During NETosis, activated neutrophils release their chromatin and granular content and form a web-like structure from strands of DNA that functions as a trap for infectious agents in the circulation (30). Secretion of neutrophil-derived proteases, such as neutrophil elastase (NE) and myeloperoxidase (MPO), contributes to a locally elevated concentration of antimicrobial substances and hence enables efficient destruction of pathogens. Both nucleic acids and the associated histones are potent inducers of platelet activation and therefore exert a prothrombotic effect with platelet aggregation and fibrin deposition as a result. So how can neutrophils form these extracellular traps? Current knowledge suggests that NETosis can occur either as a cell death-associated mechanism or in a vesicular-dependent manner where the neutrophil survives and continues to function after NET formation (referred to as "vital NETosis"). In the case where NETosis results in neutrophil death, the suggested process is dependent on chromatin decondensation, degradation of the nuclear membrane, and cellular lysis with associated release of chromatin and granular contents into the extracellular space. Nuclear decondensation is initiated by epigenetic modifications of histones, citrullination (i.e., arginine converted into citrulline), mediated by the enzyme peptidyl arginine deiminase 4 (PAD4). PAD4 has proven to be required for NETosis to be initiated and neutrophils in PAD4-deficient mice lack ability to form NETs (31, 32). Degradation of the nuclear membrane is driven by NE, which has to be translocated to the nucleus for this purpose (33). Furthermore, isolated neutrophils deficient in MPO fail to form NETs, suggesting that MPO is required for NETosis (34). In contrast to vital NETosis, which has been described as a quick event, the process of lytic NETosis takes several hours to complete (30). In addition, a recently identified process of programmed cell death, necroptosis, was earlier this year implicated as an additional mechanism for NETosis (35). Necroptosis is associated with inflammation and has been suggested to be involved in inflammatory conditions such as Crohn's disease (36). The mechanism for necroptosis-associated NETosis was shown to depend on activation of the mixed lineage kinase domain-like protein (MLKL) for membrane degradation and subsequent cell death (35). The process of vital NETosis was first described a few years ago (30, 37). During infectious conditions, vital NETosis occurs upon stimulation of TLRs by both gram-negative and gram-positive bacteria, and involves nuclear envelope blebbing and vesicular trafficking of DNA to the extracellular space (37). The process leaves the cell membrane intact and allows the neutrophil to continuously exert its classical function *via* protease release and phagocytosis. Whether both cell death-associated and vital NETosis occur in individuals with cancer is still not clear.

It has been reported that only a fraction of all neutrophils are capable of forming NETs (30). How to distinguish these specific neutrophils with capacity for NETosis is still not clear. It has been suggested that the ability to form NETs is related to aging of the neutrophil, a process paralleled with upregulation of CXCR4 on the cell surface (38). Interestingly, the same study demonstrates that the aged neutrophil population is expanded under pathological conditions. It was recently suggested that the lifespan of a neutrophil may be significantly longer than previously reported and that the average human neutrophil remains in the circulation for more than 5 days (39). Therefore, the population of neutrophils that form NETs may be larger than previously expected. However, the finding of an extended lifespan of neutrophils beyond 1 or 2 days has been questioned (40). A vast amount of studies further support that neutrophils indeed are more heterogeneous than earlier presumed. For example, migration of neutrophils has previously been described as a one-way transfer from the circulation into the tissue. However, several publications now report observations of reversed migration of tissue-resident neutrophils back into the vasculature (41–43). Furthermore, a polarization similar to that of macrophages has been suggested for neutrophils with a division into antitumorigenic neutrophils (N1) and protumorigenic neutrophils (N2) (44). Sagiv and colleagues recently demonstrated that cancer is associated with a switch in neutrophil phenotype towards a low-density neutrophil type with more immature appearance and less lobulated nuclei (45). This subpopulation of neutrophils was suggested to be protumorigenic, as compared to high-density neutrophils with an antitumorigenic function. How this relates to NETosis was not discussed in the paper. However, low-density neutrophils have previously been isolated from patient with SLE, an autoimmune disease characterized by NETosis (46, 47). These cells were further verified to be highly prone to undergo NET formation (48), suggesting that the protumorigenic neutrophils identified by Sagiv and colleagues are indeed a potential source of NETs. Studies of NETosis in autoimmune disease have suggested a role for proteinase 3 (PR3) in NET induction (49). In small-vessel vasculitis, antineutrophil cytosplasmic antibodies (ANCA), and specifically those directed against PR3, were demonstrated to induce NETosis (50). Whether PR3 stimulation mediates NETosis also in malignant disease is not yet known.

### THE ROLE OF PLATELETS IN NETosis

LPS, a component of the cell wall in bacteria, is an inducer of NETosis during infectious disease (27). However, bacteriaderived LPS is not a general cause of NETosis in individuals with cancer, unless the patient suffers from bacterial infection. So how can a tumor induce NETosis? While some mechanisms for tumor-induced NETosis have been described, there are possibly others that remain to be identified. A summary of identified mechanisms can be found in **Figure 1**. The first report of NETs in cancer appeared a few years ago and demonstrated that presence of a tumor primed neutrophils to undergo NETosis (25). The authors suggested that G-CSF was a critical factor for tumor-induced NETosis in mice with cancer. The importance of G-CSF was recently confirmed in another study, where tumors expressing high levels of G-CSF were demonstrated as more powerful inducers of NETosis than tumors expressing low levels of G-CSF (51). Furthermore, inhibition of G-CSF by injection of anti-G-CSF antibodies efficiently suppressed NETinduced vascular dysfunction in distant organs of mice with mammary carcinoma. It is however likely that additional factors are involved in induction of tumor-associated NETosis. The cytokine IL-8, frequently expressed by various tumor cells, has, for example, been described as a NET-inducing factor and was recently demonstrated to be crucial for tumor-induced NETosis (5, 52).

inducers of NETosis. This effect is mediated *via* direct binding of P-selectin on activated platelets and PSGL-1 on neutrophils. Stimulation of platelets *via* toll-like receptor 4 (TLR4), by LPS during infectious disease or tumor-derived factors, possibly fibronectin ED-A, may further contribute to platelet-induced NETosis. NETs further stimulate platelet activation and thrombosis due to externalized chromatin and localization of TF and factor XII.

As mentioned, activated platelets can regulate NET induction, although NETosis can also occur independent of platelet interaction, as exemplified by PMA stimulation. An important interaction for this effect seems to be the binding of P-selectin on activated platelets to PSGL-1 on neutrophils. It was recently demonstrated that platelets from P-selectin-deficient mice failed to induce NETosis, while platelets from mice with increased P-selectin levels were more prone to induce NETs upon co-culture with neutrophils (53). Platelets have previously been described as sensors during infectious disease for the severity of an infection, where LPS binding to TLR4 on the platelet surface is essential to determine whether NETosis should be initiated (27). Although LPS-induced TLR4 activation does not take place as a result of a tumor, it is possible that other tumor-derived factors can activate platelets *via* TLR4. For example, it has been shown that a tumorassociated splice variant of fibronectin, extradomain A (ED-A), can bind to TLR4 (54). Interestingly, studies in mice recently demonstrated that signaling *via* TLR4 on platelets by fibronectin ED-A promotes platelet aggregation and arterial thrombosis (55). Whether this effect is mediated *via* NETosis was not addressed, but the possibility is briefly discussed in the paper. Furthermore, these studies were not performed in a cancer setting, and the relevance of fibronectin ED-A for cancer-associated thrombosis still remains to be determined.

The importance of platelets for NET induction is obviously not a tumor-specific phenomenon. However, it has been known for more than a century that individuals with cancer suffer from increased risk for thrombotic disease – a fatal consequence of enhanced platelet activation (56, 57). The hyperactive state of platelets in malignant disease has been attributed to the fact that many tumors express Tissue Factor (TF), which leads to thrombin formation, coagulation, and platelet activation (58). Enhanced platelet activation in cancer patients does not only contribute to thrombosis but also to malignant progression by promoting processes such as tumor angiogenesis and metastasis (59). The increased platelet activation in cancer patients could therefore be a contributing factor to enhanced NETosis during malignant disease.

### OTHER TYPES OF PLATELET– NEUTROPHIL INTERACTIONS

While the specific interplay between platelets and neutrophils in formation of NETs was quite recently discovered, interactions between platelets and neutrophils were described much earlier. Already 50 years ago, the phenomenon of platelets adhering to neutrophils was described and referred to as platelet satellitism (60–64). These platelet–neutrophil complexes were observed in a number of pathological conditions, but their contribution to disease was not clear. Interestingly, a case study from 1975 described platelet–neutrophil aggregation in a patient with invasive prostate cancer, but the cause or significance of the finding was not further explored (65). Today, complex formation between platelets and neutrophils are known to occur and contribute to a wide variety of pathological conditions, such as asthma, ulcerative colitis, sepsis, rheumatoid arthritis, and acute coronary syndrome (66–75). By which mechanism do platelet–neutrophil complexes form? Initial platelet–neutrophil aggregation is mediated mainly by binding of the surface receptor P-selectin on activated platelets to neutrophil PSGL-1 and results in activation of the neutrophil (76–79). Thereafter, integrin receptors are important for continuous platelet–neutrophil interactions. For example, Gp1b-IX-V and alpha-IIb-beta-3 (GpIIb/IIIa), *via* fibrinogen, mediate binding to integrin alpha-M-beta-2 (Mac-1) on the neutrophil, while integrin alpha-L-beta-2 (LFA-1) on neutrophils can adhere to platelets *via* ICAM-2 (80–83). Platelets also facilitate leukocyte adherence to the endothelium *via* the same interactions, for example, upon damage to the vessel wall when direct adherence of leukocytes to endothelial cells is compromised and platelets function as a bridging factor (81). The ability of platelets to regulate neutrophil function is not limited to NETosis. It has been demonstrated that platelets promote initiation of inflammation by regulating neutrophil crawling, an effect dependent on signaling *via* PSGL-1 (84). Activated platelets can also promote neutrophil degranulation and phagocytosis (27, 85). Moreover, platelet-derived soluble CD40L promotes formation of reactive oxygen species (ROS) in neutrophils, which contributes further to the antimicrobial effect (86). There are also evidence showing that interaction between platelets and neutrophils promote metastasis by formation of an early metastatic niche (87). This study by Labelle et al. demonstrated that granulocyte recruitment to the metastatic site is mediated by platelet-derived CXCL5 induced by contact with tumor cells and signaling *via* the CXCR2 receptor on granulocytes. If the interaction between platelets and granulocytes is blocked with a CXCR2 antibody, metastatic seeding is significantly impaired. This study further highlights the importance of platelets as critical regulators of neutrophil function.

### CONSEQUENCES OF TUMOR-INDUCED NETosis

What are the consequences of NETosis in individuals with cancer? The data presented so far suggest that tumor-induced NETosis may be a promoter of cancer-associated pathology. A couple of studies show that NETs may directly contribute to malignant progression. For example, Cools-Lartigue and colleagues showed that infection-induced NETs contribute to metastasis by sequestration of tumor cells in the circulation of mice with cancer (88). This suggests an increased risk for metastasis if cancer patients are affected by infectious disease. Recently, direct cancer-promoting effects were further demonstrated in a study where NETs were suggested to contribute to tumor relapse after surgery in patients with metastatic colorectal cancer. While this study did not address tumor-induced NETosis directly but rather NETosis induced by surgical stress, it still highlights the possibility that NETs could contribute to tumor progression and relapse (89). This finding is in line with an earlier study, suggesting that presence of NETs in tumor biopsies correlated with relapse in patients with Ewing sarcoma (26). Besides direct effects on malignant progression, tumor-induced NETosis further contributes to systemic pathological effects of cancer. For example, NETs have been suggested to promote cancer-associated deep vein thrombosis (25). Hence, the interaction between neutrophils and platelets in NETosis is not limited to platelet-induced of NET formation, but NETs can also stimulate platelet activation – adding yet an important aspect to the complex interplay between platelets and neutrophils. The procoagulant effect of NETs is primarily mediated *via* the negatively charged DNA inducing the intrinsic pathway of coagulation (90) and by histones contributing to thrombin formation (91). Moreover, both TF and factor XII, inducers of the extrinsic and intrinsic coagulation pathways, respectively, can be found in NETs (92–94). Furthermore, it was recently demonstrated that NETosis contributes to impaired vascular function and systemic inflammation in organs that are not sites for tumor growth, such as heart and kidneys, in mice with mammary carcinoma and insulinoma (51). When mice were treated with DNase I to dissolve NETs, vascular function was restored and inflammation abolished. Hypoperfusion of the renal vasculature and associated inflammation are indicators of renal insufficiency – a frequent issue in cancer patients with mortal consequences (95–97). Whether suppression of NETosis could prevent renal insufficiency in individuals with cancer remains to be explored. NETosis was connected to both thrombosis and vascular dysfunction in a study published earlier this year (98). Analysis of blood and post-mortem tissues from ischemic stroke patients revealed that a high number was affected by known or occult cancer and that this could be associated with formation of arterial microthrombi with presence of NETs in various organs. Altogether, these studies suggest that tumor-induced NETosis is connected to poor prognosis in cancer patients. It is however likely that the consequences of tumor-induced NETosis are not limited to those described today, but more reports on this phenomenon should be expected.

