Boy Helder

Tertiary Lymphoid Structures in Rheumatoid Arthritis: NF-κB-Inducing Kinase-Positive Endothelial Cells as Central Players.

Tertiary lymphoid structures (TLSs) in chronic inflammation, including rheumatoid arthritis (RA) synovial tissue (ST), often contain high endothelial venules and follicular dendritic cells (FDCs). Endothelial cell (EC)-specific lymphotoxin β (LTβ) receptor signaling is critical for the formation of lymph nodes and high endothelial venules. FDCs arise from perivascular platelet-derived growth factor receptor β(+) precursor cells (preFDCs) that require specific group 3 innate lymphoid cells (ILC3s) and LTβ for their expansion. Previously, we showed that RA ST contains ECs that express NF-κB-inducing kinase (NIK), which is pivotal in LTβ-induced noncanonical NF-κB signaling. We studied the relation between NIK(+) ECs, (pre)FDCs, and ILC3s with respect to TLSs in RA ST. TLS(+) tissues exhibited a significantly increased expression of genes involved in noncanonical NF-κB signaling, including NIK, and immunohistochemical analysis revealed that NIK was almost exclusively expressed by ECs. ILC3s were present in human RA ST in very low numbers, but not differentially in TLS(+) tissues. In contrast, TLS(+) tissues contained significantly more NIK(+) ECs and perivascular platelet-derived growth factor receptor β(+) preFDCs, which correlated significantly with the quantity of FDCs. We established a strong link between NIK(+) ECs, (pre)FDCs, and the presence of TLSs, indicating that NIK(+) ECs may not only be important orchestrators of lymph node development but also contribute to the formation of TLSs in chronic inflammation.

FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis in rheumatoid arthritis

IntroductionThe FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis plays a fundamental role in proliferation and differentiation of dendritic cells (DCs). As DCs play an important role in rheumatoid arthritis (RA) immunopathology we studied in detail the Flt3L/CD135 axis in RA patients.MethodsThe levels of Flt3L in (paired) serum and synovial fluid (SF) were quantified by enzyme-link immunosorbent assay (ELISA). Expression of Flt3L and CD135 in paired peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) was quantified by fluorescence-activated cell sorting (FACS). The expression of Flt3L, CD135 and TNF-Converting Enzyme (TACE) in synovial tissues (STs) and in vitro polarized macrophages and monocyte-derived DCs (Mo-DCs) was assessed by quantitative PCR (qPCR). CD135 ST expression was evaluated by immunohistochemistry and TACE ST expression was assessed by immunofluorescence. Flt3L serum levels were assessed in RA patients treated with oral prednisolone or adalimumab.ResultsFlt3L levels in RA serum, SF and ST were significantly elevated compared to gout patients and healthy individuals (HI). RA SF monocytes, natural killer cells and DCs expressed high levels of Flt3L and CD135 compared to HI. RA ST CD68+ and CD163+ macrophages, CD55+ fibroblast-like synoviocytes (FLS), CD31+ endothelial cells or infiltrating monocytes and CD19+ B cells co-expressed TACE. IFN-γ-differentiated macrophages expressed higher levels of Flt3L compared to other polarized macrophages. Importantly, Flt3L serum levels were reduced by effective therapy.ConclusionsThe Flt3L/CD135 axis is active in RA patients and is responsive to both prednisolone and adalimumab treatment. Conceivably, this ligand receptor pair represents a novel therapeutic target.

Synovial IL-21/TNF-producing CD4+ T cells induce joint destruction in rheumatoid arthritis by inducing matrix metalloproteinase production by fibroblast-like synoviocytes

Bone and cartilage destruction is one of the key manifestations of rheumatoid arthritis (RA). Although the role of T helper (Th)17 cells in these processes is clear, the role of IL‐21–producing cells T cells has been neglected. We sought to investigate the role of IL‐21 in RA by focusing on the functional characteristics of the main producers of this cytokine, synovial CD4+IL‐21+ T cells. We show that the frequency of both synovial fluid (SF) CD4+IL‐21+ or CD4+IL‐21+TNF+ T cells in patients with RA was significantly higher compared with patients with psoriatic arthritis (PsA). The frequency of peripheral blood (PB) IL‐21+CD4+ T cells in patients with RA positively correlated with disease activity score 28 (DAS28), serum anticyclic citrullinated peptide (anti‐CCP) antibodies and IgM‐rheumatoid factor (IgM‐RF). IL‐21 levels in RA SF were associated with matrix metalloproteinase (MMP)‐1 and MMP‐3. Related to this, IL‐21 induced significantly the secretion of MMP‐1 and MMP‐3 in RA synovial biopsies. Sorted SF CD4+IL‐21+ T cells significantly induced the release of MMP‐1 and MMP‐3 by fibroblast‐like synoviocytes (FLS) compared with medium or CD4+IL‐21− T cells in a coculture system. Neutralization of both IL‐21 and TNF resulted in significantly less production of MMP by FLS. The results of this study indicate a new role for synovial CD4+IL‐21+TNF+ T cells in promoting synovial inflammation/joint destruction in patients with RA. Importantly, IL‐21 blockade in combination with anti‐TNF might be an effective therapy in patients with RA by inhibiting MMP‐induced inflammation/joint destruction.

