Background
Neutrophils are the most abundant immune cells and the fastest recruitment cells in infected or inflammatory sites, so they constitute the first line of immune defense of the human body. They have a variety of immune functions, including phagocytosis, production of reactive oxygen species (ROS), degranulation and formation of NETs [
1]. NETs have been originally described as the host defense mechanism that used to capture or kill pathogenic microorganisms for neutrophils. It is a kind of reticular structure where neurophils are released to extracellular after stimulation and activation. With DNA as its skeleton, it is embedded with proteins such as histone (histone, H), myeloperoxidase (myeloperoxidase, MPO), neutrophil elastase (NE), cathepsin G (CG) and protease 3 (PR3) and so on, which have bactericidal and permeability-increasing effects and some of them were modified after transformation in the process of forming NETs [
2,
3]. In fact, inhibition of NETs formation increases the sensitivity of mice and humans to bacterial infections [
4,
5]. However, increasing evidence shows that NETs also contribute to the aggravation of inflammation [
6], the occurrence of autoimmune diseases [
7], the metastasis and development of cancer [
8] and so on. Due to new detection and imaging methods, the exploration of NETs have expanded from in vitro observation to in vivo organ level, and engaged in many specific disease areas, such as systemic lupus erythematosus, vasculitis, diabetes, thrombosis and lung injury [
9‐
12]. Recent studies have found that NETs were related to intestinal diseases. On the one hand, it can prevent bacterial translocation and promote the repair of intestinal mucosal injury and plays an important role in maintaining the stability of intestinal epithelium [
13]. On the other hand, excessive NET formation can also destroy intestinal mucosal barrier function, damage intestinal epithelium, and play a key role in the pathological process of a variety of intestinal diseases [
6,
14]. Therefore, in this review, we describe the latest findings regarding NETs related to intestinal infection, intestinal inflammation, inflammatory bowel disease (IBD) and cancer and we hope to clarify the disease mechanism of drug treatment and develop new diagnosis and treatment strategies.
In 1996, Takei H first described NETs as a new type of programmed cell death, which was different from apoptosis with nuclear pyknosis and cytoplasmic vacuolization, and different from cell necrosis that maintains the integrity of nuclear membrane. It’s a special mechanism of cell lysis and death, in which neutrophils show morphological changes, nuclear membrane rupture, nuclear components released into the cytoplasm, and finally the plasma membrane broken, resulting in the formation of NETs outside the cell [
15]. In 2004, Brinkmann V redefined NETs as a microbial mechanism involved in cell death, namely activated neutrophils amplify the effectiveness of their antibacterial particles by producing a large network of DNA fibers wrapped in protein particles in a concentrated area, which contributes to forming physical barrier to prevent the spread of microorganisms. They also initially discovered that NETs may have harmful effects on the host and thereby stimulate autoimmunity, which opens a new field of neutrophil biology [
2]. In the early stage, the process of NET formation is called NETosis [
16]. In the latest expert review, it was emphasized that NETosis could not include all forms of NETs release, and it was recommended to avoid the use of the term "NETosis" or only in cases where neutrophil death was apparent, preferably using NETs formation [
17]. Like many host protection mechanisms, NETs may also be a double-edged sword that can promote or prolong innate and acquired immune responses to a variety of diseases [
6,
18]. Plenty of stimulation, such as physical and chemical stimulation [
19,
20], inflammatory cytokines such as C5a and IL-8 [
21,
22], and various pathogenic microorganisms and their derivatives [
23,
24], determine the different mechanisms of the formation of NETs.
