Alcohol-Induced Lysosomal Damage and Suppression of Lysosome Biogenesis Contribute to Hepatotoxicity in HIV-Exposed Liver Cells

Although the causes of hepatotoxicity among alcohol-abusing HIV patients are multifactorial, alcohol remains the least explored “second hit” for HIV-related hepatotoxicity. Here, we investigated whether metabolically derived acetaldehyde impairs lysosomes to enhance HIV-induced hepatotoxicity. We exposed Cytochrome P450 2E1 (CYP2E1)-expressing Huh 7.5 (also known as RLW) cells to an acetaldehyde-generating system (AGS) for 24 h. We then infected (or not) the cells with HIV-1ADA then exposed them again to AGS for another 48 h. Lysosome damage was assessed by galectin 3/LAMP1 co-localization and cathepsin leakage. Expression of lysosome biogenesis–transcription factor, TFEB, was measured by its protein levels and by in situ immunofluorescence. Exposure of cells to both AGS + HIV caused the greatest amount of lysosome leakage and its impaired lysosomal biogenesis, leading to intrinsic apoptosis. Furthermore, the movement of TFEB from cytosol to the nucleus via microtubules was impaired by AGS exposure. The latter impairment appeared to occur by acetylation of α-tubulin. Moreover, ZKSCAN3, a repressor of lysosome gene activation by TFEB, was amplified by AGS. Both these changes contributed to AGS-elicited disruption of lysosome biogenesis. Our findings indicate that metabolically generated acetaldehyde damages lysosomes and likely prevents their repair and restoration, thereby exacerbating HIV-induced hepatotoxicity.


Introduction
Since the emergence of antiretroviral therapy, a significant decline in AIDS-related mortality among people living with HIV (PLWH) precipitated an upsurge in non-AIDS-related mortality (NARM). Comorbidities implicated in NARM among PLWH are cardiometabolic disorders, renal disease, mental illness, and liver disease [1]. While cardiometabolic disease ranks as the leading comorbidity among PLWH [2], liver disease remains the leading injury for NARM, accounting for approximately one-fifth of all cases [3][4][5]. This makes the management of liver disease a priority for HIV care delivery. Pathogenesis of HIV-related liver comorbidities is exacerbated by "second hits", such as alcohol or co-infection with hepatotropic viruses [6]. However, the mechanism(s) by which alcohol induces liver injury during HIV infection is/are not entirely clear, which prevents improving a holistic treatment strategy. Thus, an understanding of the mechanisms of HIV-and alcohol-induced hepatotoxicity is the goal of this study.
We recently showed that in HIV-infected liver cells, ethanol and/or its metabolite acetaldehyde cause accumulation of HIV proteins to induce oxidant stress and subsequent apoptotic cell death [7]. This may lead to detrimental consequences and promote the development of end-stage liver diseases since HIV-particle-bearing apoptotic hepatocytes are engulfed by hepatic stellate cells (HSC) and macrophages (Mph), to induce profibrotic and proinflammatory events, leading to the progression of liver fibrosis [7].
In untreated liver cells, lysosomes degrade HIV proteins, thereby preventing their intracellular accumulation and subsequent oxidant stress [7,8]. While lysosome dysfunction by ethanol has been reported [9,10], it has not been implicated in the pathogenesis of HIV/alcohol-induced liver injury. Here, we characterized the contribution of acetaldehyde, the primary oxidation product of ethanol, and its ability to impair lysosome function in HIV-exposed liver cells. First, we investigated whether the alcohol-induced decline in cathepsin activities was accompanied by decreased lysosome numbers. Second, we determined whether acetaldehyde-induced lysosome leakage triggered the cathepsin decline. Lysosome damage/rupture causes the release of cathepsins and other hydrolases into the cytosol. While the etiology of lysosome damage and subsequent leakage are diverse, ROS production induces such damage [11]. Here, we used an in vitro acetaldehyde-generating system (AGS) that continuously generates physiologically relevant amounts of acetaldehyde and mimics ethanol metabolism in HIV-infected Huh 7.5 (also known as RLW) cells that overexpress cytochrome P450 2E1 (CYP2E1), but not alcohol dehydrogenase (ADH). AGS generates acetaldehyde by using ethanol as the primary substrate of yeast alcohol dehydrogenase in the presence of nicotinamide adenine dinucleotide (NAD + ) as a co-factor. Exposure of cells to the AGS elevates ROS levels in RLW cells since ethanol is part of AGS, and RLW cells generate ROS by CYP2E1 upon ethanol exposure [7]. Hence, we postulated that in HIV-exposed RLW cells, the decrease in cathepsin activities is due to lysosome leakage triggered by enzymatically generated acetaldehyde and oxidant stress, which mimics ethanol metabolism in primary hepatocytes. Such leakage is detrimental if lysosome repair/replacement is inhibited as well. The activity and intracellular localization of Transcription Factor EB (TFEB), which activates genes that encode lysosomal proteins and lysosome biogenesis [12], is disrupted by chronic ethanol administration [13,14]. Here, we sought to determine whether HIV and AGS prevent TFEB translocation from cytosol to the nucleus of HIV-infected RLW cells. TFEB nuclear translocation activates genes involved in lysosome biogenesis. TFEB nuclear translocation/gene activation is prevented by its phosphorylation [15], by reduced proteasomal degradation in the cytosol [16], and by impaired TFEB trafficking; an increased expression of ZKSCAN3, a transcriptional repressor of autophagy [17], also affects lysosomal gene activation. We hypothesized that exposure of hepatocytes to HIV and ethanol and its metabolite, acetaldehyde, induces oxidant stress to trigger lysosome leakage and lysosome biogenesis suppression. The latter suppression blocks intracellular replacement of damaged lysosomes causing cytotoxicity during combined HIV and alcohol/acetaldehyde exposure to liver cells.

