D.M. Roeleveld

The role of the Th17 cytokines IL-17 and IL-22 in Rheumatoid Arthritis pathogenesis and developments in cytokine immunotherapy

Over the past few years, the importance of Interleukin (IL)-17 and T helper (Th)17 cells in the pathology of Rheumatoid Arthritis (RA) has become apparent. RA is a systemic autoimmune disease that affects up to 1% of the population worldwide. It is characterized by an inflamed, hyperplastic synovium with pannus formation, leading to bone and cartilage destruction in the joints. By the production of effector cytokines like IL-17 and IL-22, the T helper 17 subset protects the host against bacterial and fungal infections, but it can also promote the development of various autoimmune diseases like RA. Hence, the Th17 pathway recently became a very interesting target in RA treatment. Up to now, several therapies targeting the Th17 cells or its effector cytokines have been tested, or are currently under investigation. This review clarifies the role of Th17 cells and its cytokines in the pathogenesis of RA, and provides an overview of the clinical trials using immunotherapy to target this particular T helper subset or the two main effector cytokines by which the Th17 cells exert their function, IL-17 and IL-22.

In vivo imaging of antileukemic drug asparaginase reveals a rapid macrophage mediated clearance from the bone marrow

The antileukemic drug asparaginase, a key component in the treatment of acute lymphoblastic leukemia, acts by depleting asparagine from the blood. However, little is known about its pharmacokinetics, and mechanisms of therapy resistance are poorly understood. Here, we explored the in vivo biodistribution of radiolabeled asparaginase, using a combination of imaging and biochemical techniques, and provide evidence for tissue-specific clearance mechanisms, which could reduce the effectiveness of the drug at these specific sites. Methods: In vivo localization of 111In-labeled Escherichia coli asparaginase was performed in C57BL/6 mice by both small-animal SPECT/CT and ex vivo biodistribution studies. Mice were treated with liposomal clodronate to investigate the effect of macrophage depletion on tracer localization and drug clearance in vivo. Moreover, macrophage cell line models RAW264.7 and THP-1, as well as knockout mice, were used to identify the cellular and molecular components controlling asparaginase pharmacokinetics. Results: In vivo imaging and biodistribution studies showed a rapid accumulation of asparaginase in macrophage-rich tissues such as the liver, spleen, and in particular bone marrow. Clodronate-mediated depletion of phagocytic cells markedly prolonged the serum half-life of asparaginase in vivo and decreased drug uptake in these macrophage-rich organs. Immunohistochemistry and in vitro binding assays confirmed the involvement of macrophagelike cells in the uptake of asparaginase. We identified the activity of the lysosomal protease cathepsin B in macrophages as a rate-limiting factor in degrading asparaginase both in vitro and in vivo. Conclusion: We showed that asparaginase is rapidly cleared from the serum by liver-, spleen-, and bone marrow–resident phagocytic cells. As a consequence of this efficient uptake and protease-mediated degradation, particularly bone marrow–resident macrophages may provide a protective niche to leukemic cells.

Higher efficacy of anti-IL-6/IL-21 combination therapy compared to monotherapy in the induction phase of Th17-driven experimental arthritis

Th17 cells and their cytokines are linked to the pathogenesis of rheumatoid arthritis, a chronic autoimmune disease characterized by joint inflammation. Th17 development is initiated by combined signaling of TGF-β and IL-6 or IL-21, and can be reduced in the absence of either IL-6 or IL-21. The aim of this study was to assess whether combinatorial IL-6/IL-21 blockade would more potently inhibit Th17 development, and be more efficacious in treating arthritis than targeting either cytokine. We assessed in vitro Th17 differentiation efficacy in the absence of IL-6 and/or IL-21. To investigate in vivo effects of IL-6/IL-21 blockade on Th17 and arthritis development, antigen-induced arthritis (AIA) was induced in IL-6-/- x IL-21R-/- mice. The therapeutic potential of this combined blocking strategy was assessed by treating mice with collagen-induced arthritis (CIA) with anti-IL-6R antibodies and soluble (s)IL-21R.Fc. We demonstrated that combined IL-6/IL-21 blocking synergistically reduced in vitro Th17 differentiation. In mice with AIA, absence of IL-6 and IL-21 signaling more strongly reduced Th17 levels and resulted in stronger suppression of arthritis than the absence of either cytokine. Additionally, anti-IL-6/anti-IL-21 treatment of CIA mice during the arthritis induction phase reduced disease development more potent than IL-6 or IL-21 inhibition alone, as effective as anti-TNF treatment. Collectively, these results suggest dual IL-6/IL-21 inhibition may be a more efficacious therapeutic strategy compared to single cytokine blockade to suppress arthritis development.

