Age-related immune clearance of hepatitis B virusinfection requires the establishment of gut microbiotaHan-Hsuan Choua,b,1, Wei-Hung Chienb,c,1, Li-Ling Wub, Chi-Hung Chenga,b, Chen-Han Chungb, Jau-Haw Hornga,Yen-Hsuan Nid, Hong-Tai Tsenge, Dafei Wuf, Xuemei Luf, Hurng-Yi Wangb,g,h,2, Pei-Jer Chenb, and Ding-Shinn Chenb,i,2

aDepartment of Microbiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan; bGraduate Institute of Clinical Medicine, College ofMedicine, National Taiwan University, Taipei 100, Taiwan; cDepartment of Medical Education, Chung-Shan Medical University Hospital and School ofMedicine, Chung-Shan Medical University, Taichung 402, Taiwan; dDepartment of Internal Medicine, National Taiwan University Hospital, National TaiwanUniversity, Taipei 100, Taiwan; eDepartment of Immunology, College of Medicine, National Taiwan University, Taipei 100, Taiwan; fLaboratory of DiseaseGenomics and Individualized Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China; gInstitute of Ecology andEvolution, National Taiwan University, Taipei 106, Taiwan; hResearch Center for Developmental Biology and Regenerative Medicine, National TaiwanUniversity, Taipei 100, Taiwan; and iGenomics Research Center, Academia Sinica, Taipei 115, Taiwan

Contributed by Ding-Shinn Chen, January 1, 2015 (sent for review September 23, 2014)

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

liver tolerance | Toll-like 4 receptor | temporal-temperature gelelectrophoresis | chronic hepatitis B | Kupffer cells

The hepatitis B virus (HBV) is one of the most common in-fectious agents worldwide. According to the World Health

Organization, more than one-third of the world’s population (2billion people) have been infected with HBV, and 240 millionpeople are chronic carriers (1). These chronic HBV carriers havea higher probability of developing hepatic cirrhosis and hepa-tocellular carcinoma than the general population and constitutea 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 toeliminate or cure infected hepatocytes; however, the mechanismbehind this impairment remains unclear. Numerous studies havesuggested that the genetic constitution of the host is a criticalfactor in determining the outcomes of HBV infection (3, 4).Genetic variations in genes that code for cytokines, such as IFN-γand tumor necrosis factor-α (TNF-α) (5), cytotoxic T-lymphocyteantigen 4 (6), and HLA (7, 8), have been suggested to influencethe chronicity or clearance rates of HBV infection. Recentgenome-wide association studies have demonstrated that certainSNPs near HLA-DP loci are associated with persistent HBV in-fection (9, 10).Although the genetics of the host are critical, the age of HBV

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

Mounting evidence has revealed that commensal bacteriaresiding in the gastrointestinal (GI) tract regulate a host’s im-mune response. Although the liver is not in direct contact withlive commensals, constant exposure to microbe-derived ligandsand metabolites through the gut–liver axis likely shapes liverimmunity (15, 16). Liver sinusoid endothelial cells (LSECs) re-spond to Gram-negative bacteria-derived lipopolysaccharides(LPSs) through Toll-like receptor (TLR) 4 by the secretion ofinterleukin-10 (IL-10), which induces T-cell tolerance (17, 18).Kupffer cells (KCs) are the largest population of tissue-residentmacrophages, and their activation through the LPS and TLR4pathway 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 im-munity activation. Mouse KCs pretreated with bacterial CpG-DNA exhibited significantly increased IL-6 production in responseto subsequent LPS challenge in vitro (21). Using a mouse model,signaling through the CpG-DNA/TLR9 pathway enabled hepaticexpansion 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, KCsand LSECs were observed to override the tolerance phenotypeand promote CD4+ T-cell proliferation in the presence of TLRligands, such as LPS and CpG-DNA (23). The interaction between

Significance

Ninety-five percent of adult-acquired infections lead to spon-taneous 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, adaptiveimmune response is considered essential for HBV clearance, butthe mechanisms to generate a favorable response remain elu-sive. Here, we show that, while 12-wk-old C3H/HeNmice clearedHBVwithin 6 wk postinjection (wpi), their 6-wk-old counterpartsremained HBV-positive at 26 wpi. Sterilization of gut microbiotafrom 6 to 12 wk of age using antibiotics prevented mice fromrapidly clearing HBV. This model is valuable to the study of livertolerance and enables the investigation of the mechanisms in-volved in effective control of HBV.

