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Objective Intestinal microbiota are associated with the development of inflammatory arthritis. The aim of this study was to dissect intestinal mucosal immune responses in the preclinical phase of arthritis and determine whether the presence of Th17 cells, beyond involvement of the cytokine interleukin-17 (IL-17), is required for arthritis development, and whether the involvement of Th17 cells in arthritis depends on the composition of the host microbiota. Methods Mucosal T cell production of IL-17, interferon-γ, tumor necrosis factor α (TNFα), IL-22, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was analyzed by flow cytometry and Luminex assay before arthritis onset in mice immunized to develop collagen-induced arthritis (CIA). Pathogenic features of arthritis in mice with CIA and mice with antigen-induced arthritis were compared between Th17 cell-deficient (CD4-Cre+ Rorcflox/flox ) and Th17 cell-sufficient (CD4-Cre- Rorcflox/flox ) mice. In addition, the impact of intestinal microbiota on the Th17 cell dependence of CIA was assessed. Results Lamina propria CD4 T cells were activated before the onset of arthritis in mice with CIA, with marked up-regulation of several cytokines, including IL-17A, TNFα, and GM-CSF. CD4-Cre+ Rorcflox/flox mice showed a specific reduction in intestinal mucosal levels of Th17 cells and partially reduced levels of IL-17-producing CD8 T cells. However, total levels of IL-17A, mostly produced by γδ T cells and neutrophils, were unaffected. The severity of arthritis was significantly reduced in Th17 cell-deficient mice, suggesting that Th17 cells have additional, IL-17A-independent roles in inflammatory arthritis. Accordingly, antigen-stimulated T cells from Th17 cell-deficient mice produced less IL-17A, IL-17F, and GM-CSF. Importantly, the dependence of CIA on the involvement of Th17 cells was mitigated in the presence of an alternative microbiome. Conclusion These data from murine models suggest that activation of mucosal immunity precedes the development of arthritis, and also that Th17 cells have a microbiota-dependent role in arthritis. Therefore, a microbiome-guided stratification of patients might improve the efficacy of Th17-targeted therapies.

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease hypothesized to promote inflammation via cleavage of protease-activated receptor 1 (PAR1) and PAR2. KLK6 levels are elevated in multiple inflammatory and autoimmune conditions, but no definitive role in pathogenesis has been established. Here, we show that skin-targeted overexpression of KLK6 causes generalized, severe psoriasiform dermatitis with spontaneous development of debilitating psoriatic arthritis-like joint disease. The psoriatic skin and joint phenotypes are reversed by normalization of skin KLK6 levels and attenuated following genetic elimination of PAR1 but not PAR2. Conservation of this regulatory pathway was confirmed in human psoriasis using vorapaxar, an FDA-approved PAR1 antagonist, on explanted lesional skin from patients with psoriasis. Beyond defining a critical role for KLK6/PAR1 signaling in promoting psoriasis, our results demonstrate that KLK6/PAR1-mediated inflammation in the skin alone is sufficient to drive inflammatory joint disease. Further, we identify PAR1 as a promising cytokine-independent target in therapy of psoriasis and psoriatic arthritis.

Item does not contain fulltext Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-kappaB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.

Background:Intestinal microbiota plays a prominent role in shaping the T cell immune response. Increasing evidence suggests that the gut microbiota is perturbed in patients with RA, and a variety of animal models demonstrated involvement of (mouse) microbiota in arthritis development. This underlines the necessity of understanding whether and how indigenous human NORA-associated microbiota may trigger RA.Objectives:To comprehensively investigate the intestinal mucosa cytokine production and DC, T and B cell responses to human gut microbiota associated with new-onset RA.Methods:We utilizedin vitrocultures of mucosal-like DCs (differentiated from bone marrow cells) and primary splenic DCs, as well asex vivocultures of healthy human intestinal biopsies, cultured in the presence of heat-killed fecal microbiota from either NORA or control donors. Furthermore, we performed studies in humanized mice carrying intestinal NORA microbiota, to study the effect on immune response during homeostasis and upon joint inflammation during collagen-induced arthritis (CIA).Results:In 24h DC cultures, NORA fecal microbiota more potently induced the expression of co-stimulatory molecules CD40 and CD80, and this enhanced DC maturation was partially mediated through TLR4 as demonstrated using the TLR4 antagonist TAK242. Interestingly, HC and NORA fecal microbiota differentially induced IL-12 and IL-6 production, with significantly enhanced IL-6 and reduced IL-12 secretion by the NORA microbiome. Furthermore, inex vivocultures of human ileum biopsies, the production of IL-1 and IL-33, as well as IL-23/Th17 cytokines IL-23, IL-22, and GM-CSF, were significantly increased by NORA-derived microbiome. Interestingly, in the small intestine lamina propria (SILP) of NORA-colonized mice, we observed enhanced Th17 polarization, increased innate GM-CSF expression and higher B cell CD40 and IgA levels during homeostasis. To study whether colonization with HC and NORA microbiota alters arthritis development, humanized mice and controls (mock, autologous, HC and NORA microbiota) were used in a CIA experiment. Macroscopic scoring of the arthritis severity at weekly intervals demonstrated that arthritis severity was significantly enhanced in NORA-colonized mice compared to HC-colonization and mock controls.Conclusion:Our data reveal that NORA microbiota, in addition to the previously described Th17 differentiation, induce higher levels of GM-CSF and B cell IgA in LP and have increased potential to aggravate arthritis through the activation of TLR4.References:[1]Scher et al., eLife 2013; Maeda Y et al., Arthritis & rheumatology 2016; Zhang X et al., Nature medicine 2015; Chen J et al., Genome Med 2016Disclosure of Interests:Marije Koenders: None declared, Heather Evans-Marin: None declared, Joyce Aarts: None declared, Parvathy Girija: None declared, Rebecca Rogier: None declared, Sergei Koralov: None declared, Julia Manasson: None declared, Peter van der Kraan: None declared, Shahla Abdollahi-Roodsaz: None declared, Jose Scher Consultant of: Novartis, Janssen, UCB, Sanofi.

Endogenous nucleic acids and their receptors may be involved in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). As the role of the DNA sensing Toll‐like receptor (TLR) 9 in RA is unclear, we aimed to investigate its involvement in the pathogenesis of autoimmune arthritis using three different experimental models of RA. The data obtained revealed involvement of TLR9 in the T cell‐dependent phase of inflammatory arthritis. In rats with pristane‐induced arthritis (PIA), TLR9 inhibition before disease onset reduced arthritis significantly and almost completely abolished bone erosion. Accordingly, serum levels of IL‐6, α‐1‐acid‐glycoprotein and rheumatoid factor were reduced. Moreover, in TLR9−/− mice, streptococcal cell wall (SCW)‐induced arthritis was reduced in the T cell‐dependent phase, whereas T cell‐independent serum‐transfer arthritis was not affected. Remarkably, while TLR7 expression did not change during in vitro osteoclastogenesis, TLR9 expression was higher in precursor cells than in mature osteoclasts and partial inhibition of osteoclastogenesis was achieved only by the TLR9 antagonist. These results demonstrate a pivotal role for TLR9 in the T cell‐dependent phases of inflammatory arthritis and additionally suggest some role during osteoclastogenesis. Hence, endogenous DNA seems to be crucially involved in the pathophysiology of inflammatory autoimmune arthritis.

