The role of neutrophils and NETosis in autoimmune and renal diseases

Abstract

Systemic autoimmune diseases are a group of disorders characterized by a failure in self-tolerance to a wide variety of autoantigens. In genetically predisposed individuals, these diseases occur as a multistep process in which environmental factors have key roles in the development of abnormal innate and adaptive immune responses. Experimental evidence collected in the past decade suggests that neutrophils — the most abundant type of white blood cell — might have an important role in the pathogenesis of these diseases by contributing to the initiation and perpetuation of immune dysregulation through the formation of neutrophil extracellular traps (NETs), synthesis of proinflammatory cytokines and direct tissue damage. Many of the molecules externalized through NET formation are considered to be key autoantigens and might be involved in the generation of autoimmune responses in predisposed individuals. In several systemic autoimmune diseases, the imbalance between NET formation and degradation might increase the half-life of these lattices, which could enhance the exposure of the immune system to modified autoantigens and increase the capacity for NET-induced organ damage. This Review details the role of neutrophils and NETs in the pathophysiology of systemic autoimmune diseases, including their effect on renal damage, and discusses neutrophil targets as potential novel therapies for these diseases.

Neutrophils — the most abundant type of white blood cell in humans — have crucial roles in the innate immune response and act as a first line of defence against invading microorganisms¹. Neutrophils target microorganisms through a number of processes including degranulation (the release of granular antimicrobial peptides such as myeloperoxidase (MPO), neutrophil elastase and matrix metalloproteinases (MMPs)), phagocytosis and degradation via synthesis of reactive oxygen species (ROS) inside phagolysosomes, and microbial trapping by extrusion of a meshwork of chromatin bound to granular peptides termed neutrophil extracellular traps (NETs)².

NETs are structures composed of externalized, concentrated, antimicrobial molecules that can trap, immobilize, inactivate and kill microorganisms, and activate other immune cells³. They are generated and released during a distinct process of cell death called NETosis (FIG. 1), which differs from apoptosis and necrosis. NETosis is an important step of the innate immune response, and can be triggered by both infectious and ‘sterile’ stimuli (for example cytokines⁴, monosodium uric acid crystals⁵, cholesterol crystals, autoantibodies⁶ and immune complexes⁷). In addition to triggering innate immune responses, neutrophils regulate the adaptive immune response⁸. Indeed, neutrophils engage with B cells^9,10, T cells^11,12 and antigen presenting cells in lymphoid organs^13,14. NETosis also contributes to the activation of the immune system during inflammation^15,16. As discussed in this Review, these effects are mediated, at least in part, through protease-mediated effector functions and by modulating intercellular signal transduction.

Figure 1. NETosis pathways and potential therapeutic targets.

Stimulation of neutrophil receptors by microorganisms or sterile stimuli leads to release of calcium (Ca⁺) from the endoplasmic reticulum (ER), which results in activation of protein kinase C (PKC) and assembly of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex and/or mitochondrial activation, leading to generation of reactive oxygen species (ROS). Other cellular sources might also be important contributors of ROS. This ROS generation is followed by myeloperoxidase (MPO)-dependent migration of granular neutrophil elastase (NE) to the nucleus where it cleaves histones. In addition, peptidylarginine deiminase (PAD) 4 is activated and induces histone citrullination, which contributes to chromatin decondensation. Finally, the nuclear membrane is degraded and a mixture of chromatin and granular proteins is extruded from the cell. Extracellular DNase eventually degrades neutrophil extracellular traps (NETs). Modulation of critical steps in NET formation and degradation (shown by blocking arrows) might be beneficial for the treatment of autoimmune disorders. C5aR, C5a receptor; G-CSF: granulocyte colony stimulating factor; G-CSF-R, G-CSF receptor; FcγR, Fc γ receptor; TLR, Toll-like receptor.

Systemic autoimmune diseases are characterized by a breakdown in self-tolerance to a wide range of auto-antigens. Certain autoimmune conditions such as systemic lupus erythematosus (SLE)¹⁷, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV)¹⁸, rheumatoid arthritis¹⁹ and antiphospholipid antibody syndrome (APS)²⁰ can affect the kidney.

Many of the molecules externalized through NETosis (for example MPO, double-stranded (ds)DNA, histones) are recognized autoantigens in systemic autoimmunity, suggesting that aberrant NET formation might be important for the initiation of autoimmune responses in susceptible individuals. In support of this hypothesis, neutrophils from patients with various autoimmune diseases are more prone than those from healthy controls or patients without autoimmune diseases to undergo NETosis and, in turn, various autoantibodies can promote the release of NETs. This relationship between NETosis and autoantibodies could therefore promote a vicious cycle whereby autoantigen release by NETs leads to autoantibody formation, which promotes further release of antigenic material.

In this Review, we address the contribution of neutrophils and NETosis to host defence and their important role in innate and adaptive immunity. We also discuss the evidence supporting the potential involvement of neutrophils and NETs in the development of various systemic autoimmune disorders that affect the kidneys and consider how various neutrophil functions and NET formation and/or clearance could be modulated to treat autoimmune disorders.

