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- MATERIALS AND METHODS
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.
- Top of page
- MATERIALS AND METHODS
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.