The classical pathway of NET formation requires ROS
PMA stimulated neutrophils produce NADPH oxidase-2 (NOX-2)- dependent reactive ROS which lets neutrophils release NE referring to the nucleus, where it partially degrades specific histones and MPO, driving chromatin depolymerization independent of its enzyme activity [
25‐
27], causing nuclear DNA moving and releasing out of the cell, and forming a reticular structure [
27‐
29]. It has also been reported that some special stimulation (including immune complexes) touched neutrophils, mitochondrial ROS (rather than NOX-2-derived ROS) can drive similar NETs formation with the assistance of Ca2+ [
20,
30,
31]. ROS, whether mediated by NOX or mitochondria, seems to be essential for the formation of NETs. Those NET formation takes a long time, usually lasts for several hours or even exceeds its lifespan and continues to resist the invasion of bacteria. When it causes neutrophils death, it is called "suicidal NETosis" [
16]. Interestingly, when neutrophils are stimulated by GM-CSF, C5a or LPS, mitochondrial DNA (mtDNA), not nuclear DNA is ejected out of the cell to form NETs under ROS-dependent condition [
32]. In this process, the lifespan of neutrophils is not affected. These results give neutrophil mitochondria a new role not only as a ROS generator, but also as a provider of DNA in the process of NET formation. In addition, recent studies have shown that optic atrophy1 (OPA1), a mitochondrial inner membrane protein, is essential to the process of NET formation, and its deficiency causes dysfunction in the release of DNA from the nucleus of human neutrophils, thus unable to form NETs [
33].
Pathway of ROS-independent NET formation
Although many studies have shown that PMA stimulation can easily form ROS-dependent lytic NETs, the mice model of skin infection with
Staphylococcus aureus or
Candida albicans shows that NET formation can be rapidly formed independent of ROS [
34,
35]. In the process of the NET formation, NE translocation to the nucleus and chromatin depolymerization occur without obvious rupture of the nuclear membrane. The protein-modified chromatin was packaged into vesicles merging with the plasma membrane and nuclear DNA was released outside the cell through the vesicle transport mechanism without destroying the plasma membrane [
18,
36]. Even though how these vesicles were released is not clear, it is clear that NETs formation leads to neutrophils maintain plasma membrane integrity without affecting their lifespan, and have the ability to move, chemotaxis, and phagocytize pathogens [
37]. The process without ROS or NADPH oxidase is uniquely rapid, lasting about 5 to 60 min and requires strict supervision, which was triggered by toll-like receptors (TLR)2 or C3 complements. Importantly, NETs released without neutrophil death maintain their normal function [
35,
38]. Recent studies suggest that there is a new mechanism of NETs formation independent of ROS, that is, bacterial toxins induce pores on the membrane of host neutrophils [
39,
40]. As we all know, the NET formation is a process independent of caspase-3. Interestingly, it has been reported that NE and caspase-11 can process gasdermin D so that its pore-forming N-terminal forms pores in the nuclear and granular membranes as well as the plasma membrane. As mentioned above, it will promote NE migrate to the nucleus with the help of gasdermin D on the nuclear membrane, which may be due to the induction of NETs by calcium channel activation [
41,
42].
Several different signaling pathways have been reported to play functional roles in NET formation. The first of these pathways is that PMA, LPS and various bacteria stimulation activate protein kinase C (PKC) through TLR and G protein related receptor (GPCRs), and then phosphorylate Raf kinase and activate Raf-MEK-ERK pathway [
29,
43]. ERK phosphorylation NADPH oxidase complex and leads to the production of ROS. At the same time, neutrophils release NE destroying F-actin and MPO. ROS acts as a secondary messenger and promotes the transfer of NE and MPO from cytoplasmic granules to the nucleus, which catalyzing the interpretation of histone and leading to nuclear chromatin depolymerization [
28,
44]. During this period, it is not clear how NE translates to the nucleus. Some studies have shown that nuclear membrane disintegration mediated by CDK4/6 may play an important role in NE nuclear translocation [
45]. When neutrophils were activated, Ca2 + was released into the cytoplasm in response to the receptor agonist activating phospholipase C (PLC). PLC hydrolyzes phosphatidylinositol 4-diphosphate (PIP2) on the cell membrane to produce second messenger inositol triphosphate (IP3) and diacylglycerol (DAG), which contributes to the activation of intracellular Ca2+ and PKC respectively [
46]. In fact, Ca2+ -assisted peptidylarginine deiminase 4 (PAD4) in the cytoplasm converts the positive charged arginine into neutral citrulline in the histone, destroying the electrostatic interaction of DNA- histones, weakening the skeleton structure and stability of chromatin, and finally leading to chromatin condensation [
47]. In short, nuclear chromatin condensation can be achieved not only via post-translational modification of histone, namely PAD4-mediated histone citrullination [
48] or acetylation [
44,
49], but also NE-mediated histone hydrolysis [
27,
29,
50].
