Therapeutic Targeting of Neutrophil Extracellular Traps Improves Primary and Secondary Intention Wound Healing in Mice

Abstract

BackgroundNeutrophils are the first responders in wound healing after injury that mediate pro- and anti-inflammatory activities i.a. through the formation of extracellular traps (NETs). However, excessive NETs presence in wound tissue can cause local hyperinflammation and -coagulation resulting in delayed wound healing. To improve wound healing, we aimed to examine the role of NETs and DNase1 on primary and secondary wound healing.MethodsThe study included 93 C57BL/6 mice, with 3 different genotypes: wildtype, Pad4-, and DNase1-Knockout (KO). Pad4-KO mice show limited NETs formation, while DNase1-KO mice cannot disintegrate them. All 3 genotypes were included in (1) a laparotomy group and (2) a thermal injury group. Animals in both groups either received DNase1 or a vehicle i.p. post wound induction and wound assessment and euthanasia were conducted. Laparotomy and burn scars were assessed using the stony brook scar evaluation scale and modified Yeong scale respectively. Tissue was analyzed histologically using H&E staining. Ly6g, Collagen I and III, SMA, and Fibrinogen were visualized and neutrophils activation (NE, MPO) and NETs (H3cit) formation assessed.ResultsAll animals survived with no complications. DNase1 treatment led to a significantly improved scar appearance in both groups, which was also seen in Pad4-KO mice. In the laparotomy group DNase1 improved collagen deposition and fibrin concentration was significantly reduced by DNase1 treatment. Markers of neutrophil activation were significantly reduced in the treatment and Pad4-KO group. In the thermal injury group wound closure time was significantly reduced after DNase1 treatment and in the Pad4-KO group. Even though inflammation remained high in the thermal injury model over time, neutrophil activation and NETs formation were significantly reduced by DNase1 treatment compared to controls.DiscussionPrimary and secondary intention wound healing is improved by targeting NETs through DNase1 treatment or genetic KO, as assessed by wound closure time and scar appearances. Additionally, wound stability was not affected by DNASE treatment. The results suggest that overall wound healing is accelerated and DNase1 appears to be a promising option to reduce scar formation; which should be evaluated in humans.

Severity of thermal burn injury is associated with systemic neutrophil activation

“…Targeting neutrophil function in post burn injury might serve as an alternative treatment option in severe cases with exacerbated neutrophil activation. Beneficial effects of anti-NETs therapy have already been demonstrated in various neutrophil-mediated inflammatory diseases and conditions, including wound healing 60 , inflammatory arthritis, pulmonary fibrosis, inflammatory bowel disease, and sepsis 61 . Determining the full therapeutic potential of NETosis inhibition following burn trauma will merit future investigations.…”

The emerging roles of neutrophil extracellular traps in wound healing

“…The most common treatment is DNase I, which dismantles the scaffold of NET structures, and recombinant human DNase I is cost-effective with no known adverse effects. Upon systemic DNase I administration, diabetic mice show reduced wound areas, enhanced re-epithelialization, and accelerated wound healing [ 7 , 84 , 96 ]. However, DNase I has little effect on histones, elastase, or other components bound to NETs, which may be released into the bloodstream by the simple destruction of the NET scaffold, resulting in proteolytic tissue injuries [ 29 ].…”

“… H3cit and wound aera decreased [ 12 ] Tripeptide (Thr-Asp-F-amidine) Topically Wound closure and re-epithelialization accelerated [ 98 ] DNase 1 Pulmozyme 10 mg/kg i.p. Superior scar scores and wound closure time [ 96 ] Dornase alfa 10 μg i.v. 50 μg i.p.…”

Inflammatory Microenvironment of Skin Wounds

“…Neutrophils not only inhibit healing by releasing soluble mediators and excessive ROS but they also secrete particles with pro-inflammatory microRNAs (miR-23a and miR-155) that can cause tissue damage (12). Wong et al showed the deletion of key NETosis enzymes, or the inhibition of the "Nod-like receptor protein (NLRP3) inflammasome-NETs" inflammatory loop, can improve angiogenesis and accelerate wound healing (13). The neutropenia model can accelerate the speed of wound epithelial closure without changing the overall quality of the wound healing process.…”