AUTREV-01693; No of Pages 8 Autoimmunity Reviews xxx (2015) xxx–xxx

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Review

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Boris Pinegin a, Nina Vorobjeva b,⁎, Vladimir Pinegin c a

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Article history: Received 3 February 2015 Accepted 12 March 2015 Available online xxxx

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Keywords: Neutrophils Neutrophil extracellular traps NETosis Alarmins Psoriasis Atherosclerosis LL-37/DNA complex

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The pathogenesis of many autoimmune diseases is initially based on a redundant or prolonged activation of the innate immune system. It was suggested that an excessive activation of the innate immunity is often the result of a chronic inflammatory process in the organism. This inflammation can be induced by exogenous and endogenous alarm factors, or alarmins. We believe that the recently discovered neutrophil extracellular traps, or NETs, completely meet the criteria of alarmins. This review summarizes current knowledge concerning the general characteristics of NETs, their antimicrobial properties, and their role in the development of chronic inflammatory processes that underlie the pathogenesis of psoriasis and atherosclerosis. Studies on the NETosis can provide the foundation for developing new diagnostic methods and effective treatment of chronic inflammatory and autoimmune diseases. © 2015 Elsevier B.V. All rights reserved.

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1. Introduction . . . . . . . . . . . . . . . . . . . . . 2. General characteristics of NETs . . . . . . . . . . . . . 3. Antimicrobial activity of NETs . . . . . . . . . . . . . 4. The concept of NETs as alarm signals . . . . . . . . . . 5. The role of NETs in the pathogenesis of psoriasis . . . . . 6. The role of NETosis in the pathogenesis of atherosclerosis. 7. Conclusions . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . Uncited reference . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

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The basis of pathogenesis of many autoimmune diseases initially is a redundant or prolonged activation of the innate immune system, resulting in an excessive activation of adaptive immunity. An excessive

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Institute of Immunology, Federal Medical Biological Agency, Kashirskoe Shosse 24/2, 115478 Moscow, Russia Biology Faculty, Lomonosov Moscow State University, Lenin Hills 1/12, 119991 Moscow, Russia I.M. Sechenov First Moscow State Medical University, Moscow, Russia

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Neutrophil extracellular traps and their role in the development of chronic inflammation and autoimmunity

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⁎ Corresponding author. Tel.: +7 495 4371773, +7 9168208837 (mobile). E-mail addresses: [email protected] (B. Pinegin), [email protected] (N. Vorobjeva).

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activation of the innate immunity is often the result of a chronic inflammatory process in the organism. Exogenous and endogenous factors may induce this process. Exogenous factors include the most conserved structures of microorganisms, i.e. pathogen-associated molecular patterns (PAMPs) [1]. Endogenous factors are usually substances synthesized and secreted from cells under the influence of various damaging agents including PAMPs or appearing as a result of impaired cellular metabolism. These endogenous factors are called alarmins [2,3], and they can cause sterile inflammation.

http://dx.doi.org/10.1016/j.autrev.2015.03.002 1568-9972/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Pinegin B, et al, Neutrophil extracellular traps and their role in the development of chronic inflammation and autoimmunity, Autoimmun Rev (2015), http://dx.doi.org/10.1016/j.autrev.2015.03.002

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2. General characteristics of NETs

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In 2004, Zychlinsky and coauthors [4] discovered that neutrophils were able to kill pathogens outside the cells by releasing chromatin fibrils, or neutrophil extracellular traps. Since neutrophils lose their viability in the process of trap formation, Steinberg and Grinstein [9] denoted this form of neutrophil cell death as “NETosis” in 2007. It should be noted that trap formation has also been shown for eosinophils [10], mast cells [11], and monocytes/macrophages [12]. Since chromatin is released into the extracellular milieu not only by neutrophils but also by other types of cells, the broader term used for this mechanism is ETosis (from Extracellular Traps, ETs). NETs have a unique ultrastructure. Their framework is formed by chromatin filaments ~15–17 nm in diameter [4] consisting of modified nucleosomes [13]. This framework is dotted with globular structures about 50 nm in diameter [4,5,14]. Chromatin filaments are composed of DNA and histones, where histones account for 70% of the total proteins of the traps. The composition of filaments includes nuclear protein amphoterin HMGB1 (high-mobility group box 1), which is a part of the chromatin of the intact cell. Globular structures consist of components of primary and secondary granules of neutrophils, such as neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G, proteinase 3, bactericidal proteins: BPI (cationic bactericidal/permeabilityincreasing protein), calgranulin, α-defensins, lactoferrin, a fragment of the protein cathelicidin hCAP18 — the peptide LL-37, and pentraxin PTX3. Among the components of the tertiary granules, NETs include matrix metalloproteinase-9 (MMP-9) and peptidoglycan recognition protein-S (PGRP-S) [15–17]. Surprisingly, NETs may not only have the morphology of elongated thin filaments. They can also be cloud-like structures that occupy a 10–15-fold greater area compared to the initial cell size. NETs are formed because of a unique form of cell death: an initial loss of all intracellular membranes is followed by the disintegration of the cytoplasmic membrane. To date, little is known about the mechanisms of NETosis. Nevertheless, neutrophils are known to undergo major morphological modifications in the process of NETosis. Several minutes after activation, the cells lie flat, being tightly attached to the substrate. During the next hour the nucleus loses its lobules, chromatin is decondensed, and the inner and outer leaflets of the nuclear membrane separate. The disintegration of the granules occurs simultaneously. Within another hour, the nuclear membrane breaks up into separate vesicles, while the nucleoplasm and cytoplasm merge into a homogenous mass. Finally, the cells become rounded and seem to be contracted until

