Aldose reductase regulates doxorubicin-induced immune and inflammatory responses by activating mitochondrial biogenesis.

We have recently demonstrated that aldose reductase (AR) inhibitor; fidarestat prevents doxorubicin (Dox)-induced cardiotoxic side effects and inflammation in vitro and in vivo. However, the effect of fidarestat and its combination with Dox on immune cell activation and the immunomodulatory effects are not known. In this study, we examined the immunomodulatory effects of fidarestat in combination with Dox in vivo and in vitro. We observed that fidarestat decreased Dox-induced upregulation of CD11b in THP-1 monocytes. Fidarestat further attenuated Dox-induced upregulation of IL-6, IL-1ß, and Nos2 in murine BMDM. Fidarestat also attenuated Dox-induced activation and infiltration of multiple subsets of inflammatory immune cells identified by expression of markers CD11b+, CD11b+F4/80+, Ly6C+CCR2high, and Ly6C+CD11b+ in the mouse spleen and liver. Furthermore, significant upregulation of markers of mitochondrial biogenesis PGC-1α, COX IV, TFAM, and phosphorylation of AMPKα1 (Ser485) was observed in THP-1 cells and livers of mice treated with Dox in combination with fidarestat. Our results suggest that fidarestat by up-regulating mitochondrial biogenesis exerts protection against Dox-induced immune and inflammatory responses in vitro and in vivo, providing further evidence for developing fidarestat as a combination agent with anthracycline drugs to prevent chemotherapy-induced inflammation and toxicity.

IL-17a exacerbates hepatic ischemia-reperfusion injury in fatty liver by promoting neutrophil infiltration and mitochondria-driven apoptosis.

Hepatic ischemia-reperfusion (IR) injury is a critical issue during liver transplantation (LT). Recent studies have demonstrated that IL-17a contributes to IR injury and steatohepatitis. However, the underlying mechanism is not understood. This study aimed to examine the role of IL-17a on hepatic IR injury in fatty liver and to investigate the underlying mechanisms. The correlation between serum IL-17a levels and liver function was analyzed in LT patients receiving fatty (n = 42) and normal grafts (n = 44). Rat LT model was applied to validate the clinical findings. IL-17a knockout (KO) and wild-type mice were fed with high-fat diets to induce fatty liver and subjected to hepatic IR injury with major hepatectomy. Frequency of circulating neutrophils and IL-17a expression on PBMCs were analyzed by flow cytometry. Mitochondrial outer membrane permeabilization (MOMP) was examined by a living intravital image system. Serum IL-17a was elevated after human LT, especially with fatty grafts. The aspartate aminotransferase and alanine transaminase levels were increased in recipients with fatty grafts compared with normal grafts. In rat LT model, the intragraft IL-17a expression was significantly higher in fatty grafts than normal ones post-LT. KO of IL-17a in mice notably attenuated liver damage after IR injury in fatty liver, characterized by better-preserved liver architecture, improved liver function, and reduced neutrophil infiltration. MOMP triggered cell death after hepatic IR injury in a caspase-independent way via IL-17a/NF-κB signaling pathway. KO of IL-17a protected the fatty liver against IR injury through the suppression of neutrophil infiltration and mitochondria-driven apoptosis.

Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signaling in virus-infected hepatocytes.

BACKGROUND & AIMS: Selective elimination of virus-infected hepatocytes occurs through virus-specific CD8 T cells recognizing peptide-loaded MHC molecules. Herein, we report that virus-infected hepatocytes are also selectively eliminated through a cell-autonomous mechanism. METHODS: We generated recombinant adenoviruses and genetically modified mouse models to identify the molecular mechanisms determining TNF-induced hepatocyte apoptosis in vivo and used in vivo bioluminescence imaging, immunohistochemistry, immunoblot analysis, RNAseq/proteome/phosphoproteome analyses, bioinformatic analyses, mitochondrial function tests. RESULTS: We found that TNF precisely eliminated only virus-infected hepatocytes independently of local inflammation and activation of immune sensory receptors. TNF receptor I was equally relevant for NF-kB activation in healthy and infected hepatocytes, but selectively mediated apoptosis in infected hepatocytes. Caspase 8 activation downstream of TNF receptor signaling was dispensable for apoptosis in virus-infected hepatocytes, indicating an unknown non-canonical cell-intrinsic pathway promoting apoptosis in hepatocytes. We identified a unique state of mitochondrial vulnerability in virus-infected hepatocytes as the cause for this non-canonical induction of apoptosis through TNF. Mitochondria from virus-infected hepatocytes showed normal biophysical and bioenergetic functions but were characterized by reduced resilience to calcium challenge. In the presence of unchanged TNF-induced signaling, reactive oxygen species-mediated calcium release from the endoplasmic reticulum caused mitochondrial permeability transition and apoptosis, which identified a link between extrinsic death receptor signaling and cell-intrinsic mitochondrial-mediated caspase activation. CONCLUSION: Our findings reveal a novel concept in immune surveillance by identifying a cell-autonomous defense mechanism that selectively eliminates virus-infected hepatocytes through mitochondrial permeability transition. LAY SUMMARY: The liver is known for its unique immune functions. Herein, we identify a novel mechanism by which virus-infected hepatocytes can selectively eliminate themselves through reduced mitochondrial resilience to calcium challenge.

