Development and Application of a Functional Human Esophageal Mucosa Explant Platform to Eosinophilic Esophagitis

Abstract

There is an increasing prevalence of esophageal diseases but intact human tissue platforms to study esophageal function, disease mechanisms, and the interactions between cell types in situ are lacking. To address this, we utilized full thickness human donor esophagi to create and validate the ex vivo function of mucosa and smooth muscle (n = 25). Explanted tissue was tested for contractile responses to carbachol and histamine. We then treated ex vivo human esophageal mucosa with a cytokine cocktail to closely mimic the Th2 and inflammatory milieu of eosinophilic esophagitis (EoE) and assessed alterations in smooth muscle and extracellular matrix function and stiffening. We found that full thickness human esophagus as well as the individual layers of circular and longitudinal muscularis propria developed tension in response to carbachol ex vivo and that mucosa demonstrated squamous cell differentiation. Treatment of mucosa with Th2 and fibrotic cytokines recapitulated the majority of the clinical Eosinophilic Esophagitis Diagnostic Profile (EDP) on fluidic transcriptional microarray. Transforming growth factor-beta-1 (TGFβ1) increased gene expression of fibronectin, smooth muscle actin, and phospholamban (p < 0.001). The EoE cocktail also increased stiffness and decreased mucosal compliance, akin to the functional alterations in EoE (p = 0.001). This work establishes a new, transcriptionally intact and physiologically functional human platform to model esophageal tissue responses in EoE.

Figure 1

Induced force responses in human esophageal smooth muscle. Contractile forces were generated in response to carbachol under isometric conditions in (a) freshly isolated intact tissue rings (n = 3 donors) and (b) muscularis proria rings after storage at 7 °C for 24 hours (4th donor). Arrows indicate carbachol addition. Carbachol induces concentration-dependent force responses in: (c) isolated circular smooth muscle rings, (d) longitudinal smooth muscle bundles (n = 4, cultured for 1 day), and (e) mucosal strips (n = 4, cultured for 1 day). (f) Histamine (H) stimulates LSM bundle contraction independently of carbachol (C) and forces are attenuated by isoproterenol (I) (4 bundles, cultured for 3 days). (g) Induced tension responses (1 μM carbachol) in microdissected MM (p = 0.25, n = 8, cultured 3–5 days).

Figure 2

Tension versus strain measurements to determine stiffness. After culture for 3 days, tension measurements were collected from intact mucosa during sequential cycles (a,b) of stepwise lengthening followed by stepwise shortening of the same esophageal mucosa strip. Lengthening was reversed at a force threshold set to avoid tissue damage. Shortening was reversed as the force approached zero. The step tracings indicate the length (L, right y-axis) at which the tissue was held for 2 minute intervals as length was changed in 0.72 mm increments. (a) In strain cycle 1, tissue tension (black line) but not length (red line) reached baseline on stepping down and (b) this remained the case in strain cycle 2. (c) 1 μM carbachol increases tension and shortening to less than the starting length. (d) In strain cycle 3 the tissue shortens to the starting length. (e) Integrated tension measurements plotted as a function of strain. (f) Plots generated from panel e data by subtracting cycle 2 from cycle 1 (basal) or cycle 3 (induced). (g) LSM bundle tension versus strain plots collected after culture for 10 days. (h) Contribution of smooth muscle to tissue tension in in basal and induced states for the LSM in panel g.

Figure 3

Stiffness and induced tension in ex vivo esophagus tissues. (a) Stiffness exponent k for strain cycle curves fit by tension = A(L/L₀)^k for basal (cycle 1), extracellular matrix (cycle 2), and carbachol induced (cycle 3) tissue stiffness (*p < 0.05 vs Cycle 1, ⁺p < 0.05 vs. Cycle 2.) and (b) the corresponding 1 μM carbachol-induced tension response (*p < 0.05 vs mucosa and lsmb). The data are pooled from samples cultured for 1–14 days (mean 5.3 days for mucosa, 5.2 days for LSM and 4.6 days for CSM) before measuring stiffness. where n represents the number of donors used for each tissue. *p < 0.05 vs Cycle 1, ⁺p < 0.05 vs. Cycle 2.

Figure 4

Properties of esophagus ex vivo. (a) Regeneration of superficial squamous cells of the stratum spinosum on day 7 after gentle scraping on day 0 (both untreated). (b) E-cadherin staining demonstrates squamous epithelial differentiation in full thickness esophageal mucosal explants cultured ex vivo for 14 days in vehicle or Th2 cytokine cocktail. 14 days of Th2 cytokine cocktail causes disruption of the epithelial barrier as occurs in the in vivo EoE active disease state. (c) Gene expression of phospholamban (PLN) and fibronectin (FBN) in LSM over 4 weeks of TGFβ1 treatment (***p < 0.0001). (d) Collagen1 staining in human esophageal mucosa following 14 days of vehicle or Th2 cocktail treatment (black line: epithelium, blue line lamina propia). Scale bars represent 50 (a) and 100 (d) microns if not otherwise labeled.

Figure 5

Comparison of the esophageal transcriptome in vehicle treated (blue) and Th2 cytokine cocktail (red). Color range is based on log2 normalized intensity (a). Three-dimensional plot containing five paired samples (red = cocktail, blue = vehicle) derived from principal component analysis of the genes shown in the heat map in order assess the geometric distance between the samples (b). Volcano plot of genes in the EDP with FDR p < 0.05, fold change >2 (red, left panel) following treatment with Th2 cytokine cocktail (c). Venn diagram comparing the number of genes identified as dysregulated (>5 fold change) in both an organ donor esophagus with eosinophilia and samples treated with the cytokine cocktail. Unique and overlapping genes are listed in the right panel (d). Mucosa was treated for 72 hours (one paired sample) to 7 days (4 paired samples).

Figure 6

Stiffening esophageal mucosa ex vivo with the Th2 cocktail. (a) Th2 cytokine cocktail shifts the tension versus strain to the left indicating shortening and mucosal stiffness. (b) Basal stiffness exponent k is significantly increased in mucosal strips treated with Th2 cytokine cocktail. (c) Treatment with Th2 cytokine cocktail reduces mucosal compliance.