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Abstract
Objectives The RNA epitranscriptomic modification known as N6-methyladenosine (m6A) represents a novel mechanism of gene regulation that is poorly understood in human autoimmune diseases. Our research explores the role of this RNA m6A modification in salivary gland epithelial cells (SGEC) and its impact on the pathogenesis of Sjögren’s disease (SjD).
Methods SGECs from SjD patients and controls were analysed for m6A writers METTL3 and METTL14 expression using RNA-seq, quantitative PCR and immunohistochemistry. Functional assays assessed the impact of METTL3 knockdown or pharmacological inhibition on proinflammatory gene expression and immune cell interactions (using transwell and coculture systems). Mechanistic studies examined METTL3-mediated m6A modifications in double-stranded RNA (dsRNA) formation through immunofluorescence. Unsupervised clustering identified patterns of interferon activation in salivary glands and their correlation with m6A writers.
Results METTL3 and METTL14 were elevated in SGEC from SjD patients in comparison to controls. Paradoxically, inhibiting METTL3 increased proinflammatory gene expression, enhancing SGEC’s ability to attract immune cells and activate B cells. Conversely, inhibiting the eraser FTO had the opposite effect. METTL3-mediated m6A modifications prevented dsRNA formation and IFN signalling activation. SGEC from SjD showed insufficient METTL3 upregulation compared with controls in response to inflammatory triggers, indicating a limited capacity to regulate the inflammatory response. SjD patients with elevated disease activity and higher interferon signature exhibit reduced METTL3 expression.
Conclusions Impairment of m6A modifications in SGEC in response to inflammatory triggers favour the formation of dsRNA, potentially amplifying the interferon loop and contributing to SjD pathogenesis.
- Autoimmune Diseases
- Cytokines
- Inflammation
- Sjogren's Syndrome
- Autoimmunity
Data availability statement
Data are available in a public, open access repository. Data are available on reasonable request. NS-SV-TT-AC and NS-SV-TT-DC cells are available by MTA from the National Heart, Lung and Blood Institute (NHLBI) and the National Institute of Dental and Craniofacial Research (NIDCR). STM2457, STM2120 and STM3006 are under MTA from STORM THERAPEUTICS LTD. All other reagents are commercially available. RNAseq data generated in this study are available on reasonable request, rami.bechara@universite-paris-saclay.fr. RNAseq data from these studies GSE23117 (48), GSE154926 (49), GSE157159 (50), GSE173808 (51) are publicly available.
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Data availability statement
Data are available in a public, open access repository. Data are available on reasonable request. NS-SV-TT-AC and NS-SV-TT-DC cells are available by MTA from the National Heart, Lung and Blood Institute (NHLBI) and the National Institute of Dental and Craniofacial Research (NIDCR). STM2457, STM2120 and STM3006 are under MTA from STORM THERAPEUTICS LTD. All other reagents are commercially available. RNAseq data generated in this study are available on reasonable request, rami.bechara@universite-paris-saclay.fr. RNAseq data from these studies GSE23117 (48), GSE154926 (49), GSE157159 (50), GSE173808 (51) are publicly available.
Footnotes
Handling editor Josef S Smolen
FT and AA-H contributed equally.
Contributors Conceptualisation: RB, AA-H and FT; Methodology: FT, AA-H, HS, JP, PM, ER, SE-S-S, LF, SL, LB, CM, AD, SV, GN, XM and RB; Investigation: FT, AA-H, HS, JP, PM, ER, SE-S-S, LF, SL, LB, CM and RB; Writing–original draft: RB; Writing–review and editing: AA-H, FT, HS, JP, PM, ER, SE-S-S, LF, SL, LB, CM, AD, SV, GN and XM; Funding acquisition: RB; Supervision, RB. RB was responsible for the overall content as the guarantor. All authors reviewed and approved of the final manuscript.
Funding This work received funding from the ANR-JCJC Program (ANR-22-CE15-0031) by the French National Research Agency, the France 2030 program (ANR-11-IDEX-0003) from the OI HEALTHI initiative at Université Paris-Saclay and the UPS IDEX AAP TECHNICS 2023 (R23097LL) grant from the HEADS Graduate School at Université Paris-Saclay and from FRM for the project PREMSS (R23073LL). AA-H is the recipient of a PhD fellowship from Arthritis Foundation Courtin, Arthritis R&D (CIFRE 2023/005). HS is the recipient of the M2 fellowship from OI HEALTHI at Université Paris-Saclay (IDEX HTI 2021-06).
Competing interests XM received an honorarium for consultancy advice on Sjögren’s disease from Argenx, BMS, GSK, Novartis and Otsuka. The rest of the authors declare that they have no relevant conflicts of interest.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.
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