Next, the rupture of the nuclear membrane is essential for the removal of nuclear chromatin out of the cell, and is also related to the source of NETs DNA. Amulic et al. reported the involvement of nuclear lamin A/C in NET formation [
45]. They found that cyclin-dependent kinase 4 and 6 (CDK4/6) can control NET formation by regulating lamellar disintegration and rupture of nuclear membrane caused by phosphorylation of laminin A/C. In addition, some microscopic analyses showed that nuclear lamin B was also involved in the formation of NETs [
51,
52]. The phosphorylation and dissociation of lamin B mediated by protein kinase C alpha (PKC α) is the reason for the disintegration / rupture of nuclear envelope. Lamin B is extruded from the ruptured nuclear membrane with depolymerized nuclear chromatin and can be decorated on the surface of extracellular NETs [
52]. Interestingly, chromatin condensation and nuclear swelling are the main physical forces driving nuclear membrane rupture [
53], which may provide a molecular basis for lamin kinase-mediated decomposition of the nuclear layer into the disintegration of nuclear membrane protein networks. Nuclear expansion forms a physical force from the inside out, which drives the disintegration of the nuclear membrane to expand until the whole nuclear membrane breaks, squeezing out the depolymerized nuclear chromatin. Finally, neutrophils release DNA outside the cell, and various proteins with bactericidal activity are connected to the DNA skeleton to form NETs.
NETs in intestinal infection
There are many species of bacteria, fungi and several protozoan parasites associated with the induction of NET formation in the human intestinal tract [
28,
54]. There is no doubt that the defense function of NETs against the invasion of pathogenic microorganisms [
2]. However, Crane et al
. proposed that in the model of bacterial enteritis, NETs help enteropathogenic
Escherichia coli (EPEC)and Shiga-like
Escherichia coli (STEC)attach to the intestinal mucosa by enhancing the biofilm function of microorganisms [
55]. These findings hint that NETs play a dual role in intestinal infections. However, its mechanism has not been fully elucidated. Table
1 summarizes the production or changes of NETs when tissues or cells are infected by different microorganisms and the effects of their main components on tissues or cells. More studies are needed to elucidate whether the changes in NETs during infection are due to the infection itself or to experimental manipulation.
Table 1
Summary of the included studies of NETs in intestinal infection
Gram-positive bacteria (staphylococcus) | Vivo, vitro | NET formed rapidly and NETs are released from the nucleus | NETs DNA | DNase I | NETs are crucial to contain an acute invasive infection in vivo | NET formation is tightly regulated and requires both Tlr2 and C3 | |
Enteropathogenic and Shiga-Toxigenic Escherichia coli(EPEC and STEC) | Vivo | Firstly reported the formation of DNA NETs in vivo in the intestinal tract. EPEC and STEC infections stimulate the formation of extracellular DNA NETs alonely | NETs DNA | DNase I | NETs were acting as an non-antibacterial host defense. Additing DNase provided protection against the intestinal tissue damage caused by EPEC infection | | |
methicillin-resistant S. aureus(MRSA) | Vivo(WT mice,NE−/− and PAD4−/−mice | Intravenous infection with MRSA, leading to rapid a neutrophil-dependent NET formation within the liver sinusoids | ExtDNA、NE and histones | DNase I | Neutrophil recruitment and subsequent NET release destroyed the tight junction between endothelial cells and is associated with profound liver injury | But the effectiveness of DNase might be limited in terms of removal of the most dangerous NET components and advocates for inhibition of NET production | |
Afa/Dr Diffusely Adhering Escherichia coli | Vitro | NET production by PLB-985 cells infected with the Afa/Dr wild-type (WT) E. coli strain C1845 | NET-bound proteases | DNase I | NETs is actively involved in the antibacterial response of PLB-985 cells against enterovirulent WT C1845 bacteria. But PLB-985-derived NETs might directly contribute to Caco-2/TC7 epithelial cell damage via NET-bound proteases | | Marin-Esteban et al. [ 57] |
