Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, fibrosis, and immune dysregulation. In SSc pathogenesis, circulating monocytes can be recruited to the skin and differentiate into macrophages. This possibly contributes to the activation and conversion of resident fibroblasts into myofibroblasts, driving fibrosis and skin thickness. However, how these cells exert their functions and via which fibroinflammatory mediator(s) is not fully understood. Objectives: To investigate the role of monocytes in mediating myofibroblast contraction and activation using an innovative 3D collagen hydrogel model. Methods: For our 3D skin model we cocultured human primary dermal fibroblasts with either peripheral blood mononuclear cells (PBMCs) or MACS-sorted CD14+ monocytes in a 3D collagen type 1 hydrogel. Subsequently, monocyte-driven tissue contraction was measured over time. We performed immunostainings for CD68, fibroblast activation protein (FAP), and alpha-smooth muscle actin (α-SMA) to evaluate monocyte/macrophage and fibroblast activation. To investigate the signaling pathways involved in the observed myofibroblast activation, we measured transcription factor-driven luciferase production using reporter constructs in the same dermal fibroblasts. The activity of the following reporter constructs was determined after 24 hours: Sis-Inducible Element (SIE); SMAD-Binding Element (SBE); Nuclear Factor of Activated T-cells 5 Response Element (NFAT-5), and NFκB Response Element (NFκB). Results: After 60 hours of co-culture, hydrogel plugs containing fibroblasts + monocytes displayed a strong spontaneous contraction, seen by a (approx. 80-90%) decrease in the area of the plugs. Hydrogels containing only fibroblasts did not contract. Hydrogels with fibroblasts + monocytes contracted as fast as fibroblasts + PBMCs, but depletion of CD14+ cells from PBMCs slowed down contraction, showing that monocytes strongly activated fibroblasts. The expression of FAP and α-SMA by fibroblasts increased in monocyte-containing hydrogels, compared to fibroblasts cultured without monocytes. Furthermore, CD68 expression also increased in co-culture, indicating enhanced monocyte differentiation/activation into macrophages by fibroblasts. Evaluating which intracellular pathway lead to fibroblast activation, we observed that SIE and NFκB reporter fibroblast constructs were strongly elevated in the presence of monocytes. However, we could not observed enhanced TGF-β activity using the SBE reporter. Together, our results suggest that inflammatory mediators like IL-6, IL-1 and S100A8/9 may contribute to the monocytes-driving myofibroblast contraction. Conclusion: This study highlights the importance of monocytes and the SIE and NFκB intracellular signaling pathways in myofibroblast contraction in a 3D skin model, contributing to understanding the basic mechanisms of these cells in SSc skin fibrosis and thickness. REFERENCES: [1] van Caam, Arjan et al. "Unraveling SSc Pathophysiology; The Myofibroblast." Frontiers in immunology vol. 9 2452. 13 Nov. 2018, doi:10.3389/fimmu.2018.02452. [2] Al-Adwi, Yehya et al. "Macrophages as determinants and regulators of fibrosis in systemic sclerosis." Rheumatology (Oxford) vol. 62,2 (2023): 535-545. Acknowledgements: Grant #2023/04897-7, São Paulo Research Foundation (FAPESP). Disclosure of Interests: None declared.

Background Systemic sclerosis is characterized by vasculopathy, fibrosis and autoimmune inflammation. Adaptive immune and NK cells express co-stimulatory molecules to regulate effector responses in inflammatory conditions. CD7 is a co-stimulatory molecule induced by IFN-γ signaling on T and NK cells that is downregulated upon residency in tissues such as skin [1]. Objectives Hypothetically, CD7 is upregulated in IFN-γ driven conditions, allowing targeted depletion of pathogenic cells. In this study, we analyzed expression, regulation and targeting of CD7 in SSc tissues and in a patient treated with anti-CD7 immunotoxin (CD7-IT). Methods To identify disease-specific activation markers we compared status of T cell activation in SSc skin compared to blood. To do so, single-cell RNA sequencing datasets containing 60,000 skin and blood immune cells from 109 SSc patients versus 65 healthy donors were analyzed. Twentyone-color flow cytometry (n=50) and 8-color multiplex skin immunofluorescence staining (mIF) (n=30) were used for validation and spatial localization. CD7-IT was used to induce subset-specific depletion in vitro, and ex vivo skin explants. Fibroblasts and immune cells were co-cultured in 3D collagen hydrogel to test effect of targeted immune cell depletion on fibroblast contractility. A single patient was treated with immunotoxin on compassionate use basis. Results Sc-RNAseq analysis showed that SSc skin is characterized by increased infiltration of CD7+ cells corresponding to CD8+GZMB+ and CD56+GZMB+ cytotoxic T and NK cells. Immunohistochemistry also showed that SSc lesional skin exhibits significantly higher infiltration of these CD7+ (T and NK) cells than non-affected skin (Figure 1). Expansion of CD7+ cytotoxic T cells was also observed in SSc blood. A CD7-targeting IT effectively depleted 85% of CD7+ cytotoxic T and NK cells in vitro, and in ex vivo skin. Strikingly, depletion of CD7+ cells prevented fibroblast activation and contraction in a 3D fibroblast-immune cells co-culture assay. Notably, also in vivo administration of CD7-IT significantly eliminated CD7+ cell subsets in patient's blood and skin. Conclusion Together, we show strong presence of CD7 on SSc immune cells and that targetting this molecule depletes T and NK cell populations with potential disease modifying results. Our results pave the way for a novel approach in halting SSc related fibrosis. Reference [1]Wang T, Huang C, Lopez-Coral A, et al. K12/SECTM1, an interferon-γ regulated molecule, synergizes with CD28 to costimulate human T cell proliferation. J Leukoc Biol. 2012;91(3):449-459. doi:10.1189/jlb.1011498 Acknowledgements: NIL. Disclosure of Interests None Declared.Figure 1Strong presence of CD7+ cytotoxic T and NK cells in systemic sclerosis skin

