Human esophageal myofibroblasts increase squamous epithelial thickness via paracrine mechanisms in an in vitro model of gastroesophageal reflux disease

Abstract

The pathogenesis of esophageal injury in gastroesophageal reflux disease (GERD) is incompletely understood. We modeled exposure of human esophageal myofibroblasts (HEMFs) to gastroesophageal reflux by repeated treatment with pH 4.5 and pH 4.5 bile salts and determined the effects on the epithelium in a 3D organotypic-like air-liquid interface model. Total, basal and supra-basal thickness of the epithelium were measured and immunostaining for p63, for basal (CK 14) and supra-basal (CK 4) squamous differentiation markers, and for cell proliferation (PCNA) were performed. Epithelial cell proliferation in response to HEMF conditioned media was also assessed in 2D culture. In the 3D organotypic model, total epithelial thickness increased similarly with pH 4.5 and pH 4.5 bile salt treated versus untreated and bile salt treated HEMF conditioned media. Epithelial p63 immunostaining was increased and multilayered. There was expansion of the CK14+ basal and CK4+ supra-basal layers in the epithelium established with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs versus untreated HEMF conditioned media. PCNA + cells per μm of tissue were unchanged in the basal layer across all treatment conditions while PCNA + cells per total DAPI + cells were decreased. In 2D culture, basal epithelial proliferation decreased with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs compared to conditioned media from untreated HEMF conditioned media. Secreted factors from HEMFs treated with acidic stimuli encountered in GERD increase epithelial thickness compared to secreted factors from untreated HEMFs and expand both basal and supra-basal layers. Our findings demonstrate for the first time paracrine regulation of the squamous epithelium from acid stimulated HEMFs. The effects of secreted factors from acid treated HEMFs on basal cell proliferation in this model and the mechanism mediating the increase in epithelial thickness merit further investigation.

Fig 1. Effect of conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs on the esophageal squamous epithelium in 3D OTC-ALI.

pH4.5 bile salt treated HEMF conditioned media increases total epithelial thickness vs untreated serum-free, EGF-free HEMF conditioned media (cSFMM). (A). Representative images of three independent, reproducible experiments are shown. Scale bar represents 50 μm. (B). Box and whisker plot of data from three independent experiments is shown (minimum, maximum, lower and upper quartile, and median are shown in the box and whisker plot). Mean values are indicated by X. Total epithelial thickness is increased in the squamous epithelium established with conditioned media from pH 4.5 or vs pH 4.5 bile salt treated HEMFs vs conditioned media from untreated HEMFs or HEMFs treated with bile salts alone (mean values 53.0 μm vs 53.1 μm vs 20.0 μm and 19.4 μm, p<0.005 for cSFMM vs pH 4.5 cSFMM and pH 4.5 bile salt cSFMM; p<0.005 for bile salt cSFMM vs pH 4.5 cSFMM and pH 4.5 bile salt cSFMM).

Fig 2. Basal cell marker p63 expression in the squamous epithelium in 3D OTC-ALI in response to conditioned media from untreated, pH 4.5 and pH4.5 bile salt treated HEMFs.

p63 (pink/red) immunostaining is shown for the epithelium from 3D-OTC established with conditioned media from untreated, pH 4.5, and pH 4.5 bile salt treated HEMFs. p63 signal is multi-layered in the squamous epithelium established with conditioned media from pH 4.5 and pH 4.5 bile acid treated HEMFs. Arrows point to p63 stained cells and yellow arrows indicate multi-layered cells. Representative images and quantification of three independent, reproducible experiments are shown. Quantification of p63 was done per total length (μm) of tissue available for histologic analysis and also per DAPI+ cells. Box and whisker plot of data from three independent experiments is shown (minimum, maximum, lower and upper quartile, and median are shown in the box and whisker plot). Mean values are indicated by X. An increase in p63+ cells/μm is observed in the epithelium established with conditioned media from pH 4.5 treated HEMFs (separate box and median) and a trend in increase expression with conditioned media from pH 4.5 bile salt treated HEMFs compared to untreated HEMFs (cSFMM and pH 4.5 bile salt conditioned media boxes overlap, but median of pH 4.5 bile salt conditioned media remains outside of cSFMM box). When p63 quantification is performed per DAPI+ cells, an increase in the number of p63+ cells is observed in the squamous epithelium established with conditioned media from pH 4.5 and from pH 4.5 bile salt treated HEMFs compared to conditioned media untreated HEMFs.

