A review of alcohol-pathogen interactions: New insights into combined disease pathomechanisms

Abstract

Progression of chronic infections to end-stage diseases and poor treatment results are frequently associated with alcohol abuse. Alcohol metabolism suppresses innate and adaptive immunity leading to increased viral load and its spread. In case of hepatotropic infections, viruses accelerate alcohol-induced liver hepatitis and fibrosis, thereby promoting end-stage outcomes, including cirrhosis and hepatocellular carcinoma (HCC). In this review, we concentrate on several unexplored aspects, which illustrate the combined effects of viral/bacterial infections and alcohol in disease development. Here, we overview alcohol-induced alterations implicated in immunometabolism as a central mechanism impacting metabolic homeostasis and viral pathogenesis in Simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) infection. Furthermore, in hepatocytes, both HIV-infection and alcohol activate oxidative stress to cause lysosomal dysfunction and leakage and apoptotic cell death, thereby increasing hepatotoxicity. In addition, we discuss the mechanisms of hepatocellular carcinoma and tumor signaling in hepatitis C virus (HCV)-infection. Finally, we analyze the studies aimed at reviewing and describing the immune derangements in hepatotropic viral infections for the development of novel targets and strategies to restore effective immunocompetency in alcohol-associated liver disease (ALD). In conclusion, alcohol exacerbates pathogenesis of viral infections, contributing to chronic course of infections and poor outcomes, but the mechanisms behind these events are virus-specific and depend on virus-alcohol interactions, which are not the same in various infections.

Introduction

Development of chronic infections, their progression to end-stage diseases and poor treatment outcomes are frequently associated with alcohol abuse. In fact, alcohol exacerbates the courses of viral and bacterial infections and decreases the sensitivity of infectious agents to anti-viral medications and antibiotics. The combined effects of virus and alcohol on host cells include epigenetic changes and are responsible for post-translational protein modifications detrimentally affecting cell function and even promoting cell death. Many of these changes are attributed to oxidative stress. Viruses, by themselves, may induce oxidative stress in permissive cells and compromise innate immune anti-viral protection mechanisms. Alcohol metabolism also suppresses innate immunity leading to increased viral load and spread of infection, thereby potentiating oxidative stress. The pathogenic consequences of this viral potentiation depend on the cytopathogenic properties of the virus. Cytopathic viruses will kill infected cells (such as HIV in T-cells), whereas non-cytopathic viruses (such as Hepatitis C virus (HCV) and Hepatitis B virus (HBV)) require activation of adaptive immunity to clear infected cells. Alcohol metabolism suppresses these adaptive immune responses impairing their ability to eliminate infected cells. The subsequent accumulation of virus in these cells also increases oxidative stress and worsens the pathology. In addition to its ability to exacerbate the pathogenesis of viral infections, alcohol itself causes tissue and organ damage, and viral infections frequently exacerbate this process. Several viral infections accelerate alcohol-induced liver fibrosis and chronic hepatitis, thereby promoting end-stage outcomes, including cirrhosis and HCC. In viral hepatitis, alcohol as a second hit promotes inflammation and pro-fibrotic changes. The topic of infection-alcohol interactions is very broad. In this review, we will concentrate on several aspects, which illustrate the role of viral/bacterial infections and alcohol in disease progression.

Alcohol-Associated Metabolic Dyshomeostasis: Potential Link to Enhanced Viral Pathogenesis

One important example of viral-alcohol interactions occurs during infection with simian immunodeficiency virus (SIV). Studies using the nonhuman primate model of chronic binge alcohol administration (CBA) and simian immunodeficiency virus (SIV) infection have identified a significant interaction of alcohol with pathogenesis of SIV infection. CBA is a daily intragastric administration of alcohol (30% EtOH; 2.5 g/kg/day over a 30 min period) producing average peak blood alcohol concentrations of 50 mM (≈230 mg/dL) for three months prior to infection with SIV and continued throughout the study. This reflects the interactions of heavy alcohol consumption in persons at risk and those living with HIV. CBA results in early gut immunopathogenesis, increased susceptibility to infection following intra-rectal inoculation of male and female macaques, increased plasma viral load at set point, a period where viral load is considered to be predictive of disease progression, and significantly shortened time to development of end stage disease in untreated macaques (Amedee et al., 2014, Bagby et al., 2006, Molina et al., 2008, Poonia et al., 2006, Poonia et al., 2005). Other studies also demonstrated that CBA alters skeletal muscle (SKM) homeostasis, increases proteasomal activity, promotes a profibrotic milieu, decreases antioxidant capacity and decreases regeneration potential in SIV-infected macaques (Dodd et al., 2014, LeCapitaine et al., 2011, Simon et al., 2017, Simon et al., 2015). These events are associated with a marked decrease in insulin response to glucose and disposition index, measured by an intravenous glucose tolerance test. However, this metabolic dyshomeostasis was not associated with overt changes in basal plasma insulin or glucose (Ford et al., 2016). The data collected from male macaques collectively reports a working model, in which binge alcohol consumption in the infected host leads to increased viral replication, lymphocyte turnover, dysbiosis, and gut barrier leak. This results in translocation of toxins and bacterial products into the systemic circulation and promotes immune activation, leading to a state of immune exhaustion and senescence, as well as chronic inflammation (Patel et al., 2015). All these factors contribute to tissue injury and the development of comorbidities.

