| [Objective] Interferon-y inducible protein 10 (IP-10, CXCL10) is a member of the non-ELR CXC chemokine family and a ligand for the CXCR3 receptor, chemoattracting for NK cells, activated T cells, and dendritic cells. The mRNA and protein levels of IP-10 in PBMCs, sinusoidal endothelium and plasma are all increased in patients infected hepatitis B. In IP-10-/- mice, immune responses to infection by neurotropic mouse hepatitis virus are reduced, concomitant with decreased CD4+and CD8+ lymphocyte trafficking to the brain and reduced production of inflammatory factors. Therefore, the induction of IP-10 is an important event for the development of hepatitis B. However, the molecular basis of how IP-10 is regulated for the induction and function exertion still remains unknown. In the present study, we explored the IP-10 expression in HBV-infected liver tissues and then defined underlying molecular mechanisms for HBx-induced IP-10 expression and leukocytes migration by cell line models.[Methods]1. IP-10 expression detection in HBV-infection patientsTwelve patients (8 male,4 female; age range 32-65 years, average 48±9) with chronic hepatitis B who underwent surgery treatment for cancers, seven patients with non-HBV infection underwent surgery for cancers and five normal control Hepatic hemangioma liver tissues were selected from the Union Hospital of Tongji Medical College for IP-10 detection in the liver tissues by real-time PCR and immunohistochemistry.2. Plasimds transfection and IP-10 expression in HepG2 cells HepG2 cells were transfected with relative plasmid (pBlue-HBV, pCMV-HBs, pCMV-HBc, pCMV-HBx,) and the supernatants IP-10 expressin was detected by ELISA.3. IP-10 promoter luciferase reporter assaysA series of 5'deletions and NF-κB mutation of IP-10 luciferase promoter plasmids were constructed and co-transfected with pCMV-HBx, respectively, into HepG2 cells. Luciferase activity was measured 48 hrs after transfection.4. Electrophoretic Mobility Shift AssayNuclear extracts transfected relative plasmids were incubated with the labeled NF-κB1 binding oligonucleotides in the presence or out the presence of either an unlabeled wild type NF-κB binding probe or a mutated probe and then were separated on 6% polyacrylamide gels with 0.5×TBE buffer and transferred to a nylon membrane. A chemiluminescent detection of membranes was scanned by Image Station 4000R.5. Chromatin immunoprecipitation assayHepG2 cells transfected with relative plasmids were cross-linked histones to DNA using 1% formaldehyde and sonicated to shear the chromatins. The sonicated chromatins were incubated with anti-p65 antibody, anti-p50 antibody or an isotype control IgG for 2 hrs. The DNA isolated from the chromatin-antibody complex was subject to PCR amplification and agarose gel electrophoresis.6. Confocal Laser Scanning MicroscopyHepG2 cells transfected relative plasmids were cultured on coverslips. After fixed with ice-cold methanol, cells were blocked with 1% bovine serum albumin and probed with anti-p65 antibody. Signals were visualized with FITC-conjugated second antibody and micrographs were acquired with a BX51 system equipped with DP70.7. Migration Assay Migratory activity was quantified using 24-well Transwell inserts(5-μm pore size). PBLs (1×105) in 200μl of RPMI medium were added to the upper chamber.1 ml culture supernatant from the cells transfected with relative plasmids was added to the lower chamber. The chambers were incubated for 4 hrs at 37℃in 5% CO2, then the Transwell inserts were removed and the migration cells were HE stained and counted.