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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.

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.

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.

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.

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.

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

Background and objectives Seronegative joint diseases are characterised by the lack of autoantibodies and rheumatoid factor, potent biomarkers for disease activity in seropositive arthritis. Promising alternative biomarkers in seronegative arthritis are the Damage Associated Molecular Patterns (DAMPs), S100A8 and S100A9, proteins specifically expressed by infiltrating phagocytes. In this study we explore the biomarker potential of S100A8/S100A9 to asses joint inflammation and damage in IL-1Ra-/- mice, a mouse model for seronegative arthritis in which serum autoantibodies are not correlated to disease activity. Materials and methods Serum of IL-1Ra-/- and BALB/c mice was collected every two weeks and swelling in the hind paws was macroscopically scored. Serum levels of IL-1β, IL-6 and TNFα were measured by Luminex technology and S100A8/A9 complex levels by ELISA. Hind paws were isolated and histologically scored for cell influx, cartilage damage and bone erosion, sections were also stained for S100A9 expression. Synovial S100A8 was imaged by injection of anti-S100A8-Cy7 antibody and 24 h p.i. fluorescent images were acquired in the IVIS Lumina optical imaging system. Results Starting at week 8 up to week 16, serum levels of S100A8/A9 in IL-1Ra-/- mice were significantly increased (1640 ± 1008 ng/ml) compared to BALB/c mice (429 ± 191 ng/ml, P = 0.005) and strongly correlated to joint swelling (r = 0.740, P Conclusions High levels of serum S100A8/A9 correlate to joint inflammation and destruction in experimental seronegative arthritis and local synovial expression of S100-DAMPs can be monitored in vivo by molecular imaging. These findings underline the potential of S100-DAMPs as a serum and imaging biomarker for disease severity in seronegative arthritis.

Background and objectives Interleukin-1 receptor antagonist deficient (IL-1Ra -/- ) mice spontaneously develop a T cell-driven autoimmune arthritis, which depends on the presence of commensal microbiota and Toll-like receptor 4 (TLR4). The aim of this study was to elucidate the role of IL-1 receptor signalling and TLR4 in defining the intestinal microbiota and the associated mucosal and systemic immune response during arthritis. Methods 16S rRNA 454-pyrosequencing meta-genome analysis was used to define intestinal microbial communities in BALB/c wild type (WT), IL-1Ra -/- and IL-1Ra/TLR4 double knock-out (DKO) mice. IL-1Ra -/- mice were treated with antibiotics and re-colonised with segmented filamentous bacteria (SFB). T cell differentiation and cytokine production was assessed in small intestine lamina propria (SI-LP) and draining lymph nodes. Results IL-1Ra -/- mice had a significant reduction in microbial diversity compared to WT mice. Interestingly, the species diversity was restored in IL-1Ra/TLR4 DKO, suggesting that IL-1R-driven skewing of bacterial diversity depends on TLR4. IL-1Ra -/- mice had greatly increased intestinal Th17 levels, which significantly correlated with arthritis scores. Relevance of intestinal microbiota in arthritis was underlined by significant long-term suppression of arthritis by one-week oral treatment with broad-spectrum antibiotics. Re-colonisation of antibiotic-treated IL-1Ra -/- mice by SFB, a potent intestinal Th17 inducer, was sufficient to cause full-blown arthritis. Absence of TLR4 resulted in the sustained reduction of arthritis in IL-1Ra -/- mice. TLR4 is known to play a major role in recognition of Gram-negative bacteria. Interestingly, treatment with tobramycin, specifically eliminating Gram-negative bacteria, significantly reduced arthritis severity. SI-LP mononuclear cells from IL-1Ra -/- TLR4 -/- mice ex vivo cultured with PMA and ionomycin produced substantially less IL-17. In addition, production of IL-23, IL-1β and IL-6 upon ex vivo stimulation with intestinal microbial antigens was reduced in the absence of TLR4. This suggests that TLR4 plays a role in microbiota-induced production of cytokines involved in intestinal Th17 differentiation. Conclusions Our data suggest that activation of TLR4 by commensal intestinal microbiota drives arthritis in IL-1Ra -/- mice via intestinal IL-1, IL-23, IL-6production and subsequent Th17 induction. Understanding the mechanisms linking the intestinal T cell response with arthritis may help identifying novel therapeutic targets in rheumatoid arthritis.

