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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Alcohol Clin Exp Res. 2021 Nov 23;46(1):40–51. doi: 10.1111/acer.14740

Ethanol attenuates presentation of cytotoxic T-lymphocyte epitopes on hepatocytes of HBV-infected humanized mice

Murali Ganesan 1,2, Weimin Wang 3, Saumi Mathews 3, Edward Makarov 3, Moses New-Aaron 1,4, Raghubendra Singh Dagur 1,2, Antje Malo 5, Ulrike Protzer 5,6, Kusum K Kharbanda 1,2, Carol A Casey 1,2, Larisa Y Poluektova 3, Natalia A Osna 1,2
PMCID: PMC8799491  NIHMSID: NIHMS1755987  PMID: 34773268

Abstract

Background & Aims:

3.5% of the global population chronically infected with Hepatitis B Virus (HBV) are under a high risk of end-stage liver disease outcomes, further potentiated by alcohol. However, the mechanisms behind the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs).

Methods:

We used Fumarylacetoacetate hydrolase (Fah)−/−, Rag2−/−, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2) positive hepatocytes. These mice were HBV-infected and fed control and ethanol diets. Isolated hepatocytes were ex vivo exposed to HBV 18–27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers.

Results and Conclusions:

We found that ethanol feeding attenuated HBV core 18–27-HLA-A2 complex presentation on infected hepatocytes due to suppression of proteasome function and ER stress induction, which diminishes both processing of HBV peptides and trafficking of HBV-MHC class I complexes to hepatocyte surface. This ethanol-mediated decrease in MHC class I-restricted antigen presentation of CTL epitope on target hepatocytes reduced CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes.

Keywords: HBV, Ethanol, Hepatocytes, Antigen Presentation

Introduction

Worldwide, approximately 370 million people are chronically Hepatitis B Virus (HBV) infected, and every year, 780,000 people die due to complications from HBV infection (WHO, 2017, Comber et al., 2014). The end-stage liver disease outcomes include liver cirrhosis and hepatocellular carcinoma in about 40% of chronically infected patients (Comber et al., 2014). These outcomes are worsened in patients with alcohol use disorder (AUD), and combined insults of alcohol and HBV increase incidence of such manifestations as steatosis, fibrosis, cirrhosis and HCC (Sayiner et al., 2019). However, the mechanisms are not clear yet.

HBV, which is not a cytopathic virus, infects only hepatocytes and persists in these cells without killing them. Adaptive immune response by HBV-specific CD8+ T cell (cytotoxic T-lymphocytes-CTLs) is mainly involved in the clearance of HBV-infected hepatocytes and disease pathogenesis (Gehring and Protzer, 2019). Thus, in chronic HBV-infection, viral clearance depends on the host’s immune response to viral antigens presented on the surface of infected hepatocytes (Eddleston and Mondelli, 1986). For effective clearance, HBV-specific CTLs recognize their cognate antigenic peptides in the context of major histocompatibility complex (MHC) class I molecules to induce infected cell elimination, via early non-cytolytic killing inflammatory cytokines (mainly, IFNγ) release (non-cytolytic) followed by direct killing of HBV-expressing targets (cytolytic mechanism), thereby limiting the spread of infection (Murata et al., 2018). Since alcohol exposure promotes HBV persistence, there is a possibility that alcohol exposure affects either functional properties of effector CTLs or target HBV-infected hepatocytes, or both. While dysregulation of both cell-mediated and humoral immunity is a consequence of long-term alcohol use (Barve et al., 2002, Geissler et al., 1997), less is known about alcohol-induced dysfunctions in target hepatocytes, which helps them avoiding CTL-mediated clearance. This made us concentrate on the alcohol-modulated display of HBV-MHC class I complexes on hepatocytes recognized by epitope-specific CTLs.

For presentation of viral peptide-MHC class I complex on hepatocyte surface, viral antigens need to be processed to peptides of certain length by both constitutive proteasome and immunoproteasome (IPR) (Arellano-Garcia et al., 2014). Then with the help of transporters associated with antigen processing (TAPs), these antigenic peptides are transported to endoplasmic reticulum (ER) for MHC class I molecules loading (Bitzer et al., 2016). These complexes are trafficked via the trans-Golgi to the plasma membrane, where they are supposed to be recognized by the T-lymphocyte cell receptor (TCR) on CTL (Fritzsche and Springer, 2013).

Recently, we have shown in vitro that alcohol metabolite, acetaldehyde suppresses HBV core 18–27 peptide-MHC class I complex presentation on HBV-transfected HepG2.2.15 cells via disruption of the peptide processing by proteasome, induction of ER stress and Golgi fragmentation (Ganesan et al., 2019, Ganesan et al., 2020). However, such studies have never been done on PHH isolated from in vivo HBV-infected humanized mice fed control and ethanol diets. In addition, ex vivo studies testing the ability of HBV-infected PHH from these mice to be recognized and eliminated by HBV core 18–27-HLA-A2-specific CTLs have never been performed.

