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Soluble tumor necrosis factor (sTNF) is an important inflammatory mediator and essential for secondary lymphoid organ (SLO) development and function. However, the role of its transmembrane counterpart (tmTNF) in these processes is less well established. Here, the effects of tmTNF overxpression on SLO architecture and function were investigated using tmTNF-transgenic (tmTNF-tg) mice. tmTNF overexpression resulted in enlarged peripheral lymph nodes (PLNs) and spleen, accompanied by an increase in small splenic lymphoid follicles, with less well-defined primary B cell follicles and T cell zones. In tmTNF-tg mice, the spleen, but not PLNs, contained reduced germinal center (GC) B cell fractions, with low Ki67 expression and reduced dark zone characteristics. In line with this, smaller fractions of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells were observed with a decreased Tfh:Tfr ratio. Moreover, plasma cell (PC) formation in the spleen of tmTNF-tg mice decreased and skewed towards IgA and IgM expression. Genetic deletion of TNFRI or –II resulted in a normalization of follicle morphology in the spleen of tmTNF-tg mice, but GC B cell and PC fractions remained abnormal. These findings demonstrate that tightly regulated tmTNF is important for proper SLO development and function, and that aberrations induced by tmTNF overexpression are site-specific and mediated via TNFRI and/or TNFRII signaling.

TNF is important in immune-mediated inflammatory diseases, including spondyloarthritis (SpA). Transgenic (tg) mice overexpressing transmembrane TNF (tmTNF) develop features resembling human SpA. Furthermore, both tmTNF tg mice and SpA patients develop ectopic lymphoid aggregates, but it is unclear whether these contribute to pathology. Therefore, we characterized the lymphoid aggregates in detail and studied potential alterations in the B and T cell lineage in tmTNF tg mice. Lymphoid aggregates developed in bone marrow (BM) of vertebrae and near the ankle joints prior to the first SpA features and displayed characteristics of ectopic lymphoid structures (ELS) including presence of B cells, T cells, germinal centers, and high endothelial venules. Detailed flow cytometric analyses demonstrated more germinal center B cells with increased CD80 and CD86 expression, along with significantly more T follicular helper, T follicular regulatory, and T regulatory cells in tmTNF tg BM compared with non-tg controls. Furthermore, tmTNF tg mice exhibited increased IgA serum levels and significantly more IgA+ plasma cells in the BM, whereas IgA+ plasma cells in the gut were not significantly increased. In tmTNF tg 3 TNF-RI2/2 mice, ELS were absent, consistent with reduced disease symptoms, whereas in tmTNF tg 3 TNF-RII2/2 mice, ELS and clinical symptoms were still present. Collectively, these data show that tmTNF overexpression in mice results in osteitis and ELS formation in BM, which may account for the increased serum IgA levels that are also observed in human SpA. These effects are mainly dependent on TNF-RI signaling and may underlie important aspects of SpA pathology.

Spondyloarthritis (SpA) is characterized by inflammation and new bone formation. The exact pathophysiology underlying these processes remains elusive. We propose that the extensive neoangiogenesis in SpA could play a role both in sustaining/enhancing inflammation and in new bone formation. While ample data is available on effects of anti-TNF on angiogenesis, effects of IL-17A blockade on serum markers are largely unknown. We aimed to assess the impact of secukinumab (anti-IL-17A) on synovial neoangiogenesis in peripheral SpA, and how this related to changes in inflammatory and tissue remodeling biomarkers. Serum samples from 20 active peripheral SpA patients included in a 12 week open-label trial with secukinumab were analyzed for several markers of angiogenesis and tissue remodeling. Synovial biopsies taken before and after treatment were stained for vascular markers. Serum levels of MMP-3, osteopontin, IL-6 (all P P

