Recognition of HBV antigens and HBV DNA by dendritic cells

Guang-Ying Cui and Hong-Yan Diao

Hangzhou, China

Recognition of HBV antigens and HBV DNA by dendritic cells

Guang-Ying Cui and Hong-Yan Diao

Hangzhou, China

BACKGROUND:Hepatitis B virus(HBV) is a hepatotropic, noncytopathic, DNA virus which can cause acute and chronic infection. Viral persistence is associated with a weak or absent specific immune responses to HBV, particularly the cellular immune response. Dendritic cells (DCs) are professional antigen-presenting cells with a unique T cell stimulatory aptitude that play a crucial role in the instruction of adaptive immune responses upon infection. An impaired function of DCs was suggested by recent studies to account for the T and B cell hyporesponsiveness in chronic HBV infection. This review summarizes recent insights into the recognition of HBV antigens by DCs.

DATA SOURCES:Studies were identified by searching MEDLINE and/or PubMed for articles using the key words "hepatitis B virus (HBV)", "dendritic cells", "C-type lectins", "mannose receptor", "toll-like receptor", and "dendritic cell-specific intercellularadhesion-molecule-3 grabbing nonintegrin (DC-SIGN)" up to December 2009. Additional papers were identified by a manual search of the references from the key articles.

RESULTS:DCs play an important role in the progress of hepatitis B, especially in the recognition of HBV. There are three main ways of recognition of HBV antigens by DCs. First, HBV DNA can be recognized by DCs through toll-like receptor 9 (TLR9) which activates the NF-κB signal pathway and p38 MAPK to up-regulate the expression of interferon (IFN) regulatory factor 7 (IRF-7) in a manner independent of typeiIFN signaling, resulting in secretion of typeiIFN and inflammatory cytokines, and induction of DC maturation and the adaptive immune response. Second, HBc/HBeAg cannot be recognized by DCs, but DNA or ssRNA encapsulated within HBcAg can be internalized by DCs through TLRs. Third,HBsAg can be internalized by DCs through the mannose receptor, which lacks the ability to induce DC maturation without the assistance of DC-SIGN. Meanwhile, there is some cross-talk among the three mechanisms, which induces an effective anti-viral response or HBV persistence.

CONCLUSIONS:On the basis of these recognition processes, methods have been used to enhance the efficacy of DC-based vaccine against HBV and have been useful in the clinical application of HBV vaccine therapy. But the interactions between HBV antigens/HBV DNA and DCs are not clear, and cross-talk between TLRs and various ligands makes HBV antigen recognition by DCs more complicated. More efforts should be made to define the mechanisms and develop effective vaccines and therapies.

(Hepatobiliary Pancreat Dis Int 2010; 9: 584-592)

dendritic cells; hepatitis B virus antigen; HBV DNA; toll-like receptor; mannose receptor

Introduction

Hepatitis B virus (HBV) infection is a global health concern and an economic burden. WHO reported that about 2 billion people worldwide are infected with the virus and about 350 million live with chronic infection. An estimated 600 000 persons die each year due to the acute or chronic consequences of hepatitis B. HBV infection leads to a broad spectrum of clinical manifestations including fulminated hepatic failure, cirrhosis, and hepatocellular carcinoma. It is now widely accepted that appropriate humoral and cellular immune responses are necessary for clearance of HBV infection.[1]And viral persistence is associated with weak or absent specific immune responses to HBV, particularly the cellular immune response. Studies have shown that CD8+T cells display poor proliferation and effector functions in chronic HBV-infected patients.[2,3]

Dendritic cells (DCs) have long been recognized as "professional'' antigen-presenting cells (APCs) with the potent capacity to initiate primary immune responses,and serve as a major link between innate and adaptive immunity.[4-7]DCs originate from the bone marrow and are able to capture and present antigens to the cells of the adaptive immune system. After sampling antigens, they migrate to the secondary lymphoid organs, where they initiate and regulate T and B cell immune responses by expressing high levels of lymphocyte costimulatory molecules and major histocompatibility complex molecules, and secreting biologically active molecules.[8,9]Because of these properties, DCs are being considered as an efficient means of immunization against viral infections such as HIV, herpes simplex virus, and papillomavirus.[10]And impaired function of DCs was suggested to account for the T and B cell hyporesponsiveness in chronic HBV infection by recent studies demonstrating the HBV infection of monocytederived DCs and a reduced expression of co-stimulatory molecules leading to impaired T cell allostimulation.[11]