The binding of SLE autoantibodies to mitochondria.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by immune complexes. Because these complexes contain mitochondrial components, we assessed the presence of antibodies to whole mitochondria (wMITO) using an ELISA in which mitochondria from mouse liver are bound to microtiter plates pre-coated with poly-l-lysine. Studies with this ELISA demonstrated that SLE plasmas contain abundant anti-wMITO activity. While digestion with DNase 1 did not affect anti-wMITO activity, adsorption of plasma on DNA affinity columns could reduce binding activity. Assay for anti-mitochondrial antibodies (AMA) by immunofluorescence and an ELISA with the M2 antigen (2-oxo-acid dehydrogenase protein complex) showed a low frequency of positivity, indicating that AMA and anti-wMITO are distinct specificities. In the study of 204 patients with SLE, the levels of anti-wMITO were higher in active SLE and correlated with levels of anti-DNA. These findings suggest that anti-wMITO can form immune complexes with mitochondria which may drive pathogenesis.

rt269I Type of Hepatitis B Virus (HBV) Leads to HBV e Antigen Negative Infections and Liver Disease Progression via Mitochondrial Stress Mediated Type I Interferon Production in Chronic Patients With Genotype C Infections.

Hepatitis B virus infection is a serious global health problem and causes life-threatening liver disease. In particular, genotype C shows high prevalence and severe liver disease compared with other genotypes. However, the underlying mechanisms regarding virological traits still remain unclear. This study investigated the clinical factors and capacity to modulate Type I interferon (IFN-I) between two HBV polymerase polymorphisms rt269L and rt269I in genotype C. This report compared clinical factors between rt269L and rt269I in 220 Korean chronic patients with genotype C infections. The prevalence of preC mutations between rt269L and rt269I was compared using this study's cohort and the GenBank database. For in vitro and in vivo experiments, transient transfection using HBV genome plasmid and HBV virion infection using HepG2-hNTCP-C4 and HepaRG systems and hydrodynamic injection of HBV genome into mice tails were conducted, respectively. This report's clinical data indicated that rt269I vs. rt269L was more significantly related to HBV e antigen (HBeAg) negative serostatus, lower levels of HBV DNA and HBsAg, and disease progression. Our epidemiological study showed HBeAg negative infections of rt269I infections were attributed to a higher frequency of preC mutations at 1896 (G to A). Our in vitro and in vivo studies also found that rt269I could lead to mitochondrial stress mediated STING dependent IFN-I production, resulting in decreasing HBV replication via the induction of heme-oxygenase-1. In addition, we also found that rt269I could lead to enhanced iNOS mediated NO production in an IFN-I dependent manner. These data demonstrated that rt269I can contribute to HBeAg negative infections and liver disease progression in chronic patients with genotype C infections via mitochondrial stress mediated IFN-I production.

MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo.

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.

Hepatic Mitochondrial Dysfunction and Immune Response in a Murine Model of Peanut Allergy.