Chronic immune activation-mediated alteration in energy homeostasis one of the principal mechanisms leading to end-stage organ injury. This metabolic instability was associated with alcohol induced alterations in endocrine pancreas function, decreased functional SKM mass, adipose tissue inflammation and fibrosis, and an anti-retroviral (ART)-associated increase in gluconeogenic and lipogenic capacity (Dodd et al., 2014, Ford et al., 2018, LeCapitaine et al., 2011, Simon et al., 2015). A salient mechanism underlying whole body metabolic instability in end-stage HIV-infection appears to involve alterations in genes regulating mitochondrial function in the skeletal muscle. This impacts critical functions in preserving energy homeostasis including oxidative stress, mitophagy, apoptosis related signaling, and mitochondrial biogenesis (Duplanty et al., 2017). In addition, myoblasts isolated from ART-treated SIV infected macaques, regardless of CBA administration, had decreased mitochondrial maximal oxygen consumption rate (OCR) suggesting mitochondrial dysfunction (Duplanty et al., 2018). Though there were no significant changes in maximal OCR, extracellular acidification rate (ECAR), an indicator of glycolytic function, was higher in myoblasts isolated from SIV-infected female macaques. In addition, myoblasts isolated from CBA-administered macaques had increased glycolytic capacity, as measured after addition of oligomycin, an ATP synthase inhibitor, indicating a glycolytic phenotype, which could be due to less efficient or unhealthy mitochondria (unpublished data from the Molina’s laboratory). Parallel in vitro studies using naïve myoblasts exposed to 50 mM alcohol for 3 days show that direct alcohol exposure results in a contrasting shift in metabolic phenotype. This may be associated with the impaired myoblast differentiation as shown by the close correlation between ECAR and fusion index and the number of myotubes per field (Levitt et al., 2020).

This shift in myoblast metabolic phenotype is associated with alterations in skeletal muscle gene expression. There was a significant increase in the gene expression of PPARG coactivator 1 alpha (PPARGC1A), a master regulator of mitochondrial biogenesis, and of Nuclear Respiratory Factor 2 (GABPB1), and a marked decrease in gene expression of estrogen related receptor alpha (ESRRA) and peroxisome proliferator activated receptor delta (PPARD) due to SIV-infection. These changes in genes regulating mitochondrial function suggest an attempt at a mitohormetic response; a process wherein reactive oxygen species (ROS) produced by mitochondria act as signaling molecules to initiate a cascade of cellular events that ultimately protect the cells from harmful effects. The subsequent consequences are an adaptive response (stress defense mechanism).

Our recent findings suggest similar changes in SKM expression of genes regulating mitochondrial function in a subset of persons living with HIV (PLWH) enrolled in our clinical study. Furthermore, the data from the Molina laboratory also show that increased scores on the alcohol use disorder inventory test (AUDIT) and elevated two-hour glucose levels in an oral glucose tolerance test (OGTT) are associated with an increase in mitochondrial volume and a proton leak, an indicator of mitochondrial damage. Both parameters indicate poorer mitochondrial health with at-risk alcohol use and metabolic instability (Levitt et al., 2021).

These findings lend support to the hypothesis that SKM alterations in expression of genes regulating mitochondrial function, and mitochondrial respiratory function contribute to systemic metabolic dyshomeostasis and pose the question whether similar alterations in immune cell energetics may result from chronic alcohol and SIV infection. Specifically, it is not clear whether alcohol alters immunometabolism in a way that contributes to disease pathogenesis. Gene expression analysis from the Molina laboratory showed that CBA increases mitofusin 2, superoxide dismutase 2, and GABPB1 expression in peripheral blood mononuclear cells (PBMCs), indicating a true mitohormetic response. This can be seen by the increased expression of genes critical for mitochondrial repair and antioxidative response. These alterations in expression of genes regulating mitochondrial function in PBMCs appear to be associated with increased plasma viral load and viral replication in CBA/SIV-infected macaques. These early findings support the hypothesis that PBMC energetics or immunometabolism may impact viral replication, and consequently disease pathogenesis. The question of whether mitohormesis drives viral replication or viral replication drives mitohormesis has not yet been addressed.

In summary, alcohol-induced alterations are implicated in immunometabolism as a central mechanism impacting metabolic homeostasis and viral pathogenesis (Figure 1).