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the cytoplasmic membrane is broken; then the cell contents are excreted to the exterior and form bundles of thin filaments, i.e. NETs. On the molecular level, NET formation is a gradual process with several successive steps: (1) ROS generation; (2) transport of neutrophil elastase, and, subsequently, myeloperoxidase from the granules to the nucleus; (3) histone modification, and, finally, (4) disruption of cytoplasmic membrane and release of chromatin. Of much interest are the details of these steps, because they constitute a unique mechanism of cell death, i.e. NETosis. It has been shown that ROS are necessary for NETosis. The most powerful inducer of NETosis is phorbol 12-myristate 13-acetate (PMA), and it is also the most potent inducer of ROS generation. ROS are probably responsible for the oxidative modification of DNA, proteins and other macromolecules, which make them more susceptible to the influence of neutrophil enzymes [18]. In neutrophils, ROS are formed during a “respiratory burst” involving the NADPH oxidase complex [19]. This multicomponent enzyme complex is assembled during cell activation on the cytoplasmic membrane and on the membranes of the specific granules of neutrophils. It performs the electron transfer from NADPH located in the cytoplasm to molecular oxygen across the membrane. The involvement of ROS in NET formation has been proven pharmacologically using diphenyleneiodonium and other inhibitors of NADPH oxidase, which nearly completely abolish NETosis [20]. Besides, research on the neutrophils of patients with chronic granulomatous disease (CGD) revealed that this pathology is due to mutations in NADPH oxidase subunits resulting in the assembly of a nonfunctional or lowly functional enzyme complex, which is unable to synthesize ROS. People with such mutations suffer from recurrent infections in their lifetime [21,22], and their neutrophils do not form neutrophil traps [20]. However, it has been shown that the addition of H2O2 to the neutrophils of CGD patients restores the ability to release NETs [20]. Molecules involved in signal transduction from the receptors to NADPH oxidase were revealed by rigorous inhibition analysis carried out in Zychlinsky's laboratory [23]. It has been shown that the activation of NETosis by PMA is accompanied by the induction of the Raf/MEK/ERK signaling pathway [23], as well as the Rac2 (a small GTPase of the Rho-family)-mediated pathway [24]. Another feature of NETosis is the loss of chromatin segregation into eu- and heterochromatin [20]. This process involves neutrophil elastase and myeloperoxidase, the enzymes of primary (azurophilic) granules. Both enzymes move from the granules into the nucleus at the earliest stages of NETosis. NE is the first to be transported into the nucleus, where it catalyzes the cleavage of the linker histone H1 and modifies the core histones [25]. It has been shown that elastase is extremely important for trap formation, because mice deficient in this enzyme were incapable of producing NETs [25]. MPO migrates into the nucleus later, and its function is associated with intensification of chromatin decondensation, probably due to the synthesis of hypochlorous acid [25]. It should be noted that patients with mutations in the MPO gene cannot form valid NETs [26]. In addition to partial cleavage of histones by elastase and MPO, another modification intensifies chromatin decondensation. Peptidylarginine deiminase 4 (PAD4) induced in the neutrophil after proper activation catalyzes deimination of arginine residues that yields citrulline in three out of the four core histones, which results in their weaker binding to DNA. There is evidence that histones are citrullinated in decondensed chromatin [27–29]. The role of this process in NETosis was demonstrated pharmacologically for cell lines forming a limited number of NETs. It has been shown that autophagy is necessary for NETosis, and this mechanism follows NADPH oxidase activation in the information transfer pathway [30]. Recently, a unique mechanism of NET formation was described, where the cytoplasmic membrane remained intact and the cells preserved viability [31]. Such a mechanism of NET formation was denoted vital NETosis, while a conventional NETosis resulting in cell death was called suicidal NETosis. It should be noted that vital NETotic

