LAUSR

Career situation of first and presenting author Student for a master or a PhD. Introduction Endothelial cells (EC) are important contributors to inflammation via expression of inflammatory mediators, including chemokines and adhesion molecules. Production of these inflammatory mediators can be induced via canonical and NF-κB-inducing kinase (NIK)-dependent noncanonical NF-κB signalling. The ligands activating these pathways are well studied, but less is known about the cells producing ligands that can activate NF-κB signalling in EC. Objectives To study the effects of soluble factors produced by activated memory T (Tm) cells on NF-κB dependent inflammatory activation of EC. Methods CD4+CD45RO+ Memory T cells were isolated from healthy PBMC using MACS sorting and cultured in medium containing anti-CD3 and anti-CD28 for 72 hour, after which supernatant was harvest. Human umbilical cord EC (HUVEC) were stimulated with 50% Tm supernatant (Tm sup). After 72 hour of Tm sup stimulation HUVEC protein and RNA was harvest and NF-κB signalling and downstream expression of inflammatory mediators was analysed using qPCR and Western Blot. Culture supernatant was analysed by ELISA to detect presence of inflammatory mediators. To repress canonical NF-κB signalling an inhibitor of IKKβ (iIKKβ) was used and to repress NIK-dependent NF-κB signaling an inhibitor of NIK (iNIK) was used. Results Stimulation with Tm sup led to activation of both canonical NF-κB signalling, indicated by increased levels of phosphorylated (p)-IκBα, and noncanonical NF-κB signalling, indicated by increased p100 to p52 processing. HUVEC stimulated with Tm sup had increased mRNA levels of all tested inflammatory mediators compared to non-treated cells. Gene expression of chemokines (CXCL1, CXCL5, IL6, IL8 and GM-CSF) after Tm sup stimulation was significantly reduced after treatment with iIKKβ and to a lesser, but still significant, extent after treatment with iNIK. Interestingly, treatment with iIKKβ also led to a reduction in mRNA levels of the adhesion molecules VCAM-1 and ICAM-1, while this effect was minimal after iNIK treatment. In addition, treatment with either IKKβ or iNIK led to a significant reduction in CXCL5 in the culture supernatant of HUVEC stimulated with Tm sup. Conclusions This study provides new insights into the cellular interactions leading to production of inflammatory mediators by EC. Our findings demonstrate that activated Tm cells factors produce factors that can cause NF-κB-dependent inflammatory activation of EC. Targeting canonical NF-κB signaling and, although to a lesser extent, NIK-dependent NF-κB signaling reduces inflammatory activation of the endothelium. Disclosure of Interest None declared.