Introduction: Alcohol-induced thickening of the gut mucosal layer and increased expression of goblet cell gel-forming mucins, such as mucin-2 (MUC2) are associated with disruptions to the gut barrier in alcoholic liver disease (ALD). Interest in drugs that can target gut mucins in ALD has grown; however to date, no studies have examined the properties of drugs on expression of gut mucins in models of ALD. We previously demonstrated that at 10 mg/kg/day, the drug fenretinide (N-[4-hydroxyphenyl] retinamide [Fen]), a synthetic retinoid, mitigates alcohol-associated damage to the gut barrier and liver injury in a murine model of ALD. Methods: In this study, we specifically sought to examine the effects of Fen on gut goblet cells, and expression of mucins, including MUC2 using a 25-day Lieber-DeCarli model of chronic alcohol intake. Results: Our results show that chronic alcohol intake increased gut-mucosal thickening, goblet cell numbers, and mRNA and protein expression of MUC2 in both the ileum and colon. Alcohol intake was associated with marked decreases in ileal and colonic Notch signaling, levels of Notch ligands Dll1 and Dll4, and increases in the expression of Notch-associated genes indispensable for goblet cell specification, including Math1 and Spdef. Interestingly, ileal and colonic expression of KLF4, which is involved in terminal differentiation of goblet cells, was reduced in mice chronically fed alcohol. Coadministration of alcohol with Fen at 10 mg/kg/day significantly reduced alcohol-associated increases in ileal and colonic mucosal thickening, ileal Muc2, colonic Muc2, Muc5ac and Muc6 mRNAs, and goblet cell numbers. We also found that Fen strongly prevented alcohol-mediated suppression of the Notch ligand Dll1, Notch signaling, and alcohol-induced increases in expression of Notch-associated goblet cell specification genes in both the ileum and colon. In the absence of alcohol, Fen treatments alone at 10 mg/kg/day had no effects on any of the goblet cell-related endpoints. Conclusion: These data show for the first time that the drug Fen possesses mucosal layer-modulating properties in response to chronic alcohol abuse. These data warrant further preclinical examination of Fen given the need for anti-ALD drugs and emerging evidence of a role for intestinal goblet cell mucins in the progression of ALD.

A convincing body of evidence shows that chronic alcohol intake has multiple negative effects on the gut (reviewed in [1]), including microbial dysbiosis and disruption of the gut barrier that can promote alcoholic liver disease (ALD) [2, 3]. Gut mucins, synthesized by goblet cells, play an integral role in maintenance of barrier function and protection against gut pathogens (reviewed in [4]). For reasons that remain incompletely understood, alcohol intake alters gut mucin composition, increases mucin synthesis and mucosal thickening, which are associated with human and murine ALD [5-7]. Conversely, mice lacking intestinal MUC2, the major gel-forming mucin [8, 9], are protected against alcohol disruptions to the gut barrier and development of ALD [5], suggesting that MUC2 itself may be involved in the progression of ALD. It was recently shown that mice lacking MUC2 are also protected against a related liver pathology, nonalcoholic fatty liver disease (NAFLD) [10]. Given the emerging roles for mucins in these liver diseases and some cancers [11], interest has grown in the potential of MUC2 and goblet cells as novel drug targets for ALD, and other mucin-associated pathology [11, 12].

Fenretinide (N-[4-hydroxyphenyl]) retinamide (Fen) is a synthetic retinoid known for its anticancer, and recently for its antidiabetic properties [13-15]. We previously demonstrated that when concomitantly administered during chronic alcohol intake, Fen at 10 mg/kg/day prevents disruptions to gut barrier function and mitigates endotoxemia and liver damage in a murine model of ALD [16]. We also reported, consistent with previous studies in models of type 2 diabetes [15, 17], that Fen at 10 mg/kg/day is nontoxic and shows no adverse effects or hepatotoxicity when administered to both untreated and alcohol (EtOH)-treated mice [16]. Therefore, in this study, we sought to examine the effects of Fen at 10 mg/kg/day on expression of gut secretory mucins, including MUC2, in the distal intestine (ileum) and proximal colon of mice chronically fed EtOH for 25 days.