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Alarmins include cytokine IL-1α and amphoterin HMGB1 (high mobility group box 1) that appear at the early and later stages of inflammation, respectively; the neutrophil antimicrobial peptides, cathelicidin and defensins; a large group of S100 proteins; factor HDGF (hepatomaderived growth factor); heat shock proteins (HSPs); uric acid crystals; annexins and highly glycosylated peptides, galectin, thymosin, nucleoline. The products of DNA and RNA decomposition, as well as some structural proteins of the extracellular matrix also belong to alarmins. Alarmins include neutrophil extracellular traps, or NETs, which represent a fundamentally new phenomenon in the physiology of neutrophils [4,5]. Recently, NET release has been found in systemic lupus erythematosus (SLE), ANCA-vasculitis, type II diabetes, atherosclerosis, rheumatoid arthritis, psoriasis, and gout. This review highlights the functional activity of NETs and their role in the development of chronic inflammatory processes underlying the pathogenesis of psoriasis and atherosclerosis. The choice of these diseases is based on the fact that psoriasis is not just a skin disease. A systematic study of various biological markers, including immunological, revealed a close relationship between psoriasis and atherosclerosis, the development of which is associated with the initiation of chronic inflammation induced by various exogenous and endogenous alarmins [6,7].

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It is widely believed that both suicidal and vital NETosis perform an antimicrobial function. It is assumed that NETs released by activated neutrophils entrap and kill invading microorganisms. Hence, neutrophils continue to protect the host not only intracellularly similar to phagocytosis, but also extracellularly. In vitro and in vivo experiments revealed an adhesion of gram-negative and gram-positive bacteria, as well as fungi, to DNA fibrils [4,32,36–38]. Attachment to DNA fibrils considerably restricts the dissemination of bacteria in the body. Systemic administration of DNase abolishing NET formation during experimental Staphylococci-infection, significantly accelerated the release of bacteria from the entry site, increased their number in the bloodstream, and reduced the lifespan of animals [33]. It is known that the virulence of bacteria depends on their ability to synthesize nucleases. The presence of nucleases significantly increases, and, vice versa, the inhibition of the nucleases dramatically reduces the virulence of bacteria. In the latter

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case bacteria become an easy prey for neutrophil extracellular traps [39, 40]. The entrapment of bacteria and fungi by NETs is undeniable. However, it is still an open question whether NETs can kill captured germs. Several researchers observed the death of pathogenic microorganisms entrapped in NETs [22,32,40]. As noted above, NETs contain a large number of bactericidal proteins and enzymes capable to kill bacteria, fungi and enveloped viruses. This ability is characteristic of classic bactericidal agents, such as cationic peptides LL-37, α-defensins, and protein BPI, as well as enzymes NE and MPO [4]. However, there were a number of studies which failed to reveal the death of the trapped microorganisms. The addition of DNase to captured bacteria caused the decomposition of NETs and the release of the trapped bacteria. It was established that 100% of these bacteria survived [41]. The lack of microbicidal properties of NETs has been explored theoretically. Cationic peptides LL-37 and α-defensins completely lose their microbicidal properties in the extracellular milieu under the influence of serum proteins, mainly apolipoproteins. The same concerns neutrophil enzymes with microbicidal properties, in particular NE. There is a large amount of protease inhibitors in the blood plasma, which should completely suppress the functional activity of enzymes [42]. Supposedly, the experiments which showed the microbicidal activity of NETs were carried out in a serum-free medium [31]. Therefore, the question of the microbicidal properties of NETs remains open. However, NETs indisputably can capture microorganisms and prevent their dissemination in the body. In this regard, NETs undoubtedly play a positive protective role for the host. 4. The concept of NETs as alarm signals

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neutrophils were capable to carry out the main functions, chemotaxis and phagocytosis [32–34]. The main difference between vital NETosis and suicidal NETosis is concerned with the nature of their inducers. Vital NETosis is induced due to the recognition of microbe ligands, PAMPs, by neutrophil receptors and their helpers, platelets. Several types of vital NETosis have been described. In the first type, vesicles filled with nuclear DNA bud out under the influence of Staphylococcus aureus. These vesicles pass through the cytoplasm and extrude into the extracellular milieu, where they burst and release chromatin fibrils. In the other type of vital NETosis, vesicles fuse with the cytoplasmic membrane excreting DNA fibrils into the matrix without a membrane perforation. These fibrils have low proteolytic activity and are not of mitochondrial origin. Such types of vital NETosis continue for 5 to 60 min and do not require ROS [32]. It was shown that opsonized gram-positive bacteria induce vital NETosis by activating TLR2 and complement receptors. Such activated neutrophils almost completely lose the nucleus and remain alive. This kind of NETosis is induced by Staphylococci infections and occurs in the skin. In this case, slowly moving neutrophils that release chromatin fibrils and capture large areas were detected in capillaries. A large number of un-nuclear alive neutrophils have been detected in human abscesses caused by gram-positive bacteria [33]. In another type of vital NETosis, platelets expressing TLR4 are involved. Interaction of the platelets with gram-negative bacteria or LPS causes their activation and subsequent association with neutrophils adhering to the vessel wall. The neutrophils bind to the platelets via integrin LFA-1 (CD11a), which is significantly expressed during cell activation. The binding of the neutrophils to platelets causes further neutrophil activation and NET release. If the blood flow is slow, NETs retain their structure. Such a type of NETosis was found in the capillaries of lungs and spleen sinusoids [34]. If stimulated under certain conditions NETs can be formed by the mitochondrial DNA. Priming of neutrophils with GM-CSF and subsequent stimulation with LPS (or complement component C5a) caused about 80% of NETosis. A combination of two fluorescent dyes was used that were specific to nuclear and mitochondrial DNA. It was shown that the filaments obtained were composed of mitochondrial DNA. It was also shown that perinuclear structures, probable analogues of mitochondria in neutrophils, excrete this DNA. With the aforementioned type of stimulation, ROS generation is necessary for NET release. This fact was proven by the addition of a NADPH oxidase inhibitor, diphenyleneiodonium, which caused a complete blockade of NETosis. In addition, neutrophils of CGD patients cannot form NETs after stimulation with GM-CSF/C5a. Surprisingly, the neutrophils that released NETs retained viability, and moreover, exhibited an increased survival rate compared to intact cells [35].

