Therapeutic efficacy of Tyro3, Axl, and Mer tyrosine kinase agonists in collagen-induced arthritis.

OBJECTIVE: Hyperactivation of innate immunity by Toll-like receptors (TLRs) can contribute to the development of autoinflammatory or autoimmune diseases. This study evaluated the activation of Tyro3, Axl, Mer (TAM) receptors, physiologic negative regulators of TLRs, by their agonists, growth arrest-specific protein 6 (GAS-6) and protein S, in the prevention of collagen-induced arthritis (CIA). METHODS: Adenoviruses overexpressing GAS-6 and protein S were injected intravenously or intraarticularly into mice during CIA. Splenic T helper cell subsets from intravenously injected mice were studied by flow cytometry, and the knee joints of mice injected intravenously and intraarticularly were assessed histologically. Synovium from mice injected intraarticularly was evaluated for cytokine and suppressor of cytokine signaling (SOCS) expression. RESULTS: Protein S significantly reduced ankle joint swelling when overexpressed systemically. Further analysis of knee joints revealed a moderate reduction in pathologic changes in the joint and a significant reduction in the number of splenic Th1 cells when protein S was overexpressed systemically. Local overexpression of GAS-6 decreased joint inflammation and joint pathology. Protein S treatment showed a similar trend of protection. Consistently, GAS-6 and protein S reduced cytokine production in the synovium. Moreover, levels of messenger RNA for interleukin-12 (IL-12) and IL-23 were reduced by GAS-6 and protein S treatment, with a corresponding decrease in the production of interferon-γ and IL-17. TAM ligand overexpression was associated with an increase in SOCS-3 levels, which likely contributed to the amelioration of arthritis. CONCLUSION: This study provides the first evidence that TAM receptor stimulation by GAS-6 and protein S can be used to ameliorate arthritis when applied systemically or locally. TAM receptor stimulation limits proinflammatory signaling and adaptive immunity. This pathway provides a novel strategy by which to combat rheumatoid arthritis.

Type I interferons might form the link between Toll-like receptor (TLR) 3/7 and TLR4-mediated synovial inflammation in rheumatoid arthritis (RA).

BACKGROUND: Rheumatoid arthritis (RA) has been associated with an increased risk of infections, but the underlying pathways have not yet been identified. Toll-like receptors (TLR) probably play a role in synovial inflammation and may also contribute to the understanding of the role of infections in RA. OBJECTIVES: To investigate if the synovial expression of TLR3 and TLR7 in RA correlates with that of inflammatory cytokines, and to assess whether this has functional consequences for local cytokine production and to study potential links between the TLR3/7 axis and TLR4 in RA synovium. METHODS: Immunohistochemistry was used to study the expression of TLR3, TLR7, interferon alpha (IFNalpha), tumour necrosis factor alpha (TNFalpha) and interleukins IL1beta, IL12, IL17 and IL18 in RA synovium obtained by arthroscopy from 34 patients with RA. Monocytes, monocyte-derived dendritic cells (MoDCs) and RA synovial fibroblasts were stimulated via TLR3 (poly-IC) and TLR7 (loxorubin), after which IL1beta, IL6 and TNFalpha were measured by Luminex bead array technology. Following preincubation with IFNalpha, IL1beta and IL18, TLR3 and TLR7 mRNA expression was assessed using real-time PCR. Cytokine production after preincubation with IFNalpha and subsequent TLR stimulation was measured. RESULTS: Synovial TLR3/7 expression was co-expressed with IFNalpha, IL1beta and IL18, but not with TNFalpha, IL12 and IL17. Stimulation of TLR3/TLR7 on monocytes, MoDCs or synovial fibroblasts led to secretion of type I IFN but no biologically active IL1beta or IL18 could be detected. Type I IFNalpha increased TLR3/7 mRNA expression whereas IL1beta and IL18 did not. In spite of the fact that the mRNA level of TLR4 remained unchanged, IFNalpha enhanced the response to TLR4 agonists, a phenomenon that was clearly more marked in patients with RA. CONCLUSION: Type I interferons are highly co-expressed with TLR3/TLR7 in RA synovium. They enhance TLR3/TLR7-mediated cytokine production and also TLR4-mediated responses.

Stimulation of chondrocyte-mediated cartilage destruction by S100A8 in experimental murine arthritis.

