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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

The liver is considered as a unique lymphoid organ favoring the induction of immune tolerance, rather than immunity. Biologists and clinicians alike have a long-standing interest in how the liver induces systemic immune tolerance, but the mechanism has not yet been well elucidated. Here, we employed hepatitis B virus (HBV)-carrier mice generated by hydrodynamically injecting phosphor-adeno-associated virus/HBV1.2 plasmid as a model for adult chronic HBV infection, which we found were unable to respond to hepatitis B surface antigen vaccination. Humoral tolerance induced in HBV-carrier mice could be transferred into Rag1−/− mice, because anti-HBV immunity in immunologically reconstituted Rag1−/− mice was inhibited by adoptive transfer of splenocytes from HBV-carrier mice. Humoral tolerance needed at least 7 days for induction and persisted to 3 months after a single HBV plasmid injection. Kupffer cell (KC) depletion or interleukin (IL-10) deficiency broke this humoral tolerance, and exogenous injection of IL-10 could effectively induce this tolerance. Conclusion: KCs in HBV-carrier mice expressed more IL-10 and mediated the systemic tolerance induction in an IL-10-dependent manner. This previously undescribed humoral tolerance regarding HBV infection will help to explore new approaches to reverse liver-sustained systemic immune tolerance in liver disease. (Hepatology 2014;59:443-452)

Abbreviations
Ab

antibody

anti-HBe

hepatitis B e antibody

anti-HBs

hepatitis B surface antibody

APC

antigen-presenting cell

CFA

Complete Freund's Adjuvant

FCM

flow cytometry

GC

germinal center

HBcAg

hepatitis B c antigen

HBeAg

hepatitis B e antigen

HBsAg

hepatitis B surface antigen

HBV

hepatitis B virus

HDI

hydrodynamic injection

IgG

immunoglobulin G

IL

interleukin

IM

intramuscular

IRMA

immunoradiometric assay

IV

intravenous

KC

Kupffer cell

MNC

mononuclear cell

OVA

ovalbumin

pAAV

phospho- adeno-associated virus

PCR

polymerase chain reaction

SEM

standard error of the mean

Tg

transgenic

Treg

regulatory T cell

WT

wild type.

The liver is a unique organ that is distinctly immune privileged and can also induce systemic immune tolerance, called “liver tolerance”.[1-3] The following clinical and experimental evidence support the existence of liver tolerance. Hepatotropic pathogens (hepatitis B virus [HBV], hepatitis C virus, and malaria) are likely to establish persistent or chronic infection in infected patients, and these carriers usually become unresponsive to potent vaccination.[4-6] Also, the liver plays a critical role in inducing oral tolerance not only to pathogenic invaders, but also to foods, which contain “nonself” components, including endotoxin, bacterial metabolites, and food antigens.[7-9] Experimental evidence in animal models suggests that self protein from other organs can induce systemic immune tolerance if artificially expressed in the liver.[10] More interesting, it was reported more than 40 years ago that donor skin, kidney, and heart grafts were preferentially protected from rejection in immunologically mature pigs that also received liver allografts.[11] Though the tolerogenic features of the liver have been recognized for decades, the precise mechanisms by which it participates in inducing systemic tolerance are not fully understood.

The liver promotes antigen-specific tolerance by a variety of mechanisms, including clonal deletion and induction of regulatory T cells (Tregs). Whereas clonal deletion mechanisms maintain central tolerance, Tregs are considered to be critical players in maintaining peripheral tolerance by suppressing cell activation, cytokine production, and antibody (Ab) production.[12] Interestingly, tolerogenic antigen-presenting cells (APCs) in the liver also play a critical role in promoting tolerance, but how they induce the systemic tolerance is currently unclear.[13] In addition, there are very few reports demonstrating in detail the time-kinetic relationship between liver antigen expression and systemic tolerance induction, including how soon the tolerance could be established and how long the tolerance could persist.