Pseudomonas aeruginosa | Vivo and vitro | P. aeruginosa induces the production of NETs in vitro and in vivo and quickly responds and defends against the DNA and histone mediated-antibacterial effects of NETs by stabilizing the outer membrane | ExtDNA | DNase I, Mg2+ and Ptase | DNA backbone of NETs contributes to their bactericidal function. But the spermidine and arn surface modifications contribute to resisting a broad range of antimicrobial components present within NETs, which may protect P. aeruginosa from NET-induced oxidative damage | Produced new immune escape mechanisms by sensing and defending against NETs | |
Nonpathogenic WT Escherichia coli and its several isogenic mutant | Vivo(Lcn KO,mpoko and Nox2KO mice) and vitro | Ent-producing Escherichia coli can NETs. Lcn2-deficient BMDNs generated more PMA-induced NETs than did WT BMDNs | | | The inhibition of neutrophil ROS and NET responses and impairs neutrophil function by enterobactin may confer a survival advantage to Ent-producing Escherichia coli | Showed the production of siderophore by E. coli and other bacteria may be a key mechanism that allows them to evade NET-mediated killing | |
Vibrio cholerae | Vivo and vitro | V. cholerae induces NET formation and degrades NETs by the activity of two extracellular nucleases Dns and Xds | ExtDNA | | Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs and enhanced survival of V. cholerae in the presence of NETs, leading to intestinal inflammation | Ecluded a new evidence that the innate immune response impacts the colonization of V. cholerae | |
Entamoeba histolytica trophozoites | Vitro | Neutrophils that were interacted with E. histolytica trophozoites released NETs. And the presence of both nuclear and mtDNA was detected. NETs were generated independently of NOX2-derived ROS | Histone H4, MPO, NE and decondensed DNA | DNase I, GSK484 and PMSF | NETs caught, immobilized and fragmented E. histolytica trophozoites | NETosis occurs rapidly and depends on the viability of amoebas. But mechanism the NETs formation triggered by this parasite and its role in protection or pathogenesis of amoebiasis not yet clarified | Ventura-Juarez et al. [ 65] DíAZ-GODíNEZ et al. [ 66] |
Candida albicans | Vivo and vitro | Both opsonized and unopsonized C. albicans induce NET formation. NET formation in peritoneal cavity after C. albicans infection | NE | BB-CI-Amidine and GSK484 | Neutrophil killing of unopsonized C. albicans requires dectin-2-mediated NET formation and the NETs restrains C. albicans spread from peritoneal cavity to kidney | Unopsonized C. albicans-induced NET formation is independent of NADPH oxidase and mitochondrial ROS and is dependent on PAD4 and dectin-2 enzymatic activity | Wu et al. [ 34] Branzk et al. [ 68] |
Aspergillus fumigatus | Vivo and vitro | Reutrophils released NETs in response to A. fumigatus hyphae or large aggregated A. fumigatus conidia but failed to form NETs in response to small single conidia | NE | AREG (inhibitor of NE) | NETs not only were irrelevant in protecting these mice against the yeast-locked hgc1ΔC. albicans strain but also were detrimental to the host when present in large amounts | Selective NETosis was independent of the expression of molecules on the surface of fungi or the enzymatic activity of fungi and was regulated only by differences in microbe size | |
NETs and bacterial clearance
NETs have the proteolytic activity of NE. In infectious inflammation, such as methicillin-resistant
Staphylococcus aureus (MRSA) infection, it has been observed that NETs can destroy the tight junction between endothelial cells and increase vascular permeability [
56]. Moreover, this experiment shows that the absence of essentia MRSA toxins still caused NETs production and the consequent liver damage, which reminds us that in the future treatment of infectious diseases should not only remove bacterial toxins but prevent NETs formation in order to completely alleviate infectious injury. Diffusely adherent
Escherichia coli expressing Afa/Dr fimbriae strain C1845 could also induce NETs to damage the F-actin cytoskeleton of human enterocyte-like cells and destroy the intestinal epithelium and that this deleterious effect is prevented by inhibition of protease release [
57]. In order to avoid the capture of NETs, bacteria also adapt to other escape mechanisms. For example,
Pseudomonas aeruginosa is more resistant to NETs than
Staphylococcus aureus or
Escherichia coli, resulting in less NET formation. There are several factors. Firstly,
P. aeruginosa is the production of a microbial secreted DNase that degrade NETs DNA, which in turn restrict NETosis through non-representational mechanisms. Alternatively, DNA can induce expression of the