Neutrophils and NETosis: basic principles

Host-defence

Neutrophils are typically short-lived with a circulating half-life of approximately 1–8 h²¹; however, some reports suggest that their lifespan can extend to over 5 days²², especially once activated^23,24. They are characterized by a multilobulated nucleus, as well as distinct types of cytoplasmic granules packed with microbicidal molecules and oxidative enzymes, which are an important armamentarium for host defence. Neutrophils develop in the bone marrow and are released in the circulation as terminally differentiated cells²¹. Once they are recruited to sites of injury or infection, a multistep inflammatory response is initiated in which β₂-integrins and kindlin-3 on circulating neutrophils promote their adhesion to the activated vascular endothelium²⁵, followed by their extravasation and migration towards inflammatory foci, ultimately leading to the destruction of foreign microorganisms²⁶.

Neutrophil granules have been classified into three types: azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules^27,28. Azurophilic granules can be distinguished from the other types by their uptake of basic dye azure A, owing to their acid mucopolysaccharide content, and contain MPO, azurocidin, bacterial permeability-increasing protein, cathepsin G, defensins, elastase and human neutrophil peptides. Secondary granules contain cathelicidin antibacterial peptide (also known as antibacterial peptide LL-37), lactoferrin and neutrophil gelatinase-associated lipocalin. Tertiary granules contain peptidoglycan recognition proteins. Lysozyme is present in all three types of granule. Together these antimicrobial granular proteins have an important role in the activation of innate and adaptive immunity^29,30.

When they come in contact with a pathogen, neutrophils undergo a respiratory burst, a crucial phenomenon in host defence, during which activation of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex leads to superoxide synthesis³⁰. Superoxide and its downstream products, hydrogen peroxide and hypochlorous acid, have critical antimicrobial roles³⁰. Other important sources of ROS in the neutrophil include mitochondria³¹ and peroxisomes³². Neutrophils can also deploy complex enzymatic and non-enzymatic antioxidant defence systems, which involve enzymes such as catalase, superoxide dismutase and glutathione peroxidase. A balance between oxidant and antioxidant activities in the neutrophil is therefore required to maintain homeostasis of the immune system.

Neutrophils also have an important role as regulators of the adaptive immune response. Uptake of apoptotic neutrophils by dendritic cells can enhance levels of dendritic antigen presentation^13,14. Neutrophils can both stimulate and suppress T-cell responses. Indeed, neutrophils cross-prime CD8⁺ T-cells in a major histocompatibility complex class I dependent manner and activate γδ T cells through cross-presentation of bacterial antigens¹¹. Conversely, proteases like elastase and cathepsin G, contained in neutrophil granules, inhibit the production of T-cell stimulating cytokines, including IL-2 and IL-6 (REF. 12). Neutrophils can also downregulate the ζ chain of the T-cell receptor by producing ROS and secreting arginase, which leads to T-cell arrest³³. Lastly, neutrophils express PD-L1, which promotes interferon-dependent PD-1-mediated T-cell apoptosis³⁴. With regard to B cells, neutrophils synthesize cytokines crucial for B-cell development, such as B-cell activating factor (BAFF)⁹ and A proliferation-inducing ligand (APRIL)¹⁰. In the spleen, neutrophils can act as B-cell helpers in a T-cell independent manner³⁵.

Complexes of extracellular DNA and cathelicidin or HMGB1 can form stable structures that activate phagosomal Toll-like receptor (TLR) 9 in monocytes and dendritic cells^15,36. Interestingly, histones in NETs also activate TLR2 and TLR4¹⁶, suggesting that NETs stimulate the immune system through various ligands and receptors. NETs can also prime T cells; however, this interaction is not clearly defined³⁷. Lastly, NETs participate in the defence against pathogens by promoting timely and enhanced thrombus formation, which prevents microorganism dissemination^38–41.

Neutrophils in resolution of inflammation

In addition to immune system activation, NETs might help to control inflammation by modulating the function of monocyte-derived dendritic cells by promoting the expression of type 2 T helper (T_H2) cytokines and down-regulating the synthesis of T_H1 and T_H17 cytokines⁴². The presence of NETs during dendritic cell maturation diminishes their capacity to induce T-lymphocyte proliferation and polarization. NETs also promote the resolution of neutrophilic inflammation by degrading cytokines and chemokines and by disrupting neutrophil recruitment and activation via serine proteases in acute neutrophilic reactions such as gout⁴³.

NET formation and clearance

NETs target many pathogens, including Staphylococcus aureus^44,45, Streptococcus pyogenes⁴⁶, Shigella flexneri³, Mycobacterium tuberculosis⁴⁷, Aspergillus fumigatus⁴⁸, Toxoplasma gondii⁴⁹ and HIV⁵⁰. Although the molecular pathways leading to NET formation remain to be fully characterized, the process of NETosis is known to involve calcium release from the endoplasmic reticulum of neutrophils, activation of PKC, assembly of the NADPH-oxidase complex, and generation of ROS^30,51. These events are followed by MPO-dependent migration of elastase from granules to the nucleus^52,53, where it cleaves histones to elicit chromatin decondensation⁵⁴. Concomitantly, protein-arginine deiminase type-4 (PAD4; also known as peptidylarginine deiminase IV) substitutes the positively charged histone arginine residues with neutrally charged citrulline residues in the nucleus, which decreases the electrostatic interaction between histones and DNA, and thus promotes nuclear decondensation⁵⁴. Eventually, the nuclear and granular membranes are degraded, leading to the mixing of chromatin and granular proteins in the cytoplasm, followed by extrusion of this meshwork from the cell and neutrophil lysis⁵².