In the present study, using an HBV-carrier mouse model of persistent HBV infection created by hydrodynamically injecting an engineered, replication-competent HBV DNA into C57BL/6 mice, we found that HBV-carrier mice acquire humoral tolerance, because they lost specific immune responsiveness to peripheral intrasplenic or –muscular (IM) HBV vaccination assessed by the inability of HBV-carrier mice to produce anti-HBs (hepatitis B surface antibody) Abs. Our data suggest that humoral tolerance needed at least 7 days for induction and persisted to 3 months after in HBV-carrier mice. Humoral tolerance was induced in HBV-carrier mice in a Kupffer cell (KC)- and interleukin (IL)−10-dependent manner, because KC depletion, IL-10 deficiency, or KC derived from IL-10−/− mice impairs HBV-induced immune tolerance.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
Mice

Male C57BL/6 mice (6-8 weeks old) were purchased from the Shanghai Experimental Animal Center (Shanghai, China). IL-10−/− mice (B6.129P2-Il10tm1Cgn/J) were purchased from The Jackson Laboratory (Bar Harbor, ME). Rag1−/− mice were obtained from the Model Animal Research Center (Nanjing, China). All mice were maintained under specific pathogen-free conditions and used according to the guidelines for experimental animal use from the University of Science and Technology of China (Hefei, China).

HBV-Carrier Mouse Model

The HBV-carrier mouse model was established by hydrodynamic injection of 6 μg of phospho-adeno-associated virus (pAAV)/HBV1.2 plasmids as previously described.[14] Serum hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and anti-HBs Ab levels were determined using commercially available immunoradiometric assay (IRMA) kits (Beijing North Institute of Biological Technology, Beijing, China). Serum HBV DNA was evaluated using a commercially available real-time polymerase chain reaction (PCR) kit (Ke Hua, Shanghai, China).

Immunohistochemistry

Liver samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Liver sections were stained for hepatitis B c antigen (HBcAg) using rabbit Abs against HBcAg (Dako, Carpinteria, CA), followed by biotinylated anti-rabbit immunoglobulin G (IgG) and streptavidin/horseradish peroxidase conjugates (Zhongshan Goldenbridge, Beijing, China). Stains were developed with a 3,3′-diaminobenzidine kit (Vector Laboratories, Burlingame, CA).

Cell Isolation, Adoptive Transfer, and Immunization

Single-cell suspensions of splenocytes and liver mononuclear cells (MNCs) were prepared essentially as previously described.[15] KC isolations were performed as previously described.[16, 17] In most experiments, 2 × 107 splenocytes or 5 × 106 KCs were intravenously (IV) transferred into naïve mice. The recipients were immunized with an HBsAg vaccine (HBsAg/Alum; Kangtai, Shenzhen, China) or HBsAg (HyTest Ltd., Turku, Finland) in a Complete Freund's Adjuvant (CFA) emulsion. All mice were immunized with 1 μg of HBsAg per mouse in a total of 50 μL in volume, and serum anti-HBs levels were measured at the indicated time points.

Reverse Transcription and Real-Time PCR Analysis

After sorting KCs from control or HBV-carrier mice, the total procedure of analyzing IL-10 messenger RNA was performed as described previously.[18]

Flow Cytometry

The following Abs were used for flow cytometry (FCM; all from BD Biosciences, San Diego, CA): fluorescein-isothiocyanate–conjugated Abs against CD19 (clone ID3), F4/80 (clone BM8; eBioscience, Inc., San Diego, CA), and Gl7 (clone Gl7); phycoerythrin-conjugated Abs against Fas (clone Jo2); PerCP-Cy5.5-conjugated Abs against B220 (clone RA3-6B2); and CD3 (clone 145-2C11). After blocking with rat IgG to saturate rat Fc receptors, cells were stained with the indicated fluorescently labeled monoclonal Abs for surface antigens, following a standard protocol. Stained cells were analyzed using a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) flow cytometer, and data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).

