Neutrophils in Rheumatoid Arthritis: Breaking Immune Tolerance and Fueling Disease

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Review

Neutrophils in Rheumatoid Arthritis: Breaking Immune Tolerance and Fueling Disease Liam J. O’Neil1,* and Mariana J. Kaplan1,* Rheumatoid arthritis (RA), a common autoimmune disease, is characterized by a highly coordinated inflammatory response that involves innate and adaptive immunity. One of the hallmarks of RA is an immune response directed at citrullinated peptides that are specifically targeted by anticitrullinated protein antibodies (ACPAs). Among the various mechanisms by which neutrophils may promote immune dysregulation in RA, their ability to extrude neutrophil extracellular traps has recently been implicated in the development of ACPAs. In the synovium, neutrophils interact with resident fibroblast-like synoviocytes to endow them with antigen-presenting cell capabilities and an inflammatory phenotype. Further understanding how neutrophils modulate autoimmunity and tissue damage in RA may lead to the development of novel effective therapies.

Highlights Neutrophils impact the various stages of RA pathogenesis and natural history, from contributing to the loss of immune tolerance to driving synovial joint inflammation. The RA synovial microenvironment is highly conducive to the formation of NETs that externalize citrullinated proteins that have the potential to act as autoantigens and activate immune and resident cells in the synovium. Synovial neutrophils interact with fibroblast-like synoviocytes to promote proinflammatory cytokine release, MHC-dependent antigen presentation, and generation of autoantibodies.

Rheumatoid Arthritis and the Disruption of Self-Tolerance Rheumatoid arthritis (RA) is a common autoimmune disease of unknown etiology that primarily targets the synovial joints, which can lead to disability if not properly treated. RA is considered a systemic disease and, in addition to the synovial joints, can affect skin, lungs, and the vasculature [1]. While the precise pathogenic mechanisms underlying the development of RA remain unclear, fundamental research breakthroughs in understanding inflammatory mechanisms triggered in this disease have facilitated improved treatments, better patient outcomes (see Clinician’s Corner), and further elucidation of the involved pathways that drive autoimmunity. Like in other autoimmune diseases, it is well established that the onset of RA is preceded by a phase of quiescent autoimmunity termed preclinical RA (pre-RA; see Glossary) [2]. This period is characterized by the generation of antibodies against self-proteins. Specifically, many of the RA autoantibodies recognize modified proteins; among them, peptides that have undergone a post-translational modification termed citrullination [anticitrullinated protein antibodies (ACPAs)] are preferentially targeted. Citrullination of proteins is carried out by peptidylarginine deiminases (PADs), a family of five isozymes (PAD1–4 and PAD6) [3]. In addition, antibodies directed against the Fc portion of immunoglobulin, rheumatoid factor (RF), and against proteins carrying other post-translational modifications, such as carbamylation (anti-CarP) [4], develop during pre-RA. Epitope spreading appears to be an important event during pre-RA, where there is rapid expansion and diversification of the autoantibody repertoire [5] and this predicts shorter time to clinical disease onset [6]. This transition to clinical RA from pre-RA is considered the result of a complex interplay between genetic risk and environmental triggers. There is a well-established link between the human leukocyte antigen (HLA) DRB1 locus, termed the shared epitope, and susceptibility to RA [7]. Other single-nucleotide polymorphisms (SNPs) associated with RA include

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Neutrophil and NET-associated biomarkers have the potential to guide clinical treatment decisions and improve patient care. Targeting neutrophil-produced cytokines, chemokines, and NET formation are novel treatment targets that may improve outcomes for RA patients.

1

Systemic Autoimmunity Branch, Intramural Research Program, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA

*Correspondence: [email protected] (L.J. O’Neil) and [email protected] (M.J. Kaplan).

https://doi.org/10.1016/j.molmed.2018.12.008 Published by Elsevier Ltd.

