Abstract 109: IL-1ß-Induced Ceramide Synthesis Accentuates NETosis and Inflammation in Abdominal Aortic Aneurysms

Abstract

Objective: We have previously shown that circulating neutrophils are required for experimental aortic aneurysm (AA) formation via a non-MMP2/9-mediated mechanism. In other experiments we documented that IL-1β signaling is required for AA formation. Therefore, we hypothesized that increased levels of IL-1β in AA triggers inflammatory ceramide synthesis and thus extracellular trap (NET) formation (NETosis) by neutrophils, thereby augmenting inflammation. Methods: Immunohistochemical techniques were used to identify neutrophils and NETs. Ceramides from human AA and control aortas from transplant donors were quantified using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Neutrophils isolated from human blood were treated with IL-1β to determine if IL-1β induces ceramide synthesis and NETosis in vitro. Experimental AAs were induced by elastase perfusion in C57BL/6 wild-type (WT) and IL-1β knock-out (KO) mice and cellular content of AA was analyzed using flow cytometry. Groups were compared using two-tailed unpaired t-test. Results: Immunohistochemical staining showed that neutrophils were scattered or grouped near lymphoid-like structures in the adventitia of human AA. Three-dimensional confocal imaging showed NETs in the adventitia of human AA. Importantly, the levels of long chain ceramides C16 and C24:1 were higher (2.6 and 1.8-fold, respectively), whereas, the levels of short chain ceramides C4, C6 and C8 were lower (4.3, 3.9 and 3.5-fold, respectively) in human AA compared to controls. In isolated neutrophils, IL-1β treatment induced synthesis of long chain ceramides and discharge of NETs. Neutrophils and NETs were also detected in aortic media at day 7 after induction of AA in WT mice. On day 7 following elastase perfusion, despite the increase in number of circulating immune cells, AA size was smaller (91.08±11.7% vs 53.8±5.4%, p=0.03), and, infiltration of neutrophils (12179±2697 vs 3046±628, p=0.01) and B cells was lower in the aorta of IL-1β KO mice compared to WT mice. Conclusions: These data document for the first time the presence of NETs in human AAs. Furthermore, the data suggests that neutrophils augment inflammation in AA by increasing pro-inflammatory long chain ceramide synthesis and NETosis in response to IL-1β.