TLRs may contribute to the progression of rheumatoid arthritis through recognition of microbial or hostderived ligands found in arthritic joints. Here, we show that TLR2 and TLR4, but not TLR9, are involved in the pathogenesis of autoimmune arthritis and play distinct roles in the regulation of T cells and cytokines. We investigated the involvement of TLR2, TLR4, and TLR9 in the progression of arthritis using IL-1 receptor antagonist-knockout (IL1rn -/-) mice, which spontaneously develop an autoimmune T cell-mediated arthritis. Spontaneous onset of arthritis was dependent on TLR activation by microbial flora, as germ-free mice did not develop arthritis. Clinical and histopathological evaluation of IL1rn -/-Tlr2 -/-mice revealed more severe arthritis, characterized by reduced suppressive function of Tregs and substantially increased IFN-γ production by T cells. IL1rn -/-Tlr4 -/-mice were, in contrast, protected against severe arthritis and had markedly lower numbers of Th17 cells and a reduced capacity to produce IL-17. A lack of Tlr9 did not affect the progression of arthritis. While any therapeutic intervention targeting TLR2 still seems complicated, the strict position of TLR4 upstream of a number of pathogenic cytokines including IL-17 provides an interesting potential therapeutic target for rheumatoid arthritis.

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 ␣ (TNF␣), 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 (TNF␣ 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.

Objective. Degeneration of extracellular matrix of cartilage leads to the production of molecules capable of activating the immune system via Toll-like receptor 4 (TLR-4). The objective of this study was to investigate the involvement of TLR-4 activation in the development and progression of autoimmune destructive arthritis.Methods. A naturally occurring TLR-4 antagonist, highly purified lipopolysaccharide (LPS) from Bartonella quintana, was first characterized using mouse macrophages and human dendritic cells (DCs). Mice with collagen-induced arthritis (CIA) and mice with spontaneous arthritis caused by interleukin-1 receptor antagonist (IL-1Ra) gene deficiency were treated with TLR-4 antagonist. The clinical score for joint inflammation, histologic characteristics of arthritis, and local expression of IL-1 in joints were evaluated after treatment.Results. The TLR-4 antagonist inhibited DC maturation induced by Escherichia coli LPS and cytokine production induced by both exogenous and endogenous TLR-4 ligands, while having no effect on these parameters by itself. Treatment of CIA using TLR-4 antagonist substantially suppressed both clinical and histologic characteristics of arthritis without influencing the adaptive anti-type II collagen immunity crucial for this model. Treatment with TLR-4 antagonist strongly reduced IL-1␤ expression in articular chondrocytes and synovial tissue. Furthermore, such treatment inhibited IL-1-mediated autoimmune arthritis in IL-1Ra ؊/؊ mice and protected the mice against cartilage and bone pathology.Conclusion. In the present study, we demonstrate for the first time that inhibition of TLR-4 suppresses the severity of experimental arthritis and results in lower IL-1 expression in arthritic joints. Our data suggest that TLR-4 might be a novel target in the treatment of rheumatoid arthritis.Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology associated with chronic inflammation of peripheral joints. Today it is generally accepted that proinflammatory cytokines play an important role in the pathogenesis of RA (1); however, the mechanisms of initiation and perpetuation of the inflammatory cascade in RA are still unknown.Toll-like receptors (TLRs) are a family of pattern recognition receptors that are involved in the recognition of conserved pathogen-associated molecular patterns (2). Ligand binding to TLRs initiates a signaling cascade that leads to the activation of the NF-B and interferon regulatory factor 3 transcription factors and MAPKs, which in turn promote the production of inflammatory cytokines, chemokines, and tissuedestructive enzymes and the expression of costimulatory molecules on antigen-presenting cells (APCs). These costimulatory molecules provide a second signal to T cells to initiate the adaptive immune response (3,4

