Extracellular vesicles: intercellular mediators in alcohol-induced pathologies

Abstract

Though alcoholic liver injury plays the primary role in direct alcohol-related morbidity, alcohol consumption is also interlinked with many other diseases in extra-hepatic tissues/organs. The mechanism of alcoholic tissue injury is well documented, however the mechanisms that affect extra-hepatic tissues have not yet been well defined. Extracellular vesicles (EVs) such as exosomes and microvesicles, have been identified as key components of alcohol-induced extra-hepatic effects. We have reviewed the recent findings on the potential impact of alcohol-modified EVs/exosomes production and their downstream effects on extra-hepatic tissues. In this review, we discuss the available information on the cross-talk between hepatocytes and immune cells via EV/exosomal cargos (miRNA, mRNA, protein, etc.) in alcoholic liver diseases. We also discuss the effects of alcohol exposure on the contents of EVs/exosomes derived from various extra-hepatic tissues and their associated pathological consequences on recipient cells. Finally, we speculate on other potential EV/exosomal agents that may mediate alcohol-induced tissue damage.

Graphical Abstract

Alcohol can alter contents of extracellular vesicles (EVs) (e.g. exosomes) such as miRNAs, protein, cytokines, etc. in hepatic and extra-hepatic cells. The transfer of these alcohol modified EVs to nearby or distant cells can play vital role in inflammatory pathways in alcohol induced pathogenesis/comorbidities.

Introduction

According to the World Health Organization (WHO), alcohol-related complications pose a major public health challenge to the world population, contributing to approximately 5% of the global burden of disease and 6% of total deaths annually [1]. Alcoholic liver injury, which includes hepatitis and cirrhosis, majorly contributes to this problem. In addition to liver damage, excessive alcohol consumption can cause severe damage to the central and peripheral nervous systems, the gastrointestinal tract, the heart and vascular systems, and the endocrine and immune systems [2]. The WHO reports that drinking alcohol is associated with more than 60 non-communicable diseases, including cancer. Moreover, recent evidence points to a causative association between alcohol intake and infectious diseases such as HIV-1, tuberculosis, and pneumonia [1]. For many years, investigators have undertaken research studies to understand these alcohol-induced hepatic and extra-hepatic complications [3, 4]. However, the molecular and cellular mechanisms of alcohol-induced toxic effects follow a multiplicity of pathways. Despite tremendous advancements in the study of alcohol metabolism and its effects, the complete mechanism(s) by which alcohol causes tissue injury, especially extra-hepatic complications, are still poorly understood.

Extracellular vesicles as intercellular messengers

One potential phenomenon that is likely to play a role in alcohol-associated pathologies, but has yet to be extensively studied, is the formation and release of extracellular vesicles (EVs). EVs were first identified more than 50 years ago by two different groups [5, 6]. At that time, they were referred as “platelet dust” or “pro-coagulant platelet-derived particles”. These vesicles, small membrane-bound micro-particles containing proteins, RNA, and other biomolecules from their donor cell, have only attracted mainstream scientific attention relatively recently. There are mainly three distinct types of EVs- exosomes, microvesicles, and apoptotic bodies, which are secreted from eukaryotic cells [7]. These are categorized based on the size, mechanism of biogenesis, and their biological contents. Exosomes are produced from multivesicular bodies via endosomal pathways, which include internal budding and exocytosis [8]. Multivesicular bodies contain intraluminal vesicles, which contain cellular proteins, lipids, RNAs, etc. [9]. Inside cell cytoplasm, the intraluminal vesicles are formed by components of the endosomal-sorting-complex-required-for-transport (ESCRT) machinery, lipids, and tetraspanins (e.g. CD63, CD81, etc.). When the multivesicular bodies dock and fuse with the plasma membrane, the intraluminal vesicles are released as exosomes [9]. The size of exosomes typically varies from 30–150 nm. On the other hand, microvesicles are generated as a result of outward budding off the plasma membrane and the size ranges typically from 100–1000 nm. The biological contents of microvesicles are quite similar to that of exosomes, the mechanism of biogenesis being the major distinction between these two EV types. Apoptotic bodies are largest among all the EVs (size ranges from 1–5 μM), and are produced as a result of cell fragmentation/blebbing during programmed cell death. Apoptotic bodies are sometimes referred to as apoptosomes [10]. In general, EVs have a short half-life (from a few minutes to ~ 6 h after their release into the circulation), probably because of their subsequent uptake into the recipient cells [9, 11]. In the current review, EV has been used as a generic term, which encompasses exosomes, microvesicles, and apoptosomes. However, it is important to note that the role of exosomes has been discussed in more detail in the latter part of this manuscript due to their greater clinical and biological implications.