Targeting non-canonical nuclear factor-κB signalling attenuates neovascularization in a novel 3D model of rheumatoid arthritis synovial angiogenesis

Objective. Angiogenesis is crucial in RA disease progression. Lymphotoxin &bgr; receptor (LT&bgr;R)-induced activation of the non-canonical nuclear factor-&kgr;B (NF-&kgr;B) pathway via NF-&kgr;B-inducing kinase (NIK) has been implicated in this process. Consequently, inhibition of this pathway may hold therapeutic potential in RA. We describe a novel three-dimensional (3D) model of synovial angiogenesis incorporating endothelial cells (ECs), RA fibroblast-like synoviocytes (RAFLSs) and RA synovial fluid (RASF) to further investigate the contributions of NF-&kgr;B in this process. Methods. Spheroids consisting of RAFLSs and ECs were stimulated with RASF, the LT&bgr;R ligands LT&bgr; and LIGHT, or growth factor bFGF and VEGF, followed by quantification of EC sprouting using confocal microscopy and digital image analysis. Next, the effects of anginex, NIK-targeting siRNA (siNIK), LT&bgr;R–Ig fusion protein (baminercept) and a novel pharmacological NIK inhibitor were investigated. Results. RASF significantly promoted sprout formation, which was blocked by the established angiogenesis inhibitor anginex (P < 0.05). LT&bgr; and LIGHT induced significant sprouting (P < 0.05), as did bFGF/VEGF (P < 0.01). siNIK pre-treatment of ECs led to reductions in LT&bgr;R-induced vessel formation (P < 0.05). LT&bgr;R–Ig not only blocked LT&bgr;- or LIGHT-induced sprouting, but also RASF-induced sprouting (P < 0.05). The NIK inhibitor blocked angiogenesis induced by LT&bgr;, LIGHT, growth factors (P < 0.05) and RASF (P < 0.01). Conclusion. We present a novel 3D model of synovial angiogenesis incorporating RAFLSs, ECs and RASF that mimics the in vivo situation. Using this system, we demonstrate that non-canonical NF-&kgr;B signalling promotes neovascularization and show that this model is useful for dissecting relative contributions of signalling pathways in specific cell types to angiogenic responses and for testing pharmacological inhibitors of angiogenesis.

A8.19 Non-lymphoid CD103+ dendritic cells are required for the initiation of collagen-induced arthritis

Background and Objectives Dendritic cells (DCs) are essential for the initiation of synovial inflammation, but it is unclear which subset of DCs performs this task. We have shown that FMS-like tyrosine kinase 3 ligand (Flt3L)-dependent DCs are important for disease development in a mouse model for RA. However since Flt3L-/- mice have reductions in all conventional (c)DCs it remained unclear which subset is required for disease induction. Materials and Methods CIA was induced in Flt3L-/-, Batf3-/- (mice lacking both CD103+ and CD8a+DCs) and WT C57/BL6 littermates. In vitro and in vivo uptake and migration was performed using bone marrow (BM)-DCs and dermal DCs. Antigen presentation was studied in vivo by adoptive transfer of CFSE-labeled OT-I or OT-II T cells + OVA in Flt3L-/- and WT mice and in vitro by culturing BM-DCs with OT-I and OT-II cells. T cell proliferation was analysed by CFSE dilution. To study BM-DC function qPCR array (Dendritic and Antigen Presenting Cell PCR Array- Qiagen) for 84 genes was performed. To test the potential of a Flt3 inhibitor (CEP701) in the prevention of CIA, an in vivo study was performed injecting CEP701 before the onset of disease in DBA-1 mice. Results Flt3L-/- mice were susceptible to innate K/BxN arthritic model and arthritic T cell transfer. Batf3-/- mice lacking both CD103 + and CD8a + DCs were resistant to CIA, demonstrating that CD11b + and monocyte-derived DCs (moDCs) were not sufficient to induce CIA. The amount of DCs carrying antigen reaching the LN in Flt3L-/- mice was reduced compared with WT. Uptake and migratory capacity was similar in Flt3L-/- BM-DCs compared to WT BM-DCs. CEP701 (a Flt3L inhibitor) treatment prevented CIA induction, and reduced dramatically CD103 + DCs in the lymph nodes and synovium. Human CD141hi DCs, homologues of mouse CD103 + DCs, were present and increased in inflamed rheumatoid arthritis (RA) synovium. Conclusions Antigen presentation in Flt3L-/- mice is impaired. As CD103 + DCs are absent in Flt3L-/- mice and are important in (cross)-presenting antigens our data reveals a crucial role for CD103 + DCs in the induction of CIA. As a consequence of CD103 + DC absence Flt3L-/- mice showed a reduction in T cell activation in particularly reduced CD8 T cell responses. Hence this study identified non-lymphoid CD103 + DCs as the main subset orchestrating the initiation of cell-mediated immunity in arthritis. Targeting this DC subset might be of interest in individuals at risk of developing autoimmune disorders.

