Expression of Toll-like receptor (TLR) 2, TLR3, TLR4 and TLR7 is increased in rheumatoid arthritis synovium and regulates cytokine production by dendritic cells upon stimulation of TLR specific pathways

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by inflammation of the synovial joints, which leads to joint destruction. Dendritic cells (DC) are specialized antigen presenting cells, which are activated by 'pathogen associated molecular patterns' via toll-like receptors (TLRs). TLR3 and TLR7 are important in the recognition of viral RNA, whereas TLR2 and TLR4 are involved in the recognition of bacterial components and host-derived molecules, including heat shock proteins, fibronectin, hyaluronic acid. TLR and DC are critical in the regulation of immune responses.

To determine the expression of TLR2, TLR3, TLR4 and TLR7 in synovial biopsies and to evaluate the potential differences in DC maturation and cytokine production upon TLR-mediated triggering of cells from RA patients and healthy controls.

The presence of TLR2, TLR3, TLR4 and TLR7 in synovial tissue, from RA patients, osteoarthritis patients and healthy controls was studied with immunohistochemistry techniques. Furthermore, monocyte-derived dendritic cells from RA patients and healthy controls were cultured for 6 days and subsequently stimulated via specific TLR pathways; TLR2 (pam₃cys), TLR3 (poly(IC)), TLR4(LPS) and TLR7 (R848). We determined the expression of cell surface markers (CD14, CD80, CD83, CD86, MHCI and MHCII) by flow cytometry and tumour necrosis factor alpha, IL-6, IL-10 and IL-12 production was measured by multiplex cytokine bead arrays.

TLR2, TLR3, TLR4 and TLR7 expression was markedly increased in synovial tissue from RA patients, compared with synovial tissue from osteoarthritis patients and healthy controls. After stimulation via specific TLR pathways, flow cytometry showed a clear DC maturation upon stimulation via all TLR pathways without any difference between RA cells and cells from healthy controls. However, cytokine production by DC from RA and controls was clearly different. RA DC produced increased levels of the proinflammatory cytokines tumour necrosis factor alpha and IL-6 upon stimulation of TLR2 or TLR4. In contrast, such differences were not noted after TLR3 and TLR7 stimulation. Triggering of TLR3 and TLR7 pathways resulted in increased production of IL-12, which was equal between RA and control DC. Intriguingly, simultaneous stimulation of TLR3/4, TLR3/7 and TLR4/7 pathways resulted in a marked synergy with respect to the production of proinflammatory mediators.

These results point out that despite a comparable DC phenotype, RA cells produce higher cytokine levels upon stimulation of TLR2 and TLR4 compared with healthy controls. The combination of specific TLR stimulations resulted in a distinguished synergy in cytokine production. Here, we postulate the hypothesis that the persistence of viral material in synovial tissue triggers the TLR3 and TLR7 pathways, which subsequently results in inflammation and release of endogenous ligands, resulting in TLR4 stimulation that leads to a vicious circle of synovial inflammation.

Affiliations

1. Department of Rheumatology, Rheumatology Research and Advanced Therapeutics, University Medical Center Nijmegen, The Netherlands
   • MF Roelofs
   • , LAB Joosten
   • , S Abdollahi-Roodsaz
   • , AWT van Lieshout
   • , TRDJ Radstake
   •  & WB van den Berg

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