Age-related immune clearance of hepatitis B virus infection requires the establishment of gut microbiota

Significance

Ninety-five percent of adult-acquired infections lead to spontaneous clearance, whereas >90% of exposed neonates and ∼30% of children aged 1–5 y fail to resolve hepatitis B virus (HBV) and develop chronic infection. A strong, diverse, adaptive immune response is considered essential for HBV clearance, but the mechanisms to generate a favorable response remain elusive. Here, we show that, while 12-wk-old C3H/HeN mice cleared HBV within 6 wk postinjection (wpi), their 6-wk-old counterparts remained HBV-positive at 26 wpi. Sterilization of gut microbiota from 6 to 12 wk of age using antibiotics prevented mice from rapidly clearing HBV. This model is valuable to the study of liver tolerance and enables the investigation of the mechanisms involved in effective control of HBV.

Abstract

A unique feature of hepatitis B virus (HBV) infection in humans is that viral clearance heavily depends on the age of exposure. However, the reason for this remains unclear. Here we show that gut microbiota contribute to the age dependence of HBV immunity in a hydrodynamic transfection mouse model. Although adult (12-wk-old) C3H/HeN mice cleared HBV within 6 wk postinjection (wpi), their young (6-wk-old) counterparts remained HBV-positive at 26 wpi. Sterilization of gut microbiota from 6 to 12 wk of age using antibiotics prevented adult mice from rapidly clearing HBV. Young mice with the Toll-like-receptor (TLR) 4 mutation (C3H/HeJ) exhibited rapid HBV clearance. The results suggest that an immuno-tolerating pathway to HBV prevailed in young mice, before the establishment of gut bacteria, through a TLR4-dependent pathway and that the maturation of gut microbiota in adult mice stimulated liver immunity, resulting in rapid HBV clearance.

The hepatitis B virus (HBV) is one of the most common infectious agents worldwide. According to the World Health Organization, more than one-third of the world’s population (2 billion people) have been infected with HBV, and 240 million people are chronic carriers (1). These chronic HBV carriers have a higher probability of developing hepatic cirrhosis and hepatocellular carcinoma than the general population and constitute a substantial burden on health care systems (2).

The chronicity of HBV infection has been attributed to the impairment of HBV-specific immune responses that fail to eliminate or cure infected hepatocytes; however, the mechanism behind this impairment remains unclear. Numerous studies have suggested that the genetic constitution of the host is a critical factor in determining the outcomes of HBV infection (3, 4). Genetic variations in genes that code for cytokines, such as IFN-γ and tumor necrosis factor-α (TNF-α) (5), cytotoxic T-lymphocyte antigen 4 (6), and HLA (7, 8), have been suggested to influence the chronicity or clearance rates of HBV infection. Recent genome-wide association studies have demonstrated that certain SNPs near HLA-DP loci are associated with persistent HBV infection (9, 10).

Although the genetics of the host are critical, the age of HBV exposure is a more important nongenetic factor that determines the incidence of becoming chronic HBV infection. Ninety-five percent of adult-acquired infections lead to spontaneous clearance, whereas more than 90% of exposed neonates and ∼30% of children aged 1–5 y fail to resolve HBV and develop chronic infection (11, 12). It is postulated that “liver tolerance” and “immune immaturity” to HBV result in high viral persistence in the early stage of life (13, 14), but that the maturation of the liver immunity environment in late age empowers HBV clearance. However, this maturation process has not been clarified.

Mounting evidence has revealed that commensal bacteria residing in the gastrointestinal (GI) tract regulate a host’s immune response. Although the liver is not in direct contact with live commensals, constant exposure to microbe-derived ligands and metabolites through the gut–liver axis likely shapes liver immunity (15, 16). Liver sinusoid endothelial cells (LSECs) respond to Gram-negative bacteria-derived lipopolysaccharides (LPSs) through Toll-like receptor (TLR) 4 by the secretion of interleukin-10 (IL-10), which induces T-cell tolerance (17, 18). Kupffer cells (KCs) are the largest population of tissue-resident macrophages, and their activation through the LPS and TLR4 pathway induces the secretion of immunosuppressive mediators, i.e., IL-10 (19) and transforming growth factor-β (TGF-β) (20). Nevertheless, commensal signaling can also promote liver immunity activation. Mouse KCs pretreated with bacterial CpG-DNA exhibited significantly increased IL-6 production in response to subsequent LPS challenge in vitro (21). Using a mouse model, signaling through the CpG-DNA/TLR9 pathway enabled hepatic expansion of HBV-specific cytotoxic CD8+ T cells which, in turn, eradicated HBV-infected hepatocytes during chronic infection, leading to viral clearance (22). During microbial infection, KCs and LSECs were observed to override the tolerance phenotype and promote CD4+ T-cell proliferation in the presence of TLR ligands, such as LPS and CpG-DNA (23). The interaction between commensals and the liver significantly influences hepatic physiology and determines the outcomes of pathophysiologic responses to challenges. The age dependence of HBV clearance thus results from the development of gut microbiota, which begin to colonize the GI tract after birth.