Fig 3. Immunofluorescence for basal and supra-basal differentiation markers and basal/supra-basal thickness of the squamous epithelium in 3D OTC-ALI established with conditioned media from untreated, pH 4.5, and pH 4.5 bile salt treated HEMFs.

Basal (red, CK 14) and supra-basal (green, CK 4) immunofluorescent staining shown in panels A-C for the epithelium in 3D OTC established with conditioned media from untreated (A), pH 4.5 treated (B), and pH 4.5 bile salt treated (C) HEMFs. Overlap between basal (red, CK 14) and supra-basal (green, CK 14) differentiation markers is visible in the epithelium established with conditioned media from pH 4.5 (B) treated HEMFs. Representative images of three independent, reproducible experiments are shown. (D and E) Box and whisker plot of data from three independent experiments is shown (minimum, maximum, lower and upper quartile, and median are shown in the box and whisker plot). Mean values are indicated by X. (D) Quantification of basal epithelial thickness as defined by CK14 immunostaining shows an increase in mean basal thickness of the epithelium established with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs (mean values: 7.1 μm vs. 14.2 μm and 15.4 μm, p< 0.05; ranges of CK14 + cell layer thickness in cSFMM, pH 4.5 cSFMM, and pH 4.5 bile salt samples are 6.6–8.0 μm; 11.2–16.5 μm; and 11.8–18.4 μm, respectively) (E) Quantification of supra-basal thickness as defined by CK4 immunostaining shows an increase in mean supra-basal thickness of the epithelium established with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs (mean values: 12.3 μm vs 38.1 μm and 32.8 μm, p< 0.05; ranges of CK4 + cell layer thickness in cSFMM, pH 4.5 cSFMM, and pH 4.5 bile salt samples are 9.5–13.8 μm; 25.5–50.1 μm; and 22.7–43.5 μm).

Fig 4. Proliferative marker PCNA expression in the squamous epithelium in 3D OTC-ALI in response to conditioned media from untreated, pH4.5 and pH4.5 bile salt treated HEMFs conditioned media.

PCNA immunostaining (pink/red) is limited to the lowest layer cells in the squamous epithelium established with conditioned media from untreated, pH4.5 treated, and pH4.5 bile salt treated HEMFs. Quantification of PCNA + cells per μm of tissue length and per DAPI+ cells are shown in the graphs. Quantification of PCNA per μm of tissue length is similar across all conditions. Quantification of PCNA per DAPI+ cells demonstrates a decrease in PCNA expression in epithelial cells established with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs vs untreated HEMFs. Box and whisker plot of data from three independent experiments is shown (minimum, maximum, lower and upper quartile, and median are shown in the box and whisker plot). Mean values are indicated by X.

Fig 5. Esophageal epithelial basal cell proliferation in response to untreated and acid and acidic bile salt treated HEMF conditioned media.

(A) Fluorescence proliferation assay. Epithelial cells (STR, 600 cells/well of 96 well plate) were treated with conditioned media of untreated HEMFs (cSFMM) or conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs, at 1:1, 1:2, 1:4; and 1:8 dilutions in SFMM for 48 hrs. Controls were undiluted keratinocyte serum-free media (KSFM, ThermoFisher Scientific) and serum-free, EGF-free myofibroblast media (SFMM). * p < 0.05 vs. cSFMM. Proliferation was assessed with alamarBlue assay. (B) Luminescence proliferation assay. STR were plated overnight (2000 cells/well of 96 well plate) and then cultured for 96 hours with KSFM, or with SFMM, cSFMM, or conditioned media from HEMFs treated with pH 4.5 or pH 4.5 bile salts, all diluted 1:1 in KSFM, as described in the methods. On day 4, CellTiter-Glo assay reagents was added per manufacturer’s instructions and luminescence read. * p < 0.05 vs. KSFM and SFMM; ** p < 0.05 vs cSFMM. In panel A, ** is p < 0.05 vs cSFMM for the indicated dilution.