Background/Purpose: Reduction of adenosine deaminase 2 (ADA2) activity due to autosomal recessive loss of function mutations in the CECR1 gene results in a systemic illness known as DADA2 characterized in part by early-onset stroke, vasculitis, and clinical response to TNF-inhibitors. Adenosine, which is extracellularly degraded by ADA2, modulates inflammation via four different adenosine receptors (ARs). Neutrophils and a subset of neutrophils known as low-density granulocytes (LDGs) have been implicated in the pathogenesis of small-vessel vasculitis through the formation of neutrophil extracellular traps (NETs). The study objective was to determine whether neutrophils and NETs play a pathogenic role in DADA2.

Methods: Neutrophils and LDGs were isolated from healthy volunteers, patients with DADA2, and their family members. NETs were quantified and visualized by fluorescence microscopy. Immunofluorescence was performed against citrullinated-histone H4 and macrophage markers to detect NETs and macrophages in affected tissue from a patient with DADA2. Neutrophils were incubated with adenosine +/- ADA2 enzyme and resultant NET formation was quantified. Pharmacologic approaches were utilized to determine the specific receptor(s) and pathways that mediate NET formation by adenosine. ELISA quantified TNF-alpha release in supernatants from macrophages incubated with NETs.

Results: An abundance of circulating LDGs prone to spontaneous NET formation were observed during active disease in DADA2 and were significantly reduced after remission induction by anti-TNF therapies. Increased circulating LDGs were identified in unaffected heterozygous carriers of CECR1 mutations. In vivo evidence demonstrated NETs and macrophages in affected gastrointestinal tissue in a patient with DADA2. Adenosine triggered NET formation by engaging A1 and A3 adenosine receptors and through ROS- and PAD4- dependent pathways. Adenosine-induced NETosis was inhibited in the presence of recombinant ADA2, A1/A3 AR antagonists, or an A2A agonist. M1 macrophages incubated with NETs from patients with DADA2 released significantly increased amounts of TNF-alpha. Treatment with IL-1Ra (anakinra) or an A2A AR agonist decreased nuclear translocation of NFkB and pro-inflammatory cytokines-induced by NETs in macrophages. ­

Conclusion: Neutrophils may play a pathogenic role in DADA2. LDGs and NETs are observed during active disease. Adenosine can trigger NET formation, and deficiency of ADA2 may enhance adenosine-mediated NETosis. M1 macrophages produce TNF-alpha when exposed to NETs from patients with DADA2, potentially explaining the efficacy of anti-TNF therapies in this disease. Modulation of adenosine-mediated NET formation may constitute a novel and directed therapeutic approach in the treatment of DADA2.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophil-extracellular-traps-are-induced-by-adenosine-and-stimulate-release-of-tnf-alpha-from-macrophages-in-deficiency-of-adenosine-deaminase-2-dada2/