Renoud J. Marijnissen

The anti-CD20 antibody rituximab reduces the Th17 cell response

OBJECTIVEnRituximab has been shown to be successful in the treatment of rheumatoid arthritis (RA), and this unexpected finding indicates that B cells have an important role in this disease. The present study was undertaken to investigate the mechanism of action of rituximab in RA.nnnMETHODSnTwelve patients with active RA were treated with rituximab. Disease activity was evaluated using the 28-joint Disease Activity Score. Synovial biopsy samples obtained at baseline and 12 weeks after treatment initiation were analyzed by microarray, quantitative polymerase chain reaction, and immunohistochemistry. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and from 4 patients with X-linked agammaglobulinemia were stimulated with the Th17-inducing stimulus Candida albicans, and the response in the presence and absence of rituximab was examined.nnnRESULTSnIn RA patients, rituximab reduced expression of retinoic acid-related orphan receptor γt and interleukin-22 (IL-22) and numbers of Th17-positive cells in synovial tissue, and this correlated with better clinical outcome. Rituximab did not affect tumor necrosis factor α (TNFα), Th1 cell, or Treg cell responses. Rituximab strongly reduced in vitro IL-17 and IL-22 production induced by C albicans. This effect was not observed in PBMCs from patients with X-linked agammaglobulinemia.nnnCONCLUSIONnRituximab reduced the local Th17 response in RA patients, whereas it did not influence Th1 cell, Treg cell, or TNFα responses. The decreased Th17 response was associated with reduced inflammation and better clinical outcome. Moreover, inhibition of the Th17 response by rituximab was lost in the absence of B cells, providing evidence that the effects of rituximab are due to B cell depletion. These data demonstrate an unexpected role of B cells in the development of Th17 responses, which could possibly lead to B cell-based strategies for the treatment of Th17-related autoimmune diseases.

Interleukin-1 drives pathogenic Th17 cells during spontaneous arthritis in interleukin-1 receptor antagonist–deficient mice

OBJECTIVEnInterleukin-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.nnnMETHODSnT 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.nnnRESULTSnCompared 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.nnnCONCLUSIONnIncreased 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.

A1.22 IL-22 Drives the initiation and augmentation of TH17-dependent experimental arthritis

Background Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that leads to progressive destruction of cartilage and bone. IL-22 and IL-22-producing T helper cells are elevated in RA patients, suggesting a role for this cytokine in the pathogenesis of this disease. Interestingly, IL-22 is a dual cytokine with both proinflammatory and anti-inflammatory properties, and therefore its exact role in RA pathology requires further investigation. Objectives The aim of this study was to explore the role of IL-22 in the initiation and augmentation of a spontaneous model of experimental arthritis by using gene knockout mice and neutralizing antibodies for IL-22. In addition, we aimed to determine the influence of IL-22 on T cell subsets and cytokines in our mouse model. Methods IL-1Ra-deficient mice develop spontaneous arthritis due to excess IL-1 signalling, and we previously demonstrated the importance of IL-17 and Th17 cells in this model (Koenders, Arthritis Rheum 2008). To investigate the role of IL-22 in this Th17-dependent arthritis model, we compared IL-1Ra-/- x IL-22WT mice to mice lacking both IL-1Ra and IL-22. Joint swelling was scored weekly, and mice were sacrificed at the age of fifteen weeks. In addition, IL-1Ra-deficient mice were treated for 4 weeks with anti-IL-22 neutralizing antibodies after the onset of arthritis to inhibit disease progression. Results IL-1Ra-/- mice also deficient for IL-22 showed reduced incidence of arthritis, as well as reduced joint swelling and bone erosion. In addition, Th1, Th17, and regulatory T cell numbers were decreased in spleen and lymph nodes of mice lacking IL-22. Furthermore, IL-1Ra-deficient mice treated with anti-IL-22 antibodies after the clinical onset of arthritis showed reduced progression on inflammation and significant inhibition on bone erosion. This indicates that not only the onset but also the progression of arthritis is dependent on IL-22. Interestingly, reduced IL-17 serum levels were found after IL-22 blocking, suggesting a not previously observed feedback loop of IL-22 on Th17 cells. Conclusion These findings suggest that the Th17 cytokine IL-22 plays an important role both in initiation and augmentation of experimental arthritis, and might therefore be an interesting new target in RA treatment.