There are at least two subsets of DCs: myeloid (mDCs) and plasmacytoid (pDCs). mDCs are the classic DCs characterized by the presence of CD11c, CD11b, CD13, and CD33, and the absence of surface expression of lineage markers.[12]mDCs are the primary APCs; they home preferentially to non-lymphoid tissues, where they are specialized in the uptake of invading pathogens and activation of naive T cells. After engagement of TLR2 and TLR4, they rapidly secrete proinflammatory cytokines.[13]On the other hand, pDCs are characterized by the presence of CD123 (IL-3 receptor a-chain), blood dendritic cell antigen-2 (BDCA-2) and BDCA-4, and without the lineage markers. They produce large amounts of typeiinterferons (IFNs), especially IFN-α, in response to viral challenge or other inducers such as cytidine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODNs).[12-14]The release of typeiIFNs initiates a cascade of events that eventually leads to viral clearance. Furthermore, IFN-α acts as a potent regulator of the innate and adaptive immune responses,[15]and is used for the treatment of chronic HBV patients, but the response rate is only about 30%.[16]

Properties of peripheral blood DCs from patients with chronic HBV infection

Studies have uncovered the reduced expression of costimulatory molecules, the impaired cytokine secretion, and the lower allostimulatory capacity of DCs from chronic HBV carriers compared to healthy controls.[8,10,17-20]However, these views were challenged by recent studiesin vitro. These studies demonstrated that the alloreactive and antigen-specific T cell stimulatory capacities of DCs from chronic HBV carriers are intact.[21,22]Tavakoli et al[22]indicated that circulating total DCs, mDCs or pDCs are not reduced in chronic HBV carriers. And in DC populations, both viral DNA and viral mRNA are undetectable by RTPCR, arguing against viral replication in DCs. Some researchers indicated that functional impairment of DCs in hepatitis B may be due to micro-environment changes in the liver and lymphoid organs after HBV infection, affecting the maturation of DCs and consequently affecting the functions of antigen presentation, leading to a lower level of specific cell-mediated immunity, and even defective specific cytotoxic T-lymphocytes (CTLs).[19,23]This seems reasonable; a functional defect of DCs would not be compatible with the general immunocompetence of chronic HBV carriers.[18,21]Thus, autologous DCs might represent fully functional and useful tools for future immunotherapy.

Interactions between DCs and HBV

Innate immune activation not only precedes adaptive immune activation, but also regulates the infectious process. And DCs form the pivotal link between the innate and the adaptive immune systems, by controlling the initiation of a diverse set of effector functions, which are suitable for the elimination of a wide range of pathogens. Thus direct interactions between DCs and HBV may explain the apparently impaired function of DCs and the ineffective anti-viral response resulting in HBV persistence in chronic HBV-infected patients.[24]

As is well known, HBV primarily infects hepatocytes. But more importantly, bothin vitroandin vivodata indicate that the virus also interacts with DCs. Some studies demonstrated the presence of HBV DNA in blood DCs from chronic HBV-infected patients.[21,23]Recently, Op den Brouw et al[24]showed that HBsAg is internalized by blood-derived mDCsin vitro, leading to impaired mDC function. What is more, consistent with those findings, this impaired mDC function improves upon viral load reduction.[25]

DCs have many surface receptors, including the wellcharacterized opsonic receptors for g-immunoglobulins,[26]the complement receptors, such as complement receptor 3 (CR3),[27,28]and an array of non-opsonic pattern recognition receptors (PRRs), such as the C-type lectin receptors (CLR) and TLR, which have evolved to recognize pathogenassociated molecular patterns (PAMPs),[29]including lipids, carbohydrates and proteins, mediating interaction with the host cell to regulate the immune response. Direct recognition of PAMPs is mediated by PRRs such as TLR and CLR. They differ in the recognition of antigens. TLR recognizes cell-wall components of bacteria, fungi, andprotozoa at the cell surface or nucleic acid structures of bacteria and viruses in a specialized endosomal compartment, while CLR binds to carbohydrate structures associated with pathogens.[30]Now we outline the recognition of HBV antigens by DCs.

Recognition of HBV DNA by DCs

Different classes of PRRs share PAMPs for recognition in the same or different cell types.[31]For example, RNA viruses are sensed by TLR7 on pDCs[32,33]and retinoic acid-inducible gene-I-like receptors in other cell types.[34,35]DNA viruses are sensed by TLR9[36-40]and the cytosolic DNA sensor,[41-44]and flagellin is sensed by TLR5[45]and IPAF-NAIP5.[46-49]HBV belongs to the family of hepadnaviruses. The HBV genome is a relaxed ring of partially double-stranded DNA of approximately 3200 base pairs.[50]These findings give us a clue that HBV DNA might be recognized by TLR9, but there is no evidence to establish this directly.