#### THERAPEUTIC TARGETING OF NETosis IN INDIVIDUALS WITH CANCER – WHAT ARE THE OPTIONS?

The role of tumor-induced NETs as potential promoters of malignancy and associated complications, such as thrombosis and systemic inflammation, suggests that therapeutic approaches to suppress NETosis might be beneficial for cancer patients. Several potential strategies could be considered for this purpose. Treatment with DNase I, a strategy to degrade extracellular DNA strands, would be an option to dissolve already formed NETs.

#### REFERENCES

DNase I is already in clinical use for treatment of patients with cystic fibrosis, which indicates its safety as a drug (99). Another option would be to prevent NETosis by inhibition of PAD4, an enzyme required for initiation of NETosis (32). Specific PAD4 inhibitors, with capacity to prevent formation of NETs from both human and murine neutrophils, were recently developed (100). A third alternative approach would be treatment with heparin, which function to destabilize NETs by extraction of histones (29). Heparin has long been used in the clinic for its anticoagulative effects and is therefore well established as a therapeutic method. Based on current knowledge about NET induction described in this review, intervening with the P-selectin/PSGL-1 interaction could be yet a potential therapeutic strategy. An important issue to address to enable clinical use of NET targeting approaches is whether there are risks with NET inhibition. A few studies performed in mice lacking PAD4 and hence unable to form NETs have been published but with various results. While increased susceptibility to bacterial infection was described as a consequence of PAD4 deficiency in one study, other studies reported that mice lacking PAD4 are not more sensitive to infections. Instead, it was suggested that PAD4-deficient mice are protected against septic chock (31, 32, 101). Further research is needed to fully explore the potential risks with therapeutic approaches targeting NETs.

The existing data on tumor-induced NETosis strongly indicate that targeting NETs could be beneficial for cancer patients. NETs, originally identified as a defense against severe infectious disease, seem rather to have a negative influence during malignant disease by promoting mortal processes such as thrombosis, systemic inflammation, and cancer relapse. With this in mind, NETs could provide excellent targets for future anticancer therapies, with capacity to suppress processes contributing to the absolute majority of cancer-related deaths.

#### AUTHOR CONTRIBUTIONS

A-KO and JC performed the literature search and wrote the manuscript.

### FUNDING

Funding was provided by grants from The Swedish Cancer Society (#11 0653) to A-KO and from Magnus Bergvalls foundation (2015-01164) to JC.

systemic lupus erythematosus induces vascular damage and synthesizes type I IFNs. *J Immunol* (2010) 184:3284–97. doi:10.4049/jimmunol.0902199

and platelet-independent mechanisms. *Arterioscler Thromb Vasc Biol* (2014) 34:1977–84. doi:10.1161/ATVBAHA.114.304114

anticancer drug management: the renal insufficiency and anticancer medications (IRMA) study. *Cancer* (2007) 110:1376–84. doi:10.1002/ cncr.22904

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Olsson and Cedervall. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Neutrophil Extracellular Traps in Pulmonary Diseases: Too Much of a Good Thing?

#### *Bárbara Nery Porto1 \* and Renato Tetelbom Stein2*

*<sup>1</sup> Laboratory of Clinical and Experimental Immunology, Infant Center, Institute of Biomedical Research, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil, 2 Laboratory of Pediatric Respirology, Infant Center, Institute of Biomedical Research, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil*

Neutrophil extracellular traps (NETs) arise from the release of granular and nuclear contents of neutrophils in the extracellular space in response to different classes of microorganisms, soluble factors, and host molecules. NETs are composed by decondensed chromatin fibers coated with antimicrobial granular and cytoplasmic proteins, such as myeloperoxidase, neutrophil elastase (NE), and α-defensins. Besides being expressed on NET fibers, NE and MPO also regulate NET formation. Furthermore, histone deimination by peptidylarginine deiminase 4 (PAD4) is a central step to NET formation. NET formation has been widely demonstrated to be an effective mechanism to fight against invading microorganisms, as deficiency in NET release or dismantling NET backbone by bacterial DNases renders the host susceptible to infections. Therefore, the primary role of NETs is to prevent microbial dissemination, avoiding overwhelming infections. However, an excess of NET formation has a dark side. The pathogenic role of NETs has been described for many human diseases, infectious and non-infectious. The detrimental effect of excessive NET release is particularly important to lung diseases, because NETs can expand more easily in the pulmonary alveoli, causing lung injury. Moreover, NETs and its associated molecules are able to directly induce epithelial and endothelial cell death. In this regard, massive NET formation has been reported in several pulmonary diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, respiratory syncytial virus bronchiolitis, influenza, bacterial pneumonia, and tuberculosis, among others. Thus, NET formation must be tightly regulated in order to avoid NET-mediated tissue damage. Recent development of therapies targeting NETs in pulmonary diseases includes DNA disintegration with recombinant human DNase, neutralization of NET proteins, with anti-histone antibodies and protease inhibitors. In this review, we summarize the recent knowledge on the pathophysiological role of NETs in pulmonary diseases as well as some experimental and clinical approaches to modulate their detrimental effects.

Keywords: neutrophil, neutrophil extracellular traps, NETs, pulmonary diseases, lung infection, respiratory infection, bacteria, viruses

### INTRODUCTION

Neutrophils are key players in microbial killing, being the first immune cells to achieve the site of injury or infection (1). Therefore, neutrophils act as the first line of defense against microorganisms through phagocytosis, release of reactive oxygen species (ROS), and degranulation (2). Aside from these traditional mechanisms, neutrophils are also able to extrude DNA lattices,

#### *Edited by:*

*Marko Radic, University of Tennessee, USA*

#### *Reviewed by:*

*Patrizia Rovere Querini, Vita-Salute San Raffaele University, Italy Anna M. Blom, Lund University, Sweden*

> *\*Correspondence: Bárbara Nery Porto<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 15 June 2016 Accepted: 02 August 2016 Published: 15 August 2016*

#### *Citation:*

*Porto BN and Stein RT (2016) Neutrophil Extracellular Traps in Pulmonary Diseases: Too Much of a Good Thing? Front. Immunol. 7:311. doi: 10.3389/fimmu.2016.00311*

called neutrophil extracellular traps (NETs), which entrap and facilitate the killing of bacteria, fungi, protozoa, and even viruses (3–8). NETs are composed of decondensed chromatin fibers coated with antimicrobial proteins, such as histones, neutrophil elastase (NE), myeloperoxidase (MPO), and α-defensins (3, 7). Besides being expressed on NET fibers, NE and MPO also regulate NET formation (9). Differently, the participation of NADPH oxidase-derived ROS in NET release seems to be a matter of time of stimulation. While ROS are required to NET generation in time points beyond 1 h after stimulation (10, 11), a very rapid process (5–30 min) of NET extrusion has been reported to be ROS-independent in response to *Staphylococcus aureus* and *Candida albicans* (12, 13). Furthermore, histone deimination by peptidylarginine deiminase 4 (PAD4) is a central step to NET formation (14). Additionally, the release of these DNA threads requires autophagy and activation of p38 MAPK and the Raf-MEK-ERK signaling pathways (15–17). However, it is important to keep in mind that the specific cell components and signaling cascades may vary depending on the stimulus (18).

The primary role of NETs is to prevent microbial dissemination because of its stringy structure, and to kill pathogens due to the high local concentrations of antimicrobial molecules (19). However, these attributes make NETs potentially detrimental to the host. The pathogenic role of NETs has been described for many human diseases, infectious and non-infectious (20), being particularly important to lung diseases. Netting neutrophils in the lung tissue are able to disturb microcirculation and NETs produced in the pulmonary alveoli can expand easily, filling the lungs, as is the case for cystic fibrosis (CF) (19, 21). Therefore, NET formation must be tightly regulated. In this review, we summarize the recent knowledge on the pathophysiological role of NETs in pulmonary diseases as well as some experimental and clinical approaches to modulate their detrimental effects.

#### CYSTIC FIBROSIS

Cystic fibrosis is a fatal hereditary disorder resulting from mutations in the CF transmembrane conductance regulator (CFTR) anion channel (22). This anion channel is responsible for the transport of chloride ions across the epithelial layer of the airways, which is necessary for the production of thin, freely flowing mucus. Therefore, the lungs of CF patients produce large amounts of thick mucus, leading to an obstruction of the airways and colonization by bacteria (23). Typically, CF infants are rapidly colonized by *Haemophilus influenzae* or *S. aureus*, or both. Over time, *Pseudomonas aeruginosa* represents the main bacterial pathogen infecting CF lungs (23, 24). Due to these frequent infections, there is a massive neutrophil infiltration to the lungs and development of chronic inflammation (25, 26). The chronic and progressive lung disease accounts for morbidity and mortality of CF patients (25).

Cystic fibrosis sputum constituents include DNA, NE, MPO, and other neutrophil proteins (27), as it has been shown that bronchoalveolar lavage fluid (BAL) from CF infants presented high concentrations of DNA, which correlated with neutrophil numbers in BAL (28). However, the great amounts of extracellular DNA in CF sputum were considered to be from necrotic neutrophils and lung tissues (29). More recently, several studies have demonstrated that NETs and NET-associated proteins are present in CF sputum (30–35). Marcos and coworkers quantified free DNA levels in airway fluid from CF patients and found that those patients with poor pulmonary function presented higher levels of extracellular DNA compared to patients with mild lung disease (36), indicating that the accumulation of NET–DNA in the airways contributes to airflow obstruction in CF. Moreover, analysis of CF sputum samples revealed that elevated levels of macrophage migration inhibitory factor (MIF), a potent proinflammatory cytokine, correlated with poor pulmonary function, and MIF was able to induce NET formation (33). Although many of the microorganisms that colonize CF airways have been shown to induce NET formation directly (4, 6, 12, 37, 38), pro-inflammatory cytokines and neutrophil chemokines present in CF lungs are also able to stimulate NET release (30, 33), thus perpetuating the inflammation.

Neutrophil recruitment and NET production in the lungs would be key events to fight against invading microorganisms, but their mission accomplishment is profoundly compromised in CF airways as patients often suffer chronic infections. Together with the failure in killing the bacteria, the excessive release of extracellular DNA accounts for biofilm formation by *P. aeruginosa*, and NETs act as a proinflammatory component of biofilms (39). Furthermore, over the time of infection in CF airways, *P. aeruginosa* is able to acquire resistance to NET-mediated killing (38), probably due to its hypermutability, a well-described mechanism for *P. aeruginosa* adaptation within CF lungs (40–42). In addition, it has been recently demonstrated that sub-inhibitory concentrations of LL-37, a NET component, triggers *P. aeruginosa* mutagenesis in chronic infections (43). Interestingly, *P. aeruginosa* triggers the release of the eicosanoid hepoxilin A3 by lung epithelial cells, which induces neutrophil transepithelial migration and is a natural inducer of NET formation (44, 45). Thus, the excessive release of NETs coated with proteases, together with the colonizing bacteria may worsen pulmonary inflammation and dysfunction. Besides NETs being able to directly induce endothelial and epithelial cell death *in vitro* through histones (46), MPO and NE expressed on NET fibers could exacerbate lung pathology through the destruction of connective tissue and degradation of endothelial cell matrix heparan sulfate proteoglycan (47, 48). Moreover, it has been shown that NE cleaves host proteins at the site of inflammation (49). Additionally, histones are highly cytotoxic to endothelial cells *in vitro* and are lethal in mice (50). Altogether, these findings highlight the need to target the massive NET release in CF.

The current therapy to improve CF symptoms is the administration of recombinant human DNase I (pulmozyme/dornase alpha) (51). DNase inhalation is one of the successful treatments for CF, as it improves lung function and reduces infectious exacerbations (52); however, it is not effective for all CF patients. Therefore, alternative therapeutic options are desired. Dubois and colleagues have demonstrated that DNase administration to CF sputum dramatically increased its elastase activity (53). Thus, the combined administration of DNase and an elastase inhibitor could be useful to avoid the devastating effects of excessive proteases in CF lungs. There are also candidate drugs to inhibit NET release, such as chloroquine and PAD4 inhibitors, however neither of these molecules has been evaluated in animal models of CF.

### ASTHMA

Asthma is a chronic heterogeneous inflammatory disorder of the airways characterized by airway inflammation and reversible airflow obstruction (54–56). Asthmatic subjects present periods of stable condition that alternate with severe episodes of exacerbations, leading to the impairment of lung function (57). Asthma symptoms include recurrent wheezing, coughing, and shortness of breath (55). This very complex disease is caused by multiple environmental factors that act in combination with hundreds of susceptibility genes (55). Asthma has been seen for a long time as an eosinophilic disease (56); however, in recent years, it has become evident that some asthmatics have a prominent neutrophilic inflammation in the lungs (58). Patients with neutrophilic asthma usually present a severe form of the disease that does not respond to the classical treatment with glucocorticoids (59, 60). In addition, glucocorticoid administration to neutrophilic asthmatics could aggravate lung inflammation, since glucocorticoids can prolong neutrophil survival (61). It has been described that neutrophils recruited to the lungs of atopic asthmatic patients generated NETs colocalized with elastase (62). In some patients, the number of neutrophils and NET-releasing neutrophils exceeded the number of eosinophils in the lungs. In this study, Dworski and colleagues also demonstrated that eosinophils infiltrating the airways of atopic asthmatics were able to release eosinophil extracellular traps (EETs), which colocalized with eosinophil granule proteins, such as major basic protein (MBP) and eosinophil cationic protein (ECP). Similar to the first study reporting the release of EETs from viable eosinophils (63), the DNA actively released by eosinophils in asthmatic lungs was from mitochondrial origin, and not nuclear (62). Interestingly, allergen challenge did not increase EET or NET formation in the airways of asthmatic subjects (62). Thus, what would be the role of EETs and/or NETs in the pathogenesis of asthma? And what would be the cause of EET/NET release in asthma? Taking into consideration the high concentrations of proteases anchored in extracellular DNA traps, one can assume that these enzymes could contribute to epithelial and endothelial cell damage, a hallmark of asthma. On the other hand, the formation of DNA lattices could protect the host against possible infections secondary to cell damage. Currently, these and many other questions regarding DNA traps formation in allergic diseases are still open for debate. More recently, it has been demonstrated that eosinophils from asthmatic mice release EETs decorated with eosinophil peroxidase (EPO) with no signs of cell death (64), indicating that DNA release is an active process. In addition, recombinant human DNase treatment of asthmatic mice improves lung resistance and decreases oxidative stress in the lungs, providing a potential antioxidant effect on asthma (65, 66). Accordingly, the combined use of recombinant human DNase therapy together with the current treatments (such as inhaled glucocorticoids) for severe acute asthma may prove effective in decreasing sputum viscosity, as it has been shown in specific case reports (67, 68).

