Insights into the pathogenic roles and targeted therapy of neutrophil extracellular traps in inflammatory bowel disease

Table 1 A summary of studies revealing increased presence of neutrophil extracellular traps in inflammatory bowel disease patients and dextran sulfate sodium-induced colitis

Subjects	Sampling location	Detection method	Key findings	Region	Ref.
UC (n = 28), CD (n = 23), HCs (n = 12)	Peripheral blood	In vitro NET induction	(1) Sera from UC/CD enhanced patient/control NET formation in vitro; (2) IgG from PR3-ANCA-positive IBD enhanced control NET formation in vitro; and (3) DNase I decreased procoagulant activity in vitro	Harbin, Heilongjiang Province, China	He et al[30]
UC (n = 9), CD (n = 9), HCs (n = 12)	Colonic biopsies; peripheral blood	Immunohistochemistry, immunofluorescence, Western blot. In vitro NET induction, 3% DSS-induced colitis	(1) Over-expression of colocalized PAD4, NE, MPO, and citH3 in inflamed colon of UC; (2) IFX treatment diminishes NETs in UC; and (3) PAD4 inhibitor attenuates DSS induced-colitis in mice	Rome and Milan, Italy	Dinallo et al[31]
UC (n = 24), CD (n = 24), controls (n = 10, IC and HCs)	Peripheral blood, colonic mucosal biopsies	Immunofluorescence, ELISA, in vitro NET induction; 3.5% DSS-induced colitis	(1) Elevated cfDNA and MPO-DNA complexes in active UC or CD; (2) NET deposition in the colon, impaired NET degradation in the plasma of IBD; (3) Increased serum cf-DNA and enhanced NET formation in DSS-induced colitis in mice, attenuated by DNase perfusion or anti-Ly6G antibody; (4) NET degradation protects mice against DSS-induced colitis and colitis-associated tumorigenesis; and (5) NETs enhance the procoagulant activity in vitro. DNase blunt prothrombotic effects	Harbin, Heilongjiang Province, China	Li et al[32]
UC/CD (n = 51)	Peripheral blood	Immunofluorescence, ELISA. In vitro NET induction, 3.5% DSS-induced colitis, Western blot	(1) Increased MPO-DNA complexes in blood; (2) Enhanced NET formation in vitro; (3) NETs enhance procoagulant activity in vitro; and (4) DNase I protects against DSS-induced colitis	Harbin, Heilongjiang Province, China	Cao et al[33]
Pediatric IBD: UC (n = 6), CD (n = 6). Pediatric HCs (n = 2)	Biopsies from the small bowel and colon	Immunofluorescence	Increased, colocalized MPO, chromatin and histones, and NE in biopsies of pediatric IBD	Tel Aviv, Israel	Gottlieb et al[34]
UC (n = 14), CD (n = 11), HCs (n = 17). Others (n = 29, including 8 CA, 8 GCA and 13 IBS)	Fecal samples	Meta-proteomics: LC-MS	Increased NE, MPO, azurocidin, and cathepsin G in stool of UC and CD	Magdeburg and Hannover, Germany	Lehmann et al[35]
UC (n = 23), CD (n = 11), infectious colitis (n = 15), HCs (n = 25)	Peripheral blood neutrophils, sera, and colonic tissue	Immunofluorescence, Western blot; ELISA, PCR, flow cytometry; in vitro NET induction	(1) Increased, colocalized NE and citH3 in biopsies of active UC; (2) Increased MPO-DNA complexes in blood of UC; (3) Sera/ex vivo culture media from UC enhances control NET formation in vitro; and (4) Activation of REDD1/autophagy/NETs/IL-1β pathway in UC	Alexandroupolis, Greece	Angelidou et al[36]
UC (n = 10), HCs (n = 10)	Mucosal colon biopsies from noninflamed tissue	Proteomics (LC-MS); histology (light microscopy and confocal microscopy)	Increased lactoferrin, MPO, NE, calprotectin, neutrophil defensin 3 in colonic mucosa of UC	Silkeborg, Denmark	Bennike et al[38]