Among all the EVs, exosomes in particular, have become a new buzzword in the last decade, due to their relatively tightly regulated biogenesis and packaging processes. Interestingly, when they first emerged on the scene in the late 1980s, exosomes were thought to be the “garbage cans of the cell”, i.e. their function was assumed to be removal of unnecessary proteins and other cellular debris [12]. Dr. Rose Johnstone from McGill University, who was among the first to identify exosomes, believed that they could act as key regulators in cellular processes, though exosomes research has gained significant momentum only in the last decade [13, 14]. Due to advances in exosome isolation techniques and proteomic analysis, emerging evidences have revealed that these once-called “dumpsters of cells” are crucial mediators of both cell-to-cell communication and disease pathogenesis [12].

Recent findings show that EVs/exosomes can shuttle a plethora of key biological agents such as microRNA (miRNA), mRNA, lipids, proteins, and other molecules through biological fluids to both nearby recipient cells and those at distant sites [15, 16]. Due to tightly regulated mechanisms of cargo sorting and process of vesicular biogenesis, the cargo of EVs/exosomes generally varies between healthy and diseased populations, suggesting a role for EVs/exosomes in mediating cellular communication and disease progression [17]. EVs have been shown to play significant roles in the innate immune response, tumor progression, angiogenesis, and other processes, and recent data intriguingly suggest that alcohol can modulate the EV/exosomal pathway in both hepatic and extra-hepatic tissue systems, potentially exacerbating these effects [18–20]. This phenomenon can potentially help us to decipher the missing ‘pieces of the puzzle’ of the broad spectrum of alcohol-induced toxic outcomes in various tissues. In this review, we have attempted to summarize the latest discoveries relating to the effect of alcohol on secretion of EVs, with a major focus on exosomes, specific packaging of biomolecules and their transport via EVs/exosomes, and the potential impact of alcohol-modified EVs/exosomes on the recipient cells.

Alcohol-induced EVs/exosomes from hepatic cells in cell-cell communication

The liver is the principal organ responsible for the metabolism of most xenobiotic compounds, including ethanol, often estimated to metabolize up to 90% of imbibed ethanol [21]. It is equipped with high concentrations of the three major ethanol-metabolizing enzymes: alcohol dehydrogenases (ADHs), cytochrome P450 2E1 (CYP2E1), and catalase. Each of these enzymes metabolizes ethanol to acetaldehyde, which causes DNA and protein adducts that greatly contribute to the risk of liver cancer [22]. Additionally, the CYP2E1 enzyme, in the process of metabolizing ethanol to acetaldehyde, produces superoxide anion and hydrogen peroxide. These reactive oxygen species (ROS) are agents that have long been known to cause oxidative liver damage [21, 23]. These common metabolic pathways of ethanol are generally well understood. Even so, alcohol still acts as a major risk factor in many disease conditions aside from the widely heralded phenomenon of alcohol-induced liver injury. Since the ethanol molecule displays tremendous biological reactivity, the consequences of alcohol abuse can be far-reaching and complex. Therefore, alcohol-induced extra-hepatic complications remain a major area of interest today. Recent studies strongly point to the potential involvement of a defined group of biological nanovesicles, namely exosomes, as a key player in modulating the deleterious effects of alcohol in different tissue systems. Cho et al. [24] reported that exosomes obtained from mice with acetaminophen-induced liver injury can cause hepatotoxicity in recipient naïve primary hepatocytes as well as in mice. Exosome treatment caused elevation of plasma reactive oxygen species in mice and increased the expression of proteins associated with apoptotic signaling pathway such as phospho-JNK/JNK, Bax, and cleaved caspase-3 in the recipient hepatocytes. Alcohol-induced liver toxicity may follow a similar exosome–mediated communication pathway. In the following section, we attempt to summarize the findings from recent publications regarding changes to liver-derived EVs/exosomes upon alcohol exposure and their potential role in cellular communication, especially between hepatocytes and immune cells such as monocytes.

Hepatic EVs/exosomes in alcoholic hepatitis

Alcoholic hepatitis (AH) is one of the most devastating conditions associated with heavy alcohol intake, manifesting as acute inflammation of the liver [25]. AH pathogenesis follows a multifactorial pathway that involves intricate interplay between metabolism of alcohol, liver damage, and inflammation. The identification and role of different inflammatory mediators such as tumor necrosis factor α (TNFα) and interleukin-1 β (IL-1β) in AH and gut microbiome-derived lipopolysaccharides (LPS) have been studied extensively [26]. Damage to hepatocytes has been recently shown to be a prerequisite of alcohol-induced liver inflammation [27]. However, many facets of this pathogenesis remain unclear. For example, the exact mechanism of cross-talk between hepatocytes and immune cells (such as monocytes, macrophages, T-cells, etc.) was unknown until recently. Accumulating evidence suggests the potential role of exosomal miRNAs in drug-induced liver, kidney, and muscle injury [28–31].

