Background/Purpose: A strong crosstalk exists between endoplasmic reticulum (ER) stress and synovitis. Several ER chaperone proteins, besides their function in protein folding, can play a role in cell survival enhancing inflammation and immunogenicity once ER stress occurs in pathological conditions. This research aims at localizing and quantifying ER stress proteins (BiP, HYOU1, MANF, PDIA4, GANAB, HSP90B1, TXNDC5, DNAJB11, LMAN1, ERP29 and CALR) in inflamed and non-inflamed synovial membranes, as well as assessing their cellular localization. It also investigates the modulation of these ER protein expressions in fibroblast-like synoviocytes (FLS) under ER stress, profibrotic and proinflammatory conditions.

Methods: Immunohistochemistry was performed on formalin-fixed paraffin-embedded synovial tissues from patients with osteoarthritis (OA; n=9), chronic pyrophosphate arthropathy (n=7), and rheumatoid arthritis (n=8), exhibiting continuous degree of inflammation. These results were confirmed using the collagenase-induced osteoarthritis (CIOA) mouse models. Protein localization and semi-quantification were done using QuPath. Imaging mass cytometry (IMC) was performed on the biopsies but were conjugated with metal antibodies recognizing synovial cells (FLS, immune cells, …) and the 11 ER stress proteins, followed by laser desorption and time-of-flight mass spectrometry to detect and quantify the spatial distribution of multiple proteins simultaneously. FLS were isolated from OA patient synovium post-surgery and stimulated in vitro with tunicamycin (0.1–10 µg/mL, 1–7 days) to induce ER stress. TNF-α and TGF-β were used to mimic inflammatory or fibrotic conditions. Protein expression and secretion were assessed by western blot on cell lysates and supernatants. Fibroblasts markers (CD55 and CD34) expression was also evaluated by immunofluorescence.

Results: ER stress proteins were mostly restricted to the lining layer in mildly inflamed synovium and infiltrated the whole sCollynovium when the inflammation was severe. Their differential expression was confirmed in a CIOA mice model. This spatial distribution mirrors the fibroblast phenotype shift, with CD55⁺ (lining) fibroblasts predominating in non-inflamed OA and declining with inflammation, while CD34⁺ (sublining) fibroblasts, enriched in inflamed OA and RA, become prominent in the sublining. IMC confirmed the co-expression of ER stress proteins with CD55⁺ fibroblasts in the lining layer of non-inflamed OA, and with CD34⁺ fibroblasts in the sublining layer of inflamed OA. ER stress proteins do not co-express with lymphocytes markers. In vitro, these proteins were observed under basal conditions in FLS, which expressed CD55 and CD34. ER stress induction increased their intracellular levels and, for some, their secretion. Inflammatory (TNF-α) and profibrotic (TGF-β) stimuli further enhanced their expression.

Conclusion: These findings suggest that fibroblasts infiltrating the synovial membrane—particularly CD34⁺ cells—may play a central role in the pathogenesis of synovial inflammation, acting as secretory cells of the chaperone proteins which may exhibit pro-inflammatory and pro-fibrotic properties.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/coexpression-and-infiltration-of-endoplasmic-reticulum-stress-proteins-together-with-cd34-fibroblast-like-synoviocytes-in-human-inflamed-synovial-membranes/