Neutrophils in the Pathogenesis of Rheumatic Diseases

Abstract Rheumatic diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), are a group of auto-inflammatory disorders associated with substantial morbidity and mortality. One unifying feature of these diseases is the presence of abnormal neutrophils exhibiting dysregulated neutrophil extracellular trap (NET) release, reactive oxygen species (ROS) production, degranulation, and pro-inflammatory cytokines secretion. Moreover, the release of autoantigens associated with NETs promotes the generation of autoantibodies and a breakdown of self-tolerance, thereby perpetuating inflammation and tissue injury in these patients. In recent years, targeted therapies directed at neutrophilic effector functions have shown promising results in the management of rheumatic diseases. In this review, we will highlight the emerging roles of neutrophils in the onset and progression of rheumatic diseases, and further discuss current and future therapeutic approaches targeting the pathogenic functions of neutrophils, which can modulate inflammation and hence improve patients’ survival and quality of life.


Introduction
Rheumatic diseases comprise a set of heterogeneous autoimmune disorders affecting predominantly the joints, tissues, and organs. Among them, the most common pathologiesincluderheumatoidarthritis(RA),systemic lupus erythematosus(SLE),andvasculitides. [1] Over the past few decades, accumulating evidence points to the involvement of aberrant and abnormal neutrophils in the initiation and perpetuation of rheumatic diseases. In this review, we will provide an overview of the emerging roles of neutrophils in rheumatic diseases. We will discuss current knowledge on how neutrophils contribute to disease pathogenesis, and further highlight emerging neutrophil-based strategies for effectivediseasemanagement.

Effector Functions of Neutrophils
Neutrophilsexpressavarietyofpatternrecognitionreceptors (PRRs) involved in the direct sensing of pathogenand danger-associated molecular patterns (PAMPs and DAMPs),includingToll-likereceptors(TLRs),C-typelectin receptors (CLRs), Nod-like receptors (NLRs), and RIG-like receptors (RLRs). These receptors enable them to respond almostinstantlytopathogeninvasionanddiverseinflammatorystimuliinthetissueenvironment.Uponreceptoractivation, a series of complex and diverse signal transduction pathwayswillbetriggered,whichculminateinthemounting ofantimicrobialimmuneresponses,suchasphagocytosis, reactive oxygen species (ROS) production, degranulation, and neutrophil extracellular trap (NET) release. [6] Although these processes are pertinent for the elimination of invadingmicroorganisms,prolongedandexaggeratedactivation of neutrophils could lead to hyperinflammation and tissue damage, indicating the importance of tight regulation to ensure a delicate balance between protective and pathological immune responses.

Degranulation
Neutrophils can secrete an array of effector molecules encapsulated in granules into the extracellular environment andphagosomestodestroyinvadingpathogens.Fourdistinct granule subsets, namely the primary or azurophilic granules,thesecondaryorspecificgranules,thetertiaryor gelatinase granules, and the secretory vesicles, are found in neutrophils. Azurophilic granules are packed with peptidesandproteinswhichconferpotentanti-microbialactivity throughoxidativeaswellasnon-oxidativemeans,including myeloperoxidase(MPO),defensins,bactericidal/permeability-increasingprotein(BPI),cathepsinG,elastaseandserine proteases.Granule-derivedMPOcatalyzestheformationof hypochlorousacid(HOCl)andothercytotoxicoxidants,while BPI targets Gram-negative bacteria by binding to lipopolysaccharide (LPS) to neutralize their proinflammatory propertiesandpromotephagocytosis.Thesegranulesprimarily release their contents into the phagosome for the elimination of internalized pathogens. On the other hand, specific granulesfusepredominantlywiththeplasmamembraneto deliver their cargo extracellularly. These granules contain high levels of iron-binding protein lactoferrin, as well as antimicrobial peptides cathelicidin and LL-37. These peptides and proteins are usually stored in an inactive form in the granulesandareactivatedbyproteolyticcleavageuponsecretion. Tertiary granules contain matrix metalloproteinase 9 (MMP9), beta-2 microglobulin, and various receptors and adhesion proteins to mediate the adhesion and penetration of neutrophils as they extravasate from the endothelium intotheinflamedtissue.Secretory granules are endocytic vesicles containing membrane-associated receptors such asCR1/CR3,formylmethionyl-leucyl-phenylalanine(fMLP) receptors, and FcRs which play critical roles during early inflammation. During degranulation, receptor-mediated signaling triggers an elevated calcium signaling, which induces the granules to translocate to the phagosomal or plasmamembranethroughactincytoskeletonremodeling andmicrotubuleassembly.Followingthis,thegranuleswill tether, dock, and fuse with the lipid bilayer membrane to releasetheircontentsintothephagosomeorextracellular environment.Among the granules, secretory vesicles are mostreadilyreleasedfromneutrophils,followedbytertiary granules,secondarygranules,andfinallyazurophilicgranules. [11]