Statistical Analysis

An unpaired two-tailed Student t test was used for statistical analyses. Data are expressed as means ± standard error of the mean (SEM), and data were considered statistically significant when values reached P < 0.05. Significance was denoted as P < 0.05, P < 0.01, and P < 0.001.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
Establishment of a Model of Systemic HBV-Specific Humoral Tolerance

To experimentally mimic human HBV persistent infection, we utilized an HBV-carrier mouse model developed by hydrodynamic injection of a pAAV/HBV1.2 plasmid into C57BL/6 mice.[14] Almost all HBV-carrier mice became HBsAg positive at 5 weeks postinjection (Supporting Fig. 1A,B), and HBsAg titers correlated with serum HBV DNA copy numbers (Supporting Fig. 1C). Cytoplasmic and nucleic HBcAg was observed in livers without accompanying inflammatory injury (Supporting Fig. 1D,E), similar to HBV persistent infection in humans. In our study, HBsAg persisted for >3 months in HBV-carrier mice (data not shown). In mice previously immunized with HBsAg vaccine by the IM route, HBV persistence could not be established because HBV was eliminated within 1 week (Supporting Fig. 2A,B), serum HBeAg became undetectable, and HBcAg was undetectable in liver tissue (Supporting Fig. 2C,D) within 6 weeks after hydrodynamic pAAV/HBV1.2 plasmid injection, suggesting that viral persistence in the liver does not occur in the prepresence of specific systemic immunity.

To determine whether HBV preexistence in the liver induced systemic immune tolerance, HBV-carrier or control mice were immunized with an HBsAg vaccine. In contrast to control mice, HBV-carrier mice did not produce anti-HBs Abs in response to vaccination (Fig. 1A), showing that HBsAg vaccination did not reduce serum HBsAg and HBV DNA levels or liver HBcAg expression in HBV-carrier mice (Fig. 1B-D). Meanwhile, CD69 expression on B cells and frequency of germinal center (GC) B cells, which can differentiate into memory B and plasma cells, were significantly decreased in HBV-carrier mice, when compared to wild-type (WT) mice after HBsAg vaccination (data not shown). Despite their tolerance to HBsAg, HBV-carrier mice remained responsive to other antigens in a typical delayed-type hypersensitivity reaction, similar to control mice (Fig. 1E). These results suggested that specific humoral tolerance to HBV developed in HBV-carrier mice.

image

Figure 1. Liver HBV preexistence induces systemic immune tolerance to HBV vaccination. (A) Two weeks after HBV or control plasmid injection, HBV-carrier and control mice were immunized IM with HBsAg vaccine at days 0 and 14. Serum anti-HBs levels were monitored by IRMA. HBV-carrier mice were immunized with HBsAg vaccine or phosphate-buffered saline (PBS) twice within a 2-week interval, and serum levels of HBsAg (B) and HBV DNA (C) were examined at the indicated time points. (D) Immunohistochemical staining for HBcAg in livers of HBV-carrier mice at 6 weeks after HBsAg immunization. (original magnification: 100×). (E) HBV-carrier and control mice were injected subcutaneously with OVA in a CFA emulsion and challenged 1 week later with aggregated OVA in the footpad. OVA-specific response was determined by measuring footpad swelling at 24 and 48 hours after rechallenge. Results are presented as mean ± SEM for (A), (B), (C), and (E), and represent at least three independent experiments. n = 5-8 mice/group. ND indicates not detected. [Correction added after publication December 24, 2013: The y axis of Fig. 1C was changed from “HBV DBA (copies/mL)” to “HBV DNA (copies/mL)”]

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HBV-Specific Humoral Tolerance Could Be Transferred into Naïve Rag1−/− Mice

