Abstract 655: Notch Inhibition Reverses the Polarization of Pro-Inflammatory (M1) to Anti-Inflammatory-like (M2) Macrophages

Abstract

Introduction: Our previous studies demonstrated that loss of Notch1 signaling reduces the incidence of abdominal aortic aneurysm (AAA) by preventing the influx of pro-inflammatory M1 macrophages and that Notch1 deficiency promotes M2-polarization. In the present study, we determined if inhibition of Notch signaling could reverse the pre-existing pro-inflammatory M1 macrophages to anti-inflammatory M2 phenotype. Methods and Results: Raw cells (264.7 murine macrophage cell line) were treated with various concentrations of LPS (5, 10, 100 ng/ml) and IFN-γ (10 ng/ml) for 3h to polarize naïve macrophages into M1-phenotype. After washing, these cells were treated with either vehicle or DAPT (10 ng/ml or 25 ng/ml); a potent Notch inhibitor for 6, 12, 24 or 48h and later used for gene expression studies or functional assays. LPS pretreatment significantly increased the gene expression of M1-genes; Il6 , Il12, Cd38, Fpr2 and iNOS , whereas the expression of M2 genes; Egr2 and c-Myc was significantly decreased. Replacement of LPS with vehicle alone lowered the expression of Il6 and Il12 within 6-12h, but remained significantly higher than basal levels. Expression of iNOS , Cd38 and Fpr2 remained unchanged in response to vehicle in the LPS-pretreated macrophages. DAPT treatment further reduced the gene expression of Il6 and Il12 significantly and iNOS moderately as compared to vehicle-treated macrophages. Interestingly, Notch inhibition increased the expression of Egr2 and c-Myc genes at 24 and 48h. Similar trends with regard to M1 and M2 genes were observed in bone marrow derived macrophages from Apoe -/- and WT mice treated with LPS followed by vehicle or DAPT. Phagocytosis assay showed increased uptake of zymosan bioparticles in the macrophages treated with LPS. Replacement of LPS with vehicle for 24h did not affect the phagocytosis significantly. DAPT treatment decreased phagocytic activity of these macrophages as compared to vehicle-treated macrophages. Conclusions: Our data suggest that Notch inhibition reverses the M1-polarized macrophages towards M2-like macrophages. These novel insights in the functions of Notch signaling may have potential implications in chronic inflammatory diseases including AAA and atherosclerosis.