Reagents and Antibodies
High-glucose Dulbecco's modified eagle medium (DMEM) and fetal bovine serum were purchased from Invitrogen (Waltham, MA, USA); Trizol was from Life Technologies (Carlsbad, CA, USA); primer probes, high-capacity reverse transcription kit, and real-time polymerase chain reaction (RT-PCR) reagents were from Applied Biosystems by Thermo Fisher Scientific, Carlsbad, CA, USA. Pan-caspase inhibitor (PCI) was obtained from Ubiquitin-Proteasome Biotechnologies (UBPBio) Inc.

In Vitro Studies
Due to the paucity of primary human hepatocytes, we performed all experiments on hepatocyte-like RLW cells. These cells have reduced innate immunity and do not sufficiently oxidize (metabolize) ethanol because they lack ADH. To circumvent this limitation, we exposed RLW cells to an exogenous constant source of acetaldehyde (acetaldehydegenerating system, AGS), which consists of 0.02 EU yeast alcohol dehydrogenase (ADH), 2 mM nicotinamide adenine dinucleotide (NAD), and 50 mM ethanol. AGS is known to continuously generate a physiologically relevant amount of acetaldehyde without unwarranted toxicities [7]. HIV-1 ADA propagated on primary human macrophages was purified at University of Nebraska Medical Center (UNMC) [18]. RLW cells were pre-treated for 24 h with AGS, infected with HIV-1 ADA , and then exposed again to AGS for another 48 h. HIV was removed after overnight treatment, and the cells were washed thoroughly with 1× phosphate-buffered saline (PBS) to remove extracellular HIV.