Investigating the basis of immune activation during HBV infection is experimentally challenging because of the intrinsic difficulty of acquiring samples at the appropriate stages. Therefore, to understand the mechanisms underpinning HBV clearance or persistence, we used a nontransgenic hydrodynamic transfection mouse model. This model is able to mimic multiple features of HBV infection observed in humans, including age-dependent chronicity, genetic variations influencing the outcomes of HBV infection, and the possible interactions between them. A previous study showed that after transfection HBV plasmids lasted for more than 6 mo in C57BL/6 mice, but were rapidly cleared in BALB/c mice within 4 wk and elicited successful immunity (24), demonstrating that HBV clearance is genetically dependent.

In this study, multiple inbred mouse strains were transfected with HBV and studied. We observed different rates of HBV clearance and persistence among the strains, indicating that HBV immunity is genetically dependent. We also showed the age-dependent HBV clearance in C3H/HeN and DBA/2J mice, which mimicked natural history of HBV in humans. As the age-dependent HBV clearance developed synchronously with the postnatal establishment of gut microbiota and portal blood flow, we hypothesized that gut commensals prepared the liver immunity system to clear HBV. After sterilizing the gut bacteria of C3H/HeN mice using antibiotics, adult mice were more tolerant to HBV exposure, similar to their young counterparts. The age-dependent HBV clearance, however, disappeared in C3H/HeJ mice, a TLR4 mutant strain in which LPS-induced TLR4 signaling was abolished. These results suggested that gut microbiota contribute to the age dependence of HBV immunity because they regulate the innate immunity pathway in the liver.

Results

Strain and Age-Dependent Clearance of HBsAg After Hydrodynamic Injection.

We monitored serum HBsAg levels in young (6-wk-old) mice of various inbred strains after hydrodynamic injection (HDI). A signal/noise (S/N) ratio of 10 was set as a cutoff for HBsAg positivity (24). BALB/cJ, FVB/NJ, NOD/ShiLtJ, and 129 × 1/SvJ mice rapidly cleared serum HBsAg within 4 wk postinjection (wpi). However, certain mice in strains C57BL/6J (40%), C3H/HeN (90%), and DBA/2J (75%) failed to clear HBsAg within 8 wpi, and all CBA/caJ mice remained HBsAg-positive at 8 wpi (Fig. 1A). Therefore, genetic components at least partially contribute to HBV exposure outcomes. Chronic hepatitis B infection in humans is defined as an infection lasting longer than 6 mo. After 6 mo, the HBsAg-positive rate was 15% among the C57BL/6J mice, 25% among the DBA/2J mice, and 80% among the C3H/HeN and CBA/caJ mice. Although both the BALB/cJ and DBA/2J mice shared the MHC haplotype H2-d, they responded differently to HBV. Similarly, the immune responses of the 129 × 1/SvJ and C57BL/6J mice, which shared the MHC haplotype H2-b, differed considerably. These observations suggest that MHC haplotypes alone did not determine outcomes of HBV transfection.

Fig. 1.

The outcomes of HBV transfection by hydrodynamic injection in mice were context-dependent. Six-wk-old mice were hydrodynamically injected with 10 μg of pAAV/HBV1.2 plasmid. Serum HBsAg titers were monitored weekly, using an AXSYM system kit (Abbott Diagnostics). Positive rates of serum HBsAg (S/N > 10) were observed among eight inbred strains (A) and compared between young (6-wk-old) and adult (12-wk-old) C3H/HeN (B), DBA2/J (C), and C57BL/6J (D) mice. The sample sizes (to the left of the slash) and MHC haplotypes (to the right of the slash) are presented in parentheses.