MiRNAs play a fundamental role in regulating AH pathogenesis. One recent study reported that miR-122, miR-192, and miR-30a can act as useful diagnostic markers for AH [32]. MiR-122 is abundantly expressed in hepatocytes, but to a much lower degree in immune cells, and its function remains unclear. Momen-Heravi et al. [33] observed that, after chronic and/or binge alcohol exposure, greater numbers of exosomes that were rich in miR-122 were present in human sera. They also demonstrated that alcohol exposure increased miR-122-enriched exosome production in hepatocytes in a dose-dependent manner. Most importantly, these exosomes are horizontally transported miR-122 to monocytes, which rendered them more sensitive to LPS stimulation, inhibited heme oxygenase-1, and enhanced secretion of proinflammatory cytokines. Pre-treatment with exosomes loaded with a miR-122 inhibitor prevented this proinflammatory phenotype. In brief, this group demonstrated that exosomal transfer of miR-122 and consequent immune modulation could potentially be an alternative pathway of immune sensitization to LPS in AH pathogenesis. In addition, this study also demonstrates the potential for use of exosomes as an effective vehicle for delivering gene and RNA interference therapy in immune cells to reverse exosome-mediated deleterious effects from alcohol-exposed hepatocytes.

Hepatic EVs/exosomes in alcoholic liver disease

Alcoholic liver disease (ALD) is an umbrella term for a broad spectrum of disorders which includes AH, with or without cirrhosis, steatosis, hepatoccellular carcinoma, etc. Due to the varying spectrum of ALD, the pathophysiology is incompletely understood and hence, it is one of the leading causes of chronic liver disease [34]. A recent study by Verma et al. [35] attempted to demonstrate the mechanism by which macrophages are activated following alcohol exposure to hepatocytes in alcoholic liver disease (ALD). The authors showed that ethanol treatment significantly increased caspase-3 activation, which triggered increased EV production in hepatocytes. However, blocking of caspase-3 activation abolished the alcohol-induced EV production, which indicates that alcohol triggers enhanced vesicle production via a caspase-dependent pathway. Moreover, they also showed that cluster of differentiation 40 ligand (CD40L), a member of the TNF superfamily, was highly packaged in exosomes upon alcohol exposure inin vitro, in vivo, and ex vivo, and was associated with increased inflammatory cytokine production as well as hepatic macrophage infiltration. Blocking of CD40L reversed those phenomena, indicating that the crosstalk between hepatic and immune cells upon ethanol exposure may occur through a CD40L-mediated pathway.

A similar study conducted by Saha et al. [36] showed that EVs isolated from ALD mice had protein cargos distinct from the non-ALD mice, and that EV-induced activation of macrophages was mediated by heat shock protein 90 (Hsp90). Hsp90 is a molecular chaperone protein that has been previously shown to be involved in macrophage activation by the anti-tumor agent Taxol and by bacterial LPS [37]. This study demonstrated that upon intravenous administration of ALD-derived EVs into alcohol-naïve mice, those EVs were uptaken by recipient hepatocytes and macrophages. The EV-treated hepatocytes showed increased expression of monocyte chemoattractant protein 1 (MCP1) compared to hepatocytes treated with control EVs. MCP1 is an important chemokine that regulates recruitment of monocytes/macrophages in response to inflammation [38]. Moreover, the authors observed significant induction of Hsp90 in whole liver, and in purified hepatocytes isolated from ALD mice compared to pair-fed mice. The authors hypothesized that this elevated amount of Hsp90 was highly likely to be secreted via hepatic EVs into systemic circulation. Proteomic analysis showed that ALD EVs did indeed have higher expression of Hsp90. Furthermore, treatment of macrophages with recombinant Hsp90 resulted in a dose-dependent increase in the expression of the pro-inflammatory cytokines TNF-α and IL-1β, whereas the level of anti-inflammatory markers such as CD163 and CD206 were reduced. Inhibition of Hsp90 by a competitive inhibitor reversed this phenomenon, confirming the role of Hsp90 in macrophage activation in ALD via an EV-mediated pathway.

Hepatic EVs/exosomes in fibrogenesis

In recent years, liver-derived exosomes have also been implicated in wound healing mechanisms. For example, Nojima et al. [39] observed that in the case of ischemia/reperfusion injury or partial hepatectomy, exosomes derived from primary murine hepatocytes induced hepatocytic proliferation by fusing with recipient liver cells and delivering neutral ceramidase and sphingosine kinase 2, resulting in increased production of sphingosine-1-phosphate (S1P). S1P is a critical regulator in many pathophysiological processes, including cell proliferation [40]. The number of exosomes with proliferative effects increased after ischemia/reperfusion injury. In this scenario, hepatic exosomes appeared to play a beneficial role in response to tissue injury. Though, that is not true for all disease conditions. For example, exosomes may play an important role in tissue fibrogenesis, which is a natural physiological response to stress-induced tissue injury. However, several tissue repair processes are activated during stress conditions, and chronic insults such as prolonged ethanol exposure lead to dysregulation of the wound healing process, resulting in tissue fibrosis, scar formation, and ultimately organ failure. Alcohol metabolism triggers the release of a major fibrogenic cytokine, transforming growth factor-beta-1 (TGF-β1), and hepatic stellate cell (HSC) activation, which are key events in fibrosis progression [41]. These changes coincide with the dysregulation of global miRNA expression in liver cells, which play crucial roles in HSC functionality. Although the role of cellular miRNA-mediated HSC activation in alcoholic fibrosis has been well studied, the potential contribution of exosomal miRNAs in this phenomenon has not been delineated yet. In one recent study, Brandon-Warner et al. [42] found that alteration of a single miRNA, miR-19b, can result in a change in the expression and localization of multiple other miRNAs at the cellular and exosomal levels in an alcohol-induced hepatic fibrogenesis model. MiR-19b is part of the miR-(17–92) cluster family, and has been shown to be downregulated in activated HSCs. In addition, alcohol increased the expression of pro-fibrotic genes and decreased miR-19b level in HSCs. Interestingly, the level of miR-19b was significantly induced in plasma- and activated HSC-derived exosomes. However, the clinical relevance of the release of exosomal miR-19b in the fibrogenic pathway needs additional investigation. Figure 1 summarizes the above findings.