RNA Isolation and Real-Time PCR (RT-PCR)
RNAs encoding p53, cathepsin D, and LAMP1 were quantified by RT-PCR as previously described [7]. Briefly, total cellular RNA was isolated from cells using Trizol reagent. A two-step procedure was used, in which 200 ng RNA was reverse-transcribed to cDNA using the high-capacity reverse transcription kit. In the second step, the cDNA was amplified using TaqMan Universal Master Mix II with fluorescent-labeled primers (TaqMan gene expression systems). These were incubated in a Model 7500 qRT-PCR thermal cycler. The relative quantity of each RNA transcript was calculated by its threshold cycle (Ct) after subtracting that of the reference (GAPDH).

Whole-Cell Lysates and Nuclear/Cytosolic Fractionations
Whole-cell lysates were prepared in phosphorylation capture buffer (0.5 M EDTA, 2 M Tris, 20 mM Na 3 VO 4 , 200 mM Na 4 P 2 O 7 , 100 mM PMSF, 1 M NaF, 20% Triton X-100, and aprotinin, pH 7). The extraction of the nuclear and cytosolic components from RLW cells was performed using the NE-PER Nuclear and Cytoplasmic Extraction Kit (ThermoFisher Scientific, Carlsbad, CA, USA) based on the manufacturer's protocol.

Immunoblotting
Immunoblotting was performed as described [21,22]; blots were developed using Odyssey infrared imaging system, which was also used to quantify the protein bands [23]. Equal (20 µg) amounts of protein were loaded in each lane. Beta-Actin or TATA-binding proteins (TBP) were used as loading controls to normalize the protein band densities.

Immunofluorescence
RLW Cells (130,000/well) were seeded onto coverslips, which were inserted into each well of a 12-well plate. Cells were infected with the HIV-1 virus (Multiplicity of infection, MOI = 0.1). After overnight incubation of cells with HIV, the virus-containing medium was removed and replenished with fresh media. Cells were incubated for another 48 h of AGS treatment; cells were washed with 1× PBS, fixed with 4% paraformaldehyde for 12 min at 37 • C, permeabilized with 0.1% Triton X-100 for 3 min at room temperature, and blocked for 30 min with 1% (g/vol) bovine serum albumin (BSA) in PBS. Cells were stained to study the colocalization of LAMP1:Galectin 3, Tom20:Cathepsin-B, and quantification of LAMP1. First, cells were incubated with primary antibodies for 1 h. The cells were then washed and incubated with the mixture of Alexa-Fluor-labeled secondary antibodies for 30 min. Nuclei were stained with DAPI. The coverslips were transferred to microscope slides for imaging by using LSM 710 confocal microscope. Immunostainings and colocalization were quantified for intensity using National Institute of Health (NIH) Image J program.

Statistical Analyses
Data were analyzed using GraphPad Prism v7.03 software (GraphPad, La Jolla, CA, USA). Data from at least three duplicate independent experiments were expressed as mean ± SEM. Comparisons among multiple groups were performed by one-way ANOVA, using a Tukey post hoc test. For comparisons between two groups, we used Student's t-test. A p-value of 0.05 or less was considered significant.

AGS and HIV Exposure Lowers Cathepsin Activities and Lysosome Numbers
To understand the mechanism(s) by which HIV and acetaldehyde induce accumulation of HIV proteins in hepatocytes, we measured cathepsin B and L activities as indices of lysosomal enzyme function. The combination of AGS and HIV treatment (AGS + HIV) decreased (p < 0.05) cathepsin B and L activities ( Figure 1A,B). To determine whether lower cathepsin B and L activities by AGS + HIV exposure reflected lower lysosome numbers, we measured LAMP1 immunofluorescence intensity. Images were normalized to the number of cells in each field. LAMP1 intensity was 42% lower in AGS-treated uninfected cells, and this was further suppressed in HIV-infected cells ( Figure 1C

