Session Title: Sjögrenʼs Syndrome – Basic & Clinical Science Poster I
Session Type: Poster Session (Tuesday)
Session Time: 9:00AM-11:00AM
Background/Purpose: Primary Sjögren’s syndrome (SS) is a chronic autoimmune epithelitis characterized by the presence of autoantibodies against SS-related antigen A (SSA) and lymphocytic infiltration of exocrine glands. The underlying mechanisms in the initiation and perpetuation of SS remain to be fully elucidated. Recently, the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and pro-inflammatory cytokine Interleukin-18 (IL-18) have been implicated in the pathogenesis of SS. IL-18 serum concentrations were significantly higher in anti-SSA+ than in anti-SSA- SS patients and titers of anti-SSA antibodies were closely related with IL-18 protein levels. We hypothesized that immune complexes (ICs) containing anti-SSA antibodies (anti-SSA ICs) would lead to NLRP3 inflammasome activation and eventually to IL-18 production in an experimental model represented by the human salivary gland cell line A-253.
Methods: A-253 cells from human epidermoid carcinoma of the submaxillary gland were cultured. ICs were generated by incubation of cell culture supernatants (which contained released SSA autoantigens) with human anti-SSA antibody (OriGene Technologies, MD, USA), control human IgG and culture medium respectively for 1 hour and were stored at -80°C until used. A-253 cells were subjected to different stimulations for 4 hours. NLRP3, ASC, procaspase1 and IL-18 protein expression were measured by western blot and supernatant IL-18 level by enzyme-linked immunosorbent assay. Anti-SSA antibody and control human IgG were used at a final concentration of 80 ug/ml in all experiments. Results are presented as the mean ± S.D. The Student’s t-test was used for comparisons between two groups.
Results: A253 cells spontaneously released IL-18 and supernatant levels of IL-18 were significantly increased after stimulation with anti-SSA ICs, compared with control human IgG (p=0.021). Reactive oxygen species (ROS) scavengers, N-acetyl cysteine (NAC, 10mM), significantly decreased the secretion of IL-18 in each group (p < 0.05). More importantly, in the presence of NAC (10mM), anti-SSA ICs failed to induce IL-18 production from A-253 cells (p=0.19, compared to medium control). NLRP3 blockade by NAC were confirmed as NAC (25mM) treatment resulted in a marked decrease in the protein expression of the NLRP3 inflammasome and almost completely abolished the production of IL-18 from A-253 cells. Immunoprecipitation of NLRP3 was used to demonstrate that anti-SSA ICs potentiated coprecipitation of ASC and procaspase-1 proteins in A-253 cells. Furthermore, ICs containing anti-SSA antibodies and RNase A-digested cell culture supernatants (RNase A 100 ug/ml, incubation of 2 hours, for the removal of RNA from supernatants) decreased the amount of coprecipitated procaspase-1,secreted less IL-18 (p=0.02 compared to ICs containing no RNase A-digested supernatants) and lost the capacity to induce IL-18 production from A-253 cells (p=0.43, compared to medium control).
Conclusion: This novel study showed that ICs containing anti-SSA antibodies activated NLRP3 inflammasome and induced IL-18 secretion in A-253 cells. Endogenous RNA components of anti-SSA ICs and reactive oxygen species were responsible for this activation.
To cite this abstract in AMA style:Wu C, Li K, Yu C, Hsieh S. Autoantibodies from Sjögren’s Syndrome Enhance NLRP3 Inflammasome Activation and IL-18 Production in Human Salivary Gland Cell Line A-253 [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/autoantibodies-from-sjogrens-syndrome-enhance-nlrp3-inflammasome-activation-and-il-18-production-in-human-salivary-gland-cell-line-a-253/. Accessed May 30, 2020.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/autoantibodies-from-sjogrens-syndrome-enhance-nlrp3-inflammasome-activation-and-il-18-production-in-human-salivary-gland-cell-line-a-253/