Bacillus Calmette-Guerin-Induced Trained Immunity Is Not Protective for Experimental Influenza A/Anhui/1/2013 (H7N9) Infection in Mice

Avian influenza A of the subtype H7N9 has been responsible for almost 1,600 confirmed human infections and more than 600 deaths since its first outbreak in 2013. Although sustained human-to-human transmission has not been reported yet, further adaptations to humans in the viral genome could potentially lead to an influenza pandemic, which may have severe consequences due to the absence of pre-existent immunity to this strain at population level. Currently there is no influenza A (H7N9) vaccine available. Therefore, in case of a pandemic outbreak, alternative preventive approaches are needed, ideally even independent of the type of influenza virus outbreak. Bacillus Calmette–Guérin (BCG) is known to induce strong heterologous immunological effects, and it has been shown that BCG protects against non-related infection challenges in several mouse models. BCG immunization of mice as well as human induces trained innate immune responses, resulting in increased cytokine responses upon subsequent ex vivo peripheral blood mononuclear cell restimulation. We investigated whether BCG (Statens Serum Institut-Denmark)-induced trained immunity may protect against a lethal avian influenza A/Anhui/1/2013 (H7N9) challenge. Here, we show that isolated splenocytes as well as peritoneal macrophages of BCG-immunized BALB/c mice displayed a trained immunity phenotype resulting in increased innate cytokine responses upon ex vivo restimulation. However, after H7N9 infection, no significant differences were found between the BCG immunized and the vehicle control group at the level of survival, weight loss, pulmonary influenza A nucleoprotein staining, or histopathology. In conclusion, BCG-induced trained immunity did not result in protection in an oseltamivir-sensitive influenza A/Anhui/1/2013 (H7N9) challenge mouse model.

FRI0022 Periodontal pathogens promote autoimmune arthritis by reducing TH2 response and inducing a toll-like receptor 2-dependent TH17 phenotype

Background Epidemiologic evidence and the capability of periodontal pathogen Porphyromonas gingivalis to citrullinate mammalian proteins support a link between periodontal disease (PD) and rheumatoid arthritis (RA). Based on the relevance of pathogen-mediated Toll-like receptor (TLR) activation in shaping the T cell response, we reasoned that the interaction between PD and RA may be a direct result of T cell modulation and not necessarily dependent on citrullination capability. Objectives The aim of this study was to investigate the influence of periodontal pathogens P. gingivalis and Prevotella nigrescens, the latter lacking citrullinating enzymes, on T helper cell phenotype and the development of experimental arthritis. Methods The influence of P. gingivalis and P. nigrescens on T cell differentiation and the TLR involvement were studied using wild-type, and interleukin-1 receptor antagonist (IL-1Ra)/TLR single or double gene deficient cells. In vivo, mice with collagen-induced arthritis received five oral inoculations of either P. gingivalis or P. nigrescens every other day starting from day 14 after immunization. Joint histopathology, synovial gene expression, T cell phenotype and presence of anti-citrullinated peptide antibodies (ACPA) were analyzed on day 30. Results P. gingivalis and P. nigrescens strongly induced Th17 differentiation in a co-culture of antigen-presenting cells (APCs) with CD4+ T cells, as measured by FACS and IL-17 production. This effect was highly increased by IL-1Ra deficiency. In addition, both bacteria significantly lowered the Th2 differentiation, but were weak inducers of Th1. Using IL-1Ra-/- TLR2-/- cells, Th17 induction was found to depend on TLR2 expression on APCs; whereas the minor Th1 induction was dependent on TLR2 expression directly on T cells. TLR4 activation was not involved in Th17 differentiation induced by these bacteria. In vivo, oral inoculation with P. gingivalis and P. nigrescens significantly increased the clinical arthritis scores. Although no antibodies against cyclic citrullinated peptide (CCP)-2, citrullinated fibrinogen, a-enolase and vimentin could be detected under this condition, both bacteria substantially increased the severity of bone erosion. Collagen II stimulation of draining lymph node T cells revealed that P. gingivalis as well as P. nigrescens significantly increased antigen-specific IL-17, but not IFNγ, production. This IL-17 production strongly correlated with the intensity of bone erosion. In addition, P. nigrescens markedly suppressed the anti-inflammatory Th2/IL-4 phenotype in vivo, whereas P. gingivalis was the main inducer of local IL-1b, TNFa and Cathepsin K in synovium. Conclusions These data reveal a substantial effect of periodontal pathogens, irrespective of the capability to induce ACPA, on T cell phenotype in the context of arthritis with a remarkable impact on bone erosion. This study further supports the relevance of periodontal pathogens in the pathogenesis of RA. Disclosure of Interest None Declared

Periodontal pathogens amplify arthritic bone erosion by reducing the TH2 response and promoting a toll-like receptor 2-dependent TH17 phenotype

