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.
Table of Contents
Introduction -- Review of literature and background -- Central hypothesis and specific aims -- General materials and methods -- Redox-active trace metals (RATM) as pro-oxidants -- Effect of RATM on HMGB1 release and markers of inflamatory phenotypes -- Effect of RATM on cell proliferation and cell death -- Effect of RATM pro-oxidants on TLR4-coupled AP-1 signaling pathway -- Research summary, conclusions, and future directions -- Appendix
Degree
Ph.D. (Doctor of Philosophy)