### CHRONIC OBSTRUCTIVE PULMONARY DISEASE

Chronic obstructive pulmonary disease (COPD) is a progressive disorder of the airways characterized by persistent neutrophilic inflammation (69, 70). The disease develops following long-term exposure to external stresses, such as inhaled tobacco smoke (71–73). COPD patients are affected by recurrent bacterial and respiratory viral infections, which represent the main causes of exacerbations in these subjects. Exacerbations are associated with increased upper and lower airway and systemic inflammation (74). Patients with severe COPD present large amounts of airway neutrophils when stable, and these numbers further increase during exacerbations, which may be due to the high expression of neutrophil chemokines and chemokine receptors in airway mucosa (75). Furthermore, NE is expressed in the airway mucosa of COPD patients during severe exacerbations (75) and has a proinflammatory role by inducing the secretion of IL-8 in COPD (76). It is noteworthy that IL-8 is a potent NET inducer (3, 77). These features make COPD lungs more likely to be filled by NETs. Indeed, confocal microscopy analysis has shown that sputum from exacerbated COPD patients presents extracellular DNA, frequently entangled with bacteria (78), characterizing NETs. Moreover, NETs are present not only during COPD acute exacerbations, but also in the lungs of patients with stable disease (79–81). There is a clear correlation between the abundance of NETs in the sputum of COPD patients and disease severity – over 90% of exacerbated COPD subjects presented large amounts of NETs in their sputum compared to 45% of stable COPD subjects. In addition, the very large quantities of NETs directly correlate with the severity of airflow limitation in these patients (79). Why NETs are produced in excess in COPD and what would be the trigger for NET release are unsolved issues. Under physiological conditions, NETs would be degraded by endogenous nucleases and cleared by alveolar macrophages (82). However, COPD subjects present lower numbers of alveolar macrophages (81) and these macrophages are defective in phagocytosis (83), which may explain the persistence of NETs in the airways. Nonetheless, a recent interesting study has shown that the outcome of the interaction of macrophages and netting neutrophils depend on macrophage phenotype. M2 macrophages in contact with netting neutrophils helped to perpetuate an inflammatory response, while M1 macrophages initially released extracellular DNA and thereafter degraded DNA in a caspase-activated DNase-dependent manner (84). These findings highlight a phenotype-dependent mechanism of macrophage regulation of NET release, which reinforce the argument that a prolonged exposure to NETs may favor the development of autoimmunity. The exact role of NETs in COPD pathogenesis is uncertain, but the need for developing novel diagnostic and therapeutic strategies is clear. The treatment for COPD is very difficult, as anti-inflammatory drugs are ineffective. The most successful current treatment for COPD is long-acting bronchodilators, but no therapy reduces the progression or inhibits the inflammation (85). As NETs were implicated in disease worsening, selective inhibitors of NET formation or NET-associated proteins (such as NE, MPO, histones) may prove valuable in improving the clinical picture of the disease.

#### TUBERCULOSIS

Tuberculosis (TB) remains a major health problem for humankind. Annually, there are approximately nine million new cases and 1.5 million deaths caused by the disease (86). This chronic bacterial infection is caused by *Mycobacterium tuberculosis* and affects the lungs, promoting huge morbidity and mortality rates (86, 87). *M. tuberculosis* is usually transmitted by tiny droplets from cough or sneeze of an infected subject. Once in the lungs, the bacilli is phagocytosed and killed by alveolar macrophages. However, *M. tuberculosis* developed strategies to survive inside the macrophages. Therefore, the infection develops as a latent infection, inducing granuloma formation in the lung parenchyma. Consequently, the subject remains healthy while harboring dormant bacteria (87, 88). The key factor for the maintenance of latent TB infection is the equilibrium between the bacteria and the host immune response. TB reactivation is achieved when the immune response decreases and cannot restraint bacteria growth, inducing cell death and an increase in granulomatous lesions, as a result of inflammatory cell recruitment (88). Clinical symptoms of TB are caused by a severe impairment of lung function and by substantial morphological alterations in the lung parenchyma (87).

Although macrophages are generally viewed as the main cells involved in harboring *M. tuberculosis*, a growing body of evidence shows that neutrophils are rapidly recruited to infected lungs and can serve as bacterial reservoirs. Additionally, neutrophils were identified as the main immune cell type in sputum and BAL from active TB patients (89). Furthermore, human neutrophils are able to phagocytose *M. tuberculosis in vitro*, but fail to kill the bacilli (90). Neutrophils have been assigned to play both protective and pathological roles during active TB (91–93). As a part of their role in TB pathogenesis, neutrophils have been shown to release NETs coated with NE and histones when stimulated by two genotypes of *M. tuberculosis* (H37Rv and *M. canetti*). NETs were able to trap mycobacteria but not to kill them (94). This lack of killing ability of NETs may favor lung destruction in active TB. Another study has found matrix metalloproteinase-8 (MMP-8) expressed on NET fibers induced by *M. tuberculosis in vitro*. In addition, induced sputum from TB patients had increased amounts of NETs compared to healthy subjects and MMP-8 secretion correlated to lung tissue destruction in these patients (95). The effect of *M. tuberculosis* on NET induction might be mediated by the early secretory antigen-6 (ESAT-6), a protein secreted by *M. tuberculosis*, responsible for the escape of mycobacteria from phagosome to cytoplasm of cells (96), as ESAT-6 induces the production of NETs colocalized with MPO (97). ESAT-6 is also secreted in large quantities in the extracellular space and therefore can interact with immune cells to stimulate them and facilitate the maintenance of chronic inflammation in the lungs of TB patients (98). Importantly, neutrophils release high levels of calprotectin (S100A8/A9) within lung granulomas of patients with active TB (99), which are constituents of NETs (100). The release of calprotectin in TB could be related to NET formation, as neutrophil cytoplasmic proteins can attach to DNA fibers before being released. Urban and coworkers have shown that calprotectin can be released from neutrophils in two ways: bound to NETs and unbound (100). This could be the case for calprotectin release in the lungs of TB subjects; however whether *M. tuberculosis* induces the formation of NETs expressing calprotectin remains to be determined.

Tuberculosis is a curable disease, although the treatment is difficult, since it can take several months (6–9 months) and has different drug regimens. Currently, the first line anti-TB drugs include isoniazid, rifampin, ethambutol, and pyrazinamide, among others when necessary, according to CDC (Centers for Disease Control and Prevention – http://www.cdc.gov/tb/topic/ treatment/). Moreover, new therapies aiming to improve the treatment outcomes, shorten the duration of treatment, and reduce lung pathology in TB patients were described (101). However, no therapeutic approach aimed to specifically regulate the deleterious effects of NETs in TB lungs was reported.

#### BACTERIAL PNEUMONIA

The most common type of bacterial pneumonia is communityacquired pneumonia (CAP). CAP remains a burden worldwide, being responsible for approximately 3.5 million deaths annually (102). A total of 20–60% of CAP patients require hospitalization due to disease severity, including children under age 5 years (102, 103). The etiology of CAP is variable, depending partly on the diagnostic tools used in the population studied. Among all bacteria, *Streptococcus pneumoniae* (*S. pneumoniae*) is the most frequently identified cause of CAP, with high morbidity and mortality rates, but *H. influenzae* is also an important etiologic agent of CAP (102, 104).

Once bacterial infection is established in the lungs, neutrophils are massively recruited to the infection site, inducing a prominent inflammatory response. The clinical outcome in CAP depends on the balance between the inflammatory response and pathogen clearance (102). In this sense, neutrophils actively producing NETs during CAP might lead to potential collateral damage to the lungs. Indeed, three different strains of *S. pneumoniae* (serotypes 3, 4, and 19F) were able to induce pulmonary NET formation in mice, which correlated with the histopathologic severity. In addition, the pneumococcal capsule directly contributes to excessive NET release that paralleled with pneumonia severity in mice (105). The mechanism of NET induction by *S. pneumoniae* seems to be mediated by the pneumococcal protein α-enolase, which binds to myoblast antigen 24.1D5 on neutrophil surface and stimulates NET generation (106). However, *S. pneumoniae* appears to have evolved strategies to counteract NET-mediated killing. In an elegant study, Beiter and colleagues have demonstrated that *S. pneumoniae* expresses EndA, a membrane-localized endonuclease able to degrade NETs *in vitro* and to promote spreading of bacteria from the upper airways to the lungs and from the lungs to the bloodstream of mice. Additionally, mutant bacteria lacking EndA infect the upper airways but fail to disseminate to the lungs and bloodstream (107). Moreover, EndA is secreted into the culture medium during pneumococcal cell growth and rapidly dismantle DNA in NETs, being required for full virulence of *S. pneumoniae* during lung infection (108). Corroborating with these studies, streptococcal endonuclease has been previously implicated in disease progression (109). Besides EndA, streptococcal cells hold other important mechanisms to protect them from NET trapping and killing, such as a positive charge on their surfaces as a result of capsule expression and lipoteichoic acid d-alanylation (110). Thus, it seems that NETs released during *S. pneumoniae* infection function only to damage lung tissue, instead of having a bactericidal activity. The evidence that NETs released in response to bacterial infections can trap and inactivate viruses (8, 111) points out the utmost importance of NETs during co-infections *in vivo*. On the other hand, secondary pneumococcal infection following primary influenza intensified NET formation, but NETs did not show any bactericidal activity, only worsening lung pathogenesis (112). Altogether, these findings suggest that the nature of NET trigger is fundamental to the clearance of subsequent infections.

Non-typeable *H. influenzae* (which lacks a capsule) is an important cause of pneumonia, mainly in subjects with chronic bronchitis and COPD (113), and the persistence of NETs could worsen lung inflammation in these subjects. Viable and heat-killed *H. influenzae* induces NET release *in vitro*, in a mechanism possibly mediated by lipooligosaccharide binding to TLR-4 and Myeloid Differentiation Primary Response (MyD)-88, an adaptor protein necessary to TLR-4 signaling. Interestingly, bacteria are not killed by NET proteins and survive within NETs (114). Accordingly, it has been recently demonstrated that these bacteria evolved to express specific molecules, peroxiredoxin–glutaredoxin and catalase, which allow them to resist to host oxidants and to survive within NETs *in vivo* (115). In addition, non-typeable *H. influenzae* populations survive in biofilm communities in the airway surface, and NETs constitute an integral part of these biofilms (116). Astoundingly, it has been reported a fatal case of nontypeable *H. influenzae* infection with severe pneumonia and bacteremia in an adult found to have large amounts of NETs expressing NE and histone H3 in his sputum (117). This case highlights the association between excessive NET generation and severe respiratory infection and sepsis. More recently, it has been shown that besides NETs, non-typeable *H. influenzae* is also able to induce macrophage extracellular traps (METs) expressing MMP-12 (118). MMP-12 has been implicated as a key factor for protease imbalance and emphysema. Therefore, the release of METs together with NETs may have a detrimental role during emphysema, pneumonia, and COPD. Importantly, DNase was effective to dismantle non-typeable *H. influenzae*induced MET and NET formation (118), which could be used as a short-term adjunctive therapy to avoid the injurious effects of these extracellular traps and associated proteases during pneumonia and other lung diseases.

#### RESPIRATORY SYNCYTIAL VIRUS BRONCHIOLITIS

Respiratory Syncytial Virus (RSV) is the leading cause of acute bronchiolitis in children under age 2 years (119). Throughout the winter, RSV causes a significant number of hospitalizations, resulting in a huge burden to communities worldwide (119, 120). Due to the high infectivity of RSV, almost 70% of all children are infected with the virus during the first year of life, and by age 3, practically all children will have experienced at least one infection with this virus (121, 122). The clinical symptoms of RSV bronchiolitis include labored breathing, coughing, and wheezing (123). Microscopically, there is a massive neutrophil recruitment to the airways of infected children – these cells comprise for approximately 80% of infiltrated cells (124–127). Once in the airways, RSV is able to activate neutrophils, inducing degranulation and IL-8 secretion (128), and also to inhibit neutrophil apoptosis, through phosphoinositide 3-kinase (PI3K) and nuclear factor-κB (NF-κB)-dependent mechanisms (129). This body of evidence suggests that neutrophils may play a significant role in disease pathogenesis.

