| Hepatitis B virus (HBV) belonging to hepadnavividae, is a reverse transcription DNA virus. HBV can not only cause acute hepatitis, but also induce liver cirrhosis. In addition, HBV is closely associated with hepatocellular circinoma to severely impair humanity health. Approximately 5% acute hepatitis B patients can’t eliminate HBV, result in HBV persistent infection, and then develop into liver cirrhosis or hepatocellular circinoma. In light of HBV pathogenesis study, HBV infection involves in virus and body factors. HBV exrets restricted direct cytotoxic effect on virus infected cells. Hepatocyte injury is related to the infiltration of host immue cells and cytokines in liver local tissues. During HBV acute and chronic infections, body immmunity shows different status. Thl type immune response is predominant in acute HBV infection, in which increased Thl type cytokines promote cytotoxic T lymphocytes (CTL) to eliminate HBV. There is Thl/Th2 cytokine imblance in chronic HBV infection patient’s body, with decreased Thl type cytokines and increased Th2 type cytokines.Lysine specific demethylase 1 (LSD1) is a flavin adenine dinulcleotide (FAD) dependent monoamine oxidase. LSD1 can regualte histone methylation state dynamically, modulate the interaction between histone and other functional protein to control the activation and inhibition of gene transcription via specifically removing methyl group of single methylated and double methylated histone lysine 4 (H3K4) and H3 lysine 9 (H3K9) locus.The study aims to explore the relationship between LSD1 and Thl/Th2 cell-mediated immune imbalance to affect HBV infection elimination from both angles of hepatitis B patients and animal model, probing the function and mechanism of LSD 1 in hepatitis B virus infection. The results will provide experimemtal basis to further illustrate the pathogenesis and immune therapy target of hepatitis B.Part I The study on the function and mechanism of LSD1 in hepatitis B virus infectionObjective:To determine the relationship between LSD1 expression level in CD4+Th cells derived from peripheral blood of chronic hepatitis B patients and Thl/Th2 imbalance. To explore the effect of LSD1 mediated histone modification on CD4+Th cell differentiation of chronic hepatitis B patients. To observe how LSD1 inhibitor Tranylcypromine (TCP) affects Thl/Th2 CD4+Th cell differentiation into Thl/Th2. To investigate the function and underlying mechanism of TCP in regulating Thl/Th2 differentiation balance.Methods:248 chronic hepatitis B patients who were hospitalized in Affiliated Hospital of Nantong University from January 2011 to June 2014 and 206 healthy controls were chosen for the study. LSD1 expression level in CD4+Th cells derived from peripheral blood of chronic hepatitis B patients, IFN-y and IL-4 level in serum were observed. After TCP intervening, Thl and Th2 cell frequency, Thl type transcription regulating factor T-bet, phosphorylated signal transducers and activators of transcription (pSTATl) and dimethylation of histone 3 lysine 4 (H3K4me2) expression pattern was detected.1. According to liver function, hepatitis B virus markers (HB VM) and HBV-DNA quantitative determination results when hospitalizing,248 chronic hepatitis B patients were divided into four groups:(1) hepatitis B surface antigen (HBsAg) high level group (HBsAg>250 IU/ml) and low level group (HBsAg≤250 IU/ml); (2) hepatitis B e antigen (HBeAg) positive group and negative group; (3) alanine aminotransferase (ALT) high level group (ALT>threefold of upper limitation of detect) and low level group (ALT≤threefold of upper limitation of detect); (4) HBVDNA high load group (HBV-DNA>105 copy/ml) and low load group (HBV-DNA≤105 copy/ml). Microparticle chemiluminescence was assay utilized to detect serum HBsAg and HBeAg level. Automatic biochemical analyzer was used to detect serum ALT level. HBV-DNA load was detected by real-time quantitative polymerase chain reaction (RQ-PCR).2. Density gradient centrifugation was adopted to separate mononuclear cells, immunomagnetic beads to sort CD4+Th cells and flow cytometry to detect the purity of CD4+Th cell. Total protein of CD4+Th cells was extracted and performed western blot (WB) to detect LSD1 protein expression. Enzyme-linked immunosorbent assay (ELISA) was used to detect IFN-γ, IL-4 level in serum.3. The peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated and purified by magnetic separation anti-CD4 MACS microbeads. After activated by CD3 and CD28 monoclonal antibodies, CD4+T cells were treated with different concentrations (10,30,50,80,100 μM) of TCP. CD4-PerCP-Cy5.5/IFN-γ-FITC/IL-4-PE three-color flow cytometry was used to detect Th1, Th2 cell frequency. LSD1, Th1 type regulatory factors including T-bet, STAT1 and pSTAT1 protein levels were assayed by WB.Results:1. After separated, the CD4+ Th cell’s mean purity of each normal control cases and CHB cases were (96.9±2.3)% and (96.5±2.5)%, there was no significant difference (P>0.05). (1) The expression of LSD1 in peripheral blood CD4+Th cell of CHB group was