Background Systemic sclerosis (SSc) is a rare, severe auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells. Objectives We explored the use of FAP-tPDT to selectively target primary skin fibroblasts from SSc patients, both in 2D and 3D cultures, as well as in biopsies from fibrotic skin lesions in the murine bleomycin-induced skin fibrosis model. Methods The FAP targeting monoclonal antibody (clone 28H1) was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous out-growth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer (Ab-PS) construct, washed after 4 h and exposed to λ = 689 nm light (50 J/cm 2 at 280 mW/cm 2 ). Cell viability was measured using CellTiter Glo. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Skin fibrosis was induced in mice by 3x/week injections of 1.5 IU bleomycin intradermally on the back of the mouse. After 5 weeks the mice were sacrificed and biopsies were taken from affected and not affected skin. These were incubated with or without the FAP targeting Ab-PS construct or a control Ab-PS. After washing, the biopsies were exposed to light, incubated for 1h and subsequently formalin fixed and paraffin embedded. Sections were immuno stained for the presence of cleaved caspase-3. Results FAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with a control Ab-PS construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. In fibrotic skin biopsies from mice with bleomycin-induced skin fibrosis, an upregulation of apoptosis, as evidenced by increased caspase-3 staining (Figure 1), was observed in response to FAP-tPDT. The same treatment on biopsies of control skin did not increase caspase-3 staining, nor did incubation with a control Ab-PS construct or light alone. Figure 1. Cleaved caspase-3 staining of biopsies from the skin of mice with bleomycin-induced dermal fibrosis upon treatment with FAP-tPDT. The red arrows indicate the presence of positive fibroblasts. Conclusion Here we have shown, for the first time, the potential of FAP-tPDT for the selective targeting of pathogenic fibroblasts and treatment of fibrosis in SSc skin both in relevant patient-derived culture models as well as an in vivo model of fibrosis. Disclosure of Interests Daphne Dorst: None declared, Arjan van Caam: None declared, Elly Vitters: None declared, Birgitte Walgreen: None declared, Monique Helsen: None declared, Christian Klein Employee of: CK is an employee of Roche pharmaceutics, Shreya Gudi: None declared, Tirza Wubs: None declared, Jyoti Kumari: None declared, Przemysław Błyszczuk: None declared, Madelon Vonk: None declared, Peter van der Kraan: None declared, Marije Koenders: None declared.

BackgroundSystemic sclerosis (SSc) is a rare, severe auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells.ObjectivesWe explored the use of FAP-tPDT to selectively target primary skin fibroblasts from SSc patients, both in 2D and 3D cultures, as well as in biopsies from fibrotic skin lesions in the murine bleomycin-induced skin fibrosis model.MethodsThe FAP targeting monoclonal antibody (clone 28H1) was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous out-growth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer (Ab-PS) construct, washed after 4 h and exposed to λ = 689 nm light (50 J/cm2 at 280 mW/cm2). Cell viability was measured using CellTiter Glo. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Skin fibrosis was induced in mice by 3x/week injections of 1.5 IU bleomycin intradermally on the back of the mouse. After 5 weeks the mice were sacrificed and biopsies were taken from affected and not affected skin. These were incubated with or without the FAP targeting Ab-PS construct or a control Ab-PS. After washing, the biopsies were exposed to light, incubated for 1h and subsequently formalin fixed and paraffin embedded. Sections were immuno stained for the presence of cleaved caspase-3.ResultsFAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with a control Ab-PS construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. In fibrotic skin biopsies from mice with bleomycin-induced skin fibrosis, an upregulation of apoptosis, as evidenced by increased caspase-3 staining (Figure 1), was observed in response to FAP-tPDT. The same treatment on biopsies of control skin did not increase caspase-3 staining, nor did incubation with a control Ab-PS construct or light alone.Figure 1.Cleaved caspase-3 staining of biopsies from the skin of mice with bleomycin-induced dermal fibrosis upon treatment with FAP-tPDT. The red arrows indicate the presence of positive fibroblasts.ConclusionHere we have shown, for the first time, the potential of FAP-tPDT for the selective targeting of pathogenic fibroblasts and treatment of fibrosis in SSc skin both in relevant patient-derived culture models as well as an in vivo model of fibrosis.Disclosure of InterestsDaphne Dorst: None declared, Arjan van Caam: None declared, Elly Vitters: None declared, Birgitte Walgreen: None declared, Monique Helsen: None declared, Christian Klein Employee of: CK is an employee of Roche pharmaceutics, Shreya Gudi: None declared, Tirza Wubs: None declared, Jyoti Kumari: None declared, Przemysław Błyszczuk: None declared, Madelon Vonk: None declared, Peter van der Kraan: None declared, Marije Koenders: None declared.

