A R Noort

NF-kappaB inducing kinase (NIK) is a key regulator of inflammation-induced angiogenesis

Background In rheumatoid arthritis (RA) synovial tissue (ST) angiogenesis can be observed already in the earliest phase of disease. The chemokine CXCL12, which is induced via the non-canonical nuclear factor-kappaB (NF-kB) pathway, plays an important role in angiogenesis, lymphocyte transendothelial migration, and the homing of endothelial progenitor cells. Therefore, the non-canonical pathway, with its key mediator NF-kB inducing kinase (NIK), may play an important role in pathological angiogenesis and the perpetuation of synovial inflammation in RA.

Preferential expression of NF-κB-inducing kinase (NIK) in blood vessels of rheumatoid arthritis synovial tissue containing ectopic lymphoid neogenesis

Approximately 30% of synovial tissues (ST) derived from rheumatoid arthritis (RA) patients contain ectopic lymphoid neogenesis (ELN). NF-κB transcription factors can be activated via two distinct pathways. The non-canonical NF-κB pathway, with its key mediator NIK, may play an important role in ELN, as this pathway can be triggered by stimuli like CD40L and lymphotoxin that are abundantly present in ELN.

A2.08 Extrathymic autoimmune regulator (AIRE) expression can be induced in dendritic cells by CD40-mediated activation of noncanonical NF-κb signalling, but is impaired in primary sjögren’s syndrome

Background The nuclear factor (NF)-κB family of transcription factors has a key role in the regulation of inflammation. We have previously demonstrated that activation of noncanonical NF-κB signalling in dendritic cells (DC) via CD40 stimulation is important for the induction of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). This pathway is also involved in the expression of Autoimmune Regulator (AIRE) in the thymus, which is essential central tolerance. Peripheral extrathymic AIRE-expressing cells (mainly antigen presenting cells, including DC) can also induce tolerance. However, stimuli that induce extrathymic AIRE expression in DC are unknown. Therefore, we studied whether activation of noncanonical NF-κB signalling induces AIRE expression in monocyte-derived DC (moDC) and whether this induction is altered in moDC derived from patients with autoimmunity, using primary Sjögren’s syndrome (pSS) as a prototypic systemic autoimmune disease. Objective To study whether activation of noncanonical NF-κB signalling induces AIRE expression in moDC from healthy donors and pSS. Methods MoDC were generated by culturing monocytes for 6 days with GM-CSF and IL-4. Maturation was induced by addition of the noncanonical NF-κB stimulus anti-CD40 for 48 h. Cleavage of p100 into p52, IDO and AIRE protein was analysed by Western blot. Immunofluorescence staining for RelB was performed on cytospins of moDC. RelB protein was visualised by confocal microscopy. siRNA-mediated silencing of NF-κB-inducing kinase (NIK), the main activating kinase of the noncanonical pathway, was accomplished by neon electroporation. Results Anti-CD40 stimulation of moDC from healthy donors induced cleavage of p100 and nuclear translocation of RelB, which is indicative of active noncanonical NF-κB signalling. This was accompanied by upregulation of IDO and AIRE expression, which was inhibited by siRNA-mediated silencing of NIK. Anti-CD40 stimulation of moDC derived from pSS patients also resulted in activation of noncanonical NF-κB signalling and upregulation of IDO. However, AIRE expression was to a much lesser extent upregulated in moDC derived from pSS patients compared to healthy donor DC. Conclusion Extrathymic AIRE expression can be induced in moDC via CD40-stimulation and is regulated by noncanonical NF-κB signalling. This mechanism is impaired in pSS patients. The functional consequences of this potentially defective immunoregulatory program are currently investigated.

