Female C57BL/6J mice (age, 4–6 weeks; weight range, 16–22 g) were purchased from the Animal Center of Chongqing Medical University (Chongqing, China). All animals were housed under specific pathogen-free conditions in which the ambient temperature (23±1°C) and humidity (~35–45%) were controlled with a 12-h light/dark cycle and food and water ad libitum and treated according to the guidelines of the animal facility at the Chongqing Medical University. All experiments were approved by Chongqing Medical University and were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals in China (27).

An HBV replication-competent plasmid encoding the 1.3-fold overlength HBV genome [pcDNA3.1-HBV 1.3 (ayw subtype)] was a kind gift from Professor Ni Tang (Key Laboratory of Molecular Biology for Infectious Diseases, Institute for Viral Hepatitis, Chongqing Medical University, Chongqing, China). Corresponding control pcDNA3.1 vector was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). All plasmids were reserved at −20°C.

A total of 55 female mice were randomly divided into 11 groups, including 0 (normal mice), 1, 3, 5, 7 or 15 days after pHBV plasmid injection and 1, 3, 5, 7 or 15 days after control plasmid injection. Mice were then hydrodynamically injected with 15 µg plasmid dissolved in 1.5 ml saline solution through their tail veins within 5 sec. There were 5 mice per group in each experiment.

Peripheral blood, spleen and liver samples were collected for analysis at different timepoints following plasmid transfection. Mice were anesthetized by exposure to ether presented on a cotton ball inside a conical tube. A conical tube containing diethyl ether-soaked cotton balls was placed near the nose of each mouse without contact. The mice were fully anesthetized several minutes later, but remained alive with their hearts beating and body temperature kept constant at 37°C. The mice were then fixed and placed in a supine position, the abdominal and thoracic cavities were subsequently opened and 0.5–0.8 ml blood samples were obtained from the heart, which were collected into a tube containing the anticoagulant EDTA. The portal vein was then perfused with 5 ml saline and the liver and spleen were collected in a plate filled with iced RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) for cell and lymphocyte isolation. At the completion of the procedure, all mice were sacrificed by cervical dislocation prior to awakening from anesthesia. At the time of sacrifice, the weights of the mice had decreased to 15–19 g due to blood and tissue collection. No fixatives were applied during any of the aforementioned procedures.

On days 0, 1, 3, 5, 7 and 15 following transfection, levels of HBsAg and HBeAg in the serum were measured using cobas^® HBsAg detection kit and cobas^® HBeAg detection kit by electrochemiluminescence immunoassay (Roche Diagnostics) according to the manufacturer's protocols, with the results represented as cut-off index (COI) values. To avoid plasmid contamination, mouse serum was treated with 20 U DNAase I for ≥12 h, following which HBV DNA was extracted using a Viral DNA extraction kit (Da An Gene Co., Ltd., China), according to the manufacturer's protocol, and detected by reverse transcription-quantitative PCR (RT-qPCR) using a Roche Thermocycler (Roche Diagnostics) according to the manufacturer's protocol.

Within the same timeframe, alanine aminotransferase (ALT) levels were also measured in serum collected from the mice using a Hitachi 7600 Automatic Biochemical Analyzer (Hitachi, Ltd.).

Liver histology was determined using hematoxylin-eosin (H&E) staining. Liver tissues (6 µM sections) from pHBV-transfected mice on days 0, 1, 3, 5, 7 and 15 were fixed in 10% neutral formalin for 24 h at room temperature (RT), dehydrated using an ethanol gradient (70, 80, 90 and 100%) and embedded in paraffin. The slides were subsequently stained using H&E for 10 min at RT for histological examination.

