Abstract

Introduction

Genetic and environmental factors contribute to the development of rheumatoid arthritis (RA), a chronic, systemic inflammatory disease that attacks synovial joints and leads to increased morbidity and mortality. Various cytokines, including TNF-α and IL-17, play fundamental roles in the processes causing inflammation, joint destruction, and various comorbidities in RA(1). RA follows a natural history divided into phases initially characterized by asymptomatic autoimmunity (detection of RA-related autoantibodies (Abs)), then evolving into clinically apparent disease(2). Indeed, RA-related pathogenic autoAbs (those to citrullinated proteins (ACPAs) and rheumatoid factor (RF)) are detected years before clinical diagnosis(2).

AutoAbs to citrullinated antigens (Ags) are highly specific for RA and recognize epitopes centered by citrulline, a postranslationally modified form of arginine(3). Experimental evidence indicates that citrullination is involved in breakdown of immune tolerance and may generate neoAgs that become additional targets during epitope spreading(4). Citrullinated proteins and immune complexes containing various citrullinated Ags have increased immunogenicity and arthritogenicity, and their presence in arthritic joints correlates with disease severity. Some of the candidate citrullinated autoAgs include vimentin, antithrombin, α-enolase and fibrinogen (4–7).

The peptidylarginine deiminase (PAD) enzymes 2 and 4 likely generate these citrullinated Ags because they are expressed in myeloid cells (8) and are detected in the RA synovium closely associated with neutrophilic infiltrates (9). Increased neutrophils in RA synovial fluid (SF), particularly in early disease stages, supports a prominent role for these cells in joint damage(10). Indeed, critical roles for neutrophils in initiating and maintaining joint inflammatory processes have been described in experimental arthritis (10, 11). However, the exact roles that neutrophils play in autoAg modification and disease initiation and perpetuation in RA remain unclear.

Recent evidence suggests that, among the various mechanisms by which neutrophils cause tissue damage and promote autoimmunity, aberrant formation of neutrophil extracellular traps (NETs) could play important roles in the pathogenesis of systemic lupus erythematosus (SLE), psoriasis, small vessel vasculitis (SVV) and gouty arthropathy (12–15). NETs, released via a novel form of cell death called NETosis, consist of a chromatin meshwork decorated with antimicrobial peptides typically present in neutrophil granules(16). Of potential relevance to RA pathogenesis, NETs have the capacity to externalize proinflammatory, immunostimulatory molecules and various autoAgs (13, 14, 17). Histone citrullination, catalyzed by PAD4, appears to be a critical step in NETosis, and citrullinated histones are externalized in the NETs(18). We hypothesized that enhanced NETosis in peripheral joints, blood or other tissues, could promote initiation and perpetuation of aberrant immune responses and inflammation in RA, by externalizing citrullinated proteins and other immunostimulatory molecules. We also investigated whether autoAbs and inflammatory cytokines elevated in RA patients promote NETosis, thereby perpetuating a cycle of citrullinated autoAg generation and induction of autoimmune responses.

Results

NETosis is enhanced in RA peripheral blood (PB) and SF neutrophils and this correlates with ACPA levels and systemic inflammation

PB and SF neutrophils from RA patients display a significantly increased propensity to form NETs in the absence of added stimuli, when compared to PB control neutrophils or to SF neutrophils isolated from patients with osteoarthritis (OA) (Figures 1A, 1C). Significantly increased NETosis was observed following LPS stimulation, when compared to baseline levels, in RA and control neutrophils. Upon LPS stimulation, PB and SF RA neutrophils displayed significantly enhanced capacity to form NETs, when compared to control and OA neutrophils (Figure 1B–D). Furthermore, netting neutrophils were detected as infiltrating cells in RA synovial tissue, rheumatoid nodules and skin from RA patients affected by neutrophilic dermatoses (Figures 1E–F, S1 and S2). These observations suggest that RA neutrophils are primed to undergo NETosis in the joints and in the periphery. Evidence of enhanced NET formation was observed in unstimulated RA neutrophils within 1 h of culture, and continued to increase by 2–3 h in culture (Figure S3). A significant correlation was found between percentage of PB netting neutrophils and serum levels of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), ACPA (Figure S3) and IL-17. In contrast, RA disease duration, RF titers and counts of painful and swollen joints did not correlate with NETosis. There were no associations between use of various RA medications (disease-modifying antirheumatic drugs (DMARDs), corticosteroids and/or biologics) and percentage of neutrophils undergoing NETosis (Tables S1 and S2). These results suggest that enhanced and accelerated NET formation occurs in RA neutrophils, and is associated with presence and levels of ACPA Abs and with systemic markers of inflammation.