Here, we aimed to establish in vivo evidence of alcohol-mediated suppression of HBV peptides -MHC class I complex display on HBV-infected primary human hepatocytes (PHH). We anticipate that the decrease in MHC class I-restricted HBV peptide presentation on PHH diminishes the recognition and clearance of infected hepatocytes by CTLs. For this purpose, as a CTL epitope, we specifically chose HBV core peptide 18–27 (FLPSDEFPSV) presented on PHH in the context of human leukocyte antigen-A2 (HLA-A2) (Bertoletti et al., 1994, Maini et al., 1999). This CTL epitope is associated with viral control (Liu et al., 2012) and has been reported as having therapeutic potential (Shi et al., 2004). Our study will further identify the mechanisms by which alcohol influences HBV-infection pathogenesis to identify the targets for future treatment interventions.

2. Materials and Methods

2.1. Reagents and Media:

Hepatocyte plating and maintenance supplements along with Williams E media were obtained from Gibco by ThermoFisher Scientific (Foster City, CA). Trizol was acquired from Life Technologies (Carlsbad, CA). All RNA isolation, cDNA synthesis and RT-PCR reagents were obtained from ThermoFisher Scientific (Carlsbad & Foster City, CA). Proteasome activity substrates (Boc-LRR-AMC and Suc-LLVY-AMC) were procured from UBPBio Inc (Aurora,CO). Ac-Ala-Asn-Trp-AMC, an immunoproteasome substrate, was obtained from BostonBiochem (Cambridge, MA). For flow cytometry studies to measure the expression of HBV core peptide FLPSDEFPSV-HLA-A2, Human Fc receptor blocker (Affymetrix-eBioscinece, San Diego, CA) and HBV peptide 18–27-HLA-A2 antibody (Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE) were used. Hybridoma cells (where HBV peptide 18–27-HLA-A2 antibodies used in flow cytometry were purified from) were acquired from PHARMEXA, Inc (San Diego, CA). LDH Assay Kit (Cytotoxicity, Fluorometric) and Anti-HBV core antibody were purchased from Abcam Inc. (Cambridge, MA). The following antibodies: anti-anti-ATF, atni-peIF2α, anti-pPERK, anti-CHOP, BiP, and cleaved caspase-3 were obtained from Cell Signaling (Beverly, MA). Antibodies Anti-pIRE1 and anti-ATF6 were purchased from Novus Biologicals, LLC (Centennial, CO). LMP7, LMP2, and Anti-β actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). HBV surface antigen (HBsAg) ELISA Kit was from LifeSpan Biosciences, Inc. (Seattle, WA). Control and ethanol diet were purchased from (Lieber-DeCarli diet, Shake and Pour) Bio-Serv (New Jersey, NJ). Antibody details were mentioned in supplement.

2.2. In Vivo Methods

2.2.1. Experimental manipulations on FRG-KO mice with liver humanization:

Fumarylacetoacetate hydrolase (Fah)−/−, Rag2−/−, common cytokine receptor gamma chain knock-out (FRG-KO on C57BL/6 strain background), highly immunosuppressed FRG™ male mice deficient in T cells, B cells, and natural killer cells transplanted with human HLA-A2+ hepatocytes from a single donor (>80% humanization) were purchased from Yecuris Corporation (OR, USA) and housed in the pathogen-free animal facility at the University of Nebraska Medical Center (UNMC). Animal studies were carried out according to the guidelines for the humane care of laboratory animals as approved by the UNMC Animal Care and Use Committee (IACUC). Liver humanization was characterized by human albumin levels with no cross-reactivity to mouse albumin (ELISA, Bethyl Laboratories Inc.; TX, USA) as described previously (Dagur et al., 2018). These mice showed average human albumin levels of 7161 ± 276 μg/mL, which was not affected by ethanol feeding. Supplementary figure 1 shows the humanization and about 90–95% human cells were HBV infected. They were infected intravenously with HBV (106 genome equivalents (GE)/mouse) for 45 days using de-identified patient serum (single donor-same genotype-D) sample (UNMC clinic). After 45 days of infection, based on equal HBV titer, mice were randomized into two groups that is pair-fed with isocaloric control or 5% ethanol (EtOH) diets. For 5% of EtOH feeding, we used NIH-Gao model (Bertola et al., 2013), (ramp up stage 1%, 2%, 3%, and 4% EtOH - 2days/each %). Then 5% EtOH for 10 days, on 11th day, 8 hours before sacrifice, we gavage single dose of EtOH (5g/kg body weight). We call this post-HBV EtOH feeding since this feeding started when mice were already infected. Each group contains 5–6 mice.