Dendritic cells (DCs) are specialized APCs that can be activated upon pathogen recognition as well as recognition of endogenous ligands, which are released during inflammation and cell stress. The recognition of exogenous and endogenous ligands depends on TLRs, which are abundantly expressed in synovial tissue from rheumatoid arthritis (RA) patients. Furthermore TLR ligands are found to be present in RA serum and synovial fluid and are significantly increased, compared with serum and synovial fluid from healthy volunteers and patients with systemic sclerosis and systemic lupus erythematosus. Identification of novel endogenous TLR ligands might contribute to the elucidation of the role of TLRs in RA and other autoimmune diseases. In this study, we investigated whether five members of the small heat shock protein (HSP) family were involved in TLR4-mediated DC activation and whether these small HSPs were present in RA synovial tissue. In vitro, monocyte-derived DCs were stimulated with recombinant αA crystallin, αB crystallin, HSP20, HSPB8, and HSP27. Using flow cytometry and multiplex cytokine assays, we showed that both αA crystallin and HSPB8 were able to activate DCs and that this activation was TLR4 dependent. Furthermore, Western blot and immunohistochemistry showed that HSPB8 was abundantly expressed in synovial tissue from patients with RA. With these experiments, we identified sHSP αA crystallin and HSPB8 as two new endogenous TLR4 ligands from which HSPB8 is abundantly expressed in RA synovial tissue. These findings suggest a role for HSPB8 during the inflammatory process in autoimmune diseases such as RA.

Increasing epidemiologic evidence supports a link between periodontitis and rheumatoid arthritis. The actual involvement of periodontitis in the pathogenesis of rheumatoid arthritis and the underlying mechanisms remain, however, poorly understood. We investigated the influence of concomitant periodontitis on clinical and histopathologic characteristics of T cell–mediated experimental arthritis and evaluated modulation of type II collagen (CII)–reactive Th cell phenotype as a potential mechanism. Repeated oral inoculations of periodontal pathogens Porphyromonas gingivalis and Prevotella nigrescens induced periodontitis in mice, as evidenced by alveolar bone resorption. Interestingly, concurrent periodontitis induced by both bacteria significantly aggravated the severity of collagen-induced arthritis. Exacerbation of arthritis was characterized by increased arthritic bone erosion, whereas cartilage damage remained unaffected. Both P. gingivalis and P. nigrescens skewed the CII-specific T cell response in lymph nodes draining arthritic joints toward the Th17 phenotype without affecting Th1. Importantly, the levels of IL-17 induced by periodontal pathogens in CII-specific T cells directly correlated with the intensity of arthritic bone erosion, suggesting relevance in pathology. Furthermore, IL-17 production was significantly correlated with periodontal disease–induced IL-6 in lymph node cell cultures. The effects of the two bacteria diverged in that P. nigrescens, in contrast to P. gingivalis, suppressed the joint-protective type 2 cytokines, including IL-4. Further in vitro studies showed that the Th17 induction strongly depended on TLR2 expression on APCs and was highly promoted by IL-1. Our data provide evidence of the involvement of periodontitis in the pathogenesis of T cell–driven arthritis through induction of Ag-specific Th17 response.

In the past few years, new developments have been reported on the role of Toll-like receptors (TLRs) in chronic inflammation in rheumatic diseases. The inhibitory function of TLR10 has been demonstrated. Receptors that enhance the function of TLRs, and several TLR inhibitors, have been identified. In addition, the role of the microbiome and TLRs in the onset of rheumatic diseases has been reported. We review novel insights on the role of TLRs in several inflammatory joint diseases, including rheumatoid arthritis, systemic lupus erythematosus, gout and Lyme arthritis, with a focus on the signalling mechanisms mediated by the Toll-IL-1 receptor (TIR) domain, the exogenous and endogenous ligands of TLRs, and the current and future therapeutic strategies to target TLR signalling in rheumatic diseases.