ROS Release
Neutrophils generate a strong oxidative burst in response to various stimuli such as phagocytosis and bacterial componentsforeffectiveantimicrobialdefense.Uponactivation, NOX2complexassemblesitselfoncellularmembranessuch as plasma membrane and membranes of the phagosomes and secretory vesicles to produce large amounts of superoxide. Upon release into the extracellular environment or phagolysosome following phagocytosis, the superoxide will be spontaneously or enzymatically dismutated to hydrogen peroxide,andMPOcanfurtherconvertitintoothersecond-aryoxidantssuchasHOCl.AlthoughROScaninducedirect oxidative killing, most of their bactericidal properties stem fromtheirabilitytoaugmentpro-inflammatorycytokineproduction,degranulation,andNETosis. [12] NETosis NETsarelarge,extracellular,web-likestructurescomposed of decondensed chromatin and granule proteins (including neutrophil elastase, MPO, calprotectin, and defensins) which areextrudedfromtheneutrophilsinresponsetolargepathogens that cannot be phagocytosed. [13] (Figure 1) However, smallbacteriawhichevadephagocytosisthroughtheformation of large aggregates or interfering with phagosome maturationcanalsoinduceNETsrelease.DuringNETosis, MPO activates neutrophil elastase in a ROS-dependent manner, and this promotes their release from the azurophilicgranulesintothecytoplasmtofacilitatedegradation of the actin cytoskeleton, thereby blocking phagocytosis. NEisthentranslocatedtothenucleus,whereitdegrades histones and lamin to disrupt the chromatin packaging and nuclear envelope. In addition, protein-arginine deimi-nasetype4(PAD4)isrequiredforhistonecitrullinationby converting amine groups on arginine to ketones, thereby leading to chromatin decondensation. The decondensed chromatin, together with the damaged nuclear lamina, contributes to the destruction of nuclear envelope and the subsequent release of chromatin into the cytoplasm. [14] Activation of pore-forming protein gasdermin D protein (GSDMD)bycaspase-11hasalsobeenreportedtoinduce nuclear delobulation, DNA expansion, and plasma membrane rupture to elicit NETosis. [15] Although NETs release is a potent mechanism to combat invading pathogens, excessive production of NETs can lead to tissue damage and occlusion of vasculature. NETs can also serve as a source of autoantigens in various autoimmune rheumatic diseases,suchasRAandSLE,whichwillbediscussedin detail below.

Role of Neutrophils in Rheumatic Diseases
While neutrophils play a cardinal role in anti-microbial defense,prolongedandexcessiveactivationofneutrophils can lead to devastating consequences, such as cell lysis, tissue damage, and exacerbated inflammatory responses. Inrecentyears,agrowingbodyofevidencehasimplicated neutrophils in the onset and progression of various rheumatic diseases, including RA, SLE, and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In this section, we will outline current knowledge on the role of neutrophils in the pathogenesis of these rheumatic diseases.