Fig. 1: Effect of alcohol on liver-derived EVs.

Alcohol exposure in the liver enhances EV secretion and carries inflammatory molecules to other hepatic cells or immune cells, which trigger/exacerbate liver injury.

Alcohol and EVs/exosomes derived from extra-hepatic tissues in cell-cell communication

Alcohol not only affects the liver, but also myriad other tissues. As mentioned previously, numerous studies have focused on the effects of alcohol on hepatocytes and liver-derived exosomes. However, very few studies exist concerning EVs/exosomes derived from ethanol-treated non-hepatic cells and tissues. In the following section, we summarize the findings of those studies that have been conducted on the effect of alcohol on the secretion as well as content of EVs/exosomes derived from extra-hepatic cells. This information will contribute to our understanding of the role of alcohol in altering EV/exosome-mediated intercellular communication, which in turn may lead to novel therapies for alcohol-induced extra-hepatic toxicities.

Alcohol and EVs/exosomes in the immune system

Alcohol can increase release of EVs from immune cells such as monocytes [43]. Monocytes are differentiated into macrophages in response to various stimulus/signals. These macrophages can polarize to either M1 (proinflammatory) or M2 (anti-inflammatory) phenotypes depending upon the type of stimulus/status of the disease. Saha et al. [43] found that exosomal miRNA-27a plays a role in the process of macrophage differentiation and/or polarization. Authors have demostrated that exosomes derived from ethanol-treated monocytes stimulate naïve monocytes to differentiate into M2 macrophages, mediated through miR-27a (an M2-polarizing miRNA). The authors validated these findings in vivo: circulating EVs in the plasma of alcoholic hepatitis patients were found to have high expression of miR-27a [43]. Studying the transport of miR-27a by exosomes derived from monocytes may give insights into the role of exosomes in mediating the modulatory effects of ethanol on the mechanisms of inflammation.

Similarly, alcohol can also stimulate certain cells of the immune system to release cytokines [44] and these can be packaged in exosomes [45]. A recent publication from our own laboratory investigating the plasma and exosomal cytokine levels in individuals who abuse drugs and HIV-infected subjects found that chronic alcohol comsumption could alter the exosomal packaging of cytokines in vivo [45]. In particular, IL-10 is highly packaged in the plasma exosomes of alcohol drinkers, though whether or not the higher exosomal levels of this anti-inflammatory cytokine reflect a physiologically protective or harmful phenomenon in chronic drinkers is a matter still under investigation.

Alcohol and EVs/exosomes in epithelia and endothelia

Ethanol also increases EV production in a dose-dependent manner from endothelial cells derived from human umbilical vein (HUVECs) and dermal microvasculature (HDMECs), according to a recent publication by Lamichhane et al. [46]. Further, exposure of naïve endothelial cells to exosomes derived from ethanol-treated endothelial cells increases vascularization by down-regulating anti-angiogenic miRNA-106b and up-regulating the pro-angiogenic long non-coding RNAs MALAT1 and HOTAIR [46].

Similarly, Atienzar-Aroca et al. [47] reported that retinal pigment epithelium (RPE) cells also dose-dependently produced higher quantities of exosomes upon exposure to ethanol. Further, these exosomes contained higher levels of vascular endothelial growth factor receptor (VEGFR-1 and VEGFR-2) mRNA than control exosomes. Interestingly, when exosomes derived from ethanol-treated RPEs and untreated control cells were exposed to HUVECs, the progression of tube formation was delayed by control exosomes and enhanced by ethanol-modified exosomes. Moreover, the intracellular levels of VEGFR-1 and VEGFR-2 were also increased in recipient HUVECs upon treatment with exosomes derived from the ethanol-exposed RPEs [47]. These findings indicate that EVs can be used as therapeutic targets for alcohol-induced angiogenesis in pathological conditions.

