Birgitte Walgreen

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

OBJECTIVE Interleukin-1 receptor antagonist-deficient (IL-1Ra-/-) mice spontaneously develop an inflammatory and destructive arthritis due to unopposed excess IL-1 signaling. In this study, the role of Th17 cells and the effect of neutralization of IL-17, IL-1, and tumor necrosis factor alpha (TNFalpha) were investigated in this IL-1-driven murine arthritis model. METHODS T cells isolated from IL-1Ra-/- and wild-type (WT) mice were stained for IL-17 and interferon-gamma, with results assessed by fluorescence-activated cell sorting analysis. To investigate the contribution of IL-1 and IL-17 in further progression of arthritis in this model, mice were treated with neutralizing antibodies after the onset of arthritis. RESULTS Compared with WT mice, IL-1Ra-/- mice had similar levels of Th1 cells but clearly enhanced levels of Th17 cells; this increase in the number of Th17 cells was evident even before the onset of arthritis, in young, nonarthritic IL-1Ra-/- mice. The percentage of Th17 cells increased even more after the onset of arthritis and, similar to the serum levels and local messenger RNA levels of IL-17, the percentage of IL-17+ Th17 cells clearly correlated with the severity of arthritis. Anti-IL-17 treatment prevented any further increase in inflammation and bone erosion, whereas blocking of TNFalpha after the onset of arthritis had no effect. In contrast, neutralization of IL-1 resulted in a complete suppression of arthritis. Interestingly, this anti-IL-1 treatment also significantly reduced the percentage of IL-17+ Th17 cells in the draining lymph nodes of these arthritic mice. CONCLUSION Increased levels of Th17 cells can be detected in IL-1Ra-/- mice even preceding the onset of arthritis. In addition, the results of cytokine-blocking studies demonstrated that IL-17 contributes to the inflammation and bone erosion in this model, which suggests that IL-1 is the driving force behind the IL-17-producing Th17 cells.

Immuno-PET and Immuno-SPECT of Rheumatoid Arthritis with Radiolabeled Anti–Fibroblast Activation Protein Antibody Correlates with Severity of Arthritis

One of the most prominent cell populations playing a role in rheumatoid arthritis (RA) is activated fibroblast-like synoviocytes. Among many other proteins, fibroblast-like synoviocytes dominantly express fibroblast activation protein (FAP). Because of the high expression of FAP in arthritic joints, radioimmunoimaging of activated fibroblasts with anti-FAP antibodies might be an attractive noninvasive imaging tool in RA. Methods: SPECT and PET with 111In- and 89Zr-labeled anti-FAP antibody 28H1 was performed in mice with CIA. The radioactivity uptake in joints was quantified and correlated with arthritis score. Results: Both 111In-28H1 and 89Zr-28H1 showed high uptake in inflamed joints, being 3-fold higher than that of the irrelevant isotype-matched control antibody DP47GS, clearly indicating specific accumulation of 28H1. Uptake of 111In-28H1 ranged from 2.2 percentage injected dose per gram (%ID/g) in noninflamed joints to 32.1 %ID/g in severely inflamed joints. DP47GS accumulation ranged from 1.6 %ID/g in noninflamed tissue to 12.0 %ID/g in severely inflamed joints. Uptake of 28H1 in inflamed joints correlated with arthritis score (Spearman ρ, 0.69; P < 0.0001) and increased with severity of arthritis. Conclusion: SPECT/CT imaging with the anti-FAP antibody 111In-28H1 specifically visualized arthritic joints with high resolution, and tracer accumulation correlated with the severity of the inflammation in murine experimental arthritis. Background uptake of the radiolabeled antibody was low, resulting in excellent image quality. 89Zr-28H1 was less favorable for RA imaging because of an elevated bone uptake of 89Zr. Future studies will focus on the potential role of 28H1 as a tool to monitor therapy response early on.

Liposomal treatment of experimental arthritis can be monitored noninvasively with a radiolabeled anti-fibroblast activation protein antibody