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PTPN22, PADI2/4, FCGR2A, and STAT4, among many others [8]. Environmental exposures associated with enhanced risk of developing RA include smoking [9] and periodontitis [10]. The microbe Porphyromonas gingivalis has been vigorously studied as a possible trigger of autoimmunity in RA, as it causes periodontal inflammation and produces citrullinated antigens in a highly vascularized environment [11]. As with many autoimmune diseases, there is a sex bias in RA risk, with women being significantly more at risk than men [2]. At the onset of clinical disease, patients present with marked articular inflammation usually in the form of a symmetric polyarthritis. Leukocytes migrate to the joint where synchronized innate and adaptive responses develop, with early infiltration of neutrophils, T cells, B cells, and plasma cells that synthesize autoantibodies and form ectopic lymphoid follicles [12] within the synovium. Immune complexes (ICs) formed in the joint can activate the complement cascade and further activate/recruit leukocytes and mesenchymal cells [13]. Besides neutrophils, other innate immune cells, primarily monocytes/macrophages and natural killer cells, are recruited to the synovial lining. Ultimately, the local inflammatory milieu leads to fundamental changes in the biology of resident fibroblast-like synoviocytes (FLSs) and this promotes the formation of an hyperplastic, inflammatory, and invasive synovium termed pannus. There is emerging evidence indicating that neutrophils play a very important role in the initiation and perpetuation of RA through both direct effects on the synovium and the modulation of articular and systemic innate and adaptive immune responses [14]. Furthermore, recent work indicates that neutrophils may promote the generation of modified autoantigens, further influencing antigen-specific T cell and autoantibody responses and, potentially, epitope spreading [15]. In this review, we will provide an overview of neutrophils in health and disease and specifically, discuss their putative contribution to RA pathogenesis (Figure 1).

Neutrophils as the First Responders Neutrophils are the first cellular responders to acute pathogenic insults and are endowed with a diverse arsenal of antimicrobial mechanisms (Box 1). Neutrophils originate in the bone marrow from resident myeloid precursors in response to granulocyte colony-stimulating factor (G-CSF). Cell adhesion molecules such as integrins and selectins are considered essential in the process of neutrophil egression from the bone marrow. C–X–C chemokine receptors (CXC) are also expressed ubiquitously by bone marrow neutrophils. The balance between chemokine receptors CXCR4 and CXCR2 and their respective chemokines appear to play a fundamental role in neutrophil mobilization [16]. In the absence of inflammatory stimuli, terminally differentiated mature neutrophils patrol the bloodstream and remain in circulation for a relatively short period (6–12 h) before dying [16]. Once a neutrophil encounters a danger signal (microbes, pathogen metabolites, products of tissue damage), it will respond primarily to molecules that are classified as either pathogenassociated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) through a variety of pattern recognition receptors, including Toll-like receptors (TLRs). Interleukin-8 (IL-8) is a chemokine produced by various cell types (Th17 cells, epithelial cells, and neutrophils) that acts as the main chemokine that signals neutrophil migration toward an inflammatory environment [17]. Recent studies indicate that, upon exposure to PAMPs and DAMPs, neutrophils may display a prolonged life span, hypothetically up to several days long, as well as enhanced plasticity [18,19]. For example, neutrophils will produce different effector molecules when exposed to different conditions and they display versatile functional heterogeneity when orchestrating adaptive immune responses. Through diapedesis, neutrophils migrate from the bloodstream into affected tissues [20]. Neutrophils tether, role, and crawl 2

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Glossary Citrullination: an irreversible protein post-translational modification mediated by peptidyl arginine deaminases where an arginine residue is converted to citrulline. Fibroblast-like synoviocytes (FLSs): a specialized mesenchymal cell that is located within the synovium. These cells are responsible for collagen homeostasis in the articular joint, and play an important role in the pathogenesis of RA. Granules: intrinsic subcellular particles that play a variety of roles in innate defense including extracellular/ intracellular pathogen killing. Low-density granulocytes (LDGs): a subset of neutrophils found in malignancy, autoimmunity, and infection that are distinguished by their reduced buoyancy relative to normal dense granulocytes. Neutrophil extracellular traps (NETs): an innate mechanism of neutrophil defense where there is extrusion of DNA strands bound to cytotoxic granule and nuclear proteins to immobilize and potentially kill invading pathogens. Peptidylarginine Deiminase (PAD): a family of enzymes that mediate the irreversible posttranslational modification of arginine, converting it to citrulline. Pre-clinical RA (Pre-RA): the first stage of rheumatoid arthritis where at-risk patients develop autoantibodies to a variety of antigens. This stage is defined by the absence of clinical arthritis, and may be incidentally discovered in clinical practice. Currently there are no recommended treatments for preRA. Reactive oxygen species (ROS): a family of chemically reactive oxygen with pathogen killing capacity and a primary role in cell signaling. Shared epitope: a family of genetic risk alleles that predisposes individuals to develop RA, and more specifically ACPA-positive RA.

