Neutrophil Extracellular Traps in Pediatric Inflammatory Bowel Disease: A Potential Role in Ulcerative Colitis

Abstract

Inflammatory bowel disease (IBD), encompassing Crohn's disease (CD) and ulcerative colitis (UC), is a chronic inflammatory condition of the gut affecting both adults and children. Neutrophil extracellular traps (NETs) are structures released by activated neutrophils, potentially contributing to tissue damage in various diseases. This study aimed to explore the presence and role of NETs in pediatric IBD. We compared intestinal biopsies and peripheral blood from 20 pediatric IBD patients (UC and CD) to controls. Biopsy staining and techniques for neutrophil activation were used to assess neutrophil infiltration and NET formation. We also measured the enzymatic activity of key NET proteins and evaluated NET formation in UC patients in remission. Both UC and CD biopsies showed significantly higher levels of neutrophils and NETs compared to controls (p < 0.01), with UC exhibiting the strongest association. Peripheral blood neutrophils from UC patients at diagnosis displayed increased NET formation compared to controls and CD patients. Interestingly, NET formation normalized in UC patients following remission-inducing treatment. This pilot study suggests a potential role for NETs in pediatric IBD, particularly UC. These findings warrant further investigation into the mechanisms of NET involvement and the potential for targeting NET formation as a therapeutic strategy.

Keywords: Crohn’s disease; inflammatory bowel disease; neutrophil; neutrophil extracellular traps (NETs); prognostic factor; ulcerative colitis.

Figure 1

Elevated neutrophils and NETs in pediatric IBD tissue samples. (A) The number of neutrophils and (B) NET formation was significantly elevated in both UC (N = 9) and CD (N = 11) compared to controls (N = 9) (*** p < 0.001, ** p < 0.01). (C) Representative images of NETs in the tissue samples of patients with UC and CD. The tissues were stained with NE (green) and H3 (red). NET-forming cells are identified by their high H3 signal and NE co-localization (yellow). Scale bar: 50 μm. On the right bottom panel, there is a higher magnification of the selected area (marked by dashed lines), scale bar: 20 μm (right).

Figure 2

Neutrophil infiltration and NET formation in inflamed vs. non-inflamed intestinal tissue. (A,B) Significantly higher levels of infiltrating neutrophils were demonstrated in the inflamed intestine of UC (A) and CD (B) compared to the non-inflamed intestine (* p < 0.05, ** p < 0.01). (C,D) Significantly higher levels of NET formation were demonstrated in the inflamed intestine of UC (C) and CD (D) compared to the non-inflamed tissue (*** p < 0.001).

Figure 3

NET formation in inflamed vs. non-inflamed intestinal tissue. Representative images of NETs in the inflamed intestine of UC and CD, compared to the non-inflamed tissue. Tissues were stained with NE (green) and H3 (red). NET-forming cells are identified by their high H3 signal and NE co-localization (yellow). Scale bar: 50 μm. On the right bottom panel, there is a higher magnification of the selected area (marked by dashed lines), scale bar: 20 μm (right).

Figure 4

Increased NET formation by neutrophils from pediatric UC patients. (A) Neutrophils isolated from patients with UC at diagnosis displayed significantly elevated NET formation upon stimulation with PMA compared to both healthy controls and CD patients (** p < 0.01 and * p < 0.05, respectively). (B,C) Enzymatic activity of neutrophil elastase (NE) and myeloperoxidase (MPO) in UC and CD patients at diagnosis showed no significant difference compared to controls. (D) Representative images of NET formation in UC at diagnosis. Cells were stained with Sytox Green (green) and Hoechst 33342 (blue) dyes to identify decondensed chromatin. White arrows indicate NET-forming cells. Scale bar: 50 μm.

Figure 5

Normalization of NET formation in UC with immunomodulatory therapy. (A) Neutrophils from UC patients in remission displayed significantly lower NET formation upon stimulation compared to UC patients at diagnosis (** p < 0.01 and * p < 0.05, respectively). NET formation in remission was comparable to healthy controls (p = 0.962, ns—not significant). (B,C) Enzymatic activity of neutrophil elastase (NE) and myeloperoxidase (MPO) showed no significant difference between UC patients in remission, at diagnosis, and healthy controls. (D) Representative images confirm lower NET formation in UC patients in remission compared to diagnosis. Cells were stained with Sytox Green (green) and Hoechst 33342 (blue) dyes to identify decondensed chromatin. White arrows indicate NET-forming cells. Scale bar: 50 μm.