OP0075 Sensitive Spect/CT Imaging of Synovial Fap Expression after Anti-IL-22 Treatment in Experimental Arthritis

Background Rheumatoid arthritis (RA) synovial tissue has been demonstrated to express high levels of fibroblast activation protein (FAP) using anti-FAP-antibody 28H1. In addition, RA patients show elevated levels of IL-22 and IL-22-producing T helper cells that correlate to erosive disease, suggesting a role for this cytokine in the pathogenesis of RA. Objectives The purpose of this study was to determine the feasibility of 111In-28H1 SPECT/CT imaging of FAP-expressing synovium to monitor the therapeutic potential of neutralizing IL-22 during experimental arthritis. Methods Collagen-induced arthritis (CIA) was induced in male DBA/1J mice. Mice were treated 3 times per week with anti-IL-22 antibodies (8 mg/kg), while the control group received rat IgG1 isotype control antibodies. To monitor the therapeutic effect after 2 weeks of treatment, SPECT/CT images were acquired 24 h after injection of 111In-labeled DTPA-conjugated anti-FAP antibody, 28H1. After image acquisition, mice were euthanized and dissected. Imaging results were compared with the macroscopic arthritis scores and radiographic bone damage scores acquired by X-ray. Results Blocking IL-22 during CIA was a potent approach to prevent arthritis development, reaching a disease incidence of only 50%, versus 100% in the control group. SPECT/CT imaging using indium-labeled anti-FAP antibodies showed that joint uptake of the tracer was reduced highly significant (p=0.002) in anti-IL-22-treated mice (4.3 ± 3.4%ID/g) compared to the isotype control group (12.7 ± 3.7%ID/g) (See figure). This was confirmed by the corresponding macroscopic arthritis scores and radiographic bone damage scores that were significantly (p=0.047 and p=0.017 respectively) lower in the anti-IL-22-treated group. Besides its sensitivity, the in vivo FAP-based SPECT/CT had the great advantage to visualize sites of inflammation that were overlooked during clinical scoring, like in knee, hip, elbow and shoulder (See figure). Conclusions These findings demonstrate that IL-22 plays an important role in the development of experimental arthritis, and targeting this cytokine seems an attractive new strategy in RA treatment. Most importantly, SPECT/CT imaging of the inflamed synovium using the labeled anti-FAP antibody 111In-DTPA-28H1 can be used to specifically monitor response to therapy in an objective and quantitative way, and is potentially more sensitive in disease monitoring compared to the standard method of clinical arthritis scoring by macroscopic inspection. Disclosure of Interest D. Roeleveld: None declared, T. van der Geest: None declared, T. Nayak Employee of: Roche, C. Klein Employee of: Roche, B. Walgreen: None declared, M. Helsen: None declared, M. Hegen Employee of: Pfizer, P. Laverman: None declared, O. Boerman: None declared, M. Koenders: None declared

A germ line mutation in cathepsin B points toward a role in asparaginase pharmacokinetics

To the editor: l-Asparaginase (ASNase) is a key component of protocols used to treat acute lymphoblastic leukemia (ALL). Poorly understood interpatient differences in ASNase pharmacokinetics demand therapeutic drug monitoring to prevent patients from receiving an inadequate dose.[1][1] Although it

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.