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It seems likely that the formation of a significant NET amount indicates that the organism faces a certain problem. In this respect, this process plays a positive role. Importantly, NETs contain the nuclear protein HMGB1, peptide LL-37, α-defensins, DNA and its decomposition products, i.e. a complex of compounds with a strong biological effect. All these substances are classical alarm signals or alarmins that make DNA/ protein complexes or NETs, the classical representatives of alarmins. It was suggested that the totally exogenous PAMPs and endogenous alarmins should be classified into a large family of molecular patterns associated with damage, denoted DAMPs (damage-associated molecular patterns) [43]. More detailed information about alarmins can be found in the following review [44]. Alarmins indicate tissue or cellular damage and transmit a danger signal to neighboring or distant cells. In this respect, they are the inducers of sterile inflammation, which occurs without participation of microorganisms. Notwithstanding the role of exogenous factors, i.e., microorganisms, sterile inflammation also plays a significant role in the development of autoimmune diseases. Alarmins are characterized by the following properties [2]:

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1. They can be rapidly and passively released in the process of nonprogrammed death of the cells of any origin. 2. They can be inducibly released from immune cells without their subsequent death. 3. They can be the inducers of ligand-receptor activation of innate immune cells, followed by a probable activation of adaptive immunity. 4. They can induce a recovery of damaged tissues.

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It is apparent that the products of vital NETosis completely meet the criteria of alarmins. They are released under the influence of powerful inducers from viable neutrophils and contain substances that activate innate and adaptive immunity. Importantly, substances that activate innate immune system primarily include DNA, the main component of NETs. A large number of immune cells contain endosomal and cytosolic receptors that recognize autologous DNA. These cells include plasmacytoid dendritic cells (pDC), which contain the endosomal receptor TLR9, and monocytes/macrophages that carry cytosolic

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Psoriasis is a chronic inflammatory skin disease that affects about 1–2% of human population. Psoriasis is characterized by an intense proliferation of keratinocytes, the deterioration of their differentiation, and the infiltration of the affected skin with neutrophils and lymphocytes. Clinically, psoriasis manifests itself in the formation of papular-rash patches covered with silvery-white scales. Immunological, genetic, psychological, and environmental factors are involved in the development of this disease. In recent years, a major role in the development of psoriasis has been attributed to such cytokines as IL-17, IL-1β, IFN-γ, IL-22, and TNF-α. NETs can be involved in the initiation and maintenance of the chronic inflammatory process in psoriasis because of the following factors: 1. Formation NETosis-associated immunostimulatory complexes that consist of proteins/peptides and nucleic acid (NA); 2. Release of proinflammatory cytokines from immune cells simultaneously with NETosis; and 3. Release of a series of proteins with autoantigen properties during NETosis. Let us consider a specific role of each of these factors in the pathogenesis of psoriasis. First, it is known that peptide LL-37 can form a complex with DNA. This complex results from electrostatic interaction of the cationic amino groups of the peptide with the anionic phosphate groups of DNA. Moreover, it retains a positive charge. The resulting complex is insoluble and resistant to DNase action. Interestingly, an

