Increased Production of LIGHT by T Cells in Eosinophilic Esophagitis Promotes Differentiation of Esophageal Fibroblasts Toward an Inflammatory Phenotype

Abstract

Background & aims: Eosinophilic esophagitis (EoE) is an antigen-mediated eosinophilic disease of the esophagus that involves fibroblast activation and progression to fibrostenosis. Cytokines produced by T-helper type 2 cells and transforming growth factor beta 1 (TGFβ1) contribute to the development of EoE, but other cytokines involved in pathogenesis are unknown. We investigate the effects of tumor necrosis factor superfamily member 14 (TNFSF14, also called LIGHT) on fibroblasts in EoE.

Methods: We analyzed publicly available esophageal CD3⁺ T-cell single-cell sequencing data for expression of LIGHT. Esophageal tissues were obtained from pediatric patients with EoE or control individuals and analyzed by immunostaining. Human primary esophageal fibroblasts were isolated from esophageal biopsy samples of healthy donors or patients with active EoE. Fibroblasts were cultured; incubated with TGFβ1 and/or LIGHT; and analyzed by RNA sequencing, flow cytometry, immunoblots, immunofluorescence, or reverse transcription polymerase chain reaction. Eosinophils were purified from peripheral blood of healthy donors, incubated with interleukin 5, cocultured with fibroblasts, and analyzed by immunohistochemistry.

Results: LIGHT was up-regulated in the esophageal tissues from patients with EoE, compared with control individuals, and expressed by several T-cell populations, including T-helper type 2 cells. TNF receptor superfamily member 14 (TNFRSF14, also called HVEM) and lymphotoxin beta receptor are receptors for LIGHT that were expressed by fibroblasts from healthy donors or patients with active EoE. Stimulation of esophageal fibroblasts with LIGHT induced inflammatory gene transcription, whereas stimulation with TGFβ1 induced transcription of genes associated with a myofibroblast phenotype. Stimulation of fibroblasts with TGFβ1 increased expression of HVEM; subsequent stimulation with LIGHT resulted in their differentiation into cells that express markers of myofibroblasts and inflammatory chemokines and cytokines. Eosinophils tethered to esophageal fibroblasts after LIGHT stimulation via intercellular adhesion molecule-1.

Conclusions: T cells in esophageal tissues from patients with EoE express increased levels of LIGHT compared with control individuals, which induces differentiation of fibroblasts into cells with inflammatory characteristics. TGFβ1 increases fibroblast expression of HVEM, a receptor for LIGHT. LIGHT mediates interactions between esophageal fibroblasts and eosinophils via ICAM1. This pathway might be targeted for the treatment of EoE.

Keywords: Eosinophilia; Fibrogenesis; Fibrosis; ICAM1; Immune Regulation.

Figure 1.

LIGHT is expressed in active EoE. (A) Representative image of LIGHT⁺ cells in the epithelium (EPI) (top) and LP (bottom). Quantification of LIGHT-positive cells/μm in the (B) epithelium and (C) LP of biopsy samples from 4 normal and 6 to 8 active EoE esophagi. (D) Representative images of CD3⁺ cells in biopsy samples from 4 normal and 7 active EoE esophagi. (E) Quantification of epithelial CD3⁺ and LIGHT⁺ cells in paired biopsy samples from healthy individuals and patients with active EoE. Each dot represents an independent donor (healthy) or patient (Active EoE). Arrows point to LIGHT or CD3 stained cells. (F) LIGHT transcript abundance in 8 clusters of esophageal T cells (T1–T8) defined by Wen et al. (G) Total number of CD3⁺ cells and (H) total number of LIGHT⁺ cells in each T-cell cluster comparing cells from healthy, active EoE, or remission. (I) Comparative analysis of IL5, IL13, and LIGHT expression in T8 cells. *P < .05, **P < .01, ***P < .001.

Figure 2.

LIGHT induces a unique proinflammatory gene expression profile in esophageal fibroblasts. (A) Representative flow cytometry analysis of baseline HVEM and LTbR in healthy (n = 4) or active EoE (n = 5) esophageal fibroblasts. (B) Venn diagram of genes down-regulated (top) or up-regulated (bottom) by TGFβ1 and LIGHT compared to untreated in healthy esophageal fibroblasts (n = 4; P adj < .05; >1.5-fold). (C) Heatmap comparing the expression levels of genes associated with myofibroblast or inflammatory phenotype in esophageal fibroblasts treated with TGFβ1 or LIGHT (n = 4; P adj < .05; >1.5fold). (D–I) qRT-PCR analysis of some LIGHT-induced targets in healthy and active EoE fibroblasts. Each dot represents an independent donor (healthy, n = 3) or patient (Active EoE, n = 3–5). Differences are significant if *P < .05, **P < .01, or ***P < .001.