Backgroundand objectives Increased incidence of periodontal disease and association with development of anticitrullinated peptide antibodies (ACPA) in patients with rheumatoid arthritis suggest a possible pathologic link between the two diseases. The aim of the present study was to investigate the influence of periodontal pathogens on T helper cell phenotype and disease severity during experimental arthritis. Materials and methods The effect of periodontal bacteria Porphyromonas gingivalis and Prevotella nigrescens on T cell differentiation and the involvement of toll-like receptors (TLRs) were studied using cells from wild-type and interleukin-1 (IL-1) receptor antagonist deficient mice. Invivo, mice with collagen-induced arthritis received five oral inoculations of either P gingivalis or P nigrescens every other day starting from day 14 after immunisation. Joint histopathology, gene expression in synovium, T cell phenotype and presence of ACPA were analysed on day 30. Results P gingivalis and P nigrescens strongly induced Th17 differentiation in a co-culture of splenic antigen-presenting cells (APCs) with CD4+ T cells, as measured by fluorescence-activated cell sorting analysis and IL-17 production. This effect was enormously increased in the absence of IL-1 receptor antagonist. In addition, both bacteria significantly lowered the Th2 differentiation, but were weak inducers of Th1. Using IL-1Ra−/− cells derived from TLR knockouts, Th17 induction was found to depend on TLR2, but not TLR4, expression on APCs; whereas the minor Th1 induction was dependent on TLR2 expression directly on T cells. Invivo, oral inoculation with P gingivalis and P nigrescens significantly increased the clinical scores of arthritis. Although no antibodies against CCP2 and citrullinated fibrinogen, α-enolase and vimentin could be detected under this condition, both bacteria substantially increased the intensity of bone erosion specifically, without influencing cartilage destruction. Analysis of T helper cell phenotype in draining lymph nodes upon pan-T cell as well as collagen II stimulations revealed a significant increase of IL-17 production, but not interferon γ. The level of IL-17 induced by periodontal bacteria strongly correlated with the intensity of bone erosion. While P gingivalis was the main inducer of local IL-1β, tumour necrosis factor α and cathepsin K in synovium, only P nigrescens caused a marked reduction of Th2/IL-4 phenotype invivo. Conclusions This study reveals the modulation of T cell phenotype, in particular Th17 induction, as a relevant pathogenic characteristic of periodontal pathogens irrespective of ACPA induction or possession of citrullinating enzymes (present in P gingivalis, but not P nigrescens). The data further support the relevance of periodontal bacteria in pathogenesis of arthritis, especially with respect to bone erosion.

Increased innate immune responses by interleukin-22 contributes to the inflammatory process in rheumatoid arthritis

Backgroundand objectives IL-22 is a mediator in antimicrobial responses and inflammatory autoimmune diseases. It can be produced by innate and adaptive haematopoietic immune cells, but the expression of the receptor (IL-22R) seems to be restricted to non-haematopoietic tissue cells. Although both IL-22 and the IL-22R have been identified in the synovium of RA patients, the contribution to disease pathogenicity remains to be established. Materials and methods The authors first investigated the expression and origin of IL-22 and the IL-22R in the inflamed joints of IL-1Ra-/- mice. Furthermore, the mice were treated with neutralising anti-IL-22 antibodies. Subsequently, the authors analysed the IL-22R expressing cells in human RA synovium by immunohistochemistry (IHC) and flow cytometry. Eventually, the authors stimulated RA synovial biopsies, RA human fibroblast-like cells (RAFLS) and endothelial (HUVEC) cells with IL-22 and analysed the production of inflammatory mediators. Results In the arthritic joints of IL-1Ra-/- mice, IL-22 is mainly produced by T cells that co-expressed IL-17. Anti-IL-22 treatment of IL-1Ra-/- mice significantly reduced inflammation and bone erosion, suggesting an important role of IL-22 in joint destruction. The authors subsequently analysed the effects of IL-22 on human RA synovium. Exposure to IL-22 increased the expression of TNFα, IL-6 and IL-8. Compared to IL-17, stimulation with IL-22 induced higher levels of IL-8, and combination of the two cytokines resulted in an additional response. IHC identified endothelial cells and synovial fibroblast-like cells as the main IL-22 receptor expressing cells. Surprisingly, stimulation of RA human fibroblast-like cells and endothelial (HUVEC) cells did not result in a clear increase in the innate immune response. This in contrast to the synovial tissue, which is heavily infiltrated with inflammatory cells. Conclusions Our data support the dual role of IL-22 in pathological inflammation, showing that the functional importance of IL-22 is context dependent. Current SCID studies with neutralising IL-22 antibodies will have to prove the therapeutic potential of IL-22 in RA.

