The Journal of Clinical Investigation   REVIEW

Bite of the wolf: innate immune responses propagate

autoimmunity in lupus
Sarthak Gupta and Mariana J. Kaplan
Systemic Autoimmunity Branch, Intramural Research Program, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NIH, Bethesda, Maryland, USA.

The etiopathogenesis of systemic lupus erythematosus (SLE), a clinically heterogeneous multisystemic syndrome that
derives its name from the initial characterization of facial lesions that resemble the bite of a wolf, is considered a complex,
multifactorial interplay between underlying genetic susceptibility factors and the environment. Prominent pathogenic factors
include the induction of aberrant cell death pathways coupled with defective cell death clearance mechanisms that promote
excessive externalization of modified cellular and nuclear debris with subsequent loss of tolerance to a wide variety of
autoantigens and innate and adaptive immune dysregulation. While abnormalities in adaptive immunity are well recognized
and are key to the pathogenesis of SLE, recent findings have emphasized fundamental roles of the innate immune system in
the initiation and propagation of autoimmunity and the development of organ damage in this disease. This Review focuses
on recent discoveries regarding the role of components of the innate immune system, specifically neutrophils and interferons,
in promoting various aspects of lupus pathogenesis, with potential implications for novel therapeutic strategies.

Introduction predisposed hosts, this imbalance in dead cell handling can con-
Systemic lupus erythematosus (SLE) is an autoimmune syndrome tribute to modification and externalization of nucleic acids and
of unclear etiology that predominantly affects women and dispro- other autoantigens, to promotion of innate and adaptive immune
portionately afflicts minorities (1). Lupus derives its name from dysregulation, to the development of autoantibodies that predom-
the Latin word for wolf, and early descriptions of this disease used inantly recognize nucleic acids and/or proteins binding to nucle-
the term to describe the facial lesions that look like a wolf ’s bite. ic acids, and to the formation of immune complexes (ICs) that
SLE has pleiotropic clinical manifestations and profound clinical deposit in various organs and promote damage (3). Autoantibody
heterogeneity, making its diagnosis and treatment very challeng- formation can precede clinical disease by many years, suggesting
ing. Indeed, what we call SLE may be driven by heterogeneous that immune dysregulation is an early event in disease pathogene-
pathways of immune dysregulation that eventually converge into sis (4). Notably, aspects of immune dysregulation characteristic of
a loosely shared clinical phenotype. SLE affects many organ sys- SLE have been described in unaffected relatives (5), further sup-
tems, including skin, kidneys, brain, and the vasculature. Char- porting a role for both genetic and environmental factors in sus-
acteristically, SLE presents with periods of clinical and serologic ceptibility to autoimmunity.
flare interspersed among phases of clinical remission, which can The innate and adaptive arms of the immune system are
occur spontaneously or modulated by exposure to environmen- altered and almost every immune cell type becomes dysregulated
tal stimuli, such as ultraviolet light or infections. Recent advanc- in SLE and its various murine models (3, 6). The role of the adap-
es in immunomodulatory/immunosuppressive treatments have tive immune system in SLE has been thoroughly studied and can-
improved morbidity and mortality in SLE. However, as patients not be overemphasized, and various current therapeutic strategies
live longer, prolonged inflammation and exposure to enhanced target these immune responses (7). In contrast, the role of various
oxidative stress promote the development of chronic complica- components of the innate immune system in SLE pathogenesis is
tions, including increased incidence of premature cardiovascu- less well characterized, but recent work emphasizes their crucial
lar disease (CVD) (2). Furthermore, the ability to predict which role in initiating and perpetuating autoimmunity in this disease.
patients will respond to specific medications or develop end-organ Abnormalities in phenotype and function of monocytes, mac-
complications remains an area of need to optimize SLE outcomes. rophages (8, 9), myeloid and plasmacytoid DCs (10, 11), baso-
SLE develops in a multistep process in which genetic, epigen- phils (12), neutrophils (13), NK cells, and γδ T cells (6) have been
etic, and environmental factors can promote aberrant cell death described in SLE. Furthermore, dysregulation of fundamental
and ineffective clearance of dead cell debris (3). In genetically innate immune strategies, such as the complement pathway, syn-
thesis of and response to IFNs, and mechanisms of neural regula-
tion of immune responses, contributes to autoimmunity and tis-
Conflict of interest: The NIAMS has collaborative research agreements with MedIm- sue damage in SLE (3, 14–16). In addition, genetic variations that
mune/AstraZeneca, Pfizer, and Bristol Myers Squibb. MJK is on the scientific advisory
alter various components of innate immune responses have been
boards of Neutrolis, Inc. and Cytrill.
Copyright: © 2021, American Society for Clinical Investigation.
reported to contribute to the risk and/or severity of SLE (Table 1).
Reference information: J Clin Invest. 2021;131(3):e144918. This Review focuses on the putative roles that the crosstalk
https://doi.org/10.1172/JCI144918. between aberrant cell death (particularly the formation of neutro-

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Table 1. Genes that modulate the innate immune system and are associated with SLE risk

