Correspondence Bor-Luen Chiang, Department of Medical Research, National Taiwan University Hospital, Floor 7, No. 7, Chung-Shan S. Road, Taipei, 100, Taiwan. Tel: 886 2 2312 3456 ext. 67302; fax: 886 2 2311 9087; e-mail: email@example.com
Hepatitis B virus surface antigen (HBsAg) is a major antigen of hepatitis B virus (HBV). Dendritic cells (DC) of HBV carriers have been reported to exhibit functional impairment. In this study, the role of HBsAg on mice bone marrow-derived dendritic cells and immune responses in vivo was studied. The immune modulatory function of HBsAg was explored by using mice bone marrow-derived dendritic cells in vitro and also by examining an ovalbumin (OVA) specific immune response in vivo. Treatment of dendritic cells with HBsAg resulted in enhanced cell surface expression of cluster of differentiation (CD) 80, CD83, CD86, and major histocompatibility complex (MHC) class II, and enhanced production of interleukin (IL)-12 p40 and IL-12 p70. Treatment of dendritic cells with HBsAg resulted in decreased T cell secretion of IL-5 by OVA stimulation. In addition, the results showed stronger OVA-specific immunoglobulin (Ig) M and weaker IgG responses in mice sera when they had been immunized with OVA and co-injected with HBsAg. It was also found that the mice exhibited significant enhancement of anti-OVA IgG2a antibody (Ab), as well as marked inhibition of IgG1 Ab production. In cellular immune responses, IL-5 production was significantly decreased and interferon (IFN)-γ increased in the group co-injected with HBsAg. On the other hand, the induction of lymphoproliferative response to OVA stimulation in spleen cells was decreased in the HBsAg co-injected group. These results demonstrate that HBsAg can affect the differentiation of T helper (Th) cells, which might provide a strategy for improving its prophylactic and therapeutic efficacy.
The hepatitis B virus is one of the most prevalent human pathogens. More than 350 million people worldwide are chronically infected and around 65 million of them die from cirrhosis or liver cancer eventually. Moreover, in both acutely and chronically infected persons, cellular and HBsAg responses are impaired despite the presence of high titers of HBsAg in sera (1). Chronic HBV carriers are unable to initiate an adequate immune response to HBsAg. Defects in the antigen-presenting activity of DCs, rather than functional defects in T or B cells, are held responsible for the induction of HBV persistence (2, 3). The mechanisms by which HBV establishes persistent infection remains unclear. Both the reason for the high titers of HBsAg, and the possible advantages of its production, remain elusive.
DCs are powerful APCs with the primary function of capturing, processing, and presenting antigens to naive T cells (4, 5). Although fully mature DCs show strong surface expression of MHC class II and co-stimulatory molecules (CD80 and CD86), they have a reduced capacity to internalize antigens (6). The maturation of DCs is critical for the induction of Ag-specific T lymphocyte responses and might be essential for the development of human vaccines relying on T cell immunity.
Immune modulation by HBsAg of cellular and immunological responses during HBV infection has received scant attention. Some studies suggest that HBsAg interacts with one or more receptors on antigen-presenting cells such as monocytes, macrophages and DCs (7). According to other studies, the tendency towards atopy is increased in hepatitis B virus carriers (8). It has likewise been speculated that large amounts of HBsAg might induce T cell anergy, leading to decreased antibody-mediated neutralization of HBV and generalized hyporesponsiveness toward pathogens. One cross-sectional survey showed a decreased risk of atopic disease in individuals vaccinated against hepatitis B virus (9). The possible mechanisms are improved antigen presentation by APCs and virus elimination by increased IL-12 secretion (10). However, a recent study has demonstrated that whole recombinant yeast-HBsAg induce protective HBsAg-specific Th 1 and Th2 immune responses (11). These data suggest, nonetheless, that HBsAg act as an immune modulator.
HBsAg, one of the major antigens of hepatitis B virus, is secreted from infected hepatocytes. Unfortunately, the role of HBsAg in immune modulation has not been well studied. In the present study, we explored the immune modulatory function of HBsAg by using mice bone marrow-derived dendritic cells in vitro and examining an OVA specific immune response in vivo. We demonstrated that HBsAg induces maturation of BMDCs and promotes Th1 immune responses. BMDCs expressed higher MHC class II and costimulatory molecules in the presence of HBsAg. Furthermore, increased IL-12p40 and p70 were secreted by BMDCs co-cultured with HBsAg. HBsAg decreased the production of IL-5 cytokine induced by OVA stimulation. Using an in vivo OVA immunization model, we showed that HBsAg altered OVA-induced Th2 immune response towards Th1. Collectively; our findings suggest that HBsAg could act as an immune modulator and play a critical role in initiating immune response to HBV vaccine.