To investigate whether humoral tolerance in HBV-carrier mice was mediated by tolerized immune cells and was therefore transferable to naïve mice, splenocytes from control or HBV-carrier mice were adoptively transferred into Rag1-deficient (Rag1−/−) recipient mice, which were then vaccinated with HBsAg. Adaptive immunity was successfully established in Rag1−/− mice (Supporting Fig. 3). As expected, control splenocytes successfully produced serum anti-HBs Abs after HBsAg vaccination and controlled HBsAg and HBeAg levels after pAAV/HBV1.2 plasmid injection in Rag1−/− recipient mice; however, splenocytes from HBV-carrier mice did not produce anti-HBs Abs, and serum HBsAg and HBeAg levels were significantly higher than control splenocytes (Fig. 2A-C), indicating that splenocytes transferred from HBV-carrier mice did not confer immunity to HBsAg in recipient mice. This transferred humoral tolerance was antigen specific, because anti-ovalbumin (OVA) IgG production was not inhibited (Fig. 2D). Similar results were obtained for anti-HBs Ab production when donor splenocytes were isolated at weeks 2 and 12 postinjection from HBV-carrier mice, suggesting that HBV-specific humoral tolerance lasted at least 12 weeks in HBV-carrier mice (Fig. 2E).

image

Figure 2. HBV-specific tolerance could be transferred into naïve Rag1−/− mice. (A) At day 4 after splenocytes from control or HBV-carrier mice (4w) transfer, recipient Rag1−/− mice were immunized with HBsAg vaccine and serum anti-HBs Ab levels were measured. Next, these Rag1−/− mice were hydrodynamically injected with pAAV/HBV1.2 at day 14 after HBV vaccination, and serum HBsAg (B) and HBeAg (C) levels were determined at the indicated time points. (D) Rag1−/− mice received an IV transfer of splenocytes from HBV-carrier or control mice, followed by a subcutaneous immunization of OVA in CFA emulsion 1 day later. One week later, serum anti-OVA IgG levels were determined by enzyme-linked immunosorbent assay. (E) Treatment was performed as (A) donor splenocytes from control or HBV-carrier mice at week 2 or 12 after HBV plasmid injection. Results are shown as mean ± SEM (n = at least 5/group) and represent one to three independent experiments.

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The Kinetic Relationship of Liver HBV Persistence With Humoral Tolerance

To further test the kinetic relationship of liver HBV persistence with humoral tolerance, HBV-carrier mice 1 day, 1 week, and 12 weeks after pAAV/HBV1.2 injection received IM or intrasplenic immunization with HBsAg, respectively (Fig. 3A). Similar to the result shown in Fig. 1, HBsAg vaccination neither reduced serum HBsAg nor developed the production of anti-HBs Ab in these HBV-carrier mice, which became HBV persistent already for 12 weeks (Fig. 3B,C). The same results could be observed when the mice kept their HBV persistence for 1 week (Fig. 3D,E). However, when the time point of HBsAg vaccination was shortened to 1 day after HBV plasmid injection, HBV-specific tolerance could not be established because HBsAg decreased and anti-HBs Ab could be detected, indicating 1 day was too short to establish the tolerant state (Fig. 3F,G). To explore the exact time point when splenocytes acquired the ability to inhibit anti-HBV immunity in HBV-carrier mice, splenocytes isolated at different time points from HBV-injected mice were transferred into recipient Rag1−/− mice. At day 10 after HBV-plasmid injection, splenocytes from these mice exerted the greatest inhibitory effect on anti-HBs production (Fig. 4B{FIG4) and GC B-cell generation (Fig. 4C), though the splenocytes from day 7 HBV-plasmid-injected mice also exhibited strong inhibitory effects, indicating that the time required for splenocytes to induce humoral tolerance toward HBsAg was approximately 7-10 days after pAAV/HBV1.2 injection.

image

Figure 3. HBV tolerance persists at least 12 weeks in HBV-carrier mice. (A) Treatment was performed as described in (B)-(G). (B) Twelve weeks, (D) 1 week, or (F) 1 day after pAAV/HBV1.2 injection, HBV-injected mice were immunized IMy with 1 μg of HBsAg vaccine or phosphate-buffered saline (PBS) at days 0 and 14. (C) Twelve weeks, (E) 1 week, or (G) 1 day after pAAV/control or pAAV/HBV1.2 injection, HBV-injected and control mice received a single intrasplenic immunization with 1 μg of HBsAg/CFA at day 0. Serum HBsAg or anti-HBs levels were monitored by IRMA at indicated times. Results are presented as mean ± SEM and represent at least two independent experiments (n = 5-10 mice/group).