Reports also exist of a non-lytic process of NET formation, termed ‘vital’ NETosis. This process is characterized by the formation of an anuclear — but still viable — cell or by selective extrusion of mitochondrial DNA; these processes and their implications in health and disease remain to be fully characterized^55,56.

NETs are eventually degraded by DNase 1, an endonuclease that cleaves chromatin within NETs⁵⁷. NET degradation can also occur by engulfment of NETs by macrophages through an active endocytic process that leads to the lysosome-mediated degradation of these lattices⁵⁸.

Systemic autoimmune diseases

Systemic autoimmune diseases develop as multistep processes through a complex interplay between genetic and environmental factors, including infections, ultraviolet light (in the case of SLE), smoking (in rheumatoid arthritis), and certain medications (in the case of SLE and AAV)⁵⁹. These factors can induce cellular damage through various mechanisms and increase the exposure of the immune cells to autoantigens⁶⁰. Subclinical immune dysfunction leads to a preclinical phase of autoantibody synthesis⁶⁰. Eventually, clinically apparent disease develops, along with detectable evidence of immune dysregulation⁶⁰.

In diseases characterized by autoantibody development, responses to antigens are fairly specific. Autoantibodies to dsDNA and to RNA-binding proteins are characteristic of SLE¹⁷, whereas autoantibodies that target MPO and proteinase-3 (PR3) are predominantly observed in patients with AAV¹⁸. Individuals with rheumatoid arthritis develop autoantibodies (anti-citrullinated protein antibodies; ACPAs) that target citrullinated intracellular and extracellular antigens, such as vimentin, enolase, and fibrinogen¹⁹. APS is characterized by antibodies to β2 glycoprotein-I (β2GP1)²⁰.

Dysregulation of apoptosis and/or clearance of apoptotic material has been proposed to be an important source of modified and/or externalized autoantigens in autoimmune diseases⁶¹. This view is perhaps oversimplified, given that various other cell-death mechanisms (including necroptosis, pyroptosis, NETosis, autophagic- related cell death and caspase-independent cell death)⁶² could act as sources of modified autoantigens. In fact, nuclear material released from NETs could be more immunogenic than apoptotic material. The proximity of the genomic DNA to ROS during NETosis promotes nucleic acid oxidation. Oxidised DNA externalized in NETs, which is more resistant than non-oxidised nucleic acids to degradation by nucleases such as TREX-1, activates the cGAS–STING intracellular sensing pathway, leading to enhanced type I interferon synthesis and immune dysregulation^57,63.

Patients with systemic autoimmune diseases have a markedly enhanced risk of developing cardiovascular atherosclerotic diseases⁶⁴ and thromboembolic events^65,66, which could be mediated by a proatherogenic^67,68 and prothrombotic⁶⁹ effect of neutrophils and NETs. Many autoimmune disorders also involve the kidneys and neutrophils might have a prominent role in inflammation, tissue vasculopathy and fibrosis in the renal manifestations of these diseases⁷⁰.

NETosis and autoimmune disorders

Systemic lupus erythematosus

SLE is a systemic autoimmune disease that predominantly affects women of childbearing age and involves multiple organs¹⁷. Although the exact aetiology is unclear, the development of SLE is multifactorial and can be attributed to disruptions in innate and adaptive immunity and aberrant responses to nuclear self-antigens. Patients with SLE have an increased production of and/or response to type I interferons, a process considered critical in SLE pathogenesis⁷¹. Furthermore, SLE is characterized by accelerated atherosclerosis and an increased risk of premature cardiovascular diseases⁶⁴.

Patients with SLE display a distinct granulocyte population, called low-density granulocytes (LDGs), which are found in the peripheral blood mononuclear cell fraction following density separation of whole blood^72–74. LDGs are a subset of pathogenic granulocytes that synthesize more proinflammatory cytokines, including type I interferons, than their regular-density counterparts and are toxic to endothelial cells^73,75. Importantly, LDGs have an enhanced capacity to form NETs at baseline in vitro that remains unchanged even in the presence of PMA, a strong NET inducer, unlike their normal density or healthy counterparts, suggesting that LDGs might already be maximally stimulated in vivo⁷⁵ (FIG. 2). These NETs contain higher levels of autoantigens and immunostimulatory molecules, such as LL-37, MMP9 and dsDNA^15,36,76,77, and are more immunostimulatory than are NETs generated by healthy control neutrophils⁷⁷.

Figure 2. NETs in low-density granulocytes.

Low-density granulocytes from a patient with systemic lupus erythematosus show spontaneous formation of neutrophil extracellular traps (NETs). Immunofluorescence imaging reveals NETs as strands of DNA (blue (labelled with Hoechst stain)) with neutrophil granular proteins (red (myeloperoxidase)). Image obtained by Monica Purmalek, Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, USA.

In addition to the presence of LDGs with enhanced capacity to undergo NETosis, NETs are not properly cleared from the circulation in a substantial proportion of patients with SLE. In some patients, this defect in NET clearance might result from complement activation within NETs or from modifications (oxidation) of nucleic acids externalized by the NETs^63,76,78. In other patients the defect might be due to the presence of inhibitors of DNase 1, or of anti-NET antibodies, which hamper the access of the DNase 1 to the NETs⁵⁷. This inability to degrade NETs has been associated with a higher incidence of lupus nephritis in patients with SLE⁵⁷.

