Inhibition of TGF-β Signaling Suppresses Th17 Differentiation and Promotes Treg Numbers but does not Reduces Experimental Arthritis

ObjectivesTGF-β is an important growth factor to promote the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). The potential of TGF-β as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. We investigated the effect of TGF-β inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. MethodsMurine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 was studied. Synovial biopsies were cultured in the presence or absence of SB-505124. Experimental arthritis was induced in C57Bl6 mice and treated daily with SB-505124. FACS analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction.ResultsSB-505124 potently reduced murine Th17 differentiation by decreasing Il7a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by synovial explants. In vivo, SB-505124 reduced Th17 levels, while increased levels of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction.ConclusionsBlocking TGF-β signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, SB-505124 treatment does not suppress experimental arthritis.

Inhibition of TGF-β Signaling Suppresses Th17 Differentiation and Promotes Treg Numbers but Does Not Reduce Experimental Arthritis.

ObjectivesTGF-β is an important growth factor to promote the differentiation of T helper 17 (Th17) as well as regulatory T cells (Treg). Due to its dual role, the potential of TGF-β as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. In this study, we investigated the effect of TGF-β inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. MethodsMurine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 on Th17 differentiation was studied. RA synovial biopsies were cultured for 24h in the presence or absence of SB-505124. Experimental arthritis models were induced in C57Bl6 mice, and were treated daily with SB-505124. FACS analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction.ResultsSB-505124 potently reduced murine Th17 differentiation by decreasing Il7a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by RA synovial explants. In the Th17-driven arthritis model, SB-505124 reduced Th17 levels, while increased levels of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction in this model.ConclusionsBlocking TGF-β signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, SB-505124 treatment does not suppress experimental arthritis, and is therefore not an adequate way to target Th17-driven inflammation.

The Activation Fragment of PAR2 Is Increased in RA and Modulated in Response To Anti-IL-6R Treatment

Osteoarthritis (OA) and rheumatoid arthritis (RA) are serious and painful diseases. Protease-activated receptor 2 (PAR2) is involved in the pathology of both OA and RA including roles in synovial hyperplasia, cartilage destruction, osteophyogenesis and pain. PAR2 is activated via cleavage of its N-terminus by serine proteases. In this study a competitive ELISA assay was developed targeting the 36-amino acid peptide that is cleaved and released after PAR2 activation. Technical assay parameters including antibody specificity, intra- and inter-assay variation (CV%), linearity, accuracy, analyte stability and interference were evaluated. PAR2 pro-fragment release was confirmed after in vitro cleavage of PAR2 recombinant protein and treatment of human synovial explants with matriptase. Serum levels of 22 healthy individuals, 23 OA patients and 15 RA patients as well as a subset of RA patients treated with tocilizumab were evaluated. The PAR2 antibody was specific for the neo-epitope and intra-inter assay CV% were 6.4% and 5.8% respectively. In vitro cleavage and matriptase treated explants showed increased PAR2 pro-fragment levels compared to controls. In serum, PAR2 pro-fragment levels were increased in RA patients and decreased in RA patients treated with tocilizumab. In conclusion, PAR2 may be a potential biomarker for monitoring RA disease and pharmacodynamics of treatment.

AB0082 INHIBITION OF TGFβ SIGNALING USING SB-505124 BLOCKS TH17 DIFFERENTIATION AND RESTORES THE TH17/TREG BALANCE IN VIVO, BUT DOES NOT SUPPRESS EXPERIMENTAL ARTHRITIS

