Neutrophil Extracellular Traps: Inflammation and Biomaterial Preconditioning for Tissue Engineering

Abstract

Tissue injury initiates a tissue repair program, characterized by acute inflammation and recruitment of immune cells, dominated by neutrophils. Neutrophils prevent infection in the injured tissue through multiple effector functions, including the production of reactive oxygen species, the release of granules, the phagocytosis of invaders, and the extrusion of neutrophil extracellular traps (NETs). However, these canonical protective mechanisms can also have detrimental effects both in the context of infection and in response to sterile injuries. Of particular interest to biomaterials and tissue engineering is the release of NETs, which are extracellular structures composed of decondensed chromatin and various toxic nuclear and granular components. These structures and their dysregulated release can cause collateral tissue damage, uncontrolled inflammation, and fibrosis and prevent the neutrophil from exerting its prohealing functions. This review discusses our knowledge of NETs, including their composition and morphology, signaling pathways, inhibitors, and contribution to inflammatory pathologies, as well as their role in the resolution of inflammation. In addition, we summarize what is known about the release of NETs as a preconditioning event in the response to biomaterials and highlight future considerations to target the neutrophil response and enhance biomaterial-guided tissue repair and regeneration.

Impact statement

Neutrophil extracellular trap (NET) release is an active process programmed into the neutrophil's molecular machinery to prevent infection. However, the release of NETs on biomaterials appears to be a significant preconditioning event that influences the potential for tissue healing with largely detrimental consequences. Given their contribution to inflammatory pathologies, this review highlights the role of NETs in the response to biomaterials. Together, the studies discussed in this review suggest that biomaterials should be designed to regulate NET release to avoid maladaptive immune responses and improve the therapeutic potential of tissue-engineered biomaterials and their applications in the clinical setting.

Introduction

An injury to a tissue or organ initiates a tissue repair program that is initially characterized by inflammation and the local recruitment and accumulation of immune cells at the wounded site.¹ Independent of injury location, neutrophils are rapidly recruited to the damaged area and dominate the acute response. The early and robust recruitment of neutrophils are essential for preventing pathogen dissemination when barriers such as the skin or mucosa are damaged because their multiple effector functions efficiently kill bacteria. These effector functions include the production of reactive oxygen species (ROS), the release of granules, the phagocytosis of invaders, and the extrusion of neutrophil extracellular traps (NETs).² NETs, which are extracellular structures composed of decondensed chromatin and various nuclear and granular components, were initially characterized in the context of host defense as a mechanism to locally concentrate the noxious antimicrobial cargo of the neutrophil and kill trapped pathogens.³ However, it is now well established that the recruitment of neutrophils and the release of NETs occur both in the context of infection and in response to sterile injuries,^4,5 such as the implantation of a biomaterial or tissue-engineered construct.^6–8 Moreover, dysregulated neutrophil recruitment and NET release can cause collateral tissue damage and uncontrolled inflammation, which are particularly detrimental to biomaterial-guided tissue regeneration and repair. This review discusses our knowledge of NETs and their role in inflammatory pathologies, including cancer, thrombosis, and fibrosis, and the resolution of inflammation. In addition, we discuss what is known about NETs in response to biomaterials and highlight future considerations for immunomodulatory biomaterial design to control the neutrophil response and enhance tissue repair and regeneration.

The Biology of NETs

The composition and morphology of NETs

NETs are extracellular structures composed of a physical network of DNA nanofibers that are decorated by bound proteins derived from several compartments within the neutrophil. Although an initial description included only 25 proteins,⁹ more recent proteomic analyses have identified nearly 700 proteins within NETs with the vast majority implicated in autoimmunity and inflammatory disorders.¹⁰ In addition, most investigations into the protein cargo of NETs indicate that the proteomic profiles change slightly depending on the stimuli and whether neutrophils are used from healthy donors or a disease state.^10–13 Therefore, while some differences likely exist when NETs are released in response to biomaterials, the most abundant proteins in NETs substantially overlap independent of disease state or stimulus, suggesting their relevance in biomaterial-induced NET release. In this review, we present a nonexhaustive list of the most important NET components to consider for tissue repair and regeneration (Table 1) and recommend that interested readers reference the proteomic studies for a complete list of NET components.^{9–11, 13–15}

Table 1.

Neutrophil Extracellular Trap-Bound Protein Cargo and Their Significance in Tissue Engineering

Protein	Significance in tissue engineering
Cytoskeletal
β-actin^9–12,15	DAMP that activates some immune cells¹⁶ Physiologic inhibitor of DNase^17,18
Vimentin^10,11,13,15	Proinflammatory stimulus susceptible to citrullination¹⁹ Stabilizes activated platelets complexed with plasminogen activator inhibitor 1²⁰ Exacerbates epithelial damage²¹
Cytosolic
PAD4¹³	Citrullinates proteins leading to inflammatory responses¹¹
S100-A8 protein^9–11,15	DAMP that activates some immune cells and endothelial cells²² Proinflammatory stimulus that triggers neutrophil chemotaxis and adhesion²³
S100-A9 protein^9–11,15	DAMP that activates some immune cells and endothelial cells²² Proinflammatory stimulus that triggers neutrophil chemotaxis and adhesion²³
S100-A12 protein^9,10,15	DAMP that activates some immune cells and endothelial cells²² Proinflammatory stimulus that triggers neutrophil chemotaxis and adhesion²³
Granular
Cathepsin G^9,10,13,15	Activates MMPs²⁴ Promotes platelet activation and aggregation²⁵
MMP-8^11,13	Degrades ECM components²⁶
MMP-9^10,11,13,15	Degrades ECM components²⁶
MPO^9,10,12,15	Generates ROS²⁷
NE^10,11,13,15	NE-DNA complexes inactivate plasminogen²⁸ Degrades all ECM components²⁹ Activates MMPs³⁰
NGAL^10,11,13	Stabilizes MMP-9 to enhance activity³¹
Nuclear
Histone 2A^9–11,13,15	DAMP that activates proinflammatory responses from immune cells³²
Histone 2B^9–12,15	DAMP that activates proinflammatory responses from immune cells³²
Histone 3^9–11,13,15	DAMP that activates proinflammatory responses from immune cells³²
Histone 4^9–12,15	DAMP that activates proinflammatory responses from immune cells³²
HMGB1^10,13	Induces NET release and proinflammatory responses⁴
Non-neutrophilic
Tissue factor	Promotes thrombin generation³³ Contributes to thrombosis through thrombin generation^5,34

