Pro-inflammatory role of extracellular nicotinamide phosphorybosiltransferase in cystic fibrosis

TITLE: Pro-inflammatory role of extracellular nicotinamide phosphoribosyltransferase in cystic fibrosis Objectives Although the recent introduction of the triple combination of Cystic Fibrosis Transmembrane Regulator (CFTR) modulators Elexacaftor/Tezacaftor/Ivacaftor (ETI) has improved Cystic Fibrosis (CF) care, the impact of ETI on CF airway epithelial cell inflammation requires further investigation. Previous studies have shown elevated Matrix Metalloproteinase 9 (MMP9) plasma levels in CF patients compared to healthy subjects. Notably, in patients responding positively to ETI, MMP9 levels significantly decreased, whereas non-responders showed no variation. Stress and inflammation are known to drive the immune cells to release extracellular Nicotinamide Phosphoribosyltransferase (eNAMPT) which act as pro-inflammatory cytokine, eventually promoting MMP9 production. This suggests a potential link between MMP9 modulation and eNAMPT activity. Our aim is to explore this relationship to identify new anti-inflammatory therapeutic targets for CF. Methods Plasma eNAMPT from CF patients and healthy controls was quantified by Enzyme-Linked Immunosorbent Assay (ELISA). Peripheral Blood Mononuclear Cells (PBMCs) from healthy donors were treated with three recombinant eNAMPT forms (wild type, H247A mutant, and K68 mutant) for 24 hours. MMP9 expression was evaluated by immunoblotting and its proteolytic activity by zymography. Results ELISA analysis showed significantly higher plasmatic eNAMPT levels in CF patients than in healthy donors. Therefore, healthy PBMCs were treated with recombinant eNAMPT: the immunoblotting analysis revealed an increase in MMP9 expression after 24 hours of treatment compared to the untreated condition. Consistently, zymography analysis confirmed enhanced MMP9 activity in the supernatants of treated PBMCs. Conclusions This study proposes, for the first time, a correlation among eNAMPT, MMP9 and CF, offering new insights into the mechanisms underlying the therapeutic responses, particularly for patients unresponsive to current CFTR-modulating therapies.