ABSTRACT
Hepatitis B virus (HBV) infection is a major public health concern. The clearance of HBV may involve cytotoxic T-lymphocyte (CTL) activity and T helper type 1 reactions. Exosomes generated from dendritic cells (DCs) can induce immunological responses capable of eradicating viruses. However, exosomes loaded with antigens have not yet demonstrated therapeutic potential in HBV infection. Therefore, the present study aimed to investigate the antiviral effects of DC-derived exosomes (Dexs) loaded with ubiquitinated HBV core antigen (Dexs-Ub-HBcAg). Murine bone marrow-derived DCs were loaded with a recombinant lentivector encoding the ubiquitinated form of HBcAg. High-purity Dexs were generated using differential velocity centrifugation. Splenic T-lymphocytes were stimulated with Dexs-Ub-HBcAg and the specific T-cell-mediated immune responses were examined. Cytokine expression was analyzed using enzyme-linked immunosorbent assays. T-lymphocyte proliferation was detected using a Cell Counting Kit-8 assay and HBcAg-specific CTL activity was determined using a lactate dehydrogenase release assay. The results revealed that Dexs-Ub-HBcAg effectively stimulated T-cell proliferation and induced the activation of antigen-specific CTLs to exhibit HBcAg-specific CTL immune responses in vitro. These results suggest the potential of Dexs-Ub-HBcAg for development as a future therapeutic option for the elimination of HBV.
ABSTRACT
MicroRNA (miR)-219a-5p has been implicated in the development of numerous progression of carcinoma and autoimmune diseases. However, whether miR-219a-5p is involved in the pathogenesis of inflammatory bowel disease (IBD) remains elusive. In this study, we demonstrated that miR-219a-5p expression was significantly decreased in the inflamed intestinal mucosa and peripheral blood (PB)-CD4+ T cells from patients with IBD. Proinflammatory cytokines (e.g., IL-6, IL-12, IL-23 and TNF-α) inhibited miR-219a-5p expression in CD4+ T cells in vitro. Lentivirus-mediated miR-219a-5p downregulation facilitated Th1/Th17 cell differentiation, whereas miR-219a-5p overexpression exerted an opposite effect. Luciferase assays confirmed that ETS variant 5 (ETV5) was a functional target of miR-219a-5p and ETV5 expression was significantly increased in the inflamed intestinal mucosa and PB-CD4+ T cells from IBD patients. ETV5 overexpression enhanced Th1/Th17 immune response through upregulating the phosphorylation of STAT3 and STAT4. Importantly, supplementation of miR-219a-5p ameliorated TNBS-induced intestinal mucosal inflammation, characterized by decreased IFN-γ+ CD4+ T cells and IL-17A+ CD4+ T cells infiltration in the colonic lamina propria. Our data thus reveal a novel mechanism whereby miR-219a-5p suppresses intestinal inflammation through inhibiting Th1/Th17-mediated immune responses. miR-219a-5p might be a target for the treatment of IBD.
Subject(s)
Colitis/immunology , Inflammatory Bowel Diseases/immunology , MicroRNAs/genetics , Th1 Cells/immunology , Th17 Cells/immunology , Adult , Animals , Cells, Cultured , Colitis/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Disease Models, Animal , Female , Gene Expression Regulation , Humans , Inflammatory Bowel Diseases/genetics , Male , Mice , Mice, Inbred BALB C , Middle Aged , Transcription Factors/genetics , Transcription Factors/metabolism , Trinitrobenzenesulfonic AcidABSTRACT
Hepatitis B virus (HBV) infection is a global public health problem. T helper (Th)1associated cytokines are involved in HBV clearance during acute and persistent infection. In our previous study, it was demonstrated that lentiviral vectors encoding ubiquitinated hepatitis B core antigen (LVUbHBcAg) effectively transduced dendritic cells (DCs) to induce maturation, which promoted T cell polarization to Th1 and generated HBcAgspecific cytotoxic T lymphocytes (CTLs) ex vivo. In the present study, HBV transgenic mice were immunized with LVUbHBcAgtransduced DCs and HBcAgspecific immune responses were evaluated. Cytokine expression was analyzed by ELISA. T lymphocyte proliferation was detected with a Cell Counting Kit8 assay and HBcAgspecific CTL activity was determined using a lactate dehydrogenase release assay. The expression levels of p38mitogenactivated protein kinase (p38MAPK), phosphorylated (p)p38MAPK, cJun Nterminal kinase (JNK) and pJNK were detected by western blot analysis. The results demonstrated that LVUbHBcAgtransduced DCs significantly increased the Th1/Th2 cytokine ratio, and effectively reduced the levels of serum hepatitis B surface antigen (HBsAg), HBV DNA, and liver HBsAg and HBcAg. Furthermore, the LVUbHBcAgtransduced DCs upregulated the expression of pP38MAPK and pJNK in T lymphocytes. In conclusion, the present study indicated that LVUbHBcAgtransduced DCs generated predominant Th1 responses and enhanced CTL activity in HBV transgenic mice. Activation of the P38MAPK/JNK signaling pathway may be involved in this induction.