DAMP, damage-associated molecular pattern; ECM, extracellular matrix; HMGB1, high-mobility group box 1; MMP, matrix metalloproteinase; MPO, myeloperoxidase; NET, neutrophil extracellular trap; NGAL, neutrophil gelatinase-associated lipocalin; PAD4, peptidyl arginine deiminase 4; ROS, reactive oxygen species.

The composition of NET-derived proteins can be grouped into four major categories: cytoskeletal proteins, cytosolic proteins, nuclear proteins, and granular proteins. Of the cytoskeletal proteins found in NETs, actin and vimentin are significant in tissue healing. Actin is an evolutionarily conserved damage-associated molecular pattern (DAMP) recognized by some immune cells¹⁶ that act as a physiologic inhibitor of DNase,^17,18 which can degrade NETs. Therefore, the presence of actin in NETs can contribute to immune activation and the resistance of NETs to degradation, leading to aberrant NET accumulation and collateral tissue damage.¹² Similarly, vimentin, which can be secreted by activated neutrophils³⁵ and macrophages,³⁶ can promote proinflammatory responses and is susceptible to citrullination, an enzymatic modification intimately tied to neutrophil-driven autoimmune disorders and NET release.¹⁹ Moreover, it can stabilize activated platelets complexed with plasminogen activator inhibitor 1 and exacerbate epithelial damage,^20,21 hindering tissue healing.

Of the array of cytosolic proteins found in NETs, the S100 proteins and peptidyl arginine deiminase 4 (PAD4) can have a significant impact on tissue healing for biomaterial applications. The S100 proteins, a set of calcium-binding proteins, have a broad range of functions in inflammation and immune homeostasis.³⁷ When they are released into the extracellular space, S100 proteins function as DAMPs to activate immune cells and endothelial cells.²² S100A8 and S100A9, together known as calprotectin,³⁸ and S100A12 are abundantly expressed in neutrophils and bind to Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) to initiate proinflammatory responses implicated in chronic neutrophil-driven inflammation.^23,39 Likewise, PAD4 is an enzyme that not only citrullinates histones to facilitate chromatin unraveling during NET release⁴⁰ but also citrullinates other extracellular proteins, contributing to autoantibody generation in inflammatory pathologies.^11,41 Therefore, while an inflammatory response is necessary for tissue healing, the highly localized expression of S100 proteins and PAD4 on NETs can lead to overactivation of an inflammatory response and drive chronic responses rather than resolution.

Majority of the NET protein cargo are granular proteins with diverse enzymatic activity.¹⁰ The two granular proteins commonly associated with NET formation are NE and myeloperoxidase (MPO). Most of the proteolytic activity of NETs is attributed to NE,¹⁵ which can target and degrade all extracellular matrix (ECM) components²⁹ and activate matrix metalloproteinases (MMPs).³⁰ NET-bound MPO on the other hand exerts its affects through the generation of ROS,²⁷ and elevated levels are associated with poor prognosis in several cardiovascular disease states.⁴² Additional granular proteins found in NETs include MMP-8 and MMP-9, which can also contribute to ECM degradation,²⁶ and neutrophil gelatinase-associated lipocalin (NGAL), the stabilizer of MMP-9.^31,43 The serine protease cathepsin G is also found in NETs and can process MMPs into their active form²⁴ and promote platelet activation and aggregation.²⁵ Together, this milieu of granular proteins contributes to NET-driven inflammation through the excessive degradation of the ECM and activation of proinflammatory signals.

NETs also contain proteins of nuclear origin, including histones and chromatin-bound proteins. Histones are of particular importance in inflammation and tissue healing because they are recognized as DAMPs, leading to proinflammatory responses from immune cells.³² Moreover, histones have been shown to directly induce epithelial and endothelial cell death that contributes to tissue destruction.⁴⁴ In addition, nonhistone nuclear protein high-mobility group box 1 (HMGB1) is also a DAMP with diverse functions in inflammation and immunity.⁴⁵ NET-bound HMGB1 has been shown to aggravate systemic lupus erythematosus,⁴⁶ potentially participate in acute gouty inflammation,⁴⁷ and induce NET release and proinflammatory responses in liver injury.⁴ As such, these NET-bound nuclear proteins participate in the regulation of the inflammatory microenvironment with predominantly proinflammatory effects.