Gene Name Putative mechanism References

FCGR (2A, 3A, 3B) Fcγ receptor Antibody and immune complex signaling 132, 199
NCF2 Neutrophil cytosol factor 2 Defects in NOX-mediated ROS 111
IFIH1 (MDA5) Interferon induced with helicase C domain 1 (encoding melanoma RNA sensing, IFN signaling pathway 134
differentiation–associated protein 5)
TNFAIP3 (A20) Tumor necrosis factor-α–induced protein 3 Disruption of protein’s deubiquitinase activity, increased NET formation 104
through PAD4 modulation
PTPN22 Protein tyrosine phosphatase, nonreceptor type 22 Neutrophil activation, enhanced NET formation through PAD4 activation 3, 132
DNASE1L3 Deoxyribonuclease γ Defective nucleic acid degradation 120, 184
TREX1 Three prime repair exonuclease 1 Defective nucleic acid degradation 3
IRF5 Interferon regulatory factor 5 IFN signaling pathway 107, 132
IRF7 Interferon regulatory factor 7 IFN signaling pathway 132, 199
IRF8 Interferon regulatory factor 8 IFN signaling pathway 199
ITGAM Integrin αM Defective leukocyte adhesion and migration, enhanced IFN synthesis 132
TYK2 Tyrosine-protein kinase 2 IFN signaling pathway 132
STAT4 Signal transducer and activator of transcription 4 IFN signaling pathway 105, 132
TLR7 Toll-like receptor 7 Endosomal TLR, RNA sensing, IFN signaling pathway 199
TLR8 Toll-like receptor 8 Endosomal TLR, RNA sensing, IFN signaling pathway 199
TLR9 Toll-like receptor 9 Endosomal TLR, DNA sensing, IFN signaling pathway 199

phil extracellular traps), dysregulation of neutrophils, and the IFN as discussed below, play prominent roles in SLE pathogenesis (30,
pathway plays in the pathogenesis of SLE and its associated compli- 31). In particular, dysregulation in neutrophil cell death and the
cations, in the context of current and potential therapeutic targets. role of NETs in SLE, as discussed later, display high immunogenic
and proinflammatory potential.
SLE and the aberrant handling of cell death
Dysregulation in cell death pathways, defective clearance of dying Neutrophils, NETs, and SLE pathogenesis
cells and their products, and aberrant response to this cell debris by Neutrophil functions and plasticity. Over the past decade, neutro-
various immune cell subsets are considered fundamental aspects phils have reemerged as presumed culprits in various aspects of
of SLE pathogenesis (Figure 1). This process has been recently SLE pathogenesis (Figure 1 and ref. 13). As the first responders
reviewed in detail (17). Cell death is associated with modifica- against invading microorganisms (32), neutrophils develop and ter-
tion and redistribution to the cell surface and extracellular space minally differentiate in the bone marrow and are then released into
of many ubiquitously expressed autoantigens, which may affect the circulation, where they patrol and sense danger signals (32, 33).
immunologic self-tolerance. Dysregulation in many forms of cell Under conditions of homeostasis, neutrophils are also found in the
death, including apoptosis, necrosis, necroptosis, pyroptosis, and lung, spleen, and liver, and they follow tightly regulated circadian
neutrophil death through formation of neutrophil extracellular rhythms that allow for disarming strategies prior to their migration
traps (NETs), may have diverse effects on immune responses and to various tissues (33, 34). Whether disruptions in neutrophils’ nor-
tissue damage in the context of autoimmunity (17–21). It is import- mal circadian rhythm modulate autoimmune responses remains to
ant to consider that whether an elicited immune response to the be determined. In addition to their antimicrobial roles, neutrophils
dead cell becomes tolerogenic or immunogenic is determined not are responsible for fundamental homeostatic functions, including
only by whether cells die or not, but also by which cells die, and roles in coagulation, angiogenesis, resolution of inflammation, and
how and where they die (17). A combination of genetic (Table 1), tissue repair (33, 35–39). Some of these neutrophil properties are
epigenetic, and environmental factors may be involved in driving induced and modulated by specific tissues, with profound neutro-
aberrant cell death, impaired clearance, and/or altered immuno- phil intertissue heterogeneity in the steady state (33). Under condi-
logic response to the cell death products, including presentation tions of injury or infection, neutrophils display their antimicrobial
of modified autoantigens (Figure 1 and refs. 17, 19, 22–25). Vari- and proinflammatory strategies in tissues (33, 40), including the
ous mouse models that fail to clear apoptotic cells develop lupus- release of neutrophil-derived serine and matrix metalloproteinas-
like autoimmunity (26–28), and non-phagocytosed dead cells are es (MMPs) that cleave extracellular matrix components, thereby
detected in SLE germinal centers, indicative of impaired clearance disrupting existing tissue architecture (41). Neutrophils use a com-
(29). These autoantigens may be presented by germinal center plex armamentarium of strategies to immobilize and kill microbes,
follicular DCs to B and T lymphocytes in secondary lymphoid including phagocytosis and bacterial degradation through synthe-
tissues, resulting in loss of tolerance. The formation of ICs con- sis of ROS; release of antimicrobial peptides from granules; and the
taining autoantibodies that recognize nuclear and/or cytoplasmic synthesis of NETs, which are lattices of chromatin bound to gran-
material released from dead cells can lead to potent downstream ule peptides that have the ability to immobilize and potentially kill
inflammatory effects, including the synthesis of type I IFNs that, various microorganisms (42–44).

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Figure 1. Role of neutrophils, NETs, and IFNs in SLE pathogenesis. Various stimuli can trigger neutrophils to undergo neutrophil extracellular trap (NET)
formation. NETs, in turn, can externalize self-antigens, including oxidized DNA and/or DNA–antimicrobial peptide complexes that can be presented to
antigen-presenting cells (APCs) and activate plasmacytoid DCs (pDCs) to synthesize type I IFNs. NETs have the ability to activate the NLRP3 inflam-
masome in macrophages, resulting in increased release of IL-1 and IL-18, which further prime neutrophils to undergo NET formation and perpetuate tissue
damage. Different exogenous and endogenous stimuli can promote type I IFN generation. The synthesis of type I IFNs further modulates other APCs,
tissue-resident cells, and T and B cell functions. NET products and IFNs modulate T cell responses and can also activate B cells to undergo class switching
and secrete autoantibodies against a wide range of self-antigens. DNA–antimicrobial peptide complexes (like LL37-DNA) released from NETs have the abil-
ity to directly activate B cells via TLR9 and promote autoantibody generation. NETs directly stimulate T cells by decreasing their activation threshold via
Zap70-mediated phosphorylation of the T cell receptor (TCR). Activated T cells release IL-17 and other proinflammatory cytokines that can result in endo-
thelial cell damage as well prime neutrophils to undergo further NET formation and migrate to inflamed tissues. NETs and IFNs can promote direct tissue
damage and vascular inflammation through their effect on endothelial cells and platelets. APRIL, a proliferation-inducing ligand; BAFF, B cell activating
factor; BLyS, B lymphocyte stimulator; LDG, low-density granulocyte.