Author contributions: H.-Y.W., P.-J.C., and D.-S.C. designed research; H.-H.C., W.-H.C., L.-L.W.,C.-H. Cheng, C.-H. Chung, J.-H.H., Y.-H.N., H.-T.T., D.W., and X.L. performed research; Y.-H.N.,H.-Y.W., and D.-S.C. contributed new reagents/analytic tools; H.-H.C., W.-H.C., L.-L.W.,H.-Y.W., and P.-J.C. analyzed data; and H.-H.C., H.-Y.W., P.-J.C., and D.-S.C. wrotethe paper.

The authors declare no conflict of interest.1H.-H.C. and W.-H.C. contributed equally to this work.2To whom correspondence may be addressed. Email: chends@ntu.edu.tw or hurngyi@ntu.edu.tw.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1424775112/-/DCSupplemental.

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commensals and the liver significantly influences hepatic physiologyand determines the outcomes of pathophysiologic responses tochallenges. The age dependence of HBV clearance thus resultsfrom the development of gut microbiota, which begin to colonizethe GI tract after birth.Investigating the basis of immune activation during HBV in-

fection is experimentally challenging because of the intrinsic dif-ficulty of acquiring samples at the appropriate stages. Therefore,to understand the mechanisms underpinning HBV clearance orpersistence, we used a nontransgenic hydrodynamic transfectionmouse model. This model is able to mimic multiple features ofHBV infection observed in humans, including age-dependentchronicity, genetic variations influencing the outcomes of HBVinfection, and the possible interactions between them. A previousstudy showed that after transfection HBV plasmids lasted formore than 6 mo in C57BL/6 mice, but were rapidly cleared inBALB/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 HBVclearance and persistence among the strains, indicating that HBVimmunity is genetically dependent. We also showed the age-dependent HBV clearance in C3H/HeN and DBA/2J mice, whichmimicked natural history of HBV in humans. As the age-dependentHBV clearance developed synchronously with the postnatal estab-lishment of gut microbiota and portal blood flow, we hypothesizedthat gut commensals prepared the liver immunity system to clearHBV. After sterilizing the gut bacteria of C3H/HeN mice usingantibiotics, adult mice were more tolerant to HBV exposure, similarto their young counterparts. The age-dependent HBV clearance,however, disappeared in C3H/HeJ mice, a TLR4 mutant strainin which LPS-induced TLR4 signaling was abolished. These resultssuggested that gut microbiota contribute to the age dependence ofHBV immunity because they regulate the innate immunity pathwayin the liver.

ResultsStrain and Age-Dependent Clearance of HBsAg After HydrodynamicInjection.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 HBsAgpositivity (24). BALB/cJ, FVB/NJ, NOD/ShiLtJ, and 129 × 1/SvJmice 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 contributeto HBV exposure outcomes. Chronic hepatitis B infection inhumans is defined as an infection lasting longer than 6 mo. After 6mo, the HBsAg-positive rate was 15% among the C57BL/6J mice,25% among the DBA/2J mice, and 80% among the C3H/HeN andCBA/caJ mice. Although both the BALB/cJ and DBA/2J miceshared the MHC haplotype H2-d, they responded differently toHBV. Similarly, the immune responses of the 129 × 1/SvJ andC57BL/6J mice, which shared the MHC haplotype H2-b, differedconsiderably. These observations suggest that MHC haplotypesalone did not determine outcomes of HBV transfection.To test the effect of age on HBV exposure outcomes, adult

(12-wk-old) CBA/caJ, DBA/2J, C3H/HeN, and C57BL/6J micewere administered HDI, and their serum HBsAg titers weremonitored. In contrast to young mice, the adult C3H/HeN micecleared serum HBsAg at 5 wpi (Fig. 1B). The accelerated HBsAgclearance in adult mice was also observed among the DBA/2J(Fig. 1C) and C57BL/6J (Fig. 1D) mice. The substantial dif-ferences in the young vis-à-vis adult mice implied that outcomesof HDI in the C3H/HeN, DBA/2J, and C57BL/6J mice werestrongly influenced by developmental stages analogous to HBVinfection in humans. Moreover, these results suggested that,among these mouse strains, liver immunity system maturity mightplay 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 in-teraction between commensal gut bacteria and host immunity(25, 26). Microbiota are introduced into the gut from the envi-ronment after delivery, and a stable gut microbiota profile isgradually established. The timing of this establishment coincideswith improved HBV clearance. We thus examined whether in-testinal microbiota were associated with the outcomes of HBVtransfection in C3H/HeN mice because adult and young mice inthis strain exhibited the most considerable difference after HDI.C3H/HeN mice between 5 and 12 wk of age were gut-sterilizedusing a well-established antibiotic (ABX) mixture protocol (27,28). The body weights of the ABX mice dropped by ∼20% in thefirst week of antibiotic treatment and subsequently recovered(Fig. 2A). This reduction in body weight was attributed to de-creased water intake (Fig. S1); water intake among the mice was20% of normal levels 3 d after the start of antibiotic treatment