Besides proteins from defined neutrophil compartments, proteins that are not of neutrophil origin can also bind to NETs and play an important role in the response to NETs in tissue healing. The most noted example in literature is tissue factor, the primary initiator of the extrinsic coagulation cascade. Although some reports suggested that neutrophils can synthesize and secrete tissue factor,^34,48 more recent proteomics studies did not find tissue factor bound to NETs isolated from ultrapure populations of neutrophils,^{9–11, 13–15} suggesting that NET-bound tissue factor is not of neutrophil origin. Nonetheless, NET-bound tissue factor promotes thrombin generation³³ and has been shown to contribute to thrombosis in acute myocardial infarction as well as in antineutrophil cytoplasmic antibody-associated vasculitis.^5,34 Therefore, the ability of NETs to bind active tissue factor could be especially detrimental to biomaterial applications with blood-contacting biomaterials.

The diverse NET-bound proteins concentrate in NETs based on their interaction with the charged DNA fibers, but the source of DNA from within the neutrophil is subject to debate. NET formation was originally described by the release of nuclear chromatin,³ but since the initial description, an additional type of NETs characterized by the release of mitochondrial DNA has been described.^49,50 In one example, mitochondrial NETs were released from viable neutrophils after priming with granulocyte-macrophage colony-stimulating factor and stimulation of TLR4 or complement factor 5a receptor.⁴⁹ Eosinophils are known to release mitochondrial DNA through a similar mechanism.⁵¹ In another example, mitochondrial NET release was identified after traumatic injury and trauma surgery.⁵⁰ Most neutrophil biologists now accept that NETs can be composed of nuclear or mitochondrial DNA, which may impact the NET protein cargo, but the physiologic and mechanistic distinction remains unclear.⁵² As such, there is potential that both nuclear and mitochondrial NETs are released in response to biomaterials, but this has yet to be specifically investigated. In our work, when abundant NETs are observed on electrospun biomaterials, there is an absence of intact nuclei, suggesting the NETs are primarily of nuclear origin.^8,53

NETs are typically described as having a cloud-like morphology that can grow beyond the size of a neutrophil (Fig. 1). However, the morphology can vary between cloud-like structures and elongated fibrous extrusions, but these differences are attributed to agitation during sample preparation rather than physiologically relevant, morphological differences.⁵² The cloud-like morphology of NETs is likely related to their indiscriminate release from the cell, thought to be driven by entropic chromatin swelling, which has been observed both in vitro and in vivo using live microscopy.^12,54–56 On our electrospun biomaterials imaged by fluorescence microscopy, NETs appear to randomly cover the surface of the biomaterial, which others have similarly observed.^7,8,57 Given their volume and ability to cover surfaces, it is easy to imagine that the extensively decorated DNA fibers can precondition a proinflammatory environment when released in response to stimuli other than invading pathogens.

FIG. 1.

Fluorescent micrographs of NETs on electrospun biomaterials. Although they appear two dimensional when viewed from (A) the top in epifluorescence microscopy, NETs are 3D structures with height and volume, which is evident when viewed in (B) a Z-stack of fluorescent micrographs. In these images, human neutrophils were cultured on the electrospun biomaterial for 3 h. Extracellular chromatin is stained red with STYOX orange, cell membrane is green with Alexa Fluor™ 488-conjugated wheat germ agglutinin, and nuclei are blue with DAPI. (A) Scale bar is 100 μm. (B) Scale bar in the z-axis is 55 μm. 3D, three dimensional; NET, neutrophil extracellular trap.

Stimuli and pathways of NET release

The release of NETs is a distinct, coordinated process that requires sequential steps to facilitate chromatin unraveling and ultimately the extrusion of NETs.⁵⁸ During nuclear NET release, the nuclear and granular membranes disintegrate, the nucleus decondenses, and the DNA mixes with cytoplasmic and granular components. The process culminates with rupture of the cell membrane and release of the protein-decorated NET into the extracellular space. However, the signaling pathways and involvement of critical enzymes, such as NADPH oxidase 2 (NOX2) production of ROS to free NE and MPO from granules,⁵⁹ activation of nuclear factor kappa B (NF-κB),⁶⁰ and PAD4 citrullination of histones to facilitate chromatin unraveling,⁴⁰ vary greatly depending on the stimuli acting on the neutrophil. In addition, the kinetics of NET release have been reported to vary from 10 min to 24 h and are also stimuli dependent,⁶¹ which further emphasizes the importance of studying NET release within a specific disease state or lack thereof. Multiple nonphysiological and physiological infectious, inflammatory, and immunologic stimuli are documented in the literature to trigger in vitro and in vivo NET release. In this review, we highlight the stimuli and known signaling pathways most relevant for biomaterial and tissue engineering applications (Fig. 2). Interested readers should reference Hoppenbrouwers et al. for a thorough list of stimuli, their studied concentration ranges, and their impact on NET release with corresponding references.⁶¹

FIG. 2.