NETs are typically generated and released through a distinct rial (61). This process is followed by degradation of nuclear and
programmed inflammatory cell death process (Figure 2 and refs. granule membranes and extrusion of NETs into the extracellular
13, 43), although mechanisms of NET formation that do not result space (Figure 2). Gasdermin D, a pore-forming protein considered
in cell death have been described (45, 46). NETs contain many a key executioner of pyroptosis, has been implicated in certain
potent antimicrobial molecules that can stimulate other neu- forms of NET formation (62, 63) but not in NETs formed in lupus
trophils and immune cells (43, 47, 48). Besides microbial stimu- animal models (64).
li, NETs are induced by sterile inflammatory stimuli, including The role of aberrant NET formation and clearance in various
platelets (49, 50), cytokines (51), uric acid and cholesterol crystals homeostatic and pathogenic conditions is an area of increased
(52, 53), various autoantibodies (54–56), and ICs (55, 57, 58). While interest. Compared with males, neutrophils from healthy young
the exact mechanisms that lead to NET formation continue to be adult females display enhanced ability to form NETs, both sponta-
further refined, this process can occur through several pathways neously and under microbial or sterile inflammatory stimuli (65).
that may or may not involve the NADPH oxidase (NOX) pathway This suggests that, in women, neutrophils have a higher propensity
of ROS formation and/or mitochondrial ROS synthesis (55, 59). to generate and externalize modified autoantigens. In individuals
Once initiated, posttranslational modifications of histones — with genetic predisposition to autoimmunity, this enhanced abil-
including citrullination by the peptidylarginine deiminase (PAD) ity to form NETs may contribute to breaking tolerance and may
family of enzymes, particularly PAD4 (60) — promote changes in explain, in part, why women are more prone to develop autoim-
electrostatic interactions of DNA and histones that disrupt chro- munity but also mount more robust responses to infectious agents
matin’s structure and promote decondensation of nuclear mate- than men. Female neutrophils are also more mature, activated,

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Figure 2. Pathways of NET formation and targets for therapies. Stimulation of neutrophils in an individual genetically predisposed to SLE by various
stimuli (microbial, autoantibodies, cell products, etc.) mobilizes calcium from the endoplasmic reticulum that results in activation of protein kinase C
(PKC) and NADPH and/or mitochondrial ROS production. This leads to migration of granule protein to the nucleus and activation of peptidylarginine deimi-
nase-4 (PAD4), which induce citrullination, resulting in changes in electrostatic interactions of DNA and histones, which disrupt chromatin’s structure and
promote decondensation of nuclear material. This process is followed by degradation of nuclear and granule membranes with mixture of granular protein
with chromatin and eventual extrusion of NETs into the extracellular space. Gasdermin D (GSDMD) is implicated in some forms of NET formation, in which
ROS-mediated release of neutrophil elastase (NE) processes GSDMD, which further facilitates release and activation of NE. GSDMD also localizes to the
plasma membrane to form pores that promote cell lysis to release NETs. Possible therapies targeting critical steps in this pathway (represented by blunt
arrows) may be beneficial for SLE. FcγR, Fcγ receptor; IFNAR, type I IFN receptor; JAK, Janus kinases; MPO, myeloperoxidase; TLR, Toll-like receptor.

and readily primed to respond to danger signals than neutrophils ligand (APRIL) (79, 80), molecules that are highly relevant to B
from male counterparts, properties that appear to be regulated by cell development and function.
sex hormones (66). Several neural pathways activated by microbes or endogenous
Neutrophils regulate other innate and adaptive immune cells stimuli modulate neutrophil biology (16) through various neuro-
in fundamental ways (Figure 1 and refs. 67, 68). These cells can peptides that inhibit neutrophil function, including calcitonin
stimulate and suppress T cell responses in context-dependent gene–related peptide and somatostatin (81). Adenosine, a purine
manners (69). Various neutrophil granule proteases inhibit T nucleoside released by neutrophils at sites of inflammation, has
cell cytokine synthesis, including IL-2 and IL-6 (70). Neutro- complex effects on neutrophil biology, including NET formation,
phils can downregulate the ζ chain of the T cell receptor through which depend on the concentration of and signaling through its
synthesis of arginase and ROS, promoting T cell arrest (71), and four receptors (82–84).
they can express PD-L1 to induce IFN-dependent PD-1–medi- Attempts at characterizing neutrophil diversity in recent years
ated T cell apoptosis (72, 73). Conversely, neutrophils activate have highlighted the presence of neutrophil plasticity and neutro-
γδ T cells through cross-presentation of microbial antigens and phil subsets based on transcriptional, epigenetic, proteomic, and
cross-prime CD8+ T cells in an MHC class I–dependent manner functional analyses that suggest diverse physiological and patho-
(74, 75). Through T cell–independent mechanisms, splenic neutro- genic roles (Table 2 and refs. 39, 85–90). In healthy individuals,
phils function as B cell helpers (76) and are important sources of B several distinct circulating neutrophil subsets were recently identi-
cell activating factor (BAFF) (77, 78) and a proliferation-inducing fied based on single-cell transcriptomic analysis (66). It remains to