A B

C D Fig. 1. The outcomes of HBV transfection by hydro-dynamic injection in mice were context-dependent.Six-wk-old mice were hydrodynamically injected with10 μg of pAAV/HBV1.2 plasmid. Serum HBsAg titerswere 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) andadult (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) arepresented in parentheses.

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and returned to normal levels after 10 d. The amount of gutbacteria, assessed according to the level of 16S ribosomal DNAin stools, was relatively constant among the adult mice, whereasa roughly 100-fold reduction was observed in the ABX mice 1 wkafter treatment; this reduction lasted throughout the entire ex-posure period (Fig. 2B). ABX mice also showed an enlargementof cecum commonly observed in germ-free mice (Fig. S2). Afterthe removal of antibiotics, the amount of stool bacteria in theABX mice returned to levels similar to those of the adult miceafter 4 d [2 d postinjection (dpi)].Temporal-temperature gel electrophoresis (TTGE) was used

to compare the gut microbiota profiles of undisturbed andantibiotic-treated mice, based on the GC contents of variousmicrobes. The TTGE profiles of an untreated mouse variedconsiderably between 6 and 8 wk of age and stabilized after 9 wkof age (Fig. 2C), suggesting that gut microbiota were establishedbetween birth and 11 wk of age and thereafter reached equilib-rium. Similar patterns of microbiota establishment were ob-served in another three untreated mice (Fig. S2). In the ABXmice, gut microbiota were nearly undetectable during treatmentand appeared 4 d after antibiotic treatment was stopped (2 dpi);however, 4 wk after treatment was stopped, bacterial diversity inthe ABX mice did not return to the levels of untreated adultmice (Fig. 2D and Fig. S2).

Effects of Antibiotics on Serum and Intrahepatic HBV Clearance.Adult, ABX, and young C3H/HeN mice (Fig. 3A) were hydro-dynamically injected with 10 μg of pAAV/HBV1.2 plasmid tomonitor their responses against HBV. Serum HBsAg levels (Fig.3B) and viral titers (Fig. 3C) were similar between the groups at1 wpi, demonstrating that the initial HBV transfection efficiencywas similar. In the adult mice, serum HBV DNA and HBsAglevels quickly decreased. Levels of HBV DNA decreased belowdetection levels (103 copies/mL) at 4 wpi, and levels of HBsAgdecreased 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) ofthe ABX and 80% (8 of 10) of the young mice remained HBsAg-positive. Repeat ABX experiments showed similar results inserum HBsAg levels and persistence rate (Fig. S3).To further characterize intrahepatic viral transcriptions and

protein expressions in different mouse groups, liver samples werecollected at different time points and assayed using Northern and

Western blotting. HBV transcripts, including 3.5-kb pregenomicand 2.4/2.1-kb surface mRNAs, were detected in the livers of micefrom all three groups at 3 dpi (Fig. 3E). In addition, the intra-hepatic expression of HBcAg and HBsAg were also confirmed.The transcripts and protein expressions became undetectable afterthe clearance of serum HBsAg in all individuals examined. Formice in ABX and young groups, which failed to clear serumHBsAg at 47 wpi, intrahepatic HBV transcripts as well as HBcAgand HBsAg were still positive. Serum HBsAg is strongly corre-lated with intrahepatic viral transcription and protein expression.The results clearly showed that gut sterilization reduced the im-mune 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) wasdetected in all adult mice 1 or 2 wk after the clearance of serumHBsAg. However, at 26 wpi, only six of nine HBsAg-negativeABX mice produced anti-HBs, and anti-HBs were not detectedin HBsAg-negative young mice (Table 1). Antibiotic treatmentthus not only affected serum HBsAg clearance, but also impairedantibody production. To further investigate the immune responseagainst HBV, we examined the frequency of HBcAg-specific IFNγ-producing T cells in the splenocytes using an IFNγ enzyme-linkedimmunospot (ELISPOT) assay because the response of T cells toHBcAg stimulation is correlated with the resolution of HBVinfections. Adult mice exhibited a significantly higher number ofHBcAg-specific IFN-γ–secreting cells (418 cells per 106 spleno-cytes) than the other two groups (Fig. 3F); this strong responseagainst HBcAg was maintained through 47 wpi. Regardless of theirHBsAg status, the ABX and young mice induced IFN-γ–secretingcells at levels similar to those of the negative control. These resultssuggested that less effective adaptive humoral and cellular immuneresponses among the ABX mice increased HBV persistence.