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insoluble complexes resistant to RNase are also formed as a result of interaction between peptide LL-37 and RNA [50,51]. Surprisingly, the level of LL-37 in keratinocytes of psoriatic patients is significantly increased [47,50]. In the affected skin of such patients peptide LL-37 forms complexes with DNA or RNA. Typically, these complexes co-localize with DC, forming numerous cell clusters located in the epidermis. Moreover, the LL-37/DNA complex co-localizes with pDC, while LL-37/RNA complex with mDC. It was shown that peptide LL-37 collocates with TLR9 inside the endosomes of pDC in the affected skin. A peptide with a molecular weight of 4.5 kDa was isolated from the affected skin, which induced a significant synthesis of IFN-α in the culture of pDC. However, the addition of anti-LL-37 antibodies to the culture of pDC completely abolished the stimulatory effect of this peptide. Amino acid analysis of the isolated peptide showed a complete coincidence of its composition with that of LL-37 [50,51]. The molecular mechanism of interaction of the LL-37/DNA complex with DC was investigated in detail. When added to the culture of DC, the positively charged complex LL-37/NA binds to the anionic groups of the proteoglycan of membrane rafts and enters the cytoplasm due to endocytosis. Thereafter, the LL-37/DNA and LL-37/RNA complexes use different routes in different types of DC. When added to the culture of pDC, complex LL-37/DNA enters TLR9-containing endosomes, where it induces IFN-α synthesis. It should be noted that LL-37 has unique properties among cationic peptides, because neither α-defensins (the cationic peptides of the primary granules of neutrophils), nor β2 -defensins (the main bactericidal cationic peptide of psoriatic skin) induce the synthesis of type I IFNs (type I interferons) in the culture of pDC. Contrarily, the addition of LL-37/RNA complex to the culture of mDC causes its entry into TLR7-containing endosomes and induces the synthesis of TNF-α and IL-6 [52]. The interaction of LL-37/DNA complex with keratinocytes is significantly different from that with DC. The complex of LL-37 with genomic DNA, as well as that with CpG oligodeoxynucleotides (CpG ODN) does not induce the synthesis of type I IFNs in keratinocytes. This is probably due to the structural features of the cytoplasmic membrane of keratinocytes. However, pretreatment of the cells with LL-37 followed by a stimulation with genomic DNA or CpG ODN induces a powerful synthesis of type I IFNs in keratinocytes which is associated with TLR9 activation [53]. Apart from LL-37, another cationic protein, SLPI (secretory leukocyte protease inhibitor), can form a complex with DNA. The main target for SLPI is an enzyme of neutrophil primary granules, neutrophil elastase, that plays a crucial role in chromatin decondensation during NETosis. In the affected parts of the psoriatic skin, the DNA/NE complex was found in association with SLPI. It was shown in in vitro experiments that the DNA/NE/SLPI complex is a powerful inducer of the synthesis of type I IFNs in the culture of pDC. According to the data of the RNAimethod the type I IFNs-synthesis was associated with the activation of TLR9 in pDC. It has been shown recently [54] that LL-37/DNA complex is not the main inducer of type I IFNs-synthesis in the skin. It should be mentioned that LL-37 peptide is synthesized in the skin by keratinocytes. In contrast, pDCs are primarily located in the dermis, where large amount of neutrophils, the main producers of the peptide, accumulates. The authors cited suggest that the role of DNA/NE/SLPI complex in the induction of type I IFNs in psoriasis is more significant, than the role of the LL-37/DNA complex [54]. These data provide evidence that the psoriatic SLPI protein does not only play an antiinflammatory role, which restricts the destructive activity of proteases. It performs an immunostimulatory function, that can produce both positive and negative effects. Second. Proinflammatory cytokines, Th1, Th17 and Th22, produced by the respective subpopulations of T-lymphocytes, play a leading role in the pathogenesis of psoriasis. Cytokine IL-17 which is synthesized by Th17-cells, plays an important role under normal and pathological conditions. In a normal organism this cytokine enhances antiinfection immunity by raising the activity of bactericidal peptides, including LL-

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receptors DAI, DHX9, DHX36, PolIII, Ku-70, LRRFP1, IFI16, and AIM2. The recognition of DNA by these receptors leads to the activation of the respective signal pathways and the synthesis of type I IFNs, as well as of a number of pro-inflammatory cytokines. Interestingly, an excessive synthesis of type I IFNs underlies the pathogenesis of autoimmune and autoinflammatory diseases [45,46]. The healthy organism has mechanisms which protect the receptors of innate immune cells from contact with DNA. In particular, the DNA recognizing domains of TLR9 are located on the inner surface of pDC endosomes and, therefore, they are unavailable for DNA. Besides, a powerful arsenal of endonucleases is located in the extracellular milieu that includes DNase 1. It should be noted that, in the healthy organism, the DNA is always presents in a small amount outside the cells, in particular as a result of necrotic cell death. DNA can also “leak” from apoptotic cells as a result of inopportune clearance by macrophages in the process of efferocytosis. However, due to the DNase action, this DNA is cleaved almost immediately, as soon as it exits from the cell. Apart from the aforementioned mechanisms of DNA protection, there is one more unique mechanism of preserving the functionally active DNA. This mechanism is based on the formation of an autoDNA/LL-37 complex. Peptide LL-37, the product of cathelicidin hCAP18 (human cationic antimicrobial protein) cleavage, is synthesized in neutrophils, keratinocytes, and epithelial cells of the respiratory tract as an inactive precursor with a molecular weight of 18 kDa. Cathelicidin hCAP18 consists of a N-terminal catelin-like domain and a C-terminal cationic antimicrobial domain. In the process of leukocyte exocytosis, proteinase 3 cleaves cathelicidin to form cationic peptide LL-37 that is composed of 37 amino acid residues and two leucine residues on the N-terminal side. As for keratinocytes, the serine proteinase kallikrein carries out the cleavage of inactive precursor to form active LL-37 peptide. It is known that peptide LL-37 is involved in protecting the skin against infection. It was shown that a reduction in its level in atopic dermatitis results in the development of pyoderma [47]. Any skin damage causes an increase in the level of LL-37, which promotes the healing of the injury. The protective effect of LL-37 is due to its ability to stimulate angiogenesis, keratinocyte proliferation, the chemotaxis of neutrophils and monocytes, and to its bactericidal effect [48,49]. In parallel, peptide LL-37 is involved in the initiation of many chronic inflammatory processes, owing to its ability to form a complex with nucleic acids and some proteins.