Liquid Ethanol Diet

Male, wild-type C57BL/6J mice (8–9-week-old) (n = 20) were fed a Lieber-DeCarli liquid alcohol diet as described [16]. Please see extended Materials and Methods for all experimental details.

Fenretinide Reduces Ileum and Colon Mucosal Thickness and Goblet Cell Numbers

We used Periodic Acid-Schiff (PAS) and alcian blue staining to examine the gut mucosal layer thickness and mucin density, as previously described [18, 19]. Mucosal layer PAS staining showed that, compared to pair-fed mice, EtOH-fed mice had ∼2.5-fold increases in mucosal thickness in the ileum (shown in Fig. 1a [b vs. a, red arrows], c) and ∼2-fold increases in the colon (shown in Fig. 1f, [b vs. a, red arrows], h). Accordingly, we also found that in EtOH-treated compared to pair-fed mice, villus total PAS staining density was 3-fold and 2.3-fold higher in the ileum and colonic tissue, respectively (shown in Fig. 1d, i). Moreover, alcian blue staining, which stains for goblet cell total mucins, showed that ileum and colon, goblet cell numbers (shown in Fig. 1b and g [b vs. a, yellow arrows], e and j), and goblet cell-mucin density, increased 2–2.5-fold (shown in Fig. 1e) and 3.2–3.7-fold (shown in Fig. 1j), respectively, in EtOH-treated compared to pair-fed mice. In contrast, ileal-mucosal thickness was reduced by 44% in EtOH + Fen mice versus EtOH-fed mice (shown in Fig. 1a [d vs. b, red arrows], c). Similarly, ileal PAS staining density (shown in Fig. 1d), goblet cell number and mucin intensity (shown in Fig. 1e), were similarly reduced by 42%, 31%, and 36% respectively, in EtOH + Fen compared to EtOH-treated mice. We also found that EtOH + Fen mice showed reductions in colonic mucosal thickness by 34% (shown in Fig. 1f[d vs. b, red arrows], h), as well as reductions in PAS staining (shown in Fig. 1i), goblet cell numbers and mucin intensity, by 57%, 46%, and 34%, respectively (shown in Fig. 1j), compared to EtOH-treated mice. We found no differences in intestinal mucosal thickness, PAS staining, goblet cell number, or mucin intensity in pair-fed + Fen mice, compared to the pair-fed group (shown in Fig. 1a–j).

Fig. 1.

Fenretinide reduces ileal and colonic mucosal thickness and goblet cell numbers. a, b Representative images of PAS- and AB-stained goblet cells (yellow arrows) in the ileum and (f, g) the colon. c, d Quantification of mucosal thickness (red arrows) and PAS staining in the ileum and (h, i) the colon. e, j Quantification of goblet cell numbers (light blue bars) and AB optical intensity (dark blue bars) in the ileum and colon. All image magnification is ×100; scale bar = 50 μm. All data error bars represent ± SD, with **p< 0.01, ***p< 0.001, ****p< 0.0001. ns, not significant; AB, alcian blue; PAS, Periodic Acid–Schiff.

Fig. 1.

Fenretinide reduces ileal and colonic mucosal thickness and goblet cell numbers. a, b Representative images of PAS- and AB-stained goblet cells (yellow arrows) in the ileum and (f, g) the colon. c, d Quantification of mucosal thickness (red arrows) and PAS staining in the ileum and (h, i) the colon. e, j Quantification of goblet cell numbers (light blue bars) and AB optical intensity (dark blue bars) in the ileum and colon. All image magnification is ×100; scale bar = 50 μm. All data error bars represent ± SD, with **p< 0.01, ***p< 0.001, ****p< 0.0001. ns, not significant; AB, alcian blue; PAS, Periodic Acid–Schiff.