Figure 1:

Alcohol, HIV/SIV, and antiretroviral therapy (ART) act as cellular stressors producing alterations in mitochondrial signaling leading to adaptive and maladaptive mitochondrial responses. Alterations in mitochondrial gene expression in peripheral blood mononuclear cells (PBMC) and skeletal muscle (SKM) myoblasts isolated from chronic binge alcohol treated simian immunodeficiency virus infected non-human primates suggest a mitohormetic response to cellular stressors. These mitochondrial alterations are associated with increased plasma viral load and viral replication; decreased SKM functional mass; increased disease pathogenesis; and increased morbidity and mortality. Our findings implicate alcohol-induced immunometabolism alterations are central mechanisms impacting whole body metabolic homeostasis and viral pathogenesis.

Current needs and future directions

These findings underscore the need to mechanistically understand how alcohol-mediated bioenergetic adaptations dysregulate cellular functions and increase the risk for comorbidities in SIV/HIV infection. Studies in PLWH with dysglycemia provide evidence for changes in expression of genes regulating biogenesis, dynamics, and lipid handling. Whether these changes functionally contribute to the observed poor bioenergetic health remains to be established. Examining whether aerobic exercise intervention improves metabolic health in PLWH with at-risk alcohol use is urgent. At the cellular level, understanding whether alcohol-mediated mitohormetic responses in immune cells impacts on SIV/HIV viral replication, CD4 T cell phenotype, and proinflammatory cytokine production is critical to better understand the persistence viral reservoirs. Finally, systematic studies integrating basic mechanisms and lifestyle, or therapeutic strategies will lay the groundwork for improving systemic metabolic health.

HIV, Alcohol and Liver

Further evidence of the ways that alcohol potentiates the consequences of HIV infection and interferes with HIV therapy have been obtained from cell culture models of infection. Anti-retroviral therapy (ART) significantly increased life expectancy in PLWH. The prevalence of alcohol abuse is twice as high in PLWH than in the general population, and thus, the combination of HIV and exposure to alcohol appears to play a leading role in non-AIDS-related morbidity in PLWH individuals where approximately 20% of individuals have some degree of liver disease development (Price and Thio, 2010, Falade-Nwulia and Thio, 2011, Kaspar and Sterling, 2017).

As found recently, exposure of hepatocytes in culture to ethanol metabolites induces HIV protein accumulation leading to hepatocyte death (Ganesan et al., 2019). In the same study, it was shown that the intracellular degradation of HIV proteins involved proteasomes and lysosomes, and the exposure to the lysosome inhibitor, bafilomycin, caused stabilization of HIV gag proteins in alcohol-treated hepatocytes as well as acetaldehyde-generating system (AGS)-treated hepatoma cells (Huh7.5-CYP designated as RLW cells). ASG consists of yeast alcohol dehydrogenase (ADH), NAD⁺ (as a co-factor) and 50 mM ethanol (as a substrate). This system described in (Ganesan et al., 2019) allows enzymatically and continuously producing about 200 μM acetaldehyde, which corresponds to acetaldehyde concentration in supernatants from ADH-expressing VA-13 cells naturally metabolizing ethanol.

Subsequent studies showed that acetaldehyde affects HIV entry into hepatocytes. As previously reported, there are several receptors for HIV entry into hepatocytes, which do not express the CD4 receptors critical for HIV entry in T-lymphocytes. These candidates mainly include the co-receptors, CXCR4 and CCR5 (Balasubramanian et al., 2003, Vlahakis et al., 2003) binding gp120 HIV protein. In experiments from the Osna laboratory used HIV_ADA, which does not bind to the CXCR4 receptor. However, they discovered that maraviroc, a CCR5 receptor blocker, fully suppressed the expression of HIV gag RNA in hepatocytes exposed to AGS (unpublished observations). The blocking effects of maraviroc has also been shown for HIV-infection in hepatocytes with no ethanol metabolite exposure (Kong et al., 2012), and in HIV-induced HCC development (Ochoa-Callejero et al., 2013). While HIV penetration into hepatocytes is a known fact (Crane et al., 2012, Xiao et al., 2008, Kong et al., 2012, Fromentin et al., 2011, Fromentin et al., 2010), the role of ethanol metabolites in the regulation of hepatocytes permissiveness to HIV was investigated in a very few papers.