OBJECTIVE: To investigate whether S100A8 is actively involved in matrix metalloproteinase (MMP)-mediated chondrocyte activation. METHODS: S100A8 and S100A9 proteins were detected in inflamed knee joints from mice with various forms of murine arthritis, using immunolocalization. Murine chondrocyte cell line H4 was stimulated with proinflammatory cytokines or recombinant S100A8. Messenger RNA (mRNA) and protein levels were measured using reverse transcriptase-polymerase chain reaction and intracellular fluorescence-activated cell sorting (FACS). Breakdown of aggrecan on the pericellular surface of the chondrocytes was measured using VDIPEN and NITEGE antibodies and FACS, and breakdown in patellar cartilage was measured by immunolocalization. RESULTS: S100A8 and S100A9 proteins were abundantly expressed in and around chondrocytes in inflamed knee joints after induction of antigen-induced arthritis or onset of spontaneous arthritis in interleukin-1 (IL-1) receptor antagonist-knockout mice. Stimulation of chondrocytes by the proinflammatory cytokines tumor necrosis factor alpha, IL-1beta, IL-17, and interferon-gamma caused strong up-regulation of S100A8 mRNA and protein levels and up-regulation to a lesser extent of S100A9 levels. Stimulation of chondrocytes with S100A8 induced significant up-regulation of MMP-2, MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5 mRNA levels (up-regulated 4, 4, 3, 16, 8, and 4 times, respectively). VDIPEN and NITEGE neoepitopes were significantly elevated in a concentration-dependent manner in chondrocytes treated with 0.2, 1, or 5 microg/ml of S100A8. (VDIPEN levels were elevated 17%, 67%, and 108%, respectively, and NITEGE levels were elevated 8%, 33%, and 67%, respectively.) S100A8 significantly increased the effect of IL-1beta on MMP-3, MMP-13, and ADAMTS-5. Mouse patellae incubated with both IL-1beta and S100A8 had elevated levels of NITEGE within the cartilage matrix when compared with patellae incubated with IL-1beta or S100A8 alone. CONCLUSION: These findings indicate that S100A8 and S100A9 are found in and around chondrocytes in experimental arthritis. S100A8 up-regulates and activates MMPs and aggrecanase-mediated pericellular matrix degradation.

The expression of toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells.

OBJECTIVE: To evaluate the expression of Toll-like receptors (TLRs) 3 and 7 in synovium and to study potential differences in the maturation and cytokine production mediated by TLR-2, TLR-3, TLR-4, and TLR-7/8 by dendritic cells (DCs) from rheumatoid arthritis (RA) patients and DCs from healthy controls. METHODS: Synovial expression of TLR-3 and TLR-7 in RA was studied using immunohistochemistry. Monocyte-derived DCs from RA patients and healthy controls were cultured for 6 days and subsequently stimulated for 48 hours via TLR-mediated pathways (lipoteichoic acid, Pam(3)Cys, and fibroblast-stimulating lipopeptide 1 for TLR-2, poly[I-C] for TLR-3, lipopolysaccharide and extra domain A for TLR-4, and R848 for TLR-7/8). Phenotypic DC maturation was measured using flow cytometry. The secretion of tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), IL-10, and IL-12 was measured using the Bio-Plex system. Cell lines expressing TLR-2 and TLR-4 were used for the detection of TLR-2 and TLR-4 ligands in serum and synovial fluid from RA patients. RESULTS: TLR-3 and TLR-7 were highly expressed in RA synovium. All TLR ligands elicited phenotypic DC maturation equally between DCs from RA patients and those from healthy controls. TLR-2- and TLR-4-mediated stimulation of DCs from RA patients resulted in markedly higher production of inflammatory mediators (TNFalpha and IL-6) compared with DCs from healthy controls. In contrast, upon stimulation of TLR-3 and TLR-7/8, the level of cytokine production was equal between DCs from RA patients and those from healthy controls. Remarkably, both TLR-3 and TLR-7/8 stimulation resulted in a skewed balance toward IL-12. Intriguingly, the combined stimulation of TLR-4 and TLR-3-7/8 resulted in a marked synergy with respect to the production of inflammatory mediators. As a proof of concept, TLR-4 ligands were increased in the serum and synovial fluid of RA patients. CONCLUSION: TLRs are involved in the regulation of DC activation and cytokine production. We hypothesize that various TLR ligands in the joint trigger multiple TLRs simultaneously, favoring the breakthrough of tolerance in RA.