Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometer-sized (30–150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells [referred as HepAD38 (dox⁻)-exo] and from HBV nonreplicating cells [referred as HepAD38 (dox⁺)-exo]. A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox⁻)-exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox⁻)-exo had enhanced proteolytic activity compared with HepAD38 (dox⁺)-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox⁻)-exo induced a significantly lower level of IL-6 secretion compared with IL-6 levels from HepAD38 (dox⁺)-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of proteasome subunit proteins by HepAD38 (dox⁻)-exo might modulate the production of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus providing a new perspective on the role of exosomes in HBV-host interaction.

Background and Aims: HBsAg serves as an important immune-modulatory factor in chronic hepatitis B. One aspect of such modulation may act through monocytes, which are the major Ag-presenting cells taking up HBsAg. There is evidence for the encapsulation of hepatocellular microRNAs (miRNAs) by HBsAg particles, while its pathobiological significance is unclear. Here, we characterized the miRNA profile in patients with chronic hepatitis B and probed their association with liver inflammation. Approaches and Results: We collected plasma from patients that are treatment-naive with chronic hepatitis B (n = 110) and quantified total/HBsAg-enveloped miRNAs by qRT-PCR and plasma cytokines by ELISA. The biological effects of HBsAg-delivered miRNAs in monocytes were evaluated using multiple approaches. The clinical significance of candidate miRNAs and cytokines was corroborated in patients with HBV-associated advanced liver diseases. The plasma miRNA profile showed 2 major clusters, one significantly associated with HBsAg titer and the other correlated with liver inflammation. Among HBsAg-carried miRNAs, miR-939 displayed the most significant correlation with IL-8. Mechanistically, miR-939 in subviral particles enters monocytes and significantly augments IL-8 production through the mitogen-activated protein kinase (MAPK) p38 signaling pathway. Finally, the findings that miR-939 positively correlated with IL-8 level and inflammation/fibrosis stage in the cohort of HBV-associated advanced liver diseases support its causative role in the progression of liver diseases. Conclusions: HBsAg particles carry hepatocellular miRNAs, including miR-939, which enter monocytes and alter their functional status, such as IL-8 secretion. Our findings demonstrate that the HBsAg-miR-939-IL-8 axis may play a crucial role in HBV-induced hepatic necro-inflammation and the progression of advanced liver diseases.

Background and aims: Hepatitis B virus (HBV) causes chronic hepatic infection, leading to various advanced liver diseases. Currently, there is still a lack of effective non-invasive biomarkers for evaluating hepatic inflammation and fibrosis. Circulating microRNAs (miRNAs) serve as key regulators and potential biomarkers in the progression and pathogenesis of HBV-associated progressive liver diseases. Here, we characterized the miRNA profile in chronic hepatitis B (CHB) patients and probed their association with liver inflammation and fibrosis. Approaches and results: We profiled 34 candidate miRNAs in serum from a well-characterized HBV Disease Continuum Cohort, comprising 165 individuals in the discovery set (42 healthy controls, HC; 40 CHB; 42 cirrhosis; 41 hepatocellular carcinomas, HCC) and 143 in an independent testing set. Serum miRNA levels were quantified by qRT-PCR. Supervised clustering and correlation analyses revealed distinct miRNA expression profiles across disease stages. Statistical analyses included logistic regression, and ROC/AUC evaluation with 5-fold cross-validation and external validation. Unsupervised clustering and correlation with histological G/S staging revealed stage-specific miRNA signatures: miR-224-5p, miR-125a-5p, and miR-15a-5p peaked in cirrhosis and strongly correlated with fibrosis stage (miR-224-5p: r = 0.606, p = 1.5E-12), while miR-200a-3p and miR-939-5p were predominantly upregulated in HCC. Critically, miR-224-5p emerged as a robust non-invasive biomarker for cirrhosis, with exceptional diagnostic accuracy (AUC = 0.973 in discovery; 0.906 in external validation), significantly outperforming APRI (0.803) and FIB-4 (0.809), and remained the sole independent predictor in multivariable analysis (p = 0.009). For HCC detection, the miR-200a-3p/AFP combined model achieved outstanding performance (AUC > 0.9), substantially improving upon AFP alone (0.737). Bioinformatic prediction of targets (297 for miR-224-5p; 616 for miR-200a-3p) highlighted associated in cancer- and senescence-related pathways; however, these associations are in silico and require experimental validation. Conclusion: We identify miR-224-5p as a fibroinflammatory activity indicator for early cirrhosis detection and miR-200a-3p as a synergistic enhancer of AFP for non-invasive HCC diagnosis, establishing a dual miRNA signature that spans the HBV disease continuum and addresses critical gaps in current risk stratification. These findings highlight the potential of specific serum miRNAs as non-invasive biomarkers for monitoring disease progression and improving the differential diagnosis during the process of HBV-related liver diseases.