Background Intestinal microbiota have been associated with psoriatic and rheumatoid arthritis. One of the major effects of microbiota is the induction of mucosal T helper 17 (Th17) cells. We therefore reasoned that the efficiacy of Th17-targeted therapies in arthritis may depend on the host microbiota. Previous studies focused on the role of the cytokine interleukin-17A (IL-17), rather than Th17 cells, by using IL-17 inhibitors or IL-17-deficient mice. Therefore, the role of Th17 cells, which produce multiple pro-inflammatory mediators in addition to IL-17, is not yet fully understood. Objectives The aim of this study was to determine the role of Th17 cells, beyond the cytokine IL-17, in arthritis, and to investigate whether Th17 cells are differentially involved in arthritis depending on the microbiota present. Methods We established conditional Th17-deficient mice, which exhibit a CD4-Cre-induced floxing of a part of the Rorc allele that encodes the Th17 master regulator RORγt. We compared the development of collagen-induced arthritis in Th17-deficient (CD4-Cre+ Rorcflox/flox) and -sufficient (CD4-Cre- Rorcflox/flox) littermate mice, either colonized with known Th17 cell inducers segmented filamentous bacteria (SFB) or harboring the SFB-free Jackson microbiota. The abundance of Th1 and Th17 cells and the production of IL-17, IFNγ and GM-CSF were quantified by flow cytometry and multiplex cytokine assay. Results CD4-Cre+ Rorcflox/flox mice had significantly lower Th17, but similar Th1 cell abundance, in intestinal lamina propria compared with Cre- littermate controls. Surprisingly, the total amount of IL-17A production by all lamina propria cells during arthritis was rather increased in Th17-deficient mice, with CD8+ T cells and Gr1+ neutrophils being the main alternative sources of IL-17. Despite this increased total IL-17 levels, conditional Th17-deficient mice developed a less severe arthritis compared with Th17-sufficient mice when intestinal microbiota comprised SFB. This suggests a role for Th17 cells in inflammatory arthritis distinct from IL-17. Accordingly, synovial inflammation, cartilage destruction and proteoglycan depletion were reduced in SFB-colonized Th17-deficient mice. While the production of IL-17 by joint-draining lymph node cells stimulated with PMA and ionomycin was similar between Th17-sufficient and –deficient mice, cells from the latter group produced significantly less IL-17 upon antigen-specific stimulation with type II collagen. Furthermore, the production of GM-CSF, another Th17 cell-derived cytokine, was significantly lower in the lymph nodes of Th17-deficient mice, an effect associated with the protection against arthritis. Importantly, substitution of the intestinal microbiota with SFB-free Jackson microbiota resulted in the loss of Th17 cell dependency of arthritis as Th17-sufficient and –deficient mice showed similar disease progression under this condition. Conclusions These data suggest that Th17 cells may mediate inflammatory arthritis partly through IL-17-independent mechanisms. Our observations also suggest that the involvement of Th17 cells in arthritis depends on the microbiota subset present in the host. Therefore, a microbiome-guided stratification of rheumatoid or psoriatic arthritis patients might improve the efficacy of Th17 (or IL-17)-targeted therapies. Disclosure of Interest None declared

Background The composition of intestinal microbiota is perturbed in patients with new-onset rheumatoid arthritis (RA) and those with chronic RA. However, it is not known whether the changes in the intestinal microbiome precede the development of arthritis or are rather a consequence of the inflammatory processes. Furthermore, while both germ-free condition and administration of oral antibiotics prevent arthritis in mice, it is unclear whether modulation of the intestinal microbiota after the onset of arthritis may still suppress the disease. Objectives We aimed to assess potential alterations of the intestinal microbiome in the preclinical phase of inflammatory arthritis, and to determine the efficacy of microbiota modulations in the treatment of established disease in mice. Methods We sequenced fecal bacterial 16S rRNA to define the intestinal microbiome in mice before immunization with collagen and 21 days later before the booster injection for the induction of collagen-induced arthritis (CIA). To assess the efficacy of microbiota modulation during arthritis, mice with ongoing CIA were treated orally by a broad-spectrum antibiotic cocktail for one week to partially eliminate the intestinal microbiota. T cell differentiation and production of cytokines in intestinal lamina propria and joint-draining lymph nodes were assessed by flow cytometry and Luminex. Arthritis was assessed macroscopically and by histology. Serum-transfer arthritis induced by intraperitoneal injections of arthritogenic K/BxN mouse serum was used as a control, T cell-independent, model. Results The preclinical phase of inflammatory arthritis in mice was characterized by marked changes in the intestinal microbiome, represented by a significant increase of the phylum Bacteroidetes and a decrease of Firmicutes and Proteobacteria. Among the most abundant bacterial families, S24–7 and Staphylococcaceae were expanded, whereas Lachnospiraceae were reduced during the immune priming phase of CIA. Several operational taxonomic units associated with S24–7 family increased, while those assigned to Lachnospiraceae and Ruminococcaceae decreased in the intestinal microbiota before the clinical onset of arthritis. The abundance of intestinal lamina propria Th17 cells significantly correlated with the severity of CIA; however, intestinal Th1 cells were not correlated with the disease. Elimination of intestinal microbiota in mice with ongoing CIA specifically suppressed intestinal Th17 cell differentiation without affecting Th1 and Treg cells. Importantly, elimination of intestinal microbiota suppressed Th17 cell differentiation and IL-17 production in joint-draining lymph nodes, and reduced the severity of established CIA. In contrast, the T cell-independent serum-transfer arthritis was not affected by this strategy. Conclusions These observations suggest that perturbations of the intestinal microbiome may precede the development of inflammatory arthritis. Similar studies are warranted in human pre-RA or at-risk individuals to shed light on the functional role of the microbiome in the development of RA. Our studies also suggest that modulation of the intestinal microbiota after the onset arthritis may still provide opportunities to treat inflammatory arthritis. Disclosure of Interest None declared

Background/objectives Both germ-free condition and administration of oral antibiotics before the onset of arthritis modulate T cell differentiation and attenuate arthritis in mice. However, it is not known whether and how the modulation of intestinal microbiota after the onset of arthritis may influence the disease. Here, we investigated the involvement of commensal intestinal microbiota in the progression of established arthritis in both T cell-dependent and -independent mouse models. Materials and methods Mice with established collagen-induced arthritis (CIA) as well as mice with K/BxN serum-transfer arthritis were treated orally with broad-spectrum antibiotics for one week to partially eliminate intestinal microbiota. 16S rRNA gene high-throughput sequencing was performed to assess the effect of arthritis and antibiotic treatment on the gut microbiota composition. Arthritis was assessed macroscopically and by histology. Differentiation of Th1, Th17 and regulatory T (Treg) cells and production of their prototypic cytokines in intestinal lamina propria and joint-draining lymph nodes were assessed by flow cytometry and Luminex cytokine array. Results Induction of arthritis as such resulted in significant alterations in the gut microbiota. Antibiotic treatment of CIA mice eliminated the majority of the gut microbiota, while the microbiota composition of control mice remained relatively stable. Partial elimination of intestinal microbiota during ongoing CIA specifically suppressed intestinal Th17 cell differentiation without affecting Th1 and Treg cells. Accordingly, production of IL-17, but not IFNγ, IL-4 and IL-10, by lamina propria lymphocytes was significantly diminished in antibiotic-treated mice. Importantly, elimination of intestinal microbiota resulted in suppressed Th17 cell differentiation in joint-draining lymph nodes and reduced the severity of established CIA. In contrast, antibiotic treatment did not influence disease severity in the T cell-independent K/BxN serum-transfer arthritis. Intriguingly, the abundance of intestinal Th17 cells strongly correlated with the severity of arthritis in the CIA mice. However, elimination of intestinal microbiota after disease onset did not affect the development of anti-collagen type II auto-antibodies. Conclusion These observations suggest that modulation of commensal intestinal microbiota during established arthritis specifically suppresses Th17 differentiation and dampens T cell-mediated arthritic processes. Our study supports the notion that inflammatory signals provided by the gut microbiota continue to promote arthritis after its onset.