OP0213 CD40-Mediated Activation of Noncanonical NF-κB Signalling in Dendritic Cells Induces Extrathymic Autoimmune Regulator (AIRE) Expression, Which Is Impaired in Primary Sjögren's Syndrome

Background The nuclear factor (NF)-κB family of transcription factors has a key role in the regulation of inflammation. We have previously demonstrated that activation of noncanonical NF-κB signalling in dendritic cells (DC) via CD40 stimulation is important for the induction of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) [1]. This pathway is also involved in the expression of Autoimmune Regulator (AIRE) in the thymus, which is essential central tolerance. Peripheral extrathymic AIRE-expressing cells (mainly antigen presenting cells, including DC) can also induce tolerance. However, stimuli that induce extrathymic AIRE expression in DC are unknown. Therefore, we studied whether activation of noncanonical NF-κB signalling induces AIRE expression in monocyte-derived DC (moDC) and whether this induction is altered in moDC derived from patients with autoimmunity, using primary Sjögrens syndrome (pSS) as a prototypic systemic autoimmune disease. Objectives To study whether activation of non-canonical NF-κB signalling induces AIRE expression in moDC from healthy donors and pSS. Methods MoDC were generated by culturing monocytes for 6 days with GM-CSF and IL-4. Maturation was induced by addition of the non-canonical NF-κB stimulus anti-CD40 for 48 hours. Cleavage of p100 into p52, IDO and AIRE protein was analyzed by Western blot. Immunofluorescence staining for RelB was performed on cytospins of moDC. RelB protein was visualized by confocal microscopy. siRNA-mediated silencing of NF-κB-inducing kinase (NIK), the main activating kinase of the noncanonical pathway, was accomplished by neo electroporation. Results Anti-CD40 stimulation of moDC from healthy donors induced cleavage of p100 into p52 and nuclear translocation of RelB, which is indicative of noncanonical NF-κB signalling. This was accompanied by upregulation of IDO and AIRE expression, which was inhibited by siRNA-mediated silencing of NIK. Anti-CD40 stimulation of moDC derived from pSS patients also resulted in activation of noncanonical NF-κB signalling and upregulation of IDO. However, AIRE expression was to a much lesser extent upregulated in moDC derived from pSS patients compared to healthy donor DC. Conclusions Extrathymic AIRE expression can be induced in moDC via CD40-stimulation and is regulated by noncanonical NF-κB signalling. This is impaired in pSS patients. The functional consequences of this potentially defective immunoregulatory program are currently investigated. References Tas, S.W., et al., Noncanonical NF-kappaB signaling in dendritic cells is required for indoleamine 2,3-dioxygenase (IDO) induction and immune regulation. Blood, 2007. 110(5): p. 1540–9. Acknowledgement This work was supported by a fundamental research grant of the Dutch Arthritis Foundation. Disclosure of Interest None declared

A6.21 NF-κB-inducing kinase (NIK) is expressed in synovial endothelial cells in early arthritis patients and correlates with markers of inflammation

Background and objectives The NF-κB family of transcription factors is strongly involved in synovial inflammation. We have previously shown that NF-κB-inducing kinase (NIK) is a key regulator of inflammation-induced angiogenesis in rheumatoid arthritis (RA) synovial tissue (ST). Here, we investigated synovial NIK expression in early arthritis patients and in autoantibody-positive individuals at risk for developing RA. Materials and methods ST biopsies were obtained via arthroscopy from 154 early arthritis patients (arthritis duration <1 year) with different diagnoses and from 54 IgM-rheumatoid factor and/or anti-citrullinated peptide antibody-positive individuals at risk for developing RA, without any evidence of arthritis. ST was stained for NIK and the endothelial cell (EC) marker vWF. Additionally, measures of disease activity (ESR, CRP, joint swelling) were collected and contrast-enhanced MRI was performed in a subset of these patients. Results In early arthritis patients, NIK was predominantly expressed in EC of small blood vessels. Furthermore, NIK expression correlated with ESR (r = 0.184; p = 0.024), CRP (r = 0.194; p = 0.017), joint swelling (r = 0.297; p < 0.001), synovial cellular markers (CD68+ lining and sublining macrophages r = 0.585; p < 0.001 and r = 0.728; p < 0.001, respectively, CD3+ T cells r = 0.733; p < 0.001 and CD22+ B cells r = 0.264; p = 0.040), MRI effusion (r = 0.665; p < 0.001), MRI synovitis (r = 0.632; p < 0.001) and MRI total score (r = 0.569; p < 0.001). In 18.5% of autoantibody-positive individuals ST NIK+EC were present, but this was not predictive for the development of arthritis. Conclusions NIK+EC are present in the earliest phase of synovial inflammation and may be indicative of high angiogenic activity in the inflamed ST. Therefore, NIK+EC may play an important role in the persistence of synovitis. Collectively, our data underscore the importance of angiogenesis in synovial inflammation and identify NIK as a potential therapeutic target in arthritis.