MATERIALS AND METHODS
HBsAg was purified from serum of hepatitis B patients by SDS-PAGE (Chemicon International Serologicals, Temecula, CA, USA) to a purity of greater than 99%. HSA was purified from human serum by SDS-PAGE (Sigma-Aldrich, St. Louis, MO, USA) to a purity of greater than 97%. OVA was Grade V (Sigma Chemical, St. Louis, MO, USA).
Preparation of bone marrow derived-dendritic cells
Bone marrow derived-dendritic cells were prepared as described previously (12). Briefly, bone marrow cells from the femurs and tibias of 6–8 week female BALB/c mice were depleted of red cells by using RBC lysis buffer. Approximately 1.5 × 106 cells were placed in 24 well plates in 1 mL medium supplemented with recombinant murine granulocyte-macrophage colony-stimulating factor (750 U/mL) and IL-4 (1000 U/mL, Pepro Tech, Rocky Hill, NJ, USA). The culture medium was RPMI-1640 medium supplemented with 5% heat-inactivated FCS, 4 mM L-glutamine, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2), 50 μM 2-mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin (37°C/5% CO2). On day 3, half of the medium was changed. On day 5, the BMDCs were collected for further analysis. Different doses of HBsAg were added to the BMDC cultures for a further 48 hr.
Production of the cytokines IL-10, IL-12 p70, and IL-12 p40 was measured by ELISA in the supernatants of dendritic cells. Supernatants were analyzed for IL-5 produced by activated T cells after 3 days of culture. (R&D Systems, Minneapolis, MN, USA).
Flow cytometric analysis for surface markers
Dendritic cells were harvested and washed with cold buffer (PBS containing 2% FCS and 0.1% sodium azide). Cells were then incubated in cold buffer and subsequently stained with FITC or PE-labeled mAbs (anti-mice CD86, CD80, CD83, MHC class II or relevant isotype controls; BD PharMingen, Tokyo, Japan) according to the manufacturer's instructions. DC surface marker expression was analyzed using the CellQuest program, dead cells being excluded.
Autogenic mixed leukocyte reaction
BMDCs were obtained as described above. A single-cell suspension was prepared from splenocytes according to standard laboratory procedures. CD4+T cells were purified from suspension using anti-mouse CD4+ magnetic particles (BD Biosciences, Franklin Lakes, NJ, USA). The autogenic CD4+T cells thus obtained were distributed at 2 × 105 cells per well and incubated for 3 days in the presence of 1 × 105 stimulated BMDCs.
Mice and immunization
Female BALB/c mice were bred and maintained in the animal center of the College of Medicine, National Taiwan University. Animal care and handling protocols were approved by the Animal Committee of National Taiwan University. Only female mice of 6–8 weeks were used for intraperitoneal injection. In brief, groups of five mice were anesthetized and injected intra-peritoneally with different agents on three occasions: on days 0, 14, and 28. Group 1 was injected with 200 μL PBS and served as a negative control; group 2 with 50 μg OVA in PBS; group 3 with 50 μg OVA and 5 μg HBsAg in PBS; group 4 with 50 μg OVA and 50 μg HBsAg in PBS; group 5 with 50 μg OVA and 50 μg HSA in PBS; and group 6 with 50 μg HBsAg in PBS.
Determination of antibody concentrations
Serum samples were collected on days 0, 28, and 35 and analyzed for the presence of OVA-specific Abs. Micro-titer plates were coated with 10 μg OVA. After incubation with 200 μL of 1% BSA in PBS in each well overnight at 4°C to prevent non-specific binding, 100 μL dilutions of test sera were added to each well and incubated overnight at 4°C. After the samples had been washed with PBS, bound anti-OVA isotype Abs were detected with biotin-conjugated goat anti-mouse IgM or IgG. Avidin- horseradish peroxidase (1:10000) was then added. Color was developed by adding substrate (H2O2 and tetramethylbenzadine), and absorbance at 450–540 nm was then measured. For the measurement of IgG1 and IgG2a anti-OVA isotype Abs, a method was similar to that described above was employed.