[Results]1. IP-10 expression in HBV-infected liver tissues.We analyzed the expression of IP-10 in HBV-infected liver tissues tissues, and found that the mRNA and protein levels of IP-10 were much higher in HBV positive liver cancer tissues, compared to HBV negative liver cancer tissues or normal Hepatic hemangioma liver tissues. These data suggested that IP-10 is elevated in HBV-infected liver tissue, and may play an important role in HBV-induced inflammation of the liver injury.2. HBx induces IP-10 gene expression in HepG2 cells.The protein levels of IP-10 in the supernatants in HepG2 cells were significantly increased after transfected with the full-length HBV gene (pBlue-HBV). Evaluated the effect of different HBV proteins we defined that the transfection of HBx-rather than HBs-or HBc-expressing vector increased IP-10 protein levels. By promoter luciferase activity assay, we found that IP-10 promoter was markedly activated by HBx protein in a dose-dependent manner.3. HBx-induced IP-10 expression is mediated by TRAF2/TAK1/NF-κB signaling pathwayTo investigate the mechanize of HBx-induced IP-10 expression, a series of 5' deletions of IP-10 promoter were constructed and co-transfected with pCMV-HBx, respectively, into HepG2 cells. Luciferase assay suggest that the sequence between nt -190 to-96 is critical for activation of IP-10 promoter by HBx protein. Coincidently, two NF-κB binding sites (NF-κB1, and NF-κB2) were found in this region. Afer mutation the two NF-κB binding sites, we confirmed that the NF-κB 1 binding site is required for the activation of IP-10 promoter regulated by HBx protein. The addition of NF-κB inhibitor SN50, blocks the effect of HBx on IP-10 induction. In parallel, the increase of NF-κB subunits p65 and p50 in HepG2 cells also augments IP-10 expression.Next, we asked whether the activated NF-κB bound to IP-10 promoter directly. For this purpose, we performed EMSA and CHIP assay. Results showed the DNA binding activity of NF-κB was significantly increased in the cells transfected with pCMV-HBx detected by a biotin-labeled NF-κB1 binding oligonucleotides probe in the IP-10 promoter (-125 to-102) and a 177-bp DNA fragment in IP-10 promoter was amplified by PCR from DNA which isolated from cells transfected with pCMV-HBx and then immunoprecipitated with anti-p50 or anti-p65 antibody. These data suggested that the NF-κB directly binds to the NF-κB1 binding site in the IP-10 promoter.To further defined the mechanism of HBx activated NF-κB activation. In this regard, by western blot, promoter luciferase activity and confocal assay we found after transfection of HBx plasmid into HepG2 cell line, the phosphorylation of TAK1 and IKKa was observed in HBx plasmid group. By addition TAK1 inhibitor or TAK1 siRNA, both NF-κB activity and IP-10 promoter activity were reduced. TRAF family member TRAF2 knockdown also impaired the NF-κB and IP-10 activity induced by HBx. Consistently, the inhibition of the nuclear translocation of NF-κB subunit p65 was defined after knockdown of TRAF2 or TAK1. Taken together, our data suggested that HBx-induced IP-10 expression might be mediated by TRAF2/TAK1/NF-κB signaling pathway.4. HBx-induced IP-10 increases migration of peripheral blood leukocytes (PBLs) We performed migration assay and found that PBLs exhibited decent increase of migratory activity in response to supernatants from cells transfected with plasmids expressing HBx, p50, or p65 comparing mock plasmids. The addition of neutralizing anti-IP-10 but not the control antibody to the supernatants significantly impaired this migration. Interestingly, NF-κB inhibitor SN50 treatment could decrease the chemotactic activity after transfection of pCMV-HBx. Together, these data suggested that HBx-induced IP-10 may mediate the migration of PBLs and NF-κB activity is involved in this process.[Conclusions] Our study found that the mRNA and protein levels of IP-10 were much higher in HBV positive liver cancer tissues, compared to HBV negative liver cancer tissues or normal Hepatic hemangioma liver tissues, and may play an important role in HBV-induced inflammation of the liver injury. Then we transfected different HBV protein expressing plasmids into HepG2 cells and explored a novel molecular mechanism of HBV infection inducing IP-10 expression, which involves viral protein HBx affecting TRAF2/TAK1/NF-κB pathway, leading to the transactivation of IP-10 promoter. Our study provides insight into the migration of leucocytes in response to HBV infection, thus causing immune pathological injury of liver. |
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