NETs can further trigger an autoimmune response in SLE by exposing cathelicidin–DNA complexes, which, through activation of endosomal TLRs, promote the synthesis of type I interferons by plasmacytoid dendritic cells^15,36 (FIG. 3). NETs generated by LDGs are enriched in oxidized mitochondrial DNA. Oxidized nucleic acids in NETs can also activate type I interferons in myeloid cells through activation of the STING pathway⁷⁸. Furthermore, cathelicidin, and perhaps other NET proteins, can activate the NLRP3 inflammasome in lipopolysaccharide-primed macrophages by interacting with their P2X7 receptor and inducing potassium efflux, which leads to release of IL-1 and IL-18. In turn, these cytokines further promote NETosis, amplifying inflammatory pathways in tissues⁷⁹ (FIG. 3). Interestingly, cathelicidin-mediated activation of the inflammasome is increased in macrophages derived from patients with SLE, compared to healthy macrophages, which could promote further inflammation and organ damage. Skin and kidneys from patients with SLE can be infiltrated by neutrophils that undergo NETosis and externalize nucleic acids in these tissues, which, in turn, is associated with increased levels of serum anti-dsDNA antibodies, supporting a direct role of NETosis in SLE pathogenesis and associated organ dysfunction⁷⁵.

Figure 3. Neutrophils and NETosis in the pathogenesis of autoimmune and renal diseases.

Upon exposure to various infectious and ‘sterile’ stimuli, neutrophils (and low density granulocytes (LDGs) in the setting of systemic lupus erythematosus (SLE)) release neutrophil extracellular traps (NETs). These NETs externalize granular peptides (such as LL-37) in complex with DNA, which, in turn, activate plasmacytoid dendritic cells (pDCs) to synthetize type I interferons (IFN), which have critical roles in SLE pathogenesis. Neutrophils also release inflammatory cytokines that activate T cells and B cells, which produce autoantibodies, potentially contributing to the development of systemic autoimmune and renal diseases. NETs promote the expression of tissue factor, which activates platelets and coagulation factors, thereby promoting thrombosis. This process might contribute to the development of renal diseases, particularly antiphospholipid antibody syndrome (APS) and ANCA-associated vasculitis (AAV). NET-bound proteins, such as metalloproteinases (MMPs) and histones, promote vasculopathy — a feature of renal disease, AAV, SLE and APS — by damaging endothelial cells. Autoantibodies, immune complexes, autoantigens, complement activation factors and cytokines can in turn cause NETosis. NET peptides stimulate NLRP3 inflammasome activation in macrophages, leading to release of IL-1 and IL-18, which promote neutrophil activation and NET formation.

Mouse models of SLE display enhanced bone-marrow NETosis and autoantibodies that recognize NET components. In these murine models, the administration of chemical inhibitors that target PADs decreased NET formation and significantly reduced endothelial dysfunction, the expression of type I interferon-associated genes and tissue damage^80–82. NADPH oxidase (Nox) 2 deficiency increased disease severity in lupus-prone mice⁸³, suggesting that inhibition of NADPH oxidase-dependent pathways might actually stimulate the immune response. These findings are similar to observations in patients with chronic granulomatous disease, a condition caused by defective phagocyte NADPH oxidase activity⁸⁴, in which the incidence of autoimmune diseases is increased. In contrast, inhibition of mitochondrial ROS synthesis, which is enhanced in LDGs from patients with SLE, ameliorates murine SLE⁷⁸. These observations suggest that the effects of NET inhibition strategies will have to be carefully characterized in the context of SLE before human studies are undertaken.

ANCA-associated vasculitis

ANCAs are associated with a distinct form of vasculitis, termed AAV, which affects small and medium vessels. AAV frequently affects the kidney, skin, upper and lower respiratory tract and peripheral nerves. Systemic AAV can be categorized into three groups, according to the localization of the vascular inflammation and the presence or absence of granulomatosis and asthma: granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA)¹⁸.

In patients with vasculitis, antibodies to MPO⁸⁵ and PR3 (REF. 86) are not only serological markers of AAV but might also activate neutrophils, leading to degranulation and production of oxygen radicals and to the development of destructive necrotizing vascular and extravascular inflammation^87,88. Indeed, MPO-ANCA and PR3-ANCA can activate neutrophils that have been primed by a proinflammatory stimuli, such as TNF⁸⁹, G-CSF⁹⁰, lipopolysaccharide⁹¹ or the complement anaphylatoxin C5a⁹², which results in activation of the alternative complement pathway⁹³. This pathway triggers an inflammatory amplification loop in the vessel wall that attracts and activates neutrophils. The activated neutrophils undergo a respiratory burst, thereby producing toxic oxygen radicals and releasing destructive enzymes^94,95.

Neutrophils from patients with AAV display enhanced NET formation in vitro^96,97. In addition, levels of NET remnants, such as MPO–DNA complexes⁹⁶, and neutrophil granular proteins, such as calprotectin (a dimer of S100-A8 and S100-A9)⁹⁸, were increased in sera from patients with AAV. High levels of NET remnants are found in patients with high AAV disease activity and high neutrophil count, and correlate inversely with levels of ANCA⁹⁹. Furthermore, immunostaining of renal biopsy specimens from patients with AAV revealed the presence of NETs and NET-associated molecules in areas of inflammation, around areas of fibrinoid necrosis in kidneys with necrotizing glomerulonephritis, and along interlobular arterial walls^96,100. These observations suggest that NET formation is involved in vascular damage and immune system activation in AAV^96,97,99,100. Vascular inflammation could be initiated and perpetuated by ANCA-induced activation of primed neutrophils and monocytes.

