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. Author manuscript; available in PMC: 2024 Jul 1.
Published in final edited form as: Alcohol Clin Exp Res (Hoboken). 2023 May 9;47(7):1297–1311. doi: 10.1111/acer.15093

Direct and indirect effects of alcohol and its toxic metabolite acetaldehyde on human esophageal myofibroblasts and epithelial cells

Atousa Khalatbari a, Joshua D Castle a, Tao Li a, Anisa Shaker a
PMCID: PMC10524176  NIHMSID: NIHMS1896565  PMID: 37128647

Abstract

Background:

Mechanisms by which alcohol increases the risk of esophageal squamous cell carcinoma remain undefined. Human esophageal myofibroblasts (HEMFs) subjacent to the squamous epithelium are directly exposed to these agents via epithelial barrier defects and indirectly via factors derived from exposed epithelium. Our aim was to investigate the cellular biology of HEMFs and HEMF-esophageal epithelial cell interactions in response to alcohol and its toxic metabolite acetaldehyde.

Methods:

An immortalized HEMF and a human esophageal epithelial cell line (Epi) were treated with alcohol (0–200mM) or acetaldehyde (0–100µM) in a cyclic fashion or incubated with supernatants collected from treated cells. Healthy cell %, reactive oxygen species (ROS), and proliferation were assessed via flow cytometry, luminescence, scratch wound and colorimetric assays respectively. 15-plex multiplex assay performed on cell supernatants was followed by IL-6 and IL-8 qRT-PCR and ELISA.

Results:

Healthy HEMF % decreased to less than 80% at 30mM alcohol and 70µM acetaldehyde, with microscopic changes at 40µM acetaldehyde. HEMF ROS was detected at 100mM alcohol and 80uM acetaldehyde. Supernatants from 30mM alcohol or 40uM acetaldehyde treated HEMFs increased Epi proliferation more thantwo-fold vs lower doses. In complementary studies, healthy Epi cell % decreased to less than 80% at 50mM and 70µM acetaldehyde, with microscopic changes at 40µM. Supernatants from Epi treated with 50mM alcohol or 40uM acetaldehyde increased HEMF proliferation more than two-fold fold vs lower doses. Multiplex assay of supernatants showed the greatest increase in concentrations of IL-6 and IL-8 in HEMFs and in Epi treated with higher doses of alcohol or acetaldehyde. Neutralization of IL-6 and IL-8 in supernatants of HEMFS and esophageal epithelial cells inhibited proliferation of Epi and HEMFs, respectively.

Conclusions:

Alcohol and acetaldehyde doses in which the majority of HEMFs and epithelial cells are healthy, elicit production of paracrine mediators with pro-proliferative effects on neighboring cells. Understanding the effect of alcohol and acetaldehyde on HEMFs and HEMF-epithelial interactions may help identify the molecular basis by which alcohol increases risk for esophageal cancer.

Keywords: human esophageal myofibroblasts, alcohol, acetaldehyde, paracrine, proliferation, esophageal epithelial cells

Introduction:

Chronic alcohol consumption remains a leading cause of morbidity and mortality, with a number of epidemiological studies showing an independent correlation between higher alcohol consumption and increased risk of esophageal squamous cell carcinoma (ESCC) (Bagnardi et al., 2015, Vanella et al., 2019). Alcohol is oxidized to acetaldehyde by oral cavity microorganisms, which then accumulates in the saliva. The esophageal mucosa, therefore, can be locally exposed not only to alcohol, but also to the damaging effects of its toxic metabolite (Liu et al., 2015, Salaspuro, 2020).

The molecular and cellular mechanisms of alcohol-induced esophageal injury and the impact of ingested alcohol and locally formed acetaldehyde remain incompletely described. A number of mechanisms including effects on epithelial penetration, proliferation, enhanced generation of oxidative stress and induction of chronic inflammation have been proposed to mediate the effects of alcohol (Matejcic et al., 2017, Liu et al., 2015, Salaspuro, 2020). An increase in intestinal barrier permeability is well described with chronic alcohol use (Wood et al., 2013). In addition, alcohol exposure lowers epithelial resistance in the rabbit esophagus thereby increasing risk of damage from acid (Bor et al., 1999). Similarly, alcohol increases the permeability of porcine non-keratinized oral mucosa (Du et al., 2000). Finally, alcohol acts as a solvent that allows for cellular penetration of noxious dietary and environmental agents (Poschl and Seitz, 2004).

Overall, alcohol-induced esophageal injuries likely reflect poorly understood, incompletely investigated complex interactions. The local effect of alcohol such as increasing epithelial permeability, raises the possibility of exposure of deeper layers of the esophageal mucosa to alcohol and locally generated acetaldehyde. Direct effects of alcohol and acetaldehyde on sub-epithelial esophageal stromal cells, an emerging cell of investigation in benign and pre-malignant states (Patankar et al., 2022) for example, have not been investigated. We have previously shown the presence of a sub-type of stromal cells immediately subjacent to the stratified squamous epithelium of the human esophagus, and based on their morphology and protein expression, termed them human esophageal myofibroblasts (HEMFs) (Gargus et al., 2015). HEMFs support squamous epithelial growth in part via paracrine mechanisms (Zhang et al., 2018). HEMFS along with other stromal cells are exposed to extrinsic (alcohol, acetaldehyde, bacteria, food antigens) and intrinsic (acid) luminal stimuli in the setting of an impaired epithelial barrier as well as to factors derived from exposed epithelium. To begin to define the molecular mechanisms of alcohol-induced injury in the human esophagus, our aim in this study was to define the direct and indirect cellular consequences of exposure to alcohol and acetaldehyde, focusing on HEMFS and on esophageal squamous epithelial cells.

2. Materials and methods

2.1. Cell culture and treatment

Previously described immortalized human esophageal myofibroblast cell line (HEMF) and an esophageal epithelial cell line, the esophageal hTERT-immortalized human epithelial cell EPC2 line (kind gift from Dr. Anil Rustgi) were used. HEMFs were grown in 6 well plates (1 × 105 cells/well) and when 75% confluent (1.2 × 106 cell/well), were treated with previously described serum-free HEMF growth media (Patankar et al., 2022) (pH 7.2) or serum-free HEMF growth media supplemented with alcohol (0–200 mM) or acetaldehyde (0–100 µM) for 8 minutes every 2 hours, a total of 4 times over the course of one day in this cyclic fashion. After each treatment, cells were washed twice with 1X PBS and incubated in serum-free myofibroblast media (pH 7.2). After the 4th treatment, cells were incubated overnight in keratinocyte serum-free media (KSFM) and harvested the next day for flow cytometry, RNA extraction, and/or supernatant collection for ELISA or treatment of epithelial cells as described below. Epithelial cells were similarly directly treated, in a background of keratinocyte serum-free media (KSFM) and harvested the next day for RNA extraction and/or supernatant collection for ELISA or treatment of HEMFs.

Microscopy (Revolve 4, ECHO,San Diego, CA) was used to screen for morphological features of cell death including cell dehydration and shrinkage and detachment from the plate surface. Cells were evaluated after each treatment cycle and the next day after overnight Incubation, at the same time points as flow cytometry, RNA extraction, and/or supernatant collection for ELISA.