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Epitope spreading

AutoanƟbody Ɵter (ACPA, RF, anƟ-CarP)

Loss of immune tolerance

Healthy

Preclinical RA

Exposure to environmental triggers at mucosal surface leads to NET forma on and increased PAD ac vity

Local intracellular and extracellular citrullina on increases citrullina on burden and poten al for autoimmunity

Clearance of NETs by local DNAses

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Figure 1. Neutrophils in Rheumatoid Arthritis (RA) Pathogenesis. The development of RA occurs over several stages (middle and bottom panels) and generally evolves over many years. At mucosal sites, cigarette smoke, inhalants, and exposure to Porphyromonas gingivalis and other microbes can induce neutrophil extracellular trap (NET) formation, peptidylarginine deiminase (PAD) activation, and the generation and externalization of citrullinated peptides. Activated neutrophils release PAD enzymes (PAD2/4) that contribute to tissue citrullination. Carriers of the shared epitope efficiently present citrullinated peptides in MHCII, which can promote antigen-specific T cell activation and the generation of autoantibodies (Pre-RA). An expansion of autoantibody diversity occurs (upper panel) prior to the onset of clinical disease. RA joint fibroblast-like synoviocytes (FLSs) exposed to NETs acquire antigen-presenting cell (APC) capabilities when they internalize NET-associated citrullinated peptides and present them to antigen-specific CD4+ T cells. Neutrophils within the joint are a source of various cytokines, including tumor necrosis factor (TNF) and interleukin-8 (IL-8). Neutrophils also degranulate and produce reactive oxygen species (ROS) that induces cartilage and collagen breakdown. Neutrophils support the proliferation of osteoclasts and B cells, leading to erosive bony lesions, ectopic germinal centers, and immune complex (IC) formation. Anticitrullinated protein antibodies (ACPAs) promote NET formation, and this propagates a vicious cycle of inflammation, recruitment of leukocytes, and release of cytokines. Abbreviations: BAFF, B cell activating factor; CCL20, chemokine (C–C) ligand 20; CCR2, chemokine (C–C) receptor 2; CXCL8, chemokine (C–X–C) ligand 8; IA, intraarticular; ICAM-1, intracellular adhesion molecule; Ig, immunoglobulin; RANKL, receptor activator of nuclear factor kappa B ligand.

by synchronous mechanisms involving integrins and selectins. Integrins and selectins interact with locally produced inflammatory chemokines to ultimately allow extravasation of the cell into target tissues [21].

Neutrophil Extracellular Trap Formation One of the most unique facets of the defense arsenal that neutrophils carry is their ability to form neutrophil extracellular traps (NETs). NETs are strands of nucleic acids bound to nuclear,

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Box 1. The Pathogen-Fighting Armory of a Neutrophil The main pathways by which neutrophils fight microorganisms are phagocytosis, degranulation, oxidative burst, and the formation of NETs. Through phagocytosis, neutrophils engulf pathogens or dead cells for ultimate disposal. Encapsulation of microorganisms into phagosomes allows the neutrophil to kill them by generating ROS or releasing granule enzymes [107]. Phagocytosis is followed by neutrophil clearance by resident macrophages. Cytoplasmic neutrophil granules are formed early in development at the promyelocyte stage. Granules are mobilized to the cell membrane to undergo exocytosis, where their protein contents are released into the extracellular space in a process called degranulation [108]. Granules are either primary (azurophilic), which have antimicrobial properties, secondary (specific), required for migration through tissue, or tertiary (secretory), responsible for cellular extravasation from the circulation [108]. Azurophilic granules contain a variety of enzymes including MPO and neutrophil elastase that are active both extracellularly and intracellularly. Molecules present in the secondary and tertiary granules, including complement activators, collagenases, and metalloproteinases function primarily in the extracellular space. ROS generation in neutrophils is mediated by the NOX pathway [109], mitochondria, and MPO [110]. Generation of superoxide (O2 ), hydrogen peroxide, and hypochlorous acid is crucial in antimicrobial responses [111]. In addition to TLRs, membranebound Fcg receptors (FcgR) transduce signals intracellularly and engage ICs, which leads to degranulation and ROS release. TNF and Type 1/2 IFNs are amongst many cytokines that can also activate proinflammatory responses in neutrophils [112]. Tight regulation of these signals is critical for preventing unregulated tissue damage, as the inflammatory response by neutrophils in the joint can lead to significant structural damage.