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image

Figure 4. Induction of HBV tolerance needs approximately 7-10 days after HBV plasmid injection. (A) Treatment was performed to (B)-(C). (B) Recipient Rag1−/− mice received splenocytes isolated at days 0, 3, 5, 7, and 10 postinjection from HBV plasmid-injected mice. (C) GC B-cell (B220+Gl-7+Fas+) frequency in draining lymph nodes was examined by FCM. Serum anti-HBs levels and FCM were performedat day 15 after cell transfer. Results are presented as mean ± SEM.

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KCs Play a Crucial Role in Inducing Liver Tolerance

Antigen-specific humoral tolerance requires help from APCs. The liver contains a large KC population, which expresses relatively low levels of costimulatory molecules, and KCs are potent inducers of immune tolerance.[19] To explore the role of KCs in inducing systemic tolerance, mice were transiently depleted of KCs by clodronate liposome treatment on day −2 preceding hydrodynamic injection of HBV plasmid. Strikingly, this treatment resulted in almost complete HBV elimination within 4 weeks (Fig. 5A), along with significantly increased serum anti-HBs Ab levels (Fig. 5B), after HBV plasmid injection in WT mice, demonstrating a failure to establish an HBV persistence resulting from losing HBV-specific humoral tolerance in KC-depleted mice. However, the same treatment did not alter serum HBsAg levels in Rag1−/− mice, ruling out the possibility that KC depletion may directly influence HBV plasmid transfer (Fig. 5C). KCs depleted at day 1, but not day 7, after pAAV/HBV1.2 injection could influence the tolerant state of HBV-carrier mice (Fig. 5D). These data indicated that KCs induced humoral tolerance at an early stage, which was consistent with the observation that establishment of humoral tolerance needs approximately 7-10 days after HBV plasmid injection (Fig. 4).

image

Figure 5. HBV persistence was broken in KC-depleted mice. (A and B) C57BL/6 mice or (C) Rag1−/− mice received a single IV injection of clodronate liposomes (125 μL) or control phosphate-buffered saline liposomes, followed by HDI of pAAV/HBV1.2 2 days later. Serum levels of HBsAg (A and C) and anti-HBs antibody (B) were determined at indicated time points after HDI. (D) C57BL/6 mice received a single IV injection of clodronate liposomes at day 1 or 7 after HDI. Serum HBsAg levels were determined at indicated time points after HDI. Results are presented as mean ± SEM and represent at least two independent experiments (n = 5-10 mice/group).

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Furthermore, splenocytes from KC-depleted, HBV-injected mice lost the capacity to adoptively deliver humoral tolerance into Rag1−/− mice, showing significantly increased anti-HBs Ab production in recipient mice after HBsAg vaccination (Fig. 6A), suggesting that KCs are crucial for initiating antigen-specific immune tolerance. To exclude the influence of other phagocytes (e.g., macrophages and dendritic cells), which were also depleted by clodronate liposomes, and to determine whether KCs were sufficient to induce tolerance, we transferred purified KCs from HBV-carrier mice into naïve mice, which were then vaccinated with HBsAg. As expected, KCs from HBV-carrier mice exerted greater inhibition on anti-HBs Ab production in recipient WT mice after HBsAg immunization (Fig. 6B).

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Figure 6. KCs from HBV-carrier mice induce humoral tolerance. (A) Splenocytes from control, HBV-carrier, or KC-depleted HBV mice were transferred into recipient Rag1−/− mice. (B) KCs from HBV-carrier or control mice were sorted and transferred IV into naïve mice, followed by immunization with the HBsAg vaccine 4 days later. Serum anti-HBs Ab levels were measured 1 week after immunization. KC purity (F4/80+) was >85%. Results are shown as mean ± SEM. Results were repeated twice, with 3-6 mice/group.

