Fibrostenotic eosinophilic esophagitis might reflect epithelial lysyl oxidase induction by fibroblast-derived TNF-α

Abstract

Background: Fibrosis and stricture are major comorbidities in patients with eosinophilic esophagitis (EoE). Lysyl oxidase (LOX), a collagen cross-linking enzyme, has not been investigated in the context of EoE.

Objective: We investigated regulation of epithelial LOX expression as a novel biomarker and functional effector of fibrostenotic disease conditions associated with EoE.

Methods: LOX expression was analyzed by using RNA-sequencing, PCR assays, and immunostaining in patients with EoE; cytokine-stimulated esophageal 3-dimensional organoids; and fibroblast-epithelial cell coculture, the latter coupled with fluorescence-activated cell sorting.

Results: Gene ontology and pathway analyses linked TNF-α and LOX expression in patients with EoE, which was validated in independent sets of patients with fibrostenotic conditions. TNF-α-mediated epithelial LOX upregulation was recapitulated in 3-dimensional organoids and coculture experiments. We find that fibroblast-derived TNF-α stimulates epithelial LOX expression through activation of nuclear factor κB and TGF-β-mediated signaling. In patients receiver operating characteristic analyses suggested that LOX upregulation indicates disease complications and fibrostenotic conditions in patients with EoE.

Conclusions: There is a novel positive feedback mechanism in epithelial LOX induction through fibroblast-derived TNF-α secretion. Esophageal epithelial LOX might have a role in the development of fibrosis with substantial translational implications.

Keywords: Eosinophilic esophagitis; TGF-β; TNF-α; coculture; fibrosis; lysyl oxidase.

Figure 1:. LOX expression is increased in patients with active EoE.

qRT-PCR was performed to evaluate LOX mRNA expression from human esophageal biopsies. LOX mRNA expression is increased in patients with Active EoE (n=33) compared to normal (n=34), and inactive EoE (n=30) (A). ANOVA was performed with multiple comparisons. IHC staining for LOX was performed with representative images shown (B). Scale bar represents 100μm. Histogram representing average LOX score (0–3) for normal (n=5), active EoE (n=6) and inactive EoE (n=4). *** p<0.001 *p<0.05

Figure 2:. LOX expression increased in fibrostenotic EoE.

RNA from EoE biopsies was isolated and those patients with disease complications (stricture or endoscopically removed food impaction) and qRT-PCR was performed. Those with complications (n=22) were found to have enhanced LOX expression compared to those with uncomplicated disease (n=39) (A). LOX expression was significantly increased in patients with fibrostenotic endoscopic findings (rings, narrowing) (n=12) compared to in patients with inflammatory endoscopy findings (furrows, exudates, normal) (n=31) (B). Receiver operator characteristics representing LOX expression and fibrostenotic complications. **p=0.01, *p<0.05

Figure 3:. EoE milieu and TNFα upregulate LOX expression in esophageal epithelial cells.

EPC2-hTERT cells were treated with the indicated cytokines for 24 hours and evaluated by PCR (A) and Western Blot (B). Densitometry was performed on Western blots. Numbers under each band represent the signal intensity relative to NT set as 1. EPC2-hTERT cells were treated with increasing doses of TNFα (C). Human and murine esophageal epithelial cells were isolated and grown in matrigel to support organoid formation. Organoids were stimulated with TNFα and qRT-PCR was performed for LOX in human and murine organoids (D) and (E) respectively. IHC for LOX was performed on murine organoids stimulated with TNFα (F). Scale bar represents 50μm. Data represents ≥2 biologic replicates with similar results. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; NT- no treatment.

Figure 4:. Conditioned media enhances LOX expression in vitro.

Schematic of experimental design (A). Media from esophageal epithelial cells was placed upon esophageal fibroblasts. After 24 hours, fibroblast conditioned media was placed on fresh epithelial cells in the presence of TNFα inhibitory antibody. After 24 hours of stimulation, qRT-PCR was performed for LOX (B). Data represent 3 biologic replicates with similar results.

Figure 5:. Co-culture of esophageal fibroblasts and epithelial cells induces LOX expression.

FEF-tdTomato and EPC2-GFP were grown together for 24 hours and separated by FACS (A). qRT-PCR for LOX was performed on EPC2-hTERT (B) after co-culture and shown relative to monoculture (co-culture/mono-culture). This was validated with two independent primary esophageal epithelial cell cultures derived from patient biopsies, EPC-A and EPC-B. Anti-TNFα antibody was added to co-culture and qRT-PCR was performed for LOX (C). qRT- PCR for TNFα was performed on EPC2-hTERT (D) and FEF3 (E) after coculture. Data represent 3 biologic replicates with similar results. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Figure 6:. TNFα stimulation of TGFβ contributes to LOX expression.

TNFα stimulation of esophageal epithelial monolayer induces TGFβ expression (A), SMAD activity as measured by luciferase assay (B), increased SMAD phosphorylation (C), and nuclear localization of pSMAD3 (D). Inhibition of SMAD with SIS3 inhibited LOX production in TNFα stimulated EPC2hTERTs by immunoblot (E). Addition of anti-TGFβ antibody (1D11) to TNFα stimulated EPC2-hTERTs abrogates LOX expression (F). Isotype antibody used as control did not stimulate LOX expression (data not shown). Numbers under each band represent the signal intensity relative to NT set as 1. Data represent ≥2 biologic replicates with similar results. **p<0.01, ***p<0.001; NT- no treatment.