IHC staining procedures were conducted according to the manufacturer's protocols. The main steps were as follows: Liver specimens (6 µM sections) from pHBV-transfected mice on days 0, 1 and 5 were paraffin-embedded. Following de-paraffinization, rehydration using an ethanol gradient (95 and 80%) and antigen retrieval in a 0.1% trypsin solution at 37°C for 20 min, endogenous peroxidase was quenched using 3% H₂O₂ and unspecific binding was blocked using 2.5% goat serum (Cell Signaling Technology, Inc.) for 20 min at RT. Mouse anti-HBsAg primary monoclonal antibody (1:100, dilution; cat. no. ZM-0122) or mouse anti-HBcAg primary antibody (1:100, dilution; cat. no. ZM-0421; both ZSGB-BIO) was subsequently added, followed by incubation overnight at 4°C. The samples were then incubated with horseradish peroxidase-conjugated goat anti-mouse polymer (Elivision™ plus Polyer HRP (Mouse/Rabbit) IHC Kit; cat. no. KIT-9902) for 30 min at room temperature (Fuzhou Maixin Biotech Co., Ltd., China), followed by treatment with 3,3′-diaminobenzidine (DAB; Elivision Super; Fuzhou Maixin Biotech Co., Ltd., China). Yellow or brown dye in hepatocytes indicated positive staining. The percentage of positively stained cells were determined by counting in five random high-power fields using an Olympus optical light microscope (Olympus Corporation), where there were ≥100 cells in each field (magnification, ×400).

Liver lymphocytes were isolated as previously reported (28). Briefly, mice were anaesthetized with diethyl ether and the portal vein was perfused with 5 ml saline until the liver became pale in color. The liver was then cut into small pieces, and incubated in RPMI 1640 solution (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 0.05% collagenase IV (cat. no. C5138; Sigma-Aldrich; Merck KGaA) and 0.01% DNAase I (cat. no. D5025; Sigma-Aldrich; Merck KGaA) at 37°C for 30 min, after which the pieces were pressed through a 200-gauge stainless steel mesh. Following centrifugation at 50 × g (4°C, 10 min, the precipitate was discarded) and again at 500 × g (4°C, 10 min, the supernatant was discarded), the remaining cell pellet was resuspended in 3 ml RPMI-1640 solution (Gibco; Thermo Fisher Scientific, Inc.) and overlaid onto a 33% Percoll solution (cat. no. 17-0891-01; Pharmacia Biotech; GE Healthcare, USA), followed by centrifugation at 800 × g for 30 min at room temperature. The supernatant was then aspirated, and the red blood cells (RBC) were lysed using a 0.75% NH₄Cl solution. After subsequent washing with PBS, the liver lymphocytes were prepared for immediate FACS analysis.

Mouse spleen tissues were smashed and dissociated thoroughly using two glass slides with rough surfaces smeared beforehand with a RBC lysis buffer (0.75% NH₄Cl solution). After RBC lysis for 10 min, the cell suspension was filtered through a 70 µM filter (BD Biosciences) to obtain a single-cell suspension. The suspension was then washed with PBS and the lymphocyte-enriched spleen cells were prepared for later use.

The following fluorochrome-conjugated mAbs were used according to the manufacturer's protocol: Purified anti-mouse CD16/CD32 (1:100 dilution; cat. no. 14-0161-81; eBioscience; Thermo Fisher Scientific, Inc.); peridinin-chlorophyll-protein complex-conjugated hamster anti-mouse CD3e (1:50 dilution; cat. no. 553067; BD Biosciences); phycoerythrin (PE)-conjugated hamster anti-mouse γδ TCR (1:50 dilution; cat. no. 553178; BD Biosciences); PE-Cy™7-conjugated anti-mouse CD69 (1:50 dilution; cat. no. 25-0691-81; eBioscience; Thermo Fisher Scientific, Inc.); allophycocyanin (APC)-conjugated rat anti-mouse CD25 (1:50 dilution; cat. no. 558643; BD Biosciences); fluorescein isothiocyanate (FITC)-conjugated hamster anti-mouse γδ TCR (1:100 dilution; cat. no. 553177; BD Biosciences); PE-conjugated anti-mouse IFN-γ (1:100 dilution; cat. no. 12-7311-81; eBioscience; Thermo Fisher Scientific, Inc.); PE-Cy™7-conjugated rat anti-mouse TNF-α (1:100 dilution; cat. no. 557644; BD Biosciences); PE-Cy™7 anti-mouse NK1.1 (1:50 dilution; cat. no. 552878; BD Biosciences); FITC-conjugated anti-mouse CD4 (1:100 dilution; cat. no. 553047; BD Biosciences); and APC-Cy™7-conjugated anti-mouse CD8 (1:50 dilution; cat. no. 557654; BD Biosciences).