Aside from the mechanisms mentioned above, we have recently demonstrated that RSV particles and one of its membrane-bound glycoproteins are capable of inducing NET formation by human neutrophils (130). RSV Fusion protein mediates the fusion of virus with the host cell and it is essential for viral replication both *in vivo* and *in vitro* (131), being considered the primary target for vaccine and antiviral drug development. RSV F protein induces the release of NETs coated with MPO and NE through Toll-like receptor (TLR)-4 activation. Moreover, F protein stimulates ROS generation and MAPK phosphorylation, and these signaling pathways are necessary to F protein-induced NET formation (130). Data in the literature regarding the role of NETs in viral diseases are conflicting (132). We hypothesized that the excessive production of NETs could fill the lungs and impair lung function, worsening inflammation in young children and babies affected by RSV infection. Indeed, analysis of bronchoalveolar fluid cytology samples from children with severe RSV lower respiratory tract infection revealed the presence of NETs expressing NE and citrullinated histone 3 (citH3) (133). Furthermore, the infection of calves with bovine RSV induced an extensive release of NETs colocalized with dense cellular plugs containing shed epithelial cells and large amounts of neutrophils, which obstructed the airways (133). These recent studies indicate that NETs contribute to the airway obstruction and immunopathology observed in children and animals infected with RSV.

Despite extensive research efforts, there is no RSV vaccine currently available. Nevertheless, monoclonal antibodies targeting the RSV fusion protein have been developed and they passively protect against RSV challenge in an animal model and reduce the severity of infection in premature and newborn babies (134, 135). However, the humanized monoclonal antibody against RSV F protein is only used in high-risk groups, such as preterm infants and those suffering from cardiovascular diseases or immunosuppression (134). In addition, ribavirin is an antiviral drug used to treat severe RSV bronchiolitis due to its anti-replicative activity, but it presents a high cost and is administered only to high-risk infants (136). Moreover, the use of recombinant human DNase in the management of severe RSV bronchiolitis has been previously reported. The administration of nebulized DNase to young babies with complicated bronchiolitis was able to immediately improve the clinical signs and chest radiograph, and even led to the resolution of atelectasis (137, 138). In contrast, in infants with mild RSV bronchiolitis, recombinant DNase therapy did not reduce the length of hospital stay or the duration of supplemental oxygen (139). Thus, DNase seems to be a useful therapeutic option in the treatment of infants who develop atelectasis due to severe RSV bronchiolitis.

#### INFLUENZA VIRUS INFECTION

Influenza A virus is responsible for regular outbreaks, whose severity may vary among the population. While the influenza pandemic that started with the Spanish flu in 1918 killed approximately 50 million people worldwide, the pandemic influenza A H1N1 2009 virus has affected more than 214 countries and caused nearly 18,449 deaths (140, 141). The clinical features of influenza infection include fever and upper respiratory symptoms, such as cough, runny nose, and sore throat (141). To date, there is little information about clinical complications of influenza A infection, but they appear to be similar to those of seasonal influenza, including sinusitis, otitis media, pneumonia, bronchiolitis, seizures, toxic shock syndrome, and secondary bacterial pneumonia with or without sepsis. Among subjects with high risk for complications are those at extremes of age and those with pre-existing medical conditions (141).

The characteristic feature of acute lung inflammation following influenza virus infection is the excessive infiltration of neutrophils in the lungs (142, 143), and CXCR2 seems to be the major receptor mediating neutrophil recruitment during this infection (144). Neutrophils have been demonstrated to play both protective and detrimental roles during influenza virus infection (143, 145, 146). Among the harmful roles played by neutrophils is the excessive production of NETs in the lungs of animals infected with influenza A H1N1 virus. NETs expressing histones and MMP-9 were found entangled with alveoli, causing increased alveolar capillary damage and obstruction of the small airways, thus confirming the link of these DNA lattices with lung damage (146). Furthermore, NET formation stimulated by influenza A infection is dependent on histone deimination by PAD4 (147). In addition, NET release induced by influenza virus is potentiated by the cathelicidin LL-37 (148), which has been shown to facilitate the formation of NETs (149). Paradoxically, the antimicrobial protein expressed on NETs, α-defensin-1, is able to directly inhibit influenza replication through the inhibition of protein kinase C (PKC) in infected cells (150); however the expression of α-defensins on NETs induced by this virus has yet to be demonstrated. The expression of α-defensins on NETs could inactivate the virions sequestered in NET fibers and consequently prevent them from reaching the target cells in the lungs. Thus, although antimicrobial proteins expressed on NETs have the ability to inactivate the virus and to prevent spreading, they are also able to inflict damage to host cells and tissues due to their cytotoxic properties.

Currently, influenza treatment relies on the administration of two groups of antiviral drugs, the adamantanes and neuraminidase inhibitors. Zanamivir and oseltamivir are neuraminidase inhibitors active against both influenza A and B, and are approved for the prevention and treatment of influenza in the United States. Supportive care of uncomplicated cases of influenza includes administration of fluids and rest (141). To date, there is no study describing the effect of DNase treatment on the outcome of influenza infection in animal models.

#### TRANSFUSION-RELATED ACUTE LUNG INJURY

Transfusion-related acute lung injury (TRALI) is a serious complication of blood transfusion (whole blood or blood components) that develops within 6 h of transfusion and is characterized by hypoxemia, respiratory distress, and pulmonary infiltrates (151, 152). Currently, TRALI is the most important cause of transfusion-related morbidity and mortality (152). Histological analysis revealed lung edema, capillary leucostasis, and massive neutrophil infiltration (153). TRALI development requires the presence of antileukocyte antibodies in the transfused product, and antineutrophil antibodies have been linked to the most severe cases of TRALI (154). These antibodies activate recipient's neutrophils, inducing their sequestration in the pulmonary capillaries and consequently tissue injury (155).

In an elegant study, Thomas and coworkers have found NET biomarkers (DNA, nucleosomes and MPO) in the serum of patients with documented TRALI (156). In addition, in a fatal case of TRALI neutrophils with decondensed nuclei were detected in lung vessels together with abundant extracellular histones and MPO (157). In a mouse model of TRALI, DNA streaks colocalizing with citrullinated histone H3 were found in alveoli outside blood vessels (156). Moreover, platelets also accumulate in the lungs of mice with TRALI, being required for injury development (158). In this model, platelets were shown to induce NET formation during TRALI (157). As a vicious cycle, histones expressed on NETs may activate platelets (159), which in turn induce further NET release, promoting coagulation and thrombi formation in the lungs. Accordingly, the pretreatment of mice with a histone-blocking antibody decreased lung edema, lung vascular permeability, and even mortality. This treatment also reduced NET generation detected in plasma, indicating that extracellular histones may help to spread NETs in the body (157). Furthermore, intranasal administration of DNase provided several benefits to mice undergoing TRALI, such as improvement of blood oxygenation, reduction in lung edema and vascular permeability, impairment of NET formation, and platelet sequestration in the lungs (156, 157). These studies support the argument that NETs are formed and play a critical role in the pathogenesis of TRALI and may be a promising target for therapeutic approaches.

### MECHANICAL VENTILATION

Mechanical ventilation is a supportive intervention and a key feature of intensive care for patients with acute respiratory failure, including those with severe RSV bronchiolitis, pneumonia, or influenza infection (160, 161). However, it can be potentially injurious to the ventilated lung, inducing the so-called ventilatorassociated lung injury (VALI), which contributes to morbidity and mortality in those patients (162). Furthermore, animal models of acute lung injury have been developed and characterize an experimental insult to a normal lung and therefore were named ventilator-induced lung injury (VILI) (162).

Neutrophils have been implicated as central cells in the pathogenesis of both VALI and VILI. It has been described that the early phase of VILI involves the release of several proinflammatory cytokines and chemokines, whereas the late phase is characterized by the infiltration of a lung-marginated neutrophil pool (163, 164). However, more recently Choudhury and coworkers demonstrated that injurious mechanical ventilation induced a prominent neutrophil recruitment to the lung at the very early stage of VILI, before the development of physiological signs of lung injury. The infiltration of neutrophils in the course of VILI was dependent on L-selectin engagement but independent of CD18 (165), indicating that immune mechanisms mediate neutrophil recruitment and activation during mechanical ventilation. Moreover, lung-derived soluble mediators appear to have a pathogenic role in an isolated perfused lung model of VILI (166). In line with this evidence, the chemokine receptor CXCR2 and its ligands, CXCL1 (KC) and CXCL2/3 (MIP-2), were shown to play a significant role in mediating neutrophil recruitment and promoting lung inflammation in VILI (167). Accordingly, short periods of mechanical ventilation in preterm infants induce an overproduction of the pro-inflammatory cytokines TNF and IL-1β, neutrophil chemokines IL-8 and MCP-1, and MMP-9 (168, 169). These inflammatory mediators may work together to induce a massive neutrophil infiltration to ventilated lungs and to stimulate NET release in response to mechanical ventilation in those patients. So far, IL-8, TNF, and IL-1β were shown to promote NET release in different experimental settings (10, 170, 171). In fact, excessive NET formation has been recently implicated in the pathogenesis of VILI. A double-hit model of intratracheal LPS challenge followed by high tidal mechanical ventilation induced a prominent lung injury in mice, with high amounts of NETs, decreased lung compliance and release of pro-inflammatory cytokines (172). The mechanism of NET formation during VILI seems to rely on the simultaneous engagement of G protein-coupled receptors (GPCR) and Mac-1 (CD11b), by the platelet-derived CCL5/ CXCL4 heterodimer and a β2-integrin ligand, respectively (173). Surprisingly, these two studies showed opposing results regarding the role of NETs during VILI. Rossaint and coworkers found that DNase treatment of mice after induction of VILI was protective, as treated mice showed an improved gas exchange and reduced NET markers in the blood; whereas Yildiz and colleagues did not find a significant impact of DNase treatment on lung injury induced by VILI. There is at least one possible explanation for these differences: in the study of Yildiz and colleagues, the lungs of mice were already filled with neutrophils at the early stage of VILI due to LPS instillation, which could not be counteracted by DNase. Whereas in the study of Rossaint and coworkers, neutrophils infiltrated the lungs in the course of VILI, in this case a sterile inflammation. Although the outcome of DNase treatment in VILI is an issue for debate, there is no doubt that excessive NET formation accounts for the pathogenesis of acute lung injury.

### OTHER PULMONARY DISEASES AND NETs

Besides the pulmonary diseases aforementioned, there are other disorders or syndromes affecting the lungs, in which NETs may play harmful roles as well.

Acute lung injury following severe sepsis is a common clinical consequence with significant morbidity and mortality rates (174), as the lung is the most sensitive target organ during systemic inflammation (175). Czaikoski and collaborators have recently shown that NETs are produced systemically in mice with cecal ligation and puncture (CLP) model of sepsis. The excessive release of NETs was directly correlated to heart, liver, and lung injury, as rhDNase plus antibiotics treatment of septic mice drastically decreased organ damage (176). Additionally to extracellular DNA measurement, NETs were observed in alveolar spaces and pulmonary capillaries of septic mice (177). Furthermore, higher concentrations of cell-free NETs were present in the serum of septic patients who developed severe acute respiratory distress syndrome (ARDS) compared to healthy controls (176), extending the experimental observations in mice to the clinical setting. Mechanistically, platelet TLR-4 is essential for NET induction within hepatic sinusoids and pulmonary capillaries of septic mice (178). Interestingly, NETs retained their integrity under flow conditions and were able to trap bacteria in septic blood. Therefore, platelets may serve as a platform for neutrophil activation and NET production, which can trap and kill pathogens but also induce disseminated organ injury during severe sepsis (178).

Another lung disorder featuring neutrophil-induced injury is interstitial lung disease (ILD). Actually, ILD are a group of diffuse parenchymal lung disorders characterized by pulmonary fibrosis. ILD can be frequently associated with a specific environmental exposure or an underlying connective tissue disease (179). Activated neutrophils were found increased in BAL from patients with idiopathic pulmonary fibrosis and were associated with early mortality (180). Interestingly, patients with ILD complications due to autoimmunity showed elevated levels of circulating cellfree NETs and plasma LL-37 (a NET component), together with a decreased DNase activity (181), suggesting that the prolonged exposure to NETs is involved in the pathogenesis of ILD. *In vitro*, NETs have been demonstrated to promote the activation of lung fibroblasts and differentiation into myofibroblast phenotype. Moreover, these fibrotic effects were significantly decreased after degradation of NETs with DNase (182). Consistently, these findings were supported by the detection of NETs in close proximity to alpha-smooth muscle actin-expressing fibroblasts in biopsies from patients with fibrotic ILD (182). This effect is very likely to be mediated by NE, since NE directed both lung fibroblast proliferation and myofibroblast differentiation *in vitro* (183). In addition, a NE inhibitor attenuated pulmonary fibrosis induced by bleomycin in mice via inhibition of TGF-β1 and inflammatory cell recruitment to the lungs (184). Altogether, these studies point to a key role of NETs in the development of ILD of different etiologies.

goal of NETs is to protect the host from invading microorganisms through their sticky nature and the high concentrations of antimicrobial proteins. However, these characteristics make NETs potentially detrimental to host cells and tissues. Excessive NET formation enhances mucus viscosity, filling the lungs, and impairing lung function. NET proteins are highly cytotoxic and can induce endothelial and epithelial cell death and cause the disruption of host proteins and cellular matrix.