2.2.2. Hepatocyte Isolation and Culture:

Primary hepatocytes were isolated from livers of HBV+ control- and EtOH-fed mice by a modified collagenase perfusion technique as described and used previously (Casey et al., 1987, Tworek et al., 1996). For CTL cytotoxic killing assay, hepatocytes were suspended in William’s media as plating media and seeded onto collagen-coated 96-well plates. After 4 hr at 37oC, cells were washed with William’s maintenance media and then incubated in the same media. The latter incubations were conducted in the presence or absence of HBV-specific 18–27 transduced T cells. Additional portions of hepatocytes were used for HBV 18–27 peptide-MHC class I complex flow cytometry analysis. Remaining aliquots were washed in cold PBS, and the pellets were stored at −70 °C for RNA, DNA and immunoblotting analysis.

2.2.3. Serum Enzymes, Triglycerides and Glutathione Measurement:

The clinical laboratory at the VA NWIHCS analyzed the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), both of which are serum enzymes. The Folch extraction method was used to extract and saponify hepatic lipids as previously described (Rasineni et al., 2019). Then triglycerides were quantified spectrophotometrically using the Infinity Triglycerides reagent (Thermo Fisher Scientific, Middletown, VA). Glutathione levels and GSH/GSSG ratio were measured using the enzymatic recycling method (Tietze, 1969).

2.2.4. Flow Cytometry Analysis:

The presentation of HBV peptide 18–27-HLA-A2 complex was measured as previously described (Ganesan et al., 2019). Flow buffer was used to wash cell cultures and then a cell pellet was obtained via centrifugation. Human Fc receptor was added to cell pellet and then incubated for 20 minutes on ice. Then HBV peptide 18–27-HLA-A2 primary antibody was added and incubated on ice 1 hour. After removal of primary antibody and subsequent washing, Alexa Fluor 647 secondary antibody was added for and incubated for 30 mins on ice. In order to perform flow cytometry analysis, the cells were fixed in 2% paraformaldehyde. Using a BD LSR2 flow cytometer, data was collected and then analyzed by using BD FACSDiva™ Software v6.0.

2.2.5. Proteasome Activity:

As previously reported by our laboratory (Ganesan et al., 2019), Proteasome chymotrypsin-like (Cht-L), trypsin-like (T-L) and immunoproteasome activities were detected by fluorometric assay.

2.2.6. Immunoblotting (western blot):

Immunoblotting has been done in hepatocytes as previously described (Ganesan et al., 2019). Antibodies used in this study were mentioned in supplementary table S1.

2.2.7. RNA, DNA isolation, Real-time PCR (RT-PCR) and ddPCR:

RT-PCR was used to measure HBV RNA level in hepatocytes as described previously. (Ganesan et. al., 2019). ddPCR was used to quantify HBV DNA levels as previously described (Ganesan et. al., 2020) using the following primers and probes: HBV sense (5′-CGA CGT GCA GAG GTG AAG-3′), antisense (5′-CAC CTC TCT TTA CGC GGA CT-3′) primers, and HBV probe (5′-/56-FAM/ATC TGC CGG/ZEN/ACC GTG TGC AC/3IABkFQ/−3′).

2.2.8. HBsAg Sandwich ELISA:

ELISA using LSBio Kit (LifeSpan Biosciences, Inc, Seattle, WA) was used to measure HBV surface antigen (HBsAg) levels in serum.

2.3. Ex-Vivo study Methods:

2.3.1. HBV-CTLs-induced cytotoxicity:

HBV specific T-cell receptor (TCR) bearing T-cells, (T cells stably expressing high-affinity HBV core–specific 18–27-HLA-A2 T-Cell receptor) obtained from Institute of Virology, München, Germany, were used as CTL effectors (Wisskirchen et al., 2019) and HBV-infected hepatocytes isolated from control and alcohol fed were used as target cells. Target cells were seeded at a cell density of 5 × 104 cells per well in 96-well plates. Effector cells were incubated with hepatocytes at the effector and target (E/T) ratio 10:1 at 37 °C, 5% CO2 for 8 h. The specific CTL activity was measured using LDH Assay Kit for lactate dehydrogenase (LDH) release according to the manufacturer’s instructions. The fluorescence values were recorded at Ex/Em = 535/587 nm.

2.4. Statistical Analyses

The data was expressed as mean values ± standard error. A one-way analysis of variance (ANOVA) was performed on the multiple groups of this study in, and comparison was made possible using a Tukey post-hoc test. A Student’s t-test was used to compare two groups at a time. Results were considered significant at a probability value of 0.05 or less.