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Table 2. Neutrophil subsets described in the literature

Neutrophil subset Description Features Function

Low-density granulocytes Identified in various autoimmune and Colocalize with mononuclear cells upon density Proinflammatory, with enhanced ability to form
(LDGs) autoinflammatory diseases gradient separation NETs and damage the vasculature
LDGmature Characterized in SLE CD10+; less transcriptionally active; increased Associated with vascular damage and coronary
expression of IFN and neutrophil activation genes; plaque formation
multilobulated nucleus; spontaneous NET formation
LDGimmature Characterized in SLE CD10–; transcriptionally active; round/bilobed nucleus; Enhanced degranulation
enhanced degranulation; decreased chemotaxis
and phagocytosis
N1 Tumor-associated hypersegmented neutrophils TNF-α; ICAM-1; FAS; hypersegmented; Proinflammatory with antitumor cytotoxic activity
identified in association with factors in the ROS-dependent cytotoxicity
tumor microenvironment
N2 Tumor-associated neutrophils seen in majority CCL2; CCL5; circular nuclei; T cell inactivation; Antiinflammatory subset with tumorigenic effects
of untreated tumors matrix degradation
CD16dimCD62Lbright Immature neutrophils identified in experimental CD11clo; CD11blo; CD54lo Decreased ability to opsonize bacteria and
human models of endotoxemia generate ROS
CD16brightCD62Ldim Hypersegmented neutrophils identified in CD11chi; CD11bhi; CD54hi Increased capacity to produce ROS and suppress
experimental human models of endotoxemia T cell proliferation
CD177+ Predictor of relapse in ANCA-associated CD177+ Unclear
vasculitis
IFNhi Found in healthy subjects Increased expression of IFN-stimulated genes Unclear; higher ISG expression in women
in RNA sequencing than men

be better defined whether this heterogeneity is genetically driven healthy control neutrophils at the transcriptomic, epigenetic,
and/or secondary to differences in microbiome composition, circa- and functional levels (19, 85, 86, 90). LDGs are key drivers of the
dian rhythms, hormonal challenges, transmigration, senescence, characteristic type I IFN gene signature of SLE and contribute to
and/or specific tissue microenvironments. Identifying distinct synthesis of type I IFNs and other proinflammatory cytokines.
neutrophil subsets that play prominent roles in disease states is Lupus LDGs differ in protein content and have distinct modula-
key in selectively targeting pathogenic granulocytes without ham- tion of their cytoskeleton compared with normal-density control
pering important homeostatic functions of the neutrophil com- and lupus neutrophils. This translates into differences in modula-
partment as a whole. In this regard, the identification of a distinct tion of biomechanical properties that enhance their retention in
subset of proinflammatory neutrophils, called low-density granulo- microvasculature mimetics, suggesting that these cells may be
cytes (LDGs) because of their tendency to fractionate with PBMCs more prone to become trapped in organs like the lung and induce
during density separation of whole blood, has contributed to high- tissue damage (90). LDGs are not a homogenous population and
lighting the putative role of neutrophil heterogeneity and dysregu- display transcriptional, epigenetic, and functional heterogeneity,
lated neutrophil death in autoimmune diseases (85, 86, 91). comprising two main subpopulations of intermediate-mature and
NETs display many putative proinflammatory properties that immature neutrophils, with various degrees of chromatin accessi-
will be discussed in detail in the next section. In addition to their bility and distinct differences in transcription factor motif analy-
role in activation of the immune system, neutrophils and NETs sis (Table 2 and ref. 86). These two LDG subsets differ in various
contribute to resolution of inflammation by degrading chemokines functional readouts, including phagocytosis, responses to type
and cytokines and activating serine proteases that decrease neu- I IFN stimulation, NET formation, and chemotaxis. Overall, the
trophil recruitment (92). NETs can enhance Th2 cytokine expres- bulk of SLE LDGs do not represent immature neutrophils, and
sion, decrease Th1/Th17 cytokines (93), and induce both pro- and most of them have enhanced proinflammatory roles. Further-
antiinflammatory effects on DCs (Figure 1 and refs. 30, 94). Neu- more, they do not display suppressive capabilities characteristic of
trophils and NETs play a role in clearance of damaged endothelial myeloid-derived suppressor cells (97).
cells and remodeling of senescent vessels in some tissues (95). LDGs isolated from subjects with SLE (85) and other auto-
Role of neutrophils, LDGs, and NETs in SLE pathogenesis. A immune and autoinflammatory conditions (54, 56, 83, 98–100)
plethora of phenotypic and functional abnormalities have been exhibit enhanced propensity to form NETs and damage endothe-
described in lupus neutrophils (Figure 1), including increased lial cells, striated muscle cells, and other targets (19, 56). NETs
cell death, impaired phagocytosis, and dysregulated oxidative also promote endothelial dysfunction by inducing vascular leak-
activity (19, 47, 55, 85, 86). In SLE bone marrow, neutrophils con- age and endothelial-mesenchymal transition through their ability
tribute to type I IFN synthesis and B cell dysregulation through to degrade vascular endothelial cadherin and activate β-catenin
BAFF-mediated effects (96). signaling, with implications for lupus nephritis (101). LDG NETs
Lupus LDGs are considered a distinct neutrophil subset with are more immunostimulatory and proinflammatory than NETs
pathogenic roles. They differ from normal-density lupus and generated by other stimuli, with a higher abundance of modified