Stimuli and signaling pathways of NET release most relevant for biomaterials and tissue engineering applications. Question marks indicate pathways that have not been extensively investigated. PMA is the most widely used nonphysiological NET stimulus requiring PKC, NOX2, and PAD4 for NET release.^54,62 LPS, which is relevant in the context of infection, signals through JNK and also requires NOX2 and PAD4.⁶³ The signaling pathways for IL-8, IL-1β, and TNF-α are not as well studied, but recent data indicate they do not require NOX2 for NET release.^64,65 Data suggest IL-8-induced NET release requires calcium flux,⁶⁶ whereas TNF-α signals through TAK1.⁶⁵ Activated platelets can prime neutrophils for NET release through binding of P-selectin to PSGL-1 and directly stimulate NET release through HMGB1 binding to RAGE, although the signaling pathway has not been elucidated.^67–69 Biomaterials can also induce NET release, but the receptors and signaling pathways have yet to be evaluated and likely vary depending on the type of biomaterial. Finally, requirements for NF-κB activation and mobilization of NE and MPO are dependent on the stimuli, but have been found to participate in NET release.^60,70–73 CXCR2, CXC chemokine receptor 2; HMGB1, high-mobility group box 1; IL-1R1, interleukin 1 receptor type 1; IL-8, interleukin 8; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa B; NOX2, NADPH oxidase 2; PAD4, peptidyl arginine deiminase 4; PKC, protein kinase C; PMA, phorbol-12-myristate 13-acetate; PSGL-1, P-selectin glycoprotein ligand 1; RAGE, receptor for advanced glycation end products; TAK1, transforming growth factor beta-activated kinase 1; TNF-α, tumor necrosis factor alpha; TNFR, tumor necrosis factor receptor.

The most frequently used NET stimulus for in vitro studies is phorbol-12-myristate 13-acetate (PMA), a plant-derived compound that bypasses receptor engagement and actives protein kinase C (PKC) to initiate its signaling cascade.^3,11,74 Downstream of PKC, PMA-induced NET release requires NOX2 production of ROS and may activate PAD4.^54,62 The benefit of PMA as an in vitro stimulus is that it consistently triggers NET formation with reported 100% efficacy across a broad range of concentrations.⁶¹ However, the detriment of PMA is that it is not physiologically relevant since it does not play a role in in vivo processes. Therefore, it is now recognized that the use of PMA as an in vitro stimulus for NET formation should be approached with caution, especially in the context of biomaterials and the tissue repair program. If the goal is to study the neutrophil response during acute inflammation in an in vitro model or to evaluate potential inhibitors of NET release, PMA should not be used as a stimulus since it does not play a physiological role in acute inflammation. Alternatively, if the goal is to stimulate maximum NET release for the purpose of quantifying NET formation in vitro, PMA may be an appropriate positive control. For example, our group uses PMA to represent 100% NET formation in our in vitro assay for quantification of biomaterial-induced NETs, and we express our results as a percentage of NET release relative to PMA.^75,76

Similar to PMA, lipopolysaccharide (LPS) is widely used as an in vitro NET stimulus and may or may not be appropriate in the context of biomaterials and tissue regeneration.^{3,11,62,77,78} LPS has been shown to signal through TLR4 and c-Jun N-terminal kinase (JNK), although one group has demonstrated a TLR4-independent mechanism, and requires NOX2 production of ROS and PAD4 for NET release.^63,75,79 Derived from the outer membrane of Gram-negative bacteria, LPS is composed of a lipid A component, a core containing an oligosaccharide, and an O-antigen that is the major basis for bacterial serotyping.⁸⁰ Thus, if NET release is being studied in an infected tissue environment, LPS may be an appropriate physiological control for assessing the efficacy of inhibitors. Unfortunately, contradicting data exist in the literature with some groups reporting LPS and neutrophil interactions trigger NET release, while others report that they do not.^77,81,82 These different results can be primarily attributed to variability of the O-antigen between different types of LPS, which has been found to selectively regulate NET formation as a consequence of bacteria serotyping.⁷⁹ Moreover, the same study illustrated differential regulation in vitro under serum- and platelet-free conditions, indicating that both the type of LPS and culture conditions should be carefully considered when studying LPS-induced NET release.

In addition, several proinflammatory cytokines and chemokines are efficient inducers of NET release and are important to consider for biomaterial-guided tissue regeneration. Interleukin 8 (IL-8), the archetypal neutrophil chemoattractant secreted by damaged cells and neutrophils, has been shown to stimulate NET release at low concentrations, but has no effect at high concentrations.^{3,58,64,82,83} Likewise, interleukin 1 beta (IL-1β) is released during acute tissue injury and can exacerbate tissue damage, which can be partially attributed to its ability to stimulate NET release.^48,84,85 Tumor necrosis factor alpha (TNF-α) is also rapidly released after tissue injury to enhance immune cell recruitment and stimulates NET formation.^64,85,86 While the signaling pathways for these proinflammatory mediators have been debated, recent data suggest that IL-8, IL-1β, and TNF-α signal through their respective receptors and trigger NET release in an ROS-independent manner.^64,65 Given their involvement in acute inflammation, IL-8, IL-1β, and TNF-α should be considered important NET stimuli in tissue regeneration applications.

Activated platelets are a source of multiple growth factors and inflammatory mediators, which can activate neutrophils and trigger the release of NETs.^67,68,87 Alternatively, resting platelets cannot stimulate NET release,⁸¹ suggesting that the upregulated expression of certain platelet receptors after activation and secretion of growth factors and inflammatory mediators is critical. In fact, platelet P-selectin has been found to prime neutrophils for NET release through binding to P-selectin glycoprotein ligand 1 (PSGL-1),⁶⁷ and platelet presentation of HMGB1 after activation also stimulates NET formation through RAGE in a NOX2-independent mechanism.^68,69 The ability of activated platelets to stimulate NET release is intriguing for biomaterial-guided tissue regeneration since platelet-rich plasma and platelet-derived growth factors have been found to resolve inflammation and enhance tissue healing in multiple tissue engineering studies.⁸⁸ It is possible that the high concentration of growth factors within platelet-rich plasma overcomes the negative consequences of NET release in these scenarios, but this has not been investigated.

