Loss of Endothelial TSPAN12 Promotes Fibrostenotic Eosinophilic Esophagitis via Endothelial Cell-Fibroblast Crosstalk

Abstract

Background & aims: Eosinophilic esophagitis (EoE) can progress to fibrostenosis by unclear mechanisms. Herein, we investigated gene dysregulation in fibrostenotic EoE, its association with clinical parameters and specific pathways, and the functional consequences.

Methods: Esophageal biopsies from subjects with EoE were collected across 11 Consortium of Eosinophilic Gastrointestinal Disease Researchers sites (n = 311) and 2 independent replication cohorts (n = 83). Inclusion criteria for fibrostenotic EoE were endoscopic rings, stricture, and/or a history of dilation. Endoscopic, histologic, and molecular features were assessed by the EoE Endoscopic Reference Score, EoE Histology Scoring System, EoE Diagnostic Panel, and RNA sequencing. Esophageal endothelial TSPAN12 expression and functional effects on barrier integrity and gene expression were analyzed in vitro.

Results: TSPAN12 was the gene most correlated with fibrostenosis (r = -0.40, P < .001). TSPAN12 was lower in fibrostenotic EoE and correlated with EoE Endoscopic Reference Score, EoE Diagnostic Panel, and EoE Histology Scoring System (r = 0.34-0.47, P < .001). Lower TSPAN12 associated with smaller esophageal diameter (r = 0.44, P = .03), increased lamina propria fibrosis (r = -0.41, P < .001), and genes enriched in cell cycle-related pathways. Interleukin (IL)-13 reduced TSPAN12 expression in endothelial cells. Conversely, anti-IL-13 therapy increased TSPAN12 expression. TSPAN12 gene silencing increased endothelial cell permeability and dysregulated genes associated with extracellular matrix pathways. Endothelial cell-fibroblast crosstalk induced extracellular matrix changes relevant to esophageal remodeling.

Conclusions: Patients with fibrostenotic EoE express decreased levels of endothelial TSPAN12. We propose that IL-13 decreases TSPAN12, likely contributing to the chronicity of EoE by promoting tissue remodeling through fibroblast-endothelial cell crosstalk.

Keywords: Endothelium; Eosinophil; Eosinophilic Esophagitis; Fibrosis; Transcriptome.

Figure 1.. TSPAN12 most highly dysregulated gene for fibrostenotic EoE.

A, Negative log10 FDR P value of the Spearman correlation between the fibrostenotic score (total score of rings and stricture) and a diagnostic subset of genes from the EoE transcriptome (EDP). Red indicates a positive correlation and blue indicates a negative correlation. The dashed line indicates an FDR P value of 0.05. B-E, Peak esophageal eosinophil counts and TSPAN12 expression are plotted by groups for EoE disease activity (active, inactive), age (adults, children), gender (male, female), and EoE phenotype (NF, non-fibrostenotic [blue]; F, fibrostenotic [red]) for the Discovery cohort (CEGIR, active and inactive EoE). Data are mean ± SEM; markers represent individual subjects. NS, *P < .05, and **P < .01 using the Mann-Whitney U test. CEGIR, Consortium of Eosinophilic Gastrointestinal Disease Researchers; EoE, eosinophilic esophagitis; EDP, EoE Diagnostic Panel; FDR, false-discovery rate; GWAS, genome-wide association study; NS, not significant; HPF, high-power microscopic field.

Figure 2.. TSPAN12 associates with EoE diagnostic parameters and fibrostenotic features.

A, Associations between TSPAN12 expression and diagnostic parameters of gene expression, endoscopic, and histologic platforms. TSPAN12 expression levels correlate with peak esophageal eosinophils per HPF (upper left), total score from EREFS (upper right), EDP (lower left), and HSS (lower right). Markers represent individual subjects. B, Associations between TSPAN12 expression and stricture diameter (left, distal; right, proximal). Markers represent individual subjects. C, Associations (Spearman r values) between TSPAN12 expression and HSS domains (EI, eosinophilic inflammation; EA, eosinophilic abscess; ESL, eosinophilic surface layering; SEA, surface epithelial alteration; BZH, basal zone hyperplasia; DIS, dilated intercellular spaces; DEC, dyskeratotic epithelial cells; LPF, lamina propria fibrosis) for grade and stage are shown. ****P < .001. D, Functional enrichment analysis of the genes that strongly correlated with TSPAN12 (1,495 genes). Heatmap based on absolute Spearman r values between TSPAN12 and genes assessed by means of RNA sequencing. Shown are the 10 most significant terms by functional enrichment analysis in the following categories: Molecular Function (upper left), Biological Processes (upper right), Cellular Component (lower left), and Pathway (lower right). The x-axes represent the negative log10 FDR P value. EoE, eosinophilic esophagitis; EDP, EoE Diagnostic Panel; EREFS, EoE reference score; HSS, EoE Histology Scoring System; HPF, high-power microscopic field; FDR, false-discovery rate.

Figure 3.. IL-13 decreases TSPAN12 expression in endothelium.

