NETs are present in DADA2-affected tissue and adenosine induces NET formation through NOX- and PAD-dependent pathways. (A) Hematoxylin-and-eosin (H&E) staining of small bowel tissue obtained from a patient with DADA2 shows neutrophilic infiltration in the wall of mesenteric arteries. (B) Neutrophil (arrows) infiltration in the appendix of a patient with DADA2. Red represents neutrophil elastase and blue is Hoechst. Scale bar, 100 µm. (C) Detection of NETs in a biopsy from a patient with DADA2. Red represents citrullinated histone H4 and blue is Hoechst. Scale bar, 100 µm. (D) LDGs were identified in patients with DADA2. LDGs per milliliter were significantly more abundant in patients with DADA2 during periods of disease activity (active, n = 4; remission, n = 10). LDGs from control (Ctrl; n = 2) and patients with SLE (n = 9) were used for comparison. (E) Adenosine levels were measured in plasma from patients with DADA2 (n = 9) and controls (n = 4). (F-G) Control neutrophils were incubated with different concentrations of adenosine (Ado) for 3 to 4 hours. Immunofluorescence shows that adenosine induces NET formation in control neutrophils, blue represent Hoechst; red is myeloperoxidase (MPO). Scale bar, 50 µm. Results are expressed as percentage of NETs (number of NETs/total number of neutrophils + NETs). **P < .01, Mann-Whitney U test. Results are the means ± standard error of the mean (SEM) of 4 independent experiments. (H) Control neutrophils were incubated in the presence or absence of NOX inhibitor (DPI; 5µM) or pan-PAD inhibitor (Cl-amidine, Cl-am; 20 µM) and stimulated with 16 µM adenosine (Ado). Phorbol myristate acid (PMA; 100 ng/mL) and calcium ionophore (Io; 2.5 µM) were used as positive controls. Results are expressed as percentage of NETs (number of NETs/total number of neutrophils + NETs). **P < .01, ***P < .001, Mann-Whitney U test. Results are the means ± SEM of n = 6. DMSO, dimethyl sulfoxide.