Acetaldehyde suppresses HBV-MHC class I complex presentation on hepatocytes via induction of ER stress and Golgi fragmentation

Abstract

Alcohol consumption worsens hepatitis B virus (HBV) infection pathogenesis. We have recently reported that acetaldehyde suppressed HBV peptide-major histocompatibility complex I (MHC class I) complex display on hepatocytes, limiting recognition and subsequent removal of the infected hepatocytes by HBV-specific cytotoxic T lymphocytes (CTLs). This suppression was attributed to impaired processing of antigenic peptides by the proteasome. However, in addition to proteasome dysfunction, alcohol may induce endoplasmic reticulum (ER) stress and Golgi fragmentation in HBV-infected liver cells to reduce uploading of viral peptides to MHC class I and/or trafficking of this complex to the hepatocyte surface. Hence, the aim of this study was to elucidate whether alcohol-induced ER stress and Golgi fragmentation affect HBV peptide-MHC class I complex presentation on HBV+ hepatocytes. Here, we demonstrate that, while both acetaldehyde and HBV independently cause ER stress and Golgi fragmentation, the combined exposure provided an additive effect. Thus we observed an activation of the inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α, but not the phospho PKR-like ER kinase-phospho eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein arms of ER stress in HBV-transfected cells treated with acetaldehyde-generating system (AGS). In addition, Golgi proteins trans-Golgi network 46, GM130, and Giantin revealed punctate distribution, indicating Golgi fragmentation upon AGS exposure. Furthermore, the effects of acetaldehyde were reproduced by treatment with ER stress inducers, thapsigargin and tunicamycin, which also decreased the display of this complex and MHC class I turnover in HepG2.2.15 cells and HBV-infected primary human hepatocytes. Taken together, alcohol-induced ER stress and Golgi fragmentation contribute to the suppression of HBV peptide-MHC class I complex presentation on HBV+ hepatocytes, which may diminish their recognition by CTLs and promote persistence of HBV infection in hepatocytes.NEW & NOTEWORTHY Our current findings show that acetaldehyde accelerates endoplasmic reticulum (ER) stress by activating the unfolded protein response arms inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α but not phospho PKR-like ER kinase-p eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein in hepatitis B virus (HBV)-transfected HepG2.2.15 cells. It also potentiates Golgi fragmentation, as evident by punctate distribution of Golgi proteins, GM130, trans-Golgi network 46, and Giantin. While concomitantly increasing HBV DNA and HBV surface antigen titers, acetaldehyde-induced ER stress suppresses the presentation of HBV peptide-major histocompatibility complex I complexes on hepatocyte surfaces, thereby promoting the persistence of HBV infection in the liver.

Keywords: ER stress; Golgi; HBV; acetaldehyde; hepatocytes.

Graphical abstract

Fig. 1.

Effects of acetaldehyde-generating system (AGS) and hepatitis B virus (HBV) on unfolded protein response (UPR) mRNA markers in HepG2 and HepG2.2.15 cells. Cells were either treated or not treated with AGS for 72 h and then harvested in TRIzol for RNA isolation. Real-time PCR (RT-PCR) analysis was done for spliced X-box binding protein 1 (sXBP-1) (A), uXBP-1 (B), ER degradation-enhancing-α-mannosidase-like protein 1 (EDEM1) (C), immunoglobin-binding protein (BiP) (D), and activation transcription factor (ATF)4 (E), and C/EBP homologous protein (CHOP) (F) GAPDH was used as an internal control. Data are from 3 independent experiments presented as means ± SE. Bars marked with the same letter are not significantly different from each other; bars with different letters are significantly different (P ≤ 0.05).

Fig. 2.

Effects of acetaldehyde-generating system (AGS) and hepatitis B virus (HBV) on unfolded protein response (UPR) protein expression in HepG2 and HepG2.2.15 cells. Cells were either treated or not with AGS for 72 h. Then protein expressions were detected by immunoblotting in cell lysates. A: phospho- and total inositol-requiring enzyme 1α (IRE1α). C: cleaved activation transcription factor (ATF)6α. B and D: quantification of immunoblotting bands. E: phospho-PKR-like ER kinase (PERK), phospho eukaryotic initiation factor 2α (peIF2α), ATF4, and C/EBP homologous protein (CHOP). F: immunoglobin-binding protein (BiP). G: cleaved caspase-3. H: nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione peroxidase 1(GPx1). Equal (20 µg) amounts of protein were loaded in each lane. β-Actin was used as an internal control. Data are from 3 independent experiments presented as means ± SE. Bars marked with the same letter are not significantly different from each other; bars with different letters are significantly different (P ≤ 0.05).

