Background/Purpose: Gastroesophageal reflux disease (GERD) is a very common manifestation of scleroderma (SSc), affecting as high as 90% of patients, second only to Raynaud’s phenomenon. GERD is also a very common in the general population, not dissimilarly from Raynaud’s phenomenon. The acid induced injury and repair of the distal esophageal mucosa is known to be a risk factor for metaplastic transformation of the epithelium (Barret esophagus) and in turn, increases the risk of esophageal cancer. Nonetheless, it is not known whether the cellular and molecular events induced by acid injury may have a role in the loss of immunological tolerance and/or other aspects of the pathogenesis of SSc. Here we aimed to study the morphological and molecular changes induced by acid exposure of esophageal cells to inform research on their role in the pathogenesis of SSc.

Methods: Normal squamous epithelial cells (Het1A) were exposed to acidified media (pH 4) contains 100μM bile salts for 10 minutes/day for five consecutive days. Media was acidified through the addition of 1M hydrochloric acid. Bile salts mix comprised glychocolic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid and deoxycholic acid. After 5 days of bile acid (BA) exposure cells grown in full media until day 21 to access their recovery from BA. Co-culture of healthy and SSc derived fibroblasts and Het1A cells was performed by Transwell assays and exposed to BA as described above. Twenty-four hours after the last treatment Het1A cells were lysed for protein and RNA analyses.

Results: Oesophageal epithelial cells exposed to acid lost their cobblestone appearance and acquired mesenchymal characteristics (change in orthogonal diameters ratio P< 0.0001). Further, cells lost the usual cell-cell junction. These changes were confirmed by qPCR and immunofluorescence which showed increased expression of EMT markers vimentin (p=0.001) and N-cadherin (p=0.05). Transcriptome analysis identified 3313 genes differentially expressed in the cells after chronic BA exposure (p< 0.05, t test). GO pathway analysis indicated upregulation of response to interferons, type I interferon signalling pathway, response to interleukin-1, compared with the control. qRT-PCR confirmed significant increased expression of STAT1 (p< 0.0001), OAS1 (p=0.0004), ISG15 (p=0.002) and IFIT1 (p< 0.0001). Co-culture of epithelial cells with SSc fibroblasts enhanced these changes with significant upregulation of both type I IFN and EMT genes compared to heatlhy fibroblasts. Following 16 days of further culture from last BA insult, showed normalisation of Type I IFN response vs persistent and enhanced EMT including increased expression of vimentin (p=0.001) and N-cadherin (p=0.0004).

Conclusion: Challenge with BA has a direct effect on the morphology and transcriptome of esophageal epithelial cells including proinflammatory and profibrotic gene expression. Co-culture with SSc fibroblasts enhances this response suggesting that as well in the context of a permissive genetic background, a common condition such as acid reflux may contribute to the pathogenesis of SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/acid-reflux-triggers-type-i-interferon-and-persistent-epithelial-mesenchymal-transition-in-esophageal-epithelial-cells-a-novel-microenvironment-contribution-to-the-pathogenesis-of-systemic-sclerosis/