Background Accumulating evidence indicates the relevance of intestinal microbiota in shaping the immune response and supports its contribution to the development of autoimmune diseases. Prebiotic non-digestible oligosaccharides are known to selectively support growth of commensal Bifidobacteria and Lactobacilli and adjust the microbiota composition. Objectives The aim of this study was to assess the efficacy of microbiota modulation using non-digestible oligosaccharides as a therapeutic approach for T cell-dependent autoimmune arthritis. Methods IL-1 receptor antagonist (IL-1Ra) deficient mice spontaneously developing an autoimmune T cell-dependent arthritis were used for this study. To examine the feasibility of microbiota modulation as a therapeutic approach during established disease, IL-1Ra -/- mice which had already developed arthritis under conventional microbial status were orally fed a prebiotic diet containing short-chain galacto- and long-chain fructooligosaccharides (scGos:lcFos, 9:1). Disease progression was monitored and intestinal and systemic T cell differentiation was studied. Multiplex 454 pyrosequencing of fecal bacterial 16S rRNA was used to asses changes in composition of microbiota. Results Oral treatment of arthritic IL-1Ra -/- mice with scGoslcFos significantly suppressed the progression of arthritis. Gene expression of T-bet and RORγt, the Th1 and Th17-related transcription factors, in lymph nodes draining the arthritic joints was significantly reduced in the group receiving the scGoslcFos diet. Furthermore, dual-energy X-ray absorptiometry scanning revealed that a prebiotic diet containing scGoslcFos significantly improved bone mineral density and tended to increase bone mineral content in arthritic IL-1Ra -/- mice. High-throughput pyrosequencing revealed a that scGoslcFos has a profound effect on relative abundance of bacteria in different taxa. The most notable alterations concerned a significant increase in Lactobacilli and a strong decrease in the genus Turicibacter . Interestingly, intestinal gene expression of the Treg-related transcription factor FoxP3 as well as anti-inflammatory cytokine IL-10 were increased with scGoslcFos. Accordingly, small intestine lamina propria lymphocytes of mice receiving the scGoslcFos diet produced significant higher levels of IL-10 upon ex vivo stimulation with PMA and ionomycin. Production of IL-4 and IFNγ also tended to be increased, while production of TNFα, IL-6 and IL-17 was not affected by the prebiotic diet. Conclusions Our data suggest that scGoslcFos suppresses arthritis progression, potentially through induction of anti-inflammatory cytokines such as IL-10 and IL-4. Suppression of disease progression using dietary intervention with prebiotic scGoslcFos may be applicable as a therapeutic approach to suppress autoimmune arthritis. Disclosure of Interest None declared

Background Mice deficient in interleukin-1 receptor antagonist (IL-1Ra -/- ) spontaneously develop a T cell-driven autoimmune arthritis, which we previously showed to depend on the presence of commensal microbiota. Recent findings suggest alteration of intestinal microbiome in new-onset rheumatoid arthritis (RA). Objectives The aim of this study was to investigate the role of IL-1 receptor signaling and Toll-like receptor (TLR) 2 and TLR4 in defining the intestinal microbiota and the associated mucosal and systemic immune response during arthritis. Methods Multiplex 454 pyrosequencing of fecal bacterial 16S rRNA was used to define intestinal microbial communities in BALB/c wild type (WT), IL-1Ra -/- and IL-1Ra/TLR double knock-out (DKO) mice. For gene sequencing analysis, a customized workflow based on Quantitative Insights Into Microbial Ecology (QIIME version 1.2) was adopted. T cell differentiation was assessed in small intestine lamina propria (SI-LP) and spleen using flow cytometry and gene expression was assessed by qPCR. Results IL-1Ra -/- mice had a significant reduction in microbial diversity compared to WT mice. Both alpha diversity (number of unique taxonomic entities) and phylogenetic diversity (PD) whole tree (based on taxonomic distance) were significantly diminished in IL-1Ra -/- mice. Interestingly, the loss of species diversity was absent in IL-1Ra/TLR4 DKO, but not IL-1Ra/TLR2 DKO mice, suggesting that IL-1R-driven skewing of bacterial diversity depends on TLR4. IL-1Ra -/- mice exhibited significantly increased abundance of the genus Helicobacter and reduced Prevotella ( p =0.008 and p =0.004, respectively). Importantly, significant alterations in the genera Xylanibacter , Prevotella , Streptococcus , and Ruminococcus were markedly normalized in TLR4, but not TLR2, deficient mice, identifying a role for TLR4 in IL-1 mediated shifts in microbial community. In line with the relevance of intestinal microbiota in mucosal T cell polarization, IL-1Ra -/- mice had greatly increased Th17 in SI-LP. Interestingly, SI-LP Th17% significantly correlated with arthritis score. Although intestinal mRNA expression of IL-1 itself remained unaltered, IL-23p19 expression was increased. Both IL-1b and IL-23 were significantly diminished in IL-1Ra/TLR4 DKO mice, suggesting a TLR4-mediated regulation. These mucosal responses paralleled systemic response and arthritis development, since Th17% and associated genes such as RORγt were increased in IL-1Ra -/- and reduced in IL-1Ra/TLR4 DKO mice which had less severe arthritis. Conclusions These data suggest a TLR4-mediated regulation of intestinal microbiome, and mucosal and splenic immune responses controlling arthritis severity, potentially through an IL-1 and IL-23 dependent mechanism. Understanding the molecular and cellular mechanisms linking the intestinal T cell response with arthritis may help identifying novel therapeutic targets in RA. Disclosure of Interest None declared

Contains fulltext : 154313.pdf (Publisher’s version ) (Open Access) In autoimmune diseases, a disturbance of the balance between T helper 17 (Th17) and regulatory T cells (Tregs) is often observed. This disturbed balance is also the case in rheumatoid arthritis (RA). Genetic predisposition to RA confers the presence of several polymorphisms mainly regulating activation of T lymphocytes. However, the presence of susceptibility factors is neither necessary nor sufficient to explain the disease development, emphasizing the importance of environmental factors. Multiple studies have shown that commensal gut microbiota is of great influence on immune homeostasis and can trigger the development of autoimmune diseases by favoring induction of Th17 cells over Tregs. However the mechanism by which intestinal microbiota influences the Th cell balance is not completely understood. Here we review the current evidence supporting the involvement of commensal intestinal microbiota in rheumatoid arthritis, along with a potential role of Toll-like receptors (TLRs) in modulating the relevant Th cell responses to trigger autoimmunity. A better understanding of TLR triggering by intestinal microbiota and subsequent T cell activation might offer new perspectives for manipulating the T cell response in RA patients and may lead to the discovery of new therapeutic targets or even preventive measures.