Neutrophil extracellular traps and DNases orchestrate formation of peritoneal adhesions

Abstract

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.

Keywords: Biological sciences; Cell biology; Health sciences; Immunology.

Graphical abstract

Figure 1

The absence of DNases precipitates the formation of adhesions (A and B) Animals with targeted deletion of Dnase1 and even more Dnase1l3 displayed significantly higher Leach and Nair adhesion scores than controls. In mice with PAD4-KO adhesions were reduced compared to controls (C) Adhesions are reflected by significantly increased thickness of the parietal peritoneum in the area of the peritoneal injury. (D) Representative HE images of DNases knockout mice compared to controls and shams. (E) Representative images of abdominal adhesions in the DNases knockout mice compared to controls and shams. I is marking the intestine. Data shown as mean ± SD. Statistics: ANOVA with Dunnett’s correction or Kruskal-Wallis test with Dunn’s correction.

Figure 2

DNASE1L3 affects wound healing by inducing intensive collagen deposition (A and E) DNases appear not to affect SMA in short- and long-term which is a marker of wound contractility. (B, C, F, G, and I) Collagen 1:3 ratio and collagen alignment are used to access wound maturation. In Dnase1l3 knockout mice very high levels of collagen I and III were to be found. Even after three weeks, the collagen fibrils remained aligned in parallel. This pattern is often associated with an immature wound and fibrosis. (D and H) Fibrin remained the same in all groups. Data shown as mean ± SD. Statistics: Kruskal-Wallis test with Dunn’s correction.

Figure 3

Topical NTR-10 significantly reduces the formation of adhesions (A‒C) The Leach and Near score were used to access the formation of adhesions at day 21. The most effective treatment option was NTR-10. (D and E) The thickness of the parietal peritoneum was significantly reduced in mice that received topical DNase treatment when compared to controls. (E) Representative images of the peritoneal thickness. I is marking the intestine. Data shown as mean ± SD. Statistics: ANOVA with Dunnett’s correction or Kruskal-Wallis test with Dunn’s correction.

Figure 4

Dornase alfa reduces the formation of adhesions but maintains wound healing To test various typical clinical scenarios, mice were subjected to anastomosis of the small intestine (A) deserositation (B) and thermal injury (C) Treatment with dornase alfa prevented the formation of adhesions in all three scenarios almost completely. Additionally, it did not affect the rate of incision hernia, suture insufficiency, peritonitis, or mortality but rather improved it. Data shown as mean ± SD. Statistics: Kruskal-Wallis test with Dunn’s correction.

Figure 5

Human adhesions show NETs, DNase1l3, CCR2, and CD68 expression (A) Paraffin section of a human adhesion shows expression of the characteristic NET proteins myeloperoxidase (MPO, red) and neutrophil elastase (NE, green). (B) Isotype control for MPO and NE from (A). (C) HE staining of the same human adhesion. (D) Human adhesion also shows expression of DNase1l3 (red). (E) Isotype control for the staining of DNase1l3 in (D). (F and H) the monocyte marker CCR2 (red) and the macrophage marker CD68 (red) are also expressed in human adhesions. (G and I) control for the staining of CCR2 and CD68, respectively. (A,B,D,E, F, G, H, I) DNA was counterstained using DAPI. The size bar represents 100 μm.

Figure 6

Human adhesions contain post-translationally modified fibrin High resolution mass spectrometry analysis with subsequent peptide and post-translational modification analysis was performed on surgical samples of human adhesions. (A) An unmodified peptide containing the thrombin cleavage site was only detected in one sample and corresponded to the thrombin cleavage site in the beta chain as indicated by the blue square. (B) Peptides identified corresponding to the fibrinogen beta chain in different samples of human adhesions. These peptides were either unmodified (w/o, blue box), oxidized (ox, orange box), citrullinated (cit, yellow box), or carbamylated (carb, gray box). The cleavage sites for thrombin are marked in red, and the major cleavage sites for plasmin in green. Note that the adhesions contain many peptides that had been oxidized, citrullinated, and carbamylated posttranscriptionally. Many of these are clustered near the plasmin cleavage sites. ∗ samples were fixed and subjected to antigen retrieval before mass spectrometry analyses. No major differences between fixed and unfixed samples were observed in the peptide analysis.