Background Abnormalities in T lymphocyte populations are associated with the pathogenesis of many autoimmune diseases, such as systemic sclerosis (SSc). Various studies report on the aberrations of different T cell cytotoxic (CTL) and helper (Th) subsets that appear to be linked with inflammatory and/or fibrotic manifestations of patients with SSc. Since T cells seem to play a pivotal role in the pathophysiology of SSc, targeting the pathogenic T cell subsets might be a promising therapeutic option. Objectives Here we set out to comprehensively compare T lymphocyte phenotypes between SSc patients and healthy donors. We further test the in-vitro efficacy of a combination of anti-CD3/CD7 immunotoxins (CD3/CD7-IT), that have been developed to eliminate activated CD4+ and CD8+ T cells, to study specific sensitivity of T-cell subpopulations to CD3/CD7-IT. Methods 30 SSc patients and 15 age and sex matched healthy donors were included. Of these patients, lymphocyte populations were quantified by 17-parameter flow cytometry of peripheral blood mononuclear cells (PBMCs) to identify CD4+ T helper cells (Th1, Th2, Th17, T peripheral helper), CD8+ naïve, memory, effector CTLs and senescent/exhausted subsets. We next developed a cell killing assay to evaluate the effect of T cell depletion. To address this, patients’ (N=6) PBMCs were first activated for 24 hours in the presence of phytohemagglutinin, followed by CD3/CD7-IT addition for 48 hours. Subset-specific T cell depletion was assessed by using a combination of CellTiter-Glow luminescent cell viability assay and multi-parameter flow cytometric (FCM) quantification of CD3/CD7-IT-induced cell death. Results Frequencies of effector CD8+ CTLs,Th2 and T peripheral helper cells were elevated in SSc patients compared to healthy controls. Furthermore, SSc patients exhibited lower percentages of the anti-fibrotic Th1 subset. A striking expansion of the senescent CD4+CD28- and CD8+CD28- populations was noted in patients, while these subsets were barely detectable in healthy controls. In-vitro adittion of anti-T cell immunotoxins effectively depleted 50 % of patients’ CD8+ T cells (including the CD8 effector subset) and 62% of CD4+ T cells (including Th2 and T peripheral helper cells). No difference in cytolytic sensitivity between different T cell subsets was observed. Conclusion Our findings demonstrate that deep FCM immunephenotyping reveals pathophysiological differences in peripheral T cell subsets of SSc patients. Strikingly, the developed cytolytic assays show that CD3/CD7-IT is able to target the potential disease-associated T cell subsets in an in-vitro setting. References Not applicable Disclosure of Interests None declared

Introduction The pathophysiology of systemic sclerosis (SSc) is closely linked to overactive TGFβ signaling. TGFβ is produced and circulates in latent form, making its activation crucial for signaling. This activation can be mediated via integrins. We investigated the balance between active and latent TGFβ in serum of SSc patients and investigated if this correlates with integrin expression on monocytes. Methods A TGFβ/SMAD3- or BMP/SMAD1/5-luciferase reporter construct was expressed in primary human skin fibroblasts. Both acidified and non-acidified sera of ten SSc patients and ten healthy controls were tested on these cells to determine total and active TGFβ and BMP levels respectively. A pan-specific TGFβ1/2/3 neutralizing antibody was used to confirm TGFβ signaling. Monocytes of 20 SSc patients were isolated using CD14+ positive selection, and integrin gene expression was measured using qPCR. Integrin expression was modulated using rhTGFβ1 or a small molecule inhibitor of TGFBR1: SB-505124. Results SSc sera induced 50% less SMAD3-reporter activity than control sera. Serum acidification increased reporter activity, but a difference between healthy control and SSc serum was no longer observed, indicating that total TGFβ levels were not different. Addition of a pan-specific TGFβ1/2/3 neutralizing antibody fully inhibited SMAD3-reporter activity of both acidified and not-acidified control and SSc sera. Both HC and SSc sera induced similar SMAD1/5-reporter activity, and acidification increased this, but not differently between groups. Interestingly, expression of two integrin alpha subunits ITGA5 and ITGAV was significantly reduced in monocytes obtained from SSc patients. Furthermore, ITGB3 , ITGB5 , and ITGB8 expression was also reduced in SSc monocytes. Stimulation of monocytes with TGFβ1 induced ITGA5 and ITGAV but lowered ITGB8 expression, whereas the use of the TGFβ receptor inhibitor SB-505124 had the opposite effect. Conclusion Total TGFβ serum levels are not different between SSc patients and controls, but TGFβ activity is. This coincides with a reduced expression of TGFβ-activating integrins in monocytes of SSc patients. Because TGFβ regulates expression of these integrins in monocytes, a negative feedback mechanism possibly underlies these observations.