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IL-10 Plays a Crucial Role in KC-Mediated Liver Tolerance

Previous studies have shown that IL-10 played an important role in mediating liver tolerance in different models.[20, 21] We found that the KCs in HBV-carrier mice produced more IL-10 than that in control mice, especially in the early stage after HBV plasmid injection (Fig. 7A,B). To further investigate the role of IL-10 in KC-mediated systemic humoral tolerance in our model, IL-10-deficient (IL-10−/−) mice received HBV plasmid injection and were then immunized with HBsAg vaccine. Serum HBsAg levels became negative within 6 weeks of hydrodynamically injecting HBV plasmid (Fig. 7C), suggesting that IL-10 was required for developing HBV persistence. Moreover, HBV+ IL-10−/− mice rapidly eliminated HBV within 2 weeks when triggered with a single HBsAg vaccine (Fig. 7C), and almost all IL-10−/− mice (4 of 5) became anti-HBs positive thereafter (Fig. 7D), implying that IL-10−/− mice are more sensitive to triggering humoral immune response or have more difficulty maintaining HBV tolerance. In contrast to donor splenocytes from HBV-carrier WT mice, we found that donor cells from HBV-carrier IL-10−/− mice lost the ability to suppress anti-HBs Ab production in recipient Rag1−/− mice after HBsAg vaccination (Fig. 7E). Taking these data together, we hypothesize that KCs induce humoral tolerance dependent on their IL-10 production. Donor KCs from IL-10−/− HBV-injected mice resulted in more obviously increased anti-HBs Ab production in recipient Rag1−/− mice than donor KCs from WT HBV-carrier mice, suggesting that KCs contribute to systemic humoral tolerance in an IL-10-dependent manner (Fig. 7F). Furthermore, we found that exogenous injection of IL-10 could also effectively induce humoral tolerance. Serum levels of anti-HBs decreased significantly in both soluble IL-10-injected and pLive/IL-10 plasmids-injected mice, especially in IL-10 plasmids-injected mice, which may be the result of persistent expression of human IL-10, which can also be active on murine cells (Fig. 8A-D).

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Figure 7. IL-10 plays a critical role in inducing liver tolerance in HBV-carrier mice. (A) After HBV plasmids injection, KCs were sorted to detect mRNA by quantitative reverse-transcription PCR at days 5 and 30. (B) Two weeks after HBV plasmid injection, KCs were sorted and cultured in vitro. Supernatants were collected and measured against IL-10. (C) WT or IL-10−/− mice were injected with pAAV/HBV1.2 plasmid. Two weeks later, mice were vaccinated once with HBsAg. Serum HBsAg levels were measured at indicated time points. (D) Mice were treated as described in (C). Serum anti-HBs Ab levels were measured 6 weeks after pAAV/HBV1.2 injection. (E) Splenocytes from WT, HBV-carrier, or IL-10−/− HBV mice were transferred into recipient Rag1−/− mice. (F) Treatment was performed as described in Fig. 6B, with KCs from HBV-carrier or IL-10−/− HBV mice. Data represent mean ± SEM. Results were repeated twice, and at least 5 mice were used for each group.

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image

Figure 8. Exogenous IL-10 could effectively induce humoral tolerance after HBsAg vaccination. (A) Treatment was performed as described in (B)-(D). Mice received 100 ng of murine IL-10 (i.p.) or 20 μg of pLive/IL-10 plasmids (HDI) as described. Serum levels of anti-HBs were measured at weeks 1 (B), 2 (C), and 3 (D), respectively, after HBsAg vaccination. Results are presented as mean ± SEM. i.p., intraperitoneally.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

