AB0046\u2005COMPARATIVE ANALYSIS OF TLRS AND INFLAMMASOMES GENES EXPRESSION IN DIFFERENT ETHIOLOGY ARTHRITIS AFTER STIMULATION WITH INFLAMMATORY STIMULI AND VITAMIN D

Abstract

Background: It has been shown that a variety of environmental and genetic factors, as well as deficiency of vitamin D plays a key role in outcomes of arthritis. Many studies have shown that the central feature of synovitis in rheumatoid arthritis (RA) is activated synovium fibroblasts (SF) that play a key role in expression and secretion of distinct patterns of inflammatory factors. Recent studies demonstrated that nucleotide-binding oligomerisation domain-like receptor (NLR) containing a PYRIN domain 1 (NLRP1) and NLRP3 inflammasomes as well as Toll-like receptors (TLR), may be important in pathogenesis of chronic autoimmune joint diseases such as RA and potentially in development of osteoarthritis (OA). Therefore, better understanding of the role of SF, TLRs and inflammasomes inflammatory pathways in different ethiology joint damage could make a significant contribution to the early disease prognosis, monitoring, and therapy. Objectives: A pilot study, to evaluate the effects of tumour necrosis factor α (TNFα), lipoteichoic acid (LTA), lipopolysaccharide (LPS), vitD on expression levels of TLR, inflammasomes, and vitD receptor (VDR) in human SF different ethiology knee damage: OA, RA, early arthritis (EA) (duration <12 months), healthy controls (HC) (after meniscus tear due to trauma). Methods: Synovial tissue and blood samples for vitD analysis were collected from patients undergoing joint replacement/artroscopic synovectomy surgery, following informed consent according to the permission Lithuanian Ethics Committee. The isolated cells were expanded in a monolayer and used between passages 2 and 4. The expression of VDR, TLR1, TLR2, TLR4, NLRP1, NLRP3 inflammasomes genes was analysed by qRT-PCR after 24h of stimulation with LTA, LPS, TNFα, vitD. Results: Analysis of gene expression results revealed that TNFα, LPS or LTA have no effect on TLR4 and TLR1 genes expression levels in SF. Downregulation of NLPR1 expression and upregulation of NLRP3 accompanied by enhanced expression of TLR2 was determined after stimulation with all factors, particularly TNFα. Highest upregulation of TLR2 was observed in RA and early arthritis patients, levels of other genes showed high variation between all patients, disrespectfully to diagnosis. Stimulation with TNFα resulted in 8-fold downregulation of VDR gene expression only in RA group, but not in OA, EA or HC. Stimulation with vitD had no effect on expression levels of studied genes in SFs in vitro, while the blood levels of vitD were neither associated with the ethiology of arthritis nor with VDR responses to stimulation with TNFα, LPS, LTA. Conclusion: We demonstrated downregulated expression of NLPR1, associated with increased levels of NLRP3 and TLR2 upon inflammatory stimuli in human articular SF from patients with arthritis of different ethiology. These data further support active involvement of those cells in inflammatory responses. Downregulated expression of VDR by TNFα in SF of RA patients implies altered signalling of vitD in the disease. References [1] Zao LR, et al. Mol Med Rep. 2018Apr17(4):5463-5469 [2] Guo C, et al. Clin Exp Immunol. 2018Nov;194(2):231-243 Acknowledgement: This study was supported by Sate Research Council grant S-MIP-17-12 Disclosure of Interests: Regina Sakalyte Grant/research support from: AbbVie Ivestigators Initiated Study grant., Jerroslav Denkovskij : None declared, Eiva Bernotiene: None declared, Algirdas Venalis: None declared, Gabriele Mourad: None declared, Sigita Stropuviene Grant/research support from: AbbVie Ivestigators Initiated Study grant., Narunas Porvaneckas: None declared, Vytautas Tutkus: None declared, Giedrius Kvederas: None declared, Irena Butrimiene: None declared