### CONCLUSION

Neutrophil extracellular traps formation by activated neutrophils has a crucial role in host defense against microorganisms, as deficiency in NET release or dismantling NET backbone by bacterial DNases render the host susceptible to disseminated and lethal infections (107, 185). Moreover, aggregated NETs have been shown to limit sterile inflammation by degrading cytokines and chemokines via serine proteases (186). However, an excess or persistence of NET release is potentially injurious to host organs and cells, leading to worsening or perpetuation of many diseases. The pathogenic effects of excessive NET production is especially important in pulmonary diseases due to lung architecture itself, which may favor the spreading of DNA fibers, consequently enhancing tissue damage and impairing lung function (**Figure 1**). The mechanisms underlying NET production and the boundaries between the beneficial and detrimental effects of NETs during disease state are still to be unveiled. To date, recombinant human DNase is the only treatment targeting NETs approved for a small number of pulmonary disorders. Nevertheless, a long-term DNase therapy presents side effects to patients. Hence, the quest for an ideal therapy targeting NETs and its associated proteins continues to be a challenge for scientists around the globe.

### AUTHOR CONTRIBUTIONS

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

### ACKNOWLEDGMENTS

We thank Stéfanie P. Muraro for support with figure, Natália Jaeger and Rafael S. Czepielewski for critical reading of the manuscript.

### FUNDING

This work has not been funded by any agency.

### REFERENCES

with respiratory syncytial virus bronchiolitis. *Chest* (2007) 131:788–95. doi:10.1378/chest.06-2282

platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4. *Blood* (2011) 118:1952–61. doi:10.1182/blood-2011-03-343061

for treatment. *Expert Rev Anti Infect Ther* (2011) 9:1169–78. doi:10.1586/ eri.11.141

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Porto and Stein. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The emerging Role of NeTs in venous Thrombosis and immunothrombosis

#### *Andrew S. Kimball, Andrea T. Obi, Jose A. Diaz and Peter K. Henke\**

*Section of Vascular Surgery, Conrad Jobst Vascular Research Laboratories, Department of Surgery, University of Michigan, Ann Arbor, MI, USA*

Venous thrombosis (VT), a leading cause of morbidity and mortality worldwide, has recently been linked to neutrophil activation and release of neutrophil extracellular traps (NETs) *via* a process called NETosis. The use of various *in vivo* thrombosis models and genetically modified mice has more precisely defined the exact role of NETosis in the pathogenesis of VT. Translational large animal VT models and human studies have confirmed the presence of NETs in pathologic VT. Activation of neutrophils, with subsequent NETosis, has also been linked to acute infection. This innate immune response, while effective for bacterial clearance from the host by formation of an intravascular bactericidal "net," also triggers thrombosis. Intravascular thrombosis related to such innate immune mechanisms has been coined immunothrombosis. Dysregulated immunothrombosis has been proposed as a mechanism of pathologic micro- and macrovascular thrombosis in sepsis and autoimmune disease. In this focused review, we will address the dual role of NETs in the pathogenesis of VT and immunothrombosis.

#### *Edited by:*

*Mariana Julieta Kaplan, National Institutes of Health, USA*

#### *Reviewed by:*

*Kenneth Reid, University of Oxford, UK Roberta Bulla, University of Trieste, Italy*

*\*Correspondence: Peter K. Henke<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 02 May 2016 Accepted: 02 June 2016 Published: 27 June 2016*

#### *Citation:*

*Kimball AS, Obi AT, Diaz JA and Henke PK (2016) The Emerging Role of NETs in Venous Thrombosis and Immunothrombosis. Front. Immunol. 7:236. doi: 10.3389/fimmu.2016.00236*

Keywords: NETs, extracellular DNA, venous thromboembolism, venous thrombosis, sepsis, immunothrombosis

### INTRODUCTION

Neutrophils (PMNs) have frequently been touted as the *pawns* of the immune system. As our knowledge of immune system grows and as our techniques for evaluating dynamic cell populations improve – we are learning that this could not be further from the truth. While a PMNs principle function remains as a key player in the front line of innate immunity and host defense against bacteria, they are proving to have a multifaceted role in coagulation and have also been implicated as major contributors in the pathophysiology of many systemic illnesses.

Until the early 2000s, the associations between PMN activation and systemic disease had not been well understood; but in March of 2004, Brinkmann et al. published a landmark study in *Science* (1), where they described a fragile fibrillar material extruded from PMNs in the presence of lipopolysaccharide (LPS) by transmission electron microscopy (TEM). In actuality, these fragile fibers were decondensed chromatin and DNA, as they stained strongly for DNA and histones, they were resistant to proteases, and they disappeared upon instillation of DNase. Bacteria were found to colocalize with the extruded DNA *in vivo* in a rabbit model of shigellosis and in human specimens of acute appendicitis. In summation, they demonstrated that these large webs of DNA trap bacteria and allow adjacent or connected PMNs to drive bactericidal activity with proteases and reactive oxygen species. Brinkmann et al. coined these nuclear extrusions "neutrophil extracellular traps" or NETs.

Since that time, there has been a flurry of exciting new work in the field of NET formation (NETosis). NETosis has been demonstrated to be a distinct form of cell death outside of necrosis and apoptosis (2). Also, more interestingly, NETs have been indicted in the pathophysiology of many systemic diseases, including venous thrombosis (VT) (3), sepsis (4, 5), trauma (6), cancer-related thrombosis (7), and autoimmune diseases (8–12). Despite the apparent widespread influence of NETs on disease, there remains a common theme throughout that NETs drive micro- or macrovascular thrombosis leading to ischemia and further injury (13, 14).

In this article, we will review the role of NETs in pathologic thrombosis. Specifically, we will review the findings of NET pathophysiology in murine models of VT, NETs in primate models and human studies of VT, and NETs in immunothrombosis.

#### NETs IN MURINE VT MODELS

Murine models have been essential to our understanding of the role of NETosis in the pathophysiology of thrombosis. PMNs were first shown to be essential for immune-mediated microvascular thrombosis in a murine model of glomerulonephritis, in which CD11b<sup>−</sup>/<sup>−</sup> or PMN-depleted mice were resistant to glomeruli thrombosis and renal failure (15). At that time, it was not widely recognized that NETs contributed to thrombosis; however, this changed in 2010, when Fuchs et al. showed that NETs caused platelet adhesion, activation, and aggregation (3). Stimulation of platelets with purified histones was sufficient for aggregation, and interestingly, DNase and heparin dismantled the NET scaffold and prevented thrombus formation. Brill et al. later demonstrated that NETs are principle effectors in an IVC stenosis model (16). In mice with uninterrupted IVC side-branches, levels of extracellular DNA increased in plasma 6 h after thrombus initiation. Citrullinated histone H3 (CitH3), an element of NETs' structure, was present in thrombi and was frequently associated with the Gr-1 antigen. Furthermore, immunofluorescent staining of thrombi showed proximity of extracellular CitH3 and von Willebrand factor (vWF), a platelet adhesion molecule crucial for thrombus development in this particular model.

Neutrophils, monocytes, and NETs have also been found to affect the clotting cascade in murine models of thrombosis (17– 20). For example, myeloid cells roll along the venous endothelium in a P-selectin-dependent manner and produce thrombogenic tissue factor (TF) in the IVC stenosis model (17). TF, then contributes to thrombin generation and extensive fibrin deposition along the vein wall. Despite this finding, TF alone was inadequate for thrombus propagation. Neutropenia, genetic ablation of Factor XII, and disintegration of NETs were all protective against thrombus propagation. Later, activated PMNs within the fibrin matrix were found to produce NETs that associate with secreted Factor XII, activate the intrinsic pathway, and lead to thrombus extension (17, 21). However, these conclusions were questioned, as during the same year, TF was also found to be secreted by PMNs in an inflammatory signaling and autophagy-dependent manner, with adherence to extruded NETs, activation of the extrinsic pathway, and eventual propagation of the thrombus (18, 22). Regardless, the presence of PMNs and/or monocytes at the endothelial interface has long been assumed to have a major role in VT and now appears unquestionable (17, 18, 23, 24).

Many proteins have been implicated in contributing to NETosis and thrombosis. In 2013, Martinod et al. demonstrated that the enzyme peptidyl arginine deiminase 4 (PAD4) – an enzyme essential for the citrullination and decondensation of chromatin was – not only essential for NET formation but also important for thrombus formation in the IVC stenosis model of murine VT (25). PAD4<sup>−</sup>/<sup>−</sup> mice formed IVC thrombus in less than 10% of cases of IVC stenosis at 48 h compared to 90% in C57BL/6 controls (25). This antithrombotic tendency was rescued in the PAD4−/− mice with adoptive transfer of wildtype PMNs. Conversely, in a stasis IVC ligation model of VT, either preemptive administration of DNase to wild-type mice or PAD4−/− mice did not have an effect on thrombogenesis, suggesting a model-dependent effect (19).

Other proteins that have been implicated in NET formation and NET-associated thrombosis include cathepsin G, serine proteases, PMN elastase, P-selectin, high-mobility group box protein 1 (HMGB-1), platelet glycoprotein Ib, integrin beta-2, vWF, and platelet factor 4. Pharmacological inhibition or genetic deletion of cathepsin G increases mouse bleeding time, decreases platelet activation, and decreases circulating neutrophil–platelet conjugates (26). While the serine proteases and PMN elastase have been implicated in pathologic thrombosis and have been found to colocalize with NETs (27), it was recently demonstrated that PMN elastase is neither necessary for NET formation in mice nor essential for IVC thrombosis in murine IVC stenosis model (28). Indeed, it is likely that the prothrombotic effect of the serine proteases is better explained by their inhibition of tissue factor pathway inhibitor (TFPI) (29). Signaling through P-selectin by P-selectin glycoprotein ligand 1 (PSGL-1) is known to assist in PMN migration to sites of injury or inflammation (30), but beyond that, the P-selectin axis has now been postulated to promote NETosis in the setting of activated platelets (24). However, this conclusion was recently challenged in a study that demonstrated that HMGB-1 expressed by activated platelets was individually capable of activating PMNs *via* the receptor for advanced glycosylation end-products (RAGE) to induce NETosis (31). Similarly, in 2016, an *in vitro* study demonstrated activated platelets binding to vWF through glycoprotein Ib, in turn, linked to PMNs *via* integrin beta-2 and stimulated by platelet factor 4 led to NETosis independent of P-selectin (32). While our knowledge of NET formation continues to grow, the specific proteomic cascade that leads to NETosis remains contested. These findings also underline the heterogeneity and limitations of model systems as well.

Given that thrombi consist of significant quantities of DNA combined with proteins, thrombosis resolution and associated inflammation becomes less straight forward. We have explored this topic in our laboratory by examining toll-like receptor 9 (TLR9) function in VT resolution. TLR9 is a conserved pathogenassociated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) receptor that recognizes CpG DNA repeats and alerts the immune system to invading pathogens or local damage. In an IVC ligation model of VT (complete stasis), TLR9<sup>−</sup>/<sup>−</sup> mice had significantly increased thrombus size at 2 and 8 days despite increased numbers of PMNs and monocyte/ macrophages (33). Further, TLR9−/− mice had increased apoptosis, citrullinated histones, PAD4, and neutrophil elastase; and reduced TFPI (19), suggesting that TLR9 is important for normal thrombogenesis and thrombosis resolution. Lastly, M1-like (CCR2+) monocyte/macrophages were decreased in TLR9<sup>−</sup>/<sup>−</sup> thrombi, consistent with impaired inflammatory cell influx, and this divergence was corrected with adoptive transfer of TLR9<sup>+</sup>/<sup>+</sup> bone marrow-derived monocytes with normalization of thrombus size (20). The new found composition of DNA-rich thrombi will likely have long-standing implications for future research in VT resolution.

#### NETs IN PRIMATE AND HUMAN VT STUDIES

Although first thought to only occur in pathologic states, NETosis with release of extracellular DNA has been shown to occur in healthy individuals (34). Following exhaustive treadmill or cycling exercise, circulating levels of cell-free DNA (cfDNA) and myeloperoxidase (MPO) rise, and isolated PMNs from the circulation develop swollen nuclei and emanating DNA. A concomitant rise in circulating DNase occurs, suggesting that similar to the ongoing processes of fibrin formation and fibrinolysis, NETosis may be a tightly regulated and constantly ongoing homeostatic process. Dysregulation of NETosis and its relationship to thrombosis has been recognized in a variety of clinical scenarios: NETs are present in fresh thrombi from individuals with acute myocardial infarction (31, 35), they are found in high circulating levels in patients with severe trauma and microvascular thrombosis with acute lung injury (6); and in patients with thrombotic microangiopathies (TMAs) (8). Circulating cfDNA rises 24 h following chemotherapy in breast cancer patients, corresponding to peak in thrombin–antithrombin levels (36). This has been proposed as a potential causative mechanism for the high rate of thrombotic events experienced by individuals undergoing chemotherapy.

*Ex vivo* platelet activation studies with recombinant human histones demonstrate that not all histones are created equal in NETosis: only histone 3 (H3) and histone 4 (H4) induce functional platelet response (37). Recent advances in techniques of PMN isolation (38) and recognition of NETs in human pathologic thrombus specimens have improved the ability to study NETosis in humans (39).

The link between human venous thromboembolism (VTE) events and NETosis has been established in a handful of studies to date. Analysis of balloon occlusion-induced iliac thrombosis in a baboon model of VT demonstrated increased circulating NETs at 48 h post-thrombosis and persisting through 6 days (3). Consistently, extracellular DNA markers were present in the experimental thrombus. In 2013, a human study composed of healthy controls and symptomatic patients (swelling and leg pain) with and without VT examined circulating NET markers (40). Extracellular DNA and MPO were significantly elevated in symptomatic patients with VT compared to both groups of non-thrombosed patients. A direct correlation was also seen between common predictors of thrombosis including D-dimer, Wells score, and plasma DNA, suggesting that NETs may be useful in diagnostic evaluation. Development of more accurate methods to diagnose VT is of particular interest as the current "gold standard," duplex ultrasound, is often limited in availability and in evaluation of central veins (41). In the same year, a similar case–control study of 195 individuals with and without VT examined nucleosomes and α-1 antitrypsin elastase (as a PMN activation marker) in relation to thrombosis. Levels of nucleosomes and PMN activation above the 80th percentile were associated with a threefold risk of VT (42).