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self-antigens, including oxidized nucleic acids, and immunogenic/ drial dysfunction, and NET formation in SLE. This is supported by
proinflammatory molecules such as LL37 and MMP-9 (19, 55, 102). observations that inhibitors of mitochondrial dysfunction or mito-
NETs activate the NLRP3 inflammasome in macrophages, promot- chondrial ROS generation ameliorate murine lupus (55, 112–114).
ing enhanced release of IL-1 and IL-18, a process that is heightened Similarly, the effectiveness of targeting PAD4 as a mechanism to
in lupus macrophages and can perpetuate tissue damage (103). decrease NET generation and lupus in animal models appears to
Dysregulated NET formation plays a number of putative cru- be strain and model dependent, suggesting that models associat-
cial pathogenic roles in SLE. In genetically predisposed individu- ed with more aberrant innate immune dysfunction improve with
als, key autoantigens that are externalized by NET formation may NET inhibition through PAD4 modulation (115).
be presented to the immune system. Several genetic polymor- NETs can directly modulate adaptive immune responses
phisms associated with SLE risk can enhance NET generation. For in SLE. LL37-DNA ICs activate endosomal TLRs to drive poly-
example, genetic alterations in TNFAIP3, encoding A20, disrupt clonal B cell activation and expand self-reactive memory B cells
the protein’s deubiquitinase domain, promoting upregulation (116). B cells can internalize and process NET components, and
of protein citrullination and NET formation through increased present them on class II MHC molecules to autoreactive T cells,
PADI4 (104). Increased circulating NET complexes have been promoting generation of memory T cells. NETs directly affect T
reported in SLE patients with a STAT4 risk allele (105, 106). cells by decreasing their activation threshold via Zap70-mediated
Healthy individuals who are homozygous for the major SLE risk phosphorylation of the T cell receptor, promoting enhanced T cell
haplotype of IRF5 display increased spontaneous NET formation, responses to suboptimal dosage of antigens (117). Importantly, the
supporting dysregulated neutrophil function as an early mecha- internalization of NETs through various ligand-receptor interac-
nism promoting autoimmunity (Table 1 and ref. 107). tions can also drastically alter the function of other cells, including
Oxidation of nucleic acids and certain posttranslational mod- DCs and fibroblasts (30, 58, 94).
ifications of proteins distinctly occur during NET formation and In addition to enhanced capacity of LDGs to form NETs, a
may promote enhanced immunogenicity. Both genomic and subset of SLE patients have impaired clearance of circulating
mitochondrial DNA become oxidized during NET formation. NET components, which leads to increased half-life of modified
Enhanced nucleic acid oxidation enhances the interferogenic self-antigens and their immunostimulatory effects and association
potential of NETs by extending the nucleic acids’ half-life through with higher incidence of lupus nephritis (24). Various culprits for
resistance to degradation by nucleases, thereby effectively engag- disruptions in NET clearance have been proposed, including com-
ing intracellular DNA sensors that promote type I IFN responses, plement activation within NETs, nucleic acid oxidation (108, 118,
including the cGAS/STING pathway (55, 108). Ribonuclear protein 119), DNase I inhibitors, and anti-NET antibodies (24). Genetic
ICs characteristic of SLE enhance mitochondrial ROS generation, polymorphisms that impact the function of molecules involved in
which promotes oxidation of nucleic acids and NET formation (55). removal of nucleic acids can impair NET clearance. For example,
LDG NETs also display higher levels of oxidized nucleic acids than loss-of-function variants in DNASE1L3, encoding an extracellular
NETs purified from other sources, leading to higher induction of nuclease endowed with the ability to degrade NETs, have been
type I IFN responses in target cells (55). As such, the generation of reported in familial forms of severe SLE (120).
modified nucleic acids by oxidation during pathogenic NET forma- Dysregulated NET formation/clearance appears to be oper-
tion in SLE may be a key mechanism enhancing autoantigen half- ational in vivo, as neutrophils and NETs are increased in affect-
life and induction of type I IFN and other inflammatory respons- ed lupus skin, kidneys, and placenta, in association with tissue
es, promoting loss of tolerance. LDG NETs also display enhanced inflammation and organ damage (19, 121, 122). At this point, it has
expression of ubiquitinated proteins that increase activation of not been possible to differentiate which neutrophil subsets infil-
proinflammatory responses in SLE macrophages (109). Overall, trate lupus tissues, as no distinct cell surface markers differenti-
these observations indicate that not all NETs are generated equal, ate LDGs from other lupus neutrophils. Given recent advances in
as lupus NETs are substantially more immunogenic and proinflam- differentiating these cells at the transcriptional, epigenetic, and
matory than NETs generated in other conditions. protein levels (86, 90), it may be possible in the future to expand
The exact stimuli and pathways that lead to generation of these tools to identify the role of LDGs in vivo at the tissue level.
NETs (59) may determine their role in lupus pathogenesis. For
example, lupus-prone mice that lack NOX2, an enzyme involved LDGs and NETs in vascular damage in SLE
in certain types of NET formation, develop exacerbated SLE Accumulating evidence implicates NETs in the development of
(110), and patients with chronic granulomatous disease (CGD), vascular damage and atherothrombosis (2, 19, 85, 102, 123, 124).
characterized by impaired NOX activity, have a higher prevalence NETs induce endothelial cell apoptosis through an MMP-9/
of autoimmunity. CGD patients also display an LDG subset with MMP-2 axis that is enhanced in lupus LDG NETs (102). NETs
enhanced ability to form NETs with enhanced mitochondrial ROS harm vascular smooth muscle cells through a histone-dependent
synthesis and enhanced generation of IFN responses (55). In addi- process that induces lytic cell death and promotes instability of
tion, genetic polymorphisms that impair NOX activity (NCF2) are atherosclerotic plaques (125). Various mouse models of athero-
associated with enhanced risk of developing SLE (111) and with sclerosis and vasculopathy suggest that inhibiting NET formation
accelerated lupus and enhanced NET activity in mouse models. can alter susceptibility to plaque formation/inflammation and
As such, it is possible that a dysfunctional NOX pathway promotes thrombosis (124, 126–128). Overall, dysregulated NET formation
synthesis of alternative sources of ROS (such as the mitochondria) in the context of SLE may play prominent roles in the accelerated
that lead to aberrant pathways of nucleic acid oxidation, mitochon- vascular damage characteristic of this disease.