Rheumatoid arthritis is a chronic autoimmune disorder resulting in synovial inflammation. Fibroblast activation protein (FAP) is overexpressed by fibroblastlike synoviocytes in arthritic joints. Radioimmunoimaging with an anti-FAP antibody might be used to monitor the response to therapy, thus enabling tailored therapy strategies and therapeutic outcomes. The aim of this study was to assess whether a radiolabeled anti-FAP antibody could be used to monitor the efficacy of treatment with long-circulating liposomes (LCL) containing prednisolone phosphate (PLP-LCL) in a mouse model of arthritis. Methods: Collagen-induced arthritis (CIA) was induced in male DBA/1J mice. Mice were treated with a single injection (10 mg/kg) of PLP-LCL or empty LCL as a control. SPECT and CT images were acquired 24 h after injection of 99mTc-labeled succinimidyl-hydrazinonicotinamide (99mTc-S-HYNIC)–conjugated anti-FAP antibody 28H1 at 2, 5, and 9 d after treatment. The uptake of 99mTc-S-HYNIC-28H1 in all joints was quantified and correlated with macroscopic arthritis scores. Results: Treatment of CIA with PLP-LCL significantly suppressed joint swelling. At just 1 d after treatment, the macroscopic arthritis scores had decreased by 50%. Scores decreased further, to only 10% of the initial scores, at 5 and 9 d after treatment. In contrast, macroscopic arthritis scores had increased up to 600% in untreated mice at 9 d after the injection of empty LCL. 99mTc-S-HYNIC-28H1 uptake ranged from 1.5 percentage injected dose per gram in noninflamed joints to 22.6 percentage injected dose per gram in severely inflamed joints. The uptake of radiolabeled 28H1 in inflamed joints (percentage injected dose) correlated with the arthritis score (Spearman ρ, 0.77; P < 0.0001). Moreover, the uptake of 99mTc-S-HYNIC-28H1 was slightly increased at 9 d after therapy but was not seen macroscopically, indicating that SPECT/CT imaging might be more sensitive than the macroscopic arthritis scoring method. Conclusion: SPECT/CT imaging with 99mTc-S-HYNIC-28H1 specifically monitored the response to therapy, and tracer accumulation correlated with the severity of inflammation. In addition, SPECT/CT imaging was potentially more sensitive than the macroscopic arthritis scoring method. This study showed that SPECT/CT with 99mTc-S-HYNIC-28H1 could be used to noninvasively monitor the course of CIA in mice.

Expression of TGF-β Signaling Regulator RBPMS (RNA-Binding Protein With Multiple Splicing) Is Regulated by IL-1β and TGF-β Superfamily Members, and Decreased in Aged and Osteoarthritic Cartilage

Objective RNA-binding protein with multiple splicing (RBPMS) has been shown to physically interact with Smads and enhance transforming growth factor-β (TGF-β)–mediated Smad2/3 transcriptional activity in mammalian cells. Objective of this study was to examine whether expression of RBPMS is regulated by interleukin-1β (IL)-1β and TGF-β superfamily growth factors and whether expression of RBPMS is altered during aging and experimental osteoarthritis. Methods Expression of RBPMS protein was investigated in chondrocyte cell lines of murine (H4) and human (G6) origin using Western blot analysis. Regulation of RBPMS expression in H4 chondrocytes at mRNA level was done by reverse transcriptase–quantitative polymerase chain reaction. Furthermore, characterization of Smad signaling pathways regulating RBPMS expression was performed by blocking studies using small molecule inhibitors or by transfection studies with adenoviral vector constructs (constitutive-active ALK1 and constitutive-active ALK5). Expression of RBPMS in cartilage of different age groups of C57BL/6N mice (6 months and 20 months) and in a surgically induced osteoarthritis (OA) mouse model was analyzed using immunohistochemistry. Results RBPMS was shown to be expressed in chondrocytes and cartilage of murine, human, and bovine origin. TGF-β inhibited RBPMS expression while BMP2 and IL-1β increased its expression. TGF-β-induced inhibition was blocked by ALK5 inhibitor. Overexpression of ca-ALK1 stimulated RBPMS expression. Moreover, RBPMS expression was found to be reduced with ageing and in OA pathogenesis. Conclusions Expression of RBPMS in chondrocytes is regulated by TGF-β superfamily members and IL-1β, indicating a counter-regulatory mechanism. Expression of RBPMS, in cartilage and its reduction during ageing and OA might suggest its potential role in the maintenance of normal articular cartilage.

Alteration of the intestinal microbiome characterizes preclinical inflammatory arthritis in mice and its modulation attenuates established arthritis