As shown, exposure of HIV-infected primary human hepatocytes (PHH) to ethanol or Huh7.5-CYP (RLW) cells to AGS significantly increased the expression of HIV gag RNA and the amount of HIV gag proteins (Ganesan et al., 2019). In the same study, the authors found that this expression was reversed by 4-methyl pyrazole (4MP), an inhibitor of ethanol metabolism. Furthermore, hepatocytes treated with both HIV and ethanol undergo robust apoptosis, which goes up with increased HIV multiplicity of infection (MOI) (Ganesan et al., 2019). Currently, it is not clear whether expression of HIV gag RNA or HIV gag proteins in hepatocytes or their increased levels in ethanol-exposed hepatocytes are due to ethanol-induced HIV replication or stabilization of HIV proteins. While some studies suggest a low level of HIV replication in hepatocytes (Kong et al., 2012), other publications do not confirm it (Ganesan et al., 2018, Ganesan et al., 2019). When HIV p24 expression was measured after day 1, 3 and 5 of hepatocyte infection in the presence of pan-caspase inhibitor (to prevent apoptosis in these cells), the highest amount of HIV gag protein was observed at day 1 of HIV-infection either in AGS-non-treated or AGS-pre-exposed infected cells. This high level of HIV proteins in hepatocytes after day 1 of HIV-infection sequentially induced oxidative stress based on kinetics of ROS formation and 4-HNE-adducted proteins levels, which rises by day 3-5 of infection. The latter corresponds to the kinetics of caspase 3 cleavage (Ganesan et al., 2019). Because there is a drop in amount of intracellular p24 protein by day 5 of infection, such kinetics does not support HIV replication in hepatocytes. Thus, the increase in HIV markers in AGS-exposed cells is likely not due to AGS-enhanced HIV replication, but due to HIV protein stabilization followed by oxidative stress development and apoptotic cell death.

Modulation of pH in acidic endosomal-lysosomal compartments to neutral, as demonstrated by staining of AGS treated hepatocytes with lysosensor (Dagur et al., 2021), allows more virus to survive, which serves as one of the reasons for higher expression of HIV RNA and increased amount of p24 protein in RLW cells and in hepatocytes pre-treated with ethanol metabolites. Another reason for HIV-protein accumulation might be ethanol metabolite-induced suppression of intracellular protein-degrading systems, lysosomes and proteasomes, which was mimicked by treatment with specific enzyme inhibitors (Ganesan et al., 2019). Because protein stabilizing effect was more potently obtained by treatments with the lysosome inhibitor, bafilomycin, the regulation of lysosomal status by HIV and ethanol metabolism has been further studied. As became clear from recently published studies (New-Aaron et al., 2021), HIV-infected RLW cells exposed to AGS had the highest co-localization of Galectin 3 with LAMP1, indicating intensive lysosome leakage. In addition, there was co-localization of leaked lysosomal cathepsin B with the mitochondrial outer membrane proteins, TOM20 and VDAC1 (Chacinska et al., 2009, Yamamoto et al., 2011), suggesting that after reaching mitochondria, these leaked lysosomal enzymes may cause an increased mitochondrial permeabilization (MPT) (Boya et al., 2003, Paquet et al., 2005) and subsequent apoptosis induction (Johansson et al., 2010). Activation of caspase 9 by cytochrome C leaked out from mitochondria due to increased MPT potentially activates intrinsic apoptosis in hepatocytes in the settings of HIV-ethanol metabolite exposure to hepatic cells can be restored by N-acetyl cysteine (NAC), indicating the contribution of oxidative stress to these events (New-Aaron et al., 2021). An induction of oxidative stress by both HIV and AGS observed in vitro in RLW cells was confirmed by in vivo studies in ethanol-fed, HIV-injected, liver humanized mice (Ganesan et al., 2019). In the same study, the combination of HIV and ethanol suppressed amount of LAMP1, cathepsin and proteasome activities, and induced oxidative stress as measured by Thiobarbituric acid reactive substances (TBARS) and 2’,7’ –dichlorofluorescein diacetate-quantified ROS production.

Overall, both alcohol and HIV activate oxidative stress in hepatocytes to cause lysosomal dysfunction and leakage. Leaked cathepsins may damage mitochondria and induce caspase 9 activation leading to initiation of intrinsic apoptosis as an impotent component of HIV-ethanol metabolism-induced hepatotoxicity (see Figure 2). Future directions will include the studies on the role of hepatocyte apoptotic bodies in induction of inflammasome in HSC and macrophages and activation of liver fibrosis as well as elucidation of therapeutic targets to prevent the fibrosis development.

Figure 2: HIV and alcohol promote lysosomal leakage and hepatocyte apoptosis.

HIV combined with alcohol induce oxidative stress in hepatocytes, which causes lysosomal leakage to mitochondria, with further activation of caspase 9-dependent intrinsic apoptotic pathway, which leads to hepatotoxicity.

Cell Fate Decision of Liver Tumor-Initiating Stem-like Cells Induced by HCV and Alcohol Western Diet

Another critical disease process that is specifically promoted by the combination of alcohol and viral infection is cancer development. For example, HCC is caused by both HCV infection and alcohol, and the two components together synergistically increase the cancer risk. The mechanisms of this synergy are complex but changes in tumor-initiating stem-like cells (TICs) appears to play an important role.