FRI0013 Activation of the Non-Canonical NF-κB Pathway in Dendritic Cells Upregulates Extrathymic Autoimmune Regulator (AIRE) Expression

Background The nuclear factor (NF)-κB family of transcription factors has a key role in the regulation of inflammation. Ligation of CD40 on dendritic cells can induce early production of inflammatory mediators via the canonical NF-κB pathway, as well as late expression of the of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) via non-canonical NF-κB signalling [1]. Non-canonical NF-κB signalling via CD40 is also crucially important for the expression of Autoimmune Regulator (AIRE) in the thymus. AIRE is a transcription factor that is involved in thymic negative selection of autoreactive T cells and therefore is pivotal in the establishment of central tolerance. We have recently found extrathymic AIRE-expressing cells (eTACs) that may play a role in peripheral tolerance in rheumatoid arthritis synovial tissue and glandular tissue from Sjogrens syndrome patients. eTACs are mainly antigen presenting cells, including DC, that can inactivate peripheral T cell responses. However, until now the stimuli that can induce extrathymic AIRE expression in DC are largely unknown. Objectives To study whether activation of the non-canonical NF-κB pathway stimulates AIRE expression in human monocyte-derived DC. Methods Monocyte-derived DC were generated by culturing healthy donor monocytes for 6 days with GM-CSF and IL-4. Maturation was induced by addition of LPS or the non-canonical NF-κB stimuli such as lymphotoxin, LIGHT, RANKL or anti-CD40 for 48 hours. AIRE and IDO expression was quantified by qPCR and cleavage of p100 into p52 was analyzed by Western blot. Presence of AIRE protein was analyzed by Western blot and immunofluorescence staining. Results Anti-CD40 and RANKL stimulation of DC resulted in cleavage of p100 into p52, upregulation of AIRE RNA and AIRE protein. Confocal microscopy revealed that AIRE protein was localised in the nucleus. Interestingly, this was accompanied by increased expression of the IDO in these cells. Conclusions CD40 and RANKL stimulation in DC activates the non-canonical NF-κB pathway and simultaneously upregulates AIRE and IDO expression. The functional significance of peripheral AIRE expression in DC requires further investigation, but it may be associated with immunoregulatory properties. Eventually, this knowledge may be exploited to develop new treatment modalities for patients with autoimmune diseases, including DC-based therapies. References Tas SW, Vervoordeldonk MJ, Hajji N, Schuitemaker JH, van der Sluijs KF, May MJ, et al. Noncanonical NF-kappaB signaling in dendritic cells is required for indoleamine 2,3-dioxygenase (IDO) induction and immune regulation. Blood 2007;110:1540-9. Disclosure of Interest None declared

A6.19 Non-canonical NF-κB signalling in endothelial cells promotes angiogenesis in a novel 3D model of rheumatoid arthritis synovial angiogenesis

Background Angiogenesis is a crucial mediator in rheumatoid arthritis (RA) pathogenesis. Current in vitromodels of angiogenesis focus solely on endothelial cells (EC), however, RA fibroblast like synoviocytes (FLS) also contribute to this process. Therefore, a model including both EC and RA FLS would be more representative of synovial angiogenesis. Previous work demonstrates that the non-canonical NF-κB pathway with its main regulator NF-κB inducing kinase (NIK), induces angiogenesis in EC. Yet, this still remains to be studied in a co-culture model. Objective Generate a 3D model of RA synovial angiogenesis and study effects of non-canonical NF-κB signalling, RA synovial fluid (RASF) and inhibitors on angiogenesis. Methods We developed a 3D model in which HUVEC and RA FLS labelled with green or orange cell tracker dye, respectively, were incubated overnight to form spheroids. Subsequently, spheroids were harvested and plated in a collagen solution, and medium with or without lymphotoxinα1β2 (LT) or LIGHT (activators of non-canonical NF-κB signalling) or growth factors (bFGF/VEGF) was added. After 48 h, spheroids were fixed and imaged through confocal microscopy. Cumulative EC sprout length was quantified using Leica QWin Plus software. To demonstrate NIK dependency, EC were transfected with non-targeting or NIK-targeting siRNA before incorporation into the model. In addition, spheroids were incubated with RASF, with or without inhibitors, and subsequent changes in sprout formation were measured. Results Confocal analysis of the 3D model showed spheroids formed sprouts under all conditions. LT and LIGHT caused significant increases in cumulative sprout length (p < 0.05). LTβR-induced sprout formation was significantly decreased by siRNA-mediated knockdown of NIK in EC as compared to the non-targeting controls. RASF induced sprout formation at all concentrations with a significant increase observed at 10%. This sprout formation was significantly decreased in the presence of different inhibitors of angiogenesis. Conclusion The 3D model is an effective tool for studying RA angiogenesis. This novel model incorporates two key cellular mediators of this process, EC and RA FLS, and true capillary-like structures are formed that can be easily quantified. Using this system, we have demonstrated that activation of the non-canonical NF-κB pathway induces angiogenesis in a NIK-dependent fashion. Therefore, targeting NIK may have therapeutic potential in reducing pathological angiogenesis and halting disease progression. Further studies testing the efficacy of NIK inhibitors are currently underway. Additionally, we have demonstrated that RASF is a potent inducer of angiogenesis in the 3D model and that this can be used to measure the effectiveness of pharmacological inhibitors of angiogenesis.

