:C), challenge with a TLR4 ligand (LPS), which uses TRIF, or a TLR9 ligand (CpG DNA), which uses MyD88, resulted in increased type I IFN induction in MCD compared with MCS diet–fed mice (Fig. 2B-D). TRIF serves as the sole adapter for poly(I:C)-engaged TLR3, and it also mediates TLR4/LPS-induced type I IFN production.14 TRIF-deficient mice were shown to be defective in both TLR3- and TLR4-mediated IFN regulatory factor 3 (IRF3) activation.15 These data suggest a selective impairment of type I IFN induction upon dsRNA viral [poly(I:C)] challenge in a TLR3/TRIF-independent manner. We therefore focused on dissecting the role of the helicase RNA-sensing pathways in steatohepatitis.
Abnormal MAVS Function in NASH Involves Decreased Protein Levels, Dissociation from the Mitochondria, and Impaired Oligomerization.
The adapter molecule MAVS is critical for the downstream signaling of helicase receptors, and its dysfunction impairs proinflammatory cytokine and IFN induction through the nuclear factor κB (NFκB) and IRF3 signaling pathways, respectively.8 Consistent with decreased induction of type I IFN, we found decreased levels of MAVS protein in whole liver lysates of MCD diet–fed mice compared with those of control mice (Fig. 3A). In search of possible mechanisms for decreased MAVS protein levels, we found higher mRNA expression of the PSMA7 subunit of proteasome in MCD-induced steatohepatitis (Fig. 3B). PSMA7 can negatively regulate MAVS-mediated immune responses and promotes proteosomal degradation.16 Immunoprecipitation experiments revealed increased association between MAVS and PSMA7 in fatty livers compared with livers of control mice (Fig. 3C).
The localization of MAVS to the outer mitochondrial membrane is crucial for Mda5/RIG-I activation.9 However, we found that steatohepatitis resulted in decreased mitochondria-associated MAVS protein levels compared with controls (Fig. 4A). We also observed a corresponding increase in cytosolic MAVS protein levels in MCD compared with the MCS diet–fed livers (Fig. 4B). The purity of the mitochondrial and cytosolic preparations was confirmed by the expression of mitochondrial marker Tim23 (Fig. 4A) and cytosolic β-tubulin (Fig. 4B), respectively. The ratio of the cytoplasmic/mitochondrial MAVS was significantly higher in MCD-induced steatohepatitis (Fig. 4C). These results indicated that displacement of MAVS protein from the mitochondria to the cytosol is likely related to mitochondrial damage in steatohepatitis. The transmembrane domain of MAVS is crucial for mitochondrial localization and also for dimerization of MAVS that is required for downstream signaling.9, 17 We found that in addition to impaired mitochondrial localization, there was decreased oligomerization of MAVS in steatohepatitis compared with controls (Fig. 4D).
Given the defects in poly(I:C)-triggered IFN induction in steatohepatitis (Fig. 1), we next explored the function of the MAVS adapter protein. In control mice, poly(I:C) administration resulted in displacement of MAVS from the mitochondria to the cytosol (Fig. 4A,B). In contrast, there was no increase in cytoplasmic MAVS translocation after poly(I:C) stimulation in livers of MCD diet–fed mice (Fig. 4A,B). Poly(I:C)-induced engagement of helicases and signaling through MAVS results in downstream activation and phosphorylation of IRF3.14 In livers of MCD diet–fed mice, impaired MAVS function and decreased mitochondrial association was associated with significantly reduced IRF3 phosphorylation after poly(I:C) stimulation (Fig. 4E). These data suggest that decreased association of MAVS with mitochondria at baseline may impair downstream signaling in steatohepatitis.
Mitochondrial Damage Occurs in the Fatty Liver.