3. Results

3.1. In vivo ethanol feeding induces liver injury in HBV-infected humanized liver hepatocytes:

In current study, HBV-infected ethanol-fed mice showed an increase in ALT and numerical increase in AST levels in serum when compared to control HBV-infected mice (Figure 1A, B). Here, we found that alcohol stabilized CYP2E1 (about 3-fold) when compared to HBV-infected control hepatocytes, whereas the levels of alcohol dehydrogenase (ADH) were not altered (Figure 1C). The reduced glutathione levels were decreased (P<0.05), and lipid peroxidation products (4-hydroxynonenal, 4HNE) expression was increased in ethanol-fed HBV-infected mice (Figure 1D, E). GSH/GSSG ratio was also reduced in hepatocytes of alcohol fed HBV-infected mice (supplementary Figure 2). Importantly, we noted that the hepatocytes from HBV-infected alcohol-fed mice showed about 2-fold increase in triglyceride levels (Figure 1F), which is considered as fatty liver. In addition, in this humanized FRG™ mice model, alcohol feeding significantly increased the lipid accumulation and fibrosis marker collagen (supplementary Figure 3A and B).

Fig. 1. Effects of alcohol on serum transferases and oxidative stress markers in the hepatocytes from HBV-infected humanized mice:

Fig. 1.

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(A, B) ALT and AST (serum enzymes) activities determined in serum of HBV-infected control and alcohol-fed mice. (C) Protein expressions of ADH and CYP2E1 (alcohol metabolizing enzymes) measured by immunoblotting; β-actin acted as an internal control. (D) Glutathione levels measured by spectrophotometry as described in Methods. (E) 4-hydroxynonenal (4HNE) adducts measured by immunoblotting. (F) Triglycerides measured spectrophotometrically using the Infinity Triglycerides reagent. Data are expressed as Mean ± SEM (n = 5). The data bars marked with the same alphabetical letter are considered not significantly different, while those marked with differing alphabetical letters are considered significantly different from each other (p≤0.05).

3.2. In vivo suppression of HBV core 18–27-HLA-A2 complex presentation on target PHH by ethanol decreases ex-vivo tested cytotoxic effects of CTLs:

In hepatocytes isolated from the livers of HBV-infected mice fed either control or ethanol diets, we measured HBV core 18–27-HLA-A2-complex presentation. As shown on Figure 2A, B, there was 53% suppression of HBV peptide-HLA-A2 complex presentation. In ex vivo studies of HBV+PHH and HBV core 18–27 peptide-specific CTLs, we found that only 13% hepatocytes from ethanol-fed HBV-infected mice were lysed by these CTLs vs 26% hepatocytes from mice fed control diet (Figure 2C). Importantly, in this ex vivo study, only hepatocytes were obtained from livers of control and ethanol mice, but CTLs were not exposed to ethanol, and thus, CTL function was not compromised by ethanol, which attributes all changes in cytotoxicity to ethanol-altered presentation of HBV peptide-MHC class 1 complex on hepatocytes. We observed no suppression of hepatocyte viability when cells were isolated from HBV-negative mice and exposed to the same CTLs (negative controls, not shown).

Fig. 2. Effects of ethanol on HBV 18–27 peptide-MHC class I complex presentation and ability of HBV specific 18–27 CTLs to kill hepatocytes isolated from HBV-infected control- and ethanol-fed humanized mice :

Fig. 2.

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(A) HBV-infected hepatocytes from control and alcohol-fed humanized mice were isolated by liver perfusion. Flow cytometry was used to measure the expression of HBV core peptide (18–27) FLPSDEFPSV-HLA-A2 as previously described. (B) Quantification of flow data. Data from 5 independent experiments with comparable results are portrayed as representative expression. (C) HBV 18–27 specific CTL activity was measured by lactate dehydrogenase (LDH) assay. HBV-infected hepatocytes were incubated with HBV 18–27 specific T-cell receptor (TCR) bearing T-cells for 8 h, and cytotoxicity was measured as fluorescence using LDH assay kit. CTL activity is indicated as percentage of cytotoxicity at a target:effector ratio 1:10. The data are expressed as Mean ± SEM (n = 5) are from three separate experiments. The data bars marked with the same alphabetical letter are considered not significantly different, while those marked with differing alphabetical letters are considered significantly different from each other (p≤0.05).

3.3. In vivo ethanol feeding suppresses proteasome/immunoproteasome in hepatocytes of HBV-infected mice:

We measured constitutive (ChT-L, T-L) and immunoproteasome (IPR) activities (β5i-LMP7) using fluorogenic substrates. Figure 3 A, B and C showed that all three proteasome activities were significantly (P<0.05) decreased in hepatocytes from alcohol-fed HBV-infected humanized mice when compared to HBV+ mice fed control diet. Additionally, we tested whether alcohol affects protein expression of IPR subunits. As shown on Figures 3 D, E, and F, alcohol feeding decreased the levels of IPR subunit proteins, LMP2 and LMP7 for about 42% and 38%, respectively. These immunoblotting data were further confirmed by immunofluorescence staining of LMP2 and LMP7 (supplementary Figure 4 A, B).

Fig. 3. Alcohol decreases proteasome activities and immunoproteasome subunit protein expression in the hepatocytes:

Fig. 3.