OBJECTIVE: The cytokine interleukin-21 (IL-21) can have both proinflammatory and immunosuppressive effects. The purpose of this study was to investigate the potential dual role of IL-21 in experimental arthritis in relation to Th17 cells. METHODS: Antigen-induced arthritis (AIA) and chronic streptococcal cell wall (SCW) arthritis were induced in IL-21 receptor-deficient (IL-21R(-/-) ) and wild-type mice. Knee joints, synovial tissue, and serum were analyzed for arthritis pathology and inflammatory markers. RESULTS: During AIA and chronic SCW arthritis, IL-21R deficiency protected against severe inflammation and joint destruction. This was accompanied by suppressed serum IgG1 levels and antigen-specific T cell responses. Levels of IL-17 were reduced during AIA, and synovial lymphocytes isolated during SCW arthritis for flow cytometry demonstrated that mainly IL-17+ interferon-gamma (IFNgamma)-positive T cells were reduced in IL-21R(-/-) mice. However, during the acute phases of SCW arthritis, significantly higher joint swelling scores were observed, consistent with enhanced tumor necrosis factor and IL-6 expression. Interestingly, IL-21R(-/-) mice were significantly less capable of up-regulating suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 messenger RNA. IL-21 stimulation also affected the Toll-like receptor 2 (TLR-2)/caspase recruitment domain 15 response to SCW fragments in vitro, indicating that impaired SOCS regulation in the absence of IL-21 signaling might contribute to the increased local activation during SCW arthritis. CONCLUSION: In contrast to the proinflammatory role of IL-21 in adaptive immunity, which drives IL-17+IFN+ cells and joint pathology during chronic experimental arthritis, IL-21 also has an important immunosuppressive role, presumably by inhibiting TLR signaling via SOCS-1 and SOCS-3. If this dual role of IL-21 in various immune processes is present in human disease, it could make IL-21 a difficult therapeutic target in rheumatoid arthritis.

Perturbations of the intestinal microbiome have been observed in patients with new-onset and chronic autoimmune inflammatory arthritis. However, it is currently unknown whether these alterations precede the development of arthritis or are rather a consequence of disease. Modulation of intestinal microbiota by oral antibiotics or germ-free condition can prevent arthritis in mice. Yet, the therapeutic potential of modulation of the microbiota after the onset of arthritis is not well characterized. We here show that the intestinal microbial community undergoes marked changes in the preclinical phase of collagen induced arthritis (CIA). The abundance of the phylum Bacteroidetes, specifically families S24-7 and Bacteroidaceae was reduced, whereas Firmicutes and Proteobacteria, such as Ruminococcaceae, Lachnospiraceae and Desulfovibrinocaceae, were expanded during the immune-priming phase of arthritis. In addition, we found that the abundance of lamina propria Th17, but not Th1, cells is highly correlated with the severity of arthritis. Elimination of the intestinal microbiota during established arthritis specifically reduced intestinal Th17 cells and attenuated arthritis. These effects were associated with reduced serum amyloid A expression in ileum and synovial tissue. Our observations suggest that intestinal microbiota perturbations precede arthritis, and that modulation of the intestinal microbiota after the onset of arthritis may offer therapeutic opportunities.

Item does not contain fulltext OBJECTIVE: A prominent role of Toll-like receptor 4 (TLR4) in arthritis is emerging. TLR4 is functional in immune cells and stromal cells. The aim was to investigate the involvement of TLR4 in bone marrow (BM)-derived and resident cells in arthritis. METHODS: Reciprocal sex-mismatched BM transplantation was performed between IL-1Ra(-/-)TLR4(+/+) and IL-1Ra(-/-)TLR4(-/-) double knockout animals in Balb/c background. Arthritis was assessed macroscopically and by histopathology. Immunity was evaluated by splenic cytokine production and flow cytometry in draining lymph node (DLN) cells. RESULTS: Arthritis progression was reduced to a similar extent in animals lacking TLR4 on BM-derived, resident cells or both. Histology revealed that joint inflammation was partially TLR4-dependent in either BM-derived or resident cells. TLR4 plays an additive role in BM-derived and resident cells in promoting cartilage erosion. By contrast, TLR4 was equally important in BM-derived and resident cells in mediating bone erosion. Systemically, TLR4 in both BM-derived and resident cells contributed to IL-17 production by splenic T-cells, whereas in the DLNs of arthritic joints this was not the case. Interestingly, in DLN, the dominant cells producing IL-17 were CD4 negative, and cell numbers were determined by TLR4 in the BM-derived cells. CONCLUSIONS: TLR4 is necessary in both BM-derived and resident cells for full-blown joint swelling, inflammation and bone erosion. Furthermore, TLR4 on BM-derived and tissue-resident cells show an additive effect in cartilage destruction. Interestingly, TLR4 on BM-derived and tissue-resident cells are both required for IL-17 production in spleen, but only in BM-derived cells in DLN.

Contains fulltext : 117860.pdf (Publisher’s version ) (Closed access) 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-gamma 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.

Background Seronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis. Methods Serum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra)–/– mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 and matrix metalloproteinase (MMP)-mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition, local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF489-Cy5.5, a specific tracer for activated MMPs. Results Serum levels of S100A8/A9 were significantly increased in IL-1Ra–/– mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra–/– mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs. Conclusions Expression of S100A8 and S100A9 in IL-1Ra–/– mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1121-z) contains supplementary material, which is available to authorized users.

Item does not contain fulltext The role of the gut microbiome in animal models of inflammatory and autoimmune disease is now well established. The human gut microbiome is currently being studied as a potential modulator of the immune response in rheumatic disorders. However, the vastness and complexity of this host-microorganism interaction is likely to go well beyond taxonomic, correlative observations. In fact, most advances in the field relate to the functional and metabolic capabilities of these microorganisms and their influence on mucosal immunity and systemic inflammation. An intricate relationship between the microbiome and the diet of the host is now fully recognized, with the microbiota having an important role in the degradation of polysaccharides into active metabolites. This Review summarizes the current knowledge on the metabolic role of the microbiota in health and rheumatic disease, including the advances in pharmacomicrobiomics and its potential use in diagnostics, therapeutics and personalized medicine.

Item does not contain fulltext 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 transcription-polymerase 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. 01 mei 2012

Item does not contain fulltext OBJECTIVE: To provide an intermediate step between classic arthritis models and clinical trials, the rheumatoid arthritis (RA) synovium SCID mouse model is a valuable tool for use during preclinical research. We undertook this study to investigate the validity of this humanized mouse model using anti-tumor necrosis factor (anti-TNF) and anti-interleukin-1 (anti-IL-1) treatment and to investigate the direct effect of T cells- and B cell-related therapies on the transplanted RA synovial tissue. METHODS: CB17/SCID mice were engrafted with human RA synovial tissue and systemically treated with anti-TNF, anti-IL-1, anti-IL-17, CTLA-4Ig, anti-CD20, or isotype control antibodies. RESULTS: Validation of the model with anti-TNF treatment significantly reduced serum cytokine levels and decreased histologic inflammation, whereas anti-IL-1 therapy did not show any effect on the RA synovial grafts. In mice engrafted with B cell-rich synovial tissue, anti-CD20 treatment showed clear therapeutic effects. Surprisingly, CTLA-4Ig treatment did not show any effects in this transplantation model, despite prescreening of the synovial tissue for the presence of CD3+ T cells and the costimulatory molecules CD80 and CD86. In contrast, great therapeutic potential was observed for anti-IL-17 treatment, but only when CD3+ T cells were abundantly present in the RA synovial tissue. CONCLUSION: This human RA synovium SCID mouse model enabled us to show that CTLA-4Ig lacks direct effects on T cell activation processes in the synovial tissue. Further evidence was obtained that IL-17 might indeed be an interesting therapeutic target in RA patients with CD3-rich synovial tissue. Further characterization of the RA patients' individual synovial profiles is of great importance for achieving tailored therapy. 01 juni 2012