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 differentiation in vitro , on cytokine production by human rheumatoid arthritis (RA) synovial explants, and study the effects of local SB-505124 treatment in vivo during 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 differentiation in vitro by 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

Career situation of first and presenting author Student for a master or a PhD. Introduction High interleukin-22 (IL-22) levels are detected in serum and synovial fluid of rheumatoid arthritis (RA) patients and have been shown to positively correlate with disease activity markers and more erosive disease. The role of IL-22 in autoimmunity and inflammation appears to be greatly contradictory, being both pro- and anti-inflammatory. Especially the anti-inflammatory properties of IL-22 are not well understood. We aimed to investigate the anti-inflammatory and immune-suppressive effect of IL-22 during experimental arthritis. Methods Collagen-induced arthritis was induced in DBA1 mice by immunization and booster with bovine collagen type II (CII). After booster, but before arthritis onset, IL-22 was overexpressed either locally or systemically using an adenoviral construct (AdIL-22) or Luciferase as control (AdLuc). 1 × 107 plaque-forming units (PFU) of the adenoviruses were injected intra-articularly for local overexpression, or 3 × 108 PFU was injected intravenously for systemic overexpression in immunized mice, and mice were sacrificed 10 days later. Macroscopic scoring and histological analysis was performed, and mRNA expression and protein production of various pro- and anti-inflammatory mediators was determined in synovial tissue, spleen, and serum. Results Local overexpression of IL-22 by injection of AdIL-22 in the knee joint of CII-immunized mice resulted in an unaltered arthritis incidence and severity as compared to the control virus AdLuc. Accordingly, no changes in mRNA expression or protein production were observed in CIA mice locally overexpressing IL-22. In contrast, systemic overexpression of IL-22 potently reduced disease incidence and severity, which was also confirmed by histological analysis. Systemic levels of IL-1β, IL-17, GM-CSF and MCP1 were unaltered in mice overexpressing IL-22 systemically. However, these mice showed significantly lower serum levels of IFNγ, TNFα, MIP1α, and IL-10. Interestingly, the significantly enhanced splenic SOCS3 expression was found to negatively correlate to serum TNFα and MIP1α levels, which is in line with our hypothesis that that the observed reduction in the cytokine levels is mediated in a SOCS3-dependent manner. Conclusions With this study, we revealed clear anti-inflammatory effects of IL-22 overexpression during collagen-induced arthritis, which are completely dependent on the systemic route of administration. Additionally, we were the first to show that this protective effect of IL-22 during experimental arthritis is likely orchestrated via up-regulation of the negative regulator SOCS3. Disclosure of Interest None declared.

Background Systemic sclerosis (SSc) is a severe disease characterised by auto-immunity, vasculopathy and excessive fibrosis of connective tissues. The pathophysiology of SSc is still poorly understood, but its symptoms imply a role for dysregulated transforming growth factor β (TGFβ) signalling; e.g. this cytokine is known to regulate vascular and connective tissue biology. TGFβ circulates in blood in an inactive form bound to latency associated peptide and latent TGFβ binding proteins. This latent TGFβ first has to be activated before it can become bioactive. With the use of a bioassay, TGFβ’s bioactivity can be measured in a complex mixture like serum, unlike with an ELISA which cannot take cellular activation processes into account. Objectives To determine the bioactivity of TGFβ in SSc serum compared to that of healthy control serum Methods Serum was collected of 10 SSc patients and 10 age and sex matched healthy controls. Primary human fibroblasts of 3 donors were transduced with CAGA12-luc which produces luciferase in response to TGFβ/Smad3 or BRE-luc which produces luciferase in response to BMP/Smad1/5. These cells were treated with 10% serum for 16 hour and luciferase activity was measured. To activate all TGFβ, sera were treated with 4M HCl for 1 hour at RT, after which pH was normalised with 4M NaOH. Controls were treated with HCl and NaOH simultaneously. To verify that TGFβ signalling was measured in this reporter assay, sera were treated with anti-TGFβ1/2/3 for 1 hour at RT before use. Results Control sera significantly induced reporter activity by 4.5-fold. However, SSc sera only induced a 2.5-fold increase in luciferase activity, indicating significantly lower bioactivity of TGFβ (p<0.0001). This difference was not due to a difference in total TGFβ levels; after activation of all TGFβ both HC and SSc sera induced a similar 6-fold increase in signal strength. These data show that in HC sera approximately 75% of all TGFβ is bioactive compared to only 42% in SSc. Addition of anti-TGFβ1/2/3 inhibited all CAGA12-luc reporter activity (p<0.0001) of both HC and SSc serum, and of both acidified and not acidified sera (p<0.0001), showing that our bioassay is indeed TGFβ-dependent. To investigate if reduced bioactivity is a more general phenomenon we measured BMP activity. BMP proteins are structurally closely related to TGFβ and also circulate in inactive form. Both HC and SSc sera induced a similar 8-fold increase in BRE-luc activity, and this activity was increased to a 16-fold induction after acidification for both groups. BMPs in SSc sera are thus not less bioactive. This illustrates the uniqueness of our observation on TGFβ bioactivity. Conclusions TGFβ in SSc serum is less bioactive than in control serum whereas BMPs are not less bioactive. Disclosure of Interest None declared