UC: Ulcerative colitis; CD: Crohn’s disease; HCs: Healthy control; IC: Indeterminate colitis; CA: Colon adenoma; GCA: Gastric carcinoma; IBS: Irritable bowel disease; IBD: Inflammatory bowel disease; NETs: Neutrophil extracellular traps; DSS: Dextran sulfate sodium; ELISA: Enzyme-linked immunosorbent assay; LC-MS: Liquid chromatograph-mass spectrometer; PCR: Polymerase chain reaction; IgG: Immunoglobulin G; PR3-ANCA: Anti-proteinase 3 antibody anti-neutrophil cytoplasmic antibody; PAD4: Peptidylarginine deiminase 4; MPO: Myeloperoxidase; NE: Neutrophil elastase; IFX: Infliximab; cfDNA: Circulating free DNA; citH3: Citrullinated histone H3; IL: Interleukin.

Table 2 Therapeutic targets: Inhibition of neutrophil extracellular traps to ameliorate progression of inflammatory bowel disease

Agent	Target molecule/function	Effect	Disease/animal model	Ref.
Cl-amidine	PAD inhibitor	Inhibit colon inflammation; stimulate apoptosis of inflammatory cells in vitro and in vivo	DSS-induced mouse model	Chumanevich et al[66]
Butyrate	Suppresses neutrophil migration and NETs production	Ameliorate mucosal inflammation	DSS-induced murine colitis	Li et al[69]
Cyclosporine A	Inhibit NETs via the regulating PPP	Reduce ROS-dependent NETs release via downregulating PPP and cellular ROS levels by decreasing G6PD activity directly by activating the P53 protein	DSS-induced colitis mouse model	Xu et al[70]
Hydroxychloroquine	Inhibit NET formation	HCQ inhibit the activation of MEK/ERK/ROS signaling pathway	DSS induced colitis	Jiang and Zhang[74]
Thymopentin	Inhibit NET formation	Reduced DAI, weight loss and colon shrinkage; decreased MPO, NE, citH3, PAD4, dsDNA	TNBS-induced colitis mouse model	Cao et al[79]
PAD4 genetic deficiency	Down-regulate cGAS-STING pathway	Inhibit NET formation, protect the intestinal barrier, alleviate intestinal inflammation	DSS-induced colitis mice	Sun et al[46]
CXCR1 and CXCR2	Promote UC and NET formation through neutrophil chemotaxis and PAD4-mediated pathways	Higher expression of CXCR1/CXCR2 in colon tissues of UC patients	UC; summary data from a genome-wide association study and FinnGen	Xv et al[71]
Hydrogen sulfide	Inhibit NET formation	Inhibit the expression of NF-κB and HMGB1; elevate the expression of anti-inflammatory mediator UCHL-1	TNBS-induced colitis rat model	Török et al[80]

PAD4: Peptidylarginine deiminase 4; NETs: Neutrophil extracellular traps; PPP: Pentose phosphate pathway; cGAS: Cyclic guanosine monophosphate-adenosine monophosphate synthase; STING: Stimulator of interferon genes; UC: Ulcerative colitis; ROS: Reactive oxygen species; G6PD: Glucose-6-phosphate dehydrogenase; HCQ: Hydroxychloroquine; ERK: Extracellular regulated protein kinases; MEK: Mitogen-activated protein kinase kinase; DAI: Disease activity index; MPO: Myeloperoxidase; NE: Neutrophil elastase; citH3: Citrullinated histone H3; dsDNA: Double-stranded DNA; NF-κB: Nuclear factor kappa-B; HMGB1: High-mobility group box 1; TNBS: 2,4,6-trinitrobenzene sulfonic acid; DSS: Dextran sulfate sodium.