Systemic Lupus Erythematosus
While SLE is long known to be associated with dysregulated B and T cell responses, the pathogenic role of neutro-philsinSLEhasbeenincreasinglyrecognizedoverthepast decade. SLE patients have a characteristic increase in the numbers of immature, low-density neutrophils (LDNs) in their peripheral blood, [23] andtheseSLE-derivedLDNsadopt an activated phenotype with augmented production of type I interferon (IFN), Tumor necrosis factor alpha (TNFα) and IFNγwhichcontributetodisease pathogenesis.Inaddition, enhanced apoptosis, together with the impaired clearance of these neutrophils, led to the release of autoantigens and the generation of autoantibodies such as ANCAs in the patients, therebypromoting auto-inflammation.NeutrophilsfromSLE patientsalsotendtoundergospontaneousNETosis,resulting in the release of LL-37, citrullinated histones, neutrophil elastase,andMPOwhichareattachedtotheNETchromatin fibers. [24,25] These further serve as autoantigens which can in turn activate plasmacytoid dendritic cells (pDCs) to produce large amounts of IFNα, thereby triggering a selfamplifyingpathogenicloop. [26] Moreover,NETscanmediate cardiovascularatheroscleroticcomplicationsinSLE patients by promoting endothelial damage through activation of the endothelial MMP-2. MPO and nitric oxide synthase present inNETscanfurtheroxidizehigh-densitylipoproteintomake it proatherogenic. In the vasculature, platelets can aggre-gateonandcooperatewithNETstoenhancethrombosis.As such,theaberrantfunctionsofneutrophilsplayan important part in promoting chronic inflammation and cardiovascular morbidity in SLE patients. Recently, neutrophil ferroptosis, an iron-and lipid-peroxidation-dependent programmed cell death, has been shown to drive neutropenia during SLE. In this case, autoantibodies and IFNα in the serum of SLE patientssuppressglutathioneperoxidase4(GPX4)expression through the calcium/calmodulin kinase IV (CaMKIV)/ cAMPresponseelementmodulator(CREM)αsignalingaxis, thereby enhancingtheproductionoflipid-ROStoinduceferroptosis,amajorformofneutrophilcelldeathduringSLE. [26] Anti-neutrophil Cytoplasmic Antibody-Associated Vasculitis AAV refers to a group of diseases characterized by the inflammationofsmallbloodvesselsassociatedwithnecrotizing neutrophils. Pathogenesis ofAAV is driven primarily by ANCAs targeting proteinase 3 (PR3) or MPO of neutrophils, and disease onset can be attributed to various genetic and environmental factors, including increased expression of PRTN3 gene or exposure to infectious pathogens such as Staphylococcus aureus. Upon priming by pro-inflammatory mediatorslikeTNF,IL-1β and complement C5a, neutrophils expressANCA target antigens MPO and PR3 on their cell surfacestofacilitateANCAbinding.Concurrently,FcγRs on neutrophils engage the Fc portion ofANCAs to completely activate the neutrophils. Together, these series of events promote ROS production by and degranulation of neutrophils, whichcandirectlydamagevascularendothelialcells.ANCAs alsoactivateneutrophilstoundergoNETosis,releasingMPO and PR3 autoantigens which further amplify inflammation. Moreover, NETs can mediate endothelial injury and vascular inflammation in these patients by triggering the alterna-tivecomplementpathwaytoproducecomplementfactor5a (C5a), a powerful chemoattractant for neutrophils. In addition, the presence of anti-NETs autoantibodies in AAV patients impairsthedegradationandclearanceofNETs,furthercontributingtothedamagetosmallbloodvessels.Collectively, these phenomena demonstrate the pathogenic role of neu-trophilsinAAVdevelopmentandtheirassociatedlong-term cardiovascular risk. [27][28][29]

Neutrophil-Associated Therapies for Rheumatic Diseases
Despite our growing knowledge of the pathophysiology of rheumatic diseases as well as the availability of specific biologic therapies, broad-spectrum anti-inflammatory glucocorticoids continue to be the first-line treatment for rheumatic patients.Eventhoughglucocorticoidsplaya pivotal roleinthe managementofinflammatoryautoimmune diseases, the adverse side effects associated with their prolonged usage are common and well-established. [30] Since accumulating evidencesuggeststhatneutrophilsarethe major orchestrator ofinflammationandtissuedamagein rheumaticdiseases,it maybeanattractivestrategytotarget neutrophilsandtheir effector functions for disease management (Figure 2 and Table 1).

Inhibition of NETosis
NETsplayanimportantpathogenicroleintheinitiationand progression of several rheumatic diseases as outlined above.
Hence,potentialcandidatesthatcanblockNETosisarecurrently being explored in various preclinical studies. PAD4 is an enzyme crucial for NET formation through catalyzing histone citrullination. Indeed, PAD4 inhibition using pan-PAD inhibitors like Cl-amidine or PAD4-specific inhibitors such asGSK199candisruptNETosisandhenceamelioratedisease severity in mouse models of SLE, RA, andAAV. [31][32][33] Moreover,recentstudiesrevealedthatPAD4-specificinhibitors have the advantage of few off-target effects, further supporting their utility as treatment options for rheumatic patients. [43] However,theefficacyofPAD4inhibitorsinthetreatmentof rheumatic diseases remains to be determined in clinical trials.

Inhibition of ROS Production
Sinceoxidativeburstbyneutrophilscantriggertissueinjury anddamageinrheumaticdiseases,managementof oxidative   stress has been explored as a potential therapy. Inhibition of p38 mitogen-activated protein kinase (p38MAPK) was previously shown to suppress neutrophil respiratory burst and hence prevent neutrophil activation by ANCAs in a mousemodelofAAV. [34]

Inhibition of Pro-inflammatory Cytokines Production
Given that neutrophil activation leads to pro-inflammatory cytokinessecretion,targetingthesemediatorsrepresentsa waytotreatrheumaticdiseases.Althoughbiologicstargeting pro-inflammatory cytokines (eg. Anti-TNFα) are highly efficacious in the treatment of rheumatic diseases, their development has been greatly hampered by their cost of production, route of administration, as well as safety profile due to the immunogenic nature of these biologics. [35] As such, targeting signaling pathways that function downstreamofcytokinereceptorshasbeenexploredasalternativeimmunotherapeuticstrategies.TheJAK-STATpathway plays a major role in transducing signals from a myriad of cytokines. Currently, three clinically approved small mole-culeJAK inhibitors,Tofacitinib,Baricitinib,andUpadacitinib, are in the market for RA treatment. [34,35] In addition, next-generationJAKinhibitorFilgotinibisbeingevaluatedin several phase III clinical trials for RA, and the results have been encouraging. [37,40,44]

Inhibition of Neutrophil Survival
Granulocyte-macrophagecolony-stimulatingfactor(GM-CSF) is an important growth factor that promotes neutrophil survival.