TLR9 is a typeitransmembrane protein (i.e. the N-terminal is outside the membrane) composed of three major domains and characterized by leucine-rich repeats in the ectodomain, which mediates the recognition of their respective PAMPs; there are also a transmembrane domain and an intracellular domain that is homologous to that of the IL-1R and is known as the toll/IL-1R (TIR) domain, which is required for initiating downstream signaling pathways.[31]

TLR9 was originally shown to recognize unmethylated 2'-deoxyribo (cytidine-phosphate-guanosine) (CpG) DNA motifs.[36]The 2'-deoxyribose sugar backbone of DNA is sufficient to confer signaling.[37]Given that pDCs produce vast amounts of typeiIFN in response to DNA virus infection or certain CpG-ODNs, TLR9 expressed by pDCs may serve as a sensor for virus infection.[31]Consistently, IFN-α production following infection with DNA viruses, such as MCMV, HSV-1 and HSV-2, is totally dependent on TLR9 in pDCs. It should be noted that TLR9, similar to TLR3, TLR7, and TLR8, is expressed on intracellular compartments such as endosomes, lysosomes or endoplasmic reticulum (ER).[51,52]Initially, TLR9 is first expressed on the ER in resting cells and trafficked to the endosomal compartment in response to PAMP-mediated stimulation.[31]The intracellular localization of nucleic acid-sensing TLR9 is controlled by UNC93B, a protein of 12 membrane-spanning ER. UNC93B interacts with the transmembrane regions of TLR9 on the ER and assists in the trafficking of TLR9 from the ER to the endosome.[53,54]It was confirmed that mice bearing a single missense mutation in the gene encoding UNC93B have defects in cytokine production as well as up-regulation of co-stimulatory molecules in response to TLR9 ligands.[55]More importantly, some researchers proposed that proteolysis of TLR9 within the endolysosomal compartment is essential for robust innate immune responses.[56,57]The ectodomain of TLR9 is cleaved by cathepsins, and the cleaved product can activate downstream signaling.[31]After being trafficked to the endosomal compartment and binding to DNA, TLR signaling is initiated by the ectodomain-mediated dimerization of TLRs, which then facilitates the recruitment of TIR domain-containing cytosolic adapter molecules, including four in particular--MyD88, TIRAP (also known as MyD88 adapter-like), TRIF [also known as TIR domain-containing adapter molecule (TICAM) 1] and TRIF-related adapter molecule (TRAM, also known as TICAM-2)-- to form the receptor complex (Fig.).[58,59]The ectodomain-mediated dimerization initiating TLR9 signaling is MyD88, which is used to drive NF-κB and p38 MAPK activation by recruiting the IL-1R-associated kinase (IRAK) family protein kinases IRAK4 and IRAK1 (Fig.).[31,60,61]In the involvement of NF-κB in relation to p38 MAPK, which is important for CpG DNA-triggered gene expression, the pathway of IRF-7 activates and causes IFN-α expression.[62-66]Then DCs mature and activate T lymphocytes and then initiate adaptive immunity to control inflammatory responses.[67,68]

It appears that the TLR9 signaling pathway plays an indispensable role in the elimination of pathogens or immune tolerance, because mutations of TLR9 or its signaling molecules (e.g. MyD88, IRAK4, IRF-7 and UNC93B) are linked to many inflammatory diseases, immunodeficiencies and autoimmune diseases in humans,[69-72]as well as hepatitis B.

It is known that TLR9 mRNA in pDCs in chronic HBV patients is significantly reduced (approximately 70%), in comparison to healthy controls, correlating with the impairment of IFN-α productionin vitro. Furthermore, there is a decreased expression of the TLR9 protein with a lower TLR7 expression compared to the healthy group.[73,74]Consistent with these findings, the number of pDCs in these patients is substantially reduced (approximately 30%), inversely correlated with serum ALT levels and HBV viral load. HBsAg and HBcAg are detectable by immunohistochemistry in pDCs in chronic HBV patients.[73-75]

Fig. Schematic representation of the recognition of HBV antigens by DCs. a: HBV DNA can be recognized by DCs through TLR9 which activates the MAPK signal pathway and NF-κB, resulting in secretion of typeiIFN and inflammatory cytokines, and inducing DC maturation and the adaptive immune response; b: DNA or ssRNA encapsulated within HBcAg can be internalized by DCs through TLRs; c: HBsAg can be internalized by DCs through the mannose receptor, which lacks the ability to induce DC maturation but can suppress IRF-7 expression, and then inhibit the expression of IFN-α; d: HBc/HBeAg cannot be recognized by DCs.

