Background/Purpose: Release and insufficient removal of endogenous nucleic acids may be involved in triggering harmful autoimmune reactions important in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). Nucleic acid sensing molecules, such as the endosomal Toll-like receptors (TLRs) 3, 7 and 9, have been linked to pathogenic autoimmune processes, particularly in systemic lupus erythematosus, but their role in RA is less obvious. Data previously obtained in rats with pristane-induced arthrits (PIA) suggested involvement of TLR9 in the pathogenesis of this arthritis model (Hoffmann MH et al. J Autoimmun. 2011; 36:288). Interestingly, rats with PIA develop autoantibodies associated with RA including rheumatoid factor, anti-RA33 and antibodies to carbamylated proteins (Stoop JN et al. Ann Rheum Dis 2015; 74:949). It was therefore the aim of this study to gain more insight into the role of TLR9 in the pathogenesis of autoimmune arthritis by investigating the effects of TLR9 inhibition in rats with PIA.

Methods: Arthritis was induced in DA rats with the mineral oil pristane. Rats were treated with a TLR9 antagonist or a control oligonucleotide every other day, starting one before disease induction. Arthritis was scored using established scoring systems, inflammation and bone erosion were quantified by histological analysis. Expression of TLR9 and other nucleic acid sensing TLRs was quantified by RT-PCR and Western blotting; activation (phosphorylation) of various signal transduction molecules was determined by Western blotting. Furthermore, the role of TLR9 in osteoclast differentiation and activation was investigated in vitro using an established murine bone marrow culture system.

Results: Clinical signs of arthritis were significantly (p<0.05) reduced by 30-50% in animals treated with the TLR9 antagonist. Histological analyses revealed significantly (p<0.05) diminished inflammation, cartilage degradation, bone erosion and reduced numbers of osteoclasts in joints of animals treated with the TLR9 antagonist. Furthermore, serum levels of IL-6, AGP and RF were significantly decreased and expression and activation of NF-kB in lymph nodes appeared to be reduced. However, when treatment was started after onset of arthritis TLR9 inhibition had no effect on arthritis development and severity. Remarkably, mRNA levels of TLR7 and TLR9 strongly differed in the course of in vitro osteoclastogenesis. Whereas TLR7 expression did not change throughout osteoclastogenesis, expression of TLR9 was higher in precursor cells than in mature osteoclasts and partial inhibition of osteoclastogenesis was seen when cultures were exposed to the TLR9 antagonist, whereas a TLR7 antagonist was ineffective.

Conclusion: These results suggest a crucial role for TLR9 in the T cell-dependent initiation phase of PIA and thus important involvement of endogenous DNA presumably released during apoptosis and/or necrosis (induced by pristane in lymphoid organs) in the initiation of autoimmune arthritis and during osteoclastogenesis. The relevance of these findings for human RA needs to be further elucidated in future experiments.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/crucial-roles-for-toll-like-receptor-9-in-the-pathogenesis-of-erosive-autoimmune-arthritis-and-during-osteoclastogenesis/