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Atherosclerosis (AS) is a chronic inflammatory disease involving large and medium-sized arteries, which manifests itself in the formation of nodules and plaques in the intima of blood vessels. The inflammation is initiated due to the accumulation of low density lipoproteins, which are oxidized to reactive oxygen species (ROS) in vascular walls. These radicals induce the expression of endothelial adhesion molecules on the surface of vascular walls. The first stage of the inflammatory process leads to the infiltration of the vessel wall by inflammatory cells such as neutrophils, monocytes/macrophages, and DCs. The role of NETs in the initiation and maintenance of chronic inflammatory process in AS can involve the three mechanisms that are responsible for psoriasis. Peptides LL-37 and Cramp (the murine analog of LL-37) have been identified in the atherosclerotic plaques of blood vessels in human and mice, respectively [63,64]. Feeding mice with a fat-enriched diet resulted in a significant deterioration of the vessel walls of the aorta and coronary vessels by atherosclerotic plaques. The level of peptide Cramp, co-localized predominantly with neutrophils, was dramatically increased in aortic wall of the mice; however, the number of macrophages was also increased. However, feeding Cramp−/− mice with the fat-enriched diet caused a significantly less damage to blood vessels in comparison to wild-type mice. There were significantly less neutrophils and macrophages in the aorta walls of these mice compared to the control animals. Mice with the Apoe−/− genotype were not prone to develop atherosclerotic vascular lesions. However, feeding them with the fatenriched diet in combination with the injecting of Cramp peptide led to the development of atherosclerotic vascular lesions [63,64]. It is known that the NETs are the source of LL-37(Cramp)/DNA complexes. Their presence was shown in the affected vessel walls in several studies [63,65,66]. A method of simultaneous identification of the components MPO/DNA complex has been elaborated recently [65]. The markers of necrosis and NETosis, i.e. complexes of citrullinated histones/DNA (nucleosomes) and MPO/DNA complexes, respectively, were found in the tissue sections of the coronary vessel walls of 282 patients with coronary artery diseases. Both of these complexes are detected in the blood and their level is significantly increased in severe

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etc.). These groups change the chemical nature of the amino acid residues (e.g., by converting arginine to citrulline) or modify the secondary protein structure (as exemplified by the formation of disulfide bridges). Histones undergo the most significant modifications during NETosis. For example, in the process of NET-formation, histones can undergo methylation (H4K20), acetylation (H4K5 and H4K16), and citrullination (H3 and H4). The antibodies against all these modified proteins were detected in the serum of SLE patients [61]. The citrullination of histones is a prerequisite for the creation of auto-antigens (neoantigens) during NETosis. As mentioned before, this process implicates the activation of the enzyme peptidyl arginine deaminase-4 (PAD4), which hydrolyzes the positively charged NH2groups of arginine to form neutral oxygen groups. As a result, a new amino acid, citrulline, forms extra-genomically. Citrullinated histones in NETs were detected in SLE, rheumatoid arthritis, multiple sclerosis, psoriasis, Felty's syndrome, and other diseases. However, citrullinated histones are not the only source of neoantigens in NETosis. The NE released from the primary granules of neutrophils penetrates the nucleus and cleaves histones into several peptides, which can be recognized by T- and B-cells. Antibodies against these peptides have been detected in the serum of SLE patients [62]. Research on the role of different modified during NETosis proteins and peptides has only recently been initiated. However, it has been revealed that the process of NETosis can be accompanied by the formation of large amount of autoantigens capable to induce the adaptive immune response. But the capacity to induce the autoimmune response varies depending on the individual involved. It is conditional on the genetic susceptibility of the organism and on environmental factors.