BACKGROUND: Evidence suggests a relevant role for liver and mitochondrial dysfunction in allergic disease. However, the role of hepatic mitochondrial function in food allergy is largely unknown. We aimed to investigate hepatic mitochondrial dysfunction in a murine model of peanut allergy. METHODS: Three-week-old C3H/HeOuJ mice were sensitized by the oral route with peanut-extract (PNT). We investigated: 1. the occurrence of effective sensitization to PNT by analysing acute allergic skin response, anaphylactic symptoms score, body temperature, serum mucosal mast cell protease-1 (mMCP-1) and anti-PNT immunoglobulin E (IgE) levels; 2. hepatic involvement by analysing interleukin (IL)-4, IL-5, IL-13, IL-10 and IFN-γ mRNA expression; 3. hepatic mitochondrial oxidation rates and efficiency by polarography, and hydrogen peroxide (H2O2) yield, aconitase and superoxide dysmutase activities by spectrophotometry. RESULTS: Sensitization to PNT was demonstrated by acute allergic skin response, anaphylactic symptoms score, body temperature decrease, serum mMCP-1 and anti-peanut IgE levels. Liver involvement was demonstrated by a significant increase of hepatic Th2 cytokines (IL-4, IL-5 and IL-13) mRNA expression. Mitochondrial dysfunction was demonstrated by lower state 3 respiration rate in the presence of succinate, decreased fatty acid oxidation in the presence of palmitoyl-carnitine, increased yield of ROS proven by the inactivation of aconitase enzyme and higher H2O2 mitochondrial release. CONCLUSIONS: We provide evidence of hepatic mitochondrial dysfunction in a murine model of peanut allergy. These data could open the way to the identification of new mitochondrial targets for innovative preventive and therapeutic strategies against food allergy.

Mitochondria in non-alcoholic fatty liver disease.

NAFLD is a common disease in Western society and ranges from steatosis to steatohepatitis and to end-stage liver disease. The molecular mechanisms that cause the progression of steatosis to severe liver damage are not fully understood. One suggested mechanism involves the oxidation of biomolecules by mitochondrial ROS which initiates a vicious cycle of exacerbated mitochondrial dysfunction and increased hepatocellular oxidative damage. This may ultimately pave the way for hepatic inflammation and liver failure. This review updates our current understanding of mitochondria-derived oxidative stress in the progression of NAFLD.

Dietary creatine supplementation lowers hepatic triacylglycerol by increasing lipoprotein secretion in rats fed high-fat diet.

Recent studies have shown that dietary creatine supplementation can prevent lipid accumulation in the liver. Creatine is a small molecule that plays a large role in energy metabolism, but since the enzyme creatine kinase is not present in the liver, the classical role in energy metabolism does not hold in this tissue. Fat accumulation in the liver can lead to the development of nonalcoholic fatty liver disease (NAFLD), a progressive disease that is prevalent in humans. We have previously reported that creatine can directly influence lipid metabolism in cell culture to promote lipid secretion and oxidation. Our goal in the current study was to determine whether similar mechanisms that occur in cell culture were present in vivo. We also sought to determine whether dietary creatine supplementation could be effective in reversing steatosis. Sprague-Dawley rats were fed a high-fat diet or a high-fat diet supplemented with creatine for 5 weeks. We found that rats supplemented with creatine had significantly improved rates of lipoprotein secretion and alterations in mitochondrial function that were consistent with greater oxidative capacity. We also find that introducing creatine into a high-fat diet halted hepatic lipid accumulation in rats with fatty liver. Our results support our previous report that liver cells in culture with creatine secrete and oxidize more oleic acid, demonstrating that dietary creatine can effectively change hepatic lipid metabolism by increasing lipoprotein secretion and oxidation in vivo. Our data suggest that creatine might be an effective therapy for NAFLD.

Contribution of inducible and neuronal nitric oxide synthases to mitochondrial damage and melatonin rescue in LPS-treated mice.

NOS isoform activation is related to liver failure during sepsis, but the mechanisms driving mitochondrial impairment remain unclear. We induced sepsis by LPS administration to inducible nitric oxide synthase (iNOS-/-) and neuronal nitric oxide synthase (nNOS-/-) mice and their respective wild-type controls to examine the contribution of iNOS to mitochondrial failure in the absence of nNOS. To achieve this goal, the determination of messenger RNA (mRNA) expression and protein content of iNOS in cytosol and mitochondria, the mitochondrial respiratory complex content, and the levels of nitrosative and oxidative stress (by measuring 3-nitrotyrosine residues and carbonyl groups, respectively) were examined in the liver of control and septic mice. We detected strongly elevated iNOS mRNA expression and protein levels in liver cytosol and mitochondria of septic mice, which were related to enhanced oxidative and nitrosative stress, and with fewer changes in respiratory complexes. The absence of the iNOS, but not nNOS, gene absolutely prevented mitochondrial impairment during sepsis. Moreover, the nNOS gene did not modify the expression and the effects of iNOS here shown. Melatonin administration counteracted iNOS activation and mitochondrial damage and enhanced the expression of the respiratory complexes above the control values. These effects were unrelated to the presence or absence of nNOS. iNOS is a main target to prevent liver mitochondrial impairment during sepsis, and melatonin represents an efficient antagonist of these iNOS-dependent effects whereas it may boost mitochondrial respiration to enhance liver survival.

