Neutrophil-dependent Mitochondrial DNA Release Associated With Extracellular Trap Formation in Inflammatory Bowel Disease

Background and Aims: Inflammatory bowel disease (IBD) is associated with increased circulating damage-associated molecular patterns, in particular, the highly pro-inflammatory mitochondrial DNA (mtDNA). Here, we study the importance of blood neutrophils in mtDNA release via neutrophil extracellular trap (NET) formation and mitochondrial NETosis, where neutrophils specifically expulse mtDNA as potential targetable biological pathways. Methods: We investigated the roles of A23187 (a known NET stimulant), granulocyte macrophage stimulating factor, lipopolysaccharide (LPS), and human IBD plasma in their ability to induce NET formation, mitochondrial NETosis, mtDNA, and total DNA release from human blood neutrophils; and the evidence for increased NET formation in IBD. Results: We demonstrated that NET formation resulted in significant DNA (P < .0001) and mtDNA release (P < .0001) with long DNA fragments (>1000 base pairs) with NETs containing high levels of mtDNA. Using previously described in vitro conditions for mitochondrial NETosis, granulocyte macrophage stimulating factor + LPS triggered neutrophil mtDNA release at lower levels but not NETosis. LPS alone can trigger neutrophilic DNA release without NET formation. Heterologous coculture with plasma from patients with active IBD (vs remission [n = 6/group]) were not associated with significantly higher levels of NETs and mtDNA release. During coculture with active IBD plasma (vs remission), citrullinated histone 3 (CitH3) (a NETs biomarker) levels were significantly lower (P < .001). Similarly, CitH3 levels were lower in stool supernatants of patients with active IBD vs remission (n = 19/12, P = .0001). Stool CitH3 negatively correlates with stool calprotectin, a biomarker for gut inflammation (r = −0.47, P = .03). Conclusion: Hence, although blood neutrophils remain an important source of circulating mtDNA with defined mechanisms for release via NET formation and during neutrophil activation, our data do not support excessive systemic NET formation as a dominant underpinning pathobiological process in IBD.