For surface staining, liver lymphocytes (~5×10⁵), splenic cells (~5×10⁵) or 100 µl of fresh peripheral anticoagulated blood samples were used for staining. Cells were blocked using 0.5 µg anti-CD16/32 antibody for 10 min at 4°C, after which an appropriate volume of each specific antibody was added, and the samples were incubated for 30 min in the dark at 4°C. For whole-blood staining, erythrocytes were lysed using BD™ FACS™ lysing solution (BD Biosciences) and cells were washed using PBS supplemented with 1% fetal calf serum (FCS; Gibco; Thermo Fisher Scientific, Inc.).

Intracellular cytokine staining was performed as follows: Liver lymphocytes were adjusted to ~5×10⁶ cells/ml in RPMI 1640 culture medium supplemented with 10% FCS and stimulated with 100 ng/ml phorbol myristate acetate plus 1 µg/ml ionomycin at 37°C for 4 h in the presence of the secretion inhibitor monensin (0.16 µg/ml; BD Biosciences) (29). Cells were blocked using 0.5 µg anti-CD16/32 antibody for 10 min at 4°C and then stained with anti-TCR γδ mAb for 30 min at 4°C, followed by washing with PBS and fixing in 4% paraformaldehyde. Stained cells were permeabilized using 0.1% saponin (Sigma-Aldrich; Merck KGaA) and incubated with anti-IFN-γ and anti-TNF-α for 30 min at 4°C.

Stained cells were immediately analyzed using the FACSCanto™ II flow cytometer (BD Immunocytometry Systems; BD Biosciences). Data were analyzed using FACSDiva™ 2.0 software (BD Immunocytometry Systems; BD Biosciences). Cell gating strategies were as follows: The population of cells double positive for γδ TCR and CD3 was defined as the γδ T cell subtype, which was subsequently subdivided into several subsets, including CD25⁺, CD69⁺, IFN-γ⁺ or TNF-α⁺ γδ T cells, according to their positivity in the FACS dot plots. CD3 and NK1.1 were used to measure the presence of mouse NK and NKT cells; CD3- NK1.1+ cells were defined as NK cells (left upper quadrant) and CD3+ NK1.1+ cells were defined as NKT cells (right upper quadrant) (30).

Liver tissue (~40 mg) in 1 ml TRIzol^® solution (Invitrogen; Thermo Fisher Scientific, Inc.) was homogenized using a power homogenizer and the total RNA was extracted according to the manufacturer's protocols. RNA quality was evaluated by electrophoresis and spectral analysis. Only RNA without degradation or contamination with DNA or protein was used for subsequent RT-qPCR analyses.

RNA (~1 µg) was reverse transcribed by 2 min at 70°C, 15 min at 37°C, and then 1 min at 95°C with oligo (dT) primers using the PrimeScript™ RT Reagent kit with gDNA eraser, according to the manufacturer's protocol (Takara Bio, Inc.). qPCR was then performed using SYBR^® Green quantitative PCR dye with SYBR^® Premix Ex Taq™ II, according to the manufacturer's protocol (Takara Bio, Inc.). All samples were detected in triplicate using an Applied Biosystems 7300 Real-Time PCR Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. GAPDH was used as an internal control and the data were analyzed using the 2^−ΔΔCq method (31). Primer sequences for IFN-α, IFN-β, IFN-γ, TNF-α and GAPDH used for RT-qPCR are listed in Table I.

SPSS software (version 15.0; SPSS Inc.) was used to analyze all data. Experimental data are expressed as the mean ± SD, from five experimental repeats. Following one-way ANOVA, differences between every two groups were calculated using the Least Significant Difference method. For comparison of RNA expression, significant changes were assessed as at least a 2-fold increase or a 0.5-fold decrease. Pearson correlation analysis was performed to evaluate the correlation in the percentage of γδT cells with the relative fold changes in HBsAg or HBcAg RNA expression. P<0.05 was considered to indicate a statistically significant difference.