NETosis might also induce endothelial cell damage and vasculitis in the glomerulus by concentrating active proteases, which cause direct injury to the cells. Additionally, the presentation of ANCA antigens and dsDNA enhances the ability of NETs to induce auto-antibody formation and furthers cell damage¹⁰¹. ANCA-induced NETs generated by C5a-primed neutrophils could lead to enhanced thrombosis and inflammation in AAV by promoting the expression of tissue factor, a protein required for coagulation^94,95. NETs can also present PR3 and MPO to dendritic cells¹⁰². These dendritic cells internalize NET material and, when transferred to mice, induce ANCAs against MPO, PR3 and dsDNA, leading to autoimmune vasculitis and lupus-like syndrome. In a 2014 study, serum from patients with ANCA was shown to both induce NETosis and inhibit NET degradation¹⁰³.

Rheumatoid arthritis

Rheumatoid arthritis is a chronic, systemic autoimmune disease characterized by synovial inflammation and hyperplasia, cartilage and bone destruction, auto-antibody production (including ACPAs), and systemic features, including cardiovascular and pulmonary disorders¹⁹. Although, rheumatoid arthritis rarely affects the kidney directly, it can present as focal mesangioproliferative glomerulonephritis, membranous nephropathy or rheumatoid vasculitis¹⁰⁴. Patients with long-standing, uncontrolled, inflammatory disease may develop secondary renal amyloidosis.

ACPAs — highly specific autoantibodies that target citrullinated proteins — are found in a significant percentage of patients with rheumatoid arthritis¹⁰⁵. Immune complexes containing ACPAs and citrullinated antigens display enhanced immunogenicity and arthritogenicity¹⁰⁶. Enhanced NETosis is detected in circulating and synovial-fluid neutrophils, synovial tissue, rheumatoid nodules, and skin of affected patients. This increase in NETosis positively correlates with ACPA levels and with the presence of systemic inflammatory markers in patients with rheumatoid arthritis¹⁰⁷.

Extracellular citrullinated autoantigens are found in the joints of patients with rheumatoid arthritis¹⁰⁸. This presence could be explained by the increase in NETosis, as NETs are a source of extracellular citrullinated auto-antigens in rheumatoid arthritis¹⁰⁷, and/or by the release of active PAD isoforms through NETosis, which could citrullinate extracellular histones and fibrinogen¹⁰⁹. Indeed, PAD2 and PAD4 are overexpressed in neutrophils¹¹⁰ and these enzymes are detected in the synovium of patients with rheumatoid arthritis, in close association with neutrophilic infiltrates¹¹¹.

NETs stimulate inflammatory responses in synovial fibroblasts from patients with rheumatoid arthritis, including the production of proinflammatory cytokines, chemokines, and adhesion molecules^107,112. In a predisposed host, enhanced NET formation could, therefore, conceivably promote the generation of key autoantibodies and disturbances in adaptive immunity that generate a vicious cycle leading to further promotion of NETosis and amplification of inflammatory responses in the joint and other tissues. Whether NETs have similar roles in the extra-articular manifestations of rheumatoid arthritis, including kidney damage, remains to be determined.

Antiphospholipid antibody syndrome

Primary APS is an autoimmune disease of uncertain aetiology that can affect the venous and arterial circulation, and is associated with spontaneous thrombosis and/or pregnancy loss, and high titres of autoantibodies against phospholipids (aPLs)^20,113. It is proposed that in APS, aPLs and anti-β2GP1 antibodies promote thrombosis by activating endothelial cells, platelets and monocytes. The kidneys are commonly involved in APS, with the possibility of clot formation within any part of the renal vasculature — renal arteries, glomerular capillaries and/or renal veins. Patients with APS and renal involvement may present with renal artery stenosis, renal infarction, renal vein thrombosis, thrombotic microangiopathy and/or hypertension. Renal pathology findings vary from ischaemic glomeruli with thrombosis to proliferative glomerulonephritis with immune complex deposition and inflammatory infiltrates¹¹⁴. Patients with other autoimmune diseases, primarily SLE, can also display an associated APS, known as secondary APS.

NETs are important activators of the coagulation cascade, and are integral components of arterial and venous thrombi^38,39,115. As such, neutrophils and NETosis have been implicated in the pathogenesis of APS in the past couple of years. Similar to SLE, the serum of patients with APS displays a decreased ability to degrade NETs; this defect correlates with increased levels of antibodies against NETs and neutrophil remnants¹¹⁶. As in SLE, sera and plasma from patients with primary APS had elevated levels of both cell-free DNA and NET remnants, and neutrophils from patients formed spontaneous NETs ex vivo⁶. Mechanistically, aPLs promote the release of NETs in a ROS and TLR4-dependent manner. In addition, sera and IgG from patients with APS can stimulate NET release from control neutrophils⁶. Recently, an LDG population has also been described in primary APS but their role in the pathogenesis of the disease remains to be determined¹¹⁷.

Type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) is characterized by the organ-specific autoimmune destruction of insulin-producing pancreatic β cells in genetically predisposed individuals, leading to hyperglycaemia and the need for lifelong exogenous insulin replacement therapy¹¹⁸. Renal involvement (diabetic nephropathy) is a major cause of morbidity and premature mortality in patients with T1DM¹¹⁹. Autoimmunity in T1DM has been primarily attributed to β-cell destruction by autoreactive T cells, followed by the production of autoantibodies to various β-cell antigens¹¹⁸. In the past few years, neutrophils and NETs have been implicated in the pathogenesis of T1DM^120–123. In a mouse model of T1DM, neutrophils and NETs were abundant in pancreatic islets, and inhibition of neutrophil function mitigated T1DM development in these mice¹²⁰.

