Neutrophil extracellular traps and DNases orchestrate formation of peritoneal adhesions.

Peritoneal adhesions are poorly understood but highly prevalent conditions that can cause intestinal obstruction and pelvic pain requiring surgery. While there is consensus that stress-induced inflammation triggers peritoneal adhesions, the molecular processes of their formation still remain elusive. We performed murine models and analyzed human samples to monitor the formation of adhesions and the treatment with DNases. Various molecular analyses were used to evaluate the adhesions. The experimental peritoneal adhesions of the murine models and biopsy material from humans are largely based on neutrophil extracellular traps (NETs). Treatment with DNASE1 (Dornase alfa) and the human DNASE1L3 analog (NTR-10), significantly reduced peritoneal adhesions in experimental models. We conclude that NETs serve as essential scaffold for the formation of adhesions; DNases interfere with this process. Herein, we show that therapeutic application of DNases can be employed to prevent the formation of murine peritoneal adhesions. If this can be translated into the human situation requires clinical studies.

Murine scald models characterize the role of neutrophils and neutrophil extracellular traps in severe burns.

Introduction: Severe burns cause unique pathophysiological alterations especially on the immune system. A murine scald model was optimized as a basis for the understanding of immunological reactions in response to heat induced injury. The understanding of the roles of neutrophil extracellular traps (NETs) and DNases will support the development of new surgical or pharmacological strategies for the therapy of severe burns. Methods: We studied C57BL/6 mice (n=30) and employed four scalding protocols with varying exposure times to hot water. An additional scald group with a shorter observational time was generated to reduce mortality and study the very early phase of pathophysiology. At 24h or 72h, blood was drawn and tissue (wound, liver, lung, spleen) was analyzed for the presence of NETs, oxidative stress, apoptosis, bacterial translocation, and extracellular matrix re-organization. In addition, we analyzed the transcriptome from lung and liver tissues. Results: Exposure to hot water for 7s led to significant systemic and local effects and caused considerable late mortality. Therefore, we used an observation time of 24h in this groups. To study later phases of burns (72h) an exposure time of 6s is optimal. Both conditions led to significant disorganization of collagen, increased oxidative stress, NET formation (by immunodetection of H3cit, NE, MPO), apoptosis (cC3) and alterations of the levels of DNase1 and DNase1L3. Transcriptome analysis revealed remarkable alterations in genes involved in acute phase signaling, cell cohesion, extracellular matrix organization, and immune response. Conclusion: We identified two scald models that allow the analysis of early (24h) or late (72h) severe burn effects, thereby generating reproducible and standardized scald injuries. The study elucidated the important involvement of neutrophil activity and the role of NETs in burns. Extensive transcriptome analysis characterized the acute phase and tissue remodeling pathways involved in the process of healing and may serve as crucial basis for future in-depth studies.

Markers of neutrophil activation and extracellular trap formation predict appendicitis.

BACKGROUND: Although appendicitis is one of the most frequently occurring pediatric surgery emergencies, current biomarkers for diagnosis are unspecific and have low predictive values. Neutrophils are an essential component of the innate immune system involved during appendicitis. Thus, the current study aimed to evaluate neutrophils and their activation markers in a prospective cohort study. METHODS: The study population included all children with acute abdominal pain who presented to the pediatric surgery department of 2 large clinics between July 2018 and December 2019. All enrolled subjects underwent blood sample collection with an assessment of white blood cell count, C-reactive protein, cell-free DNA, neutrophil elastase, myeloperoxidase, and citrullinated histone H3. If an appendectomy was performed, the appendix was stained for myeloperoxidase, neutrophil elastase, and citrullinated histone H3 using immunofluorescence. RESULTS: In total, 198 subjects were included in the study, of whom 133 had histological verified appendicitis. In those with appendicitis, white blood cell count and C-reactive protein showed a moderate diagnostic value for (noncomplicated and complicated) appendicitis. However, cell-free DNA (area under the curve .87) and citrullinated histone H3 (area under the curve .88) demonstrated excellent predictive power for appendicitis. Most notably, citrullinated histone H3 was able to distinguish (1) noncomplicated from complicated appendicitis, and (2) predict patient outcome. Moreover, the examined biomarkers appear to reflect tissue expression and disease severity. CONCLUSION: Markers of neutrophil activation and extracellular trap formation are excellent biomarkers for appendicitis. In particular, citrullinated histone H3 may be used to identify children with an increased risk of developing complications after appendicitis.