Neutrophil Extracellular Trap Density Increases With Increasing Histopathological Severity of Crohn's Disease

Angie L Schroder; Belal Chami; Yuyang Liu; Chloe M Doyle; Mary El Kazzi; Golo Ahlenstiel; Gulfam Ahmad; Nimalan Pathma-Nathan; Geoff Collins; James Toh; Andrew Harman; Scott Byrne; Grahame Ctercteko; Paul K Witting

doi:10.1093/ibd/izab239

Neutrophil Extracellular Trap Density Increases With Increasing Histopathological Severity of Crohn's Disease

Inflamm Bowel Dis. 2022 Mar 30;28(4):586-598. doi: 10.1093/ibd/izab239.

Authors

Angie L Schroder^{1

2}, Belal Chami^{1

2}, Yuyang Liu^{1

2}, Chloe M Doyle^{1

3}, Mary El Kazzi^{1

2}, Golo Ahlenstiel⁴, Gulfam Ahmad^{1

2}, Nimalan Pathma-Nathan^{3

5}, Geoff Collins^{3

5}, James Toh^{3

5

6}, Andrew Harman^{1

3}, Scott Byrne^{1

3}, Grahame Ctercteko^{3

5

6}, Paul K Witting^{1

2}

Affiliations

¹ The University of Sydney, School of Medical Sciences, Faculty of Medicine and Health, NSW, Australia.
² Charles Perkins Centre, The University of Sydney, NSW, Australia.
³ Westmead Institute for Medical Research, Centre for Immunology and Allergy Research, Westmead, NSW, Australia.
⁴ Western Sydney University, Westmead Clinical School and The Westmead Institute for Medical Research, Blacktown Hospital, Blacktown, NSW, Australia.
⁵ Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW,Australia.
⁶ Department of Colorectal Surgery, Westmead Hospital, NSW,Australia.

Abstract

Background: Intestinal neutrophil recruitment is a characteristic feature of the earliest stages of inflammatory bowel disease (IBD). Neutrophil elastase (NE) and myeloperoxidase (MPO) mediate the formation of neutrophil extracellular traps (NETs); NETs produce the bactericidal oxidant hypochlorous acid (HOCl), causing host tissue damage when unregulated. The project aim was to investigate the relationship between NET formation and clinical IBD in humans.

Methods: Human intestinal biopsies were collected from Crohn's disease (CD) patients, endoscopically categorized as unaffected, transitional, or diseased, and assigned a histopathological score.

Results: A significant linear correlation was identified between pathological score and cell viability (TUNEL+). Immunohistochemical analysis revealed the presence of NET markers NE, MPO, and citrullinated histone (CitH3) that increased significantly with increasing histopathological score. Diseased specimens showed greater MPO+-immunostaining than control (P < .0001) and unaffected CD (P < .0001), with transitional CD specimens also showing greater staining than controls (P < .05) and unaffected CD (P < .05). Similarly, NE+-immunostaining was elevated significantly in diseased CD than controls (P < .0001) and unaffected CD (P < .0001) and was significantly higher in transitional CD than in controls (P < .0001) and unaffected CD (P < .0001). The CitH3+-immunostaining of diseased CD was significantly higher than controls (P < .05), unaffected CD (P < .0001) and transitional CD (P < .05), with transitional CD specimens showing greater staining than unaffected CD (P < .01). Multiplex immunohistochemistry with z-stacking revealed colocalization of NE, MPO, CitH3, and DAPI (cell nuclei), confirming the NET assignment.

Conclusion: These data indicate an association between increased NET formation and CD severity, potentially due to excessive MPO-mediated HOCl production in the extracellular domain, causing host tissue damage that exacerbates CD.

Keywords: inflammatory bowel disease; myeloperoxidase; neutrophil extracellular traps.

Plain language summary

Our data show for the first time that the density of neutrophil extracellular trap formed in the bowel of Crohn’s disease patients increases with increasing disease severity, suggesting that myeloperoxidase-mediated host-tissue damage may play a role in disease pathogenesis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Crohn Disease* / pathology
Extracellular Traps* / metabolism
Histones
Humans
Neutrophil Infiltration
Neutrophils / metabolism
Peroxidase / metabolism

Substances

Histones
Peroxidase