Both AGS and HIV Enhance Lysosome Leakage
We investigated whether AGS-induced LAMP1 suppression affected lysosome membrane permeabilization and lysosomal leakage (LL) in HIV-infected and -uninfected cells. To this end, we quantified the levels of damage-regulatory autophagy modulator (DRAM)-1, which induces lysosome leakage, thereby triggering p53-dependent apoptosis [24]. Since we found that AGS treatment increased p53 mRNA levels in both infected and uninfected RLW cells (Figure 2A), we postulated that DRAM1 expressing cells would be cleared by apoptosis. In fact, it was observed that AGS and AGS + HIV exposed cells expressed a very low amount of DRAM1 ( Figure 2B,C) because DRAM1-expressing cells were unaccounted for due to apoptosis. To prevent/slow the clearance of DRAM-positive cells, we measured DRAM expression after blocking apoptosis by a pan-caspase inhibitor (PCI). In the presence of PCI, we observed 64% higher levels of intracellular DRAM in AGS-exposed HIV-infected cells ( Figure 2D,E). To further confirm these findings, we measured the co-localization of galectin 3 and LAMP1 puncta and found that AGS exposure exhibited greater co-localization of galectin 3 and LAMP1 puncta in HIV-infected cells than in uninfected cells ( Figure 2F,G), indicating that both HIV and acetaldehyde exposure contributes to lysosome leakage.

AGS and HIV Enhance Intracellular Oxidant Stress
We investigated the contribution of oxidant stress to AGS-induced decline of cathepsin activities in control and HIV-exposed RLW cells as well as downstream apoptosis. Combined AGS and HIV exposure suppressed cathepsin B and L activities and caused an increase in apoptosis, detected by ELISA as elevated levels of M30 (cleaved cytokeratin 18) in cell supernatants. M30 detection was significantly lower (p < 0.05) after treatment of cells with N-acetyl cysteine (NAC), which restored cathepsin B and L activities ( Figure 3A-C).

AGS and HIV Enhance Cathepsin Leakage and Intrinsic Apoptosis
We measured the leakage of cathepsin B from lysosomes and its translocation to the outer mitochondrial membrane, which likely damages the mitochondrial membrane. In AGS + HIV-treated cells, cathepsin B was co-localized with mitochondria, as indicated by immunofluorescent co-localization of cathepsin B and Tom20. Tom20 is a protein on the mitochondrial outer membrane that facilitates the import of mitochondrial precursor proteins into the mitochondrion [25,26] (Figure 4A,B). Furthermore, caspase 3 cleavage in AGS-exposed HIV-infected liver cells (the condition with the highest cathepsin B-Tom20 co-localization and apoptotic effects) was suppressed nearly three-fold in the presence of 20 µM caspase 9 inhibitor, while the caspase 8 inhibitor had a minimal effect on the observed apoptosis ( Figure 4C,D).

AGS and HIV Exposure Suppress TFEB Protein Expressions in Cytosolic and Nuclear Fractions of RLW Cells
Lysosome damage reportedly activates lysosome biogenesis as a compensatory mechanism to replace damaged lysosomes [27] via enhanced TFEB transcription [15]. After we separated nuclear and cytosolic fractions from the control and treated RLW cells, we observed greater amounts of immunoreactive TFEB in the cytosolic fractions of HIV-and AGS-treated cells. Both treatments significantly decreased the nuclear to cytosolic TFEB ratios compared with untreated cells (Figure 5A,B).
The purities of our nuclear and cytosolic fractions were confirmed by Western blotting by exposing both fractions to anti-α-tubulin (a cytosolic marker) and to the anti-TATA binding protein (a nuclear marker) ( Figure 5C).
Suppression by AGS of nuclear TFEB expression in HIV-infected and -uninfected cells prompted us to analyze the role of ethanol metabolism on the factors that regulate TFEB cytosolic-nuclear shuttling. As reported, mTORC1-mediated TFEB phosphorylation is an upstream event, which causes TFEB retention in the cytosol [15]. Surprisingly, we observed the downregulation of TFEB phosphorylation (pTFEB) at the mTOR-regulated serine sites (S211) in both AGS-exposed uninfected and HIV-infected cells, which decreased the pTFEB/TFEB ratio ( Figure 5D,E). To test whether microtubules regulate TFEB trafficking to the nucleus, RLW cells were exposed for 24 h to 10 µM nocodazole, a microtubule-disrupting drug. We found that TFEB nuclear translocation was attenuated by nocodazole, indicating that translocation is microtubule-dependent ( Figure 5F,G). We also found that exposure of HIV-infected and -uninfected RLW cells to AGS enhanced α-tubulin acetylation ( Figure 5H,I).