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(A) Chymotrypsin Like Activity-Cht-L, (B) Trypsin Like Activity-T-L and (C) Fluorometric assay, using substrates Boc-LRR-AMC, Ac-ANW-AMC, and Suc-LLVY-AMC, were used to detect Immunoproteasome-β5i-LMP7 activities. (D, E, F) IPR subunits, LMP2 and LMP7 proteins expression were detected by immunoblotting in cell lysates. β-actin was used as an internal control. Data are presented as Mean ± SEM (n = 5). The data bars marked with the same alphabetical letter are considered not significantly different, while those marked with differing alphabetical letters are considered significantly different from each other (p≤0.05).

3.4. In vivo effects of ethanol on mRNA expression of Unfolded protein response (UPR) genes:

We investigated the in vivo effects of alcohol on unfolded protein response and ER stress. We measured mRNA expression of three arms of UPR genes by RT-PCR. It was found that spliced X-box binding protein 1 (sXBP1), ER-degradation-enhancing-α-mannosidase-like protein (EDEM1), activating transcription factor 4 (ATF4) and the C/EBP homologous protein (CHOP) mRNA levels were significantly (P<0.05) increased by alcohol feeding when compared to hepatocytes from HBV-infected mice fed control diet (Figure 4A–D). However, we observed no significant alterations in both unspliced XBP-1 (uXBP-1) and immunoglobin binding protein (BiP) as a central regulator of the UPR stress sensors (Figure 4E, F).

Fig. 4. Effect of Alcohol on Unfolded Protein Response (UPR) mRNA markers in hepatocytes:

Fig. 4.

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Real-Time PCR analysis was done for: (A) BiP, (B) sXBP-1 (C) uXBP-1, (D) EDEM1, (E) ATF4 and (F) CHOP. GAPDH acted as an internal control. Data are presented as Mean ± SEM (n = 4). The data bars marked with the same alphabetical letter are considered not significantly different, while those marked with differing alphabetical letters are considered significantly different from each other (p≤0.05).

3.5. In vivo effects of ethanol on ER stress in hepatocytes:

Next, we tested whether ethanol feeding causes ER stress in hepatocytes from HBV-infected humanized mice. By immunoblotting, we measured protein expression of ER stress markers and found that BiP chaperone was significantly increased (about 1.5-fold) by ethanol feeding. In addition, the first arm of UPR, IRE1α was also activated (pIRE1 about 2-fold) in hepatocytes from alcohol-fed HBV-infected mice when compared to control HBV+ mice (Figure 5A–C). Next, we tested a second arm of UPR called ATF6α and found that cleaved ATF6α was significantly increased (P<0.05) by ethanol feeding (Figure 5D, E). However, we observed no alterations in the third arm of the UPR, pPERK, and peIF2α (Figure 5F). Interestingly, the downstream part of the PERK pathway (proteins ATF4 and CHOP), was significantly increased in alcohol-fed mice when compared with control HBV-infected humanized mice (Figure 5F). We checked whether alcohol feeding causes apoptosis in HBV-infected mice. We observed no apoptosis in hepatocytes from both alcohol- and control-fed HBV-infected mice based on cleaved caspase −3 immunoblotting (Figure 5 I).

Fig. 5. Effect of Alcohol on ER stress protein markers in the hepatocytes:

Fig. 5.

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Hepatocyte lysate immunoblotting detected ER stress marker protein expressions. (A) BiP, phospho- and total IRE1α; (B, C) quantification of immunoblotting bands; (D,E) cleaved ATF6α and quantification of bands. (F) phospho-PERK, phosphor-eIF2α, ATF4 and CHOP; (G, H) quantification of bands. (I) Procaspase-3 and cleaved caspase 3. β-actin was used as an internal control and we used panel-A beta actin for panel-D quantification. Data (n=4) are given as Mean ± SEM. The data bars marked with the same alphabetical letter are considered not significantly different, while those marked with differing alphabetical letters are considered significantly different from each other (p≤0.05).

3.6. In vivo ethanol feeding suppresses Interferon Stimulated Gene (ISG) expression in hepatocytes of HBV-infected humanized mice:

In the absence of CTLs and IFNγ release by immune cells, the expression of HBV markers is controlled by innate immunity can be affected by alcohol. Thus, we measured mRNA expression of IFN type 1-inducible anti-viral genes, OAS1, APOBEC and ISG15. These mRNA expressions were significantly (P<0.05) decreased in hepatocytes from alcohol-fed HBV-infected mice (Figure 6A–C).

Fig. 6. Effect of alcohol on mRNA expression of IFN-stimulated genes in hepatocytes:

Fig. 6.

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RT-PCR analysis was done for mRNA expression of IFN-induced genes. (A) 2′–5′ oligoadenylate synthetase 1 (OAS1); (B) Apolipoprotein B Editing Complex (APOBEC3); (C) Interferon-stimulated gene 15 (ISG15). GAPDH acted as an internal control. Data are given as Mean ± SEM (n = 4). The data bars marked with the same alphabetical letter are considered not significantly different, while those marked with differing alphabetical letters are considered significantly different from each other (p≤0.05).

