HBV, mitochondrial stress, and liver fibrosis: chicken or the egg

The liver, due to its critical metabolic functions, is extremely rich in mitochondria (eg, 1000–2000 in each hepatocyte) and mitochondrial dysfunction is heavily interconnected with liver damage, inflammation, and chronic liver diseases (CLD). Due to their proteo-bacterial ancestry, many mitochondrial structural components, including mtDNA, share significant similarities with bacteria. The release of molecular danger signals from the mitochondria of injured or dying cells (mitochondria-derived damage-associated molecular patterns, mito-DAMPs) is a potent trigger of inflammation.1 The interaction between mitochondria and the endoplasmic reticulum (ER) is essential for hepatocyte function, and mitochondria-associated membranes (MAMs), the contact sites between ER and mitochondria, are key regulators of mitochondrial bioenergetics and play a crucial role in metabolic flexibility and the regulation of glucose and lipid metabolisms in hepatocytes.2 Disruption of hepatic ER-mitochondria interactions triggers hepatic insulin resistance and steatosis in the context of NAFLD, which can be improved by nutritional strategies.2 Notably, steatosis itself may cause mtDNA damage, by favoring the accumulation of lipid peroxidation products.3 HCV and HBV both directly impact MAMs and mitochondria functions in infected hepatocytes.4,5 The HBV HBx protein can localize in the mitochondria, disrupt oxidative phosphorylation, interfere with the mitochondrial respiratory chain, and inhibit ATP synthesis, thus directly contributing to hepatocyte damage in cell models and hydrodynamic mouse models.5 Mitochondrial functions are also altered in vivo in exhausted HCV-specific and HBV-specific T cells, as a result of chronic inflammation and prolonged exposure to viral antigens.6,7 Less is known about the role of mitochondria, MAMs, and mitochondrial dysfunction in the development of liver fibrosis and its progression, a hallmark of all CLD that impacts patients’ morbidity and mortality. The recent demonstration that mtDNA and other mito-DAMPs released from injured hepatocyte mitochondria directly activate HSCs to drive liver scarring together with the demonstration that circulating mtDNA is markedly increased in patients with NASH and significant liver fibrosis has provided a direct link between mitochondria and fibrosis.8 It remains to be determined whether and to what extent the progression of fibrosis in patients with CLD in turn impacts mitochondrial dysfunction. In this issue of Hepatology,9 Loureiro and co-workers present the first comprehensive study that correlates mitochondrial oxidative stress and fibrosis progression in liver samples from a large cohort of 146 treatment-naive chronic hepatitis B (CHB) mono-infected patients, compared with patients with chronic hepatitis C (n=33), NASH-related cirrhosis (n=12), and healthy controls (n=24). A comprehensive study of mtDNA damage and several parameters of mitochondrial dysfunctions was performed and patients with F3–F4 advanced fibrosis (AF) were compared with patients with no/mild (F0–F1) to moderate (F2) fibrosis in each group and between the groups. The authors (AA) show that CHB patients with high viral load (HBV DNA >5 log10 IU/ml) and NASH cirrhosis with higher steatosis (>66% of hepatocytes) display the highest reduction of mtDNA levels (mtDNA/nDNA ratios <0.5), thus supporting the hypothesis that both etiologies cause mtDNA damage. mtDNA depletion can result from impaired mtDNA replication or an increased clearance of damaged mitochondria by mitophagy. Indeed, mitophagy markers (eg, PRKN and PINK1), mtUPR markers (eg, HSPD1, HSPA9, LONP1), and the biogenesis marker TFAM were all downregulated in patients with F3–F4 when compared with patients with F0–F2. These mitochondrial changes were accompanied by a decrease in the steady-state mRNA levels of the mtDNA-encoded cytochrome C oxidase subunits 1 (MT-CO1) and 2 (MT-CO2), irrespective of the etiology. The decrease was significantly higher in F3–F4 patients as compared with F0–F2 patients, and CHB patients with AF displayed the lowest MT-CO1 and MT-CO2 mRNA levels. Altogether, these results depict a progressive alteration of mitochondria functions that parallel fibrosis progression across etiologies. Although most results do not discriminate between CHB F4 and NASH F4, the AA found that the mtDNA deletions were more frequent in CHB patients with AF, as compared with NASH cirrhosis, and displayed a more complex pattern with more deletions and 3 new deletions in CHB patients with higher viral load. The specific contribution of HBV to the induction of mitochondrial stress can be related to HBx protein’s mitochondrial localization and its ability to interact with the mitochondrial voltage-dependent anion channel and to inactivate Bcl-2 and Bcl-xL. Indeed, the AA show that HepG2 cells replicating HBV or expressing wild-type HBx display an increased mitochondrial superoxide formation and mtDNA depletion. The reversion of this phenotype in cells overexpressing HBx carrying the G124L and I127A mutations that prevent HBx binding to Bcl-2 and Bcl-xL supports the role of HBx in the induction of mitochondrial dysfunction. The attenuation of mtDNA depletion by the iNOS inhibitor 1400W and the superoxide scavenger Mito-Tempo provides indirect evidence that an increased production of peroxynitrite is likely driving mtDNA damage and mitochondrial dysfunction in HBV-infected hepatocytes. However, these observations need to be confirmed in relevant HBV-infection cell models or HBV-infected liver chimeric mice. Altogether, Loureiro and coworkers provide a detailed and comprehensive characterization of mitochondrial alterations in patients with CHB, which correlate with fibrosis progression. Several questions remain open and warrant further investigation. The HCV and NASH cohorts are quite small and merit to be expanded. In particular, the observations made in the small NASH cirrhosis cohort included in the study should be expanded to the full spectrum of NAFLD and correlated with both the fibrosis stage and disease activity. The CHB cohort is larger. Hence, it will be important to correlate mitochondrial parameters not only with the serum viral load but also with relevant intrahepatic HBV biomarkers (eg, covalently closed circular DNA, 3.5-kb mRNAs, and the covalently closed circular DNA transcriptional activity). The impact on mitochondrial dysfunction of HBV suppression by nucleos(t)ide analogue treatment, HCV eradication by direct antiviral agents, and diet/weight loss in NASH patients remains unexplored. Leakage of mtDNA and other mito-DAMPs induces both autocrine and paracrine inflammatory responses in neighboring hepatocytes, macrophages, and Kupffer cells and activates HSC to favor the progression of fibrosis. A direct measurement of circulating mtDNA9 might be informative. The role of these different cell types and inflammatory cells infiltrating the liver should be addressed in patients with different etiologies. The role of HBV-induced mitochondrial dysfunction in HCS activation and fibrosis progression may be further explored in vitro by coculturing HBV-infected hepatocytes with HSCs to monitor their differentiation into collagen-secreting myofibroblasts and in vivo in HBV-infected liver humanized mice treated or not with CCL4. Finally, the hypothesis made by the AA that in some patients “mitochondrial resilience” involving adaptive mechanisms might maintain or restore mitochondrial homeostasis and limit reactive oxygen species formation and liver inflammation needs to be confirmed by experimental observations.