cytoplasmic, and granule proteins that are extruded into the extracellular space. NETs form in response to microbial and non-microbial (sterile) danger signals including platelets, ICs, autoantibodies, cytokines, drugs, and crystals (urate or calcium pyrophosphate in gout and pseudogout, respectively; cholesterol crystals in the atherosclerotic arterial wall) [22]. Several of the pathways leading to NET formation require reactive oxygen species (ROS) generated by NADPH oxidase (NOX) [23,24]. NOX-dependent NET formation is now distinguished from the NOX-independent form, the latter not requiring ROS generated by NOX but rather from other sources such as the mitochondria [25]. Indeed, NOX-independent, rather than NOX-dependent, NETs have been recently described in the plasma of RA patients and in other autoimmune conditions [26]. During NET formation, myeloperoxidase (MPO) mediates the release of granule serine proteases such as neutrophil elastase that translocate to the nucleus where they cleave histones [27] and promote chromatin decondensation. PAD4, also localized to the nucleus, citrullinates histones during NET formation, potentially promoting further nuclear decondensation [28]. During NET formation, nuclear, granule, and cytoplasmic material colocalize and the loss of cell membrane integrity promotes the extrusion of these materials as NETs into the extracellular space. The pathways that mediate NET formation are heterogenous and at least partially dependent on both the stimuli involved in their generation and in the source of the neutrophil. For example, healthy female and male neutrophils appear to have differential ability to form NETs [29]. Importantly, while NETs have essential antimicrobial properties, uncontrolled or dysregulated NET formation and/or impaired NET clearance have been proposed to play pathogenic roles in several autoimmune and chronic inflammatory diseases such as RA (Box 2).

Box 2. Dysregulated NET Formation and Clearance in Autoimmune Conditions While NET formation likely represents an important host defense mechanism, dysregulated NET formation can result in tissue damage, thrombosis, and in aberrant activation of the innate and adaptive immune system [71]. Evidence of enhanced NET formation and/or impaired clearance of NETs from circulation has been reported in several autoimmune diseases. A putative pathogenic role of NETs has been extensively investigated in SLE, where neutrophils and a specific subset called LDGs undergo increased NET formation [113]. There is also evidence of impaired NET degradation mechanisms in SLE and this imbalance may enhance the half-life of these structures in circulation and in tissues, potentially fueling aberrant immune responses. NETs can induce Type I IFN responses [114], activate inflammasomes, and promote endothelial damage [88]. Other autoimmune diseases where enhanced NET formation and/or impaired NET degradation have been reported include antineutrophil cytoplasmic antibody-associated vasculitis, antiphospholipid syndrome, psoriasis, and as discussed, RA [71].

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Neutrophils Fuel Inflammatory Arthritis and Joint Destruction in RA Neutrophils are the most abundant cell type found in RA synovial fluid [30] and are also detected in RA synovial tissue [31,32]. ACPA ICs are present in the RA joint [33] and can induce neutrophil degranulation by signaling through FcgRs [34]. Various serine proteases present in neutrophil granules play important roles in joint damage and inflammation. Neutrophil elastase, cathepsin G, and proteinase-3 can activate proinflammatory cytokines [35–37], cleave adhesion molecules [38], and modulate chemokine function [39]. Matrix metalloproteinase-8 (MMP-8) and MMP-9 promote degradation of type 2 collagen [40] in the articular cartilage and are abundant in RA synovium [41,42]. MPO also forms ROS byproducts [24] that can increase oxidative damage in the joint. Overall, the molecules present in neutrophil granules contribute substantially to inflammation and tissue damage in the RA synovial joint. Synovial joint neutrophils synthesize various molecules with important roles in autoimmune arthritis, including tumor necrosis factor (TNF) [43,44] and B cell activating factor (BAFF) [45], a TNF superfamily member that induces B cell proliferation and contributes to synovial joint autoantibody generation [46]. Neutrophils can also synthesize receptor activator of nuclear factor kappa B (NF-kB) ligand (RANKL), a TNF superfamily member responsible for osteoclast differentiation and bone erosions in RA [47,48]. Neutrophils are also a source of various chemokines and chemokine receptors that further amplify inflammation and damage [49,50]. Overall, neutrophil cytokines and chemokines in the synovial joint orchestrate a potent immune response, and in vivo murine models provide further evidence that supports a fundamental role for neutrophils in driving disease pathogenesis and manifestations. Mouse Models Implicating Neutrophils in RA Pathogenesis A number of experimental mouse models of arthritis further support a role for neutrophils in RA pathogenesis. Neutrophil-specific caspase recruitment domain family member 9 (CARD9) knockouts exposed to the K-BxN serum transfer arthritis mouse model display a 50% improvement in joint inflammation scores associated with a threefold reduction in synovial neutrophil infiltrates [51]. Neutrophil-depleted mice are also resistant to the K/BxN arthritis model [52], while antibodies that target key neutrophil chemokine receptors like CXCR1 and CXCR2 significantly ameliorate antigen-induced arthritis. Mice that lack G-CSF, a cytokine with fundamental roles in neutrophil production in their release from the blood marrow, are protected from arthritis in both a collagen-induced arthritis (CIA) and collagen antibodyinduced arthritis (CAIA) model, while similar effects have been observed with anti-G-CSF antibodies [53]. Given that neutrophils highly express PADs (particularly PAD4 and PAD2), inhibition of these enzymes has been investigated as a strategy to modulate neutrophil biology and impact NET formation. In CIA, both pan-PAD inhibitors [54] and the PAD4specific inhibitor sGSK199 [55], previously described to reduce NET formation in vivo by up to threefold [56], reduced clinical scores to near normal levels, and delayed arthritis onset by about 4 days when compared to the control group [54,57]. Despite this, PAD4 knockout mice exposed to the K-BxN arthritis model displayed no clinical difference from wild-type mice, suggesting heterogeneity in arthritis disease models and in their dependence on PAD enzymes [58]. Further studies using PAD knockout mice in other models of arthritis will provide clarity on the role of PAD isoforms in murine arthritis. Of note, any results in murine models should be interpreted cautiously as there are important functional and phenotypic differences between mouse and human neutrophils [59]. Neutrophil-Directed Therapy in RA Observations from the effectiveness of current treatment regimens in RA that have effects on neutrophil function add another layer of evidence supporting the pathogenic role of these cells Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