Close modal

Fenretinide Reduces Ileum and Colon Mucin mRNA and Protein Levels

Mice express three secreted gut mucins, MUC2, MUC5ac, and MUC6 [8, 9]. By qPCR, we found that ileal mRNA transcripts of Muc2 and Muc6 but not Muc5ac (shown in Fig. 2a–c), and colonic Muc2, Muc5ac, and Muc6 (shown in Fig. 2f–h) were significantly increased in EtOH-treated mice compared to pair-fed mice. In contrast, we found that ileum Muc2 (shown in Fig. 2a) and colonic Muc2 and Muc6 (shown in Fig. 2f, g) were reduced by approximately 40% in EtOH + Fen mice compared to EtOH-treated mice. We detected no differences in mRNA transcript levels of Muc2, Muc5ac, or Muc6 in the ileum or colon of pair-fed + Fen mice compared to the pair-fed group (shown in Fig. 2a–c, f–h).

Fig. 2.

Fenretinide reduces ileal and colonic mucin mRNA and protein levels. qPCR histograms of (a–c) ileal, and (f–h) colonic relative mRNA levels of the genes Muc2, Muc5ac, and Muc6. e, j Representative images and (d, i) quantification of ileal and colonic MUC2 IHC. Magnification, ×100; scale bar = 100 μm. All histogram error bars represent ± SD, with *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. qPCR results represent gene fold changes relative to pair-fed mice normalized against the housekeeping gene 36B4. qPCR, quantitative PCR; ns, not significant.

Fig. 2.

Fenretinide reduces ileal and colonic mucin mRNA and protein levels. qPCR histograms of (a–c) ileal, and (f–h) colonic relative mRNA levels of the genes Muc2, Muc5ac, and Muc6. e, j Representative images and (d, i) quantification of ileal and colonic MUC2 IHC. Magnification, ×100; scale bar = 100 μm. All histogram error bars represent ± SD, with *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. qPCR results represent gene fold changes relative to pair-fed mice normalized against the housekeeping gene 36B4. qPCR, quantitative PCR; ns, not significant.

Close modal

We next assessed ileal and colonic MUC2 protein expression by immunohistochemistry (IHC). We found, consistent with the mRNA increases, that ileal and colonic goblet cell MUC2 protein levels increased by 2.5-fold (shown in Fig. 2d, e [b vs. a, yellow arrows]) and 3.4-fold (shown in Fig. 2i, j [b vs. a, yellow arrows]), respectively, in EtOH-treated mice compared to pair-fed mice. In contrast, we found that compared to EtOH-treated, the ileal and colonic MUC2 protein levels were reduced by 38% (shown in Fig. 2d, e [d vs. b, yellow arrows]) and 55% (shown in Fig. 2i, j [d vs. b, yellow arrows]), respectively, in EtOH + Fen-treated mice. We did not detect any changes to ileal or colonic MUC2 protein levels in pair-fed + Fen-treated compared to pair-fed mice (shown in Fig. 2e, j [c vs. a, yellow arrows]).