A 2013 report indicates that patients with T1DM and individuals at risk of developing T1DM exhibit circulating neutropenia¹²², which is not caused by antineutrophil autoantibody production or by impaired neutrophil production or differentiation. Rather, this neutropenia appears to be related to neutrophil sequestration in pancreatic tissue¹²¹. In another study, neutropenia in patients with T1DM was associated with elevated levels and enzymatic activity of various neutrophil serine proteases and resulted from neutrophil death due to NET formation¹²³. These observations implicate neutrophils and NETs in the pathogenesis of T1DM and future studies should address whether modulation of NET and neutrophil function has potential therapeutic implications¹²³.

In addition to a potential role in the development of diabetes, NETosis might be involved in promoting the comorbidities of this disease. Patients with diabetes mellitus (type 1 or type 2) exhibit impaired wound healing and studies in humans and mice published in the past 2 years suggest that this defect could stem from enhanced NETosis^124,125.

Renal inflammatory diseases

Glomerulonephritides encompass a varied collection of disorders, including proliferative and non-proliferative glomerulonephritis, which have similar mechanisms of immune-cell-mediated kidney damage. Proliferative glomerulonephritis is associated with increased glomerular cellularity caused by immune cell influx and local immune cell proliferation¹²⁶.

Leukocytes are key players in the progression of proliferative glomerulonephritis as they influence humoral and adaptive immune responses and modulate local effector pathways, which are directly responsible for glomerular damage⁷⁰. Neutrophil aggregation is observed in proliferative glomerulonephritis of different aetiologies, including glomerulonephritis caused by the deposition of circulating immune complexes (for example, in the context of infections or autoimmune diseases), the generation of immune complexes within the kidney (such as anti-glomerular basement membrane (anti-GBM) antibodies, ANCAs, and anti-donor antibodies in transplant rejection), and/or complement dysfunction. As described in previous sections, neutrophils, NETosis and NET products, visualized by immunostaining in renal biopsy samples, are prevalent in lupus-associated glomerulonephritis⁷⁵ and in pauci-immune glomerulonephritis in patients with AAV^96,99,100. Moreover, NETs might be important sources of modified autoantigens in the kidney¹²⁷. The impaired ability of patients with autoimmune disorders to degrade NETs might further enhance the inflammatory effects of NETs and NET components^57,103. Indeed, decreased NET degradation was associated with the development of glomerulonephritis in patients with SLE¹²⁸.

In necrotizing crescentic glomerulonephritis, neutrophil serine proteases like cathepsin G, neutrophil elastase and PR3 promoted IL-1β generation and kidney injury¹²⁹. Subnephritogenic levels of anti-GBM antibodies and ANCAs amplified neutrophil recruitment and NET formation in renal tissue, and promoted subsequent development of necrotizing crescentic glomerulonephritis¹³⁰. IL-17 (REF. 131) and C-X-C motif chemokine 5 (CXCL5) have also been hypothesized to drive neutrophil recruitment to kidney tubules, also contributing to renal tissue injury in crescentic glomerulonephritis¹³². Similarly, endogenous MPO (possibly released through NETosis and/or neutrophil degranulation) promoted neutrophil-mediated renal injury, but attenuated T-cell immunity¹³³, suggesting that endogenous MPO might contribute to local glomerular damage during neutrophil-mediated glomerulonephritis, but that MPO might also attenuate the initiation of the adaptive immune response. Endogenous MPO attenuated the development of pristane-induced lupus nephritis by inhibiting the early inflammatory response and regulating the response of CD4⁺ T cells and macrophages in secondary lymphoid organs¹³⁴; however, these observations in animal models need to be interpreted with caution, given previous evidence that comparing neutrophil function between rodents and humans has several caveats and such comparisons can yield different and even opposite results^68,135,136.

Immobilized immune complexes might be involved in the induction of NET release through activation of the low affinity immunoglobulin γ Fc region receptor FcγR IIIB, and Integrin α-M (encoded by Itgam)⁷. Mice depleted of neutrophils or lacking Itgam exhibit a significant reduction in endothelial injury, glomerular thrombosis, and acute renal failure, despite the robust production of renal cytokines¹³⁷. Intriguingly, endogenous FcγR IIB negatively regulates autoimmunity in anti-MPO-associated glomerulonephritis by limiting ANCA production, T-cell responses, and neutrophil activation in mice with AAV¹³⁸. Therefore, various neutrophil FcγR receptors might have important roles in regulating antibody-mediated glomerulonephritis¹³⁹. The alternative complement pathway might also be involved in this disorder: C5a and its neutrophil receptor C5aR could constitute an amplification loop in which NETs activate the complement cascade with production of C5a that, in turn, mediates neutrophil activation and ANCA-induced glomerulonephritis⁹².

Overall, these findings suggest a potential common denominator in systemic autoimmunity whereby various immune stimuli, including autoantibodies, immune complexes and cytokines, promote the release of extracellular traps by neutrophils in a dysregulated manner. This process can amplify inflammatory responses, activate innate and adaptive immunity and promote externalization of important autoantigens, thereby promoting loss of immune tolerance.