Novel oncoprotein TBC1D15:

HCC is the second leading cause of cancer deaths worldwide. The incidence of HCC continues to rise with an estimated 40,000 new cases and 24,000 deaths per year in the US, making it a common malignancy with unmet medical needs (Venook et al., 2010). The improvement in survival with sorafenib, an FDA-approved drug for HCC, is modest with limited median survival. HCV-related HCC accounts for 50-60% of all HCC cases (Venook et al., 2010), and alcohol abuse is a risk factor for HCC in HCV/HBV or obese patients, increasing risk of the disease by 4-6 fold (Dang et al., 1995, Majumder et al., 2003, Petrosyan et al., 2016). Tumor-initiating stem-like cells (TICs) are identified in different malignancies and represent a hallmark of therapy-resistance and tumor recurrence (Petrosyan et al., 2016). However, no coherent understanding exists for the mechanisms underlying their unchecked proliferation, tumorigenic activity, and clinical intractability

Ectopic expression of TLR4 in hepatocytes by the Apo-E promoter/hepatic control region-driven expression (Dang et al., 1995) of HCV NS5A protein in transgenic (Tg) mice (Majumder et al., 2003) results in heightened activation of TLR4 by endotoxemia associated with alcoholism (Machida et al., 2009). This induces the stem cell factor NANOG, generation of NANOG+ tumor-initiating stem-like cells (TICs), and liver oncogenesis (Machida et al., 2009). The oncogenic role of TLR4 is confirmed by others in different models, establishing the importance of TLR4 expressed in hepatocytes and endotoxin derived from intestine in the genesis of HCC (Dapito et al., 2012). A key event leading to the unchecked expansion of TICs is loss of the p53 tumor suppressor (Aparicio and Eaves, 2009), which serves to prevent pluripotency and stem cell proliferation (Zhao and Xu, 2010). Cell fate determinant molecule NUMB associates with p53, protecting it from ubiquitin-mediated proteolysis catalyzed by the MDM2 E3 ubiquitin ligase (Colaluca et al., 2008). As discovered by the Machida laboratory, TICs overexpress the protein RAB7 GTPase activating protein (GAP) TBC1D15, a novel NUMB-binding protein, and this promotes aPKCζ activity, resulting in NUMB phosphorylation, NUMB dissociation from p53, and p53 degradation (Siddique et al., 2015).

TBC1D15-NOTCH Interaction:

TICs express cleaved NOTCH intracellular domain (NICD) while primary hepatocytes express full-length NOTCH1. In the Machida’s laboratory, they performed large-scale immunoaffinity purification of endogenous TBC1D15 in TICs, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify interacting proteins. This analysis indeed identified NOTCH1, 2, 3 and 4 as high confidence interacting proteins of TBC1D15. Co-IP-Western blot analysis of TIC cell lysate confirmed TBC1D15 interaction with all NOTCH isoforms in both full-length and activated NICD forms. These observations suggested that TBC1D15 may interfere with NUMB-mediated NOTCH/N1ICD degradation by binding to the same domain.

To test the functional relationship of the NOTCH pathway with TBC1D15, the loss of function approaches by knocking down TBC1D15 (TBC1D15 KD) in Huh7 cells has been used (Choi et al., 2020). The studies performed in the Machida laboratory constitute understanding of TIC’s self-renewal and tumor-initiating activity and builds upon our discovery of the novel oncoprotein TBC1D15 which promotes p53 degradation via its interaction with NUMB. The oncofetal protein TBCD1D15 has another arm of oncogenic activity via its cooperative interaction with the NOTCH pathway (Choi et al., 2020).

The originality of this study lies within the discovery of the novel oncoprotein TBC1D15 and its unique oncogenic activities involving p53 degradation and cooperation with the NOTCH pathway in TICs. The mechanistic findings have laid down the foundation for a new translational path for discovery of new therapeutic targets for alcohol-associated HCCs. Indeed, candidate small molecule antagonists have been tested for their ability to block the interaction of TBC1D15-NOTCH/NICD (Choi et al., 2020).

Taken together, the presented research described the molecular mechanisms of the oncogenic activity of TBC1D15 overexpressed in tumor cells and TICs. TBC1D15 has two major tumor-promoting pathways induced by alcohol Western diet intake: interference of the asymmetric division machinery by interacting with NuMA1 and disruption of NuMA1-LBN association. Interaction between NuMA1 and LGN is essential for asymmetric division, and interaction with RANGAP1, leading to AURKA-aPKCζ-induced NUMB phosphorylation and p53 degradation (Figure 3). This pathway involves TBC1D15 interaction with NOTCH/NICD resulting in NOTCH activation and stabilization. The prevention of liver tumor formation by hepatocyte-specific TBC1D15 deficiency or NUMB unphosphorylatable mutant overexpression in HCV NS5A Tg mice fed alcohol western diet, validated the importance of these two gene products in liver oncogenesis (Choi et al., 2020).

Figure 3. Summary of alcohol/obesity-mediated oncogenic pathways.