Mitochondrial dysfunction plays a role in the pathogenesis of NASH18 and upon mitochondrial damage, its content leaks into the cytosol, triggering diverse signaling pathways, including apoptosis.19 Thus, we hypothesized that decreased association of MAVS with mitochondria may be linked to mitochondrial damage in NASH. Indeed, mitochondrial damage was indicated by relocation of cytochrome c from the mitochondria to the cytoplasm (Fig. 5A), and by enrichment of the mitochondria with β-actin (Fig. 5B) in livers of MCD compared with MCS diet–fed mice. We further identified evidence for increased cellular damage pathways in steatohepatitis as indicated by caspase 8 (Fig. 5C) and caspase 1 (Fig. 5D) activation. Relevant to our observation of decreased MAVS in steatohepatitis, both caspase 8 and caspase 1 were shown to cleave MAVS from the mirochondria.20-22
Mitochondrial damage in NASH has been linked to excessive levels of reactive oxygen species (ROS).18 Indeed, we detected significantly increased liver TBARS levels revealing ROS-induced lipid peroxidation at baseline and after poly(I:C) stimulation in steatohepatitis (Fig. 5E). These results indicate that ROS and lipid peroxidation occur in NASH, and their production is exacerbated in response to dsRNA stimulation.
Increased Poly(I:C)-Induced Liver Damage Occurs Without Excessive Proinflammatory Cytokine Induction in Steatohepatitis.
Liver damage, indicated by steatosis and elevated ALT, is a hallmark of steatohepatitis. Here we found that a poly(I:C) challenge significantly increased liver injury in MCD diet–fed mice as indicated by tissue hemorrhage, hepatocyte degeneration (Fig. 6A), and significantly increased serum ALT levels compared with MCS control mice (Fig. 6B). Because dsRNA-induced activation of RIG-I and Mda5 leads to type I IFN induction as well as activation of NFκB and production of proinflammatory cytokines,14 we sought to evaluate whether the increased liver damage was the consequence of enhanced proinflammatory cytokine production in steatohepatitis. At baseline, MCD diet–fed mice showed increased serum (Fig. 6C) and liver mRNA levels (Fig. 6D) of tumor necrosis factor α (TNFα), interleukin (IL)-6, and IL-1β compared with MCS control mice. Whereas poly(I:C) challenge increased TNFα, IL-6, and IL-1β production both in control mice and in MCD diet–fed groups (Fig. 6C,D), the extent of proinflammatory cytokine protein (Fig. 6C) and mRNA (Fig. 6D) induction was significantly lower in mice fed an MCD diet compared with mice fed an MCS diet. These data demonstrate that proinflammatory cytokine induction is impaired in response to a dsRNA challenge and is therefore less likely to account for the increased liver damage in NASH.
Because previous studies showed a crucial role for natural killer (NK) cells in poly(I:C)-induced liver injury,23 and higher NK cell activating ligand expression has been reported in livers of NASH patients,24 we next investigated the possible role of NK cells. We found increased mRNA expression of the NK-activating ligands, Pan-retinoic acid early inducible protein-1 (Rae), Rae-1α and Mult-1 in MCD-steatohepatitis (Fig. 6E), but poly(I:C) did not induce a further increase in the expression of these ligands (Fig. 6E). Furthermore, we found that recruitment of NK cells after poly(I:C) stimulation occurred in control livers and not in steatohepatitis (Supporting Fig. 1).
Poly I:C Promotes a Switch from Apoptosis to Necrosis and Increases RIP3 Expression in Steatohepatitis.