Based on these data, some researchers attempted to use TLR9 agonists to treat chronic HBV infection. Interestingly, a TLR9 agonist, CpG-ODN, has been shown to inhibit HBV replicationin vitro.[76]Jiang et al[77]found that treatment with CpG-ODN promotes the accumulation and activation of murine hepatic natural killer T cells. CpG-ODN pretreatment aggravates liver injury and promotes the production of inflammatory cytokines via TLR9 activation in a Con A-induced fulminant hepatitis model. These data demonstrate that TLR9 stimulation promotes the accumulation and activation of hepatic CD4+natural killer T cells and suggest that TLR9 signaling is involved in the pathogenesis of human hepatitis including HBV development,[77]being consistent with the views that stimulation of TLR9 dramatically increases CTL responses,[78]and induces antiviral cytokines at the site of HBV replication.[79]Interestingly, three categories of CpG-ODNs have been identified: A, B and C,[80]but only a class C CpG-ODN successfully induces robust IFN-α production by pDCs from chronic HCV patients, suggesting that this class of TLR9 agonists have utility as a future immunotherapeutic for the treatment of chronic HCV infection.[81]

Recognition of HBcAg by DCs

The HBV core gene encodes two polypeptides.[82]Initiation of translation at the first start codon (AUG) results in a 25-kDa pre-core protein named HBeAg after removal of 19 residues of the leader sequence and 34 C-terminal amino acids. Initiation of translation at the second AUG leads to the synthesis of a 183-aa, 21-kDa protein that assembles to form 27-nm particles that comprise the virion nucleocapsid (HBcAg). Although HBeAg and HBcAg are serologically distinct, they are cross-reactive at the level of Th cell recognition because they are collinear throughout most of their primary sequence. Some researchers reported that B cells rather than non-B cell professional APCs such as DCs or macrophages (MΦ) act as the primary APCs for HBcAg.[83,84]Further evidence showed that the inability of DCs to function as APCs for exogenous HBcAg is related to a lack of uptake of HBcAg, not to processing or presentation, because HBcAg/anti-HBc immune complexes can be efficiently presented by DCs. Furthermore, full-length (HBcAg183), truncated (HBcAg149), and non-particulate HBeAgs were screened for TLR stimulation via NF-κB activation in HEK293 cells expressing human TLRs. But none of the HBc/HBeAgs activated human TLRs. Thus, the HBc/ HBeAg proteins are not ligands for human TLRs (Fig.). However, the ssRNA contained within HBcAg183 does function as a TLR7 ligand, as demonstrated at the T and B cell levels in TLR7 knockout mice (Fig.), having been identified by bacterial, yeast, and mammalian ssRNA encapsulated within HBcAg183. These studies indicate that innate immune mechanisms bridge and enhance the adaptive immune response to HBcAg and have important implications for the use of hepadnavirus core proteins as vaccine carrier platforms.[84]

Recognition of HBsAg by DCs

Several putative binding factors have been described for HBsAg, such as human serum albumin,[85]asialoglycoprotein receptor[86]and mannose-binding lectin,[87]but their exact-role in HBV attachment and uptake remains unclear.[88]First, since HBsAg is a glycoprotein,[89]viral recognition by DCs seems plausible. Second, we note that C-type lectins on DCs can recognize glycan structures expressed on pathogens.[90]Meanwhile, some studies on HBsAg internalization by mDCs showed an increased uptake of HBsAg over time.[91]Based on these findings, we might presume that the recognition of HBsAg is mediated through C-type lectins.

Chong et al[87]have demonstrated that HBsAg interacts with soluble mannose-binding lectin leading to complement activation, which could contribute to viral clearance by enhanced phagocytosis. These data may explain the potential contribution of mannose-binding lectin polymorphisms in HBV disease progression.[92]Op den Brouw et al[24]showed that the mannose receptor (MR) is involved in HBsAg recognition and uptake by DCs (Fig.). HBsAg-positive DCs are present sporadically in blood, but frequently in the liver of HBV patients. Interestingly, a positive correlation was found between HBsAg positivity and MR expression level in both liver- and blood-derived DCs. Collectively, we demonstrated that in HBV-infected patients, MR-mediated interaction between HBsAg and DCs and subsequent impairment of DCs predominantly occur at the main site of infection, the liver.