Assay for lymphocyte proliferation and cytokine secretion
To determine whether OVA-specific lymphoproliferative responses had been induced in immunized animals, their spleens were removed 2 weeks after the last intra-peritoneal injection in order to make single-cell suspensions. To perform the lymphoproliferative assay, 100 μL of 3 × 106/mL splenocytes in complete RPMI was added in triplicate to each well in 96-well flat-bottom plates. Stimulated wells received OVA at a concentration of 50 μg/mL, while control wells received cells only. Cells in all wells were cultured in a total volume of 200 μL of medium. After 4 days in culture, the cells were pulsed with [3H] thymidine (1 μCi/well) for 18 hr and harvested with FilterMate (Packard, Waltham, MA, USA). The stimulation index was calculated as the mean counts per minute of stimulated wells divided by the mean counts per minute of the control wells.
To measure cytokine concentrations, splenocytes were cultured as described above with stimulation at the same OVA concentration for 48 hr. Cell-free supernatants were harvested and assayed immediately or stored at −80°C. These supernatants were screened for the presence of IFN-γ and IL-5 using the ELISA detection systems as per the manufacturer's instructions (R&D).
Student's t-test was used to analyze the results. A P value of less than 0.05 was considered to be statistically significant.
Hepatitis B virus surface antigen induces interleukin-12p40 and interleukin-12p70 production in mice dendritic cells in vitro
To determine whether HBsAg can stimulate cytokine production by BMDCs, we assayed cytokine concentrations in the supernatants of DCs cultured with different doses of HBsAg. When murine DCs were treated with 5, 10, 20, 40, and 50 μg/mL HBsAg for 48 hr, we found that HBsAg significantly enhanced the production of IL-12 p40 and p70 (Fig. 1a and b). On the contrary, HBsAg did not enhance the production of IL-10 (data not shown). It was noteworthy that the stimulatory effect of HBsAg, both on IL-12 p40 and IL-12 p70 production, was dose-dependent.
Hepatitis B virus surface antigen increases the expression of cluster of differentiation 80, 86, and 83 and major histocompatibility complex class II molecules on the cell surface of dendritic cells
In this study, we investigated the effect of HBsAg on the development of BMDCs in vitro. BMDCs were incubated with HBsAg for 48 hr and surface expressions of CD80, CD86, CD83 and MHC class II molecules were detected by FACS. We found that HBsAg increased the expression of CD80, CD86, and MHC class II molecules on the cell surface of DCs (Fig. 2).
Effect of hepatitis B virus surface antigen on T cell cytokine production
In BMDCs, we found that HBsAg up-regulated cell surface markers and increased IL-12 production. To test whether this maturation was sufficient to modulate CD4+ T cells, OVA-pulsed BMDCs were treated with or without HBsAg. These cells were then cocultured with autogenic CD4+T cells. The production of cytokines was then analyzed. It has been established that subsets of Th cells can be distinguished by their patterns of cytokine profile: Th1 cells produce IFN-r and IL-2, whereas Th2 cell produce IL-4, IL-5, IL-10 and IL-13 (13). When CD4+T cells were incubated with OVA-pulsed DCs, production of IL-5 was observed, but production of IL-5 was not found when they were cultured with HBsAg. However, HBsAg-treated DCs inhibited OVA-induced IL-5 in the culture supernatant (Fig. 3) HBsAg stimulation did not significantly affect the secretion of IFN-γ and IL4 in the culture supernatant (data not shown).
Hepatitis B virus surface antigen modulates antibody responses to ovalbumin in vivo
To extend our in vitro findings that HBsAg can modulate DC phenotype and to establish whether immune modulation by HBsAg holds true in vivo, OVA-induced immune response in the presence of HBsAg was investigated. Groups of five mice were injected with various agents. Mice receiving 5 and 50 μg HBsAg were used for comparison. Sera obtained from each mouse at days 0, 28 and 35 were used for assessment of IgM and IgG anti-OVA Ab-responses. As shown in Figure 4a, mice given HBsAg and OVA had higher titers of OVA-specific IgM antibody compared to mice receiving PBS, OVA only or co-injection with HSA, but these differences were not significant. In contrast, we found that the mice which had been co-injected with HBsAg showed decreased anti-OVA IgG Ab titers compared to those which had received OVA only and had been co-injected with HSA (Fig. 4b). The stronger anti-OVA IgM Ab response we obtained is consistent with the high immunogenicity we found when HBsAg was co-injected in the acute phase. On the other hand, significantly lower anti-OVA IgG Ab concentrations were noted after co-injection with HBsAg, which therefore modified the humoral immune response against OVA antigen.