arn or spermidine synthesis genes in
P. aeruginosa, which in turn protect
P. aeruginosa from NET-induced oxidative damage [
58]. It suggests that
P. aeruginosa can produce new immune escape mechanisms by sensing and defending against NETs.
Escherichia coli also inhibit neutrophils producing ROS, form NETs by producing enterobactin (Ent), which is a catecholamine iron carrier used to isolate intracellular iron and unstable iron pools in neutrophils [
59], which means the production of siderophore by
E. coli and other bacteria may be a key mechanism that allows them to evade NET-mediated killing. In addition, the culture of intestinal bacteria in mice infected with
Citrobacter rotarius showed that different bacterial subsets also had the ability to mobilize neutrophil oxidative outbreaks and initiate NET formation [
60].
Staphylococcus aureus induces NETs by recruiting neutrophils during sepsis, and these NETs bind firmly to the hepatic sinusoids through histone-vWF interactions [
35]. In 2013, the interaction between
Vibrio cholerae and NETs was reported for the first time. It indicated that
Vibrio cholerae can induce NET formation when it comes into contact with neutrophils. In turn,
Vibrio cholerae secretes two extracellular nucleases Dns and Xds to rapidly degrade the DNA component of NETs in order to avoid and adapt to the presence of NETs. In other words, Dns and Xds mediate the escape of
Vibrio cholerae from NETs and reduce the extracellular activity of NETs [
61].
NETs and intestinal amebae and fungal infection
Neutrophils are important host effector cells against amebae lysis parasites (
Echinococcus histolytica trophozoites) [
62,
63]. It was demonstrated that neutrophils activated by TNF-a and IFN-c were able to kill
E. histolytica trophozoites, with MPO binding to the trophozoite plasma membrane and killing these invaders [
64]. The part that NETs play in this process is not yet known. In 2016, Ventura-Juarez et al
. for the first time identified that in vitro
, while in direct encounter with
Echinococcus histolytica trophozoites, neutrophils lost their circular morphology and integrity and variable length NET formation was observed which trapped, immobilized and fragmented
E. lysis trophozoites. After neutrophils were pretreated with deoxyribonuclease I (DNase I), despite the fact that the nuclei of neutrophils contained histones, MPO and concentrated chromatin, they did not release NETs and the
E. histolytica trophozoites did not show any damage, which indicates that released NETs from neutrophils have amebae killing effect [
65]. In addition, it was demonstrated quantitatively that neutrophils treated with amebae trophozoites not only rapidly form NETs but also emerge with the simultaneous presence of nuclear and mtDNA. It is of interest that the formation of NETs was also found to be dependent on amebae activity, as heat-inactivated or paraformaldehyde-fixed amebae failed to induce NETs and, more interestingly, no ROS production was detected during neutrophil-amebae interaction, implying that amebae -induced NETs production is non-ROS-dependent [
66].
The action of neutrophils on fungi bears a strong resemblance to that of amebae, using NETs to capture and destroy mycelium that cannot be engulfed by phagocytes [
67].
Candida albicans hyphae have the capacity to trigger the formation of NETs, which are then trapped and killed by NETs, whereby the antifungal activity of NETs is mediated by calprotectin [
68,
69]. Similarly,
Aspergillus fumigatus mycelium can provoke the formation of NETs, which trap and inhibit fungal growth, possibly due to deprivation of the essential nutrient Zn2 + required by the fungus. However, in this case, NETs are not sufficient to kill Aspergillus fumigatus, as the NETs-mediated growth inhibition is eliminated by the addition of Zn2 + [
70].
