Presentation.pptx
- 1. Prepared by Najam ul Saqib Downregulation of C9 expression in HBV-Transfected Huh7 cells, HBV- infected HepG2hNTCP cells and in chronically HBV-infected patients
- 2. To explore whether HBV could interfere with MAC formation, we first examined the expression of different MAC components (C5/C6/C7/C8/C9) in Huh7 cells transfected with full-length HBV by Real-time PCR. In the following figure, A significant reduction in C9 expression (~ 1.5-fold) was perceived in HBV- transfected Huh7 cells as compared to untransfected cells while the expression of other components of MAC remained comparable.
- 3. Background The complement system functions primarily as a first- line host defense against invading microbes, including viruses. However, the interaction of Hepatitis B virus (HBV) with the complement-components during chronic HBV infection remains largely unknown. We investigated the mechanism by which HBV inhibits the formation of cytolytic complement membrane-attack complex (MAC) and studied its impact on MAC-mediated microbicidal activity and disease pathogenesis.
- 4. Methods Blood/liver tissues were collected from chronically HBV-infected patients and controls. HepG2hNTCP cells were infected with HBV particles and Huh7 cells were transfected with full-length linear HBV-monomer or plasmids containing different HBV-ORFs and expression of complement components or other host genes were evaluated. Additionally, ELISA, Real-time PCR, Western blot, bioinformatics analysis, gene overexpression/knock-down, mutagenesis, chromatin immunoprecipitation, epigenetic studies, immunofluorescence, and quantification of serum HBV-DNA, bacterial-DNA and endotoxin
- 5. Result Among the MAC components (C5b-C9), significant reduction was noted in the expression of C9, the major constituent of MAC, in HBV-infected HepG2hNTCP cells and in Huh7 cells transfected with full-length HBV as well as HBX. C9 level was also marked low in sera/liver of chronic hepatitis B (CHB) and Immune-tolerant (IT) patients than inactive carriers and healthy controls. HBX strongly repressed C9-promoter activity in Huh7 cells but CpG-island was not detected in C9-promoter. We identified USF-1 as the key transcription factor that drives C9 expression and demonstrated that HBX-induced hypermethylation of USF-1-promoter is the leading cause of USF-1 downregulation that in turn diminished C9 transcription. Reduced MAC formation and impaired lysis of HBV-transfected Huh7 and bacterial cells were observed following incubation of these cells with C9-deficient CHB sera but was reversed upon C9 supplementation. Significant inverse correlation was noted between C9 concentration and HBV-DNA, bacterial-DNA and endotoxin content in HBV-infected patients. One-year Tenofovir therapy resulted in improvement in C9 level and decline in viral/bacterial/endotoxin load in CHB patients.
- 6. Conclusion Collectively, HBX suppressed C9 transcription by restricting the availability of USF-1 through hypermethylation of USF-1-promoter and consequently hinder the formation and lytic functions of MAC. Early therapy is needed for both CHB and IT to normalize the aberrant complement profile and contain viral and bacterial infection and limit disease progression.
- 7. Downregulation of C9 expression in HBV-transfected Huh7 cells, HBV-infected HepG2hNTCP cells and in chronically HBV-infected patients To explore whether HBV could interfere with MAC formation, we first examined the expression of different MAC components (C5/C6/C7/C8/C9) in Huh7 cells transfected with full-length HBV by Real-time PCR. As indicated in Fig. 1a, a significant reduction in C9 expression (~ 1.5-fold) was perceived in HBV-transfected Huh7 cells as compared to untransfected cells while the expression of other components of MAC remained comparable. Further, we also investigated the expression of MAC components in HepG2hNTCP cells infected with cell culture-derived HBV produced by stable HBV-replicating cells (HepG2.2.15). The results depicted a similar trend as seen in Huh7 cells, with a significant (~ 2.4 fold) downregulation in C9 expression in HBV-infected HepG2hNTCP cells relative to uninfected control cells whereas no variation was observed in the expression of C5, C6 and C8 (Fig. 1b). C7 was undetectable in hNTCP
- 8. Cont…
- 9. Cont.. We next determined by ELISA, the level of C9 in the sera of HC and chronically HBV-infected patients in different disease phases, IT, EP-/EN- CHB and IC. The clinical and demographic features of study subjects are provided in Additional file 1: Table S3. C9 was found to be significantly low in IT, EP-CHB and EN-CHB relative to IC and HC (Fig. 1c). Further, when compared between IC and HC, C9 concentration was less in IC than HC. In addition, we detected that C9 transcript level in liver biopsy tissues of CHB patients was significantly diminished than control
- 10. HBX suppresses the expression of C9 To identify the viral factors responsible for the downregulation of C9, Huh7 cells were separately transfected with plasmids expressing different HBV proteins and the expression of C9 was evaluated by Real-time PCR. C9 mRNA level was found to be substantially low (~ 4.7 fold) in HBx-transfected Huh7 cells than in cells transfected with empty- vector (Fig. 1e). In contrast, cells expressing other HBV proteins showed no significant change in the amount of C9 mRNA relative to control. The down-regulatory effect of HBX on C9 expression was further confirmed at protein levels by immunoblot analysis of cell lysate with anti-C9 antibody and a marked reduction in C9 was noted as compared to control cells (Fig. 1f). To assess the specificity of inhibitory effect of HBX on C9 expression, Huh7 was transfected with increasing amounts of HBx-expressing plasmid and a corresponding dose-dependent diminution in C9 mRNA level was observed (Fig. 1g). Collectively, the results suggest that HBX resulted in a decline in C9 expression.