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Current targets Cytokines

Future targets Cytokines and receptors

AnƟ-TNF-α monoclonal anƟbody (Adalimumab)

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AnƟ-IL-6 monoclonal anƟbody (Tocilizumab)

AnƟ-IL-17 monoclonal anƟbody (Secukinumab)

AnƟ-RANKL monoclonal anƟbody (Denosumab)

LTB4, IL-8

cDMARDs (Leflunomide, Methotrexate, GlucocorƟcoids)

Neutrophil extracellular trap formulaƟon PAD4-specific inhibitor PAD2/4 inhibitors

Neutrophil microparƟcles Neutrophil decoy microparƟcles Neutrophil ectosomes

CXCR1/2

Figure 2. Current and Future Treatment Targets for Rheumatoid Arthritis (RA) That Directly or Indirectly Mediate Neutrophil Function. Currently, widely used targeted therapy neutralize specific cytokines that drive RA pathogenesis (left). Many of these cytokines directly impact neutrophil functionality, and their efficacy is at least partially dependent on a reduction in neutrophil-mediated inflammation. Non-biologic disease-modifying antirheumatic drugs (methotrexate, leflunomide, tofacitinib, and glucocorticoids) alter neutrophil functionality through a variety of mechanisms [14,60,62]. Furthermore, targeting the cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) reduce reactive oxygen species generation and alter the migration properties of neutrophils in RA [105,106]. Various experimental treatments may impact neutrophil functionality by targeting a variety of cytokines and receptors, neutrophil extracellular trap formation, and by harnessing neutrophil microparticles (right). Inhibiting peptidylarginine deiminases (PADs) has shown promise in murine models [55]. Neutrophil ectosomes [95] may play an important antiinflammatory role in RA, while neutrophil decoy microparticles are an evolving nanotechnology that may provide a local treatment option for some RA patients [97]. cDMARDs, conventional disease-modifying antirheumatic drugs; CXCR, C–X–C chemokine receptor; GM-CSF, granulocyte colony-stimulating factor; RANKL, receptor activator of nuclear factor kappa B ligand.

in RA. Both conventional disease-modifying antirheumatic drugs and biologics have been shown to extensively modify neutrophil function (Figure 2). A recent review by Cecchi et al. [60] describes in depth the mechanisms by which standard RA therapy can impact the inflammatory capacity of neutrophils. Indeed, antimalarials, glucocorticoids, leflunomide, methotrexate, antiTNF agents, anti-IL-6 receptor antibodies, and tofacitinib (a Jak inhibitor) have been reported to have some effect on neutrophil functionality. For example, anti-TNF and anti-IL-6 receptor therapy can reduce neutrophil adhesion molecules and downregulate the production of key neutrophil chemokines [61]. Furthermore, tofacitinib prevents migration of neutrophils toward its key chemokine, IL-8 [62], and a recent study showed that it can decrease NET formation when administered in vivo to mice [63]. Figure 2 summarizes the targeted cytokines that fundamentally alter neutrophil properties in RA, and proposes some putative targets for future treatment. Further studies on medications known to be efficacious in RA are likely to yield enhanced understanding of neutrophil biology, and their functional capacity in RA.