To test the effect of age on HBV exposure outcomes, adult (12-wk-old) CBA/caJ, DBA/2J, C3H/HeN, and C57BL/6J mice were administered HDI, and their serum HBsAg titers were monitored. In contrast to young mice, the adult C3H/HeN mice cleared serum HBsAg at 5 wpi (Fig. 1B). The accelerated HBsAg clearance in adult mice was also observed among the DBA/2J (Fig. 1C) and C57BL/6J (Fig. 1D) mice. The substantial differences in the young vis-à-vis adult mice implied that outcomes of HDI in the C3H/HeN, DBA/2J, and C57BL/6J mice were strongly influenced by developmental stages analogous to HBV infection in humans. Moreover, these results suggested that, among these mouse strains, liver immunity system maturity might play a critical role in HBV clearance after HDI.

Profiles of Stool Bacteria With and Without Antibiotic Treatment.

Previous studies have suggested the existence of a strong interaction between commensal gut bacteria and host immunity (25, 26). Microbiota are introduced into the gut from the environment after delivery, and a stable gut microbiota profile is gradually established. The timing of this establishment coincides with improved HBV clearance. We thus examined whether intestinal microbiota were associated with the outcomes of HBV transfection in C3H/HeN mice because adult and young mice in this strain exhibited the most considerable difference after HDI. C3H/HeN mice between 5 and 12 wk of age were gut-sterilized using a well-established antibiotic (ABX) mixture protocol (27, 28). The body weights of the ABX mice dropped by ∼20% in the first week of antibiotic treatment and subsequently recovered (Fig. 2A). This reduction in body weight was attributed to decreased water intake (Fig. S1); water intake among the mice was 20% of normal levels 3 d after the start of antibiotic treatment and returned to normal levels after 10 d. The amount of gut bacteria, assessed according to the level of 16S ribosomal DNA in stools, was relatively constant among the adult mice, whereas a roughly 100-fold reduction was observed in the ABX mice 1 wk after treatment; this reduction lasted throughout the entire exposure period (Fig. 2B). ABX mice also showed an enlargement of cecum commonly observed in germ-free mice (Fig. S2). After the removal of antibiotics, the amount of stool bacteria in the ABX mice returned to levels similar to those of the adult mice after 4 d [2 d postinjection (dpi)].

Fig. 2.

Intestinal microbiota of the C3H/HeN mice reached equilibrium between 6 and 11 wk of age. The C3H/HeN mice were divided into two groups, one of which was treated with antibiotics (ABX; n = 17; ampicillin, neomycin, metronidazole, and vancomycin) and one of which was not (adult; n = 10). ABX mice received antibiotics in their drinking water between 5 and 12 wk of age, and their body weights (A) were recorded. (B) The amount of bacteria 16S rRNA was reduced 100-fold after antibiotic treatment. (C) The profiles of intestinal microbiota, examined using TTGE, varied considerably between 6 and 9 wk of age in an untreated adult mouse and thereafter reached equilibrium. (D) The ABX mouse showed an extremely low amount of bacteria, which did not vary in the course of treatment. M: 100-bp ladder marker; dpi: days postinjection; wpi: weeks postinjection.

Temporal-temperature gel electrophoresis (TTGE) was used to compare the gut microbiota profiles of undisturbed and antibiotic-treated mice, based on the GC contents of various microbes. The TTGE profiles of an untreated mouse varied considerably between 6 and 8 wk of age and stabilized after 9 wk of age (Fig. 2C), suggesting that gut microbiota were established between birth and 11 wk of age and thereafter reached equilibrium. Similar patterns of microbiota establishment were observed in another three untreated mice (Fig. S2). In the ABX mice, gut microbiota were nearly undetectable during treatment and appeared 4 d after antibiotic treatment was stopped (2 dpi); however, 4 wk after treatment was stopped, bacterial diversity in the ABX mice did not return to the levels of untreated adult mice (Fig. 2D and Fig. S2).

Effects of Antibiotics on Serum and Intrahepatic HBV Clearance.