Subject(s)
Dendritic Cells/immunology , Hepatitis B Core Antigens/immunology , Hepatitis B virus/immunology , Hepatitis B/immunology , Animals , Cell Differentiation/immunology , Dendritic Cells/transplantation , Enzyme-Linked Immunosorbent Assay , Genetic Vectors/immunology , Hepatitis B/genetics , Hepatitis B/therapy , Hepatitis B/virology , Hepatitis B Core Antigens/administration & dosage , Hepatitis B Core Antigens/genetics , Hepatitis B virus/pathogenicity , Humans , Immunization , Lentivirus/genetics , Lentivirus/immunology , MAP Kinase Kinase 4/genetics , Mice , Mice, Transgenic , Signal Transduction/immunology , Th1 Cells/immunology , Ubiquitination , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
Dendritic cells (DCs) play a predominant role in initiating cell immune responses. Here we generated a DC-targeting lentiviral vector (LVDC-UbHBcAg-LIGHT) and evaluated its capacity to elicit HBV-specific cytotoxic T lymphocyte (CTL) responses. DC-SIGN-mediated specific transduction using this construct was confirmed in DC-SIGN-expressing 293T cells and ex vivo-cultured bone marrow cells. LVDC-UbHBcAg-LIGHT-loaded DCs were highly effective in inducing HBV-specific CTLs. Mechanistic studies demonstrated autophagy blocking led to a significant increase in apoptosis and obvious inhibition of CD8 + T cells entry into S-phase, correspondingly attenuated LVDC-UbHBcAg-LIGHT-loaded DC-induced T cell responses. This observation was supported by accumulation of pro-apoptotic proteins and the main negative cell cycle regulator-CDKN1B that otherwise would be degraded in activated T cells where autophagy preferentially occured. Our findings revealed an important role of autophagy in the activation of T cells and suggested LVDC-UbHBcAg-LIGHT may potentially be used as a therapeutic strategy to combat persistent HBV infection with higher security.
Subject(s)
Autophagy , Dendritic Cells/metabolism , Genetic Vectors/metabolism , Hepatitis B virus/immunology , Lentivirus/genetics , T-Lymphocytes, Cytotoxic/immunology , Transduction, Genetic , Up-Regulation , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Apoptosis/drug effects , Autophagy/drug effects , Autophagy-Related Protein 5/metabolism , CD8-Positive T-Lymphocytes/immunology , Cell Adhesion Molecules/metabolism , Dendritic Cells/drug effects , Genetic Engineering , Hepatitis B Core Antigens/metabolism , Humans , Lectins, C-Type/metabolism , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice, Inbred BALB C , Receptors, Cell Surface/metabolism , S Phase/drug effects , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/ultrastructure , Tumor Necrosis Factor Ligand Superfamily Member 14/metabolism , Ubiquitin/metabolism , Up-Regulation/drug effectsABSTRACT
BACKGROUND/AIMS: Developing engineered dendritic cell (DC)-targeting lentivectors (LVs) have been the target of intense research for their potential to create antigen-directed immunotherapeutics which can be safely administered to patients. In this study, we constructed a DC-directed LV (LVDC-UbHBcAg-LIGHT) as a potential vaccine to induce anti-HBV immune responses. METHODS: Specificity of LVDC-UbHBcAg-LIGHT for DCs in vivo was confirmed through live animal imaging studies. The levels of cytokine production in T cells were assessed by flow cytometry. The HBcAg-specific cytotoxic T lymphocyte (CTL) responses and antibody responses induced by direct administration of the LVs were detected by LDH release assay and ELISA respectively. The levels of serum HBsAg and HBV DNA were evaluated by Abbott kits and quantitative PCR respectively. The expression levels of HBsAg and HBcAg in liver tissues of HBV transgenic mice were examined by immunohistochemistry. In addition, molecular mechanism underlying the activation of CD8+ T cells was explored. RESULTS: Live animal imaging studies showed that following