The precise role of NETs in VT initiation, formation, and propagation, as well as optimal area to intervene in human VT remains relatively unknown. In a study evaluating unorganized, organizing, and organized thrombi from patients with either VT or PE, CD11b, and MPO positive cells were seen in organizing thrombi along with intra- and extracellular CitH3 and PAD4 (43). Unorganized and organized thrombi failed to demonstrate the similar histopathology, suggesting the predominant role of NETs during the inflammatory response and thrombus organization (43). Another study of 29 VT patients and controls demonstrated increased neutrophil adhesion and inflammatory cytokine profile among patients with residual vein obstruction and elevated D-dimer (44). *Ex vivo* studies of NETs related hypercoagulability in inflammatory bowel disease (IBD), and TMA patients have demonstrated efficacy of DNAse I in decreasing the thrombotic response, although this has yet to be shown in human VT (11, 12). To date, no clinical trial has targeted NETs in humans as a mechanism to prevent or treat VT.

#### NETs IN IMMUNOTHROMBOSIS

While the ability to form thrombus in the face of vessel injury has long been known to be essential for the maintenance of hemostasis, until recently, it had not been recognized as an intravascular mechanism of immune defense. Engelmann and Massberg recently reviewed the mechanism of NET-mediated microvascular thrombosis and proposed it as a potential biological adjunct for containing uncontrolled infection, coining this process "Immunothrombosis" (45). In their review of the topic, they proposed four mechanisms by which immunothrombosis prevents the spread of infection (1) it captures and ensnares pathogens in the microvasculature and prevents dissemination, (2) it prevents distant tissue invasion by forming microthrombi in microvessels, (3) it concentrates pathogens in one area for bactericidal killing by innate immune cells, and (4) it recruits other immune cells to the site of inflammation for further bacterial killing. Indeed, coagulation has been previously postulated to play a role in host defense against bacteria (46, 47), but if left unchecked, immunothrombosis may contribute to significant systemic pathology.

Severe sepsis accounts for 10–40% of ICU admissions in the United States and carries a mortality rate of 20–50% (48, 49). NET-related immunothrombosis, cfDNA, and histones have been implicated in the morbidity and mortality of sepsis. In *in vitro* and *in vivo* models of sepsis, LPS has been shown to activate platelets and PMNs *via* toll-like receptor 4 (TLR4) to induce NETosis (31, 50, 51). Intravascular NETs can be digested by DNase to release cfDNA and histones, and further, cfDNA obtained from human specimens has been shown to be at ~150–300 bp sizes consistent with nucleosome units (52). In the same study, cfDNA levels in peripheral blood of patients with severe sepsis were found to be highly predictive of ICU mortality. In 2009, Xu et al. demonstrated that extracellular histones led to endothelial dysfunction, organ failure, and death in animal models of sepsis (4). Specifically, histone administration to mice resulted in neutrophil activation, endothelial toxicity, acute lung injury, and microvascular thrombosis. Interestingly, antihistone antibodies and activated protein C (APC) improved mortality rates in sepsis models in these mice. In addition, NETosis has also been linked to prothrombotic activity because NET-associated enzymes break down TFPI (29), while also enhancing antifibrinolytic activity because cfDNA inhibits plasmin-mediated fibrin degradation (13, 14, 28). Further, H3 levels were found to correlate with ICU mortality and were inversely correlated with antithrombin and platelet levels (53). Structurally, NET-induced immunothrombosis leads to more sturdy thrombi with less permeability and decreased susceptibility to lysis, although this may be overcome with DNase (54). In a murine cecal ligation and puncture model of sepsis (CLP), DNase given at 6 h after injury reduced organ damage and mortality (55). Lastly, bacterial resistance to NET trapping has been demonstrated *in vitro* and has been postulated to contribute to widespread immunothrombosis in the septic host leading to disseminated intravascular coagulation (DIC) (56, 57).

In addition to its inflammatory role in sepsis, NETs and immunothrombosis have also been implicated in autoimmune disease. Phosphatidylserine (PS) and TF-bearing microparticles (MP) were found to be markedly increased in the sera of IBD patients (11). Increased TF-bearing MPs correlated with increased NETosis, markedly shortened coagulation times, and increased levels of fibrin, thrombin, and factor Xa. Similarly, in an *in vitro* study of antineutrophil cytoplasmic antibody-associated vasculitis (ANCA-AV or AAV), C5a-primed PMNs treated with ANCA IgG-released NETs and TF-bearing MPs contributing to a prothrombotic state, and interestingly, this was remedied partially by DNase treatment (58). TMAs, more commonly known as thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), and DIC, are characterized by microvascular thrombosis and coagulopathy. *In vitro* studies of TMAs demonstrate that the sera of TMA patients are incapable of degrading NETs due to a deficiency of DNase I and this may contribute to widespread microvascular thrombosis and organ dysfunction (12). Intriguingly, supplementation of TMA sera with recombinant human DNase I reestablished NET degradation capability. Lastly, PMNs from patients with antiphospholipid antibody syndrome (APS) were inclined for spontaneous NETosis and thrombin generation, and further, APS sera and APS-isolated IgG stimulated NETosis in control PMNs (59). These findings highlight the prothrombotic role of NETs in autoimmune-related immunothrombosis.

Finally, NETs have also been implicated in multiple organ failure related to severe trauma. In 2013, Abrams et al. examined a cohort of 52 patients with severe non-thoracic blunt trauma and correlated circulating histone levels with acute lung injury and sequential organ failure assessment scores (SOFA) (6). Circulating histone levels increased dramatically immediately following trauma and high levels correlated with acute lung injury, SOFA scores, endothelial injury, and coagulation activation. In the same study, with translational animal models of blunt trauma, circulating histone levels corresponded with organ edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. Multiple organ dysfunction syndrome (MODS) is a leading cause of delayed mortality following major trauma and has been correlated with circulating cfDNA levels (52). Inherently, future research should look to address NETosis and immunothrombosis as potential therapeutic targets for preventing immune-mediated MODS after severe injury.

### SUMMARY, INTEGRATION, AND POTENTIAL TRANSLATION

Over the past 12 years, since its discovery, NETosis has been catapulted to the forefront of innate immunity research. Nowhere is its effect more relevant in human disease than in its implication in immune-mediated micro- and macrovascular thrombosis (**Figure 1**). In this review and others, NETs have been shown to have a significant role in pathogenic thrombosis through platelet and PMN recruitment to the endothelial wall, subsequent activation and NETosis, and proteomic activation of the intrinsic and extrinsic coagulation cascades, ultimately leading to thrombosis (3, 13, 14, 16, 17, 31, 32, 45, 50, 60, 61).

This unique development in the etiology of immune-mediated thrombosis affords novel targets for the prevention of pathologic thrombosis in a susceptible patient (**Table 1**) (4, 25, 55, 62–70). One obvious target for the prevention of NET-mediated VTE is PAD4 inhibition (25, 60). Enzymatic inhibition of PAD4 would prevent NETosis, and hopefully, its prothrombotic effects as well; however, this may also leave the host susceptible to bacterial infection (71). DAMP-mediated NETosis by HMGB-1 is also a potential therapeutic target. HMGB-1 is released from damaged cells and expressed on the surface of activated platelets and leads to immune system activation *via* RAGE, TLR2, and TLR4 (5, 72, 73). HMGB-1-mediated PMN activation subsequently contributes to microvascular thrombosis and NETosis (31, 74). HMGB-1 is cleaved by thrombomodulin–thrombin complexes *in vivo*, and recombinant thrombomodulin is presently approved for the treatment of DIC in Japan and is in phase III trials here in the United States (5, 70).

Other targets include the components of PMN and platelet recruitment to the endothelium. Specifically, blockade of platelet alpha-granule release or endothelial Weibel–Palade body release would decrease P-selectin and vWF-mediated platelet and PMN recruitment to the endothelium decreasing NETosis (24, 32, 75, 76). Similarly, blockade of P-selectin (24), vWF A1 domain (16), glycoprotein Ib, or integrin beta-2 may reach a similar end, however, more specifically (32). Lastly, the vWF degradation enzyme, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif – member 13), may

FIGURE 1 | Diagram of theoretical mechanism of NET-mediated microvascular thrombosis in sepsis. (A) Sepsis-induced endothelial activation leading to the release of vWF (black lines) from endothelial cell Weibel–Palade bodies provoking the recruitment of leukocytes and platelets to the endothelial wall *via* vWF A1 domain–beta-2 integrin interaction (vWF– PMN interaction; orange diamond) and vWF A1 domain–glycoprotein Ib interaction (vWF–platelet interaction; green diamond). PMNs are also recruited to the endothelium *via* P-selectin-to-PSGL-1 interaction (black diamond) inducing neutrophil rolling. (B) Neutrophil and platelet activation by LPS (yellow triangle) *via* TLR4 (blue triangle). Platelet activation leading to the release of PF4 (blue square) and expression of P-selectin (black diamond) and HMGB-1 (pink diamond) further stimulate NETosis [purple lines of decondensed chromatin with attached histones (yellow circles)]. NETosis also leads to autophagy-induced release of TF, and NETs further stimulate FXII activation, inciting both the intrinsic and extrinsic coagulation cascades. (C) Thrombin activation by factor Xa leads to the conversion of fibrinogen to fibrin (yellow lines) culminating in microvascular thrombosis.

be given in a recombinant form (rADAMTS13) to degrade vWF and prevent PMN recruitment with subsequent NETosis (77). Although, each of the above-listed countermeasures may result in mild immunodeficiency, if given in a dose-dependent manner,

they may abrogate the pathologic immune-mediated thrombosis without sacrificing immune competence.

Despite the relatively recent discovery of NETs as a contributor to VTE, some long-standing traditional VTE therapies already affect NETs. Polyanionic heparin, long considered the gold standard therapy for prevention and treatment of VTE, has a secondary effect of displacing histones from chromatin (78, 79). This allows increased accessibility of nucleases to the exposed chromatin, further permitting degradation of NETs (78, 79). Similarly, aspirin, recently shown to decrease risk of recurrent VTE (80), inhibits NETosis *in vitro* by inhibition of thromboxane A2 synthesis (63, 81). Finally, clopidogrel has also been shown to decrease inflammation and platelet-mediated expression of soluble P-selectin, further decreasing PMN– platelet interactions and hence NETosis (68). It is currently unknown as to the effects of the new oral anticoagulants on NETs, although this may represent an important area for future investigation.

Thrombolysis has become a key weapon in the arsenal against pathologic thrombosis; however, not all thrombotic events are susceptible to thrombolysis with tissue plasminogen activator (tPA) (82). Indeed, the addition of DNA and histones to a fibrin matrix has been shown to make artificial thrombus more stable, more rigid, and more resistant to tPA, and this is partially remedied by DNase (83). Preliminary data from murine models of VT demonstrate inhibition of thrombus formation with DNase instillation prior to and during IVC stenosis (16, 17). In another study of limb ischemia-reperfusion injury, DNase instillation decreased tissue NETs but did not effect end tissue damage or inflammatory infiltrate (84). In spite of this, more recent studies have shown decreased inflammation, increased tissue perfusion, and improved survival with (1) late DNase treatment in cecal ligation puncture (CLP) model of murine sepsis (55), (2) concurrent DNase and tPA treatment in a rat model of myocardial ischemia (85), and (3) preemptive DNase treatment in a rat model of renal ischemia-reperfusion injury (86). Combination tPA and DNase therapy for thrombolysis of acute ischemic events has yet to be studied in human patients but will likely constitute a major area of research in the near future.

There are still many unanswered questions in the field of NETosis. What are the exact proteomic mechanisms that lead to neutrophil activation and subsequent NET formation? What is the role or RAGE, and does this have implications for diabetics? What is the intracellular cascade that leads to PAD4 induction? After NETosis and thrombosis, how are NETs naturally removed from the resolving thrombus? Do NETs play a role in the post-thrombotic syndrome? Will the addition of DNase to tPA broaden the spectrum of patients that can be treated with thrombolysis as opposed to surgery? These questions and more will be answered with time and continued diligent, meticulous, and conscientious research. We look forward to the future with great expectations for upcoming discoveries in the field of NETosis.

#### REFERENCES

### AUTHOR CONTRIBUTIONS

Dr. Andrew S. Kimball did the literature review, wrote the paper, and approved of the submission. Drs. Andrea T. Obi and Jose A. Diaz did a literature review, wrote sections of the paper, and approved of the submission. Dr. Peter K. Henke wrote and edited the paper, approved of the submission, and is responsible for the content.

#### ACKNOWLEDGMENTS

Special thanks to Whitney Townsend, Librarian at the University of Michigan Taubman Health Sciences Library, for her assistance with the comprehensive literature search.