Fenretinide Prevents Alcohol Suppression of Gut Canonical Notch Signaling

Canonical Notch signaling is indispensable for the maintenance of a balance of gut-absorptive cells (enterocytes) and secretory cells (i.e. goblet and Paneth cells) (reviewed in [20]). Therefore, we next determined the effects of EtOH and Fen treatments on ileal and colonic mRNA levels of Notch receptors, and genes involved in canonical Notch signaling. We found that ileal and colonic gene expression of Notch1 and Notch2 receptors were unchanged in all groups compared to pair-fed mice (shown in Fig. 3a, b, g, h); however, mRNA levels of the Notch target genes, Hes1 and Hey1 [20], were reduced by 2-fold and 2.4-fold in the ileum (shown in Fig. 3c, d) and 8-fold and 4.5-fold in the colon (shown in Fig. 3i, j) of EtOH-treated versus pair-fed mice. In contrast, ileum and colonic mRNA levels of Hes1 and Hey1 were unchanged in EtOH + Fen-treated mice compared to pair-fed mice, and significantly higher than in EtOH-treated mice (shown in Fig. 3c, d, i, j). Given these findings, we next measured by IHC ileal and colonic protein expression of the Notch intracellular domain (NICD), which, upon Notch activation, travels to the nucleus to regulate expression of Notch-associated genes, and therefore is an indicator of active Notch signaling [20]. Our IHC data showed strong ileal and colonic crypt cell expression and nuclear localization of NICD in pair-fed mice and pair-fed + Fen mice (shown in Fig. 3e, k yellow arrows). However, compared to pair-fed mice, EtOH-treated mice had 2–3-fold reductions of ileal and colonic NICD protein levels, which showed weak nuclear and mostly sparse cytoplasmic immunoreactivity (shown in Fig. 3e, k,[b vs. a, yellow arrows], f and l). In contrast to EtOH-treated mice, ileal and colonic protein levels of NICD were increased by ∼62% and ∼158% in EtOH + Fen-treated mice, and unchanged compared to pair-fed mice (shown in Fig. 3e, k, [d vs. b, yellow arrows], f and l).

Fig. 3.

Fenretinide prevents alcohol suppression of gut canonical Notch signaling. qPCR histograms of (a–d) ileal and (g–j) colonic relative mRNA levels of genes involved in canonical Notch signaling, Notch1, Notch2, Hes1, and Hey1. e, k Representative images and (f, l) quantification of ileal and colonic NICD IHC (yellow arrows). Magnification, ×200; scale bar = 100 μm. All histogram error bars represent ± SD, with *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. qPCR results represent gene fold changes relative to pair-fed mice normalized against the housekeeping gene 36B4. qPCR, quantitative PCR; NICD, Notch intracellular domain; ns, not significant.

Fig. 3.

Fenretinide prevents alcohol suppression of gut canonical Notch signaling. qPCR histograms of (a–d) ileal and (g–j) colonic relative mRNA levels of genes involved in canonical Notch signaling, Notch1, Notch2, Hes1, and Hey1. e, k Representative images and (f, l) quantification of ileal and colonic NICD IHC (yellow arrows). Magnification, ×200; scale bar = 100 μm. All histogram error bars represent ± SD, with *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. qPCR results represent gene fold changes relative to pair-fed mice normalized against the housekeeping gene 36B4. qPCR, quantitative PCR; NICD, Notch intracellular domain; ns, not significant.

Close modal

Along with Notch, intestinal Wnt signaling is another key pathway in maintenance of gut absorptive and secretory cells (for review [21]). However, we found mRNA expression of the canonical Wnt target genes Axin2, Tcf4, and Lgr5, an intestinal stem cell marker [22], unchanged in ileal tissue (shown in online suppl. Fig. 1a–c; see www.karger.com/doi/10.1159/000524386 for all online suppl. material). Although we found that colonic mRNA levels of Axin2 and Tcf4 were trending higher in EtOH-treated mice compared to pair-fed mice, our analysis found no significant mRNA changes to these genes or Lgr5 in colonic tissue across all groups (shown in online suppl. Fig. 1g–i). Taken together, these data show that Fen treatments opposed alcohol-mediated suppression of Notch signaling in both the ileum and colon.