The authors show that SpA synovitis comprises less sTNF and more tmTNF expression compared to RA synovitis associated with less active ADAM17. Moreover, tmTNF overexpression induces SpA-like pathophysiology in mice. tmTNF signaling through TNF-RI induced inflammation, but not osteoproliferation., TNF plays a key role in immune-mediated inflammatory diseases including rheumatoid arthritis (RA) and spondyloarthritis (SpA). It remains incompletely understood how TNF can lead to different disease phenotypes such as destructive peripheral polysynovitis in RA versus axial and peripheral osteoproliferative inflammation in SpA. We observed a marked increase of transmembrane (tm) versus soluble (s) TNF in SpA versus RA together with a decrease in the enzymatic activity of ADAM17. In contrast with the destructive polysynovitis observed in classical TNF overexpression models, mice overexpressing tmTNF developed axial and peripheral joint disease with synovitis, enthesitis, and osteitis. Histological and radiological assessment evidenced marked endochondral new bone formation leading to joint ankylosis over time. SpA-like inflammation, but not osteoproliferation, was dependent on TNF-receptor I and mediated by stromal tmTNF overexpression. Collectively, these data indicate that TNF can drive distinct inflammatory pathologies. We propose that tmTNF is responsible for the key pathological features of SpA.

Background Tumour necrosis factor (TNF) is important in immune-mediated inflammatory diseases such as spondyloarthritis (SpA). SpA patients have extensive angiogenesis in inflammatory and bone forming regions. Transmembrane (tm)TNF-transgenic (tg) mice (1) that overexpress tmTNF develop SpA symptoms, including inflammation, bone destruction and bone formation. Interestingly, these mice also develop lymphoid aggregates in the bone marrow (BM) of the axial and peripheral skeleton. Objectives Characterization of lymphoid aggregates in tmTNF tg mouse BM in the context of angiogenesis and bone formation. Methods Ankles, femora, tibiae, vertebrae and spleens from tmTNF tg mice and wild-type (WT) littermates (6 weeks, 12 weeks, and 8 months old; n=5 per age per group) were dissected and analyzed by confocal microscopy. In addition, 12 week old mice (n=5 per group) were analyzed by flow cytometry. To study the importance of TNF-R signaling in these processes, tmTNF tg mice lacking TNF-RI (tmTNF tgxTNF-RI-/-) or TNF-RII (tmTNF tgxTNF-RII-/-) (n=4 per group) were investigated. Results Immunofluorescent (IF) evaluation demonstrated that BM of tmTNF tg mice contained extensive lymphoid aggregates, both in the vertebrae and the ankles, but not in the femurs or spleen. IF microscopy demonstrated that the aggregates in the BM contain characteristics of ectopic lymphoid structures (ELS) and consisted of B220+ B cells and FDC-M1+ follicular dendritic cells that are in close proximity of MECA79+high endothelial venules (HEVs). Flow cytometric analysis revealed that most B220+ B cells are IgD+ naive B cells and that tmTNF tg vertebrae contain significantly more IgD-CD95+ germinal center B cells (P Conclusion: tmTNF overexpression in mice results in extensive lymphoid aggregates in the BM that are often organized in ELS, which may be mediated via TNF-RI signaling. These ELS might result in an increase of IgA+ plasma cells in tmTNF tg mice. Ongoing studies will look further into this and may indicate whether these findings contribute to the pathology observed in these mice. Reference: [1] Alexopoulou L, et al. Eur J Immunol1997; 27(10):2588-92 Disclosure of Interests Merlijn Kaaij: None declared, Jan Piet van Hamburg: None declared, Jasper Rip: None declared, George Kollias: None declared, Dominique Baeten Employee of: UCB Pharma, Martijn Nolte: None declared, Leonie van Duivenvoorde: None declared, Sander W. Tas: None declared

Background: Spondyloarthritis (SpA) is characterized by extensive angiogenesis, tissue remodeling and inflammation of both the enthesis and the synovial tissue. Several studies have shown a relation between angiogenic/tissue remodeling serum markers and disease progression. Secukinumab, an anti-IL-17A inhibitor, is an effective treatment in SpA, but it’s effects on serum markers of angiogenesis, tissue remodeling and inflammation are largely unknown. Objectives: The purpose of this study was to analyze several candidate biomarkers of these processes in patients with SpA treated with anti-IL-17A. Methods: Serum samples from 20 active peripheral SpA patients that were included in a 12 week open-label trial with secukinumab were analyzed for several markers with luminex technology. Study design and primary results were previously published (1). Results: Serum levels of IL-6, MMP-3, osteopontin (all P Conclusion: These results indicate that anti-IL-17A not only diminishes inflammation, but also impacts on other processes such as angiogenesis and tissue remodeling. Whether these observations are predictive of disease progression or outcome remains to be determined. References [1] van Mens, L. J. J. et al. IL-17 blockade with secukinumab in peripheral spondyloarthritis impacts synovial immunopathology without compromising systemic immune responses. Arthritis Rheumatol. (2018). doi:10.1002/art.40581 Disclosure of Interests: Merlijn Kaaij: None declared, Leonieke van Mens: None declared, Jan Piet van Hamburg: None declared, Dominique Baeten Employee of: UCB Pharma, Marleen van de Sande Grant/research support from: Research support from Janssen, Novartis, Eli Lily, Consultant for: Received consultation fees from Abbvie and Novartis, Sander W. Tas: None declared