Imaging fibroblast activation protein to monitor therapeutic effects of neutralizing interleukin-22 in collagen-induced arthritis

Objectives RA is a chronic autoimmune disease leading to progressive destruction of cartilage and bone. RA patients show elevated IL-22 levels and the amount of IL-22-producing Th cells positively correlates with the extent of erosive disease, suggesting a role for this cytokine in RA pathogenesis. The purpose of this study was to determine the feasibility of SPECT/CT imaging with 111In-labelled anti-fibroblast activation protein antibody (28H1) to monitor the therapeutic effect of neutralizing IL-22 in experimental arthritis. Methods Mice (six mice/group) with CIA received anti-IL-22 or isotype control antibodies. To monitor therapeutic effects after treatment, SPECT/CT images were acquired 24 h after injection of 111In-28H1. Imaging results were compared with macroscopic, histologic and radiographic arthritis scores. Results Neutralizing IL-22 before CIA onset effectively prevented arthritis development, reaching a disease incidence of only 50%, vs 100% in the control group. SPECT imaging showed significantly lower joint tracer uptake in mice treated early with anti-IL-22 antibodies compared with the control-treated group. Reduction of disease activity in those mice was confirmed by macroscopic, histological and radiographic pathology scores. However, when treatment was initiated in a later phase of CIA, progression of joint pathology could not be prevented. Conclusion These findings suggest that IL-22 plays an important role in CIA development, and neutralizing this cytokine seems an attractive new strategy in RA treatment. Most importantly, SPECT/CT imaging with 111In-28H1 can be used to specifically monitor therapy responses, and is potentially more sensitive in disease monitoring than the gold standard method of macroscopic arthritis scoring.

Involvement of T helper 17 cells in inflammatory arthritis depends on the host intestinal microbiota

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.

OP0329 Involvement of t helper 17 cells in inflammatory arthritis depends on the host intestinal microbiota

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

A2.07 Antagonisticregulation of IL-17 and GM-CSF during cell development ex vivo and during experimental arthritis

Background and objectives GM-CSF is a pro-inflammatory cytokine suggested to be mainly expressed by the Th17 cell subset. Combination blocking of GM-CSF and the key Th17-cytokine IL-17 has shown to be much more effective in reducing experimental arthritis compared to single cytokine blocking, revealing a potential connexion between the two cytokines. With this study, we aimed to unravel the interplay between GM-CSF and IL-17 during Th17 differentiation ex vivo and in vivo during experimental arthritis. Materials and methods We investigated whether IL-17 and GM-CSF levels were affected in a similar fashion when differentiating naïve murine CD4+ T cells ex vivo under conditions suboptimal for Th17 development. Additionally, we determined the effect of incubation with GM-CSF and IL-17 recombinant proteins and neutralising antibodies on expression of both cytokines during ex vivo Th17 differentiation. Finally, we studied the IL-17/GM-CSF interplay in C57Bl6/J mice with mBSA/IL-1β-induced experimental arthritis treated with anti-GM-CSF or anti-IL-17 antibodies. Results Interestingly, our ex vivo studies showed increased GM-CSF levels after differentiating naïve cells under conditions suboptimal for Th17 development, while IL-17 levels decreased as expected. In line with this ex vivo finding, we observed higher systemic levels of IL-17 and GM-CSF in mice with experimental arthritis after GM-CSF and IL-17 neutralisation respectively. Remarkably, no effects of incubation of naïve CD4+ T cells with IL-17 or GM-CSF recombinant proteins or neutralising antibodies during T cell differentiation were detected. This indicates that the two cytokines do not directly regulate each other’s expression but require interaction with environmental cells for their suppressive actions. Conclusion These data point towards deviating differentiation conditions and indirect antagonistic regulation of IL-17 and GM-CSF expression by CD4+ T cells, and provides further rationale for a combination blocking strategy of IL-17 and GM-CSF in treatment of Rheumatoid Arthritis.