Alcohol is a known risk factor of oral squamous cell carcinoma (OSCC). Recently Momen-Heravi et al. [48] found altered miRNA expression in plasma EVs of patients with OSCC compared to healthy patients, as well as higher total EV content. Further, stimulation of an OSCC cell line with different doses of alcohol stimulated release of EVs containing known oncogenic miRNAs, particularly miR-21. Treatment of monocytes with EVs derived from OSCC cell lines transfered this miR-21, leading to activation of the NF-kB pathway and stimulating production of MCP-1. These effects were more pronounced with the administration of EVS from OSCC cells treated with alcohol, further supporting the hypothesis that regular alcohol consumption may exarcerbate oral cancer and provoke excessive inflammation.

Alcohol and EVs/exosomes in the heart

Cardiac myocytes are not primarily secretory cells. However, they can release exosomes upon exposure to certain stimulus or treatments [49]. Malik et al. [49] observed that exosomes derived from ethanol-treated cardiac myocytes had different protein content compared with exosomes derived from hypoxia/reoxygenation-experienced cells. Specifically, several proteins in ethanol-derived exosomes were found to be mitochondrial in origin. Since mitochondrial dysfunction is known to play a major role in oxidative stress, and heavy ethanol consumption contributes to cardiomyopathy through oxidative stress [50], exosomes derived from alcohol-exposed cells are likely to transport mitochondrial elements that could contribute to oxidative stress-induced intercellular signaling. Also, as with other tissues mentioned previously, ethanol increased exosome production from the cardiac myocytes via oxidative stress. Ethanol exposure, variations in pH, low temperature (4°C), and hypoxia/reoxygenation conditions did not alter the stability and membrane permeability of the myocytic exosomes [49] suggesting that they retain their protein cargo under diverse physiological/pathological conditions.

Alcohol and EVs/exosomes in the pancreas

Pancreatic stellate cells (PSC), upon exposure to ethanol, become activated and release connective tissue growth factor (CCN2), which regulates collagen deposition and fibrogenesis, in a dose-dependent manner. In a murine model of alcoholic chronic pancreatitis, co-treatment of ethanol with the compound cerulein (an inducer of pancreatitis) induced both CCN2 and miR-21 expression. Further, it was shown that, in the activated PSCs, CCN2 not only increases collagen deposition but also induces miR-21, which in turn increases CCN2 expression by a positive feedback mechanism [51]. The authors, Charrier et al. [52], have also demonstrated that exosomes derived from PSCs package CCN2 mRNA and miR-21 proportionally to their relative cellular expression, and that these exosomes are successfully delivered to recipient PSCs. The exchange of RNA elements from one cell to another upon stimulation with ethanol indicates that chronic drinking perhaps exacerbates existing pathological conditions such as pancreatic fibrosis. Summary of these findings related to extra-hepatic tissues are presented in Figure 2.

Fig. 2: Effect of alcohol on extra-hepatic cells derived EVs.

Upon exposure to alcohol, extra-hepatic tissues such as monocytes, endothelium, epithelium, and pancreatic stellate cells release EVs, and carry cargo of various biological molecules to their respective naïve cells and/or other cells influencing their pathophysiology.

Plasma EVs/exosomes and cytochrome P450 enzymes

The presence and biological significance of drug metabolizing cytochrome P450 (CYP) enzymes in EVs especially in exosomes have not yet been investigated in detail. In cases of chronic abuse, CYP2E1 plays a crucial role in mediating alcohol-induced hepatic and extra-hepatic toxicity. A few recent studies, such as from our group, have shown that CYP2E1 is abundantly packaged in plasma exosomes in healthy and alcoholic individuals, as well as in rodents [53, 54]. Cho et al. [54], demonstrated that alcohol increased the total number of EVs and CYP2E1 expression in patients with alcoholism and in an alcohol-fed animal model. Interestingly, increased EV production and vesicular secretion of several CYP isoforms appears to be modulated by cellular CYP2E1, since these inductive effects were abolished in Cyp2e1-null mice and in the presence of an enzymatic inhibitor, chlormethiazole. Moreover, CYP2E1 activity increased oxidative and endoplasmic reticulum stress, contributing to further vesicular packaging of CYP2E1. These EV-CYP2E1 could potentially act as a biomarker for liver damage from long term alcohol exposure [54]. We have [53] observed that the level of CYP2E1 in exosomes derived from healthy human plasma was significantly higher than other CYP isoforms such as CYPs 1B1, 2A6, and 3A4. Most importantly, the exosomal CYP2E1 was metabolically active, which suggests its potential role in contributing to ethanol metabolism and associated oxidative stress and toxicity in extra-hepatic regions such as the brain. The major source of this exosomal CYP2E1 is likely to be liver cells, since the levels of CYP2E1 mRNA in the plasma exosomes were reflective of those of hepatocytes. In our own study, when plasma exosomes enriched in CYP2E1 were treated to naïve hepatocytes, they significantly increased alcohol- and acetaminophen-induced toxicity, which were rescued by treatment with a selective CYP2E1 inhibitor [unpublished observations]. This indicates that plasma exosomal CYP2E1 may have the potential to contribute to the pathophysiology in recipient cells. Further investigation is ongoing to determine whether these exosomal CYPs, especially CYP2E1, have any role in enhancing alcohol-induced pathologies.

