Lysyl oxidase regulates epithelial differentiation and barrier integrity in eosinophilic esophagitis

Abstract

Background & aims: Epithelial disruption in eosinophilic esophagitis (EoE) encompasses both impaired differentiation and diminished barrier integrity. We have shown that lysyl oxidase (LOX), a collagen cross-linking enzyme, is upregulated in the esophageal epithelium in EoE. However, the functional roles of LOX in the esophageal epithelium remains unknown.

Methods: We investigated roles for LOX in the human esophageal epithelium using 3-dimensional organoid and air-liquid interface cultures stimulated with interleukin (IL)-13 to recapitulate the EoE inflammatory milieu, followed by single-cell RNA sequencing, quantitative reverse transcription-polymerase chain reaction, western blot, histology, and functional analyses of barrier integrity.

Results: Single-cell RNA sequencing analysis on patient-derived organoids revealed that LOX was induced by IL-13 in differentiated cells. LOX-overexpressing organoids demonstrated suppressed basal and upregulated differentiation markers. Additionally, LOX overexpression enhanced junctional protein genes and transepithelial electrical resistance. LOX overexpression restored the impaired differentiation and barrier function, including in the setting of IL-13 stimulation. Transcriptome analyses on LOX-overexpressing organoids identified enriched bone morphogenetic protein (BMP) signaling pathway compared to wild type organoids. Particularly, LOX overexpression increased BMP2 and decreased BMP antagonist follistatin. Finally, we found that BMP2 treatment restored the balance of basal and differentiated cells.

Conclusions: Our data support a model whereby LOX exhibits non-canonical roles as a signaling molecule important for epithelial homeostasis in the setting of inflammation via activation of BMP pathway in esophagus. The LOX/BMP axis may be integral in esophageal epithelial differentiation and a promising target for future therapies.

Keywords: BMP; Eosinophilic esophagitis; Lysyl oxidase; Organoid.

Figure 1.

Figure 2.. LOX overexpression promotes cell differentiation in esophageal epithelium.

(A) Quantitative RT-PCR for LOX of monolayer-cultured EPC2-hTERT cells overexpressing GFP or LOX (LOX OE) (n = 3). (B) Representative images of immunoblot for LOX of the monolayer-cultured GFP and LOX OE cells. (C and D) Alterations of aberrant LOX expression in EPC2-hTERT organoids. GFP and LOX OE organoids were stimulated with or without IL-13 (10 μg/ml) from day 7 to 11 and then harvested at day 11. (C) Quantitative RT-PCR for SOX2, KRT14, TP63, IVL, FLG, and LOR of the GFP and LOX OE organoids (n = 3). (D) Representative images of hematoxylin and eosin (H&E) staining, immunohistochemistry for TP63, and immunofluorescence staining for IVL (red) and FLG (green) of the GFP and LOX organoids. DAPI (blue). Scale bar, 50 μm. Data are representative of three independent experiments and expressed as means ± SDs. Two-tailed Student’s t-test (A) and one-way analysis of variance (C) were performed for statistical analyses. *P < 0.05, **P < 0.01.

Figure 3.. LOX overexpression attenuates organoid formation capacity.

(A) Representative phase contrast images of EPC2-hTERT organoids overexpressing GFP or LOX (LOX OE) at day 11. Organoid formation rate (OFR) was assessed at day 11 (P0) and they were then passaged. OFR was assessed again at day 11 (P1). Scale bar, 50 μm. (B) OFR was defined as the number of organoids (≥ 50 μm) divided by the total seeded cells. Data are representative of three independent experiments and expressed as means ± SDs (n = 6). Two-tailed Student’s t-test was performed for statistical analyses. **P < 0.01.

Figure 4.. LOX overexpression improves epithelial barrier integrity.

(A) Quantitative RT-PCR for DSG1 and DSC1 of EPC2-hTERT organoids overexpressing GFP or LOX (LOX OE). GFP and LOX OE organoids were stimulated with or without IL-13 (10 μg/ml) from day 7 to 11 and then harvested at day 11 (n = 3). Data are representative of three independent experiments. (B) Schematic of air-liquid interface (ALI) model. GFP and LOX OE EPC2-hTERT cells were cultured in low-calcium (0.09 mM Ca2+) media for 3 days, followed by high-calcium media (1.8 mM Ca2+) for 5 days, and then brought to ALI at day 8. ALI-cultured cells were stimulated with or without IL-13 (10 μg/ml) from day 9 to 14. (C) Transepithelial electrical resistance (TEER, Ω * cm2) in the GFP and LOX OE EPC2-hTERT ALI-cultures (n = 5). (D) Representative images of hematoxylin and eosin (H&E), immunohistochemistry for TP63, and immunofluorescence staining for IVL (red), FLG (green), and DSG1 (green) of the GFP and LOX OE EPC2-hTERT ALI-cultures. DAPI (blue). Scale bar, 50 μm. Data are representative of two independent experiments and expressed as means ± SDs. One-way analysis of variance (A) and two-tailed Student’s t-test (C) were performed for statistical analyses. *P < 0.05, **P < 0.01. NT, nontreated.