Fenretinide Opposes Alcohol-Mediated Increases in Gut Goblet Cell Specification Genes

Evidence shows that Notch signaling regulates the gut pool of secretory cells through Hes1-mediated suppression of Math1 (Atoh1) and its downstream target genes involved in the development and maintenance of goblet cells in the gut [20, 23]. We next measured mRNA levels of Math1, and found that compared to pair-fed, ileal and colonic transcripts of Math1 were increased by ∼ 2.5–3-fold in EtOH-treated mice but comparatively reduced by 44% and 42% in ileal and colonic tissue, respectively, in EtOH + Fen mice (shown in Fig. 4a, i). We next measured mRNA levels of the Math1 target genes, Creb3l1, Clca1, Agr2, and Spdef, which are involved in goblet cell differentiation [20, 23, 24]. We found, consistent with the increases in gut Math1 mRNA levels, that the expression of Creb3l1 and Clca1 was increased ∼2–5-fold in the ileum and colon in EtOH-treated compared to pair-fed mice (shown in Fig. 4b, c, j, k). However, in contrast to EtOH-treated mice, transcript levels of these genes were significantly reduced by ∼ 30–50% in the ileum (shown in Fig. 4b, c), and 45–54% in the colon (shown in Fig. 4j, k) of EtOH + Fen-treated mice. Transcript levels of Agr2 were unchanged in ileal tissue of all groups (shown in Fig. 4e) but increased by >2-fold in the colon of EtOH-treated compared to pair-fed mice (shown in Fig. 4m).

Fig. 4.

Fenretinide opposes alcohol-mediated increases in gut goblet-specification genes. qPCR histograms of (a–f) ileal and (i–o) colonic relative mRNA levels of the goblet specification genes Creb3l1, Clca1, Spdef, Agr2, and Klf4. g, p Representative images and (h, q) quantification of ileal and colonic SPDEF IHC. Magnification, ×200; scale bar = 100 μm. All histogram error bars represent ± SD, with *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. qPCR results represent gene fold changes relative to pair-fed mice normalized against the housekeeping gene 36B4. qPCR, quantitative PCR; SPDEF, SAM pointed domain-containing ETS transcription factor; ns, not significant.

Fig. 4.

Fenretinide opposes alcohol-mediated increases in gut goblet-specification genes. qPCR histograms of (a–f) ileal and (i–o) colonic relative mRNA levels of the goblet specification genes Creb3l1, Clca1, Spdef, Agr2, and Klf4. g, p Representative images and (h, q) quantification of ileal and colonic SPDEF IHC. Magnification, ×200; scale bar = 100 μm. All histogram error bars represent ± SD, with *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. qPCR results represent gene fold changes relative to pair-fed mice normalized against the housekeeping gene 36B4. qPCR, quantitative PCR; SPDEF, SAM pointed domain-containing ETS transcription factor; ns, not significant.

Close modal

Evidence shows that Spdef is required for the terminal differentiation of goblet cells [25, 26]. We found that compared to pair-fed, ileal and colonic Spdef mRNA levels were elevated ~3-fold in EtOH-treated mice, but in comparison, were reduced by ∼35–50% in the ileum and colon of EtOH + Fen-treated mice (shown in Fig. 4d, l). Consistent with this, we found by IHC, that SPDEF protein levels were elevated ∼2.8–4.2-fold in ileal and colonic tissue of EtOH-treated mice compared to pair-fed, and comparatively reduced by ∼50% in EtOH + Fen-treated mice (shown in Fig. 4g, h, p, q).

Along with SPDEF, KLF4 is another transcription factor involved in the terminal differentiation of goblet cells [27, 28] and is also negatively regulated by Notch [29]. However, we found, unlike Spdef, that ileal and colonic Klf4 mRNA levels were reduced ∼2.5-fold in EtOH-treated mice but unchanged in EtOH + Fen-treated mice compared to pair-fed mice (shown in Fig. 4f, n). Using immunofluorescence microscopy, we next examined KLF4 protein levels in the gut and found that, compared to pair-fed mice, ileal and colonic tissue from EtOH-treated mice showed 2–3-fold reductions to KLF4-positive cells in villus regions with concomitantly higher levels of MUC2-positive goblet cells (shown in Fig. 5a, c [b vs. a, inset, white arrows], b and d). However, in contrast to EtOH-treated, ileal and colonic tissue from EtOH + Fen-treated mice had 46% and 78% higher levels of KLF4 in villus regions with comparatively lower levels of MUC2-positive goblet cells (shown in Fig. 5a, c [d vs. b, inset, white arrows], b and d). Compared to pair-fed, we did not detect changes to gut Math1 mRNA, or Math1 target genes (shown in Fig. 4a–e, i–m), Klf4 mRNA (shown in Fig. 4f, o) or percentages of KLF4-positive cells (shown in Fig. 5a–d) in pair-fed + Fen mice.