Candida albicans were NADPH oxidase-dependent. However, in a model of Candida albicans peritonitis, Wu et al
. found a pathway for NETs independent of NADPH oxidase and similar to the chemically activated pathway, and they also discovered that Dectin-2-mediated PAD4-dependent NET formation in vivo prevented the spread of
Candida albicans from the peritoneal cavity to the kidney [
34,
71]. Nevertheless, PAD4 also seems not always to be required for NETs formation, and Guiducci et al
. found that PAD4 is not required for antifungal immunity in mucosal and systemic
Candida albicans infections, despite the fact that
Candida albicans readily induces PAD4-dependent histone citrullination of neutrophils [
72]. While most research on NETs and fungal-associated diseases has focused on
candidiasis and
fumonis, recent studies have illustrated that NETs also act in other fungal infections. For example, NETs can be seen in corneal scrapings from patients with fungal keratitis, a vision-threatening infection caused by a variety of fungi, including
Aspergillus,
Fusarium,
Candida and
Streptomyces [
73]. The same applies in the case of
A. fumigatus conidia infection [
72]. In addition, it was recently reported that in a model of DSS-induced leaky gut lupus, intestinal fungi boosted the production of NETs, causing intestinal translocation of organic molecules and synergistically exacerbating the activity of lupus [
73].
Large pathogens such as fungi and amebae activate the release of NETs from neutrophils in a similar signaling pathway, while at the same time the pathway of NETs release varies depending on the infecting pathogen, which may be relevant for clinical cure. Yet, more mechanisms of NETs release in response to amoebic and fungal infections are poorly understood and need to be further explored in the future.
NETs and intestinal cancer
A growing number of research has shown that tumor cells and tumor microenvironments stimulate neutrophils and induces the release of NETs from various cancer types [
125‐
127]. Neutrophils are well-known mediators in tumor biology, but their role in solid tumors has been redefined by NETs. NETs have recently been detected in specimens from six different human solid tumors, including colorectal cancer (CRC), and they showed substantial individual differences in tissue density and distribution, and it was concluded that NETs were positively correlated with IL-8 and negatively correlated with tumor-infiltrating CD8 + lymphocytes [
128]. Given that platelet-derived poly P drives the release of NETs from neutrophils, et al. used biopsies of adenomas, hyperplastic polyps, IBD and healthy colon tissue were as a control study and found that in CRC, CD68 + mast cells expressing Poly P are one of the factors that stimulate the release of NETs from neutrophils, and mast cells with detectable CD68 + poly P expression could represent a potential prognostic marker for colorectal adenoma and/or carcinoma [
129].
With the growing number of more studies, the mechanisms related to NETs their actions on tumor tissues are slowly starting to be revealed, including direct effects to the cancer cells and changes in the tumor microenvironment, such as promotion of tumor growth [
130], promotion of metastasis [
131], awakening from a dormant state [
19,
132], and promotion of escape of cytotoxic immune cells [
133,
134]. Recent studies have indicated that NETs are involved in the entire invasion-metastasis cascade of tumors [
135]. NE released from NETs promotes further acceleration of colorectal tumor growth by upregulating PGC-1 through activating TLR-4 in cancer cells and enhancing mitochondrial biosynthesis [
136]. In LPS-injected CRC mice, cancer cells probably foster the NET formation by TLR9 and mitochondria-activated protein kinase signaling pathways, and the analysis of clinical data from CRC patients showed a striking relationship between the NET formation and the rate of metastasis and survival [
137]. CRC cells may translocate mutated KRAS to neutrophils via exons, thereby boosting the NET formation by modifying IL-8 and ultimately leading to CRC aggravation [
138].
In CRC models in mice, NETs are formed extensively and depletion or inhibition of NET formation can considerably lower the amount of tumor metastasis [
135]. Feedback regulation between elevated IL-8 and NETs in CRC can promote liver metastasis in CRC [
139], and NETs-associated CEACAM1 can also serve as a potential therapeutic target for the prevention of colon cancer metastasis [
140]. NETs exert a major action in colon cancer intraperitoneal metastasis via regulation of colon cancer cell migration and adhesion to extracellular matrix proteins [
138]. Using a clinical mouse model of colon cancer combined with in vivo video microscopy, Rayes et al
. confirmed that NETs facilitate the adhesion of circulating tumor cells (CTCs) to the lung and liver, thus functionally contributing to metastatic progression, whereas blocking NET formation by multiple measures markedly inhibits spontaneous metastasis [
125]. Low-density lipoproteins foster the retention of CTCs via NETs and suppress T cell-mediated antitumor responses in target organs, hence prompting postoperative tumor metastasis [
141]. Ample evidence shows that certain cancer cells have a high organismal preference for colonization and metastasis to certain distant organs, with colon cancer cells more prone to metastasize to the liver and lung [
142]. Liver metastases in patients with colon cancer are rich in NETs, which usually corresponds to the metastatic organ tropism of colorectal cancer [
143]. This was most likely connected to the NET-DNA receptor-transmembrane protein CCDC25 on the surface of cancer cells, which senses extracellular DNA and thus initiates the ILK-β-parvin pathway to enhance the motility of cancer cells. As we know, DNase I alters the function of NETs by cleaving the DNA strand. Xia et al
. established a mouse model of CRC liver metastasis using an adeno-associated virus (AAV) gene therapy vector that specifically expresses DNase I in the liver, which in turn proved that AAV-mediated DNase I gene transfer can be a safe and effective way to curb liver metastasis [
144], hinting at new therapies for CRC.