3.7. In vivo effects of ethanol feeding on expression of HBV-infection markers

We measured HBV RNA and HBV DNA by RT-PCR and ddPCR, HBsAg expression in serum by quantitative ELISA, and performed immunoblotting of HBV core in hepatocytes (Figure 7 A–E). These HBV-specific markers were up-regulated in hepatocytes isolated from the livers of ethanol-fed mice.

Fig. 7. Effect of alcohol on HBV RNA, HBV DNA, HBsAg and core protein levels in the hepatocytes:

Fig. 7.

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(A) HBV RNA levels were measured by Real-Time PCR. (B) HBV DNA levels measured by Droplet Digital™ PCR. GAPDH acted as an internal control for HBV RNA and HBV DNA. (C) Sandwich Elisa kit used to measure HBsAg levels. (D, E) Representative image of immunoblotting of HBV core protein and quantification of bands. Data are expressed as Mean ± SEM (n =4–5). The data bars marked with the same alphabetical letter are considered not significantly different, while those marked with differing alphabetical letters are considered significantly different from each other (p≤0.05).

Discussion

Immune responses to viral infections are a complex interplay between the virus, target cells, and effector cells of the immune system (Demers et al., 2013). HBV-specific CD8+ T cells recognize their cognate antigenic peptide-MHC class I receptors on target hepatocytes to induce infected cell death and inflammatory cytokine expression, which prevents the spread of infection (Khakpoor et al., 2019). The efficiency of HBV CTL epitope presentation after infection (Khakpoor et al., 2019), and the ability of CD8+ T cells to recognize HBV-infected hepatocytes have been studied in chimpanzees (Thimme et al., 2003) and on humanized chimeric mouse models (Kah et al., 2017). However, so far, the effects of alcohol on HBV core peptide 18–27-HLA-A2 complex presentation on hepatocytes and the recognition of these HBV-infected hepatocytes by CTLs has not been studied in the livers of humanized mice. Importantly, the mechanisms behind the above processes are not well-characterized. This is first in vivo study where we investigated the effects of alcohol on the CTL epitope, HBV core peptide 18–27-HLA-A2 complex presentation on HBV-infected hepatocytes and on the ex vivo recognition of this complex by CTLs to eliminate HBV+ hepatocytes. It is worth investigating this important concept because alcohol consumption negatively affects disease outcomes and increases the incidence of HBV-triggered cirrhosis and HCC (Lin et al., 2013). This study was performed on hepatocytes isolated in the livers of in vivo HBV-infected humanized mice fed control and ethanol diets. This cannot be considered as the limitation of this study because only human hepatocytes are HBV-infected. Furthermore, proteasome, immunoproteasome and ER stressed marker are also detected in non-parenchymal liver cells, and the measurements of these parameters in the whole liver tissue may not reflect the quantitative and qualitative changes identified in antigen presentation by HBV-infected hepatocytes exposed to ethanol.

Based on our previous in vitro findings on HepG2.2.15 cells, we hypothesized here that under the real HBV-infection circumstances, ethanol exposure decreases the recognition and clearance of HBV-infected hepatocytes due to suppression of the CTL epitope presentation on hepatocyte surface. To distinguish between the suppressive effects of ethanol on CTL (effector cells) activity and the presentation of CTL epitopes by target hepatocytes as a reason for reduced clearance of HBV-infected cells, we used mice humanized only by hepatocyte transplantation, in the absence of human CTLs since ethanol exposure may compromise activity of immune cells measured in vivo (Geissler et al., 1997). To exclusively focus on ethanol-induced suppression of HBV-MHC class I complex presentation on hepatocytes, we undertook ex-vivo studies, where hepatocytes, isolated from humanized mice fed control and ethanol diets, were exposed to CTLs recognizing HBV core 18–27-HLA-A2 epitope in the absence of ethanol. Following this scenario, we excluded the effects of ethanol on CTL activity, hence, reserving this aspect for future study. We plan to perform these experiments later to disclose the mechanisms behind ethanol-mediated suppression of HBV CTL function.

Here, we used the chimeric mice with high levels of humanization (Azuma et al., 2007) from HLA-A2-expessing hepatocytes, which were in vivo infected with HBV and fed control and ethanol diets in an “acute on chronic” mode (Bertola et al., 2013). After isolating hepatocytes from livers of HBV-infected mice, we quantified a known CTL epitope, HBV core peptide 18–27-HLA-A2-restricted complex (Bertoletti et al., 1994, Maini et al., 1999, Ganesan et al., 2019) presentation by using a specific antibody. Thus, we demonstrated that in our in vivo HBV-infection model, CTL epitopes (namely, HLA-A2 -loaded HBV peptide 18–27) are presented on PHH surface, and this presentation is compromised by ethanol exposure. In our previous in vitro studies on HepG2.2.15-HBV transfected cells exposed to artificial acetaldehyde-generating system (AGS), which mimics ethanol metabolism by acetaldehyde release, we also observed the reduction in HBV 18–27-HLA-A2 display on cell surface (Ganesan et al., 2019), indicating that our in vitro findings are in line with in vivo ones.