Adult, ABX, and young C3H/HeN mice (Fig. 3A) were hydrodynamically injected with 10 μg of pAAV/HBV1.2 plasmid to monitor their responses against HBV. Serum HBsAg levels (Fig. 3B) and viral titers (Fig. 3C) were similar between the groups at 1 wpi, demonstrating that the initial HBV transfection efficiency was similar. In the adult mice, serum HBV DNA and HBsAg levels quickly decreased. Levels of HBV DNA decreased below detection levels (10³ copies/mL) at 4 wpi, and levels of HBsAg decreased below the cutoff (S/N ratio = 10) at 6 wpi. However, 60% of the ABX and 90% of the young mice remained HBsAg-positive at 6 wpi (Fig. 3D). After 6 mo (26 wk), 53% (9 of 17) of the ABX and 80% (8 of 10) of the young mice remained HBsAg-positive. Repeat ABX experiments showed similar results in serum HBsAg levels and persistence rate (Fig. S3).

Fig. 3.

Intestinal microbiota promoted HBV clearance in the C3H/HeN mice. (A) Twelve-week-old mice either did (ABX; n = 17) or did not receive antibiotics (adult; n = 10), and 6-wk-old mice (Young; n = 12) were hydrodynamically injected with 10 μg of pAAV/HBV1.2 plasmid. (B) Serum HBsAg titers were monitored weekly, using an AXSYM system kit (Abbott Diagnostics). (C) Serum HBV viral titers were monitored at the indicated time points (detection limit = 10³ copies/mL). (D) Positive rates of serum HBsAg (S/N > 10) among three groups were shown. (E) Northern and Western blotting displayed the expression of HBV transcripts (3.5-kb pregenomic RNA and 2.1-/2.4-kb mRNA) and intracellular HBcAg and major HBsAg in the livers of the three groups on day 3 (3d) and weeks 19 and 47 post-HDI among serum-HBsAg (SAg)–positive (+) and –negative (−) mice. (F) HBcAg-specific IFNγ-producing cells in 1 × 10⁶ splenocytes from the adult, HBsAg-positive and HBsAg-negative ABX mice, ABX (SAg+) and ABX (SAg−), and young (SAg+) mice (n = 3) in week 47 post-HDI were assayed using IFNγ ELISPOT in the presence of 0.3 μg/mL of rHBcAg. Regardless the status of serum HBsAg, the number of IFNγ-producing cells in ABX and young mice was not significant differently from negative control. **P < 0.01.

To further characterize intrahepatic viral transcriptions and protein expressions in different mouse groups, liver samples were collected at different time points and assayed using Northern and Western blotting. HBV transcripts, including 3.5-kb pregenomic and 2.4/2.1-kb surface mRNAs, were detected in the livers of mice from all three groups at 3 dpi (Fig. 3E). In addition, the intrahepatic expression of HBcAg and HBsAg were also confirmed. The transcripts and protein expressions became undetectable after the clearance of serum HBsAg in all individuals examined. For mice in ABX and young groups, which failed to clear serum HBsAg at 47 wpi, intrahepatic HBV transcripts as well as HBcAg and HBsAg were still positive. Serum HBsAg is strongly correlated with intrahepatic viral transcription and protein expression. The results clearly showed that gut sterilization reduced the immune capacity of C3H/HeN mice to clear HBV.

Impaired Adaptive Immunity Against HBV in Antibiotic-Treated Mice.

The production of hepatitis B surface antibodies (anti-HBs) was detected in all adult mice 1 or 2 wk after the clearance of serum HBsAg. However, at 26 wpi, only six of nine HBsAg-negative ABX mice produced anti-HBs, and anti-HBs were not detected in HBsAg-negative young mice (Table 1). Antibiotic treatment thus not only affected serum HBsAg clearance, but also impaired antibody production. To further investigate the immune response against HBV, we examined the frequency of HBcAg-specific IFNγ-producing T cells in the splenocytes using an IFNγ enzyme-linked immunospot (ELISPOT) assay because the response of T cells to HBcAg stimulation is correlated with the resolution of HBV infections. Adult mice exhibited a significantly higher number of HBcAg-specific IFN-γ–secreting cells (418 cells per 10⁶ splenocytes) than the other two groups (Fig. 3F); this strong response against HBcAg was maintained through 47 wpi. Regardless of their HBsAg status, the ABX and young mice induced IFN-γ–secreting cells at levels similar to those of the negative control. These results suggested that less effective adaptive humoral and cellular immune responses among the ABX mice increased HBV persistence.

Table 1.

Percentages of HBsAg clearance (to the left of the slash) and anti-HB production (to the right of the slash) among the C3H/HeN and C3H/HeJ mice

SAb+/clear SAg	Young, %	ABX, %	Adult, %
C3H/HeN (26 wk)	0/25	35/53	100/100
C3H/HeJ (8 wk)	100/100		100/100

Parentheses are weeks post-HDI.