AGS and HIV Exposures Suppress Proteasome Activity and Enhance ZKSCAN3 Expression
Because TFEB is degraded by the proteasome, we examined whether HIV or AGS influences 20S proteasome peptidase activities. HIV infection suppressed chymotrypsin and trypsin-like activities ( Figure 6A,B). However, this effect was exacerbated in AGS-exposed HIV-infected and -uninfected RLW cells.
We explored whether acetaldehyde influenced TFEB induced activation of lysosomal genes. To this end, we measured the levels of ZKSCAN3 in RLW nuclear fractions as ZKSCANS represses TFEB-activated lysosome biogenesis. We observed a 40% rise in ZKSCAN3 expression after AGS treatment in both HIV-exposed and -unexposed RLW cells ( Figure 6C,D). To corroborate these findings, we quantified the mRNAs encoding LAMP1 and cathepsin D, two genes activated by TFEB. Both LAMP1 and cathepsin D mRNAs were lower after cells were treated with AGS. This suppression effect was even greater in AGS + HIV-infected cells ( Figure 6E).

Discussion
As revealed from our previous studies, pre-exposure of hepatocytes to the ethanol metabolite acetaldehyde sensitizes cells to HIV-induced apoptotic cell death. This was partly due to acetaldehyde-induced intracellular accumulation of HIV proteins, which occurs due to suppression of HIV protein degradation by both the proteasome and lysosomes [7]. In fact, inhibition of protein-degrading enzyme activities with specific inhibitors, MG132 and carfilzomib for proteasomes or bafilomycin and chloroquine for lysosomes, prolongs the persistence of HIV proteins in hepatocytes [7]. As reported by others, HIV proteins are degraded by proteasomes and lysosomes [28][29][30][31]. We showed that the accumulation of HIV proteins in hepatocytes was greater after treatment with lysosome inhibitors than with proteasome inhibitors. These findings underscore the importance of ethanol-and HIV-induced lysosomal dysfunction/damage for HIV protein retention, which can cause oxidative stress. In kinetic studies, we indeed observed that the accumulation of HIV proteins after exposure to AGS induces oxidative stress accompanied by ROS release, which leads to apoptotic hepatocyte death [7]. The latter event results in the formation of apoptotic bodies, which are engulfed by non-parenchymal (Kupffer) cells as we observed in some canonical hepatotropic infections, such as HCV-infection, and were able to further confirm in HIV infection [21,32].
Here, we addressed the contribution of lysosomal impairment to HIV alcohol-metaboliteinduced hepatotoxicity. Our results lead us to suggest that acetaldehyde induces lysosomal damage in HIV-infected hepatocytes and that leakage of lysosomal luminal content (cathepsins) to other cell compartments may damage adjacent organelles, which hastens hepatic cell death. For example, leakage of cathepsins to mitochondria can disrupt the mitochondrial membrane and may induce intrinsic apoptotic events [27].
We focused on the underlying mechanism(s) by which HIV, ethanol metabolite(s), and their combination impaired lysosome activity. To mimic ethanol metabolism in hepatocytes, we exposed CYP2E1-expressing RLW cells to AGS. Lysosome leakage (LL) is one mechanism that leads to a decline in lysosome hydrolytic potency. The pathogenic impact of LL in the liver has already been discussed for non-alcoholic [33,34] and alcoholic steatohepatitis [35]. It was also implicated in the liver injury of ethanol-fed rodents [35,36]. However, this is the first time it has been investigated in non-immune cells (hepatocytes) after HIV infection and treatment with ethanol metabolites. In our hands, the treatment of both HIV-infected and -uninfected RLW cells with AGS lowered the expression of LAMP1, a lysosomal membrane protein important for maintaining lysosome structural integrity. These findings indicate that acetaldehyde exposure causes lysosomal membrane instability. The decrease in lysosomal numbers and functions were, at least in part, triggered by ethanol metabolite-induced oxidative stress since lysosome instability was attenuated by treatment with the antioxidant NAC. In fact, lysosomes do not express anti-oxidative enzymes, and they are sensitive to the effects of ROS, which easily destabilize lysosomal membranes [27]. Indeed, ROS have been shown to trigger LL in alcoholic steatohepatitis [34].