The question we wanted to answer by the ex vivo part of humanized mice study is whether ethanol-induced suppression of CTL epitopes on HBV-infected hepatocytes reduces the clearance of these hepatocytes by CTLs. To address this, we incubated HBV-infected hepatocytes derived from the livers of control and ethanol-fed mice with CTLs expressing HBV core 18–27-HLA-A2 receptors and found that the cytotoxic effects of these CTLs were significantly lower when we used PHH from ethanol-fed mice. In these mice, the presentation of the HBV peptide-HLA-A2 complex on hepatocytes was also lower than in control diet fed mice. Since for the ex vivo performed cytotoxic assay, CTLs were not exposed to ethanol, the reduction of cell cytotoxicity can be attributed to suppression of CTL epitope presentation on target PHH by in vivo ethanol feeding.

The next question is whether the mechanisms of in vivo ethanol metabolism-downregulated display of CTL epitopes on PHH were similar to previously reported in HepG2.2.15 cells treated with AGS. This includes impaired processing of HBV peptides by proteasome and defective trafficking of HBV peptides-MHC class I complex to PHH surface due to ER stress upon AGS exposure in HBV-expressing HepG2.2.15 cells (Ganesan et al., 2019, Ganesan et al., 2020). In these regards, as a first step, we tested the effects of ethanol feeding on proteasome, which processes HBV core antigen to peptides for antigen presentation. Ethanol feeding to mice suppressed proteasome activities in hepatocytes. This corresponds to our previously reported findings on HepG2.2.15 cells treated with AGS since, as revealed from our in vitro studies, proteasome is able to cleave HBV core peptide 18–27 from C-extended peptides for loading to HLA-A2 groove (Ganesan et al., 2019). Ethanol-induced oxidative stress down-regulates the proteasome catalytic core activities (Bardag-Gorce et al., 2005, Osna et al., 2004). In fact, oxidative stress was induced in PHH from HBV-infected ethanol-fed mice (as evident by an up-regulated CYP2E1 expression, reduced glutathione levels and increased 4HNE adduction of proteins).

Immunoproteasome (IPR) is an intensive generator of peptides for MHC class I loading (Cascio et al., 2001), and we recently demonstrated that the IPR inhibitor, ONX-0914 significantly suppressed the HBV peptide-MHC class I complex presentation in HBV-transfected HepG2.2.15 cells (Ganesan et al., 2019). In alcohol-fed HBV-infected mice, protein expression of IFN-inducible IPR subunits, LMP2 and LMP7 was decreased. As reported by others, the attenuation of IPR function/expression by more than 25% may significantly reduce MHC class I-restricted antigen presentation (Joeris et al., 2012). Since our mice were transplanted only with human hepatocytes and had no human IFNγ, we anticipate that the decrease in IPR subunit protein expression is related to ethanol metabolism-induced suppression of IFN type 1 signaling, which may also stimulate IPR. In addition to the upregulation of IPR, IFN type 1 signaling activates anti-viral IFN sensitive gene (ISGs) expression. Thus, we tested the activation of anti-viral ISGs in PHH and found that gene expression of OAS1, APOBEC and ISG15 was decreased by ethanol feeding in HBV+ mice. This indicates that ethanol-impaired IFN type 1 signaling may play an important role both in ethanol-induced suppression of processing of HBV peptides in hepatocytes and in the reduction of innate immunity-mediated anti-viral protection. We indeed observed an increased HBV load in ethanol-fed mice and increased levels of HBV RNA, DNA, HBcAg and HBsAg in PHH, which was in line with our previous findings in HepG2.2.15 cells exposed to AGS (Ganesan et al., 2019, Ganesan et al., 2020).

The impairment of antigenic peptide processing by proteasome is not the only reason for the decreased display of HBV peptide-HLA-A2 complex on hepatocytes of ethanol-fed mice. As we mentioned earlier, proteasome-generated peptides are transported to the ER for MHC class I loading. Hence, we checked whether ethanol feeding causes ER stress in HBV-infected hepatocytes, which potentially leads to impaired trafficking of HBV peptide-MHC class I complex to the cell surface. In our in vivo study, we found that first arm of UPR gene mRNA expressions, sXBP-1, EDEM1 and protein expression of pIRE1α and BiP, as well as second arm of UPR, cleaved ATF6α protein were significantly increased in alcohol -fed HBV-infected mice. We demonstrated that alcohol induced pIRE1α increases the sXBP1s which regulate downstream target genes including ER chaperones and genes involved in ERAD, such as EDEM1. These UPR are related to ER and oxidative stress as has been shown in our in vitro study and also reported by others (Ji, 2012, Ganesan et al., 2020). The observed increase in HBsAg in alcohol-fed mice in our current study correlated with the activated pIRE1α and ATF6α pathways. As reported, this upregulates EDEM family proteins (EDEM1), thereby reducing the load of viral surface proteins and contributing to HBV persistence and chronicity (Lazar et al., 2014, Li et al., 2019). In addition, our previous in vivo and in vitro findings showed that ethanol metabolism increased 4HNE adduct formation and decreased SAM:SAH ratio (Ganesan et al., 2015, Ganesan et al., 2019). The above-mentioned factors clearly indicate that ethanol metabolism induces ER stress, which may affect the HBV peptide-MHC class I complex loading and trafficking to the hepatocyte surface in alcohol-fed HBV-infected mice.