More surprisingly, some findings support the idea that HBsAg is recognized by MR, but not by DCSIGN.[24,93]Whereas most pathogens recognized by the MR also show interaction with DC-SIGN, e.g. Mycobacterium tuberculosis and HIV.[94]The subtle differences between the ligand specificity of DC-SIGN and MR, namely the recognition of complex mannose structures versus end-standing mannose residues,[95]lead to major differences in HBsAg recognition. What is more, highly mannosylated HBV, obtained by treating HBV-producing HepG2.2.15 cells with the α-mannosidaseiinhibitor kifunensine, is recognized by DC-SIGN.[93]On the basis of these findings, it is tempting to speculate that HBV exploits mannose trimming as a way to escape recognition by DC-SIGN and thereby subvert a possible immune activation response.[93]This was confirmed by the findings that in interaction with C-type lectins MR did not induce DC maturation (Fig.).[96]Furthermore, both HBV and HBsAg can directly reduce the immunogenicity of DCsin vitro, which indicates a possible immune escape mechanism of HBV by the virus itself and/or by the production of HBsAg.[97]Consistent with these views, some researchers propose that HBsAg directly interferes with the function of pDCs through HBsAg-mediated upregulation of suppressors of cytokine signaling (SOCS)-1 expression and BDCA-2 ligation, which could partially explain how HBV evades the immune system to establish a persistent infection.[98]

We outline the interactions of HBV and DCs from three aspects (HBV DNA and DCs, HBcAg/HBeAg and DCs, HBsAg and DCs) (Fig.), which induce an effective anti-viral response or HBV persistence.[99,100]A recent study[98]found that pDCs treated with HBsAg secrete much less IFN-α than control pDCs. Further research[98]has shown that this kind of suppression is specific for TLR9, with no effects upon TLR7-mediated IFN-α secretion, by inhibiting TLR9-mediated IRF-7 expression and nuclear translocation, which is important for induction of IFN-α gene transcription (Fig.). These data about the possible relationships of HBV antigens and HBV DNA and DCs may provide some useful information about basic research and clinical treatment of chronic HBV infection.

Perspectives

IFN-α is considered a traditional treatment for hepatitis B, through the degradation of viral mRNA to direct inhibition of viral replication in hepatocytes and indirectly stimulate the immune system, but the HBV clearance rate is only 30%. Nucleotide analogues and other inhibitors of virus replication (such as lamivudine, adefovir dipivoxil) are effective in suppressing viral replication, but longterm use easily leads to the occurrence of drug-resistant mutants and the rebound of virus load. And the host immune system has not been fundamentally improved and regulated. Following the findings about the role of DCs in HBV infection, more studies have been conducted to develop a DC vaccine. Some methods are used to study DC vaccines, such as being cultured together with cytokines, pulsed with HBV antigens, and co-cultured with HBV DNA transgenic cells for the sake of antigen intake and internalization, and DC maturation to initiate adapted immunity. Some achievements have been gained to develop a new method for clearing the virus from patients with persistent HBV infection. The ability of these TLR ligands to induce antiviral cytokines at the site of HBV replication suggests that TLR activation is a powerful and novel therapeutic strategy for the treatment of chronic HBV infection.[78,79]So TLR ligands that mimic PAMPs and activate immune cells via TLRs are being developed for use in humans as a therapy against a variety of diseases or as vaccine adjuvants.[101]

Conclusion

It is widely accepted that DCs play an important role in the progress of hepatitis B, especially in the recognitionof HBV antigens. TLRs and MR, the receptors of HBV antigens in DCs, are essential to the recognition, and more importantly, regulate the immune response including HBV clearance or resistance. But the interactions between HBV antigens/HBV DNA and DCs are not clear, and cross-talk between TLRs and various ligands makes HBV antigen recognition by DCs more complicated. More efforts should be made to define the mechanisms and develop effective vaccines and therapies.

Funding:This work was supported by grants from the Major National Science & Technology Projects for Infectious Diseases (2009ZX10004-309, 2008ZX10002-007), the Fundamental Research Funds for the Central Universities (2009QNA7033) and the Science and Technology Department Foundation of Zhejiang Province (2010R10061).

Ethical approval:Not needed.

Contributors:CGY wrote the main body of the article under the supervision of DHY. DHY provided advice on medical aspects. DHY is the guarantor.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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March 16, 2010

Accepted after revision August 1, 2010

Author Affiliations: State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Cui GY and Diao HY)

Hong-Yan Diao, MD, PhD, State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Tel: 86-571-87236446; Email: diao.hy@163.com)

? 2010, Hepatobiliary Pancreat Dis Int. All rights reserved.

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