Contains fulltext : 98142.pdf (Publisher’s version ) (Closed access) Lipopolysaccharide is a major constituent of the outer membrane of Gram-negative bacteria and important in the induction of pro-inflammatory responses. Recently, novel LPS species derived from Neisseria meningitidis H44/76 by insertional inactivation of the lpxL1 and lpxL2 genes have been created with a lipid A portion consisting of five (penta-acylated lpxL1) or four (tetra-acylated lpxL2) fatty acids connected to the glucosamine backbone instead of six fatty acids in the wild-type LPS. We show that these mutant LPS-types are poor inducers of cytokines (tumor-necrosis factor-alpha, IL-1beta, IL-10, IL-RA) in human mononuclear cells. Both penta- and tetra-acylated meningococcal LPSs were able to inhibit cytokine production by wild-type Escherichia coli or meningococcal LPS. Binding of FITC-labelled E. coli LPS TLR4 transfected Chinese hamster ovary (CHO) cells was inhibited by both mutant LPS-types. Experiments with CHO fibroblasts transfected with human CD14 and TLR4 showed that the antagonizing effect was dependent on the expression of human TLR4. In contrast to the situation in humans, lpxL1 LPS has agonistic activity for cytokine production in peritoneal macrophages of DBA mice, and exacerbated arthritis in murine collagen induced arthritis model. N. meningitidis lipid A mutant LPSs lpxL1 and lpxL2 function as LPS antagonists in humans by inhibiting TLR4-dependent cytokine production but have agonistic activity in mice.

Contains fulltext : 89535.pdf (Publisher’s version ) (Closed access) OBJECTIVE: During the course of different musculoskeletal diseases, joints are progressively damaged by inflammatory, infectious, or mechanical stressors, leading to joint destruction and disability. While effective strategies to inhibit joint inflammation, such as targeted cytokine-blocking therapy, have been developed during the last decade, the molecular mechanisms of joint damage are still poorly understood. This study was undertaken to investigate the role of the Wnt pathway modulator R-Spondin 1 (RSpo1) in protecting bone and cartilage in a mouse model of arthritis. METHODS: Tumor necrosis factor alpha (TNFalpha)-transgenic mice were treated with vehicle or Rspo1. Mice were evaluated for signs of arthritis, and histologic analysis of the hind paws was performed. Moreover, we determined the effect of Rspo1 on Wnt signaling activity and osteoprotegerin (OPG) expression in murine primary osteoblasts. RESULTS: The secreted Wnt pathway modulator RSpo1 was highly effective in preserving the structural integrity of joints in a TNFalpha-transgenic mouse model of arthritis by protecting bone and cartilage from inflammation-related damage. RSpo1 antagonized the Wnt inhibitor Dkk-1 and modulated Wnt signaling in mouse mesenchymal cells. In osteoblasts, RSpo1 induced differentiation and expression of OPG, thereby inhibiting osteoclastogenesis in vitro. In vivo, RSpo1 promoted osteoblast differentiation and bone formation while blocking osteoclast development, thereby contributing to the integrity of joints during inflammatory arthritis. CONCLUSION: Our results demonstrate the therapeutic potential of RSpo1 as an anabolic agent for the preservation of joint architecture. 01 augustus 2010

Contains fulltext : 70944.pdf (Publisher’s version ) (Open Access) TLRs may contribute to the progression of rheumatoid arthritis through recognition of microbial or host-derived 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-gamma 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.

Contains fulltext : 69671.pdf (Publisher’s version ) (Closed access) OBJECTIVE: Interleukin-1 receptor antagonist-deficient (IL-1Ra-/-) mice spontaneously develop an inflammatory and destructive arthritis due to unopposed excess IL-1 signaling. In this study, the role of Th17 cells and the effect of neutralization of IL-17, IL-1, and tumor necrosis factor alpha (TNFalpha) were investigated in this IL-1-driven murine arthritis model. METHODS: T cells isolated from IL-1Ra-/- and wild-type (WT) mice were stained for IL-17 and interferon-gamma, with results assessed by fluorescence-activated cell sorting analysis. To investigate the contribution of IL-1 and IL-17 in further progression of arthritis in this model, mice were treated with neutralizing antibodies after the onset of arthritis. RESULTS: Compared with WT mice, IL-1Ra-/- mice had similar levels of Th1 cells but clearly enhanced levels of Th17 cells; this increase in the number of Th17 cells was evident even before the onset of arthritis, in young, nonarthritic IL-1Ra-/- mice. The percentage of Th17 cells increased even more after the onset of arthritis and, similar to the serum levels and local messenger RNA levels of IL-17, the percentage of IL-17+ Th17 cells clearly correlated with the severity of arthritis. Anti-IL-17 treatment prevented any further increase in inflammation and bone erosion, whereas blocking of TNFalpha after the onset of arthritis had no effect. In contrast, neutralization of IL-1 resulted in a complete suppression of arthritis. Interestingly, this anti-IL-1 treatment also significantly reduced the percentage of IL-17+ Th17 cells in the draining lymph nodes of these arthritic mice. CONCLUSION: Increased levels of Th17 cells can be detected in IL-1Ra-/- mice even preceding the onset of arthritis. In addition, the results of cytokine-blocking studies demonstrated that IL-17 contributes to the inflammation and bone erosion in this model, which suggests that IL-1 is the driving force behind the IL-17-producing Th17 cells.

Contains fulltext : 70292.pdf (Publisher’s version ) (Closed access) The pathogenesis of chronic joint inflammation remains unclear, although the involvement of pathogen recognition receptors has been suggested recently. In the present article, we describe the role of two members of the NACHT-LRR (NLR) family, Nod1 (nucleotide-binding oligomerization domain) and Nod2 in a model of acute joint inflammation induced by intraarticular injection of Streptococcus pyogenes cell wall fragments. Here, we show that Nod2 deficiency resulted in reduced joint inflammation and protection against early cartilage damage. In contrast, Nod1 gene-deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage, consistent with a model in which Nod1 controls the inflammatory reaction. To explore whether the different function of Nod1 and Nod2 occurs also in humans, we exposed peripheral blood mononuclear cells (PBMCs) carrying either Nod1ins/del or Nod2fs mutation with SCW fragments in vitro. Production of both TNFalpha and IL-1beta was clearly impaired in PBMCs carrying the Nod2fs compared with PBMCs isolated from healthy controls. In line with results in Nod1 gene-deficient mice, PBMCs from individuals bearing a newly described Nod1 mutation produced enhanced levels of proinflammatory cytokines after 24-h stimulation with SCW fragments. These data indicate that the NLR family members Nod1 and Nod2 have different functions in controlling inflammation, and that intracellular Nod1-Nod2 interactions may determine the severity of arthritis in this experimental model. Whether a distorted balance between the function of Nod1 and/or Nod2 is involved in the pathogenesis of human autoinflammatory or autoimmune disease, such as rheumatoid arthritis, remains to be elucidated.