Cancer-related thrombosis is strongly linked to poor prognosis, and patients with CRC are generally at higher risk of suffering from venous thrombosis, yet the exact mechanism remains unknown. It has been shown that platelets in CRC patients stimulate neutrophils to produce NETs, which can be inhibited by depletion of HMGB1, and that the level of NETs in the blood of CRC patients increases in parallel with cancer progression, leading to a shortened clotting time and a significant increase in thrombo-antithrombotic complexes and fibrin fibrils, compared to healthy subjects. Interestingly, when exposed to NETs from CRC patients, endothelial cells were also converted to a procoagulant phenotype. This finding reveals a complex interaction between neutrophils, platelets and endothelial cells [
145]. As well, tumor development and hypercoagulation was also found to be related to neutrophils in a mouse model of small intestinal tumors [
146]. Finding that intestinal tumorigenesis is associated with aggregation of low-density neutrophils, which have a pre-tumorigenic N2 phenotype and spontaneous NETs formation, and that elevated circulating lipopolysaccharide induces upregulation of complement C3a receptors on neutrophils and activation of the complement cascade, which consequently leads to NETs division, inducing coagulation and N2 polarization, thus promoting tumorigenesis. It lays the foundation for a new link between tumorigenic hypercoagulation, increased NETs and complement activation, thereby providing a favorable explanation for the promotion of tumor development by blood coagulation [
147]. We therefore consider that NETs potentially offer new therapeutic targets for preventing the risk of thrombosis in patients with CRC.
In addition, high levels of NETs are linked to a poorer prognosis of cancer. High levels of NETs in the blood of patients with colorectal cancer were correlated with postoperative complications and tumor recurrence rates [
135,
148,
149]. Patients with metastatic colorectal cancer have elevated NETs in tumor tissue, and greater preoperative serum MPO-dsDNA levels resulted in shorter survival time [
136]. Richardson et al
. identified a novel neutrophil phenotype in patients undergoing CRC resection, showing reduced forming of NETs, reduced apoptosis, and increased phagocytosis. In other words, the accumulation of neutrophils in the circulation as a result of damaged cell death may be of potential harm to the postoperative host and an early phenotypic switch may be desirable [
150]. However, the role of NETs for tumors is not restricted to only promoting tumor growth and metastasis; Arelaki et al
. obtained tumor tissue sections and metastatic lymph nodes from ten patients with colon adenocarcinoma and found that TF-bearing-NETs and neutrophil localization were evident, with a gradual decline in neutrophil infiltration and NETs concentration from the tumor center to the distal margins. Interestingly, NETs created in vitro impeded cancer cell growth by inducing apoptosis and/or inhibiting proliferation [
151].
Above findings showed that NETs are available as biomarkers to guide clinical diagnosis and treatment, and to assess the prognosis of cancer patients, and NETs will emerge as a new target for treatment and intervention of intestinal cancers. Table
3 summarizes the major effect of the studies describing NETs in CRC.