much higher than that of the normal control group (P<0.05). The expression of LSD1 in peripheral blood CD4+Th cell of CHB groups:HBsAg high level group was higher than HBsAg low level group (P<0.05), HBeAg positive group was higher than negative group (P<0.01), ALT low level group was higher than ALT high level group (P<0.05), HBV-DNA high load group was also higher than low load group (P<0.05). (2) Compared with healthy control group, CHB patients’ serum IFN-γ decreased (P<0.01), IL-4 changed subtlely (P>0.05) and IFN-γ/IL-4 decreased (P<0.01). IFN-γ and IFN-γ/IL-4 level in peripheral blood CD4+Th cell of CHB groups:HBsAg high level group was lower than HBsAg low level group (P<0.01). In contrast with HBeAg negative group, IFN-γ and IFN-γ/IL-4 of HBeAg positive group were far lower (P<0.01). Compared with ALT low level group, IFN-y and IFN-y/IL-4 of ALT high level group were also obviously higher (P<0.01). IFN-γ and IFN-γ/IL-4 of HBV-DNA high load group were lower than low load group (P<0.01). However, in all these groups of CHB, IL-4 level did not show significant difference (P>0.05). (3) The expression of LSD1 in peripheral blood CD4+Th cell of CHB group was negtively correlated with serum IFN-γ (P<0.01) and IFN-γ/IL-4 (P<0.01), but was not correlated with serum IL-4 (P>0.05).2. Under interfering with different concentrations of TCP:(1) When TCP concentration increased, IFN-γ positive cell frequency was markedly increased in TCP treatment groups compared with control group (P<0.05). However, IL-4 positive cell frequency had no significance (P>0.05). (2) TCP had effect on T-bet/STAT1 signaling pathway. Compare to healty control group, Thl specific transcription factor T-bet expression (P<0.01) and upstream regulatory factor STAT1 phosphoralytion level (pSTATl) markedly increased in CHB group (P<0.01). (3) Upon treatment different concentrations (10,30,50,80, and 100 μM) of TCP, global dimethylation of H3K4 in CD4+T cells increased.Conclusions:1. LSD1 expression level in CD4+Th cells derived from peripheral blood of chronic hepatitis B patients was higher than that of control group. In addition, LSD1 expression was higher in HBsAg high level, HBeAg positive, HBV DNA high load groups than that of HBsAg low level, HBeAg negative, HBV DNA low load groups respectively. ALT is presented for hepatocyte injury. LSD1 level was lower in ALT high level group than that of ALT low expression group, maybe because LSD1 low expression facilitated Thl cell differentiation, Thl upregulating cell-mediated immunity, producing effector CTL, destroying a larg number of hepatocytes, releasing excess ALT to peripheral blood.2.Thl type cytokine IFN-γ level in peripheral blood derived from chronic hepatitis B patients was lower than that of control group. In addition, IFN-γ level was negatively correlated to LSD1 expression in peripheral blood CD4+Th cells. In chronic hepatitis B patient group, IFN-γ expression in high level groups of HBsAg, HBeAg, HBV-DNA load which are presented for HBV infection degree was lower than that of low level groups, respectively. However, Th2 type cytokine IL-4 showed no change. These data suggested that LSD1 might induce Th1/Th2 imbalance in chronic hepatitis B via affecting Thl differentiation.3. After TCP interfering, IFN-y positive cell frequency was markedly increased compared to control group. Thl type specific transcription factor T-bet expression markedly increased. Upstream regulating factor pSTAT1 protein level dramatically increased. These data indicated that TCP regulated CD4+Th cell differentiate into Th1 through affecting T-bet/STAT1 signaling pathway to inhibit LSD1 activity. LSD1 substance H3K4me2 expression increased following TCP administration, showing that TCP effectively inhibited LSD1 activity.LSD1 high expression might participate in the pathogenesis of HBV chronic infection. Thus, LSD1 expression regulating maybe become immune therapy target for chronic HBV infection.Part Ⅱ The Study on the function and mechanism of down-regulation of LSD 1 on HBV infection mouse modelObjective:HBV hydrodynamic injection mouse model was treated with LSD1 siRNA and LSD1 inhibitor TCP. HBV antigens and HBV-DNA in mouse liver and serum was detected to reveal the function of LSD 1 expression or activity down-regulation on acute HBV infection mouse. By observing the LSD1 expression change in mouse spleen cells and LSD1 expression of murine bone marrow-derived dendritic cells after LSD1 siRNA transfection, observing LSD1, H3K4me2, Thl associated transcription regulating factors pSTATl and T-bet expression level in mouse spleen cells, detecting the expression of IFN-y and IL-4 derived from CD4+T cell, we explored the relationship between LSD1 enzyme activity and host immune cells Thl/Th2 differentiation balance, we aimed to discuss the relationship between LSD1 enzyme activity change and Thl/Th2differentiation balance in HBV infection mouse, as well as the effect and mechanism of inhibiting LSD1 enzyme activity on virus elimination after HBV infection.Methods:1.100 μg of recombinant