Contains fulltext : 71019.pdf (Publisher’s version ) (Closed access) OBJECTIVE: Toll-like receptors (TLRs) may activate innate and adaptive immune responses in rheumatoid arthritis (RA) through recognition of microbial as well as endogenous ligands that have repeatedly been found in arthritic joints. The objective of this study was to investigate the involvement of TLR-2 and TLR-4 in the development of chronic destructive streptococcal cell wall (SCW)-induced arthritis, in which interleukin-1 (IL-1)/IL-17-dependent T cell-driven pathologic changes replace the macrophage-driven acute phase. METHODS: Chronic SCW arthritis was induced by 4 repeated intraarticular injections of SCW fragments in wild-type, TLR-2(-/-), and TLR-4(-/-) mice. Clinical, histopathologic, and immunologic parameters of arthritis were evaluated. RESULTS: The TLR-2 dependency of joint swelling during the acute phase was shifted to TLR-4 dependency during the chronic phase. Persistent joint inflammation in the latter phase of the model was significantly suppressed in TLR-4(-/-) mice. In the chronic phase, TLR-4 actively contributed to matrix metalloproteinase (MMP)-mediated cartilage destruction and to osteoclast formation, since the expression of the MMP-specific aggrecan neoepitope VDIPEN and the osteoclast marker cathepsin K was significantly reduced in TLR-4(-/-) mice. Furthermore, TLR-4(-/-) mice expressed less IL-1beta, tumor necrosis factor alpha, IL-6, and IL-23, cytokines that are implicated in IL-17 production. Accordingly, SCW-specific IL-17 production was found to be dependent on TLR-4 activation, since T cells from arthritic TLR-4(-/-) mice produced markedly less IL-17 upon SCW stimulation, whereas interferon-gamma production remained unaffected. CONCLUSION: These data indicate the involvement of TLR-4 in the chronicity and erosive character of arthritis coincident with the antigen-specific IL-17 response. The high position of TLR-4 in the hierarchy of erosive arthritis provides an interesting therapeutic target for RA.

In the early 1990s, Janeway discussed the theory that an immune response could not occur unless antigen-presenting cells (APCs) were first activated, which he called “the immunologist’s dirty little secret.” Then, in the coup that caused the immunology community to finally take notice, and following the seminal study by Lemaitre et al (1), Janeway discovered the crucial role of pattern-recognition receptors (PRRs), which recognized evolutionary conserved molecules on infectious nonself organisms (2). Toll-like receptors (TLRs) belong to the PRR family, which was first shown to recognize microbial components, known as pathogenassociated patterns. TLRs are constitutively expressed by numerous immune cells and are designed to detect and eliminate invading pathogens by activating both innate and adaptive immune responses. Accumulating evidence indicates a role of TLRs in the recognition of “host-derived” agonists (so-called endogenous ligands or alarmins), which might be involved in various autoimmune and/or autoinflammatory syndromes. Examples of such endogenous TLR ligands are Hsp60, Hsp70, gp96, small HspB8, hyaluronic acid, and fibronectin, which are all released upon cell stress and are found in various tissues during inflammation (discussed below). TLRs are of interest to immunologists because of their important role in the initiation of immune responses. Because evidence seems to implicate innate immunity in a wide variety of rheumatic conditions that are seen in the clinical practices of rheumatologists, the question is whether TLRs deserve more of the rheumatologist’s attention as well. The role of TLR agonists in animal models of arthritis has been explored and is consistent with Janeway’s “dirty little secret.” In this light, the use of Freund’s complete adjuvant to boost arthritis has lost its magic with the discovery of TLR binding to constituents of this “miracle potion.” In this review, we discuss how recent advances in TLRassociated research have enhanced our understanding of the role of this receptor family in frequently occurring rheumatic conditions and how continued exploration of their role is likely to change the types of therapy available to battle these chronic diseases.

Contains fulltext : 168511.pdf (Publisher’s version ) (Closed access) 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.

Background Airway abnormalities and lung tissue citrullination are found in both rheumatoid arthritis (RA) patients and individuals at-risk for disease development. This suggests the possibility that the lung could be a site of autoimmunity generation in RA, perhaps in response to microbiota changes. We therefore sought to test whether the RA lung microbiome contains distinct taxonomic features associated with local and/or systemic autoimmunity. Methods 16S rRNA gene high-throughput sequencing was utilized to compare the bacterial community composition of bronchoalveolar lavage fluid (BAL) in patients with early, disease-modifying anti-rheumatic drugs (DMARD)-naïve RA, patients with lung sarcoidosis, and healthy control subjects. Samples were further assessed for the presence and levels of anti-citrullinated peptide antibodies (including fine specificities) in both BAL and serum. Results The BAL microbiota of RA patients was significantly less diverse and abundant when compared to healthy controls, but similar to sarcoidosis patients. This distal airway dysbiosis was attributed to the reduced presence of several genus (i.e., Actynomyces and Burkhordelia) as well as reported periodontopathic taxa, including Treponema, Prevotella, and Porphyromonas. While multiple clades correlated with local and systemic levels of autoantibodies, the genus Pseudonocardia and various related OTUs were the only taxa overrepresented in RA BAL and correlated with higher disease activity and erosions. Conclusions Distal airway dysbiosis is present in untreated early RA and similar to that detected in sarcoidosis lung inflammation. This community perturbation, which correlates with local and systemic autoimmune/inflammatory changes, may potentially drive initiation of RA in a proportion of cases. Electronic supplementary material The online version of this article (doi:10.1186/s40168-016-0206-x) contains supplementary material, which is available to authorized users.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by chronic joint inflammation and variable degrees of bone and cartilage erosion. Studies in animal models of arthritis provide insight into elements which can amplify destructive features. The presence of immune complexes in the joint makes arthritis more erosive. Although considerable bone erosion still occurs in the absence of FcgammaR triggering by immune complexes, through cytokine-induced RANKL and direct osteoclast activation, cartilage erosion is heavily dependent on the FcgammaR pathway. T cell factors such as IFNgamma and IL17 further amplify erosion through upregulation of the damaging FcgammaRI and stimulation of the influx of granulocytes, respectively. Apart from immune elements, environmental pressure and components of tissue damage contribute through innate pathways. Spontaneous T cell-dependent arthritis in IL1Ra-/- mice is absent under germ-free conditions, and markedly suppressed in TLR4-deficient mice. Moreover, TLR4 blocking with a receptor antagonist suppresses erosive arthritis.

Contains fulltext : 153819.pdf (Publisher’s version ) (Closed access) BACKGROUND: Infection with Coxiella burnetii can lead to acute and chronic Q fever. Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR6, nucleotide-binding oligomerization domain receptor 1 (NOD1), NOD2, and the mitogen-activated protein kinases are central in the innate immune response against microorganisms, but little is known about their role in the recognition of C. burnetii in humans. METHODS: Human peripheral blood mononuclear cells (PBMCs) were stimulated with C. burnetii Nine Mile and the Dutch outbreak isolate C. burnetii 3262. TLRs were inhibited using specific antibodies or antagonists. Additionally, the influence of human polymorphisms in TLRs and Nod-like receptors (NLRs) on C. burnetii-induced cytokine production was assessed. RESULTS: Inhibition of TLR2, p38, JNK, and ERK led to decreased cytokine responses in C. burnetii-stimulated human PBMCs. Humans with polymorphisms in TLR1 and NOD2 had reduced cytokine production, compared with humans with wild-type genotypes, after stimulation. Interestingly, polymorphisms in TLR6 led to decreased cytokine production after C. burnetii 3262 stimulation but not after C. burnetii Nine Mile stimulation. CONCLUSIONS: The TLR1/TLR2 heterodimer and NOD2 are important recognition receptors for the induction of cytokine responses against C. burnetii in humans. Furthermore, an interesting finding was the divergent recognition of C. burnetii Nine Mile and C. burnetii 3262.

Contains fulltext : 153209.pdf (Publisher’s version ) (Open Access) Generation of different CD4 T cell responses to commensal and pathogenic bacteria is crucial for maintaining a healthy gut environment, but the associated cellular mechanisms are poorly understood. Dendritic cells (DCs) and macrophages (Mfs) integrate microbial signals and direct adaptive immunity. Although the role of DCs in initiating T cell responses is well appreciated, how Mfs contribute to the generation of CD4 T cell responses to intestinal microbes is unclear. Th17 cells are critical for mucosal immune protection and at steady state are induced by commensal bacteria, such as segmented filamentous bacteria (SFB). Here, we examined the roles of mucosal DCs and Mfs in Th17 induction by SFB in vivo. We show that Mfs, and not conventional CD103(+) DCs, are essential for the generation of SFB-specific Th17 responses. Thus, Mfs drive mucosal T cell responses to certain commensal bacteria.