Table 3
Summary the major effect of the studies describing NETs in CRC
In vivo and in vitro | Murine colorectal (MC38) cells, HCT116, Hepa1-6, and Huh7 cell lines | Increased citrullinated histones and circulating MPO-DNA levels were related to poor survival of CRC patients | NETs can directly alter the metabolic programming of cancer cells to increase tumor growth and shorter survival time | |
In vivo, in vitro nd ex vivo | Human colorectal cell line HCT116 or luciferase-labeled HCT116 cells | TLR9 and the mitogen-activated protein kinase signaling pathway | LPS-induced formation of NETs in promoting the development of tumors and metastasis | |
In vitro and Ex vivo | Human acute myeloid leukemia (AML) cells, Caco-2 cells | – | Confirmed presence of NETs within the primary tumor sites of CRC and gradually dispersed to the tumor boundary, particularly to nearby metastatic lymph nodes | |
In vivo and in vitro | DKs-8(WT allele) cells and DKO-1 (KRASmutant)cells | Exoxsomes from KRAS mutant CRC increase IL-8 production and provoke NET formation | Released NETs increase CRC cells growth | |
In vivo, in vitro,and Ex vivo | Human hepatocellular carcinoma, human cell line HT29, mice cell line MC38 | Elevated tumorous interleukin (IL)-8 expression triggered by NETs and overproduced IL-8 in turn activate neutrophils towards NETs formation | Increased NETs boosted tumorous proliferation and invasion and contributed to onset of CRC liver metastasis | |
In vivo and in vitro | Human colon carcinoma cell line (HT-29), murine colon carcinoma subline with low CEACAM1 expression (MC38CC1-),murine colon carcinoma subline stably transfected with CEACAM1 long isoform[MC38CC1L] | NET-associated carcinoembryonic Ag cell adhesion molecule 1 (CEACAM1) as an essential element for this interaction | NETs can promote colon carcinoma cell adhesion, migration and metastasis | |
In vivo and in vitro | Murine Lewis Lung carcinoma cell subline H59, Murine colon carcinoma cell line MC38 | Primary colon cancer cells provoked NETs generation | Prime adhesion of CTCs to the liver and degradation of NETs decreased CRC cell adhesion and spontaneous metastasis to the liver and lung | |
In vivo and Ex vivo | human colon cancer cell line HCT116, | The transmembrane protein CCDC25 as a NET-DNA receptor on cancer cells that senses extracellular DNA and subsequently activates the ILK-β-parvin pathway to enhance cell motility | A transmembrane DNA receptor that mediates NET-dependent metastasis | |
In vivo and in vitro | Human hepatoma cell line HepG2, murine colon carcinoma MC38 | Neutrophil infiltration and NET formation reduced by adeno-associated virus (AAV) based DNase I gene therapy | Reduced liver metastasis | |
Ex vivo | Human umbilical vein endothelial cells (HUVECs) | platelets from CRC patients stimulated healthy neutrophils to extrude NETs, which could be inhibited by the depletion of HMGB1 | NETs induce the procoagulant activity PCA and promote hypercoagulable state in CRC | Zhang et al. [142] |
In vivo, in vitro,and Ex vivo | MC38 and Luciferase-expressing MC38 cells (MC38/Luc) | NET triggered HMGB1 release and activated TLR9-dependent pathways | NETs further fuel cancer cells adhesion, proliferation, migration, and invasionthe and reduce more than fourfold disease free survival | |
Ex Vitro | Systemic neutrophils were isolated from human | | Adverse patient outcomes were associated with increased preoperative NETs production | |
Conclusion and future direction
NETs, a double-edged sword in which neutrophils exert immunomodulatory effects, are involved in the occurrences of various diseases, especially intestinal diseases. On the one hand, the production of NETs by neutrophils prevents pathogenic microbial invasion and reduces intestinal damage caused by intestinal inflammation, and on the other hand, pharmacological inhibition of NET formation reduces tumor metastasis and IBD occurrence. Hence, as with any immunomodulatory approach, balancing the favorable and unfavorable aspects of NETs formation in each specific situation will be critical, and further exploration and understanding of the regulation and balance of NETs induction, inhibition, and degradation of NETs on pharmacological targets of intestinal disease without compromising the patient's immune defenses is imperative. While multiple methods for detecting NETs are available, there are no uniform criteria to directly define the occurrence of NETs, and in the future, identification of markers and other methods to assess the forming of NETs in vivo as biomarkers and targets for therapeutic interventions in different gut-related diseases is essential. Moreover, more signaling pathways and major regulators of NETs are required to be explored in clinical practice in the future so that we can benefit
more from their regulation and thus protect the intestine from damage and carcinogenesis.
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