Time- and dose-dependent inhibition of neutrophil extracellular trap formation by blocking of the interleukin-1 receptor.

Besides performing phagocytosis and degranulation, neutrophils are capable of eliminating microorganisms by releasing neutrophil extracellular traps (NETs). NET formation was found to be associated with increased mortality in sepsis. During sepsis levels of interleukin 1ß (IL-1ß), a cytokine, increases significantly and also was associated with increased mortality. Blocking of the interleukin 1 (IL-1) receptor by anakinra leads to less NET formation in gout patients. However, NET formation is crucial during infection by trapping pathogens and thereby slowing the process. Total or early blocking of cascades leading to NETs may lead to aggravation of infection in otherwise mild cases. The dose- and time-dependent effect of the IL-1 receptor antagonist anakinra was tested on spontaneous, lipopolysaccharide (LPS)-induced and phorbol-12-myristate 13-acetate (PMA)-induced formation of NETs in vitro. Quantitative detection of NETs was performed for NETspecific proteins and cell-free DNA. Immunostained microscopy imaging was used for visualization. Our study shows a dose- and time-dependent inhibitory effect of anakinra that involves the change of intracellular calcium mobilization on the formation of NETs in vitro for PMA-stimulated neutrophils but not for LPS-stimulated neutrophils. It may be useful for treatment of sepsis as part of a multimodal treatment concept, but it seems that timing and dose need to be carefully chosen.

Biliatresone induces cholangiopathy in C57BL/6J neonates.

Exposure to plant toxins or microbiota that are able to digest common food ingredients to toxic structures might be responsible for biliary atresia (BA). An isoflavonoid, biliatresone is known to effectively alter the extrahepatic bile duct (EHBD) development in BALB/c mice. Biliatresone causes a reduction of Glutathione (GSH) levels, SOX17 downregulation and is effectively countered with N-Acetyl-L-cysteine treatment in vitro. Therefore, reversing GSH-loss appears to be a promising treatment target for a translational approach. Since BALB/c mice have been described as sensitive in various models, we evaluated the toxic effect of biliatresone in robust C57BL/6J mice and confirmed its toxicity. Comparison between BALB/c and C57BL/6J mice revealed similarity in the toxic model. Affected neonates exhibited clinical symptoms of BA, such as jaundice, ascites, clay-colored stools, yellow urine and impaired weight gain. The gallbladders of jaundiced neonates were hydropic and EHBD were twisted and enlarged. Serum and histological analysis proved cholestasis. No anomalies were seen in the liver and EHBD of control animals. With our study we join a chain of evidence confirming that biliatresone is an effective agent for cross-lineage targeted alteration of the EHBD system.

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.

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.

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

Background: Neutrophils 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. Methods: The 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. Results: All 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. Discussion: Primary 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.

Markers of neutrophil activation and extracellular traps formation are predictive of appendicitis in mice and humans: a pilot study.

Appendicitis is one of the most frequent emergencies in pediatric surgery, yet current biomarkers for diagnosis are unspecific and have low predictive values. As neutrophils and extracellular traps (ETs) are an essential component of the immune defense against bacterial infections, and appendicitis is considered an inflammation reaction of the appendix, we hypothesized that neutrophil activation and NET formation play an essential role in appendicitis development and maintenance. Therefore, this pilot study aimed to establish a murine model of appendicitis and to evaluate ETs markers to diagnose appendicitis in mice and humans. The study used 20 (12 appendicitis- and 8 controls) 6-week old mice which underwent advanced appendicitis induction using a modified caecal ligation puncture procedure. During the study, cell-free DNA, neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated Histone H3 (H3cit) were assessed. Additionally, samples of 5 children with histologically confirmed appendicitis and 5 matched controls with catarrhal appendicitis, were examined for the same biomarkers. Moreover, NE, MPO, and H3cit were assessed histologically via immunofluorescence in mice and humans. All mice in the appendicitis group developed an advanced form of appendicitis with focal peritonitis. In mice and humans with appendicitis, markers of neutrophil activation and ETs formation (especially cfDNA, NE and H3cit) were significantly elevated in blood and tissue compared to controls. Ultimately, biomarkers correlated extremely well with tissue expression and thus disease severity. It appears that neutrophil activation and possibly NETs contribute to appendicitis development and biomarkers of neutrophil activation and ET formation reflect disease severity and thus could be used as biomarkers for appendicitis. However, large prospective clinical studies are needed to confirm our findings.