Objective. S100A8 and S100A9 are two Ca 2؉ binding proteins classified as damage-associated molecular patterns or alarmins that are found in high amounts in the synovial fluid of osteoarthritis (OA) patients. The purpose of this study was to investigate whether S100A8 and/or S100A9 can interact with chondrocytes from OA patients to increase catabolic mediators.Methods. Using immunohistochemistry, we stained for S100A8 and S100A9 protein, matrix metalloproteinases (MMPs), and a cartilage-breakdown epitope specific for MMPs (VDIPEN) in cartilage from OA donors. Isolated chondrocytes or explants from OA and non-OA donors were stimulated with S100A8 and/or S100A9. Messenger RNA and protein levels of MMPs, cytokines, and cartilage matrix molecules were determined with quantitative reverse transcriptionpolymerase chain reaction and Luminex techniques, respectively. For receptor blocking studies, specific inhibitors for Toll-like receptor 4 (TLR-4), receptor for advanced glycation end products (RAGE), and carboxylated glycans were used.Results. In cartilage from OA patients, the expression of S100A8 and S100A9 protein close to chondrocytes was associated with proteoglycan depletion and expression of MMP-1, MMP-3, and VDIPEN. Stimulation of chondrocytes with S100A8 and S100A9 caused a strong up-regulation of catabolic markers (MMPs 1, 3, 9, and 13, interleukin-6 [IL-6], IL-8, and monocyte chemotactic protein 1) and down-regulation of anabolic markers (aggrecan and type II collagen), thereby favoring cartilage breakdown. Blocking TLR-4, but not carboxylated glycans or RAGE, inhibited the S100 effect. The catabolic S100 effect was significantly more pronounced in chondrocytes from OA patients as compared to those from non-OA patients, possibly due to higher TLR-4 expression. Conclusion. S100A8 and S100A9 have a catabolic effect on human chondrocytes that is TLR-4 dependent. OA chondrocytes are more sensitive than normal chondrocytes to S100 stimulation.During osteoarthritis (OA), the erosion of cartilage is an important cause of pain and disability in the joint. Cartilage damage associated with OA is mainly caused by a shift in the balance between the resorptive (or catabolic) and synthetic (or anabolic) capacities of chondrocytes. Catabolic activities of OA chondrocytes are characterized by elevated release of cartilagedegrading enzymes, such as matrix metalloproteinases (MMPs), while anabolic activities of OA chondrocytes result in aggrecan and type II collagen production (1).Although OA is generally considered a disease of the cartilage, evidence is accumulating that synovial activation and proinflammatory mediators released after synovial activation might contribute to the induction of cartilage damage in OA (2-4). Arthroscopic studies suggest that synovial activation occurs in 50% of OA patients and that it is associated with more severe Supported by the Dutch Arthritis Foundation (grant 07-2-301).

The galactosaminogalactan (GAG) is a cell wall component of Aspergillus fumigatus that has potent anti-inflammatory effects in mice. However, the mechanisms responsible for the anti-inflammatory property of GAG remain to be elucidated. In the present study we used in vitro PBMC stimulation assays to demonstrate, that GAG inhibits proinflammatory T-helper (Th)1 and Th17 cytokine production in human PBMCs by inducing Interleukin-1 receptor antagonist (IL-1Ra), a potent anti-inflammatory cytokine that blocks IL-1 signalling. GAG cannot suppress human T-helper cytokine production in the presence of neutralizing antibodies against IL-1Ra. In a mouse model of invasive aspergillosis, GAG induces IL-1Ra in vivo, and the increased susceptibility to invasive aspergillosis in the presence of GAG in wild type mice is not observed in mice deficient for IL-1Ra. Additionally, we demonstrate that the capacity of GAG to induce IL-1Ra could also be used for treatment of inflammatory diseases, as GAG was able to reduce severity of an experimental model of allergic aspergillosis, and in a murine DSS-induced colitis model. In the setting of invasive aspergillosis, GAG has a significant immunomodulatory function by inducing IL-1Ra and notably IL-1Ra knockout mice are completely protected to invasive pulmonary aspergillosis. This opens new treatment strategies that target IL-1Ra in the setting of acute invasive fungal infection. However, the observation that GAG can also protect mice from allergy and colitis makes GAG or a derivative structure of GAG a potential treatment compound for IL-1 driven inflammatory diseases.