Hepatocyte apoptosis in NASH has been linked to increased susceptibility of the fatty liver to LPS challenge, whereas hepatocyte necrosis is associated with progressive liver damage.3 There is recent evidence that the mitochondria-associated MAVS can regulate apoptosis in viral infection.25 Apoptosis is triggered by way of intrinsic (involving proapoptotic protein Bim, mitochondria, cytochrome c, and caspase 9) or extrinsic (involving death receptors including TNF-related apoptosis-inducing ligand [TRAIL]) pathways that connect at the level of caspase 3 to culminate in cell death. We found increased expression of TRAIL (extrinsic apoptosis; Supporting Fig. 2A) and Bim (intrinsic apoptosis; Supporting Fig. 2B) in livers of MCD diet–fed mice. Expression of caspase 3 was also induced in mice fed an MCD diet versus mice fed an MCS diet (Fig. 7A). We found that caspase 3 activity was significantly increased by poly(I:C) in normal (MCS) livers (Fig. 7A), but not in steatohepatitis (MCD) (Fig. 7A). There were no differences in the extent of poly(I:C)-induced up-regulation of TRAIL and Bim mRNA expression (Supporting Fig. 2A,B) between MCD and MCS livers, indicating that although steatotic livers exhibit higher apoptosis at baseline, they fail to progress to tissue death due to apoptosis upon a viral challenge. Notably, the tissue damage was higher in poly(I:C)-challenged steatotic livers compared with control livers (Fig. 6B). Thus, we hypothesized that the increased poly(I:C)-induced liver damage in MCD diet–fed mice was due to necrosis rather than apoptosis. Indeed, we identified increased levels of serum HMGB1 (Fig. 7B), a marker of necrosis, in the poly(I:C)-stimulated MCD group compared with control mice.
The balance between apoptosis and necrosis is tightly regulated.26 A recently identified master regulator between apoptosis and necrosis is the protein kinase receptor-interacting protein 3 (RIP3).26 We found increased levels of RIP3 mRNA (Fig. 7C) and protein (Fig. 7D) in livers of mice fed an MCD diet compared with MCS diet–fed control mice. In control mice, poly(I:C) stimulation induced up-regulation of RIP3 protein expression 2 hours after stimulation, which returned to baseline by 6 hours (Fig. 7D); in contrast, there was sustained induction of RIP3 in steatohepatitis after poly(I:C) challenge (Fig. 7D). We further identified a positive correlation between RIP3 and liver HMGB1 (Fig. 7E) expression. Collectively, these data suggest that pathways that promote necrosis are preferentially up-regulated in steatohepatitis after a viral challenge, due at least in part to the regulatory involvement of RIP3.
Altered MAVS and RIP3 mRNA Expression in Human NASH.
To validate our observations in the mouse model of steatohepatitis, we next evaluated human livers. We found an increase of MAVS mRNA levels in livers of NASH patients compared with controls (Fig. 8A), mirroring MAVS RNA levels in the animal model of steatohepatitis (Fig. 2A). MAVS mRNA up-regulation was specific to NASH because we did not observe increased MAVS levels in hepatitis B virus infection (hepatitis B virus is a DNA virus) or in liver tumors (no viral infection detected) (Fig. 8A). We also found higher expression of PSMA7 mRNA in human NASH livers (Fig. 8B) that mirrored findings in the mouse model (Fig. 3B). Finally, we detected highly increased RIP3 mRNA levels in NASH patients (Fig. 8C) compared with controls; this was parallel to the RIP3 mRNA increase in the mouse model of NASH (Fig. 7C).
Steatosis and steatohepatitis are cofactors in the progression of liver diseases, including those of viral etiology, ischemia/reperfusion injury, and liver transplantation.2, 5 We report novel findings related to the impaired capacity of the fatty liver to respond to dsRNA and related viral challenges. First, livers with steatohepatitis failed to activate antiviral innate immune pathways to produce type I IFNs in response to a dsRNA challenge. Second, the MAVS adapter, which is required for type I IFN induction after recognition of dsRNA by the helicase receptors RIG-I and Mda5, was dissociated from the mitochondria to the cytosol and showed impaired oligomerization and function in steatohepatitis. Third, displacement of MAVS from mitochondria was associated with oxidative stress and instead of up-regulation of the apoptosis cascade, poly(I:C) promoted necrosis through increased expression of RIP3 in steatohepatitis. Fourth, dsRNA challenge resulted in increased liver damage in spite of decreased TNFα and proinflammatory cytokine induction in a diet-induced model of NASH.