In the current study, co-localization of LAMP1 and galectin 3 reported by others as an indication of increased lysosomal permeability [37] was highest in AGS-treated HIVinfected RLW cells. While the role of HIV-induced lysosomal damage has not been studied in hepatocytes before, in CD4 + lymphocytes, HIV potentiates LL by activating lysosome-colocalized protein, DRAM1, responsible for lysosome membrane permeabilization (LMP) [24]. Furthermore, DRAM1 regulates apoptosis by increasing the lysosomal localization of proapoptotic protein BAX [38] and has p53 as its downstream target [39]. Therefore, by measuring p53 gene activation in AGS + HIV-treated RLW cells, we identified a significant increase in p53 mRNA levels. Because p53-mediated apoptosis is caspase-dependent [40], to preserve DRAM1-expressing RLW cells, we tested the effects of HIV and AGS treatments on DRAM1 expression in the presence of pan-caspase inhibitor (PCI) and found increased levels of DRAM1 in these double-treated cells compared with untreated (control) cells and cells exposed to either HIV or AGS. These changes were not observed in the absence of PCI because DRAM1-expressing cells undergo robust caspase-dependent apoptosis. Thus, it is likely that by activating p53-mediated genes in hepatocytes, DRAM1 contributes to apoptosis induction, demonstrating the link between LL and apoptotic cell death.
The link between LL and HIV-AGS-induced apoptosis in hepatocytes is still unclear. When RLW cells were exposed to both HIV and AGS, leading to the highest levels of apoptotic cell death based on the results of M30 ELISA, we observed the localization of cathepsin B to mitochondria (specifically, to Tom20 [41]), indicating cathepsin B translocation to mitochondria may be pro-apoptotic. This translocation can trigger caspase 3 cleavage and subsequent apoptotic cell death via upstream activation of caspase 9 by cytochrome C's release from permeable mitochondria. In our hands, inhibition of caspase 9 by the specific blocker significantly suppressed HIV-AGS-induced caspase 3 cleavage, and this effect was more profound than the contribution of caspase 8 inhibition to caspase 3 cleavage, suggesting that activation of the intrinsic apoptotic pathway plays a major role in HIV + AGS-induced hepatocyte death. This prompts us to suggest that further investigations are warranted to study the interdependence of lysosomal and mitochondrial leakages in the pathogenesis of HIV and ethanol-metabolism-induced hepatotoxicity. However, since this is a highly complex and important area, we did not plan to intensively investigate lysosome-mitochondrial interactions in the frame of this study, but we intend to detail this crosstalk in future experiments. While in other studies, the role of LL and mitochondrial dysfunction for apoptosis induction has already been demonstrated [42][43][44], it has never been implemented into the lysosome-mitochondrion axis driving HIV-ethanolmetabolism-induced hepatotoxicity.
There are multiple compensatory mechanisms that remove damaged lysosomes by autophagy/lysophagy [45][46][47][48][49][50][51]. Major repair mechanisms are usually related to lysosome biogenesis via TFEB [45]. Here, we found that TFEB translocation from cytosol to the nucleus is necessary to activate lysosome-biogenesis-regulating genes and that this was impaired in AGS-exposed, HIV-infected hepatic cells. Indeed, the attenuation of TFEB translocation by ethanol metabolism in hepatocytes was reported before [13,[52][53][54], but not in the context of HIV liver studies.
To address the mechanisms by which AGS (mainly acetaldehyde) inhibits TFEB nuclear translocation in hepatocytes in the settings of HIV, we tested the factors which regulate TFEB stabilization in the cytosol.
A variety of proteins and kinases control the translocation of TFEB to the nucleus to activate lysosomal biogenesis [15,16,55,56]. In this regard, we explored the role of phosphorylation as a post-translational modification that determines TFEB cytosolic accumulation in acetaldehyde-treated cells. Thus, phospho-TFEB dimerizes with TFEB to promote the retention of TFEB in the cytosol [57], which we observed in AGS-exposed, HIV-infected hepatocytes. mTOR phosphorylates TFEB at multiple serine residues (S122, S142, and S211). However, despite TFEB retention in cytosol, we even found the downregulation of acetaldehyde-induced TFEB phosphorylation at S211 [15]. The partial explanation for this event might be related to the overexpression of galectins in acetaldehyde-exposed liver cells due to their enhanced damage, leading to suppression of mTOR [58]. However, while we have no evidence that acetaldehyde increases TFEB phosphorylation, some other downstream acetaldehyde-affected events (e.g., aggregation) might account for TFEB cytosolic retention.