NUMB phosphorylation and TBC1D15 are mutually required for liver tumorigenesis. NUMB phosphorylation and TBC1D15 (containing the cell fate determinant Canoe homology domain that regulates the asymmetric cell division machinery) are required for liver tumorigenesis in vivo. TBC1D15 interacts with the asymmetric division machinery to disrupt its function, initiate p53 degradation and self-renewal, and cooperates with the NOTCH signaling pathway to promote tumorigenesis.

Current needs, future directions, and research in the field of alcohol-pathogen interactions

Even though the role of NOTCH in cholangiocarcinoma is well supported (Fan et al., 2012, Sekiya and Suzuki, 2012), whether its activation contributes to alcohol-associated HCC is elusive. Transcriptomic analysis of patient HCC samples that include alcohol-associated HCCs also identifies a subgroup of patients with a signature consistent with NOTCH activation (Strazzabosco and Fabris, 2012). In particular, NOTCH1 activation due to NUMB downregulation triggered by IKKα-mediated inhibition of FOXA2, promotes tumorigenic activity of Hep3B (Liu et al., 2012). Further mechanistic studies are warranted to elucidate more fundamental mechanisms that are generalized not only alcohol-associated HCC development, but also etiology-derived HCC, including NASH-associated HCCs and HBV-associated HCCs.

Dysfunctional Immunity in Alcoholic Liver Disease: Lessons Learned From Hepatotropic Viral Infections

The most common cause of death in patients with alcohol-related liver cirrhosis (ARC) is the development of serious bacterial infections as a consequence of impaired host immune defenses (Gustot et al., 2017). In patients with alcoholic hepatitis (AH), the most inflammatory manifestation of alcohol-related liver disease (ALD), the susceptibility to infection is further heightened, with infection observed in up to 65% of cases, making it the leading cause of death and the most common precipitant for Acute-on-Chronic Liver Failure (ACLF) (Jalan et al., 2014). Current guidelines recommend intensive and early antibiotic therapy in these patients, which has led to the increasing emergence of multi-drug resistant bacteria (Chokshi, 2018, Piano et al., 2016). These infections are associated with a higher incidence of septic shock and/or rapid deterioration of liver function and death. Moreover, the use of corticosteroids to effectively dampen harmful, inflammation-mediated liver injury, can also suppress anti-pathogen immunity further, augmenting the vulnerability to bacterial infection in these patients (Chokshi, 2018, Vergis et al., 2017). As such, there is a pressing need to explore new paradigms for anti-infective therapy and host-directed immunomodulatory therapies are a promising approach. Studies understanding the immunopathogenesis of ALD are still in their infancy but there are many ‘lessons to be learned from studies delineating the central role of immune dysfunction in the development of chronic viral infections of the liver.

Studies from our group and others have shown that the compromised antiviral immunity in chronic Hepatitis B infection (CHB) is consequent to impairments in both innate and adaptive immune compartments including derangements of inhibitory checkpoint receptors (CR), which mediate dysfunctions in antiviral T-cell functionality, and an impaired interferon response (Cooksley et al., 2008, Phillips et al., 2017, Chokshi et al., 2014, Evans et al., 2008). It can be learned from these studies that antiviral cytokine-producing Hepatitis B Virus (HBV)-specific T cells are critical to control infection and activation of non–virus-specific immune cells contribute to liver damage (Evans et al., 2008, Cooksley et al., 2008). CR dysregulation was identified as a key mechanism of immune evasion and viral persistence. Indeed, the hyperexpression of the inhibitory CR programmed-death-1 (PD-1) on HBV-specific T cells and the downregulation of the inhibitory CR T-cell immunoglobulin and mucin domain 3 (TIM3) on IL-17-producing T cells, a subset known to participate in exacerbation of liver injury, result in defective antiviral immunity accompanied by a damaging inflammatory response in CHB (Cooksley et al., 2018, Evans et al., 2008). These investigations have revealed the utility of host-targeted immunomodulatory therapies such as neutralization of inhibitory CR expression (Evans et al., 2008) and the use of potent immunoregulators such as Type III interferons (interferon lambda, IFN-λ) (Phillips et al., 2017). Indeed, it has been reported that pegylated IFN-λ used as a treatment in patients with chronic hepatitis B can activate robust innate and adaptive antiviral responses in vivo (Phillips et al., 2017). Interestingly, several publications have also investigated the role of IFN-λ during HCV infection, particularly in regard to the association between IFN-λ3 polymorphisms and reduced viral clearance and response to interferon-based treatment (Boisvert and Shoukry, 2016, Scagnolari et al., 2017, Liu et al., 2015, Bruening et al., 2017).

These studies investigating the immunopathogenesis of CHB led us to explore whether similar immune dysfunctions were at play in ALD and whether similar paradigms for host-directed immunomodulatory therapies could be identified. Thus, ALD investigations were focused on T cells, neutrophils, type III interferons and expression of inhibitory CRs on innate and adaptive immune effector cells from well characterized cohorts of patients, using flow cytometry, multiplex cytokine analysis, Genome Wide Association Studies (GWAS) and RNA transcriptomic analysis. In addition, the Chokshi group developed an immunocompetent precision-cut liver slice model of ALD to assess the impact of immunomodulatory treatment strategies (Palma et al., 2019).