Career situation of first and presenting author Student for a master or a PhD. Introduction Tumour necrosis factor (TNF) is important in immune-mediated inflammatory diseases such as spondyloarthritis (SpA). Transmembrane (tm)TNF-transgenic (tg) mice1 that overexpress tmTNF develop SpA symptoms, including inflammation, ectopic lymphoid structures (ELS) in bone marrow (BM), bone destruction and bone formation. SpA patients have extensive angiogenesis in inflammatory and bone forming regions. Objectives To investigate whether there is a link between pathological angiogenesis and ELS in tmTNF tg mice in the BM. Methods Ankles, femora, tibiae, vertebrae and spleens from 6 and 12 weeks and 8 months old tmTNF tg mice and wild-type (WT) littermates (n=5 per age per group) were dissected and analyzed with confocal microscopy and analyzed 12 week old tmTNF tg and WT mice (n=4) with flow cytometry. To study the importance of TNF-R signaling, tmTNF tg mice lacking TNF-RI (tmTNF tgxTNF-RI-/-) or TNF-RII (tmTNF tgxTNF-RII-/-) (n=4 per group) were used. Results Immunofluorescent evaluation demonstrated that BM of tmTNF tg mice contained significantly more and extensive ELS. These ELS are limited to BM of the vertebrae and ankles, and are in close proximity of MECA79+high endothelial venules (HEVs). ELS predominantly consisted of B220+ B cells, of which most are IgD+ naive B cells. Preliminary flow cytometric analysis revealed a trend towards an increase in IgD-CD95+ germinal center B cells and CXCR5+PD-1+FoxP3-CTLA4- T follicular helper cells and CXCR5+PD-1+FoxP3+CTLA4+ in the vertebrae of tmTNF tg mice compared to WT littermates. Meanwhile, B cell lineages in the BM of tmTNF tg hind limbs were not altered. Furthermore, preliminary data indicates that BM and spleen from tmTNF tg mice contain more IgA+ plasma cells compared to WT littermates. tmTNF tgxTNF-RI-/- mice did not display lymphoid aggregates or HEVs in the BM, while tmTNF tgxTNF-RII-/- mice did, although to a lesser extent than tmTNF tg mice. Conclusions tmTNF overexpression in mice results in extensive ELS associated with HEVs in the BM, which is likely to be mediated through TNF-RI signaling. HEV formation may lead to persistence of inflammation in the BM which contributes to pathology. Reference Alexopoulou L, et al. Eur J Immunol 1997;27(10):2588–92 Disclosure of Interest None declared