To verify that the mouse model of acute HBV infection was constructed successfully in the present study, serum HBsAg, HBeAg and HBV DNA levels and the intrahepatic expression of HBsAg and HBcAg were measured.

On day 1 after hydrodynamic-based pHBV plasmid injection, serum tested positive for HBsAg, HBeAg, and HBV DNA (Fig. 1A). HBV markers were undetectable in mice after control plasmid transfection (data not shown). After pHBV plasmid injection, HBsAg serum levels increased from 113.9±31.1 (COI) on day 1 to a peak value of 255.3±47.6 (COI) on day 3, followed by a decrease to 5.3±1.5 (COI) on day 15. Serum HBeAg levels were the highest on day 1 at 31.1±6.9 (COI) and then decreased gradually to 1.5±0.7 (COI) on day 15 (Fig. 1A). Additionally, the serum HBV DNA load declined from an average of 3.2×10⁴ copies/ml on day 1 to virtually undetectable levels on days 7 and 15 (Fig. 1A). In terms of liver function, serum ALT levels in pHBV-transfected groups increased on day 1 and then decreased back to normal levels (<30 U/l).

Liver histopathology was evaluated on days 0, 1, 3, 5, 7 and 15 after injection (Fig. 1B). An apparent accumulation of mononuclear cells was observed in the mouse livers on day 3. On day 5, the liver structure changed with the appearance of some necrotic lesions and hepatocyte degeneration. By day 7, the liver tissue recovered back to a normal architecture, which was also maintained on day 15 (Fig. 1B).

HBsAg and HBcAg expression was subsequently examined by immunohistochemistry with DAB staining in the liver specimens isolated from pHBV-transfected mice (Fig. 1C). The average percentages of positively stained cells were determined by counting in five random high-power fields. On day 1, an average of 13.8 or 11.2% hepatocytes were staining positive for HBsAg or HBcAg, respectively. HBsAg was mainly expressed in the cytoplasm, whilst HBcAg expression was observed in both the cytoplasm and the nucleus. However, HBsAg and HBcAg expression decreased sharply on day 5 post-injection, with only 0.6 and 1.2% of hepatocytes on average displaying positive expression of HBsAg and HBcAg, respectively (Fig. 1C).

Aside from HBsAg⁺ or HBcAg⁺ hepatocytes, HBsAg and HBcAg mRNA expression were also measured in the liver tissues using RT-qPCR, where the results also showed that HBV expression was increased at day 1 (Fig. S1).

To investigate changes in γδ T cell numbers, the percentages of γδ T cells were first measured in the liver, spleen and peripheral blood samples of mice using FACS analysis, to dynamically monitor any changes following HBV plasmid injection (Fig. 2). Representative FACS dot plots for γδ T cells are shown in Fig. 2A. On day 0, the percentage of γδ T cells in the liver was 4.5±0.6% (percentage of total T cells), which was significantly higher compared with that observed in the spleen (2.1±0.3%; P<0.05) or the peripheral blood (1.2±0.6%; P<0.05). On day 1 post-injection, the percentage of liver γδ T cells increased to 8.3±2.9%, which was significantly higher compared with that on day 0 (4.5±0.6%; P<0.05) and that found in the liver samples of mice transfected with the control plasmid on day 1 (pcDNA; 3.8±0.7%; Fig. 2B). The percentage of liver γδ T cells in the pHBV-infected group gradually decreased to a level similar to that observed on day 0, while there were no significant changes in the percentage of liver γδ T cells after control plasmid transfection. Additionally, the percentages of spleen and blood γδ T cells from either the pHBV or pcDNA control groups displayed no significant changes compared with those on day 0 (P>0.05; Fig. 2C and D). In addition, the percentage of liver γδ T cells increased dramatically on day 1, which was in accordance with the highest percentage of HBsAg⁺ or HBcAg⁺ hepatocytes and the increased mRNA expression of HBsAg and HBcAg in the mouse livers (Fig. S1)