SAT0014 A Novel 3D Model of Rheumatoid Arthritis Synovial Angiogenesis to Screen New Therapeutic Compounds

Background Angiogenesis is a crucial mediator in rheumatoid arthritis (RA) pathogenesis and blocking this process is thought to have therapeutic potential in ameliorating the disease. Current in vitro models of angiogenesis focus solely on endothelial cells (EC), however, RA fibroblast like synoviocytes (FLS), as well as angiogenic mediators in synovial fluid (SF), are also important contributors to this process. Therefore, a model including both EC, FLS, and SF would be more representative of synovial angiogenesis, and could be used to screen new therapeutic compounds. Previous work demonstrates that the non-canonical NF-κB pathway, with its main regulator NF-κB inducing kinase (NIK), induces angiogenesis in EC. Yet, this remains to be studied in a co-culture model. Objectives To generate a 3D model of RA synovial angiogenesis and to screen the effects of inhibition of non-canonical NF-κB signaling and other inhibitors of angiogenesis Methods HUVEC and RA FLS were labeled with green or orange cell tracker dye, respectively, and incubated overnight to form spheroids. Subsequently, spheroids were harvested, plated in a collagen solution, and medium with growth factors or different concentrations of RASF was added. To activate non-canonical NF-κB signaling, lymphotoxinα1β2 (LT) or LIGHT was added. After 48 hours, spheroids were fixed and imaged through confocal microscopy. Cumulative EC sprout length was quantified using Leica QWin Plus software. As a proof of concept and to demonstrate NIK dependency of LT- or LIGHT-induced angiogenesis, EC were transfected with non-targeting or NIK-targeting siRNA before incorporation into the model and subsequent changes in sprout formation were measured. Results Confocal analysis of the 3D model showed that spheroids formed sprouts containing true capillary-like structures. RASF induced sprout formation at all concentrations with a significant increase observed at 10% (p<0.05). LT and LIGHT also caused significant increases in cumulative sprout length and this LTβR-induced sprout formation was significantly decreased by siRNA-mediated knockdown of NIK in EC as compared to the non-targeting controls (p<0.05). Significant decreases in sprout formation were also observed in the presence of various established inhibitors of angiogenesis. Conclusions The 3D model is an effective tool for studying RA angiogenesis. In this novel model, two key cellular mediators of this process are incorporated, EC and RA FLS, in addition to RASF, and true capillary-like structures are formed which can be easily quantified. Using this system, we have demonstrated that activation of the non-canonical NF-κB pathway induces angiogenesis in a NIK-dependent fashion. Therefore, targeting NIK may have therapeutic potential in reducing pathological angiogenesis and halting disease progression. Additional studies testing the efficacy of pharmacological NIK inhibitors are currently underway. We anticipate that this novel 3D model can be used to screen new pharmacological inhibitors of angiogenesis. Disclosure of Interest None declared

A1.78 Ectopic lymphoid neogenesis in rheumatoid arthritis: a potential role for NIK expressing endothelial cells as orchestrators of tertiary lymphoid structures