Inhibition of Neutrophil Activation and Recruitment
C5a is a product of the alternative complement pathway which mediates neutrophil recruitment and activation upon receptor binding. Avacopan, a small molecule inhibitor of the C5areceptor,hasrecentlybeenapprovedbyU.S.Foodand DrugAdministration(FDA)forthetreatmentofAAV. [42]

Inhibition of Neutrophil Ferroptosis
Neutrophil ferroptosis plays a critical role in the pathogen-esisofSLE.Indeed,inhibitionofferroptosisbyliproxstatin-1 (LPX-1) has been shown to rescue neutrophil cell death and alleviatediseaseseverityinamousemodelofSLE. [26] Outlook Advancementinthefieldofrheumatologyhasledtoconsiderable progress in disease management over the past few decades. With appropriate treatment, clinical remission has become a realistic therapeutic goal for most rheumatic patients.However,thecurrenttreatmentstrategyreliesmainly on the usage of glucocorticoids to suppress inflammation, and long-term immunosuppression is commonly associated withseriousadversesideeffectslikeinfectionandcancer.As such,abetterunderstandingofthemolecularpathwaysunderlyingeachdisease'sonsetandprogressionisnecessary for the development of next-generation targeted therapies.
In this review, we have discussed the role of neutrophils in driving rheumatic diseases and highlighted several promising pathways and signaling molecules that can be targeted to suppress their deleterious functions. However, this is a challenging endeavor since neutrophils are the first line of defenseagainstinfectionandhenceplayafundamentalrole in host immune responses. Ideally, their effector functions need to be targeted specifically under pathological conditions. Recently, our laboratory has identified Dok3 to be a key negative regulator of neutrophilic effector mechanisms, anditfunctionsinacontext-dependentmannerdownstream ofdifferentimmunoreceptorsviainteractionwithadistinctset of signal transducing molecules. [45][46][47] Thus, it is tempting to speculatethatDok3mayregulateneutrophilresponsesduring rheumatic diseases as well. A deeper understanding of theDok3signalingpathwaycouldpotentiallyrevealbinding partners which can be targeted for future treatment of rheumaticdiseases.Inaddition,lesstoxicimmunomodulatoryapproaches may be explored as therapeutic options for rheumatic diseases, as opposed to the use of broad-spectrum non-specificimmunosuppressiveagents.Forinstance,mesenchymalstemcell(MSC)exosomes,whichpossessunique immunomodulatory properties, have emerged as superior, well-tolerated candidates for therapeutics in recent years. Several preclinical studies have demonstrated their potential insuppressingjointinflammationduringRA, [48] and we have previouslyreportedtheirabilitytosuppressNETosisinneutrophils in response to complement activation. [49] However, future studies are warranted to understand the mechanistic effectofMSCexosomesonneutrophilsduringrheumaticdiseases for them to be approved as potential anti-rheumatic therapeutics.
While achieving remission represents an important milestone in the management of rheumatic diseases, the ultimate goal istodevelopacuresincerelapseislikelytooccuroncean anti-rheumatic treatment is withdrawn. [50] To tackle this goal, we will need to shift our treatment strategies from the targeting of neutrophil effector functions that elicit the immediate manifestations,totheidentificationofdiseasedriverswhich trigger the long-standing inflammation. Moving forward, studies will need to decipher the pathophysiology of rheumatic diseases which activates downstream pro-inflammatory effector pathways. The neutrophil is likely to be a key immunological driver of rheumatic diseases, and we propose future work to focus on investigating the molecular signatures of neutrophils associated with remission and cure. Single-cell RNA-seq of these cells in healthy and pathological conditionsmayyieldnewtargetsandpathwaysfordiseaseintervention. These studies are likely to reshape the landscape of rheumaticdiseasemanagementoverthenextfewyears, thus improvingthequalityoflifeforpatients.

ConflictofInterest
Kong-Peng Lam is an Editorial Board Member of the journal. This article was subject to the journal's standard procedures, with peer review handled independently of this member and his research group.