Given that TFEB is a substrate for the proteasome, we measured proteasome activities in HIV-infected and -uninfected RLW cells exposed or not to AGS and found a concomitant downregulation of chymotrypsin and trypsin-like proteasome activities. While there is no evidence of synergistic interactions between HIV and AGS, this is a novel finding because, to our knowledge, there are no studies that demonstrate the effects of acetaldehyde on TFEB in relation to proteasome activity in HIV-infected hepatocytes. The most likely scenario in ethanol-treated hepatic cells is the degradation of non-ubiquitylated TFEB by 20S proteasome because high oxidative stress causes the dissociation of 19S from 20S proteasome, thereby limiting its ability to recognize non-ubiquitinated substrates by the 26S proteasome [9]. In HIV-noninfected liver cells, we and others [7,[59][60][61][62] have previously reported that ethanol or acetaldehyde impair chymotrypsin-like and trypsin-like activities of 20S proteasome catalytic core. We cannot exclude that proteasome dysfunction under HIV-AGS cell exposure attenuates the degradation of total TFEB in the cytosolic compartment. This undegraded TFEB should translocate to the nucleus for gene activation if its trafficking is not blocked.
The trafficking of TFEB by microtubules has not been reported before. However, in hepatocytes, the translocation of some other transcription factors such as STAT3 and STAT5B from cytosol to the nucleus is suppressed by ethanol metabolism, which impairs their trafficking due to microtubule acetylation [63,64]. Here, we observed that nocodazole, an inhibitor of microtubule trafficking, suppressed TFEB translocation to the nucleus in RLW cells. These findings indicate that microtubules are necessary for TFEB translocation from cytosol to nucleus. While based on our findings, HIV by itself does not induce acetylation of microtubules, in infected and non-infected RLW cells, AGS exposure increases TFEB acetylation, which negatively affects TFEB trafficking to the nucleus, resulting in its cytosolic retention.
Overall, the alcohol-impaired lysosomal biogenesis in HIV-infected hepatic cells is multifactorial, including the reduced translocation of TFEB to the nucleus. The latter may be attributed to decreased degradation of non-ubiquitylated TFEB by 20S proteasome in cytosol combined with acetaldehyde-impaired TFEB trafficking to the nucleus. Additionally, exposure of RLW cells to AGS increases the expression of master repressor ZKSCAN3 that blocks TFEB target gene activation, thereby suppressing lysosomal genes. To our knowledge, this transcriptional repressor of lysosome genes has never been explored in the context of the combined treatment of hepatocytes with acetaldehyde and HIV.
It became clear from our results and discussion that some downstream events we addressed in this study were a result of synergistic/additive effects of HIV with acetaldehyde. However, there are a lot of effects that were related to the properties of acetaldehyde without synergizing with HIV; while not synergistic/additive, they are still very important in mimicking the role of ethanol metabolism in the pathogenesis of HIV-induced liver injury in alcohol-abusing PLWH. To sort out the events which are "specific" for HIV-infected hepatocytes only and/or are more general in ethanol-metabolite-exposed liver cells was one of the purposes of this study, and the results are summarized in Table 1. Importantly, the changes in cathepsin activities, LL-related parameters (DRAM1 expression and LAMP1-Galectin 3 co-localization) as well as caspase 3 cleavage and TFEB translocation to nucleus was higher in AGS-exposed HIV-infected than in non-infected liver cells and are the results of synergistic or additive effects from both exposures-HIV and ethanol metabolites. Table 1. Endpoints of the current study regulated by AGS only or by the combination of AGS/ethanol with HIV.