To mimic the in vivo situation in which HEMFs and epithelial cells are exposed nearly simultaneously to luminal toxins, after the 4th treatment with alcohol or acetaldehyde, recipient HEMFs or esophageal epithelial cells were then incubated overnight either in control media or in conditioned media/supernatant collected from corresponding, donor epithelial cells or HEMFs, respectively. These donor epithelial cells or HEMFs were treated with either previously established lower, non-toxic doses of acetaldehyde or alcohol (epithelial cells: 30uM acetaldehyde, 45mM alcohol; HEMFs: 30uM acetaldehyde, 25mM alcohol) or higher, toxic doses (epithelial cells: 40uM acetaldehyde, 50mM alcohol; HEMFs: 40uM acetaldehyde, 30mM alcohol). Recipient HEMFs and esophageal epithelial cells were further analyzed with flow cytometry as described below.

2.2. Flow cytometry

HEMFs and esophageal epithelial cells treated with alcohol (0–200mM) and acetaldehyde (0–100uM) were analyzed by flow cytometry (BD FACSCalibur) after overnight incubation in KSFM or incubation with conditioned media from other cell type, with the markers Annexin V to detect cellular apoptosis and propidium iodide (PI) to detect necrotic or late apoptotic cells using the Annexin V/PI double staining kit (Invitrogen Catalog Number: V13242) according to manufacturer’s instructions. Fluorescently-tagged Annexin V is a probe for membrane phosphatidylserine (PS) and PI enters cells with damaged membrane. Suspended cells were stained and immediately analyzed by flow cytometry. 10,000 cells were analyzed per measurement. Data was analyzed using Cyflogic V.1.2.1 software and the % of viable cells (PI-/Annexin V-), early apoptotic cells (PI-, Annexin V+), late apoptotic or necrotic cells (PI+, Annexin V+), and necrotic/unviable/dead cells (PI+/Annexin V-) were quantified (Rua Ede et al., 2014, Brauchle et al., 2014).

2.3. reactive oxygen species (ROS) evaluation

HEMFs and esophageal epithelial cells were grown in 96 well plates (2000 cells/well) and treated with acetaldehyde (0–100uM) and alcohol (0–100mM) in cyclic fashion as described above and ROS was measured via luminescence (ROS-GLO H2O2 Assay, Promega Corporation, Madison WI) the next morning after overnight treatment in KSFM. LPS (10 µg/ml for 6 hours) and H2O2 (10 µM) were used as positive control comparators for ROS generation.

2.4. Scratch Assay

Scratch closure of an esophageal epithelial cell line in response to supernatants collected from alcohol and acetaldehyde treated HEMFs was determined in 12 well plates (1 × 105 epithelial cells/well). Once epithelial cells were fully covering the well, a scratch was made with a sterile 300 µl pipette tip (VWR 300uL Universal Pipet Tips, VWR, Radnor PA) across the center of the well which was then incubated in the following conditions: fresh KSFM and supernatant from untreated HEMFs, HEMFs treated with 25mM and 30mM alcohol and 30uM and 40uM acetaldehyde. The center of each well was imaged with phase contrast microscopy (Revolve R4, ECHO, San Diego CA) under a 4X objective to document initial scratch size, at 18 and 24 hours. Scratch size was quantified using QuPath (https://qupath.readthedocs.io/en/stable/docs/intro/citing.html for citation format). 25 equally spaced measurements were taken for each well and averaged. The average measurement for the initial size of the scratch was then subtracted from the average measurement after 18 and 24 hours to obtain the average distance closed. Scratch closure of HEMFs was similarly determined in the setting of treatment with supernatants from alcohol and acetaldehyde treated esophageal epithelial cells.

2.5. Proliferation Assays

Proliferation of esophageal epithelial cells with supernatants of treated HEMFs was determined in 2D culture with a colorimetric assay which measures cellular metabolic activity as a reflection of cell proliferation (MTT Cell Proliferation Assay Kit (Colorimetric), BioVision, AB211091–1000, Milipitas, CA). HEMFs were treated with alcohol and acetaldehyde in the cyclic method described above. Given the incompatibility between HEMF and epithelial cell culture media, after the last treatment with alcohol or acetaldehyde, HEMFs were placed overnight in KSFM media (Patankar et al., 2022). HEMF supernatant was collected the following day and used to treat epithelial cells in 96-well plates (2000 cells/well) for 24 hour, followed by the MTT assay. Proliferation was assessed using supernatants that were and were not filtered for dead cells with similar results. Proliferation of HEMFs after treatment with epithelial cell supernatant was similarly determined. Studies were performed in the presence and absence of the following neutralizing antibodies (R&D Systems) and concentrations: IL-6 (AB-206-NA, 3.125 ng/mL), and CXCL-8/IL-8 (AB-208-NA, 312.5 ng/mL), with Goat IgG (312.5 ng/mL) as a control. Pilot studies were conducted to determine the optimal concentrations; including a dose-response studies to demonstrate that lower concentrations of IL-6 or IL-8 did not lead to inhibition.

2.6. RNA isolation and qRT-PCR

Following treatment, RNA was isolated using the RNAeasy mini kit (QIAGEN) as previously described. Briefly, RNA was converted to cDNAs using Invitrogen™ SuperScript™ reverse transcription system according to the manufacturer’s protocol. Quantitative PCR (qPCR) was performed with the isolated cDNAs using SYBR Green Master Mix. Ten microliters of reaction medium contained 2 µl of diluted cDNAs and 8 µl of 300 nM gene-specific primers and SYBR Green Master Mix (Applied Biosystems). A control analysis was performed by adding each RNA sample or water instead of the cDNA sample to the reaction medium to avoid contamination by genomic DNA or to exclude the formation of a primer–dimer. The PCR program was as follows: one cycle at 95°C for 2 min, and 40 cycles of 15 s at 95°C and 1 min at 60°C. Data collection was performed at 60°C. The melting curve was produced according to the following program: 15 s at 95°C at a ramp rate of 1.6 °C s-1, 1 min at 60°C at a ramp rate of 1.6 °C s-1, heating to 95°C at a ramp rate of 0.05 °C s-1 and held in place for 15 s. The data were continuously collected. The data were analyzed by the 2−∆∆Ct method. Expression of each sample relative to GAPDH was shown as a multiple of 10. qPCR was conducted in triplicate using at least three independent cDNAs.