Potential therapeutic modulation of NETosis

NETosis might be involved in the pathophysiology of a variety of autoimmune diseases, opening the possibility of modulating this process for therapeutic purposes (TABLE 1). In in vitro studies, inhibition of ROS production by targeting NADPH^6,44,140 or mitochondria⁷⁸, or use of ROS scavengers, such as N-acetyl cysteine (NAC), reduced NET release¹⁴¹. Interestingly, NAC reduced disease activity in patients with SLE in two separate studies^142,143 and inhibition of mitochondrial ROS decreased lupus severity in mouse models⁷⁸. MPO inhibitors, such as 4-aminobenzoic acid hydrazide¹⁴¹ or PF-1355 (REF. 144), reduce NETosis, neutrophil recruitment and levels of circulating cytokines. PF-1355 treatment reduced neutrophil accumulation in a mouse model of immune complex-mediated vasculitis and suppressed albuminuria and chronic renal dysfunction in a murine model of anti-GBM-induced glomerulonephritis. The use of such inhibitors might improve outcomes for patients with immune-complex-mediated diseases, such as SLE, vasculitis and various types of glomerulonephritis.

Table 1.

Potential therapeutics to modulate neutrophils and NETosis

Disease	Molecule	Target/Function	Effects on neutrophils	Effect on disease/clinical use
Systemic lupus erythematosus	NAC	ROS scavenger	Decreased NET release	Reduced disease activity in patients142,143
	MitoTEMPO	Mitochondrial ROS scavenger	Decreased NET release Decreased oxidation of nucleic acids in NETs leading to decreased immunogenicity and IFN responses	Reduced disease severity in murine models78
	Cl-amidine, BB-Cl-amidine	PAD inhibitors	Reduced NET formation leading to decreased IFN responses	Protection against renal, skin and vascular manifestations in mice models81,82
	Ciclosporin A, Tacrolimus	Calcineurin inhibitors	Modulation of calcium pools Reduced NETosis	Improvement of renal disease in patients152,153
	DNase 1	DNA	Enzymatic degradation of NETs	Reduction of autoantibody levels, proteinuria, delay mortality in mouse model156 Safe in phase I study, no change in serum markers of disease in patients157
	Eculizumab (anti-C5a monoclonal antibody)	C5a	Reduced NET formation and neutrophil activation	Improved survival in murine model Safe in phase I study, no change in disease activity in patients162,163
Rheumatoid arthritis	Cl-amidine	PAD inhibitor	Reduced citrullinated synovial proteins	Reduced arthritis severity in mouse model145
	Neutralizing anti-TNF antibodies	TNF	Reduced NET formation	Inhibition of NET release in vitro107 FDA approved for treatment160
	Neutralizing anti-IL-17 antibodies	IL-17	Reduced NET formation	Inhibition of NET release in vitro109 Ongoing trials to assess treatment efficacy161
	Rituximab	B cells	Decreased NET formation and neutrophil activation through decreased autoantibody production	FDA approved for treatment150
ANCA-associated vasculitis	Rituximab	B cells	Decreased NET formation and neutrophil activation through decreased autoantibody production	FDA approved for treatment151
	NDT9513727 (C5aR antagonist) Anti-C5aR mAb	C5aR inhibitor	Reduced NET formation and neutrophil activation	Reduced NET release by human neutrophils in vitro95
ANCA-induced glomerulonephritis	Anti-C5aR mAb	C5aR inhibitor	Reduced NET formation	Decreased albuminuria and glomerular neutrophil influx in murine model92
Immune complex vasculitis Anti-GBM disease	PF-1355	MPO inhibitor	Reduction of plasma MPO activity, neutrophil recruitment, NET formation	Suppression of pulmonary vasculitis and chronic renal dysfunction in mouse models144
Antiphospholipid antibody syndrome	TAK-242	TLR4 inhibitor	Reduced NET formation	Inhibition of NET release by human neutrophils in vitro6
Gout	Colchicine	Actin cytoskeleton	Destabilization of NETs or inhibition of NET formation. Improved NET degradation	Used in management of gout attacks158

ANCA, anti-neutrophil cytoplasmic antibody; GBM, glomerular basement membrane; mAb, monoclonal antibody; MPO, myeloperoxidase; NAC, N-acetyl cysteine; NET, neutrophil extracellular trap; PAD, protein-arginine deiminase; ROS, reactive oxygen species; TLR, Toll-like receptors; TNF, tumour necrosis factor.

TLR⁶ and PAD^80–82,145 inhibitors might have similar effects to those of MPO inhibitors. In vitro studies on human neutrophils showed that TLR4 inhibition with TAK-242, a small molecule, or with an anti-TLR4 blocking antibody decreased NET formation and ROS production⁶. Similarly, Cl-amidine and BB-Cl-amidine, two PAD inhibitors, decreased NET formation and protected against renal, skin and vascular manifestations in murine models of lupus^81,82. In another study, Cl-amidine reduced the severity of arthritis in a mouse model of inflammatory arthritis¹⁴⁵.