The liver has recently been considered to be an immune organ characterized by a predominance of innate immune cells that manifests not only in autonomous tolerance, but also in inducing systemic tolerance.[10, 20, 21] In this study, we employed a nontransgenic (Tg) model of HBV tolerance generated by hydrodynamic injection of pAAV/HBV1.2 plasmid into adult C57BL/6 mice,[14] which has been extensively used.[22] Liver HBV persistence could obviously suppress humoral responsiveness to peripheral HBsAg vaccination, showing the lack of anti-HBs Ab production (Fig. 3). Among the period of initiation of humoral tolerance, transient absence of KCs broke the HBV persistent state and HBsAg levels decreased significantly. Furthermore, KC-derived IL-10 played a critical role in KC-mediated induction of humoral tolerance toward HBsAg, because KCs and splenocytes from IL-10−/− mice lost inhibition on anti-HBs Ab production after HBsAg vaccination (Fig. 7). Therefore, systemic humoral tolerance was induced by persistent liver virus through a KC- and an IL-10-dependent manner.

In previous liver tolerance studies, tolerance was experimentally induced in vivo by a single protein component; however, this does not accurately model that the liver encounters numerous antigens derived from food and liver-targeting viruses. These models include the concanavalin A model,[20] the liver myelin basic protein Tg mouse model,[10] liver-targeting gene transfer of human alpha-1 antitrypsin by adenovirus,[21] and direct administration of antigen into the liver through the portal vein.[23] In our study, we used a hepatotropic virus model, and what it revealed for liver tolerance may be more in line with actual chronic HBV infection conditions. Our results further suggest that more careful attention should be paid in the field to the differences and interpretation between experiments using single protein-induced tolerance and intact virus-induced tolerance. For instance, whereas mice receiving HBcAg-mutated HBV plasmid exhibited higher and longer HBsAg expression than pAAV/HBV1.2 plasmid,[24] approximately 20%-30% of HBV-carrier mice carry HBV for >6 months, which is closer to natural HBV infection. Thus, models using single antigen-induced liver tolerance may not be appropriate to explain the mechanisms underlying intact virus-induced chronic infection. It is the first study on liver immune tolerance that uses a hepatotropic persistent virus model. The HBV-carrier mice in our study represent the best model to date for studying liver tolerance.

Using this hepatotropic persistent virus model, we first described in detail about the kinetic relationship between liver HBV persistence and establishment of HBV-specific humoral tolerance. We found the humoral tolerance could last to 3 months and would need 7-10 days for induction after HBV plasmid injection (Figs. 3 and 4). At day 7 postinjection, HBV-injected mice lost humoral immune responses after HBsAg vaccination (Fig. 3D,E). However, splenocytes isolated at day 7 postinjection still kept humoral response toward HBsAg, although the titers of anti-HBs levels and the frequency of GC B cells decreased significantly after HBsAg vaccination. These data indicated that mice that acquired humoral tolerance needed 7 days, but 10 days was necessary for splenocytes to obtain tolerant function, suggesting the tolerant mediators did not develop in the spleen and could infiltrate into splenocytes. Through KC depletion at different time points to evaluate HBV persistence, we showed that KCs played a critical role in initiating humoral tolerance within 7 days after HBV plasmid injection. Based on the fact that KCs are liver-resident cells and do not migrate into lymph nodes, we speculated that KCs promote the generation of tolerant mediators in the liver, which directly mediate humoral tolerance toward HBsAg vaccination.

To explore the tolerant mediator induced by persistent antigen in liver, different mouse models provide evidence to support the theories of “clonal deletion” or “regulatory cells.”[25, 26] Accumulating evidence suggests that the liver serves not only as a “graveyard” to kill effector immune cells,[25, 27, 28] but also as a “school” to educate regulatory immune cells to suppress immune responses[21, 25, 29] to persistent liver-expressed antigens during systemic tolerance. In our model, the mechanisms of humoral tolerance induced by liver HBV persistence may be deletion of HBV-specific clones, induction of HBV-specific regulatory cells, or both. KCs and IL-10 are indispensable in this process. We showed evidence that IL-10 was necessary in KC-initiated humoral tolerance (Fig. 7F). The precise mechanism needs further investigation.