#### FUNDING

Andrew S. Kimball is funded by an NIH T32 Research Fellowship (NIH-T32 HL076123).

chronic coronary disease. *Thromb Res* (2010) 125(1):61–6. doi:10.1016/j. thromres.2009.03.008

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Kimball, Obi, Diaz and Henke. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Neutrophil extracellular Traps in ANCA-Associated vasculitis

*Daniel Söderberg1 \* and Mårten Segelmark1,2*

*1Department of Medical and Health Sciences, Linköping University, Linköping, Sweden, 2Department of Nephrology, Linköping University, Linköping, Sweden*

A group of pauci-immune vasculitides, characterized by neutrophil-rich necrotizing inflammation of small vessels and the presence of antineutrophil cytoplasmic antibodies (ANCAs), is referred to as ANCA-associated vasculitis (AAV). ANCAs against proteinase 3 (PR3) (PR3-ANCA) or myeloperoxidase (MPO) (MPO-ANCA) are found in over 90% of patients with active disease, and these ANCAs are implicated in the pathogenesis of AAV. Dying neutrophils surrounding the walls of small vessels are a histological hallmark of AAV. Traditionally, it has been assumed that these neutrophils die by necrosis, but neutrophil extracellular traps (NETs) have recently been visualized at the sites of vasculitic lesions. AAV patients also possess elevated levels of NETs in the circulation. ANCAs are capable of inducing NETosis in neutrophils, and their potential to do so has been shown to be affinity dependent and to correlate with disease activity. Neutrophils from AAV patients are also more prone to release NETs spontaneously than neutrophils from healthy blood donors. NETs contain proinflammatory proteins and are thought to contribute to vessel inflammation directly by damaging endothelial cells and by activating the complement system and indirectly by acting as a link between the innate and adaptive immune system through the generation of PR3- and MPO-ANCA. Injection of NET-loaded myeloid dendritic cells into mice results in circulating PR3- and MPO-ANCA and the development of AAV-like disease. NETs have also been shown to be essential in a rodent model of drug-induced vasculitis. NETs induced by propylthiouracil could not be degraded by DNaseI, implying that disordered NETs might be important for the generation of ANCAs. NET degradation was also highlighted in another study showing that AAV patients have reduced DNaseI activity resulting in less NET degradation. With this in mind, it might be that prolonged exposure to proteins in the NETs due to the overproduction of NETs and/or reduced clearance of NETs is important in AAV. However, not all ANCAs are pathogenic and some might possibly also aid in the clearance of NETs. A dual role for ANCAs in relation to circulating NET levels has been proposed because a negative correlation was observed between PR3-ANCA and NET remnants in patients in remission.

Keywords: neutrophil extracellular traps, ANCA-associated vasculitis, ANCA, NET, small-vessel vasculitis, NET remnants

#### *Edited by:*

*Mariana Julieta Kaplan, National Institutes of Health, USA*

#### *Reviewed by:*

*Angelo A. Manfredi, Vita-Salute San Raffaele University, Italy Peter Korsten, University Medical Center Göttingen, Germany*

*\*Correspondence: Daniel Söderberg<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 30 April 2016 Accepted: 15 June 2016 Published: 30 June 2016*

#### *Citation:*

*Söderberg D and Segelmark M (2016) Neutrophil Extracellular Traps in ANCA-Associated Vasculitis. Front. Immunol. 7:256. doi: 10.3389/fimmu.2016.00256*

### ANTINEUTROPHIL CYTOPLASMIC ANTIBODY-ASSOCIATED VASCULITIS

Vasculitides are inflammations in the walls of blood vessels, and they can affect any organ system in the body. They are divided into broad groups based on the size of the vessels predominantly being affected. A subgroup of small-vessel vasculitides is characterized by a scarcity of immune depositions (pauci-immune) and the presence of antineutrophil cytoplasmic antibodies (ANCAs) and is referred to as ANCA-associated vasculitis (AAV) (1). AAV comprise three diseases, including granulomatosis with polyangiitis [GPA, previously known as Wegener's granulomatosis (2)], microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA, previously known as Churg–Strauss syndrome) (3). GPA and EGPA share the feature of necrotizing granulomatous inflammation of the lower respiratory tract, whereas MPA is characterized by the absence of this component. Also, GPA often affects the upper respiratory tract and can result in rhinitis, otitis, and cartilage destruction, while eosinophilia and asthma are defining features of EGPA. Renal involvement is observed in as many as 90% of the patients with MPA, compared to 80% of the patients with GPA and 45% in EGPA. All three diseases affect the skin, joints, eyes, and nerves to various extents (1, 4). There is also an increased incidence of venous thromboembolism in AAV patients, especially during active disease (5, 6). AAVs are relapsing–remitting diseases, and 50% of the patients have a relapse within 5 years of successful treatment. The mortality rate is around 80% at 1 year when left untreated, but with current treatments, the mortality rate is reduced to 25% within 5 years (7).

Autoantibodies specific for proteinase 3 (PR3) (PR3-ANCA) or myeloperoxidase (MPO) (MPO-ANCA) are found in over 90% of patients with active disease (8), and these are important as diagnostic tools. The association between PR3- and MPO-ANCAs and active disease in AAV suggests a pathogenic role for the autoantibodies, and such a role is supported by results from animal models (9, 10) and *in vitro* studies showing that PR3- and MPO-ANCAs can activate neutrophils to produce reactive oxygen species (ROS) and proteolytic enzymes (11). ANCA-induced neutrophil activation also leads to increased adhesion of the neutrophils (12) and the activation of the alternative complement pathway (13) with the generation of C5a. C5a in turn potentiates the inflammatory response by priming neutrophils and acting as a chemoattractant to recruit more neutrophils to the inflammatory site (14). However, ANCA levels do not conclusively predict relapses (15, 16), and there is an unmet need for biomarkers for this purpose.

#### NEUTROPHIL EXTRACELLULAR TRAPS

Neutrophil extracellular traps (NETs) were first described in 2004 as a means for neutrophils to trap and kill bacteria (17) and are released as a result of a programed cell death mechanism referred to as NETosis (18, 19). NETs consist of a DNA backbone and various proteins with proinflammatory characteristics, such as histones, high-mobility group box 1 (HMGB1), LL37, neutrophil elastase (NE), calprotectin (S100A8/S100A9, MRP8/14), and, interestingly, MPO (**Figure 1**) and PR3 (20, 21). All described ANCA antigens are components of NETs. NETosis depends on a cascade of events that lead to the mixing of nuclear, cytoplasmic, and granular components before the NETs are released into the surrounding matrix (18). NETosis has been shown to depend on NADPH oxidase and ROS production as well as on autophagy and histone citrullination. Peptidyl arginine deiminase 4 (PAD4), NE, and MPO have been shown to play important roles in this signaling pathway (18, 22, 23). More recently, other forms of NETosis have also been described, including NETosis with the release of mitochondrial DNA (mtDNA ETs) (24) instead of nuclear DNA and ROS-independent NETosis (25–27). Interestingly, when releasing mtDNA ETs, the neutrophils can also remain viable (24). In addition to their role as antimicrobial agents, NETs of both nuclear and mitochondrial origin have also been connected to various autoinflammatory and autoimmune diseases (28–33).

Figure 1 | Visualization of MPO in NETs from human neutrophils. Neutrophils isolated from human peripheral whole blood were cultured for 4 h at 37°C with 25 nM PMA. NETs were then visualized by immunofluorescence microscopy using a 40× objective. (A) DNA, the backbone of NETs, was labeled with DAPI (blue). (B) MPO (clone 2B11), a granulae protein within the NETs (17), was labeled with a Dylight 488-conjugated antibody (green). (C) DNA and MPO (merged) colocalized in the NETs. NETs, neutrophil extracellular traps; PMA, phorbol-12-myristate-13-acetate. Blood samples were collected after obtaining informed consent in accordance with the declaration of Helsinki, and the study was approved by the Regional Ethical Review Board in Linköping. This figure is not intended to be quantitative, but only to serve as a representative image of common prior knowledge regarding the presence of MPO in NETs (17).

 Traditionally, it has been assumed that these neutrophils die by necrosis, but in 2009, Kessenbrock et al. showed that NETs were present in the glomeruli in kidney biopsies from AAV patients (34). They reported the presence of NETs as co-localizations of DNA, histones, and the granule proteins PR3, LL37, NE, and MPO in various combinations (34). This phenomenon was later confirmed by others (35–38). Although the method for detecting NETs in glomeruli was rather similar in these studies, i.e., visualization of DNA and histones (although some looked at citrullinated histones) in combination with the granule proteins already described – one study also reported the presence of PAD4 in the NETs, which is necessary for histone citrullination (35), and another study detected LAMP2, which is also an ANCA antigen (36, 39).

Neutrophil extracellular traps have also been shown to be present in skin lesions (40, 41) and thrombi from AAV patients (38, 42). In the studies investigating NETs in skin lesions, the presence of NETs was based on extracellular MPO (40, 41) or on DNA in combination with MPO (41). The presence of NETs in thrombi was defined not only as co-localizations of DNA and MPO but also as citrullinated histones alone (38). Another study also defined NETs based only on the presence of citrullinated histones (42).

### INCREASED LEVELS OF NETs AND NET-ASSOCIATED PROTEINS IN THE CIRCULATION OF AAV PATIENTS

In addition to the presence of NETs in various lesions from AAV patients, it has been shown that these patients also have elevated levels of NETs in the circulation (34, 43–46) (**Table 1**). In these studies, NETs were defined as nucleosome/MPO complexes (34, 43, 46), total DNA or DNA/MPO or citrullinated histone 3 (H3) complexes (45), DNA/histone complexes (46), or as nuclear DNA or mtDNA (44). There are also several observations regarding circulating neutrophil components that are the main constituents of NETs. Important examples are HMGB1, calprotectin (S100A8/S100A9, MRP8/14), PR3, MPO, and NE (46–55) (**Table 1**). The study measuring calprotectin used longitudinally collected samples from the NORAM trial and found that calprotectin levels correlated with disease activity (47), and the studies measuring NE observed a correlation between NE and Birmingham Vasculitis Activity Score (i.e., disease activity) (51). However, the presence of these proteins in the circulation does not reveal whether they are released as a result of NETosis or by other mechanisms, but it was recently shown that the levels of MPO and NE correlate with the levels of DNA/MPO complexes in the circulation (46). The capability of using NETs as a biomarker to monitor disease activity in AAV has not been evident in previous studies. No study has so far measured the levels of NETs longitudinally in patients at multiple time points.

#### Table 1 | NET-associated proteins and structures present in the circulation of AAV patients.

*Numbers in parenthesis indicate referenced publication.*

*AAV ANCA-associated vasculitis; HC, healthy blood donors;* +*, increased levels; nd, not determined; PR3, proteinase 3; HMGB1, high-mobility group box 1 protein; MPO, myeloperoxidase; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; NE, neutrophil elastase.*

In some cross-sectional studies, the levels of NETs have been measured in patients during both remission and active disease, but with inconclusive results regarding their correlation with disease activity (43, 45).

### PROINFLAMMATORY ASPECTS OF NETs IN AAV

Neutrophil extracellular traps have previously been described as double-edged swords of innate immunity (56), considering that they are involved in both fighting pathogens and in contributing to autoinflammatory and autoimmune conditions. Various proinflammatory aspects of NETs in general might also be important in the pathogenesis of AAV. For example, NETs can cause endothelial damage (57–59) and can activate the alternative complement pathway (60), which, as already mentioned, plays an important role in amplifying the inflammatory process in AAV. Further, anti-histone antibodies have been shown to ameliorate experimental glomerulonephritis, emphasizing the proinflammatory aspect of histones in the NETs (61). It has also been shown that the presence of histones in NETs can contribute to thrombus formation (62) and that the presence of tissue factor (63, 64) in NETs can contribute to the generation of thrombin. In turn, it has been demonstrated that activated platelets can stimulate neutrophils to release NETs and that platelet-induced NETs propagate deep vein thrombosis in mice (65). Others have shown that HMGB1 expressed on platelets mediate the formation of platelet-induced NETs and that this process is dependent on autophagy (66), and in mice, it has been shown that plateletderived p-selectin can induce NETosis (67). Increased levels of platelet-neutrophil aggregates and soluble P-selectin have been observed in the circulation of AAV patients during active disease and to correlate with disease activity (46). Additionally, HMGB1 has also been shown to potentiate the effect of ANCAs on NET formation (68). Oxidized mtDNA ETs released from neutrophils in systemic lupus erythematosus (SLE) have been shown to possess proinflammatory characteristics (33), and the role of mtDNA in general as a danger-associated molecular pattern has been extensively described (69).

#### SPONTANEOUS NET FORMATION *IN VITRO*

Earlier studies have shown that neutrophils from AAV patients are less prone to undergo apoptosis (70), suggesting that these neutrophils are more prone to other forms of cell death. Indeed, *in vitro* studies have shown that neutrophils from AAV patients are more prone to release NETs spontaneously than neutrophils from healthy blood donors (36, 43, 71). A subpopulation of neutrophils, referred to as low-density granulocytes (LDGs), have been shown to spontaneously release NETs significantly more than normal-density neutrophils, and these LDGs haves been proposed to be the major source of NETs in AAV (71). However, the same study also showed that normal-density neutrophils from AAV patients spontaneously released more NETs than normal-density neutrophils from healthy blood donors (71). More detailed studies of LDGs in SLE have revealed that LDGs express increased levels of mRNA of various immunostimulatory bactericidal proteins and alarmins compared to normal-density neutrophils (59). It is important to note that during the various isolation procedures normally used to obtain neutrophils from peripheral whole blood, LDGs will not be included because they will be found in the fraction of peripheral blood mononuclear cells. This is important to consider in future *in vitro* studies of neutrophils and NET formation.

