ESE CR1 5 ONO CERRO DE C R1

Summary

Th17 cells have been implicated in the pathogenesis of chronic destructive arthritis. In physiological conditions these cells give protection against extracellular bacteria and fungi. Especially C. albicans is known for its potent induction of Th17 cells. First, we tried to induce pathogenic Th17 cells in vivo and study their effects on joint pathology. We used the chronic SCW-induced arthritis and investigated whether co-exposure to a small amount of yeast particles on top of the repetitive local exposure to TLR2/NOD2 agonist could specifically enhance the Th17 cell expansion and polarize the arthritis towards a more destructive phenotype (chapter 2). The i.a. injections with C. albicans resulted in a significant increase in joint inflammation, which was accompanied by enhanced cartilage erosion. In summary, we found an increase in IL-17, IFN-γ and IL-21 together with more IL-17 producing T-cells. Previous studies in Chrohn’s patients have demonstrated increased levels of antibodies directed against C. albicans, which imply a direct role for C. albicans in the disease. Furthermore, colonization of C. albicans in the gut of immunized collagen-in- duced arthritis mice increases disease severity. Since the gut is also implied to be involved in RA, investigating whether or not C. albicans is directly involved in other autoimmune disorders like RA is of great importance.

To understand the mechanism by which C. albicans induces the Th17 response, we explored the role the specific pattern recognition receptors (chapter 3). Freshly isolated PBMCs were stimulated with several pathogen-associated molecular patterns (PAMPs) and whole (heat-killed) microorganisms in the presence of human serum, without additional costimulatory factors. The fungal pathogen C. albicans was found to induce potent IL-17 responses, even more potently than several Gram-negative bacteria. Furthermore, several pathogens were able to induce IL-1 and/or IL-6, but only C. albicans could induce a clear IL-17 response. This Candida-induced IL-17 response turned out to be dependent on the mannose receptor (MR; CD206) expressed by monocytes/macrophages. Finally, this MR-induced IL-17 production was augmented by the TLR2/dectin-1 pathway. This study identified a new role for the MR being involved in triggering Th17 cells in response to C. albicans.

Previous studies from our department have clearly shown the potency of anti-IL-17 therapy in reducing cartilage destruction and bone erosions in several experimental arthritis models (1). TNF-α is the most commonly targeted cytokine in RA, but still approximately 30% of the patients do not respond to anti-TNF-α therapy. Because of the heterogeneity of the disease, more specific cytokine targeting or combination therapies might be beneficial for big cohorts of patients. IL-17 has been shown to synergize with several other cytokines in vitro, including TNF-α (2). Because of this synergy and since synovial explants of patients that did not respond to anti-TNF-α treatment (TNF-non-re-

sponders) still respond to anti-TNF-α treatment (unpublished observation) ex vivo, we explored the interaction between TNF-α and IL-17 in more detail. (chapter 4) First, by over expressing IL-17 and TNF-α in vivo, we observed that synovial IL-17 expression promotes TNF-α-induced joint pathology. Further analysis revealed that exposure to both IL-17 and TNF-α synergistically enhanced up-regulation of S100A8, IL-1β, and matrix metalloprotein- ases (MMPs). Additional in vitro experiments revealed that S100A9 deficient mice (which also lack functional S100A8 protein) were protected against IL-17/TNF-α-induced expression of cartilage NITEGE, a neoepitope that reflects ADAMTS-mediated proteoglycan cleavage. Both in vivo and in vitro experiments clearly demonstrate an important role for S100A8/S100A9 in the IL-17/TNF-α-induced cartilage destruction. Finally, neutralization of IL-17 and TNF-α signaling during established arthritis was more effective than single anti-cytokine treatments, and significantly inhibited further joint inflammation and cartilage destruction. In summary, this study showed that neutralizing IL-17 in addition to TNF-α was still effective in the late stages of arthritis, even under conditions in which blocking of IL-17 or TNF-α alone was no longer effective. Therefore, combination therapy might show great potential, especially in RA patients that do not sufficiently respond to anti-TNF-α therapy alone.

Besides the growing knowledge about the induction and expansion of Th17 cells, it became clear that IL-17 is not the only cytokine produced by Th17 cells, but also IL-21 and IL-22 might have some immunogenic and pathogenic properties. In chapter 5 and 6, we sought to explore the role of both cytokines during experimental arthritis, specifically focusing on their expression and role in T-cell driven joint pathology. IL-21 is one of the Th17 cytokines that acts on the T cells themselves via an autocrine loop, and contributes to their formation and expansion. An earlier study had shown that IL-21 was involved in the development of the collagen induced arthritis model (3). The purpose of our study (chapter 5) was to investigate the effect of IL-21R-deficiency on joint pathology in relation to Th17 cells during chronic experimental arthritis. We investigated the inflammation and joint destruction during the chronic T-cell driven stages of the antigen-induced arthritis (AIA) and the chronic Streptococcal Cell Wall (SCW) arthritis. In both models IL-21R-/- mice were protected against severe arthritis. This reduced pathology was accompanied by a decrease in serum IgG1 levels and suppressed antigen-specific T cells responses. IL-17 levels were reduced during AIA and in the SCW arthritis we observed a decline in the IL-17+ IFN-γ+ T-cells isolated from the arthritic knee joints. Both observations confirm the importance of IL-21 on the Th17/IL-17 induction. In contrast, clearly enhanced local activation and joint inflammation were observed in IL-21R-deficient mice shortly after each SCW injection. Previously, it has been shown that IL-21 could restrain TLR4 signaling in dendritic cells via a SOCS (suppressor of cytokine signaling) dependent mechanism (4). Consequently, we performed in vitro assays to explore the involvement of SOCS in the enhanced SCW response in the IL-21R-/- mice. Exposure to SCW together with IL-21