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37 and β-defensins; it also causes the influx of immunocompetent cells into inflammatory foci by inducing a synthesis of chemokines CXCL1, CXCL2, and CXCL8. Under pathological conditions, especially with autoimmune disorders, IL-17 intensifies the inflammatory processes. An increased expression of IL-17 and amount of Th17-cells was observed in rheumatoid arthritis, Crohn's disease, autoimmune uveitis, systemic lupus erythematosus (SLE), ankylosing spondylitis, multiple sclerosis, and psoriasis. In psoriasis, not only Th17-cells may be the source of IL-17. Research on the affected skin has shown that the majority of IL-17-containing cells were not T lymphocytes, but mast cells (MCs) and to a lesser extent, neutrophils. Predominantly, MCs excrete IL-17 into the exterior using the routine degranulation mechanism or by means of chromatin fibrils (denoted MCETs in the case of MCs). MCETs, in addition to the usual components of NETs, contain such MCs-specific enzymes as tryptase and chymase. It was revealed that, with MCs, such proinflammatory cytokines as IL-23 and IL-1β [55] cause a powerful MCETosis, which testifies to the presence of relevant receptors on these cells. Importantly, IL-23, IL-1β, and IL-6 also belong to the principal inducers of Th17-cell differentiation. There is much evidence of the participation of MCs and neutrophils in the pathogenesis of psoriasis. In the skin affected with rash, a large number of neutrophils and degranulated MCs were revealed. However, the number of MCs decreases sharply after successful PUVA or cyclosporine therapy [56,57]. There is a clear correlation between the mitigation of psoriasis, the development of agranulocytosis, and the subsequent normalization of the neutrophil number [58]. Razoxane, a highly effective drug for all forms of psoriasis and psoriatic arthritis, causes a dose-dependent decrease in neutrophil number [59]. A peculiarity of classical autoimmune diseases such as SLE is the presence of a specific population of neutrophils, low density granulocytes (LDGs), in the bloodstream. LDGs were isolated from the layer of mononuclear cells by gradient centrifugation in FicollHypaque. These cells are immature neutrophils, which leave the bone marrow prematurely and settle throughout the body under the influence of proinflammatory cytokines IL-8 and IL-17. These cells rapidly undergo apoptosis in the in vitro system and are prone to NETosis. Patients with SLE have an increased number of LDGs, while they are practically absent in healthy individuals [60]. The number of LDGs in the bloodstream of psoriatic patients is significantly increased compared to healthy donors. These cells have an increased ability to cause NETosis similar to those of SLE patients [55]. Thus, during NETosis all immunostimulatory components of NETs can be found in the skin, i.e. the LL-37/DNA complex, a powerful stimulator of type I IFNs, and the potent proinflammatory cytokine IL-17. However, the issue how IL-17 appears in MCs and neutrophils is still to be resolved. No appreciate levels of IL-17 mRNA either in neutrophils or in MCs (due to a small number of MCs in the blood [55]) were detected by RT-PCR. Probably, MCs and neutrophils passively accumulate IL-17, which was synthesized by other cells, in the cytoplasm. However, the ability of neutrophils to synthesize cytokines, including IL-17, was recently documented [60]. Thus, this issue needs further elaboration. Third. As mentioned above, a number of endogenous mediators with immunostimulatory properties are secreted by neutrophils, eosinophils, and MC during NETosis. Some of them, i.e. α-defensins, cathelicidin hCAP-18/LL-37, and amphoterin HMGB1, are classical alarmins. By activating the innate immune system and lowering the tolerance to autoantigens, these substances contribute to the development of an adaptive autoimmune response. It is possible to find antibodies practically against all the components of NETs in the blood of SLE patients. Except for antihistone antibodies, their clinical significance is not clear. Importantly, clinically significant autoantigens Ro, La, Smith, and RNP were not detected in NETs. In parallel with NETosis, granular and cytoplasmic proteins can undergo posttranslational modification based on the attachment of biochemically active functional groups (acetate, methyl, phosphate,

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Many autoimmune diseases are primarily based on the effects of endogenous or exogenous alarm factors that cause local damage or, more specifically, local metabolic disorders in relevant organs and tissues. Resident immune cells, macrophages, respond to this damage by the synthesis of proinflammatory cytokines and chemokines, causing the influx of neutrophils into the damaged area. Subsequently, neutrophils undergo activation and NETosis. NETs contain a powerful immunostimulating arsenal, i.e., complexes of DNA with various cationic proteins and peptides. Until now, only two of such substances have been described: the inhibitor SLPI and the peptide LL-37. There is no doubt that the number of such substances will be enlarged in the future. Complexes containing DNA with a peptide or inhibitor are endocytosed by TLR9-containing endosomes of pDC, which recognize any DNA that enters the cell. The consequence of this recognition is a powerful synthesis of type I IFNs. These cytokines have a multifaceted effect on the immune system. First of all, type I IFNs activate antigen-presenting cells (APC), macrophages, and DC. This results in an increased expression of MHC, activatory and co-stimulatory molecules, on APCs. The activation of APCs induced by type I IFNs abolishes auto-tolerance. This results in the involvement of low-affinity non-tolerized autoreactive T-cells in the immune process. Normally, they are not involved in this process. In addition, type I IFNs per se are potent activators of populations of lymphocytes including NK cells, and T and B lymphocytes [46]. A consequence of the prolonged and increased synthesis of type I IFNs is the development of autoimmune pathology. In sum, the data presented by us point to several major stages of the development of adaptive autoimmune response under the influence of exogenous and endogenous alarmins, which result in structural and functional damage to the relevant organ or a tissue (see Scheme 1). The scheme shows that type I IFNs are the central cytokines involved in the development of autoimmunity, while pDCs are the main cells producing these cytokines. The increased production of type I IFNs plays a significant role not only in pathogenesis of psoriasis, but also in SLE, small vessel vasculitis, type II diabetes, and other diseases. Recently, it has been revealed that type I IFNs and autoimmune processes