Background and Objectives Tertiary lymphoid structures (TLS) can be observed in rheumatoid arthritis (RA) synovial tissue, and often contain high endothelial venules (HEV) and follicular dendritic cells (FDC). Endothelial cell (EC)-specific lymphotoxin (LT)β receptor signalling is critical for lymph node and HEV formation and FDC arise from PDGFRβ+ perivascular precursor cells that require lymphoid tissue inducer (LTi) cells and LTβ for their expansion. RA synovial tissue contains EC expressing NF-κB inducing kinase (NIK) which is pivotal in LTβ-induced non-canonical NF-κB signalling. Therefore, we studied the presence and relation between NIK+ EC, (pre)FDC and group 3 innate lymphoid cells (ILC3) that comprise LTi cells with TLS in RA synovial tissue. Materials and Methods Microarray data were used to explore the expression levels of genes involved in NF-κB signalling in RA synovial tissue + /- TLS. Expression of NIK, PNAd, FDC-M1, RORc, and PDGFRβ was evaluated in tissues containing TLS (n = 25) and tissues without TLS (n = 15) using immunofluorescence microscopy. Results RA synovial tissues with TLS contained a significantly higher expression of non-canonical NF-κB related genes, including NIK. This was confirmed by immunohistochemistry: NIK was highly expressed in PNAd+ HEV. Interestingly, we could detect small numbers of ILC3 in RA synovial tissue, which did not clearly correlate with the presence of TLS. In contrast, tissues with TLS contained significantly more perivascular PDGFRβ+ cells correlating significantly with the amount of FDC. Conclusion We established a strong correlation between NIK+ EC, perivascular (pre)FDC and TLS. Furthermore, we demonstrate for the first time that ILC3 are present in human RA synovial tissue, but only in very low numbers and without a clear correlation with TLS. Therefore, NIK+ EC and perivascular (pre)FDC may be more important as orchestrators of TLS.

AB0115 Non-Canonical Nf-κB Signaling Enhances Angiogenesis in A Novel 3D Model of Rheumatoid Arthritis Synovial Inflammation

Background Angiogenesis plays a crucial role in the progression of rheumatoid arthritis (RA) and is considered a switch from acute to chronic inflammation. Previously, we have demonstrated that NF-κB inducing kinase (NIK) mediated activation of the non-canonical NF-κB pathway in endothelial cells (EC) induces angiogenesis. To further investigate the role of this pathway in RA angiogenesis, we developed an innovative 3D model that incorporates both EC and RA fibroblast-like synoviocytes (FLS) with the option to also add immune cells, thereby generating a model more representative of synovial angiogenesis. Objectives To develop a novel 3D in vitro model to study the interaction between RA FLS, EC and immune cells to further delineate the role of the non-canonical NF-κB pathway in pathological angiogenesis. Methods In the traditional 2D model, RA FLS and HUVEC were co-cultured with or without the non-canonical NF-κB signaling inducing stimuli lymphotoxin α1β2 (LT) and LIGHT, or VEGF as a positive control. EC were visualized through immunohistochemical staining of CD31. In the novel 3D model, spheroids of HUVEC and RA FLS, pre-incubated with green or orange cell tracker dye, were plated in a collagen solution with medium containing LT, LIGHT or bFGF/VEGF. After 48 hours, spheroids were imaged through confocal microscopy. Cumulative EC sprout length and the number of sprouts was quantified using Leica QWin Plus software. Results In the 2D model, LIGHT induced a significant increase in EC proliferation (p<0.05) and the LT-induced increase neared significance (p=0.08). Confocal analysis of the 3D model showed that spheroids containing HUVEC and RA FLS formed sprouts under all conditions. Importantly, both LT and LIGHT caused significant increases (p<0.05) in cumulative sprout length and total number of sprouts, which was comparable to that of bFGF/VEGF. Preliminary results indicate that siRNA-mediated targeting of NIK inhibits this process effectively. Conclusions We demonstrate that induction of non-canonical NF-κB signaling by LT or LIGHT enhances angiogenic responses in EC in both models. However, the new 3D model allows visualization of actual blood vessel formation and incorporation of immune cells in future studies. Based on our findings, targeting NIK may be a novel therapeutic approach to block pathological angiogenesis in RA, thereby halting disease progression. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5265