stimulation did result in a lower inflammatory response due to the upregulation of SOCS1 and SOCS3, a mechanism lacking in the IL-21R-/- mice. Nonetheless, IL-21 has a more dominant pro-destructive role driving Th1/17 cells and joint pathology during chronic experimental arthritis, even despite the suppressive role of IL-21 via SOCS during acute inflammatory responses. The data from chapter 5 suggests that anti-IL-21 therapy might have a potential value as a therapeutic in the course of RA.

Recently it was discovered that IL-22 is mainly produced by Th17 cells, but the exact role in the pathogenesis of rheumatoid arthritis was still unclear. In chapter 6, we investigated the regulation of IL-22 by Th17 cells and evaluated the potential for therapeutic blocking of IL-22 in an in vivo model. In vitro tests showed that Th17 cells produce high levels of IL-22 after exposure to IL-1 or IL-23. This production of IL-22 could be further increased after combining the two cytokines. Because of the observed role for IL-1 on Th17 cells in vitro and during the IL-1Ra model in vivo (5), we decided to select this IL-1-dependent model to test the potential of the anti-IL-22 antibody. Interleukin-1 receptor antagonist-deficient (IL-1Ra-/-) mice spontaneously develop an inflammatory and destructive arthritis, which is IL-1, IL-17 and T-cell dependent. Investigating the progression of the erosive arthritis in these mice, we only detected increased IL-22 and IL-22R in the severely and chronically inflamed synovial tissues, whereas IL-17 was already upregulated during early inflammation. Analysis of isolated single cells from the inflamed synovia revealed that IL-22 was mainly produced by IL-17-expressing T cells. Because of this late expression of IL-22, we decided to start the anti-IL-22 treatment with mice having active arthritis. Anti-IL-22 treatment significantly reduced the inflammation and bone erosion, verifying the pro-inflammatory role of IL-22 during arthritis.

Recent reports have shown that RA patients with high serum levels of IL-22 represent a group of patients suffering from a more destructive and more progressive phenotype. These findings suggest that IL-22 differentiates the more progressive RA patients, which might require a more aggressive therapy. Whether IL-22 will be a good target for treatment in RA will depend on its relationship with other cytokines. It has been described that IL-17 can promote the expression and pro-inflammatory properties of IL-22, while it impedes its regenerative properties (6). Therefore, it might be more reasonable to block the IL-17 pathway instead of the IL-22 pathway, which will decline inflammation and at the same time restore the regenerative capacities of IL-22.

Murine experimental arthritis models are widely used to study inflammatory processes, but not all of these findings can be translated directly to the patients. The best example might be the discrepancy of the role of IL-1 and TNF-α between mice and humans. The classical murine arthritis models have shown a central role for IL-1β in driving joint pathology, with a lesser potential for TNF-α (7). On the contrary, anti-TNF-α treatment is now the most frequently prescribed biologic approach for RA patients, while blocking of

IL-1β only shows modest therapeutic effects (8). In the preceding years, our group has put a lot of effort in setting up a humanized arthritis model, with the expectation to make better predictions for new therapeutics that will proceed to clinical trials. In chapter 7, we describe the validation of this model, using SCID-CB17 mice that were engrafted with human RA synovial tissue. We validated the model by treatment with anti-TNF-α antibodies and comparing anti-TNF-α to anti-IL-1β treatment. In addition, the direct effect of new and current T and B cell-related therapies were investigated. Anti-TNF treatment significantly reduced serum cytokine levels and decreased histological inflammation, whereas anti-IL-1 therapy did not show any effect on the RA synovial grafts. Next, anti-CD20 treatment only showed clear therapeutic effects in mice engrafted with B cell-rich synovial tissue. Surprisingly, CTLA4-Ig treatment, which is effective in the clinics, did not show any effects in this transplantation model. Pre-screening of the synovial tissue for the presence of CD3+ T cells and the co-stimulatory molecules CD80/86 did not alter the outcome. One of the reasons of this failure might be that CTLA4-Ig exerts its effects not locally within the synovium. Finally, great therapeutic potential was observed for anti-IL-17 treatment, however only when CD3+ T cells were abundantly present in the RA synovial tissue. In conclusion, the humanized RA SCID model can be a valuable tool for predicting responses of new therapeutics that act locally within the arthritic joint.