Introduction Systemic sclerosis (SSc) is a severe disease characterised by auto-immunity, vasculopathy and excessive fibrosis of connective tissues. The pathophysiology of SSc is still poorly understood, but its symptoms imply a role for dysregulated transforming growth factor β (TGFβ) signalling because this cytokine is known to regulate vascular and connective tissue biology. TGFβ circulates in blood in an inactive latent form bound to latency associated peptide and latent TGFβ binding proteins. This latent TGFβ has to be activated before it can become bioactive. With the use of a bioassay TGFβ’s bioactivity can be measured in complex mixtures like serum. This is not possible with an ELISA because this technique does not take (cellular) activation processes into account. Objectives To determine the bioactivity of TGFβ in SSc serum compared to that of healthy control serum. Methods Serum was collected of 10 SSc patients and 10 age and sex matched healthy controls. Primary human fibroblasts of 3 donors were transduced with CAGA12-luc which produces luciferase in response to TGFβ/Smad3 or BRE-luc which produces luciferase in response to BMP/Smad1/5. These cells were treated with 10% serum for 16 hour and luciferase activity was measured. To activate all TGFβ, sera were treated with 4M HCl for 1 hour at RT, after which pH was normalised with 4M NaOH. Controls were treated with HCl and NaOH simultaneously. To verify that TGFβ signalling was measured in this reporter assay, sera were treated with anti-TGFβ1/2/3 for 1 hour at RT before use. Results Control sera significantly induced reporter activity by 4.5-fold. However, SSc sera only induced a 2.5-fold increase in luciferase activity, indicating significantly lower bioactivity of TGFβ (p<0.0001). This difference was not due to a difference in total TGFβ levels; after activation of all TGFβ both HC and SSc sera induced a similar 6-fold increase in signal strength. These data show that in HC sera approximately 75% of all TGFβ is bioactive compared to only 42% in SSc. Addition of anti-TGFβ1/2/3 inhibited all CAGA12-luc reporter activity (p<0.0001) of both HC and SSc serum, and of both acidified and not acidified sera (p<0.0001), showing that our bioassay is indeed TGFβ-dependent. To investigate if reduced bioactivity is a more general phenomenon we measured BMP activity. BMP proteins are structurally closely related to TGFβ and also circulate in inactive form. Both HC and SSc sera induced a similar 8-fold increase in BRE-luc activity, and this activity was increased to a 16-fold induction after acidification for both groups. BMPs in SSc sera are thus not less bioactive. This illustrates the uniqueness of our observation on TGFβ bioactivity. Conclusions TGFβ in SSc serum is less bioactive than in control serum whereas BMPs are not less bioactive. Disclosure of interest None declared

Background In healthy cartilage, there is a balance between anabolic and catabolic activities of chondrocytes that maintains the functional integrity of the extracellular matrix. However, during osteoarthritis (OA), chondrocytes become more catabolically active and express increased levels of matrix degrading enzymes, such as MMPs and ADAMTSs. Increased MMP and ADAMTS activity results in a net loss of the extracellular matrix and therefore leads to cartilage damage. Previously, we found that the anti-inflammatory cytokine Interleukin 37 (IL37) is able to counter-regulate the catabolic status of chondrocytes by reducing the IL1β-driven expression of pro-inflammatory cytokines and catabolic enzymes. Objectives The goal of this study was to investigate, in human OA cartilage explants, the effect of IL37 on sulfated glycosaminoglycans (GAG) content and synthesis of extracellular matrix molecules and cartilage degrading enzymes to investigate its therapeutic potential in OA. Methods Human cartilage was obtained from eighteen OA patients undergoing total knee or hip arthroplasty. Biopsy punches of 4 mm in diameter were made to equalize explant size. After culturing overnight, explants were incubated for 48 h with three doses (1, 10 or 100 ng/ml) of recombinant-human IL37 (rhIL37). In the supernatant of the explant cultures, GAG release was measured with the DMB assay and levels of the ARGS neoepitope, which is one of the products of aggrecan degradation by ADAMTS5, were detected using Western Blot. Furthermore, gene and protein expression of extracellular matrix molecules and cartilage degrading enzymes were measured. Nitric oxide (NO), an important effector molecule that may suppress cartilage matrix synthesis, levels were measured in the supernatant of the explants culture using Griess reagens. Results Adding rhIL37 (100 ng/ml) to OA cartilage explants caused a highly significant reduction in GAG release to the supernatant of, on average, 32% in eighteen donors (Figure 1). Gene expression of the matrix molecules aggrecan and collagen type II was not affected, indicating that this effect of rhIL37 was not due to a loss of aggrecan synthesis. Another mechanism to prevent GAG release in cartilage is via inhibition of NO synthesis, but NO levels in the supernatant were comparable between rhIL37 treated groups and the control group. In contrast, after addition of rhIL37, ARGS neoeptiope levels, which reflect ADAMTS5 activity, were dose-dependently down regulated in the supernatant. Furthermore, protein analysis of the explants showed that rhIL37 reduced ADAMTS5 levels. These data indicate that IL37 interferes with the amount active matrix degrading enzymes in the cartilage matrix. However, gene expression of ADAMTS5 was not affected by rhIL37, indicating that the effect of IL37 on ADAMTS5 is post translational. Conclusions Our data show that rhIL37 reduces GAG release by OA cartilage explants. The mechanism behind this protective effect of IL37 probably runs via a reduction in ADAMTS5 abundance in the cartilage matrix, which is the main aggrecanase involved in OA This effect of IL37 on ADAMTS5 is probably post translational. Our data indicate that IL37 can maintain cartilage matrix integrity under OA conditions and is able to reduce the severity of cartilage destruction during OA. Disclosure of Interest None declared