Hepatitis B virus surface antigen induces greater concentrations of Th1 cytokine
Splenocytes were obtained from five mice per group at week 6, after the final intraperitoneal injections of the different regimens. Five hundred μL of 10 × 106/mL splenocytes was stimulated with OVA (50 μg/mL). Cytokine profiles of OVA-stimulated splenocytes were compared among groups of mice immunized with OVA with or without HBsAg co-injection. IFN-γ concentrations in the HBsAg co-injection supernatants were increased compared to those of the control group (Fig. 5a). In contrast, IL-5 concentrations were lower in the HBsAg group compared to the control group (Fig. 5b). Thus, cytokine production was affected by HBsAg co-injection, the results indicating a bias toward Th1 immunity. Co-injection with HBsAg promoted Th1 and downregulated Th2 development. The dose of HBsAg seems to be crucial; a smaller dose (5 μg) results in an increase in IFN-γ production, whereas a larger dose (50 μg) results in a decrease in IL-5 production. Higher doses of HBsAg may induce immune regulatory mechanisms and feedback effects.
IFN-γ and IL-5 production seems to be have been enhanced in splenocytes from the mice treated with OVA 50 μg + HSA 50 μg (Fig. 5), but not significantly so. HSA, a protein antigen, may induce lymphocyte activation or tolerance. The means of precise immune modulation need to be further investigated.
Ovalbumin-specific IgG isotype after immunization
The patterns of antibody isotypes produced in response to immunization are also reliable indicators of the types of cytokines produced in vivo. IgG2a is produced as a consequence of Th1 cell activation and IFN-γ secretion, whereas IL-4 enhances the production of IgG1 and suppression of IgG2a (14). We therefore measured anti-OVA IgG isotypes at week 7 after immunization in the sera of mice which had been treated with co-injection of HBsAg and HSA. Profound differences were observed in the IgG isotypes. As shown in Figure 3, co-injection with HBsAg resulted in a dramatic increase in anti-OVA IgG2a Ab (Fig. 4d), whereas titers of OVA-specific IgG1 Ab were decreased (Fig. 4c), indicating enhancement of Th1 and suppression of Th2 cell function in animals co-injected with HBsAg.
Lymphoproliferative response of spleen cells
With the aim of evaluating the capacity of HBsAg to modulate cellular responses to OVA in mouse spleen cells, we assessed the specific lymphoproliferative response against OVA 2 weeks after immunization. The effects of HBsAg against OVA on cellular proliferation were measured. We found that co-injection with HBsAg induced a decrease in lymphoproliferative response in spleen cells, as compared with OVA only and with co-injection with HSA.
Specific lymphoproliferative responses against OVA were measured at 2 weeks after the third dose in order to study the cellular response. The results showed that administration of HBsAg was able to modify the lymphoproliferative response in spleen cells 2 weeks after the last immunization (Fig. 6). The results indicate that the effect of HBsAg on the lymphoproliferative response to OVA stimulation is inhibited. The presence of HBsAg serves as an important interaction for the immune response.
HB vaccines containing HBsAg became available in the early 1980s. They are highly immunogenic and efficacious, resulting in marked declines in the carrier rate (15, 16). Moreover, they cause antiviral and immunomodulatory effects in both human and murine HBV carriers (17, 18). To clarify this, we investigated the effects of HBsAg on BMDCs in vitro, where the biological process of DC maturation is a crucial step in the initiation of an adaptive immune response (19). Adaptive immune response is regulated by a variety of extra-cellular stimuli, including cytokines; bacterial, fungal and viral products; and membrane-bound ligands (20–22). DC maturation is accompanied by changes in their morphologic, phenotypic, and functional properties. In this study, we found that HBsAg can induce maturation of DC, this maturation being characterized by increased cell surface expression of MHC class II and co-stimulatory molecules, as well as by the production of IL-12. Production of IL-12 by DCs is an early event in immune responses (23) and it provides a link between the innate and adaptive immune systems. The response plays a central role in initiating a specific T cell-mediated immune response (24, 25), driving Th1 cell activation and differentiation (26, 27) and inducing the production of IFN-γ and lytic activity (28, 29). Further, HBsAg inhibits OVA-induced Th2 cytokine production by CD4+T cells, and may favor the orientation of the response toward a Th1 phenotype.