Figure 3.

TGFβ1 increases HVEM expression and modulates LIGHT-induced gene expression in esophageal fibroblasts. (A) Representative flow cytometry histograms of HVEM and LTβR in esophageal fibroblasts from healthy donors or patients with active EoE untreated or treated with TGFβ1 for 48 hours. (B) Mean fluorescence intensity of HVEM in normal (n = 6) or active EoE (n = 10) fibroblasts. (C–E) qRT-PCR of (C, D) inflammatory molecules (ICAM-1, IL32, IRF8, CCL5, IL6, BIRC3, VCAM-1, CD74, CX3CL1, IL-33, IL-34 and CXCL5) and (E) myofibroblast and fibrosis markers (ACTA2, TAGLN, COL1A1, CTGF, PLN, and SERPINE1) in esophageal fibroblasts from healthy donors sequentially treated with TGFβ1 for 48 hours and LIGHT for 24 hours. n = 3–5; each dot represents an independent donor. Differences are significant if *P < .05, **P < .01, or ***P < .001.

Figure 4.

Cross-talk between TGFβ1 and LIGHT modulates adhesion molecule expression in esophageal fibroblasts. (A) Representative histograms and (B) mean fluorescence intensity of ICAM-1 and VCAM-1 analyzed by flow cytometry in active EoE fibroblasts treated with TGFβ1 for 48 hours or LIGHT for 24 hours, or sequentially treated with TGFβ1 and LIGHT as in Figure 4 (n = 5). Each dot represents cells from an independent patient; *P < .05. (C) Representative population analysis of α-SMA and ICAM-1 in fibroblasts from patients with active EoE treated as in A and B (n = 13). (D) Representative immunofluorescence staining of fibroblasts from patients with active EoE sequentially treated with TGFβ1 and LIGHT (n = 3). (E) Representative population analysis of α-SMA and VCAM-1 in fibroblasts from patients with active EoE treated as in A and B. (F) Representative population analysis of ICAM-1 expression within the VCAM-1^lowα-SMA^high and VCAM-1^highα-SMA^high fibroblast populations identified in E (n = 13). Differences are significant if *P < .05.

Figure 5.

Fibroblasts interact with activated eosinophils in the EoE esophagus. (A) representative contour plots of CD90 in active EoE fibroblasts untreated, or treated with 1 ng/mL TGFβ1, or treated with 50 ng/mL LIGHT. (B) Western blot analysis of vimentin, α-SMA, and ICAM-1 in fibroblasts derived from 2 healthy and 2 active EoE esophageal biopsy samples treated as in A. Representative images of healthy (n = 3) and active EoE (n = 4) esophageal biopsy samples double-labeled for (C) CD90/ vimentin, (D) vimentin/α-SMA, and (E) vimentin/EPX or their corresponding isotypes and imaged by using a fluorescence microscope.

Figure 6.

LIGHT mediates fibroblast-eosinophil interactions via ICAM-1. (A, B) Correlation of eosinophil counts in esophageal biopsy samples with (A) ICAM-1⁺ or (B) α-SMA⁺fibroblasts derived in vitro from the biopsy samples. Each dot represents data from an independent patient with active EoE (n = 13, Pearson’s correlation coefficient). (C) Representative images of H&E- (n = 8) or EPX- (n = 2) stained cocultures of active EoE esophageal fibroblasts treated with LIGHT for 24 hours, then washed and cocultured with IL5-prestimulated peripheral blood eosinophils for 6 hours. Red arrows point to eosinophil aggregates on a single fibroblast. Quantification of (D) total numbers of eosinophils or (E) eosinophil aggregates (≥4 eosinophils/fibroblast) in active EoE fibroblasts unstimulated or stimulated with 50 ng/mL LIGHT for 24 hours followed by incubation with 10 μg/mL isotype or anti–ICAM-1 antibody (MAB2146Z) and cocultured with eosinophils for 6 hours (n = 3). Quantifications show the average number of eosinophils or clusters in 6 high-power 20× fields in independent experiments with cells from different patients/donors. Differences are significant if *P < .05 or ***P < .001. eos, eosinophils.