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., Author Summary Aspergillus fumigatus is an opportunistic pathogenic fungus that primarily causes infections in the immunocompromised host. It is known that Aspergillus employs various strategies to evade immune recognition by the host's immune system. Recently, galactosaminogalactan (GAG), a new component of the Aspergillus cell wall, was discovered to have potent anti-inflammatory effects in mice making them more susceptible to Aspergillosis. In the current study we found that this anti-inflammatory property of GAG was due to its capacity to induce the potent anti-inflammatory cytokine interleukin-1 Receptor antagonist. This cytokine interferes with IL-1 signalling and thereby can reduce IL-1–induced immune responses such as T-cell responses. We also found that the induction of this anti-inflammatory cytokine by GAG correlates with increased fungal burden, and mice deficient for this cytokine were protected against aspergillosis. Additionally, we show that the capacity of GAG to induce the natural regulator of IL-1 signalling could be used in the treatment of IL-1–mediated disease such as allergy and colitis. Our study provides new insights on the immunoregulatory activity of GAG and opens up possibilities to exploit the anti-inflammatory potential of GAG as a therapy for inflammatory diseases.

Contains fulltext : 136500.pdf (Publisher’s version ) (Closed access) 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.

OBJECTIVE: Previous studies have demonstrated a protective role of Toll-like receptor 2 (TLR-2) and a proinflammatory function of TLR-4 during chronic T cell-driven arthritis. The involvement of TLRs in T cell-independent arthritic processes, however, remains unclear. This study was undertaken to determine the functional significance of TLR-2 and TLR-4 in T cell-independent immune complex-driven arthritis. METHODS: Serum-transfer arthritis was induced in wild-type and TLR-deficient mice by intraperitoneal injections of arthritogenic K/BxN mouse serum. Arthritis was assessed macroscopically and by histologic analysis. The influence of TLR-2 on macrophage cytokine profile, Fcgamma receptor (FcgammaR) expression, and response to immune complexes was determined. RESULTS: While TLR-4, unexpectedly, did not play any significant role, TLR-2 deficiency accelerated the onset and markedly increased the severity of acute immune complex-driven arthritis in mice. TLR-2 deficiency resulted in a substantial increase in joint inflammation, bone erosion, and cartilage pathology, indicating a protective function of TLR-2 in passive FcgammaR-driven disease. Ex vivo study of the macrophage inflammatory phenotype revealed increased production of tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) despite similar levels of IL-10, along with a significant increase in FcgammaR-specific response, in TLR-2-/- mouse macrophages early in the disease. Although distinct FcgammaR messenger RNA expression was not affected, cell surface protein expression of the inhibitory FcgammaRIIB in TLR-2-/- naive primary macrophages was specifically diminished, resulting in a higher proinflammatory response. Accordingly, TLR-2 stimulation specifically up-regulated FcgammaRIIB, but not the activating FcgammaR, on macrophages. CONCLUSION: TLR-2 regulates acute immune complex-driven arthritis by controlling macrophage FcgammaR response. Our findings indicate that the protective role of TLR-2 is extended beyond its previously described role in promoting Treg cells during T cell-mediated arthritis.

Background Seronegative joint diseases, including psoriatic arthritis and juvenile idiopathic arthritis, are characterized by the lack of autoantibodies, which are relevant biomarkers for predicting disease activity in rheumatoid arthritis. Promising alternative biomarkers are the Damage Associated Molecular Patterns (DAMPs), S100A8, S100A9 and the heterodimer S100A8/A9. These proteins are specifically expressed and released by infiltrating phagocytes and may therefore serve as relevant biomarkers for joint inflammation and destruction in seronegative arthritis. Objectives In this study we determined the biomarker potential of serum S100A8/A9 and in vivo imaging of synovial S100A8 to asses joint inflammation and damage in the IL-1 receptor antagonist deficient (IL-1Ra –/– ) mice, a mouse model for seronegative arthritis in which serum autoantibodies are not correlated to disease activity. Methods Serum levels of S100A8/A9 and various cytokines were monitored during arthritis development in IL-1Ra –/– mice using ELISA and Luminex and were correlated to macroscopic and microscopic parameters for joint inflammation and damage. Local S100A9 expression and matrix metalloproteinase (MMP) mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF-489-Cy7, a specific tracer for activated MMPs. Results Starting at week 8, serum levels of S100A8/A9 were significantly increased (1640 ± 1008 ng/ml at week 16) in IL-1Ra –/– mice compared to WT BALB/c control mice (429 ± 191 ng/ml, P =0.005) and strongly correlated to joint swelling (r =0.766, P P =0.0002). Next to macroscopic swelling, increased serum S100A8/A9 also correlated to microscopic cell influx (r =0.794, P –/– mice showed increased cartilage damage (r =0.687, P Conclusions These findings underline the potential of S100-DAMPs as a systemic and local biomarker in seronegative arthritis, not only for assessing inflammation but also joint damage. Disclosure of Interest None declared

Previous studies have demonstrated a protective role of Toll-like receptor 2 (TLR-2) and a proinflammatory function of TLR-4 during chronic T cell-driven arthritis. The involvement of TLRs in T cell-independent arthritic processes, however, remains unclear. This study was undertaken to determine the functional significance of TLR-2 and TLR-4 in T cell-independent immune complex-driven arthritis.Serum-transfer arthritis was induced in wild-type and TLR-deficient mice by intraperitoneal injections of arthritogenic K/BxN mouse serum. Arthritis was assessed macroscopically and by histologic analysis. The influence of TLR-2 on macrophage cytokine profile, Fcγ receptor (FcγR) expression, and response to immune complexes was determined.While TLR-4, unexpectedly, did not play any significant role, TLR-2 deficiency accelerated the onset and markedly increased the severity of acute immune complex-driven arthritis in mice. TLR-2 deficiency resulted in a substantial increase in joint inflammation, bone erosion, and cartilage pathology, indicating a protective function of TLR-2 in passive FcγR-driven disease. Ex vivo study of the macrophage inflammatory phenotype revealed increased production of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) despite similar levels of IL-10, along with a significant increase in FcγR-specific response, in TLR-2-/- mouse macrophages early in the disease. Although distinct FcγR messenger RNA expression was not affected, cell surface protein expression of the inhibitory FcγRIIB in TLR-2-/- naive primary macrophages was specifically diminished, resulting in a higher proinflammatory response. Accordingly, TLR-2 stimulation specifically up-regulated FcγRIIB, but not the activating FcγR, on macrophages.TLR-2 regulates acute immune complex-driven arthritis by controlling macrophage FcγR response. Our findings indicate that the protective role of TLR-2 is extended beyond its previously described role in promoting Treg cells during T cell-mediated arthritis.