Potential mechanisms of alcohol-induced toxicity via EVs/exosomes

Clearly, there is much that remains to be done to fill in the gaps in our current understanding of how alcohol-modified EVs/exosomes may damage extra-hepatic tissues. For example, as of yet, there are no research publications concerning the effects of ethanol on the protein or RNA contents of EVs/exosomes derived from the cells of the central nervous system. It is a mostly untapped area of study, and as such discussion of pathogenic ethanol-induced exosomal content changes is necessarily speculative. Nevertheless, there are some predictions that can be made with confidence, based upon knowledge of overlapping interactions between ethanol exposure and EV/exosomal secretion of established toxic proteins.

Cathepsin enzymes

Cathepsins are a family of lysosomal proteases that are highly expressed in many cell types, especially phagocytes such as macrophages and microglia. Under stress conditions, cathepsin enzymes (in particular cathepsins B and D) can be secreted into extracellular space, where they can cause neurotoxicity through dysregulated proteolysis [55]. For example, Amritaj et al. [56] demonstrated that the compound U18666A, which is used as an agent for inhibiting cholesterol synthesis and transport in an in vitro model of Niemann-Pick type C disease, induced expression and extracellular secretion of cathepsin D from hippocampal neurons, which contributed to cell death in neurons and fibroblasts. Fan and He [57] found that astrocytes expressing HIV Tat protein exocytose their lysosomes, releasing cathepsin B and causing neuronal death.

Both cathepsin B and cathepsin D have been investigated in Alzheimer’s disease pathology, as they both are known to process amyloid precursor protein, the pro-peptide of beta amyloid [58, 59] and have been observed to be induced in the brains of Alzheimer’s disease rodent models [60, 61] and human patients [62]. There is some evidence that these induced cathepsins may be neuroprotective in that they help to digest beta amyloid plaques [63, 64]. However, CTSB inhibition has also been shown by Hook et al. [65] to reduce beta amyloid and improve memory in a murine Alzheimer’s model, indicating that it may also contribute to neurotoxicity. Regardless of this conflicting data on their net effect, the general consensus in the literature is that extracellular secretion of cathepsins increases in cases of Alzheimer’s disease [66] and that secreted cathepsins directly cause neuronal apoptosis [56, 67, 68]. Ethanol has been shown to enhance the expression and activity of both cathepsin enzymes in multiple tissues, including the liver [69], pancreas [70], and brain [71]. The release of cathepsin enzymes likely occurs through lysosomal leakage caused by ethanol-induced oxidative stress, as high quantities of ROS can cause membrane instability of that organelle in many cell types [72, 73]. Goetzl et al. [74] observed that cathepsin D was packaged in neuron-derived exosomes secreted into plasma, and that the exosomal expression of cathepsin D was greater in patients with severe Alzheimer’s disease. Kang et al. [75] similarly observed increased expression of cathepsin D in exosomes derived from retinal epithelia found in the aqueous humor of patients with macular degeneration. Cathepsins B and D are also packaged in exosomes derived from macrophages in vitro, both of which are dose-dependently induced with ethanol exposure [unpublished observations]. With this in mind, there is good reason to expect that ethanol-induced oxidative stress may induce exosomal secretion of active cathepsin enzymes, which may have contribute to long-lasting neurodegenerative conditions.

NADPH oxidase

Another potential protein of interest is the NADPH oxidase complex (NOX). NOX is expressed in several cell types, particularly phagocytes, and when activated produces large quantities of superoxide, contributing to oxidative stress [76]. The NOX complex has been shown to be both activated and induced by ethanol exposure in neurons, microglia, and macrophages both in vitro and in murine models of alcohol abuse [77, 78]. Janiszewski et al. [79] were among the first to observe expression of NOX in exosomes and microvesicles in plasma, which was pro-apoptotic when treated to recipient endothelial and aortic smooth muscle cells. While it has never been studied, it is likely that ethanol exposure may induce secretion of EV/exosomal NOX from macrophages and microglia in excess, which could be a potent source of ROS-mediated toxicity.

Tau

Drinking alcohol is not definitively associated with disease severity or poorer prognosis in Alzheimer’s disease [80], yet oxidative stress is known to have some interaction with the pathways associated with its pathogenesis [81]. As ethanol metabolism is a potent source of ROS there remains potential for alcohol to exacerbate Alzheimer’s-associated pathologies. Indeed, it has been observed that ethanol can induce phosphorylation of tau protein, the microtubule-associated protein whose dysfunction is a hallmark of Alzheimer’s disease [82, 83]. Ethanol has also been shown by Gendron et al. [84] to induce aggregation of tau in neuroblastoma cells in vitro, and inhibit its clearance. Given that neurons can secrete tau in exosomes [85] and that microglia can propagate the spread of tau through phagocytosis and re-exocytosis [86], it is likely that chronic consumption of alcohol enhances the production and spread of phosphorylated tau aggregates via exosomes, which may contribute to the progression of Alzheimer’s disease.

