Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP) by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE)

Abstract

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl2 differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.

Fig 1. TSLP expression is restricted to the suprabasal differentiated compartment of the esophageal epithelium.

Immunohistochemistry for TSLP (A,C,E) and cytokeratin 13 (CK13) (B,D,F) in esophageal pinch biopsies from representative non-EoE control subjects, subjects with inactive EoE (<15 eosinophils per hpf), and active EoE (≥15 eosinophils per hpf). 200X magnification. Larger images represent 100X magnification. The box represents the area chosen for 200X magnification in the smaller side panel. SSE = Stratified squamous epithelium; B = Basal epithelium.

Fig 2

Calcium induces terminal differentiation of esophageal epithelial cells in-vitro A) Cytokeratin 13 (CK13) and, B) involucrin (IVL), mRNA expression in response to high calcium (1.8mM CaCl₂) media at 0, 24, 48, and 72 hours. Results are representative of three separate experiments. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 as compared to undifferentiated (0 hr) cells. Immunoblot for C) involucrin in calcium differentiated EPC2-hTERT cells. D) IHC for TSLP on organotypic cultures of esophageal epithelium. SSE = Stratified squamous epithelium; B = Basal epithelium, Sub = subepithelium.

Fig 3. Terminal differentiation of human esophageal epithelial cells enhances the inducible expression and secretion of TSLP protein in vitro.

TSLP mRNA expression in poly (I:C)-stimulated undifferentiated (A) and differentiated (B) EPC2-hTERT cells. C) TSLP protein secretion (pg/mL) by poly (I:C)-stimulated undifferentiated and differentiated EPC2-hTERT cells. D) TLR3 mRNA expression in undifferentiated and differentiated EPC2-hTERT cells. Results are representative of three separate experiments. * p<0.05, p<0.01***p <0.001, p<0.0001 as compared to undifferentiated cells.

Fig 4. Food antigens induce esophageal epithelial TSLP expression.

Quantification of TSLP mRNA expression following food antigen stimulation in undifferentiated (A) and differentiated (B) EPC2-hTERT cells. C) TSLP secretion (pg/mL) was quantified in undifferentiated and differentiated EPC2-hTERT cells. TSLP secretion in differentiated primary esophageal epithelial cells from non-EoE control subjects (N = 7) (D) and EoE subjects (N = 20) (E) following stimulation with food antigens. All cell lines were stimulated for 24 hours. (F) comparison of ova mediated TSLP expression in differentiated inactive and active EoE cells. The dashed lines represent the lower limit of sensitivity of the ELISA kit (8pg/mL). Bars represent the mean of each data set. * p<0.05, **p<0.01, ***p <0.001 as compared to unstimulated controls.

Fig 5. OVA-Induced esophageal epithelial TSLP induction is dependent upon NF-kB signaling and inhibited by budesonide in vitro.

A) differentiated EPC2-hTERT cells were pre-incubated with Bay-11 for 1hr prior to stimulation with OVA. TSLP in culture supernatant was assayed after 24 hrs. B) Differentiated EPC2-hTERT cells were stimulated with Ova in the presence or absence of budesonide (1uM) or DMSO (vehicle) for 24hrs.TSLP in culture supernatant was assayed using ELISA. ***p<0.001, ****p<0.0001, NS = not significant.