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SLE subjects demonstrate vascular inflammation, arterial dys- sclerosis characteristic of SLE. Elevated levels of IFN-regulated
function, and increased noncalcified plaque burden, which may proteins and ISGs associate with SLE vascular disease (119) inde-
explain the higher reported risk for acute coronary syndromes (2, pendent of traditional Framingham risk factors (153). Type I IFNs
123). Further supporting the role of LDGs in accelerated vascular impair endothelial function (154) and hamper vascular repair in
damage in SLE, circulating numbers of these cells, as well as an SLE (155). In lupus and atherosclerosis mouse models, type I IFNs
LDG transcriptional signature, associate with increased coronary impair vasculogenesis and promote endothelial dysfunction, ath-
plaque and aortic wall inflammation, independent of other vascular erothrombosis, and plaque progression (137). IFNs inhibit endo-
risk factors (86, 123). Furthermore, NETs can oxidize HDL and dis- thelial NO synthase and impair NO production by endothelial
rupt its atheroprotective role, in part by modifying the ability of this cells (156). Additionally, type I IFNs promote diet-induced insulin
lipoprotein to remove cholesterol from macrophages (cholesterol resistance by triggering liver accumulation of CD8+ T cells with
efflux capacity [CEC]) (129). HDL oxidation by NETs promotes subsequent glucose dysregulation and hepatic inflammation (157).
proinflammatory responses in macrophages because the modified Type I IFNs stimulate macrophage recruitment to arteries (158),
lipoprotein signals through the oxidized LDL receptor-1 (LOX-1), promote foam cell formation (159), and predict worse outcome
enhances NF-κΒ signaling, and disrupts the ability of HDL to block during myocardial infarction (160). ISGs are upregulated in bone
TLR-induced pathways (129). Overall, these observations suggest marrow neutrophil/monocyte progenitors and circulating and
pleiotropic vasculopathic roles of lupus LDGs and NETs. cardiac neutrophils soon after myocardial infarction (161). Block-
ing type I IFN signaling using an anti-IFNAR1 mAb (anifrolumab)
Pathogenic crosstalk between IFNs and decreases circulating NET complexes and improves CEC, imply-
neutrophils in SLE ing that type I IFN inhibition may improve cardiometabolic risk in
IFN pathway dysregulation plays critical roles in lupus pathogenesis SLE patients (138).
(130). A significant proportion of individuals diagnosed with SLE Other IFNs — type II (IFN-γ) and type III (IFN-λ1, IFN-λ2,
display an elevated IFN signature in peripheral blood and/or affect- IFN-λ3) — may contribute to SLE pathogenesis. Variations in cir-
ed tissues. Altered regulation of this pathway is likely an early event, culating levels of type I, II, and III IFNs and tissue responses may
since genetic polymorphisms that impact type I IFN signaling asso- help explain heterogeneity in pattern of organ involvement and
ciate with SLE risk (Table 1 and refs. 105, 131, 132). Genetically pre- IFN signature in SLE (15, 162, 163). Exogenous administration
disposed individuals may have a lower threshold to activate the type of IFN-γ can worsen human SLE (164), and elevations in IFN-γ
I IFN pathway when exposed to exogenous stimuli (viral, ultraviolet associate with disease activity. An IFN-γ signaling pathway is
light) or endogenous stimuli (dead cells, ICs, NETs) (55, 133, 134). activated in SLE PBMCs (165), and elevated IFN-γ in SLE T cells
In vitro and in vivo human and murine studies highlight the type I promotes monocyte/macrophage induction of BAFF (166). IFN-γ
IFN pathway’s importance in immune dysregulation and vascular may affect lupus B cell function and germinal center responses
and tissue damage (106, 135–139). Plasmacytoid DC activation is a (167, 168). Increases in type III IFNs have been reported in SLE,
main driver for enhanced production of IFNs, but other cell types, in correlation with disease activity (169, 170). In a lupus mouse
particularly of myeloid origin, also contribute (55, 85, 140, 141). model, type III IFNs were shown to have a pathogenic role that
IFNs enhance expression of costimulatory molecules and antigen is nonredundant relative to type I IFNs by promoting systemic
presentation by antigen-presenting cells (142) and alter B cell toler- immune dysregulation and local inflammatory effects in skin and
ance, plasma cell differentiation, immunoglobulin class switching, kidneys (171). B cells are the main immune responders to type III
antibody production, and generation of long-lasting memory B cells IFNs in humans, with potentially important implications in SLE
(143–147). Type I IFNs prolong T cell survival, inhibit T cell apopto- that require further investigation (172).
sis, enhance activity of cytotoxic T cells, suppress Treg activity, and Overall, IFNs play key roles in SLE pathogenesis, and the rele-
modify T cell polarization (142, 148, 149). vance of the various types of IFNs in explaining the heterogeneity
In the context of neutrophil dysregulation, type I IFNs can of SLE continues to be elucidated and may be of great importance
prime neutrophils to form NETs (47, 86, 150) and may enhance in the design of therapies that can target their deleterious effects
their response to TLR agonists (151). Conversely, as NETs induce (Table 3). A pathogenic crosstalk between IFNs and neutrophils
IFN production, this may lead to a vicious cycle of persistent may play fundamental roles in initiating, amplifying, and main-
inflammation (Figure 1). Compared with other immune cell types, taining various aspects of lupus immune dysregulation, as well as
LDGs display increased expression of ISGs (86) and increased in long-term outcomes driven by the development of vasculopathy
type I IFN–induced proteins (90). Healthy young adult females and end-stage organ damage (Figure 1).
display a neutrophil-specific increased ISG expression compared
with males, with associated hyperresponsiveness to type I IFNs Targeting neutrophils, NETs, and IFNs in SLE
(66). This enhanced priming of female neutrophils may lead to Given the growing evidence for a fundamental role of the patho-
increased responses to various danger signals. The neutrophils genic interplay between type I IFNs, neutrophils, and NETs in
that express enhanced ISGs represent a distinct circulating neu- SLE, there is interest in targeting them for therapeutic purposes.
trophil subset detected in healthy individuals, suggesting a puta- Decreasing the formation, release, and/or half-life of NETs can
tive critical role in antimicrobial and proinflammatory responses be attempted by a variety of strategies (Table 3). Targeting of oxi-
(Table 2 and refs. 66, 152). dative pathways implicated in NET formation using NOX (54,
Synergizing with the effects of neutrophils, type I IFNs may 172) or mitochondrial ROS (55) inhibitors, or ROS scavengers like
play a key role in triggering vasculopathy and premature athero- N-acetylcysteine (NAC) (173), has been explored in vitro and in