The last stimulator of NET release that is important for the application of biomaterials to tissue engineering and regeneration is the biomaterial itself. Our group and others have shown that a range of biomaterials stimulate NET formation, including electrospun biomaterials,^8,76 titanium,⁵⁷ carbon and polystyrene nanoparticles,⁸⁹ Teflon™,⁹⁰ and a tissue-engineered trachea.⁶ The stimulation of NET release is likely intimately related to the surface-adsorbed proteins on the biomaterial, which can vary greatly based on the surface chemistry and topography of the biomaterial.⁹¹ Because investigation of NETs on biomaterials is a new consideration in tissue engineering, much work remains to determine variability between different types of biomaterials and the signaling pathways involved. Nonetheless, no matter the stimuli, exact kinetics, or signaling pathways, acute NET release is a significant preconditioning event occurring early in the tissue repair program that modulates the potential for tissue healing.

Inhibitors of NET release

Similar to the stimuli triggering NET formation, multiple inhibitors can be used to block the release of NETs and their efficacy is dependent on the stimuli and signaling pathway. Because of the diversity of signaling pathways, it is unlikely that inhibition of one pathway abrogates all NET formation. Nonetheless, several common pharmacological targets (Table 2) exist due to their recurring involvement in NET release. While a plethora of inhibitors have been used specifically for elucidating the signaling pathways in NET release, in this study, we focus on the targets and inhibitors of NET release with physiological implications.

Table 2.

Common Targets and Inhibitors of Neutrophil Extracellular Trap Release

Target	Inhibitor	Application
PAD4	Cl-amidine	Protects against cardiac dysfunction in myocardial infarction⁹²
		Reduces vascular damage in lupus⁹³
		Suppresses colitis⁹⁴
		Reduces arthritis⁹⁵
		Reduces biomaterial-induced NET release⁵³
	GSK484	Reduces ionomycin and Staphylococcus aureus-induced NET release⁹⁶
ROS	Tempol	Reduces NET release induced by Candida albicans⁹⁷
	TSG-6	Suppresses LPS-induced NET release⁹⁸
	Luteolin	Suppresses arthritis⁹⁹
	Kaempferol	Reduces tumor metastasis¹⁰⁰
	Manuka honey	Reduces biomaterial-induced NET release⁷⁶
Other targets
NE	GW311616A	Reduces tumor metastasis⁷⁰
	Sivelestat	Rescues endotoxic shock⁷¹
	Secretory leukocyte protease inhibitor	Inhibits TNF-α and S. aureus-induced NET release¹⁰¹
MPO	Anti-MPO antibody	Reduces NET release in response to influenza virus primed epithelial cells⁷²
	AZM198	Attenuates endothelial damage⁷³
NF-κB	Acetylsalicylic acid	Inhibits TNF-α-induced NET release⁶⁰
	BAY 11-7082	Inhibits TNF-α-induced NET release⁶⁰
	Ro 106-9920	Inhibits TNF-α-induced NET release⁶⁰
JAK	Tofacitinib	Reduces vascular damage¹⁰²
Calcineurin pathway	Ascomycin	Attenuates IL-8-induced NET release⁶⁶
	Cyclosporine A	Attenuates IL-8-induced NET release⁶⁶
Autophagy	Chloroquine	Reduces hypercoagulability in cancer¹⁰³
	Chloroquine	Decreases severity of acute pancreatitis¹⁰⁴
Receptors	Anti-Mac-1 antibody	Inhibits LPS-induced NET release¹⁰⁵
	Anti-Mac-1 antibody	Inhibits NET release in response to immobilized immune complexes¹⁰⁶
	Anti-FcγRIIIb antibody	Inhibits NET release in response to immobilized immune complexes¹⁰⁶
	Anti-FcγRIIa antibody	Inhibits NET release in response to endocytosed immune complexes¹⁰⁷
	Roflumilast	Reduces IL-8-, fMLP-, and C5a-induced NET release on fibrinogen surfaces¹⁰⁸

Applications mentioning NET release were evaluated in vitro, whereas applications mentioning a physiological impact were evaluated in vivo.

FcγR, fragment crystallizable gamma receptor; IL-8, interleukin 8; JAK, Janus kinase; LPS, lipopolysaccharide; MPO, myeloperoxidase; NF-κB, nuclear factor kappa B; TNF-α, tumor necrosis factor alpha; TSG-6, tumor necrosis factor-stimulated gene 6.

The most frequently targeted enzyme for NET inhibition is PAD4 because its activity was initially described as essential for chromatin decondensation during NET release.^109,110 Widely studied inhibitors of PAD4 include Cl-amidine, a general irreversible inhibitor of PADs,^65,109–111 and GSK484, a selective reversible inhibitor of PAD4.^65,92,96 Both have been used successfully in vitro and in vivo to attenuate NET release in several pathologies.^93–95,112 Our group has even incorporated Cl-amidine into electrospun biomaterials to modulate biomaterial-induced NET release and showed that its local delivery could improve tissue integration and regeneration.⁵³ However, since NET release can occur independent of PAD4 as previously discussed,^113,114 PAD4 may not be a ubiquitous target for NET inhibition.

Downregulation of ROS has also been evaluated for blocking NET release since seminal studies in NET release used PMA, a powerful activator of NOX2 and ROS. Most studies have used ROS scavengers to quench ROS activity generated from various superoxide-generating enzymes. Examples include Tempol, a stable redox-cycling drug,⁹⁷ the anti-inflammatory protein secreted by stem cells, tumor necrosis factor-stimulated gene 6 (TSG-6),⁹⁸ and natural flavones and flavonoids.^99,100 Recently, we used Manuka honey, which contains multiple flavones and flavonoids, to attenuate electrospun biomaterial-induced NET release.⁷⁶ Nonetheless, the requirement for ROS in NET release is also debated, and recent data indicate that ROS is likely not essential for NET formation, but contributes to the process,^65,79,114 which explains why ROS inhibitors may indirectly modulate NET release.