Because HBsAg-host interactions may be more complicated in an in vivo system, we decided to investigate whether the immune response of BMDCs to HBsAg exerts immune modulation in the mouse model. To our knowledge, there are no published reports concerning modulation by HBsAg of other antigens in the mouse model. It is important to characterize this relationship in order to provide insights towards solving the immunogenicity effect of HBsAg. Therefore, the experiments presented here were designed to study the effects of HBsAg modulation on the immune response against OVA in a mouse model.
The effects of HBsAg on OVA specific immune responses were analyzed. After co-injection of HBsAg and OVA intra-peritoneally, a statistically significant change in the OVA-specific serum IgG antibody response was observed, as compared to that obtained when OVA was administered alone. This clearly demonstrates an immunogenic effect of HBsAg over OVA. Isotype analysis of the specific antibodies induced by HBsAg and OVA was also carried out by evaluating the major IgG subclasses in sera for all of the experimental groups. Interestingly, while HBsAg induced a predominantly specific IgG2a response, the IgG1 concentration was decreased. In addition, a Th1-related IgG2a pattern was not affected by co-injection with HSA. In this study, the findings of increased production of IFN-γ and decreased production of IL-5 demonstrated that cytokine production is affected by co-injection with HBsAg in such a way as to indicate a bias toward Th1 immunity. These data suggest that HBsAg co-injection promotes Th1, but downregulates the Th2 immune response. Similar results were also obtained in our previous studies (30, 31). However, these interesting observations need further investigation before these agents can be made part of anti-viral therapeutic management.
In this study, co-injection with HBsAg not only modified the level of antibody response, but also changed cellular immune responses to OVA. The lymphoproliferative response to OVA stimulation mediated by co-injection with HBsAg was decreased. Because memory lymphocytes had already been primed by OVA to Th2 immune response, the mechanism by which HBsAg induced decreased memory lymphoproliferative responses might have been the result of a combined effect of the HBsAg on the uptake of OVA by DCs and on the level of IFN-γ and IL-5 production.
Heterogeneity of immune response was observed with the presence or absence of HBsAg. Although the exact mechanism behind the co-injection regimen requires active investigation, there are several possible explanations. First, exogenous protein antigen can have access to alternative processing pathways and can present as an extended repertoire of antigenic epitopes (32). Second, the cross presentation ability of DC can be affected by Th1 cytokines. Studies have shown that oral immune regulation toward HBV envelope proteins induces a favorable increase in HBV specific T cell proliferation, cytotoxicity, and IFN-γ secreting clones, along with a significant decrease in anti- HBV IL-10 secreting T cell clones (33). We observed that HBsAg induced a Th1-biased pattern in related co-administered antigens, such as OVA in the mouse model.
It would be interesting to find out whether circulating HBsAg acts like a tolerogenic antigen in the HBV carrier, in which case injection of HBV carriers with vaccine containing HBsAg could induce a potent therapeutic effect. This question remains unanswered. One possible mechanism is that, during HBV infection, non-infectious sub-viral lipoprotein HBsAg particles are produced in large quantities by infected hepatocytes and secreted into the circulation, where concentrations are about 50 μg ∼ 300 μg/mL. The concentration of HBsAg might affect the immune responses. In this study, the effect of HBsAg on the functions of BMDCs was analyzed in vitro. The data clearly show that HBsAg can activate DCs. Second, other HBV Ag, such as HBeAg and HBcAg, may play a role in persistent infection by escaping immunologic survey. At the cellular level, studies in transgenic mice have shown that HBV viral antigen (e.g., HBeAg in the neonatal period, or HBcAg) may act as tolerogens, leading to the induction of an antigen specific suppressor T cell population (34).
In summary, the data from our experiments have significant clinical implications. The results here demonstrated the immune response enhancing ability of HBsAg, which might account for the efficacy of universal vaccination for HBV. Especially, further understanding the interaction between HBsAg and antigen presenting cells might shed light on future design of vaccine for HBV and other similar viral pathogens.
This study was supported by grants from the National Science Council of the Taiwan.