Fig. 3: The impact of neutrophils on NF-κB activity in MDM.
From: Neutrophils induce macrophage anti-inflammatory reprogramming by suppressing NF-κB activation
a LPS-induced (1 ng/ml; 20 min) p65 phosphorylation measured by (i-iv) flow cytometry (serine 529; n = 4) and (v) immunoblotting (serine 536, 529 and 276; n = 3) in MDM co-cultured with apoptotic or viable neutrophils (total time point: 30 min). b LPS-induced (1 ng/ml; 20 min) p65 nuclear translocation assessed by (i) quantitative high-content imaging and confocal microscopy (scale bars 100 μm and 7.5 μm, respectively), (ii) quantification of p65 nuclear translocation from high-content imaging (n = 8) and (iii) immunoblotting in MDM co-cultured with apoptotic and viable neutrophils (n = 3). Full images of individual stains and merged staining panels are available in supplemental data (supplemental data Fig. 1). c LPS-induced (1 ng/ml; 1 h) p65 binding to the TNF promotor in MDM co-cultured with apoptotic or viable neutrophils (n = 3). d LPS-induced (1 ng/ml; 20 min) p65 phosphorylation in alveolar macrophages (n = 3). e Phagocytosis of apoptotic neutrophils labelled with CellTracker Green and pHrodo Red by MDM at 40 min of co-culture in the presence of LPS (1 ng/ml; n = 14). f LPS-induced (1 ng/ml; 6 h) TNF release from MDM co-incubated with either apoptotic or viable neutrophils (cells), the supernatants from apoptotic or viable neutrophils cultured alone for 6 h (Sup) or the supernatants from apoptotic or viable neutrophils co-cultured with MDM for 6 h before (CoC-Sup) or after (CoC-Sup (UC) ultracentrifugation at 100,000 g for 1 h (n = 4). All n-numbers represent data derived from separate healthy donors with data plotted as mean ± s.e.m. ***P > 0.001, *P > 0.05 using paired t-test. A-N apoptotic neutrophil, V-N viable neutrophil, ser serine, Ct control, Nuc nuclear, Cyt. Ext. Ct cytoplasmic extract control, AM alveolar macrophage
