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P035 Pharmacological inhibition of the canonical WNT signalling pathway represents a potential novel therapy for fibrosis in Crohn’s disease

A. Lewis*1, A. Nijhuis1, G. Berti1, C. L. Bishop2, R. Feakins3, J. O. Lindsay4, A. Silver1

1Blizard Institute, Barts and The London School of Medicine and Dentistry, Centre for Genomics and Child Health, London, UK, 2Blizard Institute, Barts and The London School of Medicine and Dentistry, Centre for Cell Biology and Cutaneous Research, London, UK, 3Department of Histopathology, The Royal London Hospital, London, UK, 4Blizard Institute, Barts and The London School of Medicine and Dentistry, Centre for Immunobiology, London, UK

Background

Intestinal fibrosis and subsequent stricturing does not respond to current therapies and is the main indication for surgery in Crohn’s disease (CD). Complete understanding of the underlying molecular mechanisms of fibrosis is required to uncover novel therapies. Transforming Growth Factor (TGF-β) signalling promotes intestinal fibrosis in CD and cross-talk between TGF-β and the Wingless (WNT) signalling pathway contributes to fibrosis in other organs. However, the role of the WNT pathway in CD fibrosis is not well characterised. In this study, we evaluate markers of WNT signalling in stricturing CD patients and assess the ability of ICG-001, a potent WNT inhibitor that disrupts β-catenin transcriptional complexes, to inhibit TGF-β/ WNT signalling and limit fibrosis in vitro.

Methods

TGF-β/WNT cross-talk was analysed in intestinal fibroblasts (CCD-18Co cells) stimulated with TGF-β (10 ng/ml) for 48 h in the presence or absence of ICG-001 (10 μM). β-Catenin and collagen I protein levels were assessed using immunofluorescence. Molecules within the WNT pathway modulated by TGF-β were identified using a targeted qRT-PCR array containing 92 WNT genes (TaqMan Array Human WNT Pathway). The mRNA levels of TGF-β / WNT markers were analysed by qPCR in the mucosa-overlying strictured intestine in CD patients.

Results

TGF-β increased nuclear β-catenin levels (1.94 fold, p < 0.001) and collagen I expression (1.28 fold, p = 0.008) in intestinal fibroblasts. Conversely, ICG-001 (10 μM) reduced β-catenin levels (p = 0.039), and collagen I expression (p = 0.006) in fibroblasts stimulated with TGF-β1, and inhibited fibroblast remodelling (p = 0.0306) and pro-collagen-Iα1 secretion/production (p = 0.0024) in a 3D organotypic model of the intestinal mucosa. TGF-β/WNT activation was associated with increased DKK1 mRNA expression in intestinal fibroblasts (7.66-fold, p = 0.002). DKK1 mRNA levels were higher in the mucosa overlying strictured intestine in CD patients (2.183-fold, p = 0.037). Experiments to determine the direct effects of DKK1 on collagen I production in intestinal fibroblasts are now required.

Conclusion

Increased WNT signalling in fibrotic strictures contributes to the development of TGF-β-induced fibrosis in CD patients. Treatment with ICG-001 can limit WNT signalling in vitro and represents a potential novel therapy for stricturing Crohn’s disease.