Following the observation that there was an increased percentage of total liver γδ T cells following pHBV transfection, the activation and function of these liver γδ T cells were investigated further by assessing the expression of CD25 and CD69, surface markers for activation (32) and intracellular cytokines IFN-γ and TNF-α (Fig. 3A). The percentages of CD25⁺ and CD69⁺ liver γδ T cells on day 0 were found to be 2.5±0.5 and 6.5±0.9% (of total γδ T cells), respectively (Fig. 3B and C). The percentage of CD69⁺ liver γδ T cells in the pHBV-transfected group increased to 18.9±4.7% on day 1 post-injection, which was significantly higher compared with that in the day 0 group (6.5±0.9%) or the pcDNA control group (7.7±1.8%) on day 1. CD69 expression declined to 8.7±1.6% on day 3 and settled to 9.6±1.5% on day 7. The percentage of CD25⁺ liver γδ T cells was found to be gradually elevated in the pHBV-transfected group, with an increase from 3.9±1.8% on day 1 to 7.1±2.1% on day 5, followed by a drop to 1.9±0.4% on day 7. The percentage of CD25⁺ γδ T cells on day 5 in pHBV-transfected group was significantly different when compared with the percentages on day 0 (2.5±0.5%) and in the pcDNA control on day 5 (3.2±0.9%; P<0.05). For the pcDNA control group, no significant differences were found in the percentages of either CD25⁺ or CD69⁺ γδ T cells across the different time points tested (P>0.05).

As shown in Fig. 3D and E, the percentages of IFN-γ- and TNF-α-producing liver γδ T cells were 5.1±0.4% and 4.7±0.6%, respectively, on day 0. After pHBV plasmid injection, the percentage of IFN-γ⁺ γδ T cells increased on day 1 (8.6±3.7%), but this difference was not significant when compared to the day 0 group (5.1±0.4%) or the pcDNA control group on day 1 (6.1±1.9%; P>0.05). The percentage then dropped to 5.4±0.5% on day 7. As for TNF-α-producing γδ T cells in the pHBV-transfected group, no significant changes were observed across all time points (P>0.05). There were also no significant differences in the percentages of IFN-γ⁺ or TNF-α⁺ γδ T cells compared with the pcDNA control group among all these time points (P>0.05).

Since it was discovered that the number of liver γδ T cells from mice with acute HBV infection was increased, the possibility that other innate immune responses were activated in the liver was next explored. Early cytokine production is the most important activity associated with the antiviral innate immune response (33). Therefore, the mRNA expression of cytokine markers associated with the activation of the innate immune response, IFN-α and IFN-β, in addition to IFN-γ and TNF-α, were measured in liver tissues on days 0, 1, 3, 5 and 7 following injection with pHBV or the control plasmid. After pHBV injection, IFN-β mRNA expression was significantly upregulated, by an average of 6.8-fold on day 1 and by 2.5-fold on day 3 compared with that on day 0. It was also significantly higher compared with that in mice injected with the control pcDNA plasmid on days 1 (8.4-fold) and 3 (2.8-fold). IFN-β expression subsequently decreased to normal levels on days 5 and 7. No significant changes in IFN-β expression were observed in the pcDNA control group. TNF-α expression in tissues from the pHBV-transfected group was increased slightly on day 1 (1.9-fold) compared with day 0, but no significant difference was observed in IFN-α or IFN-γ mRNA expression between pHBV-transfected or control groups across all time points examined (Fig. 4).

FACS dot plots of liver CD4⁺ T, CD8⁺ T, NK and NK T cells are shown in Fig. 5A. The percentage of liver NK cells from the pHBV-transfected group on day 1 was significantly higher compared with that in the control group (Fig. 5D). No significant differences were found in the percentages of CD4⁺ T, CD8⁺ T or NK T cells between the pHBV-transfected and control groups (Fig. 5B, C and E). Notably, the percentage of liver NK T cells decreased on day 3 compared with day 0 after plasmid injection in either the pHBV or pcDNA control groups, but the observed differences were not significant (Fig. 5E).