Neutrophils, NETs, and Citrullination in RA The factors that drive protein citrullination in specific tissues in health and disease states remain to be fully determined and may be pleiotropic, including various factors such as tobacco use and infections [8]. PADs, particularly PAD2 and PAD4, are considered important mediators of synovial citrullination [3]. Both PADI2 and PADI4 have been identified as RA-risk SNPs, suggesting that these enzymes may play a role in disrupting immune tolerance [8]. ACPAs target a variety of citrullinated proteins in RA including histones, vimentin, fibrinogen, and alpha-enolase [64]. Various studies have shown that neutrophils are an important source

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of PAD2 and PAD4 [65]. However, the regulation of PAD biology in neutrophils is yet to be well characterized. Given their ability to synthesize PADs, neutrophils contribute to the generation of citrullinated autoantigens in the inflammatory environment in human RA and in mouse models of arthritis [66–68]. Indeed, NETs externalize citrullinated forms of histones and vimentin [69]. Other extracellular proteins that are targets in RA may be citrullinated by PADs from neutrophils (either in NETs or released upon stimulation), or from other cells, including pathogens [11,70]. Since PAD4 becomes activated during NET formation [71], citrullination of intracellular and extracellular proteins may occur during this cellular process and this can be quantified using citrulline-specific rhodamine probes or mass spectrometry. With these strategies a sample can be interrogated for the relative abundance and pattern of citrullination [72]. Using citrulline-specific rhodamine probes, it has been shown that the citrullination profile of NETs varies depending on type of stimulus used to generate these structures, as well as on the source of the neutrophils [73]. This variation may be in the presence or abundance of citrullinated proteins. Heterogeneity in the citrullinated cargo of NETs may potentially be involved in providing a spectrum of ACPA-inducing potential, depending on a variety of intrinsic and extrinsic factors. In this regard, NETs may be involved in the expanding loss of tolerance to citrullinated antigens in pre-RA (epitope spreading). As an additional mechanism, pore-forming pathways triggered by microbes appear to enhance protein citrullination in neutrophils, a mechanism that further ties microbial-induced immune responses in neutrophils with peptide citrullination [68]. NET Formation and Autoimmunity in RA Within the lungs, a site associated with initial stages of loss of self-tolerance in RA, increased NET complexes are found in at-risk first-degree relatives of RA patients [74]. These complexes correlate with mucosal IgA and IgG ACPA generation, suggesting that local NET formation in the airways of individuals at risk for RA may be linked to the generation of autoantibodies in the preclinical state. Periodontal disease, also linked with RA development, is associated with a profound gingival neutrophilic infiltrative response, along with local enhanced NET formation [75]. It is possible that enhanced NET formation in these tissues is an early event in RA where PADs are activated and promote generation and externalization of citrullinated autoantigens. Of note, specific polymorphisms in PTPN22, encoding for a tyrosine phosphatase, are associated with RA risk. RA neutrophils displaying this risk allele show earlier joint migration, enhanced ROS production [76], and increased PAD4-mediated citrullination with enhanced propensity to form NETs [77]. This observation suggests that certain genetic polymorphisms associated with RA risk may be linked to aberrant ability to citrullinate and form NETs. The inflammatory milieu of the RA synovium appears particularly conducive to allow neutrophils to form NETs [78]. Synovial RA neutrophils are primed to undergo spontaneous NET formation. Intriguingly, RA autoantibodies (ACPAs and RF) and various proinflammatory cytokines can induce neutrophils to extrude NETs [69]. NETs within the synovial joint engage in putative pathogenic interactions with resident cells. Synovial NETs can be internalized by resident RA FLS in a receptor for advanced glycation end products (RAGE)–TLR9-dependent axis [15] (Figure 3). This internalization of NETs by FLS induces them to synthesize enhanced levels of proinflammatory cytokines and chemokines and endows them with antigen-presenting cell capabilities through MHC Class II (MHCII) induction [15,79]. Indeed, FLSs that internalize NETs carrying citrullinated peptides can process and present these peptides to human RA antigenspecific T cells and activate them. In a humanized HLA-DR4 transgenic mouse model that recapitulates the shared epitope risk allele, intraarticular injection of FLS loaded with RA NETs leads to the development of RA-relevant autoantibodies (including ACPAs), antigen-specific T cell activation, and cartilage damage [15]. Taken together, the interaction between synovial Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

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MHCII B7-H3

IL-17R

RAGE

TLR9

TCR

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3 MHCII

MHCII an gen loading

Figure 3. Internalization and Processing of Neutrophil Extracellular Trap (NET)-Associated Proteins by Fibroblast-like Synoviocyte (FLS) Lead to Antigen Presentation of Citrullinated Peptides (cit-peptide) to Antigen-Specific CD4+ T Cells. Intraarticular NETs that contain citrullinated proteins can engage RAGE (receptor for advanced glycation end products) in the surface of FLS, leading to internalization of protein–DNA complexes (1) [15]. TLR9 mediates intracellular endosomal trafficking of these complexes (2). These complexes are processed and loaded onto MHC Class II (MHCII; 3) for subsequent presentation on the plasma membrane. Interleukin-17B (IL-17B), released during NET formation, promotes upregulation of MHCII in FLS (+). Antigen-specific CD4+ T cells engage the MHCII–antigen complex through T cell receptor (TCR) and co-stimulation signaling likely occurs through B7-H3 (4).