A variety of other signaling molecules and enzymes are involved in NET release and has been targeted to inhibit NET formation. Because most of the proteolytic activity of NETs is attributed to NE, several groups have evaluated inhibitors of NE.^62,70,71,101 Likewise, antibodies or inhibitors of MPO have been investigated due to the contribution of MPO to NET release and tissue damage.^72,73,115 Additional targets include NF-κB,⁶⁰ Janus kinase (JAK),¹⁰² the calcineurin pathway,⁶⁶ and autophagy.^103,104,116 Upstream of all of these signaling molecules and enzymes are the receptors that initiated the outside-in signaling, which have also been targeted to a lesser extent to modulate NET release. Blocking of Mac-1¹¹⁷ and fragment crystallizable gamma receptors (FcγRs)^106,107 with neutralizing antibodies has been shown to reduce NET release, and indirect inhibition of platelet-neutrophil interactions with Roflumilast was also efficacious for reducing NETs.¹⁰⁸ Together, these data indicate that although the complexity of NET release has initially complicated our ability to demarcate specific signaling pathways, numerous pharmacological targets exist to downregulate NET release in a variety of pathologies, including biomaterial-induced NET release.

NETs in Inflammatory Pathologies

As the number of studies on NETs in in vitro systems and in vivo models has grown, so too has the number of physiological implications attributed to NETs. In line with their first described function, NETs are critical in the defense against bacteria, viruses, and fungi, especially in scenarios when the skin or mucosa is damaged.^3,79,118,119 By trapping the invader in close proximity to the lytic antimicrobial compounds associated with the DNA structure, NETs efficiently kill the pathogen.^3,27 Another interesting function of NETs that is attributed to their localized, toxic proteases is their anti-inflammatory action when clustered in aggregated NETs.¹²⁰ It is believed that the large aggregation of NETs around monosodium urate crystals observed in gout actually attenuates inflammation by degrading cytokines and chemokines, which others have also shown is possible.¹²¹ Nonetheless, the anti-inflammatory action of NETs seems to be highly specific to gout and infection prevention with the vast majority of investigations finding that NETs are typically proinflammatory and central to a number of inflammatory pathologies. In this review, we will discuss the involvement of NETs in cancer, thrombosis, and fibrosis because of their relevance to biomaterials and tissue regeneration.

Cancer

Inflammation is a hallmark of cancer, and the proliferation of tumors has been described as wounds that fail to heal due to misappropriated mechanisms of inflammation in tissue regeneration.^122,123 As such, an appreciation for the involvement of NETs in cancer could give important insight into the role of NETs in biomaterial-guided tissue regeneration applications. Most of the literature indicates that NETs facilitate cancer metastasis in a number of tissue environments, including breast, liver, pancreatic, ovarian, lung, and gastroesophageal metastases.^124–128 In these studies, the NETs function as structures that attract and capture circulating cancer cells to promote their adhesion.^70,125,128 Subsequently, NETs activate cancer-associated fibroblasts, which have a similar morphology to myofibroblasts in wound healing, and promote the deposition of a fibrous stroma in the developing metastasis.^124,129,130 Aside from metastasis, the contribution of NETs to primary tumor growth is not well understood, but some have speculated that NET release may propagate tumor cell proliferation by stimulating the recruitment of additional immune cells, fueling the protumoral inflammatory environment.¹³¹ Last, surgical stress has been shown to induce NET release that is enhanced in hypoxic tumors, suggesting that the hypoxic environment created by the surgical site around a biomaterial may also increase NET release.¹³² Together, these studies indicate that NETs are important both as physical structures and activators of fibrous stroma deposition in tumor pathogenesis, resulting in misappropriated mechanisms of tissue healing.

Thrombosis

In addition to functioning as a scaffold for tumor proliferation, NETs contribute to cancer pathology by promoting thrombosis.^103,133,134 The procoagulant activity of NETs has also been observed in myocardial infarction,⁵ antineutrophil cytoplasmic antibody-associated vasculitis,³⁴ deep vein thrombosis,^74,135,136 heparin-induced thrombocytopenia,¹¹² and sepsis.⁵⁶ The thrombogenicity of NETs is attributed to their ability to bind and expose functional tissue factor^5,10,34,48 as well as promote platelet aggregation and stabilization through a positive feedback loop.^5,136,137 In addition, NETs can disrupt the endothelium,¹³⁸ which can induce a more procoagulative endothelial cell phenotype characterized by retraction of cell-cell junctions.¹³³ Thus, the ability of NETs to initiate and support the coagulation cascade through multiple mechanisms is concerning for tissue engineering, especially for blood-contacting biomaterials where thrombosis can lead to detrimental consequences.

Fibrosis

Dysregulated NET release also promotes tissue fibrosis in multiple tissue and organ environments. Similar to fibrous stroma deposition in cancer,^124,129,130 NETs activate lung fibroblasts and induce their differentiation into myofibroblasts, contributing to fibrosis.^139,140 Likewise, NETs enhance the fibrotic potential of dermal fibroblasts in systemic lupus erythematosus³³ and cutaneous fibrosis in systemic sclerosis,¹⁴¹ and correlate with scar formation in postepidural fibrosis.¹⁴² Interestingly, in the case of gout where aggregated NETs seem to have an anti-inflammatory effect,¹² the gouty tophi are surrounded by a fibrous capsule to isolate the stimuli from the microenvironment.¹⁴³ This may also explain why NETs lead to fibrosis in other scenarios where the body is attempting to wall off the inflammatory stimuli in an effort to restore homeostasis, an undesirable response in most biomaterial applications.