IL-21 induces socs-mediated suppression of TLR triggering but aggravates TH17-driven destructive arthritis

Background and objective IL-21 is an immune-regulatory cytokine that 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. Material and methods For in vitro studies, freshly isolated C57Bl6 splenocytes were stimulated with TLR2/NOD2-binding ligands in the presence or absence of IL-21. In addition, chronic Streptococcal cell wall (SCW) arthritis and antigen-induced arthritis (AIA) were induced in IL-21-receptor-deficient (IL-21R-/-) mice and wild-type (WT) controls. Results In vitro stimulation of splenocytes with TLR2/NOD2-binding SCW fragments resulted in enhanced production of IL-6 and CXCL1, but not IL-10. Interestingly, this proinflammatory response was blocked in the presence of IL-21. QPCR analysis demonstrated that IL-21 strongly induced SOCS expression, suggesting a SOCS-dependent immunosuppressive effect of IL-21 on TLR signaling. In contrast, at first sight our in vivo studies using IL-21R-deficient mice showed a proinflammatory role of IL-21 in experimental arthritis. In both SCW-induced arthritis and AIA, IL-21R-deficiency protected against severe joint inflammation and destruction. This reduced pathology in IL-21R-/- mice was accompanied by suppressed antigen-specific T cell responses, decreased serum IgG1 levels, reduced IL-17 levels in joint lavage, and lower numbers of IL-17+ IFNγ+T cells in the joint. However, during every local (re)challenge of SCW arthritis with TLR ligands, a clearly enhanced joint swelling was found in IL-21R-deficient mice. No differences were found in the expression of TLR2 and NOD2, the most important receptors for SCW. However, while the WT showed a massive upregulation of SOCS1/3 at day 4 of arthritis, IL-21R-/- mice were significantly less capable in upregulating these genes. These data suggest that impaired SOCS regulation in the absence of IL-21 signaling contributes to the increased local activation during SCW arthritis. Conclusion Despite the proinflammatory role of IL-21 in adaptive immunity, driving IL-17/IFNγ double-positive cells and joint pathology during chronic experimental arthritis, IL-21 also has an important immunosuppressive role in innate immunity by inhibiting TLR signaling via SOCS1/3. This dual role of IL-21 in various immune processes makes IL-21 a difficult therapeutic target in RA.

Dual role of IL-21 in experimental arthritis via SOCS regulation and Th17 differentiation

Objectives Interleukin-21 (IL-21) is a pleiotropic cytokine that is produced mainly by activated CD4+ Th17 cells. The purpose of this study was to investigate the role of IL-21 in joint pathology during chronic experimental arthritis, in particular the effect of IL-21 receptor (IL-21R) deficiency on innate and adaptive immune responses in vivo. Methods IL-21R−/− mice and their wild-type (WT) controls were used for this study, and two experimental arthritis models were induced: chronic streptococcal cell wall (SCW) induced arthritis and antigen-induced arthritis (AIA). Results At day 28 of SCW arthritis, histological analysis of the knee joints showed significantly reduced inflammation in IL-21R−/− mice compared to their WT controls. These IL-21R−/− mice also demonstrated suppressed serum levels of IL-6, but interestingly this proinflammatory cytokine tended to be increased in the local patella-washouts. This increased local activation in IL-21R−/− mice was studied in more detail in the early phase of SCW-arthritis. Before onset and 4 days after the first intra-articular injection with SCW fragments, the expression level of various receptors and regulators was determined by QPCR. No differences were found in the expression of TLR2 and NOD2, both crucial for a response to the injected SCW fragments. However, while the WT controls showed a massive upregulation of SOCS1/3 at day 4 of arthritis, IL-21R−/− mice were significantly less capable in upregulating these genes. This failure to upregulate SOCS expression in the joint resulted in increased local expression of inflammation and destruction markers in IL-21R-deficient mice, probably due to disturbed negative regulation of cytokine and TLR signaling pathways. Interestingly, despite the increased local activation in the IL-21R−/− mice, detailed histological analysis of the joints at day 28 of the chronic SCW-arthritis demonstrated that IL-21R-deficiency protected against cartilage proteoglycan depletion and chondrocyte death. FACS analysis of synovial cells showed a significant reduction of the percentage IL-17+ T cells. These findings were confirmed in a second model of chronic destructive arthritis, the mBSA-induced AIA. Also in this model, IL-21R-deficiency resulted in a significant reduction of joint inflammation and destruction compared to wild-type controls, again in striking contrast to the local increase in cytokine expression, but accompanied by suppressed numbers of Th17 cells. Conclusion Despite the local suppressive role of IL-21 via SOCS regulation, IL-21 has a more dominant prodestructive role driving Th17 cells and cartilage and bone pathology during chronic experimental arthritis. However, this dual role makes IL-21 a complicated target in the treatment of rheumatoid arthritis.

T cell lessons from the RA synovium SCID mouse model: synovial tissue rich in CD3 T cells lacks response to CTLA4-Ig, but is successfully treated with anti-IL-17 antibodies

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 patients individual synovial profile is of great importance to achieve tailor-made therapy.