2.7. Multiplex Assay and ELISA

Supernatant of HEMFs and esophageal epithelial cells was collected from each treatment group. A 15 plex Human Luminex Discovery Assay (R&D Systems, Minneapolis, MN) for BMP-4, CCL2/JE/MCP-1, CCL5/RANTES, CXCL4/PF4, IFN-gamma, IL-1 α/IL-1F1, IL-1 β/IL-1F2, IL-2, IL-6, IL-8/CXCL8, IL-10, IL-12/IL-23 p40, IL-17/IL-17α, TNF-α, and VEGF was performed per the kit manufacturers protocol. The plate was read using a Bio-Plex 200 (Bio-Rad Laboratories, Hercules, CA) and results were analyzed on the instrument’s included software, with protein concentrations being determined against a 7 point standard curve. Individual ELISAs for IL-6 (Human IL-6 Duoset ELISA, R&D Systems, Minneapolis MN) and IL-8 (Human IL-8/CXCL8 Duoset ELISA, R&D Systems, Minneapolis MN) were also performed according to the manufacturer’s protocol. Samples were assayed in triplicate on a 96 well plate and assessed against a seven point standard curve produced by a two-fold serial dilution with a maximum protein concentration of 2000 pg/mL and 600 pg/mL for IL-8 and IL-6 respectively. Endpoint absorbance readings were taken at 450 nm per assay manufacturer specifications using a plate reader (Spectramax iD3, Molecular Devices LLC, San Jose CA).

2.8. Statistics

All experiments were performed in at least triplicate and data presented as means ± Standard Error or Standard deviation. Data were analyzed using Student’s two-tailed type 2 t-test with Excel or ANOVA with a Tukey’s or Dunnett’s post hoc test, as appropriate with GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). A value of P < 0.05 indicated statistical significance.

3. Results

We began by evaluating the effects of increasing doses alcohol and its metabolite acetaldehyde on the viability of an esophageal myofibroblast cell line (HEMF) and an esophageal epithelial cell line (Epi). Treatments were administered in cyclic fashion to mimic in vivo chronic alcohol exposure. Cells were assessed via microscopy for cell morphology and confluency and via flow cytometry for markers of apoptosis and necrosis to determine healthy cell percentages. We then evaluated the effects of alcohol and acetaldehyde on HEMF and esophageal epithelial cell ROS generation and the effect of factors produced from alcohol and acetaldehyde treated HEMFs on epithelial cells and vice versa. We also considered the effect of simultaneous exposure of both of these cell types to alcohol or acetaldehyde. Finally, we began to identify mechanisms mediating our observations by considering the secretory profile of HEMFs and esophageal epithelial cells treated with alcohol or acetaldehyde.

HEMF viability in response to direct treatment with alcohol and acetaldehyde

HEMFs remained adherent to the plate surface and appeared healthy with their typical spindle shaped morphology in response to alcohol doses up to 25mM (Figure 1A) and began to detach from the plate surface and demonstrate lower confluency beginning with treatment with 30mM alcohol (Figure 1B). HEMFs treated with up to 30µM acetaldehyde appeared healthy (Figure 1C) with visible changes such as detachment from plate surface starting at 40µM acetaldehyde (Figure 1D). Treatment of HEMFs with higher concentrations of alcohol or acetaldehyde resulted in obvious cell loss on microscopy.

Figure 1.

Figure 1.

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Microscopic appearance of HEMFs treated with various doses of alcohol and acetaldehyde. Representative microscopic images at 4x of untreated HEMFs and HEMFs treated with alcohol doses up to 25mM (A) and at 30mM (B). Representative microscopic images of untreated HEMFs treated with acetaldehyde doses up to 30µM (C) and at 40uM. Scale bar = 1000 µm

To further refine the doses of alcohol with this treatment regimen which led to loss of cell viability, flow cytometry was performed to identify the percentages of healthy, apoptotic and necrotic HEMF cell populations. Flow cytometry showed at 30mM alcohol, the % of healthy HEMFs decreased to 79% versus 86% of total cells at 25mM alcohol and that late apoptotic and necrotic cells accounted for 15% of the total population compared to 10% at 25mM alcohol (p=0.02). The remaining cells were in early apoptotic phase (6% versus 4%, at 30 and 25mM alcohol, respectively) (Figure 2A). Exposure to increasing concentrations of alcohol continued to decreased the percentage of healthy HEMFs such that by 200mM alcohol, more than 30% of HEMFs were necrotic (Figure 2B).

Figure 2.

Figure 2.

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Percentage of HEMFs that are healthy, early apoptotic, late apoptotic/necrotic, and necrotic in response to treatment with alcohol (0–200mM). (A) Representative flow cytometry data to detect the apoptotic or necrotic effect of alcohol treatment on HEMFs. After treatment with control media, 25mM or 30mM alcohol, non-fixed HEMFs were dual stained with FITC-conjugated annexin-V and PE-conjugated propidium iodide. Flow cytometry density plots show annexin V (X-axis) and PI (Y-axis) staining of HEMFs. The right lower quadrant represents early apoptosis (annexin V positive, PI negative), the right upper quadrant represents late apoptosis (both high annexin V and PI staining), the left upper quadrant indicates necrosis (low annexin V and high PI), and the left lower quadrant indicates viable cells. (B) Percentages of HEMFs that are healthy, early apoptotic, late apoptotic/necrotic, and necrotic in response to treatment with alcohol (0–200mM) per quantification of flow cytometry data.

HEMFs treated with acetaldehyde were also evaluated by flow cytometry. At 30µM of acetaldehyde, 91% of HEMFs are healthy (only 2.68% late apoptotic and 2.48% necrotic). At 40µM of acetaldehyde there was a slight decrease in the percentage of healthy HEMFs to 90% and a more profound increase in the percentage of late apoptotic and necrotic cells from 5% to 8% compared to treatment with 30µM (Figure 3A). The percentage of healthy HEMFs did not decrease below 80% (78%) until 70µM of acetaldehyde (Figure 3B).

Figure 3.

Figure 3.

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Percentage of HEMFs that are healthy, early apoptotic, late apoptotic/necrotic, and necrotic in response to treatment with acetaldehyde (0–100µM). (A) Representative flow cytometry data to detect the apoptotic or necrotic effect of acetaldehyde treatment on HEMFs. After treatment with control media, 30mM or 40mM acetaldehyde, non-fixed HEMFs were dual stained with FITC-conjugated annexin-V and PE-conjugated propidium iodide. Flow cytometry density plots show annexin V (X-axis) and PI (Y-axis) staining of HEMFs. The right lower quadrant represents early apoptosis (annexin V positive, PI negative), the right upper quadrant represents late apoptosis (both high annexin V and PI staining), the left upper quadrant indicates necrosis (low annexin V and high PI), and the left lower quadrant indicates viable cells. (B) Percentage of healthy, early apoptotic, late apoptotic/necrotic, and necrotic HEMFs treated with 30mM and 40mM acetaldehyde.

HEMF ROS production in response to direct treatment with alcohol and acetaldehyde

To begin to investigate the mechanism of alcohol and acetaldehyde mediated cell injury we evaluated generation of reactive oxygen species (ROS). An increase in ROS was only detected in HEMFs treated with the highest doses of alcohol and acetaldehyde, at which point there is a high % of late apoptotic and necrotic cells (29% and 20%, in alcohol and acetaldehyde treated HEMFs respectively). An increase in ROS was detected in HEMFs treated with 100mM alcohol (6362 RLU to 22674 RLU, p = 0.0007) (Figure 4A) and with 80µM acetaldehyde (16,544 RLU increased to 43787 RLU, p = 0.024) (Figure 4B). ROS in HEMFs was not detected at lower concentrations of alcohol or acetaldehyde. Increases in ROS were also detected in HEMFs treated with LPS and the positive control H2O2.