Purpose: Cartilage repair using mesenchymal stem cells is a promising new strategy for the treatment of osteoarthritis. However, the inflammatory environment of the joint inhibits the repair process. Currently, it is not known through which pathways and signaling events inflammatory stimuli inhibit chondrogenic differentiation. Previously, it has been shown that signaling via the transforming growth factor β family members is crucial for cartilage formation and homeostasis. Therefore we evaluated the effect of inflammatory mediators on downstream mediators of TGFβ signaling: the R-SMAD transcription factors. Methods: Human mesenchymal stem cells were differentiated in monolayer or pellet cultures (chondrogenic differentiation medium: insulin, transferrin, selenite, transforming growth factor β1 (10 ng/ml), bone morphogenetic protein 2 (20 ng/ml)). Chondrogenic differentiation was assessed at day 7 using GAG measurements (DMMB assay). On day 3 of differentiation inflammatory stimuli (IL1β and osteoarthritis-conditioned medium (pooled from five donors)) were added. To determine the effect on TGFβ signaling the CAGA-luciferase reporter assay, micro-array expression analyses and immuno blotting for SMAD phospho-isoforms was used. Finally a human phospho-kinase antibody array (R&D) was performed on pellets treated with inflammatory stimuli to identify kinases responsible for SMAD phosphorylation. Results: Addition of IL1β and OAC-med to differentiating MSC at day 3, inhibited chondrogenic differentiation as measured by GAG deposition at day 7. Transcriptome analyses at 8 hours post stimulation with inflammatory mediators revealed upregulation of MMPs and down regulation of Aggrecan and Collagen type II gene expression. In addition we found deregulation of TGFβ-related and regulated genes. To determine if TGFβ signaling itself was affected within this time frame we used the CAGA luciferase reporter construct. Indeed the SMAD3 dependant CAGA-luciferase reporter construct was inhibited by inflammatory stimuli within 8 hours in differentiating MSC (Figure 1A). SMAD2/3 activation by TGFβ is largely determined by C-terminal phosphorylation through ALK5 (canonical TGFβ type I receptor). Surprisingly we did not find a down regulation of C-terminal phosphorylation after addition of inflammatory stimuli (Figure 1B, left panel). SMAD family members contain a highly variable linker region that can be phosphorylated by various kinases, such as GSK3β, RHO/ROCK, MAPK and CDKs. These linker modifications (1 threonine and 3 serines) are known to modulate SMAD activation and degradation. In differentiating MSC the threonine in the Smad2 linker region was not regulated by IL-1 exposure. However, stimulation with either IL1β or OAC-med did result in serine phosphorylation of the SMAD2 linker region (S245, S25, S255) within 1 hour (Figure 1B right panel). This may indicate SMAD To determine which pathways might be responsible for SMAD linker phosphorylation we performed a human phospho-kinase antibody array on pellets treated with OAC-med for 15 and 30 minutes. The MAP kinase pathway was strongly activated as evidenced by a >1,5 fold increase in activating pERK1/2 and pP38 compared to control pellets (Figure 1C). Conclusions: Inhibition of TGFβ signaling in differentiating MSC by IL1β and OAC-med appears to be mediated through SMAD serine linker modifications. The hot favourite kinases for phosphorylation of these serines are members of the MAP kinase family.

