Background: A strong crosstalk exists between endoplasmic reticulum (ER) stress and inflammation in synovium of arthritis patients. In addition to their primary role in protein folding, various ER chaperone proteins can contribute to cell survival, amplifying inflammation and immunogenicity when ER stress is triggered in pathological situations. Moreover, inflammation abrogates cell to cell contacts between macrophage-like synoviocytes, therefore disturbing the lining layer structure[1]. Simultaneously, fibroblast-like synoviocytes (FLS) take advantage of this loss of contact to infiltrate the subintima. Using a proteomic approach by mass spectrometry, eleven ER chaperone proteins (BiP, HYOU1, MANF, PDIA4, GANAB, HSP90B1, TXNDC5, DNAJB11, LMAN1, ERP29 and CALR) were identified in synovial membranes obtained from patients with osteoarthritis (OA), chronic pyrophosphate arthropathy (CPPA) or rheumatoid arthritis (RA)[2]. These studied proteins were highly correlated with an inflammatory histological scoring based on hyperplasia and leukocyte infiltration.

Objectives: This research aims at localizing and quantifying these proteins of interest by immunohistochemistry (IHC) in inflamed and non-inflamed synovial membranes. It also investigates the modulation of these ER protein expressions in fibroblast-like synoviocytes (FLS) under ER stress, pro-inflammatory and pro-fibrotic conditions.

Methods: Immunohistochemistry was performed on formalin-fixed paraffin-embedded (FFPE) biopsies obtained from synovial membranes collected at the same time as those used for the proteomic study: OA (n=9), CPPA (n=7) and RA (n=8). These synovial membranes exhibited continuous degree of inflammation throughout the three pathologies. Results were confirmed using the collagenase-induced osteoarthritis (CIOA) mouse models. FLS were provided from human synovial membrane of OA patients after prosthetic surgery for in vitro studies. FLS were stimulated with increasing doses (0.1 to 10 µg/mL) or timing stimulation (1, 3 or 7 days) of tunicamycin (Tm) to induce ER stress. Increasing timing of stimulation (1, 3 or 7 days) with TNF-α and TGF-ß were used to induce pro-inflammatory or pro-fibrotic condition, respectively. Cells extracts and supernatants were collected to assess ER chaperone proteins expression and secretion by western blotting (WB).

Results: Localization of the 11 ER chaperone proteins was mostly restricted to the lining layer in mild inflammation and in the whole synovium when the inflammation was severe. In RA synovium, all these proteins were further spotted in the sublining. Differential expression of some ER stress proteins (MANF, PDIA4, HSP90B, CALR, DNAJB11 and LMAN1) in synovitis was confirmed by using the CIOA mouse model. In vitro , all ER chaperone proteins (except BiP) were expressed in basal conditions by FLS. Tunicamycin-inducing ER stress enhanced the intracellular protein expression of BiP, HYOU1, MANF, PDIA4, HSP90B1, LMAN1 and CALR in FLS and their extracellular secretion (except for HYOU1, MANF and LMAN1). ERP29 was only detected as a secreted protein. Exposure to the proinflammatory mediator TNF-α increased the protein expression of Bip, HYOU1, MANF and PDIA4 expression, whereas induction of a profibrotic process with TGF-ß led to the upregulation of BiP, HYOU1, MANF and DNAJB11 expression in FLS.

Conclusion: This study highlights the delocalization of cells expressing ER chaperone proteins from the intima in non-inflamed synovial membranes to the subintima in the inflamed ones. Additionally, it highlights the increase of expression and secretion of some ER chaperone proteins by FLS under ER stress, pro-inflammatory and/or pro-fibrotic stimuli.

REFERENCES: [1] Culemann, S. et al. Locally renewing resident synovial macrophages provide a protective barrier for the joint. Nature 572 , 670–675 (2019).

[2] de Seny, D. et al. Proteins involved in the endoplasmic reticulum stress are modulated in synovitis of osteoarthritis, chronic pyrophosphate arthropathy and rheumatoid arthritis, and correlate with the histological inflammatory score. Sci Rep 10 , (2020).

Acknowledgements: NIL.

Disclosure of Interests: None declared.