Figure 4.

Figure 4.

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ROS generation in HEMFs in response to treatment with alcohol and acetaldehyde. HEMFs were treated with (A) alcohol (0–100mM) and (B) acetaldehyde (0–100uM) and in cyclic fashion (treated for 8 minutes, every 2 hour, a total of 4 times, over the course of one day. ROS was measured via luminescence the next morning after overnight treatment in KSFM. LPS (10 µg/ml for 6 hours) and H2O2 (10µM) were used as a positive control comparators for ROS generation. * = p< 0.05 vs 0mM alcohol or 0µM acetaldehyde.

Effect of supernatant from alcohol and acetaldehyde treated HEMFs on esophageal epithelial cells

The significance of changes in HEMF viability and the % of cells that were in late apoptotic or necrotic phases in response to treatment with 30mM alcohol and 40µM acetaldehyde remained unclear. Given the paracrine potential of HEMFs (Zhang et al., 2018, Hu et al., 2020) we were curious whether HEMFs were producing factors with paracrine potential in response to doses of alcohol or acetaldehyde in which HEMFs appeared healthy microscopically and in which the majority of cells (> 80%) were healthy by flow cytometry or at slightly higher doses of these agents. We therefore assessed the effect of supernatant collected from HEMFs treated with these lower and higher concentrations of alcohol and acetaldehyde, on the epithelium using scratch and MTT assays.

Utilizing the scratch assay, we observed that in response to incubation with supernatant collected from HEMFs treated with 25mM alcohol, the percentage of the esophageal epithelium scratch closure was 45% at 18 hr and 59% at 24 hr, similar to scratch closure of epithelium incubated with control media (43% and 54% at 18 and 24 hr, respectively) (p=NS). Incubation with supernatants collected from HEMFs treated with 30mM of alcohol, however, resulted in complete or 100% closure of the scratch by 18 hours (p=0.0001) (Figure 5). Scratch closure of the epithelium with supernatant collected from HEMFs treated with 30uM of acetaldehyde was 61% and 65% at 18 and 24 hr, respectively, similar to control conditions, while supernatant from HEMFs treated with 40µM of acetaldehyde resulted in complete closure of the scratch by 18 hours (p=0.0003) (Figure 6).

Figure 5.

Figure 5.

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Esophageal epithelial cell scratch closure with supernatants from HEMFs treated with alcohol. A. Quantification of scratch closure at 0, 18 and 24 hr after incubation with control epithelial (Epi) cell media, supernatant from untreated HEMFs, and with supernatant collected from HEMFs treated with 25mM and 30mM alcohol. B. Representative images of scratch closure at 0 and 24 hours after incubation with (B1) control epithelial cell media, (B2) supernatant from untreated HEMFs, and with supernatant collected from HEMFs treated with (B3) 25mM and (B4) 30mM alcohol. * = p<0.05 vs control epithelial cell media, untreated HEMF supernatant, and supernatant from HEMFs treated with 25mM alcohol, at similar time points.

Figure 6.

Figure 6.

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Esophageal epithelial cell scratch closure with supernatants from HEMFs treated with acetaldehyde. A. Quantification of scratch closure at 0, 18 and 24 hr after incubation with control epithelial cell media, supernatant from untreated HEMFs, and with supernatant collected from HEMFs treated with 30uM and 40uM acetaldehyde. B. Representative images of scratch closure at 0 h and 24 hours after incubation with (B1) control epithelial cell media, (B2) supernatant from untreated HEMFs, and with supernatant collected from HEMFs treated with (B3) 30uM and (B4) 40uM acetaldehyde. * = p<0.05 vs control epithelial cell media, untreated HEMF supernatant, and supernatant from HEMFs treated with 30µM acetaldehyde, at similar time points.

We then wanted to determine whether the increased rate of scratch closure in esophageal epithelial cells treated with supernatant from HEMFs treated with the higher doses of alcohol and acetaldehyde was due to an increase in epithelial cell proliferation. Conditioned media from HEMFs treated 25mM alcohol did not lead to an increase in epithelial cell proliferation in 2D culture. On the other hand, conditioned media from HEMFs treated with 30mM alcohol led to a greater than two fold increase in esophageal epithelial proliferation compared to control media (Figure 7). Supernatant collected from HEMFs treated with 30µM acetaldehyde did not increase epithelial cell proliferation in 2D culture, while supernatant from HEMFs treated with 40µM of acetaldehyde increased esophageal epithelial cell proliferation greater than 2-fold. These results are consistent with the complete scratch closure of the epithelium observed with supernatant from HEMFs treated with these higher doses of alcohol and acetaldehyde.

Figure 7.

Figure 7.

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Esophageal epithelial cell proliferation in response to supernatants from HEMFs treated with alcohol and acetaldehyde. Esophageal epithelial cells were incubated with control epithelial cell media, supernatant from untreated HEMFs, and with supernatant collected from HEMFs treated with 25mM and 30mM alcohol and with supernatant collected from HEMFs treated with 30uM and 40uM acetaldehyde. Proliferation was assessed with the MTT assay. * = p<0.05, supernatant from 30mM vs 25mM alcohol treated HEMFs and supernatant from 40µM vs 30µM acetaldehyde treated HEMFs.

Direct effect of alcohol and acetaldehyde on esophageal epithelial cell viability and the effect of supernatant from treated esophageal epithelial cells on HEMFs

Given the sub-epithelial location of HEMFs we were curious whether HEMFs would be affected by esophageal epithelial cells exposed to alcohol or acetaldehyde. Flow cytometry was performed on squamous epithelial cells treated with alcohol and acetaldehyde to identify the percentage of healthy, apoptotic and necrotic esophageal epithelial cells. The percentage of healthy esophageal epithelial cells decreased below 80% in response to 50 mM alcohol (Figure 8A). Increasing the concentration of alcohol beyond 50mM continued to increase epithelial cell necrosis, with 45% epithelial cells healthy and 26% of epithelial cells necrotic at 200 mM alcohol.

Figure 8.

Figure 8.

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Percentage of esophageal epithelial cells that are healthy, early apoptotic, late apoptotic/necrotic, and necrotic in response to treatment with (A) alcohol (0–200mM) or (B) acetaldehyde (0–100µM). After treatment esophageal epithelial cells were dual stained with FITC-conjugated annexin-V and PE-conjugated propidium iodide. Percentages shown are per quantification of flow cytometry data.

Similar to HEMFs, there was a slight decrease in the percentage of healthy epithelial cells from 91% to 90% and an increase in the percentage of late apoptotic and necrotic cells from 5% to 8% in response to 40µM acetaldehyde compared to treatment with 30µM (Figure 8B). The percentage of healthy epithelial cells did not decrease below 80% until treatment with 70µM of acetaldehyde. An increase in ROS, however, was not detected in esophageal epithelial cells between 0–100mM of alcohol (Supplemental Figure 1A) or between 0–100uM acetaldehyde (Supplemental Figure 1B).

