Background: 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 (RP). GERD is also very common in the general population, not dissimilarly from RP. 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.

Objectives: Here we aimed to study the morphological and molecular changes induced by acid exposure of esophageal cells and compare them with the transcriptomics data from esophageal biopsies in SSc, to inform research on the role of esophageal acid induced microenvironment 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 and then cells exposed to acidified media contain bile salts (at pH 4.0- hydrochloric acid, 100μM bile salts in DMSO <0.1% in KSFM supplemented with 1% FBS) for 10 minutes. Exposure was repeated for five consecutive days. 24 hours after the 5th BA exposure, cells were lysed for protein and RNA analyses. An equal volume of DMSO was added to control conditions as a vehicle. This did not exceed a 0.1% (v/v) final concentration. Transcriptome profiling by bulk RNA-sequencing (RNA-seq): 24 hours after the 5th BA exposure on Het1A cells, total RNA was extracted and RNA-seq performed. In parallel experiments, following 5 days of bile acid (BA) exposure, Het1A cells were grown in full media (KSFM 10%FBS) until day 21 to assess their recovery from BA. Co-culture of Het1A cells with healthy and SSc fibroblasts: Healthy and SSc fibroblasts grown on 0.4µm baskets and placed on top of 6-well plate containing serum starved Het1A cells. 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 protein and RNA analyses.

Results: Esophageal epithelial cells (Het1A) exposed to bile acid (BA) lost their cobblestone appearance and acquired mesenchymal characteristics (change in orthogonal diameters ratio p<0.0001). Consistent with these data, RT-qPCR and immunofluorescence showed increased expression of EMT markers vimentin (p=0.001) and N-cadherin (p=0.05). Transcriptome analysis identified 867 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 control. 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-platform comparison of Het1 transcriptome with publicly available SSc esophageal biopsy gene signatures showed a common upregulation of EDN1 , IFI44 , IL1R1 , CASP1 , CD44 , CARD10 , SMAD1 , LYN , STAT3 , IL4R , IFI16 , FCGBP , all driving the SSc intrinsic inflammatory signature, as well as genes driving the proliferative signature, such as JUNB , ITGB5 , ACTB , CDK4 , CCNL2 , GRB2 AND CCND3 , and non-inflammatory signature including VCL , IL18 , PLD1 , KDM6A , ADCY9 , SP1 , ULK1 and BRAF . GO pathway analysis of the commonly upregulated genes suggested that acid reflux may contribute to several processes detected in SSc esophageal biopsies, including regulation of immune response, leukocyte activation, response to wounding, cellular response to stress and microtubule cytoskeleton. In vitro results confirm faster wound closure with BA (p<0.0001). In vitro co-culture of Het1A cells with SSc fibroblasts enhanced both Type I IFN and EMT gene expression compared to cocultures with healthy dermal 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 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.

REFERENCES: [1] Taroni Jaclyn N, Martyanov Viktor, Huang Chiang-Ching, Mahoney J. Matthew, Hirano Ikuo, Shetuni Brandon, Yang Guang-Yu, Brenner Darren, Jung Barbara, Wood Tammara A, Bhattacharyya Swati, Almagor Orit, Lee Jungwha, Sirajuddin Arlene, Varga John, Chang Rowland W, Whitfield Michael L, and Hinchcliff Monique. Molecular characterization of systemic sclerosis esophageal pathology identifies inflammatory and proliferative signatures. 2015. Arthritis Research & Therapy. DOI 10.1186/s13075-015-0695-1.

Acknowledgements: NIL.

Disclosure of Interests: Safoura Zahed Mohajerani: None declared, Ala Altaie: None declared, Marco Minerba: None declared, Christopher Black Takeda and Dr Falk, FW Medical, Lesley-Anne Bissell Abbvie, Alfasigma, Alfasigma, Monique Hinchcliff: None declared, John Ladbury: None declared, Rebecca Ross: None declared, Francesco Del Galdo: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.