Redox-active trace metal-induced release of high mobility group box 1(HMGB1) and inflammatory cytokines in fibroblast-like synovial cells is Toll-like receptor 4 (TLR4) dependent

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Highlights

  • ROS can activate TLR4 to initiate inflammatory phenotypes in fibroblast-like synovial cells (FLS).

  • We used FLS as a model for studying the initiation of the mechanism of RA through ROS/TLR4-coupling.

  • We used redox-active trace metals as sources of ROS, which was attenuated by pretreatment with antioxidants.

  • ROS/TLR4-coupling increased release of HMGB1, cytokines, and MRP8/14, as inflammatory markers.

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune systemic inflammatory disease that is characterized by synovial inflammation and bone erosion. We have investigated the mechanism(s) by which essential trace metals may initiate and propagate inflammatory phenotypes in synovial fibroblasts. We used HIG-82, rabbit fibroblast-like synovial cells (FLS), as a model system for potentially initiating RA through oxidative stress. We used potassium peroxychromate (PPC, Cr+5), ferrous chloride (FeCl2, Fe+2), and cuprous chloride (CuCl, Cu+) trace metal agents as exogenous pro-oxidants. Intracellular ROS was quantified by fluorescence microscopy and confirmed by flow cytometry (FC). Protein expression levels were measured by western blot and FC, while ELISA was used to quantify the levels of cytokines. Trace metal agents in different valence states acted as exogenous pro-oxidants that generate reactive oxygen species (ROS), which signal through TLR4 stimulation. ROS/TLR4- coupled activation resulted in the release of HMGB1, TNF-α, IL-1β, and IL-10 in conjunction with upregulation of myeloid-related protein (MRP8/14) inflammatory markers that may contribute to the RA pathophysiology. Our results indicate that oxidant-induced TLR4 activation can release HMGB1 in combination with other inflammatory cytokines to mediate pro-inflammatory actions that contribute to RA pathogenesis. The pathway by which inflammatory and tissue erosive changes may occur in this model system possibly underlies the need for functioning anti-HMGB1-releasing agents and antioxidants that possess both dual trace metal chelating and oxidant scavenging properties in a directed combinatorial therapy for RA.

Abbreviations

RATM
redoxactive trace metals
RA
rheumatoid arthritis
FLS
fibroblast-like synovial cells
MRP8/14
myeloid-related protein 8/14
HMGB1
high mobility group box 1
iROS
intracellular reactive oxygen species
PPC
potassium peroxychromate
FC
flow cytometry
FM
fluorescence microscopy
DAMPs
damage associated molecular patterns
PAMPs
pathogen-associated molecular patterns
TLR4
Toll-like receptor 4
ELISA
enzyme-linked immunosorbent assay
LDH
Lactate Dehydrogenase
ANOVA
analysis of variance

Keywords

Fibroblast-like synovial cells
Redox active-trace metals
Rheumatoid arthritis
Oxidative stress
Inflammatory cytokines
Myeloid-related protein (MRP8/14)

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The project described was supported by Award Number DE021888 from the National Institute of Dental & Craniofacial Research to OJI. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Dental & Craniofacial Research of the US National Institutes of Health.

1

AA performed these experiments as a partial fulfillment for a doctoral degree requirement.