We next evaluated the effect of supernatant collected from human esophageal epithelial cells on HEMF scratch closure. HEMFs treated with supernatant collected from esophageal epithelial cells treated with 50mM alcohol (Supplemental Figure 2) or 40uM acetaldehyde (Supplemental Figure 3) closed the scratch within 18 hours (p<0.001) whereas scratch closure of HEMFs treated with supernatant from epithelial cells treated with lower doses of alcohol or acetaldehyde was similar to control conditions. To delineate whether the HEMF scratch closure was due to an increase in proliferation or migration, we evaluated proliferation of HEMFs treated with supernatant collected from alcohol and acetaldehyde treated human esophageal epithelial cells. Supernatant from epithelial cells treated with lower alcohol or acetaldehyde doses did not affect HEMF proliferation, while supernatant collected from epithelial cells treated with 50mM alcohol or 40uM acetaldehyde increased HEMF proliferation 2.4-fold (Supplemental Figure 4).

Simultaneous treatment of HEMFs and esophageal epithelial cells

Given that in vivo, these two cell populations are likely exposed to alcohol or acetaldehyde nearly simultaneously, we were interested in whether secretory mediators had a role in interactions between HEMFs and epithelial cells in such a setting. We attempted to mimic this real-life situation by culturing alcohol or acetaldehyde treated HEMFs with supernatant from similarly treated human esophageal epithelial cells and vice versa, culturing treated human esophageal epithelial cells with supernatant from similarly treated HEMFs.

The viability of human esophageal epithelial cells, assessed by microscopy and flow cytometry, treated with low or high doses of alcohol (Supplemental Figure 5) or acetaldehyde (Supplemental Figure 6) was unaltered in the presence of supernatant collected from HEMFs treated with alcohol or acetaldehyde. Epithelial cells treated with low doses of alcohol or acetaldehyde remained unhealthy, while epithelial cells treated with higher doses of alcohol or acetaldehyde displayed a decrease in viability. Similarly, the viability of HEMFs treated with low or high doses of alcohol were largely unaltered in the presence of supernatant collected from esophageal epithelial cells treated with alcohol (Supplemental Figure 7). HEMFs treated with low doses of alcohol remained healthy, and HEMFs treated with high doses of alcohol showed a decrease in viability. Similarly, HEMFs treated with low doses of acetaldehyde remained healthy, regardless of co-culture with supernatant derived from epithelium treated with low or high doses of acetaldehyde. However, the viability of HEMFs treated with higher doses of acetaldehyde improved in the presence of supernatant collected from esophageal epithelial cells treated with higher doses of acetaldehyde with an increase in the percentage of healthy HEMFs from 88% to 98% (Figure 9).

Figure 9.

Figure 9.

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HEMFs treated with acetaldehyde (ACT) plus supernatant (sup) from acetaldehyde treated human esophageal epithelial cells (Epi). A. Microscopic appearance of HEMFs treated with acetaldehyde plus sup from acetaldehyde treated human esophageal epithelial cells (Epi). The top panel shows HEMFs treated directly with low (30µM) dose of acetaldehyde in the absence of Epi sup (A1) and in the presence of sup collected from Epi treated with low (30µM, A2) or high (40µM, A3) doses of acetaldehyde. The lower panel shows HEMFs treated high (40µM) dose of alcohol in the absence of Epi sup (B1) and in the presence of sup collected from Epi treated with low (30µM, B2) or high (40µM, B3) doses of acetaldehyde. Scale bar = 1000 µm. B. Viability of HEMFs after treatment with low (30µM) or high (40µM) dose of acetaldehyde and sup from Epi treated with low (30µM) or high (40µM) doses of acetaldehyde. HEMFs were dual stained with FITC-conjugated annexin-V and PE-conjugated propidium iodide. Percentages of HEMF that are healthy, early apoptotic, late apoptotic/necrotic, and necrotic in response to each condition per quantification of flow cytometry data. * = p< 0.05 High ACT HEMF + High ACT Epi sup vs High ACT HEMF and vs High ACT HEMF + Low ACT Epi Sup.

Multiplex assay of supernatant from alcohol or acetaldehyde treated HEMFs and esophageal epithelial cells

The rescue of unhealthy acetaldehyde treated HEMFs limited to supernatant derived from epithelial cells treated with higher doses of acetaldehyde suggested a differential secretory pattern that was cell (HEMF or Epi), agent (alcohol or acetaldehyde), or dose (low or high) dependent. To begin to identify mechanisms underlying these observations we evaluated supernatant from HEMFs and esophageal epithelial cells treated with lower and higher doses of alcohol and acetaldehyde with a 15-plex multiplex panel of cytokine/chemokines and growth factors. There were no changes in IL-12, IL-10, IL-17A, IFN-γ, or CCL2 in supernatant of HEMFs or esophageal epithelial cells in response to treatment with alcohol or acetaldehyde at either dose. Detection of TNFα, VEGF, IL-1β, and CCL5 in the supernatant peaked at lower concentrations of alcohol or acetaldehyde and then decreased with the higher concentrations in both HEMFs and esophageal epithelial cells. Finally, detection of IL-8, IL-2, and IL-1α increased in a dose responsive manner, while IL-6 was predominantly detected in response to the higher doses of alcohol or acetaldehyde in both cell types. While this limited evaluation did not reveal a unique secretory profile based on cell type or agent, it did demonstrate that of the analytes tested, IL-6 and IL-8 had the greatest concentrations in response to higher doses of alcohol or acetaldehyde (Table).

Table.

Multiplex results of analytes tested in supernatant of HEMFs and esophageal epithelial cells treated with alcohol or acetaldehyde

Sample
HEMFs Epithelium
ctrl low ACT high ACT low ETOH high ETOH ctrl low ACT high ACT low ETOH high ETOH
Analyte (pg/ml)
CXCL4 6.0 8.0 14.0*^ 6.0 14.0 10.0 6.0 14.0 14.0 8.03
IL-8 0.1 104.3* 377.4*^ 100.8* 373.3*^ 0.0 100.1* 353.1*^ 93.3* 356.7*^
IL-6 0.0 1.2 501.3*^ 1.1 484.2*^ 0.2 1.2 438.6*^ 0.95 452.4*^
IL-12# 22.5 26.2 29.8 0.0 11.31 0.0 0.0 3.8 7.53 37.2
TNFα 0.0 24.3* 3.1*^ 23.0* 2.7*^ 0.0 21.0* 2.7*^ 20.3* 2.62*^
IL-2 0.4 3.5* 8.4*^ 3.5* 8.8*^ 0.4 3.2* 8.2*^ 2.6* 8.2*^
IL-10 0.0 0.1 0.1 0.1 0.1 0.0 0.0 0.1 0.0 0.0
IL-17α 0.0 0.3 0.3 0.3 0.3 0.0 0.0 0.3 0.0 0.3
VEGF 0.9 425.7* 26.6*^ 408.1* 25.5*^ 1.1 402.6* 24.1*^ 388.3* 24.3*^
BMP4 0.00 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.3 0.0
IFN γ 1.0 2.3 1.9 2.8* 1.6^ 0.0 1.8* 1.6* 2.3* 1.6
IL-1β 0.6 22.2* 11.9*^ 19.9* 10.9*^ 0.0 19.6* 10.5*^ 19.0* 10.5*^
IL-1α 0.00 62.6* 127.1*^ 60.3* 126.0*^ 0.0 59.3* 119.7*^ 54.9* 123.0*^
CCL5 0.8 5.2* 1.7 4.8* 1.6 0.8 4.4* 1.7 2.9* 1.7
CCL2 0.00 0.00 0.2 0.3 0.2 0.0 0.4 0.0 0.009 0.00
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Abbreviations: HEMFs: human esophageal myofibroblasts; Epithelium: human esophageal epithelial cells; Ctrl: control; low ACT (Acetaldehyde 30µM); high ACT (Acetaldehyde 40µM); low ETOH (Alcohol 25mM for HEMFs and 45mM for epithelium); high ETOH (Alcohol 30mM for HEMFs and 50mM for epithelium).