NETs in the Resolution of Inflammation

Since NETs retain their toxicity and proteolytic activity until degradation, NETs participate in the resolution of inflammation when their dismantling occurs efficiently during the acute inflammatory response, which can be skewed when their release is dysregulated.³ NETs are primarily degraded by endonucleases, DNase I, and DNase I-like 3.¹⁴⁴ Multiple studies have shown that exogenous DNase I treatment can abrogate the toxic effects of NETs in several pathologies, including thrombosis and fibrosis.^{140,142,145–147} Moreover, accelerated NET degradation by DNase I has also been shown to improve neovascularization and vascular repair after stroke,¹⁴⁶ highlighting that their degradation is central for the resolution of inflammation and progression in the tissue repair program. However, physiological concentrations of DNase I are not sufficient to completely degrade NETs, so macrophages engulf partially digested NETs in an immunologically silent manner, much like macrophage engulfment of apoptotic neutrophils.^148,149 In contrast to their silent clearance, an overproduction of NETs during inflammatory pathologies can lead to proinflammatory responses from interacting macrophages.¹⁵⁰ Together, these data indicate that a fine balance between the presence of NETs and their degradation exists to avoid inflammation and tissue damage, while propagating the tissue repair program.

NETs are also intimately related to the resolution of inflammation in their absence of release because neutrophils are then able to influence tissue healing through their active restorative functions.^151,152 Examples of prohealing functions of neutrophils include phagocytosis of debris, release of growth factors, and the initiation of angiogenesis.² The contribution of neutrophils to the resolution of inflammation in sterile tissue injuries has largely been studied in the context of ischemic injuries, where the purpose of the tissue repair program is to promptly initiate angiogenesis to restore blood flow. Angiogenesis is proceeded by neutrophil recruitment in many models, and these studies have found that neutrophil depletion significantly delays healing.^153–156 Moreover, these models have shown that neutrophils rapidly swarm to an injury within 25–40 min after its induction before clustering around the site of tissue damage, suggesting their integral involvement in tissue repair.¹⁵⁷ It is speculated that the absence of neutrophils delays proper healing in these sterile injuries because the resolution of inflammation is programmed and requires the regulated activation and deactivation of neutrophils.¹⁵⁸ Alternatively, when inflammation is triggered by an unnatural stimulus, such as high glucose in diabetes, alcohol, autoimmune antibodies, or a biomaterial, neutrophils struggle to respond appropriately, which results in maladaptive effector functions.¹⁵⁸ Taken together, NET release and the resolution of inflammation are inherent programs within the neutrophil's molecular machinery, and understanding how to regulate them with immunomodulatory biomaterials may significantly enhance tissue regeneration by avoiding inadvertent neutrophil activation.

NETs and Biomaterials for Tissue Engineering and Regeneration

Engineering immunomodulatory biomaterials for controlling the immune response is not a new concept, but the application of regulating neutrophils and NET release to enhance tissue regeneration has only been realized in recent years. As with macrophages, the interest of tissue engineers in neutrophils has increased with an increasing body of literature on neutrophil-driven angiogenesis and tissue remodeling in tumor biology.^26,159,160 As more studies revealed that NETs contribute to collateral tissue damage and chronic inflammation, our interest has grown in understanding NET release as a preconditioning event in the response to biomaterials and the tissue repair program. Given that surgical stress, hypoxia, and trauma can trigger NET release,^132,161 it is easy to conceptualize that implantation of a biomaterial and its ability to induce NET release from swarming neutrophils¹⁵⁷ can contribute to a dysregulated inflammatory environment that is not conducive to functional tissue healing. Most investigations into biomaterial-induced NET release have focused on understanding how the composition, surface chemistry, and topographical features of the biomaterial regulate NET release to avoid their adverse effects. In this review, we summarize what is known about NETs and biomaterials (Fig. 3) in thrombosis, fibrosis, and tissue integration and conclude with future considerations for tissue engineering applications.

FIG. 3.

NET release on biomaterials regulates thrombosis, fibrosis, and tissue integration. Several studies illustrate that NETs enhance thrombin generation and coagulation on biomaterials, occurring more readily on hydrophobic biomaterials,^90,162 and elevated biomarkers are found in the plasma of hemodialysis patients.¹⁶³ In addition, increased NET release on the surface of biomaterials polarizes macrophages toward an inflammatory phenotype,^57,164 upregulates fibrosis, and impairs tissue integration,^7,8,164 which has been seen in the clinical application of a tissue-engineered trachea.⁶ Finally, swift resolution of neutrophil-driven inflammation is needed for healing and an understanding of how to regulate NET release through immunomodulatory biomaterials may enhance biomaterial-guided tissue regeneration.