Objective. A crucial feature of OA is cartilage degradation. This process is mediated by pro-inflammatory cytokines, among other factors, via induction of matrix-degrading enzymes. Interleukin 37 (IL37) is an anti-inflammatory cytokine and is efficient in blocking the production of pro-inflammatory cytokines during innate immune responses. We hypothesize that IL37 is therapeutic in treating the inflammatory cytokine cascade in human OA chondrocytes and can act as a counter-regulatory cytokine to reduce cartilage degradation in OA. Methods. Human OA cartilage was obtained from patients undergoing total knee or hip arthroplasty. Immunohistochemistry was applied to study IL37 protein expression in cartilage biopsies from OA patients. Induction of IL37 expression by IL1&bgr;, OA synovium-conditioned medium and TNF&agr; was investigated in human OA chondrocytes. Adenoviral overexpression of IL37 followed by IL1&bgr; stimulation was performed to investigate the anti-inflammatory potential of IL37. Results. IL37 expression was detected in cartilage biopsies of OA patients and induced by IL1&bgr;. After IL1&bgr; stimulation, increased IL1&bgr;, IL6 and IL8 expression was observed in OA chondrocytes. Elevated IL37 levels diminished the IL1&bgr;-induced IL1&bgr;, IL6 and IL8 gene levels and IL1&bgr; and IL8 protein levels. In addition to the reduction in pro-inflammatory cytokine expression, IL37 reduced MMP1, MMP3, MMP13 and disintegrin and metalloproteinase with thrombospondin motifs 5 gene levels and MMP3 and MMP13 protein levels. Conclusion. IL37 is induced by IL1&bgr;, and IL37 itself reduced IL1&bgr;, IL6 and IL8 production, indicating that IL37 is able to induce a counter-regulatory anti-inflammatory feedback loop in chondrocytes. In addition, IL37 dampens catabolic enzyme expression. This supports IL37 as a potential therapeutic target in OA.

Purpose: Osteoarthritis (OA) is a multifactorial disease characterized by loss of articular cartilage. TGFβ is considered as a protective factor against cartilage loss in young cartilage by inducing Smad2/3 phosphorylation via the TGFβ receptor ALK5. Smad2/3 phosphorylation (Smad2/3P) lowers MMP13 expression, and prevents deleterious terminal differentiation of chondrocytes. In contrast to Smad2/3P, Smad1/5 phosphorylation, induced by BMPs but also by TGFβ, is linked to terminal differentiation and MMP13 production. Type I receptors that phosphorylate Smad1/5/8 include ALK1 and ALK3. BMP-9 signals via ALK1 and BMP-2 via ALK3, and both ligands induce enhanced glycosaminoglycan synthesis in young cartilage. BMP2 is induced in damaged cartilage while BMP9 is constitutively present in high concentrations in body fluids. If and how these factors modulate chondroprotective TGFβ signaling is still unclear. Therefore, we analyzed BMP-2 and BMP-9 induced Smad signaling and gene expression in chondrocytes, and studied their interaction with TGFβ. Methods: Primary bovine chondrocytes, isolated from the metacarpophalangeal joint of 2 year old animals, or the human G6 chondrocyte cell line were cultured to near confluency and incubated with TGFβ1, BMP-2, BMP-9 or a combination thereof. Smad phosphorylation kinetics were analyzed by specific Smad2p and Smad1/5p staining of Western blots. Expression patterns of Smad specific response genes: PAI-1 (Smad3 signaling), and ID-1, (Smad1/5 signaling) were analyzed from 0-48h by quantitative real time PCR (qPCR). Biological activity was tested with a CAGA12-luc transcriptional reporter construct that produces luciferase in response to Smad3 signaling. Adenovirally transduced cells (MOI = 5) were serum starved for 8h, and stimulated with growth factors for 20h, and luciferase activity was measured. Results: In primary chondrocytes, both BMP-2 and BMP-9 potently induced Smad1/5 phosphorylation, which peeked after 1 h and lasted up to 3 h. BMP-9 was more potent than BMP-2 in inducing Smad1/5p, as a lower dose of BMP-9 (5 ng/ml BMP-9 versus 15 ng/ml BMP-2) was required to induce maximal phosphorylation after 60 min. Remarkably BMP-9 (5 ng/ml or more) was also capable of inducing Smad2 phosphorylation, whereas BMP-2 (up to 50 ng/ml) was not. BMP-9-induced Smad2 phosphorylation lasted from 1 up to 3 hours. Moreover, in contrast to BMP-2, BMP-9 was also able to rapidly (from 2 up to 24h) induce PAI-1 transcription. Both growth factors potently induced ID-1 expression. Strikingly, co-stimulation of primary chondrocytes with TGFβ (1 ng/ml) and these BMPs revealed an even more remarkable difference between both factors on Western blot. BMP-2 was able to dose-dependently (15-50 ng/ml) inhibit TGFβ-induced Smad2 phosphorylation. On the contrary, BMP-9 dose-dependently (5-25 ng/ml) synergized with TGFβ on Smad2/3 phosphorylation. These results were reflected in biological activity, as luciferase production was increased by 50% after co-stimulation of chondrocytes with BMP-9 and TGFβ compared to TGFβ alone (p<0.01). Conclusions: In young cartilage, both BMPs show a distinct difference in Smad phosphorylation and interaction with TGFβ. Although both BMPs promote matrix synthesis (Glycosaminoglycans production), long term effects of both factors on cartilage will most likely differ due to their different effects on chondroprotective Smad2/3 signaling.