Moderate physical activity promotes basal hepatic autophagy in diet-induced obese mice.

Obesity is a known risk factor for the development of hepatic disease; obesity-induced fatty liver can lead to inflammation, steatosis, and cirrhosis and is associated with degeneration of the mitochondria. Lifestyle interventions such as physical activity may ameliorate this condition. The purpose of this study was to investigate regulation of mitochondrial and autophagy quality control in liver following Western diet-induced obesity and voluntary physical activity. Eight-week-old C57BL/6J mice were fed a Western diet (WD) or normal chow (NC, control) for 4 weeks; afterwards, groups were divided into voluntary wheel running (VWR) or sedentary (SED) conditions for an additional 4 weeks. WD-SED animals had a median histology score of 2, whereas WD-VWR was not different from NC groups (median score 1). There was no difference in mRNA of inflammatory markers Il6 and Tnfa in WD animals. WD animals had 50% lower mitochondrial content (COX IV and Cytochrome C proteins), 50% lower Pgc1a mRNA content, and reduced content of mitochondrial fusion and fission markers. Markers of autophagy were increased in VWR animals, regardless of obesity, as measured by 50% greater LC3-II/I ratio and 40% lower p62 protein content. BNIP3 protein content was 30% less in WD animals compared with NC animals, regardless of physical activity. Diet-induced obesity results in derangements in mitochondrial quality control that appear to occur prior to the onset of hepatic inflammation. Moderate physical activity appears to enhance basal autophagy in the liver; increased autophagy may provide protection from hepatic fat accumulation.

Autoantibodies in autoimmune liver diseases.

Autoimmune hepatitis is a chronic hepatitis of unknown etiology characterized by clinical, histological, and immunological features, generally including circulating autoantibodies and a high total serum and/or gamma globulin. Liver-related autoantibodies are very significant for the correct diagnosis and classification of autoimmune liver diseases (AILD), namely autoimmune hepatitis types 1 and 2 (AIH-1 and 2), primary biliary cirrhosis (PBC), and the sclerosing cholangitis types in adults and children. This article intends to review recent studies that investigate autoantibodies in autoimmune liver diseases from a microbiological perspective.

Chemokine ligand 2 and paraoxonase-1 in non-alcoholic fatty liver disease: The search for alternative causative factors.

The incidence and prevalence of non-alcoholic fatty liver disease (NAFLD) is constantly increasing. Despite this is apparently associated with the growing increase in obesity, insulin resistance and obesity-related metabolic disturbances their presence is not a necessary or sufficient condition to explain the accumulation of fat in the liver. Conversely, NAFLD is a predictor of other metabolic risks. NAFLD is currently the most frequent chronic liver disease but should not be considered benign or anecdotic because a considerable proportion of patients with NAFLD progress to cirrhosis and end-stage liver disease. Consequently, the search for alternative molecular mechanisms with therapeutic implications in NAFLD and associated disorders deserves a careful consideration. Mitochondria are possible targets as these organelles generate energy from nutrient oxidation. Some findings, generated in patients with extreme obesity and in murine models, support the notion that NAFLD could be a mitochondrial disease. This is plausible because mitochondrial dysfunction affects the accumulation of lipids in hepatocytes and promotes lipid peroxidation, the production of reactive oxygen species, the release of cytokines causing inflammation and cell death. Here we discuss basic research and mechanistic studies targeting the role of chemokine ligand 2 in liver inflammation and that of the paraoxonases in the oxidative stress. Their combination and association with mitochondrial dysfunction may uncover mechanisms underlying the progression of NAFLD and may help to identify novel therapeutic targets.

Breach of tolerance: primary biliary cirrhosis.