Loss of Age-Related HBV Clearance in the C3H/HeJ Strain with TLR4Mutation. The depletion of gut bacteria resulted in delayed HBsAgclearance. Emerging evidence has indicated that the activation ofTLR4 by LPS from intestinal Gram-negative bacteria is associatedwith numerous aspects of liver immunity. We tested the outcomesof HBV transfection on C3H/HeJ mice, a TLR4 mutant strain inwhich LPS-induced TLR4 signaling was abolished. Levels of HBsAgrapidly decreased in both young and adult C3H/HeJ mice (Fig. 4A),and both exhibited accelerated HBsAg clearance within 8 wpi

A B

C D

Fig. 2. Intestinal microbiota of the C3H/HeN micereached 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 van-comycin) and one of which was not (adult; n = 10).ABX mice received antibiotics in their drinking wa-ter between 5 and 12 wk of age, and their bodyweights (A) were recorded. (B) The amount of bac-teria 16S rRNA was reduced 100-fold after antibiotictreatment. (C) The profiles of intestinal microbiota,examined using TTGE, varied considerably between6 and 9 wk of age in an untreated adult mouse andthereafter reached equilibrium. (D) The ABX mouseshowed an extremely low amount of bacteria, whichdid not vary in the course of treatment. M: 100-bpladder marker; dpi: days postinjection; wpi: weekspostinjection.

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(Fig. 4B). In addition, regardless of age, all of the C3H/HeJ miceproduced anti-HBs within 2 wk of HBsAg clearance (Table 1).

DiscussionBased on our results, we propose a model of age-related HBVinfection, in which the establishment of gut microbiota significantlyinfluences hepatic immune responses that result in viral clearanceor persistence. Between 6 and 9 wk of age, the gut microbiota of theC3H/HeN mice matured toward equilibrium; this process appearsto play a crucial role in the development of liver immunity. Theyoung C3H/HeN mice transfected with HBV, which had notreached gut microbiota equilibrium, exhibited tolerance phenotypes,including prolonged HBsAg persistence, impaired anti-HBs pro-duction, and limited HBcAg-specific IFN-γ–secreting splenocytes.

In adult mice, the maturation of gut bacteria might transmit signalsto the liver to break liver tolerance, resulting in rapid HBV clear-ance. The depletion of gut bacteria in the adult C3H/HeNmice (ABX) restored the tolerance phenotypes as if they wereyoung mice.Liver tolerance elicited by hepatic antigen-presenting cells

facilitates the secretion of IL-10 after exposure to low levels ofbacterial debris (e.g., LPS). Under steady-state conditions, thelocal immune state within the liver might be biased toward tol-erance because of LPS/TLR4-mediated IL-10 secretion. Never-theless, liver tolerance can be overridden in the presence ofbacterial CpG-DNA, a TLR9 ligand. Liver immunity stimulationor inhibition might depend on the type and strength of the sig-nals to which hepatic cells have been exposed, and it is likely thatgut microbiota acts on the liver via both immuno-stimulatory andimmuno-tolerating pathways to influence HBV responses.When the gut microbiota was developing in the young mice,

the immuno-tolerating pathway was observed to prevail throughTLR4-dependent innate immunity. This idea coincides with arecent study that demonstrated that KC-derived IL-10 played akey role in initiating HBV tolerance in 6- to 8-wk-old C57BL/6Jmice. This tolerance phenotype was not observed in IL-10–deficientmice or in mice with transient KC depletion (29). The absence ofTLR4 might hinder the activation of KC, reducing IL-10 secretion

A D

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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 weremonitored 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 ofHBV 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) andweeks 19 and 47 post-HDI among serum-HBsAg (SAg)–positive (+) and –negative (−) mice. (F) HBcAg-specific IFNγ-producing cells in 1 × 106 splenocytes fromthe 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 usingIFNγ 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 wasnot significant differently from negative control. **P < 0.01.