Fig. 5.

Fenretinide increases ileal and colonic KLF4 positive epithelial cells. a, c Representative double IFM images of ileal and colonic tissue double-stained with antibodies against MUC2(red channel) and KLF4(green channel, white arrows). Magnification, ×100 (inset ×200), scale bar = 100 μm. (b, d) Quantification of ileal and colonic IFM optical intensity for MUC2(red bars) and KLF4(green bars). All data error bars represent ± SD, with * p< 0.05, **p< 0.01, ****p< 0.0001. IFM, immunofluorescence microscopy; KLF4, Krüppel-like factor 4.

Fig. 5.

Fenretinide increases ileal and colonic KLF4 positive epithelial cells. a, c Representative double IFM images of ileal and colonic tissue double-stained with antibodies against MUC2(red channel) and KLF4(green channel, white arrows). Magnification, ×100 (inset ×200), scale bar = 100 μm. (b, d) Quantification of ileal and colonic IFM optical intensity for MUC2(red bars) and KLF4(green bars). All data error bars represent ± SD, with * p< 0.05, **p< 0.01, ****p< 0.0001. IFM, immunofluorescence microscopy; KLF4, Krüppel-like factor 4.

Close modal

Notch suppression of Math1-regulated pathways is also necessary for development and maintenance of absorptive enterocytes and secretory Paneth cells [20, 25, 30]. However, we found that ileal and colonic mRNA levels of markers for enterocytes (Keratin 19) [31] and Paneth cells (lysozyme [Lyz1]) [20] were unchanged across all experimental groups (shown in online suppl. Fig. 1d, e, j, k). Collectively, these data demonstrate that among the intestinal cell types dependent on Notch signaling [20], EtOH and EtOH + Fen disproportionally modulated Notch signaling, and Notch-associated genes and gene pathways involved in intestinal goblet cell specification.

Fenretinide Prevents Alcohol-Mediated Reductions of Gut Notch Ligands, Dll1 and Dll4

We next measured expression of known mediators of Notch signaling in order to determine potential mechanisms behind EtOH and Fen modulation of Notch signaling in the gut. We first measured ileal and colonic mRNA levels of Psen1, the major subunit of membrane γ-secretase, which liberates NICD from the Notch receptor upon activation [20], and is used to estimate γ-secretase and Notch signaling activity in cells and tissue [32]. Our qPCR data showed that, compared to pair-fed, ileal transcripts of Psen1 were reduced by approximately 40% in EtOH-treated mice and unchanged in EtOH + Fen-treated mice (shown in online suppl. Fig. 1f); however, these changes did not reach statistical significance. In contrast, colonic Psen1 mRNA levels were unchanged across all experimental groups (shown in online suppl. Fig. 1l).

