Modulation of monocyte activity by hepatocellular MicroRNA delivery through HBsAg particles: Implications for pathobiology of chronic hepatitis B.

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.

Analysis of HBV cccDNA Minichromosome Accessibility by MNase-qPCR and High-Throughput Sequencing.

The covalently closed circular DNA (cccDNA) of the hepatitis B virus (HBV) is organized as a minichromosome structure in the nucleus of infected hepatocytes and considered the major obstacle to the discovery of a cure for HBV. Until now, no strategies directly targeting cccDNA have been advanced to clinical stages as much is unknown about the accessibility and activity regulation of the cccDNA minichromosome. We have described the method for evaluation of the cccDNA minichromosome accessibility using micrococcal nuclease-quantitative polymerase chain reaction and high-throughput sequencing, which could be useful tools for cccDNA research and HBV cure studies.

Optimized RNA interference therapeutics combined with interleukin-2 mRNA for treating hepatitis B virus infection.

This study aimed to develop a pan-genotypic and multifunctional small interfering RNA (siRNA) against hepatitis B virus (HBV) with an efficient delivery system for treating chronic hepatitis B (CHB), and explore combined RNA interference (RNAi) and immune modulatory modalities for better viral control. Twenty synthetic siRNAs targeting consensus motifs distributed across the whole HBV genome were designed and evaluated. The lipid nanoparticle (LNP) formulation was optimized by adopting HO-PEG2000-DMG lipid and modifying the molar ratio of traditional polyethylene glycol (PEG) lipid in LNP prescriptions. The efficacy and safety of this formulation in delivering siHBV (tLNP/siHBV) along with the mouse IL-2 (mIL-2) mRNA (tLNP/siHBVIL2) were evaluated in the rAAV-HBV1.3 mouse model. A siRNA combination (terms "siHBV") with a genotypic coverage of 98.55% was selected, chemically modified, and encapsulated within an optimized LNP (tLNP) of high efficacy and security to fabricate a therapeutic formulation for CHB. The results revealed that tLNP/siHBV significantly reduced the expression of viral antigens and DNA (up to 3log10 reduction; vs PBS) in dose- and time-dependent manners at single-dose or multi-dose frequencies, with satisfactory safety profiles. Further studies showed that tLNP/siHBVIL2 enables additive antigenic and immune control of the virus, via introducing potent HBsAg clearance through RNAi and triggering strong HBV-specific CD4+ and CD8+ T cell responses by expressed mIL-2 protein. By adopting tLNP as nucleic acid nanocarriers, the co-delivery of siHBV and mIL-2 mRNA enables synergistic antigenic and immune control of HBV, thus offering a promising translational therapeutic strategy for treating CHB.

An Eu (III)-functionalized covalent organic framework fluorescent probe for specific detection of Flumequine based on pore restriction and antenna effect.

The overuse of quinolone antibiotics has led to a series of health and environmental issues. Herein, we combine the distinct luminescence properties of Eu3+ with the unique structure of covalent organic frameworks (COFs) to develop a precise and sensitive fluorescent probe for detecting Flumequine (Flu) in water. Eu3+ is thoroughly anchored into the channels of COFs as recognition sites, while the synthesized probe material still maintains its intact framework structure. The unique structure of COFs provides excellent support and protection for Eu3+. Therefore, COF-Eu can rapidly bind with Flu which can transfer the absorbed energy to Eu3+ through an "antenna effect", resulting in red fluorescence. Moreover, there is a good linear relationship between Flu concentration in the range of 0-30 µM, with a detection limit of 41 nM. Simultaneously, the material maintains remarkable reproducibility, with its performance remaining almost unchanged after five cycles of use. Remarkably, the probe demonstrates excellent Flu recovery rates in real samples. This study provides a viable approach for the recognition of flumequine in the environment through a customized fluorescence detection method.