MicroRNA-21

Alcohol has also been demonstrated to alter the miRNA transcriptome in many tissues, inducing both protective and deleterious effects [87]. However, exosomal transport of ethanol-induced miRNAs has only just begun to be investigated. In particular, there is one miRNA that is known to be induced by ethanol exposure, to be packaged into exosomes, and to have a complex role in the CNS: miR-21. It has long been known that miR-21 has a role in regulating a number of genes, many of which are associated with controlling cellular growth and proliferation. Under conditions of stress, such as ethanol exposure, miR-21 can be induced, balancing tissue regeneration and repair in the liver [88, 89]. It has also been shown to improve neurological outcomes following traumatic brain injury in rats, indicating potential neuro-regenerative properties as well [90]. However, as mentioned previously [48], these same qualities make miR-21 a mediator of cancer in a number of different tissues, as it protects tumor cells by modulating inflammation and apoptosis [91]. It has been observed to be secreted in exosomes in a murine glioblastoma model and elevated in exosomes derived from the serum and spinal fluid of patients with high-grade glioma [92, 93]. In addition to being pro-cancerous, one study by Yelamanchili et al. [94] showed that miR-21 in EVs could also be directly neurotoxic through interactions with TLR7. With the knowledge that miR-21 can be induced by alcohol, and given its associations with neurotoxicity and cancer, the scientific community would be well served by greater understanding of the role of exosomal transport of miR-21, and other potentially harmful miRNA that may be induced by ethanol, in these conditions.

HIV elements

Alcohol consumption is also known to enhance HIV pathogenesis [95–97]. It is very well established that oxidative stress can induce viral replication and the expression of viral proteins, by multiple mechanisms [98, 99]. Ethanol metabolism, a significant source of oxidative stress, therefore also unsurprisingly enhances viral replication in T-cells and mononuclear cells [95, 100]. This phenomenon also occurs throughout the CNS [101] and consequently alcohol drinking is considered a risk factor for the development of HIV-associated neurocognitive disorders [102, 103]. Aside from inducing viral replication, ethanol also interferes with the metabolism of some antiretroviral drugs, further worsening disease progression in infected alcohol drinkers [104, 105]. Additionally, ethanol exposure has been shown to potentiate the neurotoxic effects of HIV proteins such as gp120 and Tat, through sensitization of the N-methyl D-aspartate receptor, leading to excitotoxic cell death [106, 107].

HIV, like some other viruses, is known to utilize exosome processing and secretion machinery (such as the ESCRT complex) to facilitate its own reproduction [108]. As a consequence of that, viral proteins and RNA are packaged and secreted in exosomes and other extracellular vesicles, even in the presence of antiretroviral therapy [109] These viral elements, such as the proteins Tat and Nef, have been shown to have a variety of pathogenic effects [98, 110, 111].Taken together, it is highly likely that the exosomal transport of HIV components is enhanced by ethanol exposure through oxidative stress-induced transcription of the viral genome, leading to increased susceptibility to intercellular viral infection, inflammation, direct neurotoxicity, and other deleterious conditions associated with HIV infection. However, this phenomenon, like the other previously suggested mechanisms of ethanol-induced toxicity mediated by exosomes, has not yet been demonstrated experimentally, and is merely speculative given the available data. This is an area in great need of additional research, as alcohol abuse and its effects on neurodegeneration are still not yet fully understood, but continue to affect millions of people worldwide. Figure 3 provides a summarized illustration of the above discourse.

Fig. 3: Potential EV/exosomal mechanisms of alcohol-induced toxicity.

In response to ethanol exposure, exosomal transport of pathogenic elements to nearby or distant cells can act as cellular messenger system, and may facilitate/enhance disease progression in cancer, HIV, liver disease, neurodegenerative disorders, etc.

Discussion

Alcohol alters the miRNA and protein content (e.g. miR-27a, miR-122, CD40L) of EVs such as exosomes from multiple tissues, having complex immunomodulatory effects. EV/exosomal RNA (miR-21, miR-19b, VEGF mRNA) and proteins (HSP90, CCN2) can be potentially involved in alcohol-induced exacerbation of existing disease conditions, such as pancreatic fibrosis, cardiomyopathy, and some cancers. We also considered some potential EV/exosomal agents that may contribute to alcohol-induced damage in other tissues, such as the central nervous system (NOX, cathepsin enzymes), as well as exosomal elements associated with other conditions that may be enhanced by alcohol exposure (phosphorylated tau and HIV protein and RNA elements). In conclusion, recent investigations, limited though they are, point to a consensus that exosomes derived from alcohol-exposed cells, irrespective of origins, can play a crucial role in disease progression by modulating biological pathways via delivery of cargo to recipient cells. This is an exciting area of research which is in need of further investigation. Table 1 provides a brief summary of the findings from the research articles that have been discussed in this review.