The studies from the Choksi group reveal widespread disruption of antibacterial immunity in ALD, primarily as a consequence of increased bacterial translocation from the gut into the systemic circulation (Sharma and Riva, 2020, Riva et al., 2020). Firstly, we found polarized monocyte responses, to chronic systemic endotoxin exposure, as key drivers of T-cell exhaustion, mediated through the upregulation of inhibitory CR PD-1 and TIM3. This resulted in the diminished production of antibacterial cytokines and suppressed antibacterial neutrophil functions (Markwick et al., 2015, Riva and Chokshi, 2018, Riva and Mehta, 2019). Notably, antimicrobial immune responses from both arms of the innate and adaptive immunity could be readily rescued through blockade of PD-1 and TIM3 with neutralizing antibodies without inducing a proinflammatory cytokine storm (Markwick et al., 2015). Recently it was also defined a novel role for soluble CRs in modulating immunity in ALD (Riva and Chokshi, 2018, Riva et al., 2021), further supporting the notion that these physiological regulatory mechanisms could be targeted to enhance antigen-specific responses without exacerbating systemic markers of hyperinflammation. Interestingly, an important role for soluble CRs has also been described in the immunopathogenesis of chronic hepatitis B (Fadriquela et al., 2021), supporting the cross-disciplinary approach to investigating host-targeted immunoregulatory mechanisms.

Given the important role of the gut in ALD, we hypothesized that antibacterial T-cell dysfunctions may correlate with gut bacterial translocation. Recently a new subset of innate-like T cells, known as Mucosal-Associated Invariant T cells (MAIT), have been described as a key component in the arsenal of antibacterial host defenses. These are unconventional T cells that only respond to bacteria-derived metabolites, are found in large numbers in the intestinal mucosa and liver and recirculate between the two sites through the peripheral blood (Riva et al., 2018). They represent a fundamental sentinel system for the homeostatic control of the gut flora and exert essential roles for the control of bacterial and viral infections (Howson et al., 2015). Given that these cells defend the host at key locations associated with ALD, it has been investigated whether they contributed to susceptibility to bacterial infection. We found that the proportions of circulating MAIT cells, their transcriptional state and their antibacterial potency were indeed profoundly compromised in ALD patients, and this was because of contact with microbial products and gut microbiota, demonstrating that the ‘leaky’ gut observed in ALD patients drives MAIT-cell dysfunction and underlies an infective antimicrobial response (Riva et al., 2018).

More recently, the role of IFN-λ has been extensively investigated for its antiviral activity during chronic HBV and HCV infections. Despite recent evidence for a central role for IFN-λ in modulating of antibacterial immunity, its role in ALD has remained unexplored (Luo et al., 2019, Rich et al., 2019, Peignier et al., 2020, Read et al., 2021). Recent soon-to-be-published data from the Chokshi laboratory identifies novel disturbances in the IFN-λ network in ALD. This shows that IFN-λ1 gene expression is abrogated in bacterially challenged neutrophils from AH patients and that carriage of Single Nucleotide Polymorphisms in the IFN-λ4 gene may be associated with increased susceptibility to infection. This data suggests that Type III interferons may represent a novel approach for immunomodulation, and their use as immunogenetic biomarkers could be an important tool for the clinical management of ALD patients (Ryan et al., 2021). The similarities between the loss of antiviral defense in chronic HBV infection and the immunoparesis observed in alcohol-related liver disease is summarized in Figure 4.

Figure 4. Commonalities in immune dysfunctions between chronic hepatitis B (CHB) and alcoholic liver disease (ALD).

(A) CHB is characterized by systemic and hepatic immune dysfunctions. HBV-specific T cells are functionally impaired, secreting low levels of the potent antiviral cytokine IFN-λ. This unfavorable response is mediated through the hyperexpression of the inhibitory immune checkpoint (CR) receptor PD-1. In contrast, the CR Tim-3 is downregulated on activated HBV-specific T cells secreting the pro-inflammatory cytokine IL-17, which contributes to liver injury. Non-HBV specific cytotoxic T cells also infiltrate the liver (in excess of HBV-specific T cells) and promote hepatic damage. Therapeutic interventions such as PD1 blockade and the administration of immunomodulators such as Interferon-lambda (IFN-λ) can restore the defective anti-HBV immune response.

(B) ALD is associated with loss of intestinal barrier integrity and increased bacterial translocation to the systemic circulation. This state of chronic endotoxemia polarizes monocyte responses and induces upregulation of CRs PD-1 and Tim-3 on T cells leading to their exhaustion, dysfunction and inability to contend with bacterial infection. Soluble CRS, namely Tim3 (sTim-3) and its ligands Galectin-9 (sGal) and CEACAM1 (sCEACAM1) are also elevated in ALD and contribute to the loss of host defenses. Neutrophils are also affected, with diminished phagocytotic and oxidative burst capacities and lower production of IFN-λ. Therapeutic interventions, including blockade of membrane form PD-1 and Tim-3 could facilitate the restoration of the anti-bacterial immunity in ALD. Further disruptions in the antibacterial immunity in ALD include the depletion of Mucosal Associated Invariant T cells (MAIT) in the circulation and the weakening of their anti-bacterial activities.

