Role of Redox-Active Trace Metal (RATM) Oxidants in the Regulation of Toll-Like Receptor 4 (TLR4) Signaling-Mediated Inflammatory Phenotype in Synovial Fibroblasts

Abstract

While the involvement of oxidant stress in rheumatoid arthritis (RA) has been suspected, the role of redox-active trace metals (RATM) as exogenous pro-oxidants in the pathogenesis of RA has not yet been investigated. Evidence suggests that oxidant-induced Toll-like receptor 4 (TLR4) activation plays a significant role in initiating “sterile” inflammation. Here, we investigate for the first time the role of RATM-induced oxidant stress in the molecular mechanism of the pathogenesis of RA. Potassium peroxychromate (PPC) (Cr⁺⁵), cuprous chloride (Cu⁺), and ferrous chloride (Fe⁺²) RATM agents were used as exogenous sources of reactive species. LPS-EK as a TLR4 specific agonist was used as a positive control for TLR4 activation. Given the importance of synovial fibroblasts in the development of RA, HIG-82, a rabbit fibroblast like-synoviocytes (FLS) cell line, was used as a model system in the studies proposed in this dissertation research. The expression of TLR4 in HIG-82 was confirmed by quantitative PCR (RT-PCR) and Western blots. Intracellular reactive oxygen species (iROS) production was visualized and quantified by fluorescence imaging microscopy and flow cytometry (FC), respectively. Activation of TLR4 signaling pathway was determined by measuring the expression of TLR4 and the downstream signaling proteins. Either ELISA kits or FC quantified levels of TNF-α, interleukin (IL-1β), and HMGB1 (as pro-inflammatory agents), and IL-10 (as an anti-inflammatory mediator) released into the culture medium. Proliferation index of FLS and examination of the effects of RATM on apoptosis and autophagy-related protein levels were quantified by FC and Western blots. We found that (1) RATM induced iROS production, which was attenuated by pretreatment.with antioxidants (2) Similar to TLR4 specific agonist LPS-EK, RATM significantly increased the activity of TLR4, which was blocked by pretreatment with TLR4 signaling inhibitor (CLI 095). (3) To our surprise, RATM increased proliferation of FLS and protected cells against apoptosis through activation of autophagy which is in agreement with the pathophysiological changes that occur in active RA. (4) RATM exogenous RS activate TLR4-mediated different down-stream signaling cascades that lead to an increased production of pro- and anti inflammatory mediators in FLS, and (5) Further studies reveal that RATM exogenous RS treatment increased the expression of all three major MAPK; (Extracellular signal-regulated protein kinase (ERK), the c-Jun N-terminal Kinase (JNK), and P38 MAPK pathways). Moreover, RATM concurrently increased the expression of AP-1 nuclear protein through TLR4 stimulation. Taken together, our findings indicate that TLR4 has mediated RATM induced inflammatory phenotype through AP-1 pathway activation in synovial fibroblasts. Therefore, oxidant stress through TLR4 activation may initiate and propagate inflammatory processes that maintain many chronic diseases. The design of dual-functioning antioxidants possessing both metal chelating and oxidative stress scavenging properties will be an essential milestone in pharmacotherapy and could help us live free of many chronic diseases. For the first time, we present evidence that supports a connection between exogenous and endogenous reactive species in enhancing inflammatory phenotype in synovial fibroblasts which is likely responsible for the initiation, propagation, and maintenance of RA.