Noncanonical NF-κB signaling in microvessels of atherosclerotic lesions is associated with inflammation, atheromatous plaque morphology and myocardial infarction

BACKGROUND AND AIMS Neovascularization is associated with atherosclerotic plaque instability and increased chance of myocardial infarction (MI). Patients with chronic inflammatory diseases (CID) have increased risk of atherosclerosis, and evidence demonstrates that NF-κB inducing kinase (NIK)-mediated noncanonical NF-κB signaling in endothelial cells (EC) is linked to inflammation and angiogenesis. Here, we hypothesized NIK may also be activated in EC of atherosclerotic lesion microvessels. METHODS Using cohorts of atherosclerotic lesions from coronary and carotid arteries, we quantified NIK expression in plaque microvessels and compared it to pathological markers, including inflammatory cell content, plaque characteristics and MI. Differences in gene transcripts were evaluated between stable and ruptured lesions. RESULTS NIK+EC were present in both coronary and carotid lesions. In CID patients, plaques with stenosis >40% had an increased number of NIK+EC and higher content of immune cells (p < .05) as compared to controls. Immune cells per NIK+EC were also greater in CID patients (p < .05), with pronounced differences as stenosis increased. In unstable lesions, NIK+EC were elevated as were EC expressing CXCL12 (p < .05). NIK+EC were increased in lesions with lipid content >40% (p < .05) and more abundant in coronary artery lesions implicated in MI (p < .05). These vessels also associated with atheromatous rather than fibrous plaque morphology (p < .05). Transcriptomic profiling demonstrated components of noncanonical NF-κB pathway were also upregulated in ruptured plaques (p < .05). CONCLUSIONS NIK+EC associate with chronic inflammation in advanced lesions and are linked to markers of local inflammation, lipid content, unstable plaque phenotype and development of MI. Therefore, targeting noncanonical NF-κB signaling may hold therapeutic potential for patients with atherosclerosis and cardiovascular disease.

Silencing NIK potentiates anti-VEGF therapy in a novel 3D model of colorectal cancer angiogenesis

Angiogenesis is essential for colorectal cancer (CRC) progression, as demonstrated by the beneficial clinical effects of therapeutics inhibiting VEGF signaling. However, alternative mechanisms of neovascularization can develop, resulting in treatment failure. Previously we demonstrated NF-κB-inducing kinase (NIK) contributes to pathological angiogenesis. Here, we investigate NIK as a therapeutic target in endothelial cells (EC) in CRC. To determine NIK expression levels in CRC tissues, we immunostained both primary colorectal tumors and tumors metastasized to the liver. Additionally, a 3D tumor-stromal cell interaction model was developed including EC, fibroblasts and CRC cells to study tumor angiogenesis. This model tested efficacy of NIK-targeting siRNA (siNIK) in EC alone or in combination with the anti-VEGF antibody, bevacizumab. Both primary CRC and liver metastases contained blood vessels expressing NIK. In patients receiving chemotherapy plus bevacizumab, immature NIK+ vessels (p < 0.05) were increased as compared to chemotherapy alone. Activation of NIK by lymphotoxin-beta receptor (LTβR) induced increases in pro-angiogenic mediators, including interleukin (IL)-6, IL-8, chemokine (C-X-C motif) ligand (CXCL)1 and CXCL5 in EC and fibroblasts, accompanied by sprouting in the 3D model, which was blocked by siNIK in EC. Treatment with bevacizumab plus siNIK in EC resulted in a synergistic effect and reduced VEGF and bFGF-induced sprouting (p < 0.05). Here, we demonstrate a role for NIK in CRC-associated angiogenesis. Targeting NIK in EC in combination with anti-VEGF antibody bevacizumab may hold therapeutic potential to increase efficiency in blocking tumor neovascularization, either to prevent treatment failure due to activation of accessory pathways such as NF-κB signaling or as a rescue treatment.