Thus, the advances that have been made over the years from studies aimed at understanding the immune derangements in hepatotropic viral infections may offer multiple clues for the development of novel targets and strategies for the restoration a state of effective immunocompetency in ALD.

In conclusion, there are multiple ways in which alcohol affects infectious disease pathogenesis. The combined effects of alcohol and viruses like HIV, HBV and HCV are mainly attributed to metabolic and immune dysfunctions and non-specific immune activation by exposure to endotoxins, intensive apoptosis of hepatocytes due to increased oxidative stress and lysosomal damage, gut-associated MAIT-cell dysfunction and polymorphisms in the IFN-λ4 gene, gut leakage and microbial translocation, which initiates induction of the signaling via TLR4 associated with activation of new oncoproteins. The signaling pathways destabilizing p53 finally leading to self-renewal of tumor-associated stem-like cells. All these events promote detrimental outcomes to end-stage diseases.

Current Needs

The immunodeficient state and profound vulnerability to bacterial infection observed in patients with severe ALD is well described. Whilst multiple impairments in the immunological armamentarium have been identified, there is an absence of targeted immunomodulatory therapies to restore a state of effective anti-pathogen immunity. There is a wealth of data, with emergence of immune-stimulating therapeutics aimed at restoring effective immunity to viral infections, such as cytokine therapy, activation of toll-like receptors and use of CAR-T-cells as examples. The field needs to look to these advances to develop ALD-specific host-targeted immunomodulatory strategies.

Selection Criteria for Cited Articles

The articles cited in this review were those publications that a) focused on both animal and human models where researchers investigated the role of alcohol on viral infections specifically HCV, HBV, HIV and SIV; b) presented original and peer-reviewed published research; and c) were in English. All citation were those that were published between 2000-2021 except one from 1995.

Abbreviations:

AGS

acetaldehyde-generating system

AH

alcoholic hepatitis

ALD

alcohol-related liver disease

aPKCζ

Protein Kinase C Zeta

APOE

Apolipoprotein E

ARC

alcohol-related liver cirrhosis

ART

Anti-retroviral therapy

AUDIT

alcohol use disorder inventory test

CBA

chronic binge alcohol administration

CCR5

C-C Motif Chemokine Receptor 5

CHB

chronic Hepatitis B infection

CR

Checkpoint Receptors

CXCR4

C-X-C chemokine receptor type 4

ECAR

extracellular acidification rate

FOXA2

Forkhead Box A2

HBV

hepatitis B virus

HCC

hepatocellular carcinoma

HCV

hepatitis C virus

HIV

human immunodeficiency virus

IFN-λ

interferon lambda

LAMP1

Lysosomal Associated Membrane Protein 1

LC-MS/MS

liquid chromatography-tandem mass spectrometry

LGN

Leucine-Glycine-Asparagine

MAIT

Mucosal-Associated Invariant T cells

NAC

N-acetyl cysteine

NICD

notch intracellular domain

NOTCH1

Neurogenic locus notch homolog protein 1

NOTCH2

Neurogenic locus notch homolog protein 2

NOTCH3

Neurogenic locus notch homolog protein 3

NOTCH4

Neurogenic locus notch homolog protein 4

Nuclear Respiratory Factor 2

(gene name GABPB1)

NSG

NOD Scid Gamma

NS5A

Non-structural protein 5A

NuMA 1

nuclear mitotic apparatus protein 1

OCR

oxygen consumption rate

OGTT

oral glucose tolerance test

PBMCs

peripheral blood mononuclear cells

PD-1

programmed-death-1

PPARGC1A

PPARG coactivator 1 alpha

PHH

primary human hepatocytes

PLWH

persons living with HIV

PPARD

Peroxisome proliferator-activated receptor,delta

RANGAP1

Ran GTPase-activating protein 1

ROS

reactive oxygen species

RLW

Huh7.5-CYP

SIV

simian immunodeficiency virus

SKM

skeletal muscle

TAD

transactivation domain

TBARS

Thiobarbituric acid reactive substances

TBC1D15

TBC1 domain family member 15

TBC1D15

TBC1 domain family member 15

TICs

Tumor-initiating stem-like cells

TIM3

T-cell immunoglobulin and mucin domain 3

TLR4

Toll Like Receptor 4

TOM20

translocase of outer mitochondrial membrane 20

VDAC1

Voltage Dependent Anion Channel 1

4HNE

4-Hydroxynonenal

4MP

4 methyl pyrazole