#

refers to IL12/IL23 p40;

*

p<0.05 vs control;

^

vs lower concentration of alcohol or acetaldehyde.

We confirmed these findings by performing qRT-PCR followed by ELISA for IL-6 and IL-8 in HEMFs and esophageal epithelial cells. In HEMFs, there was a greater than 2-fold increase in IL-6 and IL-8 mRNA expression in response to treatment with 25mM and 30mM alcohol compared to untreated HEMFs. IL-6 (0 pg/ml to 166 pg/ml, p< 0.05) protein was only detected in the supernatant in response to the higher dose of alcohol at 30mM, while there was a dose dependent increase in IL-8 protein (0 pg/ml to 72 pg/ml to 283 pg/ml, p< 0.05) (Figure 10A). Similarly, in response to acetaldehyde, there was an increase in IL-6 and IL-8 mRNA in HEMFs treated with 30µM and 40µM. IL-6 (0 pg/ml to 160 pg/ml, p< 0.05) protein was detected in the supernatant only in response to the higher dose of acetaldehyde, and there was a dose dependent increase in IL-8 (0 pg/ml to 102 pg/ml to 294 pg/ml, p< 0.05) (Figure 10A).

Figure 10.

Figure 10.

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mRNA expression and supernatant protein detection of IL-6 and IL-8 in HEMFs (A) and esophageal epithelial cells (B) treated with alcohol or acetaldehyde. mRNA expression is shown relative to GAPDH expression. Error bars indicated standard deviation, *, p < 0.05; **, p < 0.01; ***, p < 0.001

Human esophageal epithelial cells treated with 50mM alcohol had an increase in IL-8 mRNA expression (1.5 fold, p<0.05) and an increase in protein detected in the supernatant in a dose dependent manner (0 pg/ml to 101 pg/ml to 347 pg/ml, p<0.05). Although IL-6 mRNA expression remained unchanged, IL-6 protein was detected in the supernatant in response to the higher dose of alcohol (0 pg/ml to 196 pg/ml, p < 0.05) (Figure 10B). Treatment of human esophageal epithelial cells with acetaldehyde did not change IL-6 mRNA expression (p=NS) although IL-6 protein was detected in response to the higher dose of acetaldehdye (0 pg/ml to 187 pg/ml, p<0.05). IL-8 mRNA expression in human esophageal epithelial cells increased in response to treatment with 40µM acetaldehyde (2.2 fold, p< 0.001) and IL-8 (0 pg/ml to 129 pg/ml, p<0.05) protein production increased in a dose dependent manner (0 pg/ml to 93 pg/ml to 428 pg/ml) (Figure 10B).

HEMF and esophageal epithelial cell proliferation assays with neutralizing antibodies

Since IL-6 and IL-8 were the most detected factors in the multiplex assay, to determine whether they were responsible for the increase in proliferation, we inhibited them with blocking antibodies. We observed that inhibition of IL-8 and/or IL-6 in supernatant of HEMFs treated with higher doses of alcohol or acetaldehyde inhibited the increase in epithelial cell proliferation (Figure 11A). Similarly inhibition of IL-8 or IL-6 in supernatant of esophageal epithelial cells treated with higher doses of alcohol or acetaldehyde inhibited the increase in HEMF proliferation (Figure 11B). Dose-response curves confirmed that lower concentrations of neutralizing antibodies did not lead to inhibition.

Figure 11.

Figure 11.

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Inhibition of IL-6 and IL-8 in the supernatant inhibits proliferation of human esophageal epithelial cell and HEMFs. A. Human esophageal epithelial cell (Epi) proliferation was assessed with MTT assay under the following conditions: control epithelial cell media, supernatant (sup) from untreated HEMFs, or sup from HEMFs treated with 30mM alcohol or 40uM acetaldehyde, in the absence and presence control IgG antibody (312.5 ng/mL) or IL-6 (312.5 ng/mL) or IL-8 (312.5 ng/mL) neutralizing antibodies, error bars indicated standard deviation *, p < 0.05; treated HEMF sup + Anti-IL-6 or IL-8 versus treated HEMF sup IgG. B. HEMF proliferation was assessed with MTT assay under the following conditions: control media, sup from untreated Epi, sup from Epi treated with 50mM alcohol or 40uM acetaldehyde, in the absence or presence of control IgG antibody (312.5 ng/mL), IL-6 (312.5 ng/mL) or IL-8 (312.5 ng/mL) neutralizing antibodies, error bars indicated standard deviation*, p < 0.05; treated HEMF sup + Anti-IL-6 or IL-8 versus treated HEMF sup IgG.

4. Discussion

In this study, we show that alcohol concentrations ≥ 30mM increase HEMF toxicity as demonstrated by plate detachment on microscopy and the % of healthy cells dropping below 80% by flow cytometric evaluation. Microscopically, HEMFs and esophageal epithelial cells both begin to appear unhealthy at 40µM of acetaldehyde, and although by flow cytometric evaluation, 90% of cells are “healthy” at this concentration, there is a significant increase in the % of necrotic and late apoptotic cells. Interestingly, HEMFs do not generate ROS at these lower doses, and do not do so until they are exposed to much higher doses of alcohol and acetaldehyde (100mM and 80µM, respectively).

Esophageal epithelial cells treated with supernatants collected from HEMFs treated with 30mM alcohol and 40µM acetaldehyde close a scratch wound earlier and demonstrate a more than 2-fold increase in proliferation than epithelial cells exposed to supernatants from untreated HEMFs or HEMFs treated with lower doses of alcohol and acetaldehyde. Complementary observations were made with HEMFs treated with supernatants collected from esophageal epithelial cells treated with higher doses of alcohol or acetaldehyde. HEMF scratch wound was closed earlier and HEMF proliferation increased in response supernatant of human esophageal epithelial cells treated with higher doses of alcohol or acetaldehyde. These phenotypes suggest paracrine interactions between HEMFs and esophageal epithelial cells in the setting of exposure to alcohol or acetaldehyde.