NETs and FLS may contribute to the activation of pathogenic innate and adaptive immune pathways in RA that could ultimately promote immune dysregulation, amplification of inflammatory responses, and cartilage damage. Translating this into human disease, NET-related biomarkers may provide a novel strategy for monitoring disease activity and identifying patients at risk for RA complications. Of clinical interest, antimalarials can inhibit both NET formation and the internalization of NETs by FLS, indicating an additional mechanism of action of this type of drugs in RA [15,80].

Neutrophil Biomarkers in RA Neutrophil-related biomarkers that correlate with relevant clinical RA phenotypes further implicate these cells as important mediators of disease activity. For example, a peripheral 8

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blood neutrophil granule gene signature was recently reported to be associated with poor response to anti-TNF therapy in RA [81], while a high neutrophil-to-lymphocyte ratio is associated with disease flare [82]. Serum levels of the calcium-binding proteins S100A8, A9, and A12, expressed by neutrophils, have been associated with inflammation detected by ultrasonography and clinical examination in RA [83,84]. Measuring NET complexes in tissue, sera, and synovial fluid is emerging as an intriguing addition to this family of RA biomarkers and will require further validation. Serum NETs correlate with markers of RA disease activity in patients afflicted by this disease [85], and antimalarials [80] and biologic disease-modifying antirheumatic drugs reduce NET levels [85]. NETs have been shown to promote atherosclerosis in various disease models and they may play a putative role in the development of vascular disease in RA [85,86]. Standardization of the methodologies to quantify NETs in various tissues, organ, and in circulation is required to consider these measurements as biomarkers in health and disease. Despite the potential of neutrophil-related biomarkers in RA, further clinical studies are required to validate these approaches and translate them into clinical practice.

Neutrophil Heterogeneity and Low-Density Granulocytes in RA Low-density granulocytes (LDGs) are a subset of neutrophils that are typically isolated from the mononuclear interphase following gradient centrifugation [87]. Over the last few years, there has been significant interest in investigating the role of these cells in autoimmunity. In systemic lupus erythematosus (SLE), LDGs are distinguished from normal-density granulocytes (NDGs) by their gene expression signature and their ability to synthesize higher levels of proinflammatory cytokines, induce endothelial cell toxicity [88], and by their enhanced ability to form NETs and activate Type I interferon (IFN) responses through enhanced synthesis of oxidized mitochondrial ROS and oxidation of mitochondrial DNA [89,90]. LDGs have been described in other inflammatory conditions including some cancers [91], sepsis [92], and various autoimmune diseases [93]. However, with the exception of antineutrophil cytoplasmic antibody-associated vasculitis and psoriasis LDGs, it remains unclear whether the LDGs described in other conditions are phenotypically and functionally similar to lupus LDGs. The description of LDGs in RA is more recent and initial descriptions indicate that these cells are functionally different than their autologous NDGs [94]. Similar to SLE, RNA-sequencing analysis indicates that RA LDGs upregulate primary granule and cell cycle genes, potentially suggesting an immature phenotype. In contrast to SLE, no clear difference in spontaneous NET formation between RA LDGs and NDGs has been reported. RA LDGs were reported to synthesize lower amounts of ROS (20%) in the context of TNF priming and express lower levels of TNF receptor (threefold less TNFR2) [94]. Despite their ill-defined role in RA, further study of LDGs, both in arthritis and in other systemic autoimmune conditions, may provide insight into their origin, heterogeneity, plasticity, and overall functionality in inflammatory states.

Clinician’s Corner Neutrophils, members of the innate immune system, function primarily in host defense. In RA, neutrophils use these defense responses against selftissue, leading to joint and systemic inflammation. Many of the current treatment regimens for RA directly or target neutrophil indirectly dysregulation. NETs, extruded strands of DNA used to defend against pathogens, are increased in the joints and serum of patients with RA and lead to increased inflammation and damage. During an inflammatory response, neutrophils can citrullinate proteins using an enzyme called PAD. RA patients develop autoantibody responses to these specific proteins, suggesting that neutrophils may contribute to the development of RA. Evidence suggests that inhibiting PAD in mouse arthritis models delays the onset of disease and reduces inflammation. Cigarette smoking, a known risk factor for RA, has been shown to increase the citrulline burden in the lung and also promotes NET formation. Smoking cessation may thus prevent the loss of immune tolerance in RA, in part, by modulating neutrophil function.