03.18 Cd40-mediated activation of noncanonical NF-κB signalling in dendritic cells induces extrathymic autoimmune regulator (aire) expression

Background Nuclear factor (NF)-κB signalling plays an important role in the regulation of immune responses. We have previously demonstrated that CD40-mediated noncanonical NF-κB signalling in dendritic cells (DC) induces the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). Noncanonical NF-κB signalling is also important for AIRE expression in the thymus, which is essential for establishment of central tolerance. In the periphery, extrathymic AIRE-expressing antigen presenting cells (including DC) can induce or maintain peripheral tolerance. However, stimuli that induce extrathymic AIRE expression in DC are hitherto unknown. Materials and methods AIRE expressing cells were characterised by confocal microscopy in tonsil tissue and inflamed tissues from patients with rheumatoid arthritis (RA) and primary Sjögren’s Syndrome (pSS). Monocyte-derived DC (moDC), peripheral blood conventional DC (cDC) and plasmacytoid DC (pDC) were stimulated by anti-CD40 to induce NF-κB activation. Gene transcription and protein expression levels of AIRE, IDO and noncanonical NF-κB signalling components were detected by real time-PCR, Western blot and confocal microscopy. Expression of NF-κB-inducing kinase (NIK), the main activating kinase of the noncanonical pathway, was modulated by siRNA-mediated silencing. Results First, we identified AIRE expressing cells in inflamed tonsil tissue, RA synovial tissue and pSS glandular tissue and could demonstrate that these cells have DC characteristics, including MHCII, CD40 and CD11c expression. Next, we demonstrated that CD40-stimulation of moDC and pDC, but not cDC was accompanied by upregulation of AIRE expression. This upregulation was crucially dependent on noncanonical NF-κB signalling, since AIRE expression was abrogated by NIK siRNA-mediated silencing and was not affected by either inhibition or induction of canonical NF-κB signalling. Furthermore, preliminary data show that CD40-stimulated moDC from APECED (AIRE deficient) patients exhibited increased T cell proliferation in a mixed lymphocyte reaction compared to healthy individuals. Conclusions Collectively, our data demonstrate that AIRE expression in DC is critically dependent on CD40-mediated noncanonical NF-κB signalling. These findings suggest a role for peripheral AIRE expression in controlling T cell proliferation during adaptive immune responses, including the inflamed target tissues of patients with RA and pSS. These findings could potentially be exploited as a novel approach to boost peripheral tolerance and induce immune regulation.

A7.14 Identification of new inhibitors of angiogenesis in a novel 3d model of rheumatoid arthritis synovial angiogenesis

Background/objective Angiogenesis contributes to rheumatoid arthritis (RA) pathogenesis, however, many models focus solely on endothelial cells (EC). We developed a 3D-spheroid model including both EC and RA fibroblast-like-synoviocytes (FLS) to study angiogenesis associated with RA and to test efficacy of several inhibitors targeting this process. Previous work demonstrated a role for the non-canonical NF-κB pathway and its main regulator, NF-kB inducing kinase (NIK) in pathological angiogenesis. Here we aim to use the 3D model to further characterise its contribution to neovascularization. Methods Human umbilical vein EC (HUVEC) were combined with RA FLS in spheroids and placed in a collagen gel. Spheroids were stimulated with RA synovial fluid (SF), growth factors (GF), LT or LIGHT. To establish NIK dependency in EC, EC were pre-transfected with a non-targeting or NIK-targeting siRNA before incorporation into spheroids. Pharmacological inhibitors were also added to determine their ability to abrogate EC sprouting induced by 10% RASF. Sprouting was imaged by confocal microscopy and quantified using the Leica QWin Plus software. Results Spheroids formed sprouts under all conditions with significant increases observed upon stimulation with LT, LIGHT, 10% RA SF (p < 0.05) and growth factors (p < 0.01), as compared to basal levels. LT and LIGHT induced sprout formation was NIK dependent as spheroids containing HUVEC transfected with NIK targeting siRNA had reductions in vessel formation (p < 0.05) as compared to controls. Anginex blocked sprout formation induced not only by growth factors, but also by that induced by RA SF (p < 0.05). Inhibitors of the non-canonical NF-kB pathway were able to attenuate cumulative sprout growth promoted by LT, LIGHT and RA SF (p < 0.05). Conclusion The 3D model is an effective tool for studying synovial angiogenesis in that it incorporates several elements essential to the process namely EC, RA FLS and immune cell factors found in RA SF. Using this system, we have further demonstrated a role for activation of the non-canonical NF-kB pathway, and its central regulator NIK, in neovascularization associated with RA. Moreover, we have shown this method to be useful for testing inhibitors of angiogenesis and found that targeting of non-canonical NF-kB signalling to be an effective method of blocking pathological angiogenesis.

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.