In primary biliary cirrhosis (PBC), the breach of tolerance that leads to active disease involves a disruption in several layers of control, including central tolerance, peripheral anergy, a "liver tolerance effect," and the action of T regulatory cells and their related cytokines. Each of these control mechanisms plays a role in preventing an immune response against self, but all of them act in concert to generate effective protection against autoimmunity without compromising the ability of the host immune system to mount an effective response to pathogens. At the same time, genetic susceptibility, environmental factors, including infection agents and xenobiotics, play important roles in breach of tolerance in the development of PBC.

Clinical significance of autoantibodies in primary biliary cirrhosis.

Antimitochondrial, anti-gp210, anti-sp100, and anticentromere antibodies are specifically detected in primary biliary cirrhosis (PBC). In clinical practice, they are useful for the diagnosis of PBC or for evaluating disease severity, clinical phenotype, and long-term outcome. In the typical or classical form of PBC which shows slow progressive loss of small bile ducts with a parallel increase in liver fibrosis, anti-gp210 antibodies are a strong risk factor for progression to jaundice and hepatic failure, whereas the presence of anticentromere antibodies is a risk factor for progression to cirrhosis and portal hypertension. Of note, the autoimmune repertoire, which is established during the early stage of the disease process, can influence the clinical phenotype and the long-term prognosis of PBC. Because the natural course of PBC is being altered by treatment with ursodeoxycholic acid, the clinical significance of these PBC-specific autoantibodies awaits re-evaluation in various ethnicities.

Lipopolysaccharide impairs hepatocyte ureagenesis from ammonia: involvement of mitochondrial aquaporin-8.

We recently reported that hepatocyte mitochondrial aquaporin-8 (mtAQP8) channels facilitate the uptake of ammonia and its metabolism into urea. Here we studied the effect of bacterial lipopolysaccharides (LPS) on ammonia-derived ureagenesis. In LPS-treated rats, hepatic mtAQP8 protein expression and diffusional ammonia permeability (measured utilizing ammonia analogues) of liver inner mitochondrial membranes were downregulated. NMR studies using 15N-labeled ammonia indicated that basal and glucagon-induced ureagenesis from ammonia were significantly reduced in hepatocytes from LPS-treated rats. Our data suggest that hepatocyte mtAQP8-mediated ammonia removal via ureagenesis is impaired by LPS, a mechanism potentially relevant to the molecular pathogenesis of defective hepatic ammonia detoxification in sepsis.

Infiltration of inflammatory cells expressing mitochondrial proteins around bile ducts and in biliary epithelial layer may be involved in the pathogenesis in primary biliary cirrhosis.

AIMS: Serum antimitochondrial antibodies are characteristic in most patients with primary biliary cirrhosis (PBC); however, the significance of antimitochondrial antibodies in the pathogenesis of PBC remains unclear. We examined the extent and types of mitochondrial protein-expressing inflammatory cells and its association with deregulated autophagy of mitochondria in biliary epithelial lesions in PBC. METHODS: We examined the expression of pyruvate dehydrogenase complex-E2 component and a mitochondrial protein cytochrome c oxidase, subunit I in inflammatory cells in livers taken from patients with PBC (n=35) and control livers (n=64) including primary sclerosing cholangitis. Mitochondrial protein-expressing inflammatory cells were characterised by double immunofluorescence with surface markers. RESULTS: Infiltration of mitochondrial protein-expressing inflammatory cells was mainly observed in portal tracts and in the biliary epithelial layer and around the damaged small bile ducts in PBC. The extent of infiltration in portal tracts was significantly higher in PBC and early stage of chronic viral hepatitis than normal livers. The extent of infiltration around bile ducts and in biliary epithelial layer was significantly higher in early stage of PBC than control livers. Mitochondrial protein-expressing inflammatory cells included (1) CD68 and/or myeloperoxidase -positive monocytes/macrophages and (2) CD79a, CD38, CD138, IgM-positive and/or IgG-positive plasma cells. Colocalised expression of pyruvate dehydrogenase complex-E2 component and autophagy marker light chain 3ß was observed in the inflammatory cells. CONCLUSIONS: Mitochondrial protein-expressing inflammatory cells infiltrating around the damaged bile ducts and in biliary epithelial layers may be closely associated with the pathogenesis of bile duct lesion in PBC.