Whether impairing NET formation through the inhibition of PAD activity or the modulation of other pathways will have deleterious effects on host defence in humans, remains to be determined. Studies in PAD-knockout mice, however, suggest that, even in the absence of NET formation owing to the loss of PAD enzymes, other antibacterial functions of neutrophils remain intact^146,147. Importantly, observations in patients with a complete lack of molecules implicated in NETosis (including PAD)¹⁴⁸, as well as in patients with Papillon–Lefevre syndrome (an autosomal recessive genetic disorder caused by a deficiency in cathepsin C)¹⁴⁹, provide preliminary evidence that inhibition of NET formation is not overtly immunosuppressive. Whether disruption of NET degradation affects neutrophil antimicrobial functions is still unclear.

As modified histones appear to have immunomodulatory and vasculopathic roles, it is possible that strategies that block the response of tissues to histones might be efficacious for the treatment of inflammatory diseases and prevention of NET-mediated vascular damage⁵⁴. Alternatively, targetting B cells and plasma cells has been effective in autoimmune diseases, potentially owing to a reduction of autoantibody-induced NET formation^150,151.

As efficient NET induction requires the mobilization of intracellular and extracellular calcium pools, using inhibitors of calcineurin (such as cyclosporine A or tacrolimus) or of the G-protein-coupled receptor phospholipase C (such as staurosporine) might be an alternative strategy to suppress or modulate NETosis for therapeutic purposes⁵¹. In fact, ciclosporin A¹⁵² and tacrolimus¹⁵³ have shown efficacy for the treatment of various manifestations of SLE.

In addition to suppressing NET formation, another potential approach to treat autoimmune diseases is to disrupt the architecture of already formed NETs with DNase 1 and enhance their clearance. In transgenic mouse models of breast cancer and insulinoma, this approach restored perfusion, reduced injury and improved oxygenation of affected kidney and heart¹⁵⁴. Similarly, intrabronchial administration of DNase 1 reduced NETs and lung injury, and improved oxygenation in murine models of primary graft dysfunction after lung transplantation¹⁵⁵. In lupus-prone mice, administration of exogenous recombinant DNase can hamper the development of autoantibodies, reduce proteinuria and delay mortality¹⁵⁶. A phase Ib study showed that DNase was well tolerated; however, serum markers of disease were unchanged¹⁵⁷ and no follow-up studies have been conducted. The use of drugs, such as colchicine, which destabilize the actin cytoskeleton, a structure implicated in NETosis, should also be explored¹⁵⁸.

Inhibition of neutrophil recruitment and NET release by disrupting the cross-talk between kindlin-3 and β2-integrin, is also an interesting therapeutic avenue¹⁵⁹. CXCL5 could also be targeted to restrict pathogenic neutrophil infiltration in T_H17-mediated autoimmune diseases, especially crescentic glomerulonephritis, while retaining the protective function of neutrophils against invading pathogens¹³².

Inhibition of TNF and IL-17 decreases NET formation in neutrophils from patients with rheumatoid arthritis¹⁰⁷. Anti-TNF monoclonal antibodies are now the mainstay of rheumatoid arthritis treatment¹⁶⁰ and anti-IL-17 monoclonal antibodies have also shown some efficacy for the treatment of this disease¹⁶¹. Similarly, modulating IL-17 signalling, which is required for the development of lupus glomerulonephritis, with monoclonal antibodies is a potential therapeutic strategy for the treatment of SLE, especially in cases with kidney involvement¹³¹. Altering the alternative complement pathway might also yield novel therapeutic strategies such as the inhibition of NET formation by C5a-primed neutrophils. C5aR, which has an important role in ANCA-induced glomerulonephritis, is a potential therapeutic target and its inhibition could prevent NET formation from ANCA-primed neutrophils^92,95. Anti-C5 mAb therapy delayed onset of proteinuria and improved survival in murine SLE; a phase I study showed that the treatment was safe in patients with SLE, but did not result in a clear improvement in disease activity^162,163.

Conclusions

The involvement of NETs in autoimmune diseases is just beginning to be investigated. In addition to having an important role in the innate immune response against pathogens, ample evidence indicates that NETs can be generated through non-infectious stimuli in various clinical settings. In acute or chronic inflammatory disorders, enhanced NET formation and/or impaired NET degradation might be essential to trigger and propagate autoimmune responses and organ damage (FIG. 3). Understanding precisely how NET dysregulation contributes to different auto-immune diseases will likely require a better understanding of how NET composition varies following specific stimuli, how the interactions between genes and environment affect neutrophil responses and the resulting clinical phenotypes, and of the main pathways that modulate autoimmune NETosis versus microbial-induced NETosis¹⁰⁷. Indeed, differences in post-translational modifications of these auto-antigens exposed in various diseases might also contribute to the development of disease-specific autoantibodies¹⁰⁷.

Prospective studies are also needed to assess whether the presence of NETosis, NET components and/or LDGs can be used as biomarkers in patients with autoimmune diseases. Whether potential therapeutic interventions to inhibit aberrant NETosis or the production of neutrophilic proteins linked to the pathogenesis of autoimmune diseases will lead to improved treatment of these diseases without markedly decreasing host defence, also needs further examination.

Key points.

• Neutrophils may have critical roles in the pathogenesis of autoimmune diseases by forming neutrophil extracellular traps (NETs), secreting proinflammatory cytokines and by directly causing tissue damage

• NET formation externalizes autoantigens that might contribute to the pathophysiology and clinical manifestations of autoimmune diseases, either directly or by modulating other components of the immune system

• An imbalance in NET formation and degradation might increase the half-life of NET products and augment the immune response

• Novel therapies that target key pathways in neutrophil function and NET formation might improve the treatment of autoimmune diseases.