Background: Tissue-resident fibroblasts have emerged as a key cell type controlling the local tissue microenvironment in chronic and inflammatory diseases. The specific contribution of fibroblasts in the pathogenesis of Sjögren’s syndrome (SjS) remains incompletely understood.

Objectives: To characterize the inflammatory response of cultured human salivary gland-derived fibroblasts (SGF) from patients with SjS.

Methods: SGF were cultured form enzymatically digested minor salivary gland biopsies of patients with suspected SjS, including samples of patients with SjS and controls (sicca symptoms) that do not fulfil the SjS classification criteria. SGF were characterized by FACS analysis for the presence of fibroblast markers (CD90, PDPN) and the absence of epithelial (EPCAM) and lymphocyte (CD45) markers. SGF were treated with IL-1 (1 ng/µl) for 24h. Transcriptomes were analysed by RNA-sequencing (n=3/ group; Illumina NovaSeq 6000). Pathway enrichment analysis was conducted using Gene Onthology and Gene Set Enrichment Analysis (GSEA). Levels of cytokines and chemokines (BAFF, CLL2, CCL5, CX3CL1, CXCL10, IL6, IL8) in cell cultured supernatants were measured by ELISA. Proliferation of SGF was assessed by BrdU assay.

Results: SGF cultures (n=6) were positive for CD90 and PDPN and negative for EPCAM and CD45. SGF from patients with SjS (n=7) exhibited increased proliferation rates compared to SGF from sicca controls (n=5; p<0.048). We detected 81 differentially expressed genes (DEG; ± fold change > 1.5, FDR< 0.05) between SjS and sicca controls in IL1-stimulated SGF. Upregulated DEG in SjS were enriched in the biological processes (GO BP) “Signal transduction” and “Regulation of cell migration” “Positive chemotaxis” and “Mitotic cytokinesis”. Downregulated DEG in SjS were enriched in the GO BPs “Cell-cell signaling”, “Cell differentiation”, “Oxidative phosphorylation” and “Mesenchymal cell proliferation”. GSEA revealed an increased enrichment of “MYC targets” (FLB, SNRPD2), “oxidative phosphorylation” (UQCRH, UQCR10, ATP5MC2), “glycolysis”, “E2F targets”, “G2M checkpoint” (JPT1, HMGA1, SMARCC1), and “MTOR signaling” (CD9, NHERF1) in SjS SGF. We observed low levels of BAFF, CCL5, IL6 and IL8 in unstimulated SGF supernatants, with no difference between SjS and sicca controls; the levels of CCL2, CX3CL1 and CXCL10 were not detectable. Upon IL-1 stimulation, SGF exhibited a significant increase in the secretion of CCL2, IL6, and IL8 (all with p<0.0001), both in samples derived from patients with SjS (n=6) and sicca controls (n=5).

Conclusion: Our data point to an intrinsic activation of SjS SGF compared to control SGF, characterized by increased proliferation and differences in their transcriptomes in response to stimulation with IL-1. Local SGF activity in SjS may promote the vicious circle of chronic inflammation in salivary glands.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: Kerstin Klein: None declared, Ondrej Pastva: None declared, Matthias Brunner: None declared, Daniel Guggisberg: None declared, Marco Kreuzer: None declared, Larissa Moser: None declared, Marco Sprecher: None declared, Muriel Elhai: None declared, Rémy Bruggmann: None declared, Britta Maurer Boehringer-Ingelheim, GSK, Novartis, Otsuka, MSD, Novartis, Boehringer Ingelheim, Jannsen-Cilag, GSK, Novartis.