Background/Purpose: Fibroblast-like synoviocytes (FLS) are key players involved in the production of inflammatory mediators that trigger joint tissue damage in inflammatory arthritis (IA). The most important mechanism proposed for FLS activation is the NF-κB-dependent signalling pathway. Inhibitor kappa B (IκB) kinase 2 (IKK2) plays a critical role in the activation of NF-κB, resulting in transcription of several inflammation-associated genes. The aim of this research was the characterization of a constitutive IKK2 activation (IKK2ca) in FLS as a strategy to study tissue-specific inflammation in the joint.

Methods: In order to acquire new information regarding pathological pathways underlying IA, we develop an R26Stop^FLikk2ca-Prg4^CreERT2 mouse model, which expresses a constitutive active form of IKK2 restricted to Prg4-expressing cells in the joints. Cre-recombinase activation was induced by tamoxifen (TMX) intraperitoneal injection to 2-month old mice. Samples were collected after 4 weeks of TMX injection. Isolation of FLS was performed and NF-κB signalling pathway activation was analyzed by RT-qPCR and western blot. Additionally, flow cytometry was carried out to identify IKK2ca FLS population (Prg4 red cells). FLS at passages 2 to 4 were used for all experiments. R26-tdTomato-Prg4^CreERT2 and TNF^tg/0rtTA^tg/0 FLS were taken as Wild type (WT) for RT-qPCR, western blot, and FACS analysis.

Results: Preliminary results showed high expression of IKK2 in IKK2ca FLS compared with WT FLS, as well as decreased expression of IκBα in the cytoplasm because of IKK2 constitutive activation. However, we did not observe differences in RelA nuclear expression between IKK2ca and WT FLS. In addition, we identify higher expression of Il1b, Il6, Nanog, and Sox2 genes in IKK2ca than in WT FLS, which is consistent with the inflammatory state of IKK2ca FLS established in IA. Regarding FACS results, we identified a specific synovial sub-lining inflammatory population of IKK2ca-Prg4 CD14^– Thy1⁺ tdTomato⁺ FLS, which has reported as key player in IA.

Conclusion: Constitutive IKK2 activation in FLS induces an inflammatory phenotype characterized by increased expression of proinflammatory mediators, and represents an interesting strategy to study tissue-specific inflammation in the joint.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/constitutive-inhibitor-kappa-b-i%ce%bab-kinase-2-ikk2-activation-induces-an-inflammatory-state-in-fibroblast-like-synoviocytes/