Thrombosis

Since NETs amplify platelet aggregation and bind active tissue factor,^56,74 NETs can synergistically promote coagulation with platelets in response to biomaterials. Recently, Sperling et al. evaluated the effects of biomaterial surface chemistry on NET release and thrombin generation.⁹⁰ The authors found that hydrophobic biomaterials increased the release of NETs compared to hydrophilic surfaces.⁹⁰ Moreover, they demonstrated that NET release on hydrophobic Teflon™ AF resulted in elevated thrombin generation and coagulation, linking biomaterial-induced NET release and thrombogenicity to a clinically used biomaterial. Likewise, elevated biomarkers of NETs are found in the plasma of hemodialysis patients,¹⁶³ which suggests that hydrophobic hemodialysis membranes may stimulate NET release and contribute to thrombosis seen in patients undergoing dialysis.¹⁶⁵ Similar work evaluating cobalt-chromium, an alloy commonly used in cardiovascular implants, illustrated that thrombin generation and NET release occurred in the presence of platelets and can exacerbate endothelial cell dysfunction and coagulation.¹⁶² Together, these studies link NET formation to biomaterial-induced thrombosis and emphasize the importance of assessing neutrophil activation when considering the hemocompatibility of biomaterials.

Fibrosis and tissue integration

Several groups have also focused on the impact of NETs in fibrosis and tissue integration. Vitkov et al. found that NETs are released in response to rough titanium biomaterials that stimulate osseointegration,¹⁶⁶ but the impact of NETs on the inflammatory response and potential for tissue integration was not evaluated in this work. To address it, NET formation on smooth, rough, and rough-hydrophilic titanium surfaces was then studied to determine how the neutrophil response impacts macrophage polarization.⁵⁷ In these experiments, rough hydrophilic titanium biomaterials decreased proinflammatory responses and NET release, whereas the smooth and rough hydrophobic titanium surfaces increased NET release, indicating that neutrophils sense the biomaterial surface and respond differentially. The authors then showed that the neutrophil-conditioned media from the smooth and rough hydrophobic surfaces enhanced proinflammatory macrophage polarization, which was attenuated when the neutrophils were treated with NET inhibitors.

Similarly, Won et al. eloquently demonstrated that three-dimensional (3D) printed polycaprolactone biomaterials with hierarchical microchannels decreased NET release and enhanced macrophage polarization to the prohealing phenotype, which correlated with increased angiogenesis and reduced fibrosis, compared to control 3D printed biomaterials.¹⁶⁴ Our group has also shown that both the polymer and topography of electrospun biomaterials can regulate NET release and that upregulated NET release, occurring on hydrophobic polydioxanone with high surface area topography, correlates with fibrotic encapsulation.⁸ Jhunjhunwala et al. obtained similar results with implanted microcapsules, observing NETs on the surface up to 3 days after implantation, and speculated that the surface-coating NETs nucleated fibrosis.⁷ Last, in the only published clinical example to date, a decellularized cadaver trachea implanted into a 12-year-old boy was found to have NETs on the luminal surface for the first 8 weeks after surgery, which the authors postulated delayed epithelialization.⁶ Together, these data indicate that neutrophils sense the biomaterial and precondition the environment for differential immune cell activation and the subsequent tissue repair program, significantly impacting tissue integration and fibrosis.

Future directions

The key for successful tissue regeneration lies in the swift resolution of neutrophil-driven inflammation and progression in the tissue repair program. Because the importance of neutrophils in tissue repair has only been recognized in recent years, there are many unanswered questions for the development of immunomodulatory biomaterials that regulate the neutrophil response, release of NETs, and resolution of inflammation for functional tissue regeneration. Unraveling the problem of NETs begins with an understanding of the stimuli and signaling pathways involved in biomaterial-induced NET release. This may include focusing on neutrophil-biomaterial interactions through protein adsorption, crosstalk with platelets and other immune cells, and secreted inflammatory mediators in the local environment.

Likewise, an understanding of the stimuli and signaling pathways will enhance our discovery and use of relevant inhibitors or biomaterial design features to begin regulating NET release. For example, our use of Cl-amidine incorporated into an electrospun biomaterial did not completely abrogate NET release,⁵³ suggesting that another inhibitor may be more efficacious. However, one extremely important unanswered question is the appropriate level of NETs needed for functional tissue regeneration. It may be best to downregulate NET release to avoid adverse effects, but a complete absence of NETs could be detrimental since some inflammation is necessary for healing. Likewise, it will likely be important to consider the consequences of inhibiting NET release, which several groups have shown can induce anti-inflammatory neutrophil apoptosis.^167–169 Although neutrophil apoptosis results in silent efferocytosis by macrophages to resolve inflammation,¹⁷⁰ some biomaterial applications may require a persistence of viable, anti-inflammatory neutrophils to stimulate tissue healing. For example, Lin et al. recently demonstrated that noninflammatory neutrophils were required for the anastomosis of bioengineered microvessels within a biomaterial.¹⁷¹ All in all, systematic investigations into biomaterial design features, such as composition, surface chemistry, topography, and mechanical properties, will likely yield significant insight into our understanding of NET release and the healing potential of neutrophils.

Conclusion

NET release is an active process programmed into the neutrophil's molecular machinery to prevent infection. However, the dysregulated release of NETs in response to foreign or inflammatory stimuli contributes to multiple pathologies, including cancer, thrombosis, fibrosis, and biomaterial-related complications. Moreover, NET release prevents the neutrophil from exerting its prohealing potential, further impairing tissue repair. Although the body of literature is still small, the release of NETs on biomaterials appears to be a significant preconditioning event that influences the potential for tissue healing with largely detrimental consequences. More extensive evaluations of neutrophils and NET release in response to biomaterials will likely aid in the development of immunomodulatory biomaterials that avoid maladaptive immune responses and improve the therapeutic potential of tissue-engineered biomaterials and their applications in the clinical setting.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the National Science Foundation Graduate Research Fellowship Program [1451514].

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