Loss of Age-Related HBV Clearance in the C3H/HeJ Strain with TLR4 Mutation.

The depletion of gut bacteria resulted in delayed HBsAg clearance. Emerging evidence has indicated that the activation of TLR4 by LPS from intestinal Gram-negative bacteria is associated with numerous aspects of liver immunity. We tested the outcomes of HBV transfection on C3H/HeJ mice, a TLR4 mutant strain in which LPS-induced TLR4 signaling was abolished. Levels of HBsAg rapidly decreased in both young and adult C3H/HeJ mice (Fig. 4A), and both exhibited accelerated HBsAg clearance within 8 wpi (Fig. 4B). In addition, regardless of age, all of the C3H/HeJ mice produced anti-HBs within 2 wk of HBsAg clearance (Table 1).

Fig. 4.

TLR4 contributed to HBV clearance in the young C3H mice. Both the young (6 wk old; n = 11) and adult (12-wk-old; n = 7) mice with the TLR4 mutation (C3H/HeJ) exhibited rapid HBsAg decrease (A) and clearance (B) after the hydrodynamic injection of pAAV/1.2HBV plasmid.

Discussion

Based on our results, we propose a model of age-related HBV infection, in which the establishment of gut microbiota significantly influences hepatic immune responses that result in viral clearance or persistence. Between 6 and 9 wk of age, the gut microbiota of the C3H/HeN mice matured toward equilibrium; this process appears to play a crucial role in the development of liver immunity. The young C3H/HeN mice transfected with HBV, which had not reached gut microbiota equilibrium, exhibited tolerance phenotypes, including prolonged HBsAg persistence, impaired anti-HBs production, and limited HBcAg-specific IFN-γ–secreting splenocytes. In adult mice, the maturation of gut bacteria might transmit signals to the liver to break liver tolerance, resulting in rapid HBV clearance. The depletion of gut bacteria in the adult C3H/HeN mice (ABX) restored the tolerance phenotypes as if they were young mice.

Liver tolerance elicited by hepatic antigen-presenting cells facilitates the secretion of IL-10 after exposure to low levels of bacterial debris (e.g., LPS). Under steady-state conditions, the local immune state within the liver might be biased toward tolerance because of LPS/TLR4-mediated IL-10 secretion. Nevertheless, liver tolerance can be overridden in the presence of bacterial CpG-DNA, a TLR9 ligand. Liver immunity stimulation or inhibition might depend on the type and strength of the signals to which hepatic cells have been exposed, and it is likely that gut microbiota acts on the liver via both immuno-stimulatory and immuno-tolerating pathways to influence HBV responses.

When the gut microbiota was developing in the young mice, the immuno-tolerating pathway was observed to prevail through TLR4-dependent innate immunity. This idea coincides with a recent study that demonstrated that KC-derived IL-10 played a key role in initiating HBV tolerance in 6- to 8-wk-old C57BL/6J mice. This tolerance phenotype was not observed in IL-10–deficient mice or in mice with transient KC depletion (29). The absence of TLR4 might hinder the activation of KC, reducing IL-10 secretion and impeding the development of liver tolerance. Consequently, the C3H/HeJ mice with the TLR4 mutation did not exhibit tolerance and rapidly cleared HBV. Between C3H/HeJ and C3H/HeN mice, the proportion of KCs in the former is less than in the latter (Fig. S4). In addition, the rate of HBV clearance was accelerated in KC-depleted 6-wk-old C3H/HeN mice (Fig. S5). Both of the above support the idea that KC plays an important role in HBV clearance in the 6-wk-old C3H/HeN mice.