Although anti-HBs alone cannot protect hepatocytes from HBV infection by the hydrodynamic injection (HDI) method, HBeAg, as well as HBsAg, was also decreased in HBsAg vaccine-immunized mice (Supporting Fig. 2C). We speculate that not only Ab-producing B cells were activated after HBsAg vaccination, but also T cells or other immune cells were activated and involved in eliminating HBV-infected hepatocytes. Because both serum levels of HBeAg and HBsAg decreased significantly in HBV+Rag1−/− recipient mice after receiving CD19 splenocytes, but not CD19+ B cells, from HBsAg-vaccinated mice (Supporting Fig. 4).

Our HBV-carrier mice express almost all HBV components with high serum HBsAg and HBV DNA levels, but without liver inflammation or hepatitis B e antibody or anti-HBs Ab production. This indicates that the model used here is a good mimic for human chronic HBV infections in the immune-tolerant phase, which are usually observed more in childhood HBV infection. Generally, treating patients with chronic hepatitis B in the immune tolerant stage is not recommended, because antiviral (e.g., interferon-alpha and nucleoside/nucleotide analogs) or immunomodulatory therapy is ineffective in clearing HBeAg or HBV-DNA and may influence the natural disease course.[30] Our results suggest that KCs and related molecules (e.g., IL-10) are possible therapeutic targets to reverse immune tolerance in chronic infection by hepatotropic pathogens, including HBV.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
hep26668-sup-0001-suppfigure1.tif3047KsFigure 1. Establishment of an HBV persistent mouse model. (A) C57BL/6 mice were hydrodynamically injected with 6 μg of either pAAV/HBV1.2 or pAAV/control plasmid. Serum HBsAg titers were measured at the indicated time points using immunoradiometric assay (IRMA). (B) The positive detection rate of serum HBsAg levels at different time points in mice that received 6 μg of HBV plasmid. (C) Correlation between serum HBsAg titer and HBV DNA copy number. HBV DNA copy number was determined using quantitative PCR. (D) Liver sections were processed for HBcAg expression and (E) H&E staining at 6 wpi (original magnification: 100×).
hep26668-sup-0002-suppfigure2.tif1934KsFigure 2. Pre-existing anti-HBV immunity prevents HBV persistence in the liver. (A) C57BL6 mice were immunized intramuscularly with either 1 μg of HBV vaccine or control PBS within a 2-week interval; 1 week later, they were hydrodynamically injected with HBV plasmid as described for establishing HBV-carrier mice. Serum samples were collected and analyzed for HBsAg (B) and HBeAg (C) by IRMA at the indicated time points. (D) Immunohistochemical staining for HBcAg in the livers of HBsAg- or PBS-immunized mice at 6 wpi (original magnification: 100×). Data are expressed as mean ± SEM (n = 10 mice/group) and represent 3 independent experiments that gave similar results.
hep26668-sup-0003-suppfigure3.tif766KsFigure 3. Reconstitution of adaptive immune cells after cellular transfer. Splenocytes were isolated from HBV-carrier and control mice and were transferred intravenously (5 × 107) into Rag1-/- recipient mice. T and B cell percentages were shown 1 week after splenocyte transfer as determined by FACS analysis.
hep26668-sup-0004-suppfigure4.tif796KsFigure 4. Spleoncytes from HBsAg vaccinated mice decrease serum HBsAg and HBeAg levels in HBV+Rag1-/- recipient mice. Donor mice were immunized with HBsAg vaccine or PBS as control. Donor cells were divided into 4 parts: total splenocytes from control mice; total splenocytes from HBsAg vaccinated mice (anti-HBV); CD19+ cells of splenocytes purified by MACS from HBsAg vaccinated mice; and CD19- cells of splenocytes from HBsAg vaccinated mice. These cells were transferred into HBV+Rag1-/- mice respectively. Two weeks after cells transfer, the serum levels of HBsAg (A) and HBeAg (B) were detected in recipient mice. Data are expressed as mean ± SEM. The numbers above bars in Fig. B mean the number of HBeAg negative mice within total recipient mice.

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