Multiplex testing shows that supernatant of HEMFs and esophageal epithelial cells treated with doses of alcohol and acetaldehyde in which the majority of cells remain healthy contains several cytokines, chemokines, or growth factors. Because we observed an increase in HEMF or esophageal epithelial cell proliferation in response to supernatant from cells treated with higher doses of alcohol or acetaldehyde, we focused on the mediators with the highest concentration detected in response to the higher doses of alcohol or acetaldehyde. Of the analytes tested, pro-inflammatory, pro-proliferative mediators, IL-6 and IL-8 had the greatest concentrations in supernatants of HEMFs or esophageal epithelial cells treated with higher doses of alcohol or acetaldehyde. Concurrent increases in HEMF IL-6 and IL-8 mRNA expression suggest these factors are being secreted from HEMFs. In esophageal epithelial cells, however, while there is an increase in IL-8 mRNA, IL-6 mRNA remains unchanged, regardless of the agent used; suggesting perhaps spillage from necrotic cells. Neutralization studies indicate that IL-6 and IL-8 are factors in the supernatant of alcohol or acetaldehyde treated HEMFs and in alcohol or acetaldehyde treated esophageal epithelial cells that are at least partially responsible for changes in the proliferation of the other cell type. Collectively, these findings show that in response to a doses of alcohol in which viability of HEMFs and esophageal epithelial cells is reduced but in which nearly 80% remain healthy, and a dose of acetaldehyde in which > 90% of HEMFs and esophageal epithelial cells are healthy, HEMFs and human esophageal epithelial cells produce pro-inflammatory mediators with proliferative effects on each other.

We have demonstrated that alcohol and acetaldehyde have direct effects on HEMFs and esophageal epithelial cells. There are significant decreases in HEMF viability immediately starting at 30mM alcohol and in esophageal epithelial cell viability beyond 45mM alcohol. Esophageal epithelial cells appeared more resistant to alcohol than HEMFs, with a higher percentage of healthy esophageal epithelial cells versus HEMFs across most alcohol concentrations. Acetaldehyde affected HEMFs and esophageal epithelial cells similarly in terms of toxicity; while there were microscopic changes in both cell types starting at 40µM the percentage of healthy HEMFs and esophageal epithelial cells did not decrease below 80% until exposure to 70µM acetaldehyde.

Early and late apoptotic, and primary necrotic cells are on the spectrum of dying cells and were distinguished from viable, healthy cells by flow cytometry in our study. Reactive oxygen species signaling and control is a pathway common to these cell death pathways (Villalpando-Rodriguez and Gibson, 2021). HEMFs did not generate an increase in ROS at lower doses of alcohol and acetaldehyde with an increase in ROS not detected until exposure to 100mM alcohol and 80µM acetaldehyde. Esophageal epithelial cells did not generate an increase in ROS at studied doses of alcohol up to 100mM or acetaldehyde up to 100µM, at which point the percentage of remaining healthy cells is only 60%. The ROS production that was detected in both HEMFs and epithelial cells treated with lower doses of alcohol and acetaldehyde was not in excess of control conditions and likely tightly controlled as part of homeostatic state (Villalpando-Rodriguez and Gibson, 2021). Alcohol (Kany et al., 2019) and acetaldehyde (Tamura et al., 2014) modulate ROS production in a number of cell types including gastric epithelial cells. Although ROS via induction of CYP2E1 has been implicated in the development of esophageal malignancy (Linhart et al., 2014), and CYP2E1 has been described in the esophageal mucosa of alcohol drinkers (Millonig et al., 2011) generation of ROS in human esophageal myofibroblasts has not been previously investigated. Collectively, these findings suggest that the studied doses of alcohol or acetaldehyde are not driving ROS generation in HEMFs or epithelial cells at the time of evaluation; and that increase in the percentage of apoptotic and necrotic cells observed after cyclic treatment in response to doses of alcohol between 30mM and 90mM in HEMFs or by doses of 50mM and beyond in esophageal epithelial cells does not appear to be driven by significant ROS production. It remains possible that ROS is a driving factor at earlier time points.

Alcohol has previously been shown to modulate cytokine production (Kany et al., 2019), depending on the cell type and the length of exposure, with reports that acute and chronic alcohol exposure may have opposite effects (Mandrekar et al., 2009). For example, acute versus chronic alcohol use have divergent effects on TLR4 signaling. Mechanisms of the release of these signaling molecules from HEMFs treated with alcohol and acetaldehyde remains to be defined. The remaining healthy population of HEMFs could also be responsible for production and secretion of these factors, through as yet undefined signaling mechanisms in response to alcohol or acetaldehyde.

Detection of these mediators e.g. could also be due to cytokine release in the setting of regulated cell death (Place and Kanneganti, 2019), although the majority of cells treated with alcohol and acetaldehyde concentrations that elicited release of these factors were healthy. Apoptosis, pyroptosis and necroptosis are programmed or regulated forms of cell death, whereas primary necrosis is an unregulated process (Negroni et al., 2015). Pyroptosis and necroptosis in particular are additionally distinguished as lytic forms of cell death leading to cytokine release (Place and Kanneganti, 2019). Pyroptosis has previously been reported in a commercial esophageal epithelial cell line exposed to alcohol (Wang et al., 2018) and apoptotic cell-derived immunostimulatory signals have also been described to be released from early apoptotic cells (Poon et al., 2010). We have yet to explore whether alcohol and acetaldehyde are similar cell death-inducing stimuli in terms of generation of immunomodulatory signals from HEMFs or from esophageal epithelial cells used in this study.

Early apoptotic cell death is typically associated with a tolerogenic, anti-inflammatory response, while late apoptotic and necrotic cells release danger associated molecular patterns or DAMPs that signal through TLR4, triggering inflammation (Poon et al., 2010, Henry et al., 2013). We have previously shown that HEMFs express TLR4 (Gargus et al., 2015) providing an avenue of autocrine stimulation in this environment. Alternatively, plasma membrane-damaged cells where membrane barrier integrity has been compromised, which include late apoptotic and necrotic cells (Poon et al., 2010) could release these mediators with an effect on remaining healthy population of HEMFs, thereby indirectly driving an inflammatory secretory process.

Given the relatively low % of necrotic HEMFs and epithelial cells at tested concentrations of alcohol and acetaldehyde, supernatant generated from our treatment regimen cannot be characterized as necrotic, as has been described by others (Eto et al., 2021). Identification and characterization of the repertoire of inflammatory and immunosuppressive molecule(s) from dying cells in response to alcohol and acetaldehyde need further clarification. For example, Eto et al have shown release of PGE2 from dying and dead cells (Eto et al., 2021). Significance of the unique cell populations present in HEMFs treated with different doses of alcohol and acetaldehyde remains to be seen.

Direct exposure to alcohol has previously been shown to mediate an increase in epithelial cell proliferation and a decrease in differentiation (Liu et al., 2015, Shi et al., 2021). Chronic alcohol administration in rats results in an increase in esophageal epithelial proliferation that disappears with removal of the salivary glands, demonstrating both the importance of salivary glands for growth factor production and salivary gland associated acetaldehyde (Simanowski et al., 1993). On the other hand, an increase epithelial proliferation was not seen in a histological study of esophageal biopsies in chronic alcohol use, possibly due to the effect of chronic alcohol use on salivary gland function (Millonig et al., 2011). In a squamous cell carcinoma of the head and neck cell line, low concentrations of alcohol increased proliferation and decreased differentiation (Kornfehl et al., 1999). Acetaldehyde has also caused hyperplastic and hyperproliferative changes in the upper GI tract in a rat model (Homann et al., 1997).

