NETs promote NF-κB nuclear translocation and stimulation of TNF-α release from macrophages. (A) Neutrophils and TNF-α were detected in tissue from a patient with DADA2. Red represents elastase; green is TNF-α; and blue is Hoechst. Scale bar, 100 µm. Control macrophages were incubated in presence of spontaneously generated NETs from LDGs or normal dense neutrophils from DADA2 patients or lipopolysaccharide (LPS) for 45 minutes to 2 hours. (B-C) Translocation of the NF-κB subunit p65 was assessed by immunofluorescence and quantified. LPS was used as positive control. Scale bar, 10 µm. (D) Levels of TNF-α in supernatants from control and DADA2 macrophages incubated with NETs for 48 hours were analyzed by enzyme-linked immunosorbent assay (ELISA). *P < .05, **P < .01, ****P < .0001, Mann-Whitney U test. Results are the means ± SEM of 6 independent experiments. (E) Macrophages and NETs detected in appendix tissue from a patient with DADA2. Red represents citrullinated histone H4; green is a marker for macrophages; and blue is Hoechst. Scale bar, 10 µm.