AB0049 NF-κB-inducing kinase regulates LTβR-DRIVEN NF-κB signaling and inflammatory activation of endothelium

Background Sites of chronic inflammation, such as rheumatoid arthritis synovial tissue, are characterized by neovascularization and often contain tertiary lymphoid structures with characteristic features of lymphoid organs such as endothelial venules (HEV), and sometimes even true germinal centers. Ligation of the lymphotoxin (LT)-β receptor (LTβR) results in activation of both canonical and NF-κB-Inducing Kinase (NIK)-dependent non-canonical NF-κB signaling in endothelial cells (ECs) and plays a crucial role in lymphoid neogenesis. Non-canonical NF-κB signaling in ECs promotes inflammation-induced angiogenesis and triggers the development of the cuboidal HEV appearance. However, the relative contribution of the individual pathways to the acquisition of leukocyte traffic-regulating properties by ECs is less well understood. Objectives To identify the molecular pathways by which LTβR drives inflammatory activation of ECs to promote interactions with leukocytes. Methods Primary human ECs were treated with LTβ or LIGHT to activate LTβR. Induction of downstream signaling pathways was assessed by western blot analysis and NF-κB transcription factor ELISA. The expression of adhesion molecules, inflammatory cytokines and chemokines, such as CXCL1, CXCL5, CXCL8 and GM-CSF in ECs was measured by RT-qPCR and cytokine antibody arrays. EC interactions with leukocytes were determined by an adhesion assay, and EC barrier integrity was assessed by a permeability assay. To repress canonical NF-κB signaling pathway, a small molecule inhibitor of IKKβ was used, and inactivation of non-canonical NF-κB signaling was achieved with siRNAs targeting NFκB2. The role of NIK in LTβR signaling was investigated using small molecule inhibitors of NIK, siRNAs targeting NIK and adenoviral vectors encoding wild type and kinase-deficient NIK. Results LTβR triggering in ECs resulted in activation of both canonical and non-canonical NF-κB signaling pathways and induced the expression of inflammatory cytokines and chemokines (CXCL1, CXCL5, CXCL8, MCP-1, GM-CSF, CCL5). Consistent with inflammatory activation of ECs, LTβR ligation also induced adhesion of immune cells to activated endothelium and increased permeability across EC monolayers. IKKβ inhibition completely repressed LTβR-induced inflammatory activation of ECs, indicating that this process was mediated through canonical NF-κB signaling. Interestingly, inactivation of NIK with small molecule inhibitors and siRNAs significantly decreased LTβR-induced expression of inflammatory cytokines and adhesion of immune cells to endothelium, whereas silencing of NFκB2 had no effect. This suggests that the non-canonical pathway is dispensable for NIK-dependent activation of endothelial cells through the canonical NF-kB pathway. Further analyses, including silencing of NIK and NIK overexpression, demonstrated a role for NIK in activation of the canonical NF-kB pathway by amplifying IKK complex activity. Conclusions These findings suggest that in addition to its pivotal role in the non-canonical pathway, NIK can serve as an amplifier of the canonical NF-κB pathway and associated inflammatory responses in ECs mediated by LTβR ligation, which may play a role in development and maintenance of chronic inflammation. Disclosure of Interest None declared