The cytokine interleukin-21 (IL-21) can have both proinflammatory and immunosuppressive effects. The purpose of this study was to investigate the potential dual role of IL-21 in experimental arthritis in relation to Th17 cells.Antigen-induced arthritis (AIA) and chronic streptococcal cell wall (SCW) arthritis were induced in IL-21 receptor-deficient (IL-21R(-/-) ) and wild-type mice. Knee joints, synovial tissue, and serum were analyzed for arthritis pathology and inflammatory markers.During AIA and chronic SCW arthritis, IL-21R deficiency protected against severe inflammation and joint destruction. This was accompanied by suppressed serum IgG1 levels and antigen-specific T cell responses. Levels of IL-17 were reduced during AIA, and synovial lymphocytes isolated during SCW arthritis for flow cytometry demonstrated that mainly IL-17+ interferon-γ (IFNγ)-positive T cells were reduced in IL-21R(-/-) mice. However, during the acute phases of SCW arthritis, significantly higher joint swelling scores were observed, consistent with enhanced tumor necrosis factor and IL-6 expression. Interestingly, IL-21R(-/-) mice were significantly less capable of up-regulating suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 messenger RNA. IL-21 stimulation also affected the Toll-like receptor 2 (TLR-2)/caspase recruitment domain 15 response to SCW fragments in vitro, indicating that impaired SOCS regulation in the absence of IL-21 signaling might contribute to the increased local activation during SCW arthritis.In contrast to the proinflammatory role of IL-21 in adaptive immunity, which drives IL-17+IFN+ cells and joint pathology during chronic experimental arthritis, IL-21 also has an important immunosuppressive role, presumably by inhibiting TLR signaling via SOCS-1 and SOCS-3. If this dual role of IL-21 in various immune processes is present in human disease, it could make IL-21 a difficult therapeutic target in rheumatoid arthritis.

Item does not contain fulltext OBJECTIVE: Increasing evidence indicates the involvement of Toll-like receptors (TLRs) in the progression of arthritis; however, the contribution of the two signaling pathways used by TLRs, which are mediated by myeloid differentiation factor 88 (MyD88) and TRIF, remains unclear. The objective of this study was to investigate the specific roles of MyD88 and TRIF in chronic experimental arthritis and the accompanying adaptive immune responses. METHODS: Chronic arthritis was induced in wild-type, MyD88(-/-) , and Trif(lps2) (TRIF(-/-) ) mice by repetitive intraarticular injections of streptococcal cell wall (SCW) fragments. SCW-specific T cell and B cell responses, joint swelling, and histopathologic changes were analyzed during chronic arthritis. RESULTS: Both MyD88 and TRIF pathways contributed to antigen-specific T cell proliferation and antibody production, with the MyD88 pathway playing the dominant role. The severity of joint swelling and synovial inflammation, as well as the histopathologic damage to cartilage and bone, was strongly dependent on MyD88 signaling, whereas TRIF was redundant. MyD88 signaling was critical for the development of pathogenic T cell response (i.e., interleukin-17 [IL-17] production) in response to SCW antigen. Interestingly, when the T cell-dependent phase was prolonged, TRIF signaling appeared to down-regulate bone erosion, an effect accompanied by an inhibitory effect on IL-17 production. CONCLUSION: This study reveals a central role of MyD88 and a counterregulatory function of TRIF in T cell-driven arthritis. The findings provide a rationale for a pathway-specific interference in order to block the pathogenic features and to preserve or stimulate the beneficial aspects of TLR signaling. 01 juni 2012

Background Target discovery and drug development is a very time consuming and expensive process, which is partly due to the fact that preclinical findings from animal models cannot always be translated to the clinical situation. To provide an intermediate step between classical arthritis models and clinical trials, the rheumatoid arthritis (RA) synovium severe combined immunodeficiency (SCID) mouse model is a valuable tool during preclinical research. Objective First, the validity of this humanised mouse model was studied using anti-tumour necrosis factor (TNF) and anti-interleukin 1 (anti-IL-1) treatment. In addition, the direct effect of T and B cell-related therapies on the transplanted RA synovial tissue was investigated. Methods SCID-CB17 mice were engrafted with two standardised pieces of human RA synovial tissue subcutaneously on the back. After an engraftment period of 7 days, mice were systemically treated with anti-TNF, anti-IL-1, anti-IL-17, cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig, anti-CD20 or isotype controls. As readout, serum levels of human cytokines and chemokines were analysed, and synovial grafts were isolated for histological analysis. Results Validation of the model with anti-TNF treatment significantly reduced serum levels of hIL-6 and hIL-8, and histological analysis demonstrated a clear reduction in inflammation and suppressed local expression of TNF and IL-1 in the synovium. As could be expected from the outcome of clinical trials, anti-IL-1 therapy did not show any effect on the RA synovial grafts. The potential of this RA SCID model for B and T cell-related therapies was investigated using anti-CD20, anti-IL-17 and CTLA4-Ig. In mice engrafted with B cell-rich synovial tissue, anti-CD20 treatment significantly reduced serum cytokine levels and histological scores. Also for anti-IL-17 treatment great therapeutic potential was observed, but only when CD3 T cells were abundantly present in the RA synovial tissue. Surprisingly, CTLA4-Ig treatment did not show any effects in this transplantation model, even despite prescreening of the synovial tissue for the presence of CD3 T cells and the co-stimulatory molecules CD80/86. This finding suggests that to reach clinical effects CTLA4-Ig predominantly acts systemically on the immune system, and not directly on the local T cell activation in the arthritic joint. Conclusion This human RA synovium SCID mouse model enabled them to show that CTLA4-Ig lacks direct effects on T cell activation processes in the synovial tissue. Further evidence was obtained that IL-17 might indeed be an interesting therapeutic target in RA patients with CD3-rich synovial tissue. Further characterisation of the RA patients9 individual synovial profile is of great importance to achieve tailor-made therapy.

Item does not contain fulltext OBJECTIVE: Rheumatoid arthritis, which is associated with elevated levels of S100A8 and S100A9, is characterized by severe bone erosions caused by enhanced osteoclast formation and activity. The aim of the present study was to investigate the role of S100A8 and S100A9 in osteoclastic bone destruction in murine antigen-induced arthritis (AIA). METHODS: Bone destruction was analyzed in the arthritic knee joints of S100A9-deficient mice in which S100A8 protein expression was also lacking, and in wild-type (WT) controls. Osteoclast precursors from S100A9-deficient and WT mice were differentiated into osteoclasts in vitro. Additionally, precursors were stimulated with S100A8, S100A9, or S100A8/A9 during osteoclastogenesis. Receptor involvement was investigated using an anti-receptor for advanced glycation end products (anti-RAGE)-blocking antibody, soluble RAGE, or Toll-like receptor 4 (TLR-4)-deficient osteoclast precursors. The formation of osteoclasts and actin rings, the regulation of osteoclast markers, and bone resorption were analyzed. RESULTS: Bone erosions and cathepsin K staining were significantly suppressed in S100A9-deficient mice after AIA induction. However, osteoclast precursors from S100A9-deficient mice developed normally into functional osteoclasts, which excludes a role for intrinsic S100A8/A9. In contrast to the results observed with S100A9 and S100A8/A9, the addition of S100A8 during osteoclastogenesis resulted in stimulation of osteoclast formation in conjunction with enhanced actin ring formation and increased bone resorption. Analysis of the putative receptor for S100A8 in osteoclastogenesis revealed that osteoclast differentiation and function could not be inhibited by blocking RAGE, whereas the increase in osteoclast numbers and enhanced bone resorption were completely abrogated using TLR-4-deficient osteoclast precursors. CONCLUSION: These results demonstrate that S100A8 stimulated osteoclast formation and activity and suggest that both S100A8 and TLR-4 are important factors in mediating osteoclastic bone destruction in experimental arthritis.

