The immunological function of extracellular vesicles in hepatitis B virus-infected hepatocytes

Abstract

Hepatitis B virus (HBV) generates large amounts of complete and incomplete viral particles. Except for the virion, which acts as infectious particles, the function of those particles remains elusive. Extracellular vesicles (EVs) have been revealed to have biological functions. The EVs which size are less than 100 nm in diameter, were collected from HBV infected-patients. These vesicles contain, complete and incomplete virions, and exosomes, which have been recently shown to be critical as intercellular communicators. Here, the effects of the exosome, the complete, and the incomplete particles on the target cells were investigated. These particles are endocytosed by monocyte/macrophages and function primarily to upregulate PD-L1. The functions and composition of the EVs were affected by nucleotide reverse transcriptase inhibitors (NRTIs), suggesting that the EVs are involved in the pathogenesis of HBV hepatitis and clinical course of those patients treated by NRTIs.

Fig 1. Induction of immune suppression in monocytes by EVs.

(A) Extracellular vesicles (EVs) were collected by ultracentrifugation from 10 mL of culture supernatant of HepAD38 cells with or without hepatitis B virus (HBV) replication. The collected EVs were then labelled with PKH26 dye. Peripheral blood mononuclear cells (PBMCs) were then treated with 10 μg of PKH-labelled EVs for 24 hours. Uptake of EVs was evaluated by flow cytometry. (B) PBMCs treated with EVs were stained with PD-L1, PD-L2, and CD69 and analysed by flow cytometry.

Fig 2. Separation and immunosuppressive effects of exosomes, subviral particles, and virions.

(A) Extracellular vesicles (EVs) were collected by ultracentrifugation from 10 mL of culture supernatant of HepAD38 cells with or without hepatitis B virus (HBV) replication. Collected EVs were fractionated by a density gradient separation using iodixanol. Ten fractions were collected, and the presence of CD9, CD63, HBsAg, HBcAg, and HBV DNA in each fraction were analysed by western blotting and quantitative RT-PCR (representative of three experiments). (B) Peripheral blood mononuclear cells (PBMCs) were treated with exosomes, subviral particles, and virions for 24 hours. PBMCs treated with each particle were stained with PD-L1, PD-L2, and CD69 for analysis by flow cytometry.

Fig 3. The effects of NRTIs on the extracellular vesicles (EVs) secreted from HepAD38 cells with hepatitis B virus (HBV) replication.

(A) HepAD38 cells with HBV replication were treated with entecarvir (ETV), lamivudine (LMV), and tenofovir (TDF) for 9 days. Then, EVs were collected by ultracentrifugation from 10 mL of culture supernatant of HepAD38 cells treated with each NRTIs. PBMCs were treated with each EV for 24 hours and were stained with PD-L1, PD-L2, and CD69 for analysis by flow cytometry. (B–D) EVs collected from HepAD38 cells with HBV replication treated with NRTIs s were fractionated by density gradient separation using iodixanol. Ten fractions were collected, and the presence of CD9, CD63, HBsAg and HBcAg were analysed by western blotting (representative of three experiments).

Fig 4. Abundance ratio of exosome markers and viral proteins in extracellular vesicles (EVs) and intracellular space after treatment with NRTIs.

(A) HepAD38 cells with hepatitis B virus (HBV) replication were treated with entecarvir (ETV), lamivudine (LMV), and tenofovir (TDF) for 9 days. Then, EVs were collected by ultracentrifugation from 10 mL of culture supernatant of HepAD38 cells treated with each NRTIs. Collected EVs were lysed in radioimmunoprecipitation assay buffer, and protein concentration was measured using DC protein assay. Abundance ratio of CD9, CD63, HBsAg, and HBcAg in 5 mg protein from each EV lysate were examined by western blotting (representative of two experiments). (B) Abundance ratio of intracellular CD9, CD63, HBsAg, and HBcAg expression after NRTIs treatments were examined by western blotting (representative of two experiments), and α-tubulin was used as a loading control.

Fig 5. The level of pgRNA in the supernatant and intracellular space of HepAD38 cells with hepatitis B virus (HBV) replication after NRTIs treatment.

(A) Total RNA from HepAD38 cells with HBV replication after the NRTIs treatments was collected. Then, the level of pgRNA was measured by quantitative RT-PCR. The expression was normalised to that of GAPDH. (B) Viral RNA from the supernatant of HepAD38 cells with HBV replication after the NRTIs treatments was collected. Then, the level of pgRNA was measured by quantitative RT-PCR. All values are presented as the mean ± S.E.M. of three biological replicates (n = 3) (*p < 0,05).