Objective: Fibrosis is a major contributor to joint stiffness in osteoarthritis (OA). We investigated several factors associated with the persistence of transforming growth factor beta (TGF-beta)-induced fibrosis and whether these factors also play a role in OA-related fibrosis.Design: Mice were injected intra-articularly (i.a.) with an adenovirus encoding either TGF-beta or connective tissue growth factor (CTGF). In addition, we induced OA by i.a. injection of bacterial collagenase into the right knee joint of C57BL/6 mice. mRNA was isolated from the synovium for Q-PCR analysis of the gene expression of various extracellular matrix (ECM) components, ECM degraders, growth factors and collagen cross-linking-related enzymes. Sections of murine knee joints injected with Ad-TGF-beta or Ad-CTGF or from experimental OA were stained for lysyl hydroxylase 2 (LH2). The number of pyridinoline cross-links per triple helix collagen in synovium biopsies was determined with high-performance liquid chromatography (HPLC).Results: Expression of collagen alpha-1(I) chain precursor (Colla1), tissue inhibitor of metalloproteinases 1 (TIMP1) and especially procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2b (Plod2b) were highly upregulated by TGF-beta but not by CTGF. Elevated expression of Plod2b mRNA was associated with high lysyl hydroxylase 2 (LH2) protein staining after TGF-beta overexpression and in experimental OA. Furthermore, in experimental OA the number of hydroxypyridinoline cross-links was significant increased compared to control knee joints.Conclusions: Our data show that elevated LH2b expression is associated with the persistent nature of TGF-beta-induced fibrosis. Also in experimental OA, LH2b expression as well as the number of hydroxypyridinoline cross-link were significantly upregulated. We propose that LH2b, and the subsequent increase in pyridinoline cross-links, is responsible for the persistent fibrosis in experimental OA. (C) 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Background Osteoarthritis is characterized by loss of articular cartilage. The main cartilage degrading enzyme is Matrix metalloproteinase 13 (MMP13). Expression of MMP13 is positively correlated with Activin receptor-like kinase 1 (ALK1) expression. Moreover, terminal differentiation of chondrocytes is induced by ALK1 via phosphorylation of Smad1/5/8 (Smad1/5/8p). BMP-9, also known as GDF-2, has recently been characterized as a potent, high-affinity ALK1 ligand, that is present in serum in high amounts. We wanted to further investigate intracellular BMP-9 signaling in chondrocytes to clarify its role in cartilage maintenance and degradation. Objectives To study Smad1/5/8 and Smad2/3 phosphorylation kinetics in chondrocytes in response to addition of exogenous BMP-9, to investigate BMP-9’s potency on chondrocytes, and to investigate downstream signaling responses on mRNA level. Methods Both primary bovine chondrocytes, isolated from the metacarpophalangeal joint of 2 year old animals, as well as the murine H4 chondrocyte cell line were used in this study. Chondrocytes were cultured to near confluency, and subsequently incubated with BMP-9. After short term stimulation (1 h), Smad phosphorylation was analyzed by specific Smad2/3p and Smad1/5/8p staining of Western blots. After long term stimulation (24-48 h), expression of PAI-1, a downstream marker of Smad2/3 signaling, and ID-1, a downstream marker of Smad1/5/8 signaling, were analyzed by quantitative real time PCR (qPCR). Results In both the murine cell line, as well as the primary bovine chondrocytes, addition of BMP-9 resulted in phosphorylation of Smad 1/5/8, which was maximal at very low concentrations (100 pg/ml). Unexpectedly, Smad2/3p was also observed at this concentration. Further investigations showed that BMP-9-induced Smad2/3p was maximal after 1 h, at the physiological relevant concentration of 5 ng/ml. Exposure of chondrocytes to BMP9 not only resulted in both Smad1/5/8p and Smad2/3p but also in transcription of pathway specific response genes, as observed by a significant increase in PAI-1 and ID-1 mRNA levels after both 24 and 48h. In both cell types, collagen type 2A1 and aggrecan mRNA levels were also up regulated by BMP9. In the described experiments, no significant changes in MMP13 expression were observed after 48 h of BMP-9 stimulation. Conclusions In chondrocytes, BMP9 is capable of inducing both Smad1/5/8p and Smad2/3p. By doing so it can up regulate both PAI-1 and ID-1 simultaneously. BMP-9 is very potent in inducing Smad phosphorylation, as very low concentrations (pg/ml) are sufficient for signal induction. The induction of Smad2/3p and subsequent PAI-1 activity sets BMP-9 apart from other BMPs, like BMP-2 and BMP-6, that only induce Smad1/5/8 phosphorylation. The lack of MMP13 expression after BMP-9 stimulation can be explained by the induced Smad2/3p expression, as this potently blocks MMP13 transcription. Based on these results we propose that exposure of young cartilage to BMP-9 does not result in MMP13 expression due to its ability to induce Smad2/3p. However, we previously reported that in ageing cartilage the ability to phosphorylate Smad2/3 is reduced, and therefore BMP-9 exposure will most likely result in MMP13 production in old cartilage. Disclosure of Interest None Declared

s / Osteoarthritis and Cartilage 20 (2012) S54–S296 S133