HBV1.3 plasmid was hydrodynamic tail intravenously injected in BALB/C mice and established acute HBV infection mouse model. Detection of (1) At day 4, ELISA test was used to detect HBsAg expression in mouse serum. (2) Real-time quantitative PCR was performed to detect HBV-DNA level. (3) HE staining was used to observe hepatocyte change. (4) Immunohistochemistry analysis was applied to detect the distribution of HBsAg, HBcAg and HBeAg in liver tissue and confirm the construction of HBV infection mouse model.2. The BALB/C mice were randomly divided into four groups:control group (1 ml saline was tail intravenously injected); model group (recombinant HBV1.3 plasmid and same volume saline were tail intravenously injected); LSD1 siRNA group (recombinant HBV 1.3 plasmid and same volume saline containing 50 μg LSD1 siRNA were tail intravenously injected); TCP group (recombinant HBV1.3 plasmid and same volume saline containing 60μM TCP were tail intravenously injected). For the mice in each group (1) Automated chemiluminescent immunoassay was used to detect the expression of HBsAg in peripheral blood at 0 d、4 d、8 d ' 12 d; (2) Real-time quantitative PCR was performed to detect HBV-DNA load; (3) Immunohistochemistry analysis was applied to detect HBsAg expression level in liver tissue; (4) CD4+T cells were isolated in the spleen of mice with MACS to extract cell protein and WB was used to detect the expression of LSD1; (5) In vitro, cultured mouse bone marrow-derived DC was lysed to extracted the total protein and LSD1 protein expression was determined by WB. Flow cytometry (FCS) was utilized to detect MHC-Ⅱ, CD11c, CD80, CD86 molecules expression change on DC surface; (6) IFN-γ and IL-4 level in the mouse peripheral blood was detected by ELISA; Extracting CD4+T cells in the spleen of mice with MACS, some cells were used to extract protein, WB was used to detect the expression of LSD1, H3K4me2, STAT1 and T-bet in CD4+T cells, others use intracellular cytokine staining with FCS to detect the expression of cytokines IFN-γ and IL-4 in CD4+T cells. Results:1. After HBV1.3 plasmid was hydrodynamic tail intravenously injected in BALB/C mice, detection found (1) HBsAg was positive in mouse peripheral blood; (2) HBV-DNA load peaked; (3) Hepatocytes showed no obvious pathological change; (4) HBsAg was distributed widely in liver tissue without HBcAg or HBeAg, showing that HBV infection mouse model was constructed successfully.2. LSD1 siRNA and TCP groups compared with model group (1) Serum HBsAg level markedly decreased (P<0.01); (2) Serum HBV-DNA load markedly decreased (P<0.01); (3) HBsAg in liver tissue markedly decreased (P<0.01); (4) LSD1 expression in CD4+T cells derived from mouse spleen markedly decreased (P<0.05); (5) LSD1 expression in dentritic cells derived from mouse bone marrow markedly decreased (P<0.01). Marker assay showed that MHC-Ⅱ, CD11c, CD80 and CD86expression in LSD1 siRNA group increased compared with control group (P<0.01); (6) IFN-y level in peripheral blood was higher than model control group (P<0.05), IL-4 level in each group showed no significance (P>0.05); IFN-y positive cell frequency was higher than model control group (P<0.05); however, IL-4 positive cell frequency in each group showed no difference (P>0.05); Thl associated transcription regulating factor pSTAT1 and T-bet expression was higher than control group (P<0.05); LSD1 expression in model control and TCP groups was higher than that of LSD1 siRNA group (P<0.05); H3K4me2 expression in model control group was lower than that of TCP and LSD1 siRNA groups (P<0.05).Conclusions:1. Recombinant HBV1.3 plasmid hydrodynamic tail intravenously injection can simply and effectively construct HBV infection mouse model, which supplies conditions for HBV infection research. 2. Through TCP or LSD1 siRNA interfering, MHC-Ⅱ, CD80 and CD86 expression on DC surface in HBV infection mouse was increased, showing that both interfering methods could enhance DC antigen presenting capability to promote HBV elimination.3. Through TCP or LSD1 siRNA interfering, IFN-y level in peripheral blood and IFN-y positive cell frequency markedly increased, suggesting that TCP or LSD1 siRNA promoted mouse spleen Th1/Th2 differentiate to Th1 cells; Thl associated transcription regulating factor pSTAT1 and upstream regulating factor T-bet expression increased obviously, indicating that TCP or LSD1 siRNA could regulate the Th1 shift of CD4+Th cell differentiation, might via affecting T-bet/STAT1 signaling pathway to inhibit LSD1 activity.4. The results showed that LSD1 expression in model control and TCP groups was higher than that of LSD 1 siRNA group; H3K4me2 expression in model control group was lower than that of TCP and LSD1siRNA groups, suggesting that TCP interfering increased LSD1 enzyme activity to promote H3K4me2 protein expression, but not inhibit LSD1 expression.The study data provide experimental foundation and theoretical basis for epigenetic regulation involved in HBV infection elimination. |
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