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

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

1.56 Synergism between GM-CSF and IL-17 causes enhanced joint pathology via the production of IL-6 and IL-23

Background and Objective T helper-17 (Th17) cells are important mediators of inflammatory diseases, and are the main pathogenic cell type in many animal models of autoimmunity. Recent studies highlight a surprising role for T-cell derived granulocyte-macrophage colony stimulating factor (GM-CSF) in the pathogenicity of Th17 cells. We examined the mechanism by which interleukin 17 (IL-17) and GM-CSF contribute to cartilage- and bone damage of synovial joints during experimental arthritis, and provide a rationale for combination therapy in auto-inflammatory conditions. Materials and Methods Collagen-induced arthritis (CIA) was elicited in DBA/1J mice. Neutralizing antibodies to IL-17 and/or GM-CSF were administered after onset of disease for 14 days. Arthritis progression was followed by macroscopic scoring of the paws (maximum score of 12 per mouse). In addition, the effect of local over-expression of IL-17 and/or GM-CSF was studied by adenoviral transfection in naïve knee joints. Results Combined therapeutic treatment of mice early after the onset of CIA ameliorated disease progression. Macroscopic joint inflammation was significantly reduced, from a total score of 5.6 ± 0.4 for mice treated with isotype control antibodies to 2 ± 0.6 for mice treated with combination therapy. Treatment with anti-IL-17 or anti-GM-CSF alone resulted in scores of 3.4 ± 0.5 and 3.5 ± 0.4, respectively. Simultaneous blocking of GM-CSF and IL-17 was also the most effective treatment in the prevention of radiological bone damage and histological cartilage destruction. To provide further insight in local additive or synergistic effects of IL-17 and GM-CSF, overexpression of IL-17, GM-CSF or the combination was achieved with adenoviral vectors. Inflammatory infiltrate and cartilage- and bone damage developed in all groups from day 1 after adenoviral transfer, with the most severe effect observed in the combination group. On day 7, partial destruction of joint architecture was apparent in knee joints after combined overexpression of IL-17 and GM-CSF. Overexpression of GM-CSF alone induced IL-1β, which production was elevated by IL-17. Interestingly, overexpression of IL-17 alone caused a clear increase in synovial IL-6 production (179 ± 63pg/ml), which was dramatically enhanced in the co-presence of GM-CSF (1.9 ± 0.4 ng/ml). In addition, a strong synergistic effect of combined overexpression was seen on the Th17 differentiation factor IL-23. Conclusions We show that IL-17 and GM-CSF cause joint damage through synergistic effects on inflammatory mediators in synovial joints. In view of the moderate success of therapeutic IL-17 or GM-CSF blockade in rheumatoid arthritis, combined inhibition of IL-17 and GM-CSF might be an interesting option for patients that do not fully respond to inhibition of the separate cytokines. Abstract 1.56 Figure 1 (A) Macroscopic disease scores followed over time from day 21 of collagen-induced arthritis (day 0 of treatment). (B) End-point X-ray analysis of ankle joints after CIA with four different antibody treatments.

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.

Fcγ receptor-mediated influx of S100A8/A9-producing neutrophils as inducer of bone erosion during antigen-induced arthritis

BackgroundOsteoclast-mediated bone erosion is a central feature of rheumatoid arthritis (RA). Immune complexes, present in a large percentage of patients, bind to Fcγ receptors (FcγRs), thereby modulating the activity of immune cells. In this study, we investigated the contribution of FcγRs, and FcγRIV in particular, during antigen-induced arthritis (AIA).MethodsAIA was induced in knee joints of wild-type (WT), FcγRI,II,III−/−, and FcγRI,II,III,IV−/− mice. Bone destruction, numbers of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts, and inflammation were evaluated using histology; expression of the macrophage marker F4/80, neutrophil marker NIMPR14, and alarmin S100A8 was evaluated using immunohistochemistry. The percentage of osteoclast precursors in the bone marrow was determined using flow cytometry. In vitro osteoclastogenesis was evaluated with TRAP staining, and gene expression was assessed using real-time PCR.ResultsFcγRI,II,III,IV−/− mice showed decreased bone erosion compared with WT mice during AIA, whereas both the humoral and cellular immune responses against methylated bovine serum albumin were not impaired in FcγRI,II,III,IV−/− mice. The percentage of osteoclast precursors in the bone marrow of arthritic mice and their ability to differentiate into osteoclasts in vitro were comparable between FcγRI,II,III,IV−/− and WT mice. In line with these observations, numbers of TRAP+ osteoclasts on the bone surface during AIA were comparable between the two groups. Inflammation, a process that strongly activates osteoclast activity, was reduced in FcγRI,II,III,IV−/− mice, and of note, mainly decreased numbers of neutrophils were present in the joint. In contrast to FcγRI,II,III,IV−/− mice, AIA induction in knee joints of FcγRI,II,III−/− mice resulted in increased bone erosion, inflammation, and numbers of neutrophils, suggesting a crucial role for FcγRIV in the joint pathology by the recruitment of neutrophils. Finally, significant correlations were found between bone erosion and the number of neutrophils present in the joint as well as between bone erosion and the number of S100A8-positive cells, with S100A8 being an alarmin strongly produced by neutrophils that stimulates osteoclast resorbing activity.ConclusionsFcγRs play a crucial role in the development of bone erosion during AIA by inducing inflammation. In particular, FcγRIV mediates bone erosion in AIA by inducing the influx of S100A8/A9-producing neutrophils into the arthritic joint.

07.04 Partial elimination of intestinal microbiota dampens t helper 17 cell differentiation and established collagen-induced arthritis in mice

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.