subcutaneous administration of LVDC-UbHBcAg-LIGHT, no obvious luminescence signal was detected at the injection site. Immunization with LVDC-UbHBcAg-LIGHT elicited potent T cell responses in HBV transgenic mice evidenced by increased percentages of IFN-γ, TNF-α and GzmB producing CD8+ T cells as well as IFN-γ producing CD4+ T cells, improved HBcAg-specific CTL activities and antibody responses. Additionally, vaccination with LVDC-UbHBcAg-LIGHT efficiently reduced serum HBsAg, HBV DNA levels and the expression of HBsAg and HBcAg in liver tissues of HBV transgenic mice. More importantly, autophagy was induced in the activated CD8+ T cells, and the induced autophagy noticeably promoted the proliferation of T cells and decreased the frequencies of apoptotic CD8+ T cells by selectively degrading ubiquitinated apoptosis and cell cycle-associated protein aggregates. Futhermore, we confirmed the interaction between autophagosomes and ubiquitinated aggregates by confocal microscopy and immunoprecipitation analysis. CONCLUSIONS: These results demonstrated that LVDC-UbHBcAg-LIGHT provided a simple method of eliciting effective antiviral immune responses in HBV transgenic mice and might potentially be used as a therapeutic strategy to eradicate HBV with more safety and efficiency. Moreover, our results revealed a direct role of autophagy in promoting the survival and proliferation of activated CD8+ T cells.
Subject(s)
Autophagy , Genetic Vectors/metabolism , Hepatitis B Core Antigens/metabolism , Hepatitis B virus/physiology , T-Lymphocytes, Cytotoxic/metabolism , Animals , Antibodies, Viral/blood , Bcl-2-Like Protein 11/genetics , Bcl-2-Like Protein 11/metabolism , Dendritic Cells/cytology , Dendritic Cells/metabolism , Female , Genetic Vectors/genetics , Granzymes/metabolism , Hepatitis B Core Antigens/genetics , Hepatitis B Core Antigens/immunology , Humans , Interferon-gamma/analysis , Lentivirus/genetics , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/virology , Virus ReplicationABSTRACT
Predominant T helper cell type 1 (Th1) immune responses accompanied by boosted HBV-specific cytotoxic T lymphocyte (CTL) activity are essential for the clearance of hepatitis B virus (HBV) in chronic hepatitis B (CHB) patients. Ubiquitin (Ub) serves as a signal for the target protein to be recognized and degraded through the ubiquitin-proteasome system (UPS). Ubiquitinated hepatitis B core antigen (Ub-HBcAg) has been proved to be efficiently degraded into the peptides, which can be presented by major histocompatibility complex (MHC) class I resulting in stimulating cell-mediated responses. In the present study, lentiviral vectors encoding Ub-HBcAg (LV-Ub-HBcAg) were designed and constructed as a therapeutic vaccine for immunotherapy. HBcAg-specific cellular immune responses and anti-viral effects induced by LV-Ub-HBcAg were evaluated in HBV transgenic mice. We demonstrated that immunization with LV-Ub-HBcAg promoted the secretion of cytokines interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), generated remarkably high percentages of IFN-γ-secreting CD8(+) T cells and CD4(+) T cells, and enhanced HBcAg-specific CTL activity in HBV transgenic mice. More importantly, vaccination with LV-Ub-HBcAg could efficiently decreased the levels of serum hepatitis B surface antigen (HBsAg), HBV DNA and the expression of HBsAg and HBcAg in liver tissues of HBV transgenic mice. In addition, LV-Ub-HBcAg could upregulate the expression of T cell-specific T-box transcription factor (T-bet) and downregulate the expression of GATA-binding protein 3 (GATA-3) in spleen T lymphocytes. The therapeutic vaccine LV-Ub-HBcAg could break immune tolerance, and induce potent HBcAg specific cellular immune responses and therapeutic effects in HBV transgenic mice.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Hepatitis B Core Antigens/immunology , Hepatitis B Vaccines/immunology , Hepatitis B virus/immunology , Hepatitis B/immunology , Immunotherapy/methods , Animals , Cells, Cultured , Cytotoxicity, Immunologic , Female , Genetic Vectors/genetics , Hepatitis B Core Antigens/chemistry , Histocompatibility Antigens Class I/metabolism , Immunity, Cellular , Immunization , Lentivirus/genetics , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Inbred BALB C , T-Box Domain Proteins/genetics , T-Box Domain Proteins/metabolism , Ubiquitin/chemistryABSTRACT