Evidence shows that, among the five known ligands for Notch receptors, Jagged 1, 2, and Delta-like ligands (Dll1, Dll3, and Dll4) [33], Dll1 and Dll4 play a key role in development and maintenance of goblet cells in the gut [34]. Therefore, we next examined if EtOH or Fen treatments affected gut mRNA levels of Dll1 and Dll4. Our qPCR analysis showed that compared to pair-fed, EtOH-treated mice had 47% and 34% reductions to ileal Dll1 and Dll4, respectively; however, only the reductions to Dll1 were found to be significantly different (shown in online suppl. Fig. 2a, b). Similarly, we found that colonic mRNA levels of Dll1 and Dll4 were reduced by 62% and 51%, respectively, in EtOH-treated mice versus pair-fed mice (shown in online suppl. Fig. 2c, d). Ileal and colonic mRNA levels of Dll1 and Dll4 were unchanged in EtOH + Fen-treated mice compared to pair-fed (shown in online suppl. Fig. 2a–d). Moreover, compared to EtOH-treated, ileal Dll1 and both colonic Dll1 and Dll4 were significantly higher in ETOH + Fen-treated mice (online suppl. Fig. 2a–d). By immunofluorescence microscopy (IFM), we found, consistent with the patterns of NICD (shown in Fig. 3e, k), that ileal and colonic DLL1 protein was strongly expressed in crypt cells in pair-fed mice (shown in online suppl. Fig. 2e, f, yellow arrows). Moreover, in agreement with the reductions to NICD (shown in Fig. 3 e, k), and the mRNA changes of Dll1, ileal and colonic DLL1 protein was reduced by 2.5–3.2-fold in EtOH-treated mice compared to pair-fed, but comparatively increased ∼ 75–200% in the ileum and colon of EtOH + Fen-treated mice (shown in online suppl. Fig. 2e–h). Together these data strongly suggest that Fen prevention of EtOH-reductions to Notch signaling, and modulation of Notch-gene pathways in the gut, might occur by increasing endogenous Notch ligands Dll1 and Dll4.

Our data strongly suggest that gut-mucosal thickening and increases in goblet cell numbers with chronic EtOH exposure involve reductions in gut canonical Notch signaling (shown in Fig. 3e, k), and increases in the expression of goblet cell specification genes normally suppressed by Notch, including Math1, Creb3l1, and Spdef (shown in Fig. 4a–d, i–l). Paradoxically, despite increases in goblet cell numbers, we found that EtOH intake led to marked decreases to gut levels of KLF4 (shown in Fig. 5a–d), which along with SPDEF, is also involved in promoting terminal differentiation of goblet cells [20, 27]. These findings may be due to conflicting reports from a number of KLF4−/− mutant mouse models on how dispensable KLF4 is in maintaining goblet cells in the adult gut [27, 28, 34, 35].

Two previous studies examining the effects of EtOH feeding on gut secretory cell types showed disparate results [36, 37]. A study by Forsythe et al. [36] reported that feeding mice 15% EtOH (v/v) for 56 days resulted in suppression of gut canonical Notch signaling (i.e. decreases in Hes1), through inhibition of Notch1 receptor. However, this study reported increases in gut markers for enterocytes and enteroendocrine cells, but no changes to goblet cells or Wnt signaling [36]. In contrast, Park et al. [37] showed that mice fed 5% EtOH (v/v) for 10 days had increased Wnt signaling in colonic organoids, but no changes to Notch signaling. Our findings are most similar to those by Forsythe et al. [36]; however, we did not detect reductions in either Notch1 or Notch2 receptors. Moreover, our data show no evidence of modulation of Wnt, markers of enterocytes (Keratin 19), or Paneth cells (Lyz1) (shown in online suppl. Fig. 1).

The reasons for the difference across these studies and ours remain unclear, but as with other targets of EtOH exposure in ALD [2], including stem cells [38, 39], we speculate that the impact and mechanisms of EtOH modulation of gut Notch and Wnt, and shifts in various gut-cell types, are likely dependent on dose and length of EtOH exposure. Therefore, future studies of EtOH on gut-cell specification must consider the time and dose of EtOH exposure.