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coronary AS, as well as in strongly calcinated coronary arteries. A high level of nucleosome number was correlated with the risk of coronary stenosis; the NET markers MPO/DNA were correlated with the number of vessels involved in atherosclerotic lesions and the development of side effects [65]. The subsequent development of immunopathogenesis in AS is similar to that in psoriasis. In the affected areas of vessels the level of pDC is increased in addition to the presence of immunostimulatory complexes LL-37(Cramp)/DNA. The activation of pDC by the Cramp/ DNA complex causes an intense synthesis of type I IFNs in the vessel walls. The selective removal of pDC from mice dramatically reduced the development of atherosclerotic lesions. This also concerned Cramp−/− mice. Contrarily, the activation of the synthesis of type I IFNs, and of pDC significantly accelerated the development of AS lesions [64]. Interestingly, the number of pDC was reduced in the blood of patients with troponin-positive unstable coronary syndrome. This reduction was inversely correlated with disease severity. It is assumed that this decline is due to the migration of pDC into the focus of inflammation. In addition to the stimulation of immunopathological processes, neutrophil extracellular traps produce a direct toxic effect on the vascular endothelium. In a murine model, it was demonstrated that NETs secreted by LDG contain metalloproteinase-9 (MMP-9) which causes endothelial dysfunction and induces the apoptosis of these cells. This dysfunction is associated with the activation of endothelial MMP-2 by NETotic MMP-9, because the suppression of MMP-2 and NETosis completely restores the functional activity of endothelium [66].

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play a significant role in the pathogenesis of AS [67]. Important evidence for the inducing role of type I IFNs in promoting autoimmune pathology is that these diseases develop in 19% of cancer patients treated with type I IFNs for a long period of time [68]. Unfortunately, the above scheme oversimplifies the complex processes of the development of autoimmunity. Obviously, all cells of an organism capable to produce type I IFNs are involved in the activation of innate and adaptive immunity. However, it is pDCs that play a leading role at the initial stages of local activation of innate immunity. They are capable of synthesizing a 1000 times larger amount of type I IFNs than monocytes/macrophages. This scheme is somewhat arbitrary because it does not take account of other ways of activating of innate immunity during the development of the autoimmune response. It ignores the formation of an multimolecular complex, inflammasome, under the influence of alarm signals. The inflammasome is responsible for the synthesis and secretion of proinflammatory cytokine IL-1β, which is the inducer of nearly all other proinflammatory cytokines. The formation of inflammasome and the synthesis of this cytokine are of paramount importance for the pathogenesis of psoriasis [69]. Studies on the role of NETosis in the pathogenesis of autoimmune diseases are at the initial stage of their development. There are still many open questions and unresolved issues. However, it is evident the study of the role of NETosis in terms of human pathology including the induction of autoimmune diseases is the most important area of the modern medicine. It should be emphasized that NETs are a source of a large amount of autoantigens [70] in combination with

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• The pathogenesis of many autoimmune diseases is based on the effects of endogenous and exogenous alarmins. • Resident macrophages respond to alarmins synthesizing proinflammatory cytokines, which cause influx of neutrophils into the damaged area. • Neutrophils undergo activation and NETosis in the damaged tissues. • NETs contain immunostimulatory complexes of DNA with cationic peptide LL-37 or inhibitor SLPI. • Complexes containing DNA/peptide (inhibitor) are endocytosed by TLR9-containing endosomes of pDC followed by a synthesis of type I IFNs. • Type I IFNs are the central cytokines involved in the development of autoimmunity, while pDCs are the main cells producing type I IFNs.

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immunostimulatory substances which perform an adjuvant function. Indisputably, developing methods to eliminate NETs is of primary importance. In terms of practical clinical immunology, the development of simple and reliable methods for assessing vital NETosis and evaluating its diagnostic significance with respect to diseases of the human immune system hold much promise for the future.

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Please cite this article as: Pinegin B, et al, Neutrophil extracellular traps and their role in the development of chronic inflammation and autoimmunity, Autoimmun Rev (2015), http://dx.doi.org/10.1016/j.autrev.2015.03.002

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