The activity of hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) plays a predominant role in the clearance of HBV. Dendritic cells (DCs) are key antigen-presenting cells and play an important role in the initiation of immune responses. We previously verified that lentiviral vector encoding ubiquitinated hepatitis B core antigen (LV-Ub-HBcAg) effectively transduced DCs to induce maturation, and the mature DCs efficiently induced T cell polarization to Th1 and generated HBcAg-specific CTLs ex vivo. In this study, HBV-specific immune responses of LV-Ub-HBcAg in BALB/c mice (H-2Kd) were evaluated. It was shown that direct injection of LV-Ub-HBcAg increased the production of cytokines IL-2 and IFN-γ, elicited strong antibody responses, and remarkably generated a high percentage of IFN-γ+CD8+ T cells with HBV-specific CTL responses in BALB/c mice. In addition, direct injection of LV-Ub-HBcAg induced potent anti-HBV immune responses, similar to those elicited by in vitro-transduced DCs. In conclusion, the DC-based therapeutic vaccine LV-Ub-HBcAg elicited specific antibody immune responses and induced robust specific CTL activity in vivo.
Subject(s)
Dendritic Cells/immunology , Hepatitis B Core Antigens/chemistry , Hepatitis B Core Antigens/immunology , Hepatitis B Vaccines/immunology , Animals , Genetic Vectors/administration & dosage , Hepatitis B Core Antigens/genetics , Hepatitis B Vaccines/genetics , Lentivirus/genetics , Mice , Mice, Inbred BALB C , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
Chronic hepatitis B virus (HBV) infection is characterized by functionally impaired type 1 T-helper cell (Thl) immunity and poor HBVspecific Tcell responses. Ubiquitin (Ub), a highly conserved small regulatory protein, commonly serves as a signal for target proteins that are recognized and degraded in proteasomes. The rapid degradation of Ubmediated antigens results in efficient stimulation of cellmediated immune responses. Thus, the UbHBV core antigen (HBcAg)cytoplasmic transduction peptide (CTP) fusion protein was designed for specific delivery of a foreign modified antigen to the cytoplasm of antigenpresenting cells. HBV transgenic mice were used to determine whether UbHBcAgCTP would restore HBVspecific immune responses and antiviral immunity in these animals. The results demonstrated that synthesized UbHBcAgCTP not only significantly increased the levels of interleukin2 and interferon (IFN)γ compared with those in the HBcAgCTP, IFNα, UbHBcAg, HBcAg and phosphatebuffered saline groups, but additionally induced the highest IFNγ+ CD8+ Tcell numbers and HBVspecific cytotoxic T lymphocyte (CTL) responses, indicating a strong immune response. In addition, enhancement of specific CTL activity provoked by the fusion protein reduced hepatitis B surface antigen (HBsAg) and HBV DNA serum levels and diminished the expression of HBsAg and HBcAg in liver tissue of HBV transgenic mice, suggesting that there was a therapeutic effect. In conclusion, the present study provided evidence that UbHBcAgCTP activated the Th1dependent immunity, triggered functional T cell responses and subsequently inhibited viral replication in HBV transgenic mice. These observations suggested that the fusion protein may represent an innovative and promising candidate for active immunotherapy during chronic and persistent HBV.