Fig. 3.

Acetaldehyde-generating system (AGS) modulates Golgi fragmentation in HepG2.2.15 and HepG2 cells. Cells were either treated or not with AGS for 72 h and processed for staining. A–C: immunofluorescence staining of GM130, trans-Golgi network 46 (TGN46), and Giantin expression. Staining was visualized using a ×63 lens in LSM 710 confocal microscope. Pictures are shown as the representative from 3 independent experiments with similar results.

Fig. 4.

Acetaldehyde-generating system (AGS) and endoplasmic reticulum (ER) stress inducers suppress expression of hepatitis B virus (HBV) peptide-human leukocyte antigen-A2 (HLA-A2) complex on the surface of HepG2.2.15 cells. A: HepG2.2.15 cells were treated or not with AGS for 72 h, and expression of HBV core peptide (18-27) FLPSDEFPSV-HLA-A2 was measured by flow cytometry with antibody to HBV peptide-HLA-A2 followed by exposure to Alexa Fluor 647 secondary antibody. Mouse IgG2b K isotype control was used. B: expression of HBV core peptide (18-27) FLPSDEFPSV-HLA-A2 measured by flow cytometry in thapsigargin (100 nM)- and tunicamycin (10 µg)-treated HepG2.2.15 cells. Data were processed using BD LSR2 flow cytometer, analyzed by BD FACSDiva Software v6.0 D, and shown as the representative expression from 3 independent experiments with similar results. C: quantification of flow data. Bars marked with the same letter are not significantly different from each other; bars with different letters are significantly different (P ≤ 0.05).

Fig. 5.

Acetaldehyde-generating system (AGS) and endoplasmic reticulum (ER) stress inducers decrease on surface major histocompatibility complex I (MHC class I) turnover. A: HepG2.2.15 cells were treated or not with AGS for 72 h. B: HepG2.2.15 cells were treated or not with thapsigargin (100 nM) and tunicamycin (10 µg). Then surface MHC class I was removed from cell surface by low-acid wash, followed by measurement of restoration of human MHC class I by flow cytometry using anti-human leukocyte antigen-A2 (HLA-A2) antibody. Mouse IgG2b K isotype control APC was used. A representative experiment (1 out of 3 with similar results) was performed on detection of MHC class I measured by flow cytometry. C: quantification of flow data. Data are from 3 independent experiments presented as means ± SE. Bars marked with the same letter are not significantly different from each other; bars with different letters are significantly different (P ≤ 0 0.05).

Fig. 6.

Ethanol and endoplasmic reticulum (ER) stress inducers diminish expression of hepatitis B virus (HBV) peptide-human leukocyte antigen-A2 (HLA-A2) complex on the surface of hepatocytes. Here, we used primary human hepatocytes plated on polyelectrolyte multilayer (PEM) gels to prolong expression of alcohol-metabolizing enzymes, cytochrome s4502E1 (CYP2E1), and alcohol dehydrogenase (ADH). Hepatocytes were infected with HBV and exposed to 50 mM EtOH (as described in materials and methods). A: primary human hepatocytes were treated or not with either EtOH for 48 h or ER stress inducers overnight. Then expression of HBV core peptide (18-27) FLPSDEFPSV-HLA-A2 was measured by flow cytometry. Mouse IgG2b K isotype control was used. The results of representative (1 out of 3) experiment are shown. B: quantification of flow data. Tg, thapsigargin; Tn, tunicamycin. C: HBV DNA levels were measured by ddPCR, respectively. D: HBV surface antigen (HBsAg) levels were measured by Sandwich ELISA kit. Data are from 3 independent experiments presented as mean ± SE. Bars marked with the same letter are not significantly different from each other; bars with different letters are significantly different (P ≤ 0.05).

Fig. 7.

Alcohol suppresses hepatitis B virus (HBV) peptide-major histocompatibility complex I (MHC class I) presentation on HBV-infected hepatocytes via induction of endoplasmic reticulum (ER) stress and Golgi fragmentation. Alcohol metabolites (acetaldehyde) potentiate ER stress and Golgi fragmentation in HBV-expressing hepatocytes. This impairs the delivery of HBV peptide-MHC class I complex and turnover of MHC class I on infected hepatocyte surface. Consequently, it may diminish recognition of infected hepatocytes by cytotoxic T lymphocytes (CTLs), leading to chronic persistence of HBV infection.