Background:TGFβ is an important growth factor that promotes the differentiation of T helper 17 (Th17) as well as regulatory T-cells (Treg). Due to its dual role, the potential of TGFβ as therapeutic target is unclear.Objectives:In this study we aimed to investigate the effect of inhibition of TGFβ signaling with the ALK5 inhibitor SB-505124 on human Th17 differentiationin vitro, on cytokine production by human rheumatoid arthritis (RA) synovial explants, and study the effects of local SB-505124 treatmentin vivoduring innate immune and Th17-driven experimental arthritis models.Methods:Magnetic sorted naïve human T cells were differentiated into Th17 cells with CD3/CD28 activation beads, IL-2, TGFβ, IL-1β, IL-23, αIFNƳ and αIL-4 for 6 days. Human RA synovial biopsies were cultured for 24h w/o 5µM SB-505124, and supernatant was analyzed by Luminex. T cell-independent SCW arthritis and Th17-driven IL-1/mBSA arthritis were induced in C57Bl6, and mice were treated with SB-505124 by daily intra-articular injections from day 0-4.Results:SB-505124 potently reduced human Th17 differentiationin vitroby decreasing IL-17 and RORƳt gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 and TNFα protein production by human RA synovial explants. In addition, SB-505124 did not affect acute joint inflammation during SCW-arthritis (T-cell independent model). Interestingly, SB-505124 reduced Th17 levels in draining lymph nodes (dLN) during IL-1/mBSA arthritis while increased levels of Tregs were observed. Surprisingly, despite this skewed Th17/Treg balance, this did not result in suppression of joint inflammation and destruction in this Th17-driven arthritis model, whereas anti-IL-17 antibody treatment showed significant therapeutic effects.Conclusion:We revealed suppressive effects of SB-505124 on human Th17 differentiation and the Th17/Treg balance in arthritic mice. However, SB-505124 did not suppress joint inflammation and destruction. This indicates that despite the importance of TGFβ in Th17 differentiation, targeting TGFβ signaling is not enough to suppress experimental arthritis.Disclosure of Interests:None declared

Systemic Resolvin E1 (RvE1) Treatment Does Not Ameliorate the Severity of Collagen-Induced Arthritis (CIA) in Mice: A Randomized, Prospective, and Controlled Proof of Concept Study

Specialized proresolving mediators (SPRM), which arise from n-3 long-chain polyunsaturated fatty acids (n-3FA), promote resolution of inflammation and may help to prevent progression of an acute inflammatory response into chronic inflammation in patients with arthritis. Thus, this study is aimed at determining whether systemic RvE1 treatment reduces arthritis onset and severity in murine collagen-induced arthritis (CIA) and spontaneous cytokine production by human rheumatoid arthritis (RA) synovial explants. 10-week-old DBA1/J male mice were subjected to CIA and treated systemically with 0.1 μg RvE1, 1 μg RvE1, 5 mg/kg anti-TNF (positive control group), PBS (negative control group), or with a combination of 1 μg of RvE1 plus 5 mg/kg anti-TNF using prophylactic or therapeutic strategies. After CIA immunization, mice were treated twice a week by RvE1 or anti-TNF for 10 days. Arthritis development was assessed by visual scoring of paw swelling and histology of ankle joints. Moreover, human RA synovial explants were incubated with 1 nM, 10 nM, or 100 nM of RvE1, and cytokine levels (IL-1β, IL-6, IL-8, IL-10, INF-γ, and TNF-α) were measured using Luminex bead array. CIA triggered significant inflammation in the synovial cavity, proteoglycan loss, and cartilage and bone destruction in the ankle joints of mice. Prophylactic and therapeutic RvE1 regimens did not ameliorate CIA incidence and severity. Anti-TNF treatment significantly abrogated signs of joint inflammation, bone erosion, and proteoglycan depletion, but additional RvE1 treatment did not further reduce the anti-TNF-mediated suppression of the disease. Treatment with different concentrations of RvE1 did not decrease the expression of proinflammatory cytokines in human RA synovial explants in the studied conditions. Collectively, our findings demonstrated that RvE1 treatment was not an effective approach to treat CIA in DBA1/J mice in both prophylactic and therapeutic strategies. Furthermore, no effects were noticed when human synovial explants were incubated with different concentrations of RvE1.