Viral-sensing receptors include Toll-like receptor (TLR) 3 and the cytoplasmic helicase receptors RIG-I and Mda5 for dsRNA recognition, TLR7/8 for single-stranded RNA and TLR9 for sensing viral DNA.14 Here we identified a selective defect in signaling from viral dsRNA in steatohepatitis that altered both proinflammatory cytokines and type I IFNs and was associated with increased liver damage. Although TLR3, Mda5, and RIG-I all sense poly(I:C), their signaling pathways are different. Mda5 plays a key role in poly(I:C)-induced IFNβ production even in the absence of TLR3 or RIG-I.14 Ligand engagement of the helicase receptors catalyzes the phosphorylation of IκB proteins by IκB kinase complex and leads to NFκB activation, along with the phosphorylation and activation of IRF3.14 NFκB activation triggers the production of proinflammatory cytokines, whereas IRF3 phosphorylation leads to production of type I IFNs.14
The cellular source of the type I IFNs and inflammatory cytokines remains to be evaluated. Helicase receptors are expressed in several cell types in the liver, including hepatocytes, conventional dendritic cells, Kupffer cells, and NK cells.27, 28 RIG-I–like receptor expression is enhanced by poly(I:C).28 We found that hepatocytes that represent the majority of cells in the liver produce IFNβ after intracellular poly(I:C) stimulation in vitro (data not shown). The RIG-I/Mda5 pathway is also important in the conventional dendritic cells27 and NK cells,29 but less prominent in plasmacytoid dendritic cells. Thus, we speculate that hepatocytes and conventional DCs are the likely sources of type I IFN production after dsRNA challenge in the liver. Previous studies demonstrated a role of NK cells in NASH.24 Here we found evidence for increased expression of the NK cell–activating ligands PanRae, Rae1α, and Mult-1 in livers with steatohepatitis without a further increase after dsRNA stimulation. We also determined that NK cell recruitment was not triggered in livers with NASH, suggesting that the liver damage was unlikely to be NK cell–mediated after poly(I:C) challenge.
Here we demonstrated that both type I IFNs and proinflammatory cytokine induction were selectively disturbed in response to dsRNA, whereas TLR4- or TLR9-mediated pathways remained intact in steatohepatitis. This suggested that the signaling defects in fatty livers occurred upstream from the branching of the NFκB and IRF3 signaling pathways and involved a protein that is common to both pathways upon dsRNA stimulation. MAVS mediates the activation of both NFκB and IRF3 in response to viral infection.8 Here we show for the first time that total liver MAVS protein levels are decreased in steatohepatitis. Our data showed increased association of MAVS with the proteasome subunit PSMA7 in MCD-induced steatohepatitis, suggesting that proteosomal degradation could contribute to low MAVS levels. In this context, the apparent discrepancy between our finding of decreased MAVS protein and increased liver MAVS RNA could represent a compensatory feedback loop mechanism. Increased mRNA levels of MAVS and PSMA7 were also present in human livers with NASH.
Impaired MAVS function was suggested by three of our novel observations. First, MAVS levels were decreased in the mitochondria with a complementary increase in the cytosol in the mouse model of steatohepatitis compared with control mice. Second, in parallel with the MAVS dissociation from the mitochondria, we found decreased MAVS oligomerization in livers of MCD diet–fed mice compared with control mice. Third, we found impaired induction of IRF3 phosphorylation by poly(I:C) in livers with steatohepatitis.
The transmembrane domain of MAVS is crucial to the mitochondrial localization of MAVS, but is also required for the dimerization of the protein that is a crucial step during MAVS-induced immune responses.9, 17 Our novel finding on the reduced MAVS oligomerization is in accordance with the impaired function of the helicase receptor-MAVS signaling pathway.