Figure 5.. Transcriptome analysis identifies the functional roles of LOX in esophageal epithelium.

(A) Heatmap and (B) volcano plot of differentially expressed genes (DEGs) based on RNA sequencing data from EPC2-hTERT organoids overexpressing GFP or LOX (LOX OE). GFP and LOX OE organoids were cultured for 11 days and subjected to RNA sequencing analysis. Upregulated and downregulated DEGs in LOX OE organoids are shown with red and blue, respectively. (C and D) Top 5 enriched (C) and depleted (D) terms in LOX OE organoids based on Gene Ontology analysis. (E) Gene Set Enrichment Analysis based on the Pathway Interaction Database (PID). Top 10 enriched pathways in LOX OE organoids are shown. Dot size and color represent the number of core enrichment genes and normalized enrichment score (NES) in the pathway, respectively. FDR, false discovery rate.

Figure 6.. BMP signaling pathway is activated in LOX overexpressing organoids.

(A) Gene Set Enrichment Analysis for BMP pathway in the Pathway Interaction Database (PID) based on the LOX overexpressing (LOX OE) organoids RNA sequencing data. (B) Expression of BMP and BMP receptor (BMPR) genes which relevant to the BMP pathway gene set, plotted as reads per kilobase per million (RPKM) (n = 3). (C-F) Validation of the BMP activation in EPC2-hTERT organoids. GFP and LOX OE organoids were cultured with or without IL-13 (10 μg/ml) from day 7 to 11 and then harvested at day 11. (C) Quantitative RT-PCR for BMP2 and FST of the GFP and LOX OE organoids (n = 3). (D) Representative images of immunoblot for BMP2 and phospho-SMAD1/5/9 (p-SMAD1/5/9) and (E) immunohistochemistry staining for FST of the GFP and LOX OE organoids. Scale bar, 50 μm. (F) FST protein levels were quantified with the scoring described in the method (n = 10). Data are representative of three independent experiments and expressed as means ± SDs. Two-tailed Student’s t-test (B) and one-way analysis of variance (C and F) were performed for statistical analyses. **P < 0.01. FDR, false discovery rate; NES, normalized enrichment score; NT, nontreated.

Figure 7.. BMP2 treatment induces cell differentiation in esophageal epithelium.

(A and B) BMP2 treatment in monolayer culture of EPC2-hTERT cells. EPC2-hTERT cells were treated with recombinant BMP2 protein (10 μg/ml) for 72 h in high-calcium (1.8 mM Ca²⁺) media. (A) Quantitative RT-PCR for TP63, IVL, FLG, LOR, DSG1, and DSC1 in the EPC2-hTERT cells (n = 3). (B) Representative images of immunoblot for TP63, IVL, DSG1, and phospho-SMAD1/5/9 (p-SMAD1/5/9) of the EPC2-hTERT cells. (C-E) EPC2-hTERT organoids were treated with recombinant BMP2 protein (10 μg/ml) from day 7 to 11 and then harvested at day 11. (C) Quantitative RT-PCR for SOX2, KRT14, TP63, IVL, FLG, LOR, DSG1, and DSC1 in the EPC2-hTERT organoids (n = 3). (D) Representative images of hematoxylin and eosin (H&E) staining, immunohistochemistry for TP63, and immunofluorescence staining for IVL (red) and FLG (green) of the EPC2-hTERT organoids. DAPI (blue). Scale bar, 50 μm. (E) Organoid formation rate (OFR) was assessed at day 11 (P0) and they were then passaged. OFR was assessed again at day 11 (P1). OFR was defined as the number of organoids (≥ 50 μm) divided by the total seeded cells (n = 6). Data are representative of three independent experiments and expressed as means ± SDs. Two-tailed Student’s t-test (A, C, and E) was performed for statistical analyses. *P < 0.05, **P < 0.01. NT, nontreated.