Neutrophil Extracellular Traps Exacerbate Inflammatory Responses and Thrombotic Tendency in Both a Murine Colitis Model and Patients with Inflammatory Bowel Disease

Introduction: Inflammatory bowel disease (IBD) arises from a combination of genetic susceptibility and environmental factors, which trigger inappropriate mucosal inflammatory responses, the pathogenesis of which still remains exclusive. Therapeutic strategies for IBD mainly focus on controlling the active inflammation, intestinal epithelial barrier destruction and thrombotic tendency. Our group has recently demonstrated, for the first time, that patients with active IBD exhibited enhanced neutrophil extracellular traps (NETs) release, leading to a hypercoagulable state (He Z et al, Thromb Haemost 2016). However, the mechanisms by which NETs drive the pathogenesis of IBD remain unclear. The aim of this study was to further identify the novel role of NETs in the initiation and progression of IBD.

Methods: 51 consecutive patients with IBD were studied. Disease activity was assessed by using the Mayo Score (MS) for patients with (ulcerative colitis) UC and (Crohn's disease) CD. Acute disease was induced by the treatment of C57BL/6 mice with 3.5% DSS in drinking water for 6 days. For an inhibition assay, DNase I perfusion or neutrophil depletion with anti-Ly6G antibody (1A8 clone) was performed. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. ELISA was used to detect MPO-DNA complexes, TAT (thrombin-antithrombin) complexes, nucleosomes, chemokines, and cytokines.

Results: Compared to subjects with inactive UC or CD, patients with active UC or CD had significantly increased levels of cf-DNA, nucleosomes and NETs formation (MPO-DNA complexes). Neutrophils from active CD and UC demonstrated more spontaneous NET release as compared to inactive patients and controls. In DSS-induced colitis, significantly higher levels of serum cf-DNA and NETs formation were found in mice on day 4 and day 6 after DSS initiation. Western blot analysis and immunofluorescent staining of colonic tissues from mice with DSS-induced colitis showed increased NETs release and deposition. Mice treated with DNase I were protected from DSS-induced colitis, showing slighter weight loss, lower disease activity index, improved survival rate, diminished colon length shortening and decreased histologic signs of inflammation compared with controls. Furthermore, DNase I perfusion also decreased MPO levels by 62% on day 4 and by 58% on day 6, indicating DNase down-regulated neutrophil infiltration during DSS-induced colitis. The expression of Interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, CXCL2, CXCL10 and monocyte chemotactic protein-1(MCP-1) messenger RNA in colonic extracts were lower in DNase I perfused DSS-induced mice compared with saline perfused DSS-induced mice. Incubation of normal platelets with NETs from active IBD patients, but not inactive IBD patients, significantly enhanced their procoagulant activity by 32% and the ability to support fibrin formation by 42%. This effect was blocked by DNase I treatment.

Conclusions: We have extended our previous study and demonstrated that NETs constitute a central component in the initiation and progression of colitis through mediating inflammation cell infiltration, driving cytokines release and thrombotic tendency. NET degradation through DNase I perfusion protected mice from severe DSS-induced colitis. Thus, strategies focusing on the application of DNase I to accelerate the degradation of excessive NET release and deposition may offer potential therapeutic benefits for patients with inflammatory bowel disease.

Disclosure of Interest: None declared.