### ANCAs AS MEDIATORS OF NETosis

In addition to the effects already ascribed to PR3- and MPO-ANCA in terms of neutrophil activation, they are also capable of inducing NETosis (**Figure 2**) (34). Although the exact mechanism for neutrophil activation by ANCAs is not clear, full activation requires binding of autoantibodies to both Fc-receptors and to PR3/MPO on the surface of neutrophils (72). It has been suggested that neutrophil activation, in this case evaluated as ROS production by MPO-ANCA, is epitope-specific, that epitope specificity varies with disease activity and that ANCAs activate neutrophils more robustly during active disease (73). Furthermore, *in vitro* studies have shown that neutrophils from patients are more easily activated (they produce more ROS) by ANCAs (in this case PR3-ANCA) than neutrophils from healthy blood donors (74). It has previously been shown that neutrophils from AAV patients possess increased membrane expression of PR3 (75, 76), which could possibly be explained by disrupted epigenetic silencing of the PR3 and MPO gene in these patients (77). However, in the study by Ohlsson et al. the results could not be explained by increased PR3 expression on the cell surface of neutrophils from patients or the ANCA levels (74). Rather, epitope specificity and affinity seemed to be of importance for the antibodies' ability to activate neutrophils (74). It has also been shown that MPO-ANCA has higher affinity for MPO during active disease and that MPO-ANCA induces more NETs during active disease (78), and the observation that the affinity for MPO-ANCA is important for the ability to induce NETs was recently confirmed by another group (79). In summary, it seems that both epitope specificity and affinity are important for neutrophil activation by ANCAs and that at least the affinity is important for their ability to induce NETs.

### NETs: BRIDGING INNATE AND ADAPTIVE IMMUNITY

It has been shown using NETotic neutrophils from mice that MPO and PR3 can be taken up from the NETs by myeloid dendritic cells (mDCs) and that injection of NET-loaded mDCs into mice results in circulating MPO- and PR3-ANCA and development of AAV-like disease (41). The addition of DNaseI to the *in vitro* cultures prevented PR3 and MPO uptake by the mDCs from the NETs, and when mice were injected with those mDCs, the mice did not develop disease (41). In the same study, injection of mDCs cocultured with apoptotic neutrophils into mice also caused autoantibody production, but those mice did not develop AAV-like disease. These experiments indicate that NETs show higher immunogenicity than apoptotic cells and that the structural integrity of the NETs is important for transferring NETantigens to mDCs and the subsequent production of pathogenic autoantibodies. This is in line with a previous study showing that rats immunized with apoptotic neutrophils do develop ANCAs, but not disease (82). In another study, rats were immunized with NETs induced by phorbol-12-myristate-13-acetate (PMA) and propylthiouracil (PTU) (which together induced abnormal NETs that could not be degraded by DNaseI) or were given PTU orally in combination with PMA (intraperitoneal injection), and these rats developed MPO-ANCA and pulmonary capillaritis or glomerulonephritis and pulmonary capillaritis, respectively (80). This resembles the situation in humans, where over 20% of patients with Graves' disease treated with PTU develop MPO-ANCA and some also AAV-like disease (83, 84).

### NET FORMATION VS. CLEARANCE: THE IMPORTANCE OF BALANCE

The studies described earlier imply that NETs can act as a link between the innate and adaptive immune system with the production of pathogenic ANCAs. With this in mind, it might be that prolonged exposure to the proteins in the NETs due to the overproduction of NETs and/or reduced clearance of NETs is important in AAV. In line with this, it has been shown that AAV patients have reduced capacity to degrade NETs *in vitro* (78). This could possibly be due to the reduced DNaseI activity observed in these patients compared to healthy blood donors, although DNaseI activity did not correlate with disease activity. Thus, the elevated levels of NETs in the circulation of AAV patients might

also be explained by the reduced capacity to clear the NETs from the circulation. Interestingly, low levels of both PR3-ANCA and MPO-ANCA can be found in the circulation of healthy individuals (81), indicating that the presence of ANCAs does not necessarily lead to disease development. Rather, ANCAs might be part of the repertoire of natural antibodies that are important for maintaining homeostasis (85). In line with this, a dual role for ANCAs was recently suggested, where the autoantibodies in addition to inducing NET formation can also aid in the clearance of NETs (**Figure 2**) (43), possibly through opsonization and the formation of immune complexes. This hypothesis was proposed because a negative correlation was observed between PR3-ANCA and circulating NET remnants in AAV patients in remission (43). As others have shown that the pathogenicity of ANCAs seems to vary with both epitope specificity (73) and affinity (78) and

from Ref. (43) with permission from Oxford University Press.

that these parameters change with disease activity, it appears that ANCAs might play different roles at different stages of AAV. Together, these studies might suggest how and why all individuals can possess ANCAs but only some develop AAV.

#### INFECTIONS AND ANCAs

Antineutrophil cytoplasmic antibodies are common in chronic infections, such as *Pseudomonas aeruginosa* infections, in patients with cystic fibrosis, tuberculosis, HIV, and infective endocarditis (86–89). Infections are also implicated in the pathogenesis of AAV and as a trigger of relapses. Molecular mimicry, either directly (90) or indirectly through autoantigen complementarity (91), is the traditional way to explain the relationship between AAV and infection. However, infections lead to neutrophil activation, which triggers NETosis. Lipopolysaccharide-activated platelets can also activate neutrophils to release NETs (92), and this suggests an indirect way in which bacteria can contribute to NETosis as well as to the coagulation cascade and thrombosis formation discussed earlier. In sepsis, the liver sinusoids are filled with neutrophils undergoing NETosis (93), and in infective endocarditis, a role for NETs has also been described (94). ANCAs are found in up to 20% of patients with endocarditis (95), and many of these patients have symptoms resembling vasculitis, such as fever, increased CRP, weight loss, malaise, multiform skin lesions, and renal involvement (1, 96–98).

#### CONCLUDING REMARKS/DISCUSSION

This review has outlined the role of NETs in the pathogenesis of AAV. There is compelling evidence that NETs contribute to vessel inflammation directly by damaging endothelial cells and by activating the complement system and indirectly by acting as a link between the innate and adaptive immune system through the generation of PR3-ANCA and MPO-ANCA. This can lead to a vicious circle because ANCAs can activate neutrophils. However, ANCA pathogenicity is dependent on both affinity

#### REFERENCES

and epitope specificity, and there also seem to be ANCAs that are non-pathogenic and even beneficial. NETs are most probably formed at a constant rate in healthy individuals, but NET formation can become highly elevated by infections, certain drugs, and possibly by epigenetic changes as one age. Increased NET formation must be balanced by clearance mechanisms, which seem to include DNaseI and possibly autoantibodies with ANCA specificity. We hypothesize that under unfavorable circumstances some individuals (partly depending on genetics) develop pathogenic autoantibodies that can activate neutrophils thus creating a vicious circle resulting in widespread vessel wall inflammation.

#### AUTHOR CONTRIBUTIONS

DS has written most of the text, but in close collaboration with MS.

#### FUNDING

This review has been funded by Ingrid Asps Foundation, Swedish Society of Nephrology, and Swedish Renal Foundation.

adhesion. *Arthritis Rheum* (2000) 43(6):1337–45. doi:10.1002/1529-0131 (200006)43:6<1337::AID-ANR16>3.0.CO;2-M

cell death modality. *Cell Death Differ* (2011) 18(4):581–8. doi:10.1038/ cdd.2011.1

associated autoimmunity. *Blood* (2012) 120(15):3007–18. doi:10.1182/ blood-2012-03-416156

and anti-proteinase 3 antibodies. *Arthritis Rheum* (2000) 43(1):226–31. doi:10.1002/1529-0131(200001)43:1<226::AID-ANR27>3.0.CO;2-Q

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Söderberg and Segelmark. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Neutrophil Extracellular Traps Open the Pandora's Box in Severe Malaria

*Sebastian Boeltz1 , Luis E. Muñoz1 , Tobias A. Fuchs <sup>2</sup> and Martin Herrmann1 \**

*1Department of Internal Medicine 3 – Rheumatology and Immunology, Friedrich-Alexander-University Erlangen-Nürnberg (FAU) and Universitätsklinikum Erlangen, Erlangen, Germany, 2 Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany*

Keywords: severe malaria, cerebral malaria, neutrophil extracellular traps (NETs), Disseminated intravascular coagulation (DIC), vascular occlusion

### INTRODUCTION

Malaria is transmitted by mosquitoes and kills 2,000 humans in sub-Saharan Africa each day (1). Most of the victims are children younger than 5 years of age. Out of the five *plasmodium* species causing malaria, *Plasmodium falciparum* is responsible for most of the severe and fatal infections (2). Upon feeding, the female *Anopheles* mosquito inoculates sporozoites that seek out the hosts' liver within minutes. After a first replication cycle in hepatocytes for 5–8 days, rupture releases the parasite into circulation where erythrocytes are infected. During the second replication cycle *plasmodium* consumes the contents and energy reserves of the erythrocytes, changes the membrane to enable adherence to the vessel walls (3), and produces waste, including crystalline urate (MSU) and hemozoin (4). After 24, 48, or 72 h, depending on the species, the infected erythrocytes synchronously burst and release their content into the circulation and cause first clinical symptoms (**Figure 1**). The key pathogenic processes that cause severe malaria include rapid increase of infected erythrocytes, destruction of both infected and uninfected erythrocytes, acute inflammation, and microvascular obstruction. The final outcome is a reduced tissue perfusion that leads to downstream events compromising the cellular metabolism (5). We hypothesize that intravascular formation of neutrophil extracellular traps (NETs) contributes to the vasculopathy, driving severe malaria. NETs are web-like structures of highly modified chromatin and antimicrobial peptides released by activated neutrophils (6). In this article, we discuss the evidence supporting the role of NET formation in the pathogenesis of malaria and propose potential therapeutic interventions.

#### *Edited by:*

*Janos G. Filep, Université de Montréal, Canada*

#### *Reviewed by:*

*Stavros Giaglis, University of Basel, Switzerland Mohib Uddin, AstraZeneca, Sweden*

*\*Correspondence: Martin Herrmann<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 26 May 2017 Accepted: 10 July 2017 Published: 28 July 2017*

#### *Citation:*

*Boeltz S, Muñoz LE, Fuchs TA and Herrmann M (2017) Neutrophil Extracellular Traps Open the Pandora's Box in Severe Malaria. Front. Immunol. 8:874. doi: 10.3389/fimmu.2017.00874*

NETs CAUSE VASCULOPATHY IN MALARIA

NETosis has evolved as an important innate strategy for killing extracellular pathogens (7). The sticky chromatin fibers of NETs immobilize pathogens and inhibit their spread. Uric acid is to be found in infected erythrocytes as a precipitate together with high concentrations of hypoxanthine and the insoluble hemoglobin metabolite hemozoin. During rupture of infected erythrocytes, these compounds are abruptly released into the circulation. In this scenario, insoluble uric acid and hemozoin encounters the immune system as crystalline matter (4) and the former crystallizes in the sodium-rich plasma as MSU (8). These crystals are potent inducers of NETs formation, even in the presence of plasma proteins (8). Hemozoin activates leukocytes through an inflammasomedependent pathway (9). Indeed, it has been described that NETs with trapped parasites circulate in children infected with *P. falciparum* (10).

In flow chambers perfused with blood, NETs promote fibrin deposition and bind fibronectin and von Willebrand factor, important for platelet adhesion and thrombus propagation (11). NET fibers contain further procoagulant factors. Neutrophil elastase in NETs may proteolytically inactivate tissue factor pathway inhibitor (12). Furthermore, tissue factor can be deposited on extracellular

During the growth within the erythrocytes, the parasite consumes the cells' cytoplasm and energy resources (C). Replicating parasites produce and store waste-like uric acid (orange) and hemozoin (D). During the blood stage, the parasite population increases 6–20 times per cycle. If the resources of the erythrocyte are consumed, rupture of the host erythrocyte releases the parasites and their waste including uric acid (E), which tends to form MSU crystals in the sodium-rich plasma with time (F). MSU crystals induce endothelial inflammation and NET formation upon contact with circulating neutrophils (G). In healthy conditions, the endothelium is a thin, monolayered, tight structure (H). Intravascular NET autonomously narrow or even clog the post-capillary venules, a process that can be accelerated by the activation of the coagulation cascade (I). The activation of the coagulation cascade, the recruitment of platelets in direct interplay with intravascular NETs, and a plethora of pro-inflammatory mediators cause microvascular occlusion and disrupt the blood–brain barrier, respectively (J).

traps, especially those derived from eosinophils, and factor XII is reportedly present and active on NETs (13). The negatively charged DNA in NETs may provide a scaffold for factor XII activation, which is aided by platelets, but the mechanism is still elusive. Fibrin and NETs synergize to augment the microbial defense in a process referred to as immunothrombosis (14). We hypothesize that the formation of NETs and the activation of the coagulation cascade is a double-edged sword. On the one side, they form a barrier on the endothelial surface protecting it from damage by MSU crystals (15) and hemozoin. On the other side, their occurrence limits the bore diameter of capillaries and postcapillary venules. This is prone to restrict perfusion of the end organs. In worst case, intravascular NETs autonomously clog the vessels or initiate disseminated intravascular coagulation (DIC), a condition involving hemorrhage and microthrombosis that synergize in the abolishment of tissue perfusion (**Figure 1**). The inflammatory mediators released during the coupled processes of NET formation and coagulation cause opening of the neuroimmunological blood–brain barrier and precipitate the clinical disease as, often fatal, cerebral malaria.