The maturation of gut microbiota in adult mice appeared to diminish the tolerant phenotype and predominantly stimulate the immuno-reactive pathway, resulting in rapid HBV clearance. The depletion of gut bacteria from 5 to 12 wk of age prevented the further maturation of liver immunity, making the immune status of ABX mice resemble that of young C3H/HeN mice. The influence of age on liver immunity development was demonstrated using Swiss Webster mice; a study showed that the establishment of commensal bacteria between 3 and 8 wk of age directly controlled the numbers, maturation, and functional activity of KCs. The numbers of total and mature KCs (F4/80⁺MHCII⁺) in 9-wk-old ABX and germ-free Swiss Webster mice were reduced to numbers similar to 3-wk-old counterparts (30). In a transgenic mouse model, Publicover et al. demonstrated that KCs in 8- to 12-wk-old C57BL/6 mice facilitated lymphoid organization and immune priming and promoted successful immunity against HBV. In contrast, lymphoid organization and immune priming was substantially diminished in 3- to 4-wk-old and KC-depleted 8- to 12-wk-old C57BL/6 mice, leading to abrogated HBV immunity (31). Therefore, it is reasonable to speculate that changing gut microbiota profiles at various stages of gut development provides signals that affect the outcomes of immune activation in the liver and determines the transition from HBV tolerance to clearance.

The model presented in this study, and our argument that immunity against HBV varies between developmental stages, might echo the competing demands of immunity to pathogens and tolerance to antigens in the liver. The liver can either be resistant or tolerant, depending upon the type of system that is concerned (32). This study demonstrated that the outcomes of HBV transfection are context-dependent, and conclusions derived from one strain or system might not be applicable to other systems. For example, age-related HBsAg clearance was found in the C3H/HeN and DBA/2J mice, whereas both the young and adult CBA/caJ mice showed a tolerance phenotype to HBV. Although the TLR4 mutation in the C3H/HeJ mice might have contributed to its rapid HBsAg clearance compared with the C3H/HeN mice, rapid clearance among the BALB/cJ, FVB/NJ, NOD/ShiLtJ, and 129 × 1/SvJ mice, which did not have the TLR4 mutation, indicated that additional factors are involved in immune development. In addition, because the C3H/HeJ inbred strain was established half a century ago, the possibility that the enhanced clearance of HBV in this strain was due to genetic alterations other than TLR4 cannot be ruled out. Therefore, the role of TLR4 needs further validation.

At first glance, discordant results derived from various strains and treatments might seem to hamper the usefulness of this hydrodynamic model that adequately captures the multifaceted nature of HBV infection in humans. After all, the outcomes of HBV exposure are diverse, ranging from acute infection to chronic hepatitis, hepatic cirrhosis, and/or hepatocellular carcinoma, all of which depend on host genetics, immune status, and age of requiring. The use of different inbred strains to exploit one or few facets of HBV infection at a time may be a more reasonable approach. C3H/HeN and DBA/2J mice are ideal analogous models to study age-dependent HBV persistence in humans. CBA/caJ, which is tolerant to HBV at all ages, is ideal for research on liver tolerance to HBV. The differences between tolerant and resistant strains might help to shed light on the immune activation that determines the outcomes of HBV infection. In conclusion, using the hydrodynamic transfection method, we demonstrated that host genetics and age, and their interaction, are crucial in the generation of strong, diverse immune responses against HBV. This age-related tolerance model is valuable to the study of liver tolerance and enables the investigation of the mechanisms involved in effective control of HBV.

Materials and Methods

Animals.

BALB/cJ, C3H/HeN, CBA/caJ, DBA/2J, and FVB/NJ mice were purchased from the National Laboratory Animal Center, Taiwan. 129 × 1/SvJ, C3H/HeJ, and NOD/ShiLtJ Mice (129 × 1/SvJ, C3H/HeJ, and NOD/ShiLtJ) were purchased from Jackson Laboratory. The animals were kept in the National Taiwan University College of Medicine Laboratory of Animal Center in specific pathogen-free conditions. All mice were used according to guidelines for experimental animal use specified by the National Taiwan University College of Medicine.

Hydrodynamic Injection and Serum Collection.

HBV expression plasmid pAAV/HBV1.2 (genotype A) was described previously (24). Before HDI, all animals were anesthetized using ketamine (0.75 g/kg; Merial) and xylazine (60 µg/kg; Bayer) administered by intramuscular injection. Ten micrograms of pAAV/HBV1.2 dissolved in 8% body weight of PBS was injected into the tail veins of the mice. The injection time was controlled between 5 and 7 s. Approximately 150 µL of serum was collected on days 2, 7, 10, and every week following HDI until the end of the experiment. Serum HBsAg and anti-HBs were measured using an AXSYM system kit (Abbott Diagnostika). The anti-HB measurements were absolute values, whereas the HBsAg measurements were relative values. The HBsAg-positive threshold was set at an S/N ratio of 10 (24). Statistics were calculated using GraphPad Prism and Microsoft Excel.

