Background: GERD (Gastroesophageal reflux disease) is a very common manifestation of SSc, affecting as high as 90% of patients, second only to Raynaud’s phenomenon. GERD is also a very common condition of the general population, often mild, self-limiting and not associated to any other disease. The acid induced injury and repair of the distal oesophageal mucosa is known to be a risk factor for metaplastic transformation of the epithelium (Barret Oesophagus) and in turn, increases the risk of oesophageal 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.

Objectives: Here we aimed to study in detail the morphological and molecular changes induced by acid exposure of oesophageal cells to inform research on their role in the pathogenesis of SSc.

Methods: Acid treatment of oesophageal cells:

Normal squamous epithelial cells (Het1A) were grown in Keratinocyte Serum-Free Medium (KSFM) supplemented with 10% (v/v) FBS, bovine pituitary extract (BPE; 50mg/ml) and EGF (5μg/ml).

Cells were serum starved with KSFM 1%FBS for 48 hours prior to start of acid exposure. Then cells exposed to acidified media (at pH 4.0) contains 100μM bile salts within KSFM 1% FBS for 10 minutes.

Media was acidified through the addition of 1M hydrochloric acid. 100μM bile salts mix comprising of glychocolic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid and deoxycholic acid. At the end of 10 minutes experimental period, cells were washed twice in PBS and lysed for protein and RNA analyses or re-incubated in KSFM 1%FBS and were treated with a 10-minute acid exposure for five consecutive days a week.

Healthy or SSc dermal fibroblasts were cocultured with Het1A through 0.4µm baskets and exposed to Acid treatment as described above. 10 minutes acid treatments done on Het1A cells only followed by two washes for five consecutive days. 24 hours after the last treatment Het1A cells were lysed for RNA (RNAseq and qRT-PCR) and protein (Western Blot) analyses.

Fully integrated digital inverted EVOS microscope was used to visualise oesophageal cells. 12 Images from 3 technical replicates were taken using 20X objective lenses for the purpose of quantification. The quantification was carried out from 3 biological replicates.

Results: Oesophageal epithelial cells exposed to acid lost their cobblestone appearance and acquired mesenchymal characteristics (acid treated cells diameter: 5.63µm compared to control cells diameter: 1.66µm/ P-value <0.0001). Further, cells lost the usual cell-cell junction.

Transcriptome analysis identified 1255 genes differentially expressed by acute acid injury (1 day) and 1605 genes specific to the cells after 5 days of acid exposure, with 775 genes in common between acute and chronic acid exposure.

GO pathway analysis of single acid exposure and 5 days acid exposure shows upregulation of response to virus, response to interferon-gamma, type I interferon signalling pathway, response to type I interferon, positive regulation of epithelial cell migration, response to interleukin-1, response to transforming growth factor beta, compared with the control.

Co-culture with SSc fibroblasts enhanced these changes with upregulation of both Type I IFN genes such as OAS1, and IFIT1 and EMT genes.

Conclusion: Challenge with Bile acid 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 int eh context of a permissive genetic background, a common condition such as acid reflux may contribute to the pathogenesis of SSc.

REFERENCES: [1] Bile Acid induced cellular and molecular changes of Oesophageal epithelial cells may contribute to the pathogenesis of Systemic Sclerosis. A pilot in vitro study.

[2] Safoura Zahed Mohajerani, John Ladbury, Rebecca Ross, Francesco Del Galdo.

Acknowledgements: NIL.

Disclosure of Interests: None declared.