Background/Purpose: The NLRP3 inflammasome is a multi-protein complex activated in response to environmental pathogens. This results in caspase-1-dependant cleavage of pro-IL-1β and IL-18 to their active and mature form, induction of which leads to a variety of biological effects associated with infection, inflammation and autoimmunity. Cytokine Release Inhibitory Drug 3 (CRID3) is a novel inhibitory molecule thought to be active in the NLRP3 pathway. In this study, we examined the effects of Toll-like Receptor 2 on inflammasome activation and cytokine secretion. Furthermore, the role of CRID3 on the NLRP3 pathway in the RA joint was examined.

Methods: Rheumatoid Arthritis ex vivo synovial tissue biopsies were cultured in the presence or absence of synthetic Toll-like Receptor 2 agonist Pam3CSK4 (1 µg/mL). Expression of inflammasome component NLRP3 in addition to the pro and mature/active forms of IL-1β and IL-18 were assessed by Taqman PCR and ELISA. TLR-2-induced pro-inflammatory cytokine secretion (IL-6, IL-8, TNFα) was also assessed by ELISA. RA synovial biopsies were incubated in the presence or absence of CRID3 (100 nM), a novel inhibitory compound which is thought to act on or closely to NLRP3. Expression of NLRP3, IL-1β and IL-18 expression was analysed by Taqman PCR and ELISA. The effect of CRID3 on ex vivo pro-inflammatory cytokine secretion (IL-6, IL-8, TNFα) was also determined by ELISA.

Results: TLR2 stimulation significantly induced transcript levels of NLRP3 and pro IL-1β and IL-18 in RA ex vivo synovial tissue biopsies, a model which maintains cell-cell contact, preserves the synovial architecture and closely reflects the in vivo environment.  Additionally, protein expression of IL-1β and IL-18 was increased in comparison to basal control.  Pam3CSK4 significantly increased secretion of pro-inflammatory cytokines IL-6, IL-8 and TNFα. Incubation of synovial biopsies with CRID3 resulted in a decrease of pro IL1β, which was mirrored by a decrease in active IL-1β and NLRP3 expression. Spontaneous release of IL-6, IL-8 and TNFα were also reduced following CRID3 treatment.

Conclusion:  TLR2 signalling induces NLRP3 activation in the RA joint, resulting in the secretion of inflammasome related cytokines IL-1β and IL-18. Additionally, we show for the first time that CRID3 is a novel compound which inhibits inflammasome activity in RA, and subsequent induction of pro-inflammatory pathways.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/novel-compound-cytokine-release-inhibitory-drug-3-crid3-inhibits-the-nlrp3-inflammasome-in-rheumatoid-arthritis/