Parameters Effect Due To: Figures
Cathepsin B activity AGS + HIV Figure 1A Cathepsin L activity AGS + HIV Figure 1B LAMP1 immunostaining AGS Figure 1C,D LAMP1 Western blot AGS + HIV Figure 1E,F p53 mRNA AGS Figure 2A DRAM1 AGS + HIV Figure 2D,E LAMP1 + Galectin 3 immunostaining AGS + HIV Figure 2F,G Cathepsin B restoration by NAC AGS + HIV-higher magnitude Figure 3A Cathepsin L restoration by NAC AGS + HIV-higher magnitude Figure 3B M30 AGS + HIV Figure 3C Tom20 + Cathepsin B immunostaining AGS + HIV Figure 4A,B Cleaved caspase 3 AGS + HIV Figure 4C,D Nuclear TFEB/Cyto TFEB AGS + HIV Figure 5A,B PTFEB (S211) AGS Figure 5D,E Acetylated Tubulin AGS Figure 5H,I Chymotrypsin-like activity AGS Figure 6A Trypsin-like activity AGS Figure 6B ZKSCAN3 AGS Figure 6C,D LAMP1 mRNA AGS + HIV Figure 6E Cathepsin D AGS + HIV Figure 6E The proposed mechanism by which HIV and ethanol metabolism induce lysosome insufficiency contributing to hepatocyte cell death is summarized in Figure 7. (1) Combined treatment with HIV and AGS triggers ROS and acetaldehyde release, which mediates lysosome leakage; (2) HIV/ethanol metabolism triggers the release of DRAM1, which also induces lysosome leakage; (3) Cathepsin B leaked out from damaged lysosome and diffused into the mitochondrion to initiate the intrinsic apoptotic pathway; (4) Caspases 9 and 3 become cleaved, leading to hepatocyte apoptosis; (5) Alcohol metabolites inhibit lysosome biogenesis factor TFEB, hence impairing the compensation of damaged lysosomes; and (6) Both lysosome damage and impaired lysosome biogenesis lead to HIV-ethanol-metabolism-induced lysosome dysfunction, which triggers apoptosis.

Conclusions
Findings from this study revealed acetaldehyde-and HIV-induced lysosomal leakage and TFEB dysregulation as the mechanisms of oxidative-stress-induced lysosome dysfunction in liver cells, which leads to hepatotoxic events. Therapeutic prevention of HIV-alcohol-triggered hepatotoxicity may be based on the identification and detailed characterization of these possible targets.

Acknowledgments:
We acknowledge NIH NIAAA for providing us the funding in a grant (RO1 AA027189-01A1) and 1F31AA028743-01A1. We thank Madan Kumar Arumugam for providing software for analysis.

Conflicts of Interest:
The authors declare no conflict of interest.