Table 1. Percentages of HBsAg clearance (to the left of theslash) and anti-HB production (to the right of the slash) amongthe C3H/HeN and C3H/HeJ mice

SAb+/clear SAg Young, % ABX, % Adult, %

C3H/HeN (26 wk) 0/25 35/53 100/100C3H/HeJ (8 wk) 100/100 100/100

Parentheses are weeks post-HDI.

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and impeding the development of liver tolerance. Consequently, theC3H/HeJ mice with the TLR4 mutation did not exhibit toleranceand 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 abovesupport the idea that KC plays an important role in HBV clearancein the 6-wk-old C3H/HeN mice.The maturation of gut microbiota in adult mice appeared to

diminish the tolerant phenotype and predominantly stimulatethe immuno-reactive pathway, resulting in rapid HBV clearance.The depletion of gut bacteria from 5 to 12 wk of age preventedthe further maturation of liver immunity, making the immunestatus of ABX mice resemble that of young C3H/HeN mice. Theinfluence of age on liver immunity development was demon-strated using Swiss Webster mice; a study showed that the estab-lishment of commensal bacteria between 3 and 8 wk of age directlycontrolled the numbers, maturation, and functional activity ofKCs. The numbers of total and mature KCs (F4/80+MHCII+) in9-wk-old ABX and germ-free Swiss Webster mice were reduced tonumbers similar to 3-wk-old counterparts (30). In a transgenicmouse model, Publicover et al. demonstrated that KCs in 8- to 12-wk-old C57BL/6 mice facilitated lymphoid organization and im-mune priming and promoted successful immunity against HBV. Incontrast, lymphoid organization and immune priming was sub-stantially 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 micro-biota profiles at various stages of gut development provides signalsthat affect the outcomes of immune activation in the liver anddetermines 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 pathogensand tolerance to antigens in the liver. The liver can either beresistant or tolerant, depending upon the type of system that isconcerned (32). This study demonstrated that the outcomes ofHBV transfection are context-dependent, and conclusions de-rived from one strain or system might not be applicable to othersystems. For example, age-related HBsAg clearance was found inthe C3H/HeN and DBA/2J mice, whereas both the young andadult CBA/caJ mice showed a tolerance phenotype to HBV.Although the TLR4 mutation in the C3H/HeJ mice might havecontributed to its rapid HBsAg clearance compared with theC3H/HeN mice, rapid clearance among the BALB/cJ, FVB/NJ,NOD/ShiLtJ, and 129 × 1/SvJ mice, which did not have theTLR4 mutation, indicated that additional factors are involved inimmune development. In addition, because the C3H/HeJ inbredstrain was established half a century ago, the possibility that theenhanced clearance of HBV in this strain was due to genetic

alterations other than TLR4 cannot be ruled out. Therefore, therole of TLR4 needs further validation.At first glance, discordant results derived from various strains

and treatments might seem to hamper the usefulness of thishydrodynamic model that adequately captures the multifacetednature of HBV infection in humans. After all, the outcomes ofHBV exposure are diverse, ranging from acute infection to chronichepatitis, hepatic cirrhosis, and/or hepatocellular carcinoma, all ofwhich depend on host genetics, immune status, and age of re-quiring. The use of different inbred strains to exploit one or fewfacets of HBV infection at a time may be a more reasonable ap-proach. C3H/HeN and DBA/2J mice are ideal analogous modelsto study age-dependent HBV persistence in humans. CBA/caJ,which is tolerant to HBV at all ages, is ideal for research on livertolerance to HBV. The differences between tolerant and resistantstrains might help to shed light on the immune activation thatdetermines the outcomes of HBV infection. In conclusion, usingthe hydrodynamic transfection method, we demonstrated that hostgenetics and age, and their interaction, are crucial in the genera-tion of strong, diverse immune responses against HBV. This age-related tolerance model is valuable to the study of liver toleranceand enables the investigation of the mechanisms involved in ef-fective control of HBV.

Materials and MethodsAnimals. BALB/cJ, C3H/HeN, CBA/caJ, DBA/2J, and FVB/NJmicewere purchasedfrom the National Laboratory Animal Center, Taiwan. 129 × 1/SvJ, C3H/HeJ,and NOD/ShiLtJ Mice (129 × 1/SvJ, C3H/HeJ, and NOD/ShiLtJ) were purchasedfrom Jackson Laboratory. The animals were kept in the National TaiwanUniversity College of Medicine Laboratory of Animal Center in specific patho-gen-free conditions. All mice were used according to guidelines for experimentalanimal 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 animalswere 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 ofthe mice. The injection time was controlled between 5 and 7 s. Approximately150 μL of serum was collected on days 2, 7, 10, and every week following HDIuntil the end of the experiment. Serum HBsAg and anti-HBs were measuredusing an AXSYM system kit (Abbott Diagnostika). The anti-HB measurementswere absolute values, whereas the HBsAg measurements were relative values.The HBsAg-positive threshold was set at an S/N ratio of 10 (24). Statistics werecalculated using GraphPad Prism and Microsoft Excel.