In contrast, the third arm of ER stress, PERK pathway was partially affected by ethanol metabolism in HBV-infected mice, which was shown by an increase in ATF4 and CHOP mRNA and protein levels. However, we observed no activation of pPERK and peIF2α. As previously known, alcohol increased both ATF4 mRNA and protein levels (Li et al., 2016) and particularly, CYP2E1-induced oxidative stress plays an important role in ATF4 regulation (Magne et al., 2011), which was supported by our current finding on humanized mice confirming our previous in vitro findings, as well as other studies on the effects of acetaldehyde (Ganesan et al., 2020, Chen et al., 2014). The partial activation of PERK pathway in HBV-infected people with AUD should be extensively studied because an increased ATF4 and CHOP play a critical role in ER stress-induced apoptosis (Panganiban et al., 2019). However, we observed no alteration in expression of cleaved caspase-3 in HepG2.2.15 cells exposed to AGS (Ganesan et al., 2019, Ganesan et al., 2020) and in our current in vivo HBV-ethanol model, showed that an increased CHOP expression does not ultimately results in apoptosis, though it sensitizes cells to apoptosis (McCullough et al., 2001).

Overall, we conclude that in HBV-infected hepatocytes, in vivo exposure to ethanol suppresses proteasome and increased ER stress thereby decreasing HBV peptide-MHC class I expression on hepatocyte surface, which attenuates the ability of HBV specific CTLs to eliminate the virus-infected hepatocytes (Figure 8). Collectively, all these events may promote HBV-infection persistence and development of HBV-associated end-stage liver disease outcomes.

Fig. 8. Proposed model for the effects of alcohol on HBV18–27 peptide-MHC class-I complex presentation and HBV-specific CTLs in HBV infected humanized hepatocytes:

Fig. 8.

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In vivo exposure to ethanol reduces processing of antigenic peptides by proteasome and impairs HBV peptide-MHC class I complex trafficking to cell surface due to increased ER stress. This leads to alcohol-mediated reduction in HBV peptide-MHC class I expression on hepatocyte surface, which attenuates the ability of HBV -specific CTLs to eliminate the infected hepatocytes. Collectively, all these events may promote HBV-infection persistence and development of HBV-associated end-stage liver disease outcomes.

Supplementary Material

supinfo

Acknowledgement:

The authors would like to thank Dr. Thiele and his lab for performing the isolation and purification of HBV core 18–27-HLA-A2 antibody from hybridoma supernatant. Hybridoma cells were purchased from PHARMEXA, Inc. We also thank Dr. Bertoletti for providing an aliquot of HBV core 18–27-HLA-A2 antibody from his laboratory to validate the hybridoma-derived antibody.

Grant Support: Funding for this work was obtained from the K01-Mentored Research Scientist Development Award-1K01AA026864 provided by the National Institute on Alcohol Abuse and Alcoholism (NIAAA), USA.

List of Abbreviations:

ADH

alcohol dehydrogenase

ATF4

Activation Transcription Factor 4

ATF6

Activation Transcription Factor 6

APOBEC3

Apolipoprotein B Editing Complex

BiP

Immunoglobin binding protein

CHOP

C/EBP homologous protein

ChT-L

chymotrypsin-like

CTL

cytotoxic T-lymphocytes

CYP2E1

cytochrome P4502E1

EDEM1

ER-degradation-enhancing-α-mannidose-like protein

Ethanol

Etoh

ERAD

ER-associated degradation

eIF2α

eukaryotic initiation factor 2 alpha

HBV

hepatitis B virus

HLA-A2

human leukocyte antigen-A2

IB

immunoblotting

IPR

Immunoproteasome

IFNγ

interferon gamma

ISG15

Interferon-stimulated gene 15

LDH

lactate dehydrogenase

MHC I

Major Histocompatibility Complex I

OAS1

2′–5′ oligoadenylate synthetase 1

PERK

PKR-like ER kinase

PHH

Primary Human hepatocytes

STAT-1

signal transducers and activators of transcription

TAP

transporters associated with antigen processing

T-L

trypsin-like

UPR

Unfolded Protein Response

XBP1

X-box binding protein 1

Footnotes

Disclosures: The authors and contributors of this paper declare no conflict of interests.

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