Abstract Background/Aims Gastroesophageal reflux disease (GERD) is a common manifestation of scleroderma, second to Raynaud’s phenomenon. The acid induced injury of the distal oesophageal mucosa is known to increases the risk of oesophageal cancer. Nonetheless, it is unclear whether the cellular and molecular events induced by acid injury may contribute to the loss of immunological tolerance and/or other aspects of the pathogenesis of SSc. To determine the potential role of acid-induced injury as micro-environmental factor in the pathogenesis of SSc by investigating the effect of repeated acid exposure in oesophageal epithelial cells on innate immune and fibrotic response. Methods Acid treatment of oesophageal cells: Normal oesophageal epithelial cells (Het1A) were serum-starved in KSFM 1%FBS for 48 hours, then cells exposed to acidified media (pH 4.0- hydrochloric acid, 100μM bile salts) for 10 minutes daily for five consecutive days. 24 hours after the 5th BA exposure, cells were lysed for protein, RT-qPCR, and RNA-sequencing. Co-culture with healthy and SSc fibroblasts: Healthy and SSc fibroblasts grown on 0.4µm baskets above serum-starved Het1A cells. 10 minutes acid treatments done on Het1A cells for five consecutive days. 24 hours after the last treatment, Het1A cells were lysed for protein and RNA analyses. Results Het1A exposed to bile acid lost their cobblestone appearance and acquired mesenchymal characteristics (change in orthogonal diameters ratio p 0.0001). RT-qPCR and immunofluorescence imaging showed increased EMT markers: vimentin (p = 0.001) and N-cadherin (p = 0.05). Transcriptome analysis identified 788 genes differentially expressed (p 0.05, t test), with GO pathways enriched for type I interferon (IFN-I) signalling, response to TGF B and EMT, and oxidative stress in the cells after chronic BA exposure. RT-qPCR confirmed significant increased expression of STAT1 (p 0.0001), OAS1 (p = 0.0004), ISG15 (p = 0.002) and IFIT1 (p 0.0001). Cross-comparison of Het1 transcriptome with publicly available SSc oesophageal biopsy gene signatures showed a common upregulation of genes driving the SSc intrinsic inflammatory signature, proliferative and non-inflammatory signature. GO pathway analysis of the commonly upregulated genes suggested that acid reflux may contribute to several processes detected in SSc oesophageal biopsies. In vitro results confirm faster wound closure with BA (p 0.0001). Co-culture of Het1A cells with SSc fibroblasts enhanced both Type I IFN and EMT gene expression compared to cocultures with healthy dermal fibroblasts. Conclusion Challenge with BA has a direct effect on the morphology and transcriptome of oesophageal epithelial cells affecting pathways detected across the distinct molecular signatures of SSc, signatures present across the proinflammatory and profibrotic gene expression. Co-culture with SSc fibroblasts enhances this response, suggesting that in the context of a permissive genetic background, a common condition such as acid reflux may contribute to the pathogenesis of SSc. Disclosure S. Zahed Mohajerani: None. A. Altaie: None. M. Minerba: None. K. Man Suen: None. C. Black: Consultancies; Has received consultancy fees from FW Medical. Member of speakers’ bureau; Has received speakers fees from Takeda and Dr Falk. L. Bissell: Member of speakers’ bureau; being paid as a speaker (Abbvie, Alfasigma). Grants/research support; receiveing finanical grants (Alfasigma) . M. Hinchcliff: None. J. Ladbury: None. R. Ross: None. F. Del Galdo: None.
Mohajerani et al. (Wed,) studied this question.