Table 1:

Pathogenic exosomal elements in response to alcohol exposure

Donor cells	Recipient cells	Mediators	Implications	Reference
Hepatocytes	Monocytes	miR-122	Immune sensitization in AH	Momen-Heravi et al. 2015
Hepatocytes	Macrophages	CD40L	Macrophage activation in ALD	Verma et al. 2016
Hepatocytes	Hepatocytes, monocytes	Hsp90	Macrophage activation in ALD mice	Saha et al. 2018
Hepatocytes	Hepatocytes	Ceramidase and sphingosine kinase-2	Fibrogenesis	Nojima et al. 2016
Hepatic stellate cells	Hepatic stellate cells	miR-19b	Increased hepatic fibrogenesis	Brandon-Warner et al. 2016
Monocytes	Monocytes	mi-R27a	Differentiation to M2 macrophages	Saha et al. 2016
Endothelial cells	Endothelial cells	MALAT1 and HOTAIR	CD34-mediated vascularization	Lamichhane et al. 2017
Retinal pigment epithelium cells	Endothelial cells	VEGFR-1 and VEGFR-2	Vascularization	Atienzar-Aroca et al. 2016
Oral squamous cell carcinoma	Monocytes	miR-21	NF-kB activation and MCP-1 secretion	Momen-Heravi et al. 2018
Pancreatic stellate cells	Pancreatic stellate cells	CCN2 and miR-21	Collagen production and fibrogenesis	Charrier et al. 2014

EVs/exosomes are not just buzzwords in the scientific community anymore. As a result of advances in large-scale proteomic analysis since the early 2000s, research in this area has taken a major leap forward in recent years. However, we are still in the nascent phase of the EV/exosome revolution. In other words, extensive investigation is essential to establish a complete understanding of the role of EVs/exosomes in disease progression, particularly in the context of concurrent xenobiotic exposures. For example, the impact of alcohol on EV contents in alcohol-related co-morbidities is an under-studied area, despite its widespread use and well-established role in exacerbating many pathological conditions. Due to limited literature availability and the lack of well-defined target pathways of alcohol-associated co-morbidities, it is apparent that there is a critical need to find biomarkers for alcohol-induced toxicity outside of the liver. Recent reports suggest EVs/exosomes can play a crucial role in finding these desired biomarkers [112]. The circulation of EVs/exosomes throughout the body via body fluids, particularly plasma, urine, and saliva, make them appealing, because of their convenient accessibility for sample collection, sometimes referred to as “liquid biopsy” [113]. There have been several studies identifying EVs with specific contents as biomarkers for various conditions, especially cancers [114]. The ability of EVs/exosomes to migrate from their tissues of origin into wider circulation through plasma also allows for diagnosis of disease in otherwise less accessible regions of the body, such as the CNS. For example, neuronally-derived exosomes collected from plasma have been found to carry markers for Alzheimer’s disease in Alzheimer’s patients, as well as in individuals with Down syndrome [74, 115]. It is highly likely that circulating exosomes contain specific markers for alcohol-induced toxicity outside of the liver, which would provide a novel and non-invasive way of diagnosing specific morbidities in chronic alcohol drinkers that require therapeutic intervention. As discussed in this review, in a murine chronic pancreatitis model, exosomes appear to play a crucial role in pancreatic fibrogenesis, a pathological outcome that is enhanced by chronic alcohol exposure [52]. Identification of pancreatic exosomes containing high levels of the fibrogenic elements CCN2 and miR-21 in the plasma of chronic drinkers could allow for earlier diagnosis and therapy to counteract alcohol-enhanced pancreatitis and fibrosis. Considering that miR-21 is also induced in the liver during alcohol metabolism, and is furthermore a known oncogenic agent, miR-21 and other EV/exosomal ethanol-induced miRNAs may be valuable early indicators of alcohol-associated cancers [88, 89, 91–93, 116].

Conclusion

Based on the literature reports, it is evident that alcohol-induced protein and/or mRNA changes in cellular contents are consistent with the changes in their EV/exosomal cargos. Alcohol exposure modifies the contents of both hepatic and extra-hepatic EVs which are translocated to other naïve liver cells or non-hepatic cells. This intercellular transfer of biomolecules results in increased/decreased inflammatory responses in the recipient cells. EVs, especially exosomes represent the next frontier in the areas of diagnosis and therapy of a plethora of pathological conditions, many of which are enhanced or altered by concurrent substance abuse. It is vital for patient care that the role of EVs/exosomes in these conditions, and how that role changes in cases of chronic drug use, is elucidated. A more complete understanding of the interplay between EVs/exosomes and alcohol will allow for greater degrees of personalized care for the individuals who consume it or other commonly used substances such as tobacco constituents, cannabinoids, and opioids, ultimately leading to superior public health outcomes.