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Table 3. Potential therapeutics that target the IFN/neutrophil pathogenic crosstalk in SLE

Target/function (molecules) Effects on neutrophils/type I IFNs References

ROS scavenger (NAC) Decreased NET release/decreased IFN responses 173–175
Mitochondrial ROS scavengers and modulators of mitochondrial Decreased NET release; decreased intracellular and extracellular oxidation 55, 112–114
function (MitoTEMPO, idebenone, MitoQ, inhibitor of VDAC-1 of nucleic acids with decreased immunogenicity and IFN responses;
oligomerization) improvement in mitochondrial function
MPO inhibitors Decreased neutrophil recruitment, NET formation, and release of inflammatory cytokines 176
PAD inhibitors Reduced NET formation 124, 126, 127
Calcineurin inhibitors (cyclosporin A, tacrolimus) Modulation of calcium pools; reduced NET release 200
DNases Enzymatic degradation of NETs 181–183
Kindlin-3/integrin inhibitors Inhibition of neutrophil recruitment and NET release 188
C5a (eculizumab) Reduced NET formation and neutrophil activation 190, 191
B cells (rituximab, belimumab) Reduced NET formation and neutrophil activation 192
IFNAR blockade (anifrolumab) Decreased IFN signaling; reduced NET formation 138, 139, 194
JAK/STAT blockade (tofacitinib, baricitinib, filgotinib, Blockade of signaling of IFN and other proinflammatory cytokines; 106, 135, 198
upadacitinib, etc.) reduced NET formation
IL-17 (secukinumab, ixekizumab, etc.) Modulation of NET formation and neutrophil migration 186, 187
Antimalarials (hydroxychloroquine, chloroquine) Decreased NET formation; weak type I IFN inhibition through modulation 129
of endosomal TLRs and cGAS/STING

vivo. NAC decreased disease activity in SLE patients (174, 175), ease markers, perhaps owing more to the half-life of the molecule
while inhibition of mitochondrial ROS or targeting of aberrant used than to a lack of biological effects (183). DNaseIL3 is involved
mitochondrial function hampered murine lupus severity (55, 112, in degradation of extracellular DNA, like in NETs, and genetic
113). In contrast, NOX inhibition may exacerbate SLE as a result of deficiency of this enzyme in mice and humans is associated with
complex immunoregulatory effects (110). Inhibition of the func- autoantibody production and lupus features (184). Future studies
tion of various granule proteins can decrease neutrophil recruit- could assess the role of this enzyme in improving NET clearance
ment, NET formation, and inflammatory cytokine synthesis (176) in SLE. Other drugs that can affect NET structures by destabiliz-
and may be explored in future human studies. In preclinical stud- ing the neutrophil’s actin cytoskeleton have also been proposed as
ies, genetic deletion of PAD2 or PAD4 reduced ISGs, autoantibod- potential therapies (185).
ies, vascular dysfunction, and clinical manifestations in TLR7- Targeting IL-17 signaling can modulate neutrophil trafficking
dependent murine lupus, but not in other lupus models (115, 177). and NET formation and may mitigate lupus glomerulonephritis
Inhibiting PAD enzymes using small molecules also decreases (186, 187). Inhibition of neutrophil recruitment and NET release
NET formation and protects from organ damage and vascular dis- in tissues may also be accomplished by interruption of interactions
ease in various mouse models of lupus and vascular damage (124, between endothelial ligands and neutrophil-expressed kindlin-3
126, 127). Given the heterogeneity of response in murine systems, and β2 integrin and other similar modalities (188). Targeting var-
future studies in humans will lead to further clarification of the ious chemokines may also limit pathogenic neutrophil infiltration
role of PAD inhibition in SLE. in tissues (189). While inhibition of NET formation induced by
The putative deleterious effects on antimicrobial responses C5a priming is an important pathway to consider, early clinical
following NET inhibition in humans remain to be determined. trials using anti-C5 antibodies, although well tolerated, did not
PAD4-knockout mice maintain other antibacterial neutrophil promote improvements in disease activity even if they were effec-
functions even if NETs are inhibited. Even in mouse strains in tive in murine lupus (190, 191). In contrast, decreasing autoanti-
which PAD inhibition does not totally abolish NET formation, bodies and ICs through a combination of mAbs targeting CD20
the immunogenic and vasculopathic effects of these NETs are (rituximab) and BLyS/BAFF (belimumab) showed some benefits
diminished, suggesting that partial enzymatic blockade may be in lupus biomarkers and reduced circulating NETs (192), although
sufficient (177, 178). Patients with genetic disorders that lead to the benefit of combined therapy versus single therapy remains to
deficiency of molecules implicated in NET formation (PADs, be better defined.
cathepsin C) are not overtly immunosuppressed (179, 180). How- Targeting the IFN pathway holds promise for SLE man-
ever, additional preclinical and clinical studies are warranted to agement (Table 3). Blocking mAbs that target IFN-α, IFN-γ, or
assess the safety profile, in different patient populations, of strat- IFNAR1 have been studied in SLE (193). Anifrolumab, which
egies that specifically inhibit NETs, without considerably altering blocks IFNAR1, thus inhibiting the action of all type I IFNs, has
other essential neutrophil functions. shown promising results in one of two phase III trials, with a good
Degradation of already formed NETs to enhance their remov- tolerability profile and improvement in disease activity (139, 194).
al is another potential approach (181). Administration of DNase I Anifrolumab decreased NET formation in SLE subjects when com-
to lupus-prone mice was beneficial (182), but early phase Ib stud- pared with placebo and improved the function of lupus HDL and
ies of DNase I, though well tolerated, showed no changes in dis- other cardiometabolic parameters (138). These findings suggest