Stool DNA Extraction and TTGE.

Stool samples were freshly collected from each mouse every week using clean, autoclaved Eppendorf tubes and were stored at −80 °C before use. DNA was extracted from 0.4 to 0.5 g of stool using a QIAamp DNA Stool Mini Kit (Qiagen). DNA was recovered with 30 µL of elution buffer provided by Qiagen. The quality of the DNA was checked using gel electrophoresis, and the quantity of the DNA was measured using Nanodrop (Thermo Scientific).

The V3 region of the bacteria 16S ribosomal RNA (rRNA) gene was amplified using F341GC and R534 primers (33). To avoid potential amplification bias, PCR was conducted in triplicate for each sample, and the products were pooled. TTGE was carried out using the DCode Universal Mutation Detection System (Bio-Rad). Ten microliters of PCR product was mixed with 10 µL of DNA loading dye, loaded onto acrylamide gel [42% (wt/vol) urea, 10% (wt/vol) acrylamide, 1.5× TAE buffer, 1% ammonium persulfate, 0.1% TEMED], and run for 6 h. The running temperature was set between 55 °C and 70 °C.

Liver RNA Extraction and Real-Time PCR.

The mice were euthanized after anesthetization. Perfusion was performed using at least 10 mL of autoclaved PBS injected from the portal vein. Liver tissue was ground using a mortar and pestle with liquid nitrogen right after taken from the body and stored in liquid nitrogen. Liver RNA was extracted using TRIzol reagent (Life Technology) according to the manufacturer’s instructions. Primer Eub2 targeting 16S rRNA was used to measure the amount of eubacteria in the stool (34). The sample was mixed with Fast SYBR Green PCR master mix (Applied Biosystems). The primer concentration was 0.2 µM, and the final reaction volume was 20 µL. All samples were analyzed in triplicate. Ct values were determined using StepOne Software v2.1. After exportation, the data were analyzed using Microsoft Excel.

Northern Blotting.

Twenty micrograms of liver RNA was separated on 1.2% genetic technology grade gel and then transferred to a positively charged nylon membrane (Roche). After UV cross-linking for 210 s, using the CL-1000 UV cross-linker (UVP), the membrane was prehybridized at 50 °C for 1 h using DIG Easy Hyb (Roche) and then subjected to overnight hybridization, using a digoxigenin (DIG)-labeled HBx probe. Probe detection was performed using DIG-Ab (Roche) for 30 min. The signals were visualized by incubating membranes in CDP-Star (Roche) and exposing them to high-performance chemiluminescence film (GE Healthcare). Livers obtained 3 d after HDI from each group were used as a positive control of HDI, and 3.5 kb HBV plasmid DNA eluted from gel was used as a positive control for Northern blotting.

Western Blotting.

Fifty milligrams of liver tissue was lysed in RIPA buffer (50 mM Tris⋅HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 0.25% sodium deoxycholate, 0.1% SDS) containing Complete Protease Inhibitor (Roche). Subsequently, either 50 or 100 μg of protein lysates were subjected to SDS/PAGE followed by immunoblotting, using rabbit anti-HBcAg (1:1,000; generated by LTK BioLaboratories), mouse anti-HBsAg (kindly provided by Xia Ning-Shao, School of Life Science, Xiamen University), mouse anti–β-actin (1:5,000; Sigma), mouse anti-FLAG (1:1,000; Sigma), horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5,000; Promega), or rabbit anti-mouse (1:5,000; Dako). Chemiluminescence detection was performed using Immobilon Western chemiluminescent HRP substrate (Millipore).

ELISPOT.

The mouse splenocytes were subjected to an IFN-γ ELISPOT assay, using an IFN-γ ELISPOT set (BD Biosciences) according to the manufacturer’s instructions. In brief, 1 × 10⁶ splenocytes were added to a precoated ELISPOT plate and were cocultured with 0.3 μg/mL of recombinant HBcAg (rHBcAg, ID Labs) in 200 µL of RPMI medium 1640, with 10% FCS at 37 °C in 5% CO₂ for 20 h. The positive control was stimulated using 0.3 µg/mL of LPS. Following treatment of the biotin-conjugated antibodies and streptavidin–HRP, the spots were visualized by adding 3-amino-9-ethylcarbazole substrate and were analyzed using the ImmunoSpot series 5 analyzer (Cellular Technology).