Stool DNA Extraction and TTGE. Stool samples were freshly collected from eachmouse every week using clean, autoclaved Eppendorf tubes and were storedat −80 °C before use. DNA was extracted from 0.4 to 0.5 g of stool using aQIAamp DNA Stool Mini Kit (Qiagen). DNA was recovered with 30 μL ofelution buffer provided by Qiagen. The quality of the DNA was checkedusing gel electrophoresis, and the quantity of the DNA was measured usingNanodrop (Thermo Scientific).

A B

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 TLR4mutation (C3H/HeJ) exhibited rapid HBsAg decrease (A) and clearance (B) after the hydrodynamic injection of pAAV/1.2HBV plasmid.

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The V3 region of the bacteria 16S ribosomal RNA (rRNA) gene was am-plified using F341GC and R534 primers (33). To avoid potential amplificationbias, PCR was conducted in triplicate for each sample, and the products werepooled. TTGE was carried out using the DCode Universal Mutation DetectionSystem (Bio-Rad). Ten microliters of PCR product was mixed with 10 μL ofDNA 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 afteranesthetization. Perfusion was performed using at least 10 mL of autoclavedPBS injected from the portal vein. Liver tissue was ground using a mortar andpestle with liquid nitrogen right after taken from the body and stored inliquid nitrogen. Liver RNA was extracted using TRIzol reagent (Life Tech-nology) according to the manufacturer’s instructions. Primer Eub2 targeting16S 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 Bio-systems). The primer concentration was 0.2 μM, and the final reaction vol-ume was 20 μL. All samples were analyzed in triplicate. Ct values weredetermined using StepOne Software v2.1. After exportation, the data wereanalyzed 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 chargednylon membrane (Roche). After UV cross-linking for 210 s, using the CL-1000UV cross-linker (UVP), the membrane was prehybridized at 50 °C for 1 husing DIG Easy Hyb (Roche) and then subjected to overnight hybridization,using a digoxigenin (DIG)-labeled HBx probe. Probe detection was per-formed using DIG-Ab (Roche) for 30 min. The signals were visualized byincubating membranes in CDP-Star (Roche) and exposing them to high-per-formance chemiluminescence film (GE Healthcare). Livers obtained 3 d afterHDI 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 forNorthern 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 In-hibitor (Roche). Subsequently, either 50 or 100 μg of protein lysates weresubjected to SDS/PAGE followed by immunoblotting, using rabbit anti-HBcAg(1:1,000; generated by LTK BioLaboratories), mouse anti-HBsAg (kindly pro-vided by Xia Ning-Shao, School of Life Science, Xiamen University), mouseanti–β-actin (1:5,000; Sigma), mouse anti-FLAG (1:1,000; Sigma), horseradishperoxidase (HRP)-conjugated goat anti-rabbit (1:5,000; Promega), or rabbitanti-mouse (1:5,000; Dako). Chemiluminescence detection was performed us-ing 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’sinstructions. In brief, 1 × 106 splenocytes were added to a precoated ELISPOTplate 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 for20 h. The positive control was stimulated using 0.3 μg/mL of LPS. Followingtreatment of the biotin-conjugated antibodies and streptavidin–HRP, the spotswere visualized by adding 3-amino-9-ethylcarbazole substrate and were ana-lyzed using the ImmunoSpot series 5 analyzer (Cellular Technology).

ACKNOWLEDGMENTS. We thank S.-H. Yeh, P.-N. Shu, and H.-C. Yang forcritical discussions. We also thank the technical assistance of MicrobialGenomics Core Laboratory of National Taiwan University Center of GenomicMedicine for the virus and HBsAg quantification. This study was supportedby the National Science Council, Taiwan (Grants NSC-102-2321-B-002-003-,101-2321-B-002-009-, and 100-2321-B-002-029-), and NTU-CDP-104R7836 (toH.-Y.W.) and Ministry of Science of Technology, Taiwan (MOST-103-2321-B-002-001) (to P.-J.C. and D.-S.C.).

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