Item does not contain fulltext OBJECTIVE: Activated synovial fibroblasts are key players in the pathogenesis of RA by driving inflammation and joint destruction. Numerous molecules including cytokines and Toll-like receptor (TLR) ligands induce pro-inflammatory signalling and gene expression through a hierarchical network of kinases. Upstream mitogen-activated protein kinase kinase kinases (MAP3Ks) represent an attractive target for RA treatment. In this study, we sought to determine the role of the MAP3K TGF-beta-activated kinase 1 (TAK1) in cytokine and TLR-mediated signalling. METHODS: TAK1 activity was inhibited using either a small molecule inhibitor or lentivirally overexpressed kinase-inactive TAK1-K63W mutant in murine embryonic and human dermal and synovial fibroblasts. Fibroblasts were stimulated with IL-1, TNF, TLR2 or TLR4 agonists and responses were evaluated using transcriptional reporters, western blotting and analysis of gene expression of collagenases (MMP3 and MMP13), cytokines (IL-1beta and IL-6) and chemokines (IL-8 and MCP-1). RESULTS: TAK1 inhibition abrogated cytokine- and TLR-induced nuclear factor-kappaB (NF-kappaB) and Saa3-promoter reporter activation in murine and human dermal fibroblasts. In synovial fibroblasts, TAK1 regulated IL-1 and TNF-mediated NF-kappaB, but not Saa3-promoter reporter activation. Inducible mRNA expression of cytokines, collagenases and chemokines, except MCP-1, was TAK1 dependent for IL-1, TNF and TLR2 signalling. Unexpectedly, TLR4-mediated NF-kappaB reporter activation and inducible mRNA expression was fully TAK1 independent. Accordingly, NF-kappaB p65 and p38 MAPK phosphorylation was unaffected by TAK1 inhibition. CONCLUSION: In general, TAK1 crucially regulates IL-1 and TNF signalling in fibroblasts. Interestingly, TLR4 signalling is specifically TAK1 independent in synovial fibroblasts. Consequently, therapeutic TAK1 inhibition in arthropathies may not dampen the damage-associated molecular pattern-mediated TLR4 activation of synovial fibroblasts.

Objective Rheumatoid arthritis (RA) is characterised by severe bone erosions caused by enhanced osteoclast formation and activity. Significantly elevated S100A8 and S100A9 levels in serum and synovial fluid of RA patients are strongly correlated to joint destruction. The aim of the present study was to investigate the role of S100A8 on osteoclastic bone resorption in murine antigen-induced arthritis (AIA). Methods Bone destruction was analysed 7 and 21 days after AIA induction in knee joints of S100A9 −/− mice, also lacking S100A8 protein expression, and wild type controls. Bone marrow precursors from S100A9 −/− and wild type mice were differentiated into osteoclasts in vitro. Additionally, precursors were stimulated with recombinant S100A8 during osteoclastogenesis. Receptor involvement was investigated using an anti-receptor for advanced end products (anti-RAGE) blocking antibody or toll-like receptor 4 negative (TLR4 −/− ) osteoclast precursors. Experiments were analysed for the formation of tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP + MNCs), actin rings, mRNA expression levels of osteoclast markers and resorption pit formation on bone. Results Bone erosions and cathepsin K staining were significantly suppressed in S100A9 −/− mice after AIA induction. In vitro however, bone marrow-derived precursors from S100A9 −/− mice developed normally into functional osteoclasts, excluding a role for intrinsic S100A8/A9. Addition of S100A8 during osteoclastogenesis resulted in increased osteoclast formation The mRNA expression levels of osteoclast markers were not affected by S100A8 stimulation, but the formation of actin rings, essential for the bone resorptive capacity of osteoclasts, was significantly enhanced in conjunction increased bone resorption levels. The stimulatory effects of S100A8 on osteoclast maturation and function could not be inhibited by RAGE blockade, whereas the increased osteoclast numbers, actin ring formation and resorption pit formation were completely abrogated using TLR4 −/− osteoclasts. Conclusion This study demonstrates that S100A8 stimulates osteoclast formation and activity and suggests that both S100A8 and TLR4 are important factors in mediating osteoclastic bone destruction in experimental arthritis. Topic Myeloid cells and innate immune receptors.

Veillonella parvula is an anaerobic gram-negative coccus that is part of the normal flora of the animal and human mouth and gastrointestinal and genitourinary tracts. Oral V. parvula is involved in the development of early periodontal disease as well as different types of serious infections. Present data on molecular mechanisms responsible for innate immune response against Veillonella are very scanty. The aim of this study was to investigate the Toll-like receptor (TLR) pathways responsible for V. parvula lipopolysaccharide (LPS) and to identify the intracellular pathways induced by this recognition. V. parvula LPS stimulated tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) release in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Pretreatment of cells with a TLR4 antagonist significantly reduced TNF-α and IL-6 production in PBMC stimulated with either Veillonella or Escherichia coli LPS. However, V. parvula LPS was 10- to 100-fold less active than E. coli LPS for cytokine induction. TNF-α, IL-1β, IL-6, and IL-10 were released in wild-type and TLR2 −/− , but not TLR4 −/− , mouse macrophage cultures. V. parvula LPS was able to activate the human PBMC p38 mitogen-activated protein kinase (MAPK). A specific p38 MAPK inhibitor strongly inhibited V. parvula LPS-induced TNF-α, IL-1β, IL-6, and IL-10. In conclusion, V. parvula LPS is able to induce cytokine production in both human and murine in vitro models, although it is less effective than Enterobacteriaceae LPS. V. parvula LPS-stimulated cytokine induction, as well as p38 MAPK activation, are TLR4-dependent features.

Contains fulltext : 81401.pdf (Publisher’s version ) (Closed access) TLR2 plays an important role in the removal of Gram-positive bacteria; contrastingly, it also appears to have important protective effects against unrestrained inflammation and subsequent organ injury during infection and autoimmunity. We hypothesized that TLR2 tunes the phenotype of dendritic cells (DCs) activated through other TLRs, thereby fulfilling a crucial role in the modulation of the immune response. TLR2 potently inhibited TLR4- and TLR7/8-induced cytokine production by human DCs. The inhibitory effect of TLR2 on the release of TNF-alpha but not of IL-12p70 was mediated by PI3K. TLR2 inhibits the production of IL-12p70 by dampening the type 1 IFN amplification loop. When DCs were triggered with the potent synergistic combination of LPS (TLR4) and R848 (TLR7/8) in conjunction with a TLR2 ligand, a clear shift to more Th2- and Th17-prone responses in the naive and memory T cell subpopulations was observed. This shift in T cell responses was inherent to the inability of TLR2-stimulated DCs to produce IL-12p70 and was dependent on the production of IL-1 and IL-6.

Contains fulltext : 79968.pdf (Publisher’s version ) (Closed access) Toll-like receptors (TLRs) may contribute to the pathogenesis of chronic inflammatory destructive diseases through the recognition of endogenous ligands produced on either inflammation or degeneration of the extracellular matrix. The presence of endogenous TLR agonists has been reported in rheumatoid joints. In the present study, we investigated the significance of TLR2 and TLR4 activation by locally- produced endogenous ligands in the severity of joint inflammation and destruction. Local joint pathology independent of systemic immune activation was induced by overexpression of interleukin (IL)-1 and TNF in naive joints using adenoviral gene transfer. Here, we report that at certain doses, IL-1-induced local joint inflammation, cartilage proteoglycan depletion, and bone erosion are dependent on TLR4 activation, whereas TLR2 activation is not significantly involved. In comparison, tumor necrosis factor alpha-driven joint pathology seemed to be less dependent on TLR2 and TLR4. The severity of IL-1-induced bone erosion and irreversible cartilage destruction was markedly reduced in TLR4(-/-) mice, even though the degree of inflammation was similar, suggesting uncoupled processes. Furthermore, the expression of cathepsin K, a marker for osteoclast activity, induced by IL-1beta was dependent on TLR4. Overexpression of IL-1beta in the joint as well as ex vivo IL-1 stimulation of patellae provoked the release of endogenous TLR4 agonists capable of inducing TLR4-mediated cytokine production. These data emphasize the potential relevance of TLR4 activation in rheumatoid arthritis, particularly with respect to IL-1-mediated joint pathology.

Contains fulltext : 70945.pdf (Publisher’s version ) (Closed access) 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.