When coadministered with EtOH, Fen at 10 mg/kg/day prevented EtOH-associated mucosal thickening (shown in Fig. 1a, c, f, h) and increases to MUC2-positive goblet cells (shown in Fig. 1e, j) in both the ileum and colon. Mechanistically, Fen's prevention of EtOH-mediated reductions to gut Notch ligands Dll1 and Dll4 (shown in online suppl. Fig. 2a–h) could be a key molecular aspect behind the lower levels of goblet cell specification genes and mucosal thickening we observed in EtOH + Fen-treated mice (shown in online suppl. Fig. 4a, Theoretical Model). This is supported by evidence that mice lacking gut DLL1 have suppressed gut Notch signaling and increases in goblet cell numbers [34]. However, it is incompletely understood what signals modulate gut DLL1- and DLL4-positive intestinal epithelial cells (IECs) or how these IECs transmit their Notch ligands to other Notch receptor-positive IECs [40].

We are not aware of data demonstrating Notch or goblet cell-modulating properties of Fen in the gut. Analysis of the pharmacological effects of Fen on Notch signaling in tissue or cells outside of the gut has been limited to cancer cells, where evidence suggests Fen opposes Notch signaling through its growth arresting properties [41, 42]. Still, given that Fen treatments alone had no effect on Notch, or any goblet cell endpoint, suggests that the mechanisms of action of Fen on limiting the EtOH-induced increase in mucosal thickening are likely indirect, and instead involve pathways that are specifically affected by chronic EtOH exposure.

Regarding retinoid signaling, we found that mRNA levels of RARβ and Cyp26A1, two genes strongly regulated by retinoic acid receptors (RARs) [43, 44], were increased in the ileum and colon by ∼ 2–40 fold in EtOH-treated mice compared to pair-fed (shown in online suppl. Fig. 3a–d). These findings are consistent with our previous data and others reporting marked increases in retinoid target genes in the livers of EtOH-fed mice [16, 45, 46]. It is currently unclear what are the implications of the EtOH-driven increases of these RAR target genes in the gut, but further studies are warranted. We found no differences in ileal and colonic mRNA levels of RARβ and Cyp26A1 in EtOH + Fen mice versus EtOH-treated mice (shown in online suppl. Fig. 3a–d), which suggests, and is consistent with [47], RAR-independent pharmacological properties of Fen.

We recognize the limitations to these findings, and therefore, more experiments are needed, including ileal and colonic organoid isolation and ex vivo studies in EtOH- and Fen-treated mice at various time points. Furthermore, an in-depth analysis of the gut microbiome, mucosal layer compositional changes, and turnover dynamics with EtOH and Fen are warranted, as evidence suggests that gut bacteria and EtOH can alter the mucosal glycosylation signature, stability, and functions [7, 48]. As evidence has demonstrated a role for proinflammatory signaling in goblet cell mucin synthesis and biology [49], future studies of the modulation of DLL1, DLL4, and Notch by EtOH and Fen should also consider the shifts in the inflammatory cell milieu in the microenvironment of the gut mucosa, as we previously demonstrated that Fen possesses gut anti-inflammatory properties in this ALD model [16]. Nevertheless, given the novel findings that excessive intestinal mucosa may be clinically relevant to the gut injury and progression of ALD [5-7], these preliminary findings suggest that more preclinical studies of Fen are warranted in chronic alcohol abuse.

We would like to thank the Hunter College Animal Care Facility for their help in animal handling and care.

All animal studies and experimental procedures were conducted at Hunter College. This study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC)/Ethics Committee of Hunter College (protocol number approval #STRAR19). All animal experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health, Hunter College IACUC guidelines, and in accordance with the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines 2.0 for in vivo animal experiments [50].

The authors have no conflicts of interest to declare.

This research was supported by the NIGMS 5SC2GM127206-0 to Steven E. Trasino and Karen Mai and by NIAA R21AA027637 and Weill Cornell funds to Marta Melis, Xiao-Han Tang, and Lorraine J. Gudas.

Marta Melis, Xiao-Han Tang, Karen Mai, Lorraine J. Gudas, and Steven E. Trasino conducted research experiments, collected data, provided data interpretation, wrote the manuscript, and approved the final manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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Additional information

Marta Melis and Xiao-Han Tang contributed equally to this study.

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