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The Journal of Clinical Investigation   REVIEW

that suppressing the IFN/neutrophil axis may help prevent long- various environmental stimuli on IFN responses, neutrophil het-
term vascular complications in SLE and other diseases in which erogeneity, and the composition of NETs may help to explain the
these pathways are dysregulated. Several medications currently variability of SLE manifestations and the diverse autoantibody
used to treat SLE can modulate — at different levels — NET forma- responses. Future studies should focus on better defining the
tion and/or the type I IFN signature. Antimalarials­— drugs with mechanisms that regulate the cross-play between IFN and myeloid
proven benefit in SLE, including CVD benefits — can decrease cells, and downstream consequences in autoimmunity, in order to
NET formation in vitro (129) and are weak type I IFN inhibitors, identify promising therapies. This has implications for other chron-
potentially through modulation of endosomal TLRs (195) and the ic inflammatory diseases in which type I IFNs, neutrophils, and
cGAS/STING pathway (196). NETs are proposed to play important pathogenic roles.
JAKs play fundamental roles in downstream signaling of mul- Several important questions remain, and the exact role of
tiple proinflammatory cytokines, including all IFNs (197). Addi- these immune players in disease pathogenesis may only be under-
tionally, JAKs directly modulate neutrophil biology. Tofacitinib, stood if clinical trials that specifically target neutrophil dysregula-
a pan-JAK inhibitor, reduced NET formation in vitro and in vivo tion, aberrant NET formation, and the neutrophil/IFN crosstalk
and decreased disease manifestations, including vasculopathy, in SLE come to fruition. One important aspect is whether it will
in lupus-prone mice (135). The JAK inhibitor baricitinib improved be feasible to specifically target LDGs, while leaving other neu-
lupus clinical features in a phase II study (198), with phase III trophil subsets untouched. This will depend on further advances
studies ongoing (NCT03616964, NCT03616912, ClinicalTrials. in the identification of unique markers of this neutrophil subset
gov). A phase Ia/IIb clinical trial using tofacitinib in mild to mod- and a better understanding of the genetic, epigenetic, and envi-
erate SLE showed significant decreases in type I IFN signature ronmental factors that modulate granulocyte plasticity in health
and circulating LDGs and NETs, with associated improvement in and disease states. While significant advances have occurred in
vascular function and cardiometabolic parameters (106). These characterizing LDGs, the factors that promote their formation,
observations support that targeting the neutrophil/IFN crosstalk release, and pathogenicity (genetic or acquired) remain unclear.
with JAK/STAT inhibitors may modulate lupus vasculopathy and The extent to which NETs contribute to the various aspects of
should be further explored in CVD prevention in SLE (106). lupus pathogenesis and why only certain autoantibodies pro-
mote NET formation remain to be clarified. Understanding why
Concluding remarks and remaining questions different autoimmune diseases, characterized by enhanced NET
SLE is complex and heterogeneous, with clear dysfunction in most formation, are associated with distinct autoantibody responses
components of the immune system and therapeutic challenges giv- that target specific NET components will help better characterize
en pathogenic and clinical heterogeneity. Dysregulation in crucial heterogeneity of the NET protein and nucleic acid cargo and the
innate immune pathways implicated in host defense has profound role that genetics play in the adaptive immune response to these
implications in various aspects of lupus pathogenesis, including structures. This may lead to the identification of better molecular
break of tolerance, induction of IFNs and other proinflammatory candidates as therapies to hopefully improve clinical outcomes.
cytokines, aberrant adaptive immunity, and tissue damage. Accu-
mulating in vitro and in vivo evidence, using human and murine Acknowledgments
systems, supports a central role for neutrophil dysregulation in SLE This research was supported by the Intramural Research Program
pathogenesis, spanning loss of tolerance, induction and amplifica- of the National Institute of Arthritis and Musculoskeletal and Skin
tion of inflammatory pathways, tissue damage, vascular disease, Diseases of the NIH.
and cardiometabolic dysfunction. Future studies will hopefully
help to better define subsets of patients in whom neutrophil/IFN Address correspondence to: Mariana J. Kaplan, Systemic Autoim-
interactions play particularly fundamental roles, in order to design munity Branch, NIAMS/NIH, 10 Center Drive, 12N248C, Bethes-
better personalized therapeutic approaches. Further understand- da, Maryland 20892, USA. Phone: 301.496.0517; Email: mariana.
ing the effects of the interaction between genetic, epigenetic, and kaplan@nih.gov.

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