A, TSPAN12 expression in endothelial cells. UMAP plot displaying single cells, colored by shared nearest neighbor clusters and cell types from a single-cell RNA-sequencing analysis of esophageal biopsies (left). The featured plot demonstrates TSPAN12 expression, with each dot representing a single cell (right). B, Relative expression of TSPAN12 by single-cell RNA sequencing; the cluster numbers match those shown in the UMAP plot of panel A. C, Representative immunofluorescence images of TSPAN12 (green) in the esophageal cells. Nuclei was stained by DAPI (blue), and VWF was stained as a specific marker for endothelium (red). D, The TSPAN12 expression levels among the esophageal cells stimulated with or without IL-13 were assessed by qPCR and normalized to GAPDH. E, Effects of EoE-related cytokine stimulation. HEsMEC were treated with the indicated cytokines for 24 h and evaluated by qPCR for TSPAN12, CCL26, and IL8 mRNA. F-G, Dose dependency and time course of IL-13–induced TSPAN12 decrease. HEsMECs were treated with IL-13 (for the indicated dose and time) and evaluated by using qPCR (left) and western blot (right). Numbers above TSPAN12 band represent the signal intensity normalized by HSP90 and relative to the untreated (UT) set as 1. Data (E-G) are the means ± SEM of three independent experiments performed in duplicate. *P < .05, **P < .01, and ****P < .0001 compared with UT using the one-way ANOVA test followed by a Dunnett’s multiple-comparison test. UMAP, uniform manifold approximation and projection; EoE, eosinophilic esophagitis, NL, normal controls; HEsMEC, human esophageal microvascular endothelial cells; UT, untreated; DAPI, 4ʹ,6-diamidino-2-phenylindole; VWF, von Willebrand factor.

Figure 4.. IL-13 regulates loss of TSPAN12 and induces EoE-like changes in endothelial cells.

A, Volcano plot of the 42 EDP genes differentially expressed between the IL-13–stimulated endothelial cells and untreated cells. Top 5 of upregulated and downregulated genes were labeled. B, Venn diagram analysis of shared differentially expressed genes in EDP between IL-13–stimulated endothelial cells (HEsMEC) and esophageal mucosa in EoE. Spearman correlation comparing absolute fold change values for the overlapping genes regulated in similar manners in both IL-13–stimulated endothelial cells and the inflamed esophageal tissue of patients with active EoE. C, Heat diagram of 31 differentially expressed genes as determined by EDP analysis (A). Each column indicates endothelial cells that were untreated (left), stimulated with IL-13 (100 ng/mL) for 24 h (middle), or treated with IL-13 (100 ng/mL) and fluticasone propionate (FP) (100 nM) for 24 h (right). Hierarchical clustering was used to analyze data and generate heat diagrams (red, upregulated; blue, downregulated). D, Peak esophageal eosinophil counts and and TSPAN12 expression were assessed in patients with EoE by EoE phenotype treated with FP, as described. “Pre” corresponds to the beginning of the study, and “Post” corresponds to day 85 of the study. *P < .05, t test. E, Relative expression level of TSPAN12 was assessed in patients with EoE who were either treated with anti–IL-13 antibody or placebo, as described in previous study. “Pre” corresponds to the study beginning, and “Post” corresponds to study day 85. *P < .05, t test. EoE, eosinophilic esophagitis; EDP, EoE Diagnostic Panel; HEsMEC, human esophageal microvascular endothelial cells; UT, untreated; TEER, transendothelial electrical resistance; FITC, fluorescein isothiocyanate; NS, not significant; HPF, high-power microscopic field.

Figure 5.. TSPAN12 deficiency affects endothelial functions.

A, Representative qPCR (left) and western blot (right) of TSPAN12 after TSPAN12 downregulation by shRNA in HEsMEC. Numbers above TSPAN12 band represent the signal intensity normalized by HSP90 and relative to the control (shCtrl) set as 1. B-C, The effect of TSPAN12 gene silencing on the endothelial functions of barrier integrity (TEER [B] and FITC-dextran flux measurements [C]). Data were normalized to the control (shCtrl) set as 1. D, Heat diagram representing RNA-sequencing analysis of the TSPAN12-deficient cells and control cells. Differentially expressed genes (DEGs) were identified by filtering on TPM>1, moderated t-test with FDR P < .05. E, Gene ontology analysis of DEGs in the TSPAN12-deficient cells. F, Schematic of media swap experiments. G, Representative western blots demonstrating levels of collagen I and α-SMA in esophageal fibroblasts of media swap experiments. H, EDN1 (endothelin-1) expression in HEsMECs (shCtrl, shTSPAN12). qPCR (left) and ELISA of supernatants (right). I, Representative immunofluorescence staining of esophageal biopsy sections from normal control individuals and patients with fibrostenotic EoE; TSPAN12 (magenta), collagen (cyan), and VWF (red) with DAPI–stained nuclei (blue). White arrows indicate blood vessels, and white broken line indicates basal membrane. For A-C, G, and H, data are the means ± SEM of three independent experiments performed in duplicate. *P < .05, **P < .01, ***P < .001, and ****P < .0001 using the Mann-Whitney U test (H), one-way ANOVA test followed by a Sidak’s multiple comparisons test (B-C), or compared with shCtrl using one-way ANOVA test followed by a Tukey’s multiple comparisons test (A, G). DAPI, 4ʹ,6-diamidino-2-phenylindole; EoE, eosinophilic esophagitis; EDP, EoE Diagnostic Panel; HEsMEC, human esophageal microvascular endothelial cells; TEER, transendothelial electrical resistance; FITC, fluorescein isothiocyanate; FDR, false-discovery rate; shCtrl, shRNA non-silencing control; shTSPAN12, shRNA silencing TSPAN12; VWF, von Willebrand factor.