Mitochondrial dysfunction is a key component of fat accumulation, ROS generation, and the progression of inflammation in NASH.18 Thus, it is plausible that translocation of MAVS from the mitochondria to the cytosol could be a consequence of mitochondrial damage in steatohepatitis. In addition to MAVS redistribution, we found other indications of mitochondrial damage, such as cytochrome c leak from the mitochondria to cytoplasm, enrichment of mitochondria with β-actin, and increased activation of cellular damage pathways. Translocation of β-actin to the mitochondria leading to disruption of mitochondrial membrane was shown in influenza virus–stimulated macrophages.30 We found markedly elevated β-actin protein levels in mitochondrial fractions in steatohepatitis providing evidence for mitochondrial damage in NASH. In normal hepatocytes, MAVS is localized in the outer mitochondrial membrane.9 Our novel data indicate increased activation of multiple caspases, including caspase 1 and caspase 8, in MCD diet–induced steatohepatitis, suggesting a possible link between MAVS cleavage and caspase activation. Several viruses, including hepatitis C (NS3/4A protease) and hepatitis A (3ABC protease), disrupt the host antiviral response by cleaving MAVS from mitochondria.20, 21 An apoptotic cleavage of MAVS has also been described.21 In NASH, both the death receptor–induced and cellular stress–induced apoptotic pathways are involved, and apoptosis is indicated by increased caspase 3 activity and plasma cytokeratin 18 fragments.31, 32 Studies have shown that the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone prevents the cleavage of MAVS, whereas selective blockade of caspase 8, 9, or 3 was not sufficient to prevent MAVS cleavage.21 Relevant to our data, the pan-caspase inhibitor blocks both apoptotic caspases and caspase 1.21, 22 Thus, MAVS cleavage from the mitochondria in NASH is likely to be related to the increased caspase 8 and caspase 1 observed in our experiments.
Damaged proteins are degraded by proteasomes in the cytoplasm or nucleus.33 We show for the first time that MAVS protein preferentially binds to the proteasomal protein PSMA7 in fatty livers, suggesting that the damaged, cleaved MAVS protein from the mitochondria accumulates in the cytoplasm and is likely degraded by the proteasomes.
Virus-induced apoptosis requires MAVS in primary mouse fibroblasts25 and MAVS itself can induce caspase-dependent apoptosis. It has been shown that poly(I:C) initiates apoptosis through MAVS.34 However, MAVS levels were decreased in MCD diet–induced steatohepatitis in our experiments. Furthermore, we found that whereas caspase 3 was activated by dsRNA stimulation in normal liver suggesting apoptosis, there was no increase over the elevated baseline apoptosis (caspase 3 activity) in steatohepatitis. Instead, poly(I:C) induced liver necrosis and increased serum HMGB1 levels in MCD diet–fed mice. We speculate that decreased mitochondrial MAVS levels may result in impaired MAVS-dependent apoptosis after dsRNA challenge in MCD-induced steatohepatitis.
MAVS interacts with protein kinase RIP1 and facilitates NFκB activation.35 RIP1 and the protein kinase RIP3 may form a complex with TRADD, FADD, and caspase 8 that leads to RIP3 cleavage and proteolytic inactivation.36, 37 Studies have shown that RIP3 overexpression results in TNFα and nitric oxide (NO)–mediated necrosis.37, 38 RIP3 has been identified as a molecular switch between apoptosis and necrosis.26 We show for the first time that increased expression of RIP3 in MCD diet–fed mice occurs both at the mRNA and protein levels. Increased RIP3 mRNA was also present in human livers with NASH. We found a sustained increase in RIP3 expression that correlated with increased necrosis and increased serum HMGB1 levels after poly(I:C) challenge in steatohepatitis in mice. It is tempting to speculate that increased RIP3 results in an apoptosis-to-necrosis switch after a dsRNA challenge in steatohepatitis. Recent studies have suggested an association of RIP3 with the mitochondria and its regulation by ROS,38 and RIP3-induced promotion of necrosis is regulated by ROS.26 Our observations confirmed previous findings of increased ROS generation in diet-induced NASH.18 More importantly, we identified that poly(I:C) augmented ROS generation as well as RIP3 induction and necrosis in MCD-induced steatohepatitis.
In conclusion, our data demonstrate an important role for mitochondrial damage and MAVS dissociation from the mitochondria in the increased susceptibility of steatohepatitis to a dsRNA viral challenge. We report for the first time that livers with steatohepatitis fail to induce type I IFNs in response to dsRNA challenge due to dissociation of MAVS from the mitochondria and impaired oligomerization. The MAVS dissociation also leads to impaired induction of apoptosis and promotes necrosis together with increased RIP3 expression, impaired antiviral interferon response, and increased liver damage in NASH. These key findings were also reproducible in human NASH.