Background/Purpose: Endothelial cells (EC) are important contributors to inflammation via expression of inflammatory mediators, including cytokines, 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. In this study, we investigated the effects of factors produced by activated memory T (T_m) 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 presence of anti-CD3 and anti-CD28 for 72h, after which supernatant was harvested. Endothelial cells were stimulated for 72h with 50% T_m supernatant (T_m sup) after which protein and RNA was harvested followed by analysis of NF-κB signalling and downstream expression of inflammatory mediators using qPCR and western blot. Culture supernatants were analysed by ELISA for various 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 in the EC cultures.

Results: Stimulation with T_m sup led to activation of both canonical NF-κB signalling (increased levels of phosphorylated IκBα) and noncanonical NF-κB signalling (increased p100 to p52 processing). After stimulation with T_m sup EC had increased mRNA levels of all tested inflammatory mediators compared to non-treated cells. Gene expression of chemokines, cytokines, and growth factors (CXCL1, CXCL5, IL6, IL8 and GM-CSF) in T_m sup stimulated EC  was significantly reduced after treatment with iIKKβ and to a lesser, but still significant, extent after treatment with iNIK. Treatment with iIKKβ also led to a reduction in mRNA levels of the adhesion molecules VCAM-1 and ICAM-1, while this effect was less pronounced after iNIK treatment. Of note, treatment with either IKKβ or iNIK led to a significant reduction of CXCL5 in the culture supernatant of T_m sup stimulated EC. Results of bioinformatic analysis of RNA-sequencing derived data are pending.

Conclusion: This study provides new insights into the cellular interactions leading to production of inflammatory mediators by EC. Our findings demonstrate that activated T_m cells produce factors that can cause NF-κB-dependent inflammatory activation of EC. Targeting canonical NF-κB signaling via IKKβ or NIK-dependent NF-κB signaling reduces inflammatory activation of the endothelium and may be a potential novel therapeutic target.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/activated-memory-t-cells-produce-ligands-that-cause-nf-%ce%bab-dependent-inflammatory-activation-of-the-endothelium-identification-of-novel-therapeutic-targets/