Career situation of first and presenting author Post-doctoral fellow. Introduction NF-κB signaling plays a crucial role in chronic inflammatory diseases and can be activated via two pathways. Noncanonical NF-κB signaling can be induced by different signals, including transmembrane (tm)TNF ligation to TNF receptor 2 (TNFR2), which is restricted to specific cell types including endothelial cells (EC). Activation of the noncanonical NF-κB pathway in EC is involved in differentiation into high endothelial cells (HEC), which make up specialized high endothelial vessels (HEV) that recruite lymphocytes into lymph nodes (LN). tmTNF transgenic (tg) mice overexpressing tmTNF develop chronic inflammatory features, including arthritis and axial inflammation. Objectives To study LN size and composition, including stromal cell and lymphocyte subsets in tmTNF tg mice. We hypothesized that overexpression of tmTNF results in increased noncanonical NF-κB signaling in HEC leading enhanced lymphocyte recruitment. Methods Peripheral (PLN) and mesenteric LN (MLN) of 7 week old tmTNF tg mice and age-matched wild types (WT) were collected, dissected and analyzed using flow cytometry. Cellular markers were used to distinguish between different EC subsets, and B and T lymphocytes. Results Lymph nodes of tmTNF tg mice had more than two-fold increased total cell numbers compared to WT (PLN: 23,7*106±7,9*106; MLN: 24,9*106±5,3*106. WT PLN: 5,2*106±1,0*106; MLN: 10,4*106±4,6*106). Total numbers of HEC in tmTNF tg mice LN were increased (PLN: 2,3*103±0,50*103; MLN: 1,9*103±0,64*102. WT PLN: 714±393; MLN: 1.199±506), as were capillary EC (CEC) numbers (PLN: 2067±42; MLN: 2425±71. WT PLN: 1097±609; MLN: 1327±230). Notably, HEC:CEC ratio was increased in tmTNF tg mice (PLN: 1,10±0241. WT PLN: 0,66±0,006). In addition, total B and T lymphocytes in tmTNF tg mice LN were strongly increased (B lymphocytes: PLN: 10,2*106±3,5*106; MLN: 10,9*106±1,8*106. WT PLN: 2,8*106±5,8*105; MLN; 5,8*106±2,4*106. T lymphocytes: PLN: 12,8*106±4,5*106; MLN: 14,2*106±2,8*106. WT PLN: 2,5*106±3,8*105; MLN: 4,7*106±2,1*106). Conclusions Overexpression of tmTNF in mice leads to an increase in total HEC, CEC and lymphocyte numbers and an increased HEC:CEC ratio in PLN and MLN. The increase in HEC may be the result of enhanced noncanonical NF-κB signaling, enhancing lymphocyte recruitment into the LN. Consequently, interfering with noncanonical NF-κB signaling in EC may be a promising treatment strategy to dampen inflammation in immune-mediated inflammatory diseases, which is the subject of current studies. Disclosure of Interest None declared.

Introduction Spondyloarthritis is characterised by inflammation, extensive angiogenesis and pathological osteogenesis. Transmembrane (tm) TNF transgenic (tg) mice1 that overexpress tmTNF exhibit features of SpA, including chronic inflammation and pathological osteogenesis. tmTNF ligation to TNF receptor 2 in endothelial cells (ECs) can induce signal transduction pathways, that may promote these processes. Of note, angiogenesis and osteogenesis are coupled by EC differentiation towards a type H (CD31hiendomucinhi) phenotype.2 Objectives To investigate the link between pathological angiogenesis and osteogenesis in tmTNF tg mice and the potential contribution of noncanonical NF-κB signalling in ECs to this process and infiltration of immune cells into the bone marrow (BM). Methods Vertebrae and ankles from 6 and 12 weeks and 8 months old tmTNF tg mice or sex-matched non-tg littermates (n=18) were prepared by cutting 60 µm thick cryosections for confocal imaging. Results tmTNF tg mice exhibited ectopic osteogenesis which was not observed in non-tg littermates. Immunostainings showed that type H vessels are in the vicinity of the ectopic osteogenesis and osterix+ osteoprogenitors. At six weeks of age, osterix+ cells are located throughout the ectopic lesion, while at eight months, osterix+ cells are only present at the border of the lesion. Furthermore, there is increased osteogenesis and a different vessel architecture within the vertebrae of tmTNF tg mice compared to non-tg littermates that progresses with age. Non-tg littermate vertebrae only have physiological osteogenesis, which is in the metaphysis and periosteum. In addition, tmTNF tg mice also exhibit altered bone marrow (BM) architecture containing extensive lymphoid aggregates, which predominantly consisted of B220+ B cells. Conclusions tmTNF overexpression in mice leads to development of type H vessels associated with ectopic osteogenesis. In addition, extensive lymphoid aggregates develop in the BM. Current studies are aimed at identification of signalling pathways in ECs that contribute to these processes. References . Alexopoulou L, et al. Eur J Immunol1997;27(10):2588–92. . Kusumbe AP, et al. Nature2014;507(7492):323–328. Disclosure of interest None declared