Anti-inflammatory Effects of Neutrophils While neutrophils clearly contribute to enhance acute inflammatory responses, recent evidence suggests that they also have anti-inflammatory properties that are important in self-regulating and dampening inflammation. Neutrophil-derived microvesicles are small ectosomes formed from the neutrophil that contain cytosolic material and surface proteins [95]. In RA, synovial fluid microvesicles express the anti-inflammatory protein annexin A1 and seem to have cartilageprotective capabilities through direct interaction with chondrocytes [96]. Recent evidence also suggests that neutrophil membrane-coated nanoparticles that express neutrophil-specific surface receptors but cannot respond to cytokines can inhibit synovial inflammation by neutralizing key proinflammatory cytokines [97]. Furthermore, dying neutrophils release a-defensins at sites of inflammation, and these molecules can inhibit inflammatory macrophages by reducing their capacity to release TNF [98]. In other inflammatory conditions such as

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gout, high density of neutrophils in the synovial joint can lead to aggregation of the formed NETs. In contrast to nonaggregated NETs, the aggregated ones can develop anti-inflammatory potential through their ability to cleave chemokines and cytokines [99]. While aggregated NETs have not been described in RA, it is important to keep in mind their potential anti-inflammatory role in certain conditions. Both viable and apoptotic neutrophils mediate an anti-inflammatory response in macrophages through suppression of NF-kB signaling [100]. The role of myeloidderived suppressor cells, a heterogenous population of myeloid cells that suppress T cell responses, remains ill-defined in RA but may have a protective role in rodent CIA [101,102]. While the anti-inflammatory potential of neutrophils in RA remains relatively understudied, harnessing these strategies may eventually provide novel treatment opportunities for RA patients (Figure 2).

Concluding Remarks While there have been significant advances in the understanding of the role of neutrophils in RA, many gaps in knowledge remain. Better understanding of neutrophil heterogeneity and plasticity in health and disease remains a key aspect that will help in designing more targeted approaches aimed at putative pathogenic subsets (see Outstanding Questions). Additional research should be aimed at understanding how neutrophils modulate innate and adaptive immune responses in the RA joint and systemically. The pathogenic role of neutrophils in the mechanisms leading to extraarticular organ involvement (primarily lung and skin) in RA requires further investigation, particularly given the recent findings suggesting that enhanced NET formation may be an early event in the pre-RA lung [103]. Indeed, given the contribution of neutrophils to the articular manifestations of RA, more comprehensive analyses of the neutrophils’ phenotype and function in other organs in RA may lead to further understanding of the pathogenesis of disease. Given the significant increase in cardiovascular risk in RA [85], further characterizing how dysregulated RA neutrophils and NETs contribute to this complication and may be a viable therapeutic target remain to be determined [86]. Better understanding of PAD isozyme regulation, physiology, and heterogeneity in health and disease and how citrullination impacts cellular function will be required to assess if targeting PADs in neutrophils and other myeloid cells is a viable therapeutic option. PADs are also important for transcriptional regulation, amongst other key cellular processes [104], and inhibition in humans could have an unacceptable side effect profile. Prior to the transition to clinical trials, a better understanding of the biology of PAD enzymes is required. Designing targeted therapies that influence dysregulated neutrophil function and/or neutralize cytokines that promote neutrophilic inflammation or their interaction with pathogenic immune cell subsets may also add to the efficacy of the current drug armamentarium used in RA (Figure 2 and Box 1). It will also be important to asses if targeting early aberrant neutrophil responses in pre-RA may lead to prevention or delay of disease onset. Finally, the use of neutrophil and NET-associated biomarkers in RA has the potential to help guide treatment decisions and improve patient care. A collective effort to probe biological samples from prospective cohorts and clinical trials is required before we can clearly delineate which biomarkers will be the most impactful. References 1.

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Outstanding Questions Which neutrophil-related biomarkers translate into improving clinical decision-making? Further studies may help bring some of these assays into mainstream clinical practice and provide clinicians with a precision-medicine approach to clinical problems. How do neutrophils promote extraarticular manifestations in RA and does this help us understand the pathogenesis of RA and other systemic autoimmune diseases? What role do specific neutrophil subsets play in the pathogenesis of RA? These cells have been reported in RA but their role in disease remains unclear. The role of citrullination in breaking immune tolerance in RA continues to be extensively studied, but the source of pathogenic citrullination needs to be better defined.

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