We have also looked at the indirect effect of alcohol and acetaldehyde exposure, focusing on the role of mediators released by directly exposed HEMFs and esophageal epithelial cells on neighboring esophageal epithelial cells and HEMFs, respectively. The production of paracrine signaling mediators in response to these extrinsic factors demonstrates a potential mechanism underlying the proliferative abnormalities observed along the spectrum of esophageal squamous cell cancer (Taylor et al., 2013, Yu et al., 2003). Factors produced by HEMFs, including IL-6 and IL-8, in response to the lowest toxic doses of alcohol and acetaldehyde, increase basal epithelial cell proliferation modeled in the MTT assay. Similarly, esophageal epithelial cells treated with alcohol and acetaldehyde at doses in which the majority of cells are healthy, increase HEMF proliferation. IL-6 has both pro-and anti-inflammatory properties and has been implicated in gut barrier repair (Grivennikov et al., 2009). Along with its role as a chemoattractant for granulocytes, IL-8 also has proliferative effects on esophageal epithelium (Patankar et al., 2022). We had previously shown that secreted factors from HEMFs increased esophageal epithelial cell proliferation in 2D (Patankar et al., 2022) and 3D culture (Hu et al., 2020). Because collected supernatant was filtered to remove floating cells in our study, the observed effect is mediated by released mediators rather than cells themselves. Neutralizing antibodies against IL-6 and IL-8 in the supernatant of alcohol or acetaldehyde treated HEMF and esophageal epithelial cell inhibited the proliferative effect of this supernatant on esophageal epithelial cells and HEMFs, respectively. Collectively, these results indicate that IL-6 and IL-8 are factors in the supernatant of alcohol or acetaldehyde treated HEMFs and in alcohol or acetaldehyde treated esophageal epithelial cells that are at least partially responsible for changes in the proliferation of the other cell type.

To mimic the in vivo situation wherein HEMFs and esophageal epithelial cells are concurrently exposed to luminal agents, we directedly treated HEMFs and esophageal epithelial cells with alcohol or acetaldehyde and supplemented the treatment with supernatant from the complementary cell type. Interestingly, we observed that HEMFs treated with higher doses of acetaldehyde were rescued from any toxicity when treated concurrently with supernatant collected from similarly treated esophageal epithelial cells. HEMFs treated with higher doses of alcohol were not rescued by supernatant from similarly treated esophageal epithelial cells. Nor was rescue achieved in esophageal epithelial cells treated with high doses of alcohol or acetaldehyde and supplemented with supernatant from similarly treated HEMFs. These findings possibly suggested a secretory profile unique to human esophageal epithelial cells treated with high doses of acetaldehyde that can overcome the toxicity of higher doses of acetaldehyde in HEMFs. The limited multiplex analysis in this study, however, did not identify unique differences in the secretory profile of HEMFs versus epithelial cells treated with alcohol or acetaldehyde.

Collectively, these findings highlight the indirect consequences of alcohol and acetaldehyde exposure and suggest that exposure of the esophageal mucosa to alcohol and acetaldehyde has the potential to modulate HEMF-epithelial interactions via paracrine mechanisms. It remains to be seen which populations of HEMFs treated with alcohol or acetaldehyde (healthy or unhealthy) are responsible for elaboration of paracrine mediators in the supernatant that increase epithelial proliferation.

Limitations of our study include the use of immortalized cell lines for HEMFs and epithelial cells, rather than primary samples. In addition, we studied the effect of varying doses of alcohol or acetaldehyde via cyclic treatment at defined times and have not fully investigated the effect of treatment duration, although our pilot studies demonstrated similar findings at the 4 hour time point. We evaluated the effects of supernatant collected from HEMFs and esophageal epithelial cells treated with the lowest concentrations of alcohol and acetaldehyde that elicited microscopic changes. Although we suspect similar patterns with supernatant collected from HEMFs and esophageal epithelial cells treated with higher concentrations of alcohol or acetaldehyde, it remains to be seen if transcriptional effects, released mediators, and effects on neighboring cells types will persist at higher, more obviously toxic doses. Similarly, the readout or output we evaluated (cell death versus % healthy cells) that we were using may be missing effects of these toxic agents at lower doses.

Because proliferation of esophageal epithelial cells was also increased at these time points, the scratch assay performed in this study does not address the role of migration. Future studies that suppress proliferation e.g. by addition of mitomycin C (Grada et al., 2017) are necessary. Earlier time points should also be investigated. Regardless, these results are consistent with the profound increase in epithelial cell proliferation in response to HEMF secreted factors in the setting of treatment with alcohol and acetaldehyde. Finally, the secretome of alcohol or acetaldehyde treated HEMFs and esophageal epithelial cells remains largely unexplored; and although we show the results of the multiplex testing, this is a limited analysis. Mechanisms mediating our observations should be interrogated in the future, with more extensive testing.

Given the ongoing misuse of alcohol and the implications for development of esophageal malignancy, it remains important to define the mechanisms governing the esophageal cellular response to exposure to alcohol. Acetaldehyde, formed by oral bacteria, is a toxic metabolite of alcohol and also deserves consideration. Compared to esophageal epithelial cells, HEMFs appeared more sensitive to the effects of alcohol, with a lower percentage of healthy HEMFs vs epithelial cells across most concentration.

In summary, this study reveals a hitherto unknown direct effects of alcohol and its toxic metabolite acetaldehyde on esophageal myofibroblasts and human esophageal epithelial cells. Understanding the effect of alcohol on HEMFs and HEMF-epithelial interactions in this complex environment, prior to dysplasia or invasive cancer will identify the molecular basis by which alcohol increases risk for esophageal cancer and lead to novel approaches that therapeutically target a permissive tumor microenvironment.

Supplementary Material

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Acknowledgements

This work was performed at the Swallowing and Esophageal Disorders Center at the Keck School of Medicine of USC. The authors would like to acknowledge the for funding support the National Institutes of Health NIAAA/NCI grant 1R21AA028891 for funding support. Additional analytical/instrumentation support was provided by the USC Research Center for Liver Diseases (NIH P30 DK048522). The project described was also supported in part by award number P30CA014089 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

Support:

This work was performed at the Swallowing and Esophageal Disorders Center at the Keck School of Medicine of USC. The authors would like to acknowledge the for funding support the National Institutes of Health NIAAA/NCI grant 1R21AA028891 for funding support. Additional analytical/instrumentation support was provided by the USC Research Center for Liver Diseases (NIH P30 DK048522).

Footnotes

The authors do not have any conflicts of interest.

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Supplementary Materials

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NIHMS1896565-supplement-FigS6.tiff (1.6MB, tiff)
FigS7
NIHMS1896565-supplement-FigS7.tiff (1.3MB, tiff)

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