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Figure 1

Enhanced NETosis in RA neutrophils

A and B. Peripheral blood (PB) RA neutrophils undergo increased NETosis in the absence (A) or presence (B) of added stimulation (LPS for 1 h); (control, n=7; RA, n=13–14). C. SF RA neutrophils undergo increased NETosis in the absence (unstimulated) or presence of 1 h LPS stimulation, n=5/group, *p<0.05. Results represent mean ± SEM D. Representative microphotographs display enhanced LPS-induced NETosis in RA (bottom) vs. control PB neutrophils (top). NETs were visualized as structures co-staining for neutrophil elastase (green) and DAPI (blue); 40x magnification. E. and F. Multilobate inflammatory cells consistent with netting neutrophils are present in RA synovial tissue. E represents H&E stain (400X) of RA synovial tissue. F represents same area stained with anti-MPO (red) and DAPI (blue). Squares represent areas where netting neutrophils were observed. Images are representative of 3 RA patients. Statistical analysis was with two-tailed unpaired t tests except for SF experiments where paired t-test was used. Bar is 10 µm.

RA autoAbs and inflammatory cytokines induce NETosis

Some autoAbs present in the serum of patients with various autoimmune diseases, including anti-neutrophil cytoplasmic (ANCA) and anti-RNP Abs, stimulate NETosis upon neutrophil priming with inflammatory cytokines (12, 19). To test if RA-specific autoAbs similarly enhanced NETosis, we first compared the effect of RA serum and SF with control serum or OA SF, respectively. Both serum and SF from RA patients with high levels of ACPA and/or RF significantly induced NETosis in control and RA neutrophils, when compared to control sera or OA SF (Figure 2A–C). Similarly, when compared with IgG purified from healthy control sera or osteoarthritis SF, IgG fractions obtained from sera or SF from RA patients with high titers of ACPA and/or RF significantly increased NETosis in control or RA neutrophils, in the absence of additional cytokine priming (Figure 2D–E). Purified IgM RFs also potently induced NETs in control and RA neutrophils, compared to control IgM (Figure 2F). IgG isolated from ACPA-positive and/or RF-positive RA patients could bind to control and RA NETs, while IgG isolated from controls did not (Figure 2G). These observations suggest that RA autoAbs can mediate NETosis, and that autoAbs against NET components are generated in RA patients.

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Figure 2

AutoAbs present in RA serum and SF induce NETosis and bind to NETs

A–C: RA serum and SF induce significantly higher NETosis in RA autologous neutrophils (white bar) and control neutrophils (black bar), when compared to the effect of control sera or OA SF on autologous neutrophils or RA neutrophils. D–E. Similar effects were observed when comparing IgG purified from RA serum or from SF from patients with seropositive RA, compared to control serum or OA SF. F. Purified IgM RF significantly enhances NETs in RA and control neutrophils (n=3–8/group). The % of NETosis (elastase and DAPI labeled neutrophils/total neutrophils) was quantified after 1 h exposure to serum or SF. Results represent mean ± SEM; *p<0.05; **p<0.01 using two-tailed unpaired t tests. G. RA IgG isolated from patients with higher titer ACPA and/or RF binds to RA and control NETs induced by LPS. Results are representative of 3 independent experiments. Bar is 10 µm. NC: normal control; OA: osteoarthritis: RA: rheumatoid arthritis, SF: synovial fluid; RF: rheumatoid factor.

Patients with RA have enhanced levels of circulating inflammatory cytokines, including TNF-α and IL-17A. Members of the IL-17 family modulate various proinflammatory effects of neutrophils, including chemotaxis and bone marrow mobilization. However, while TNF-α can induce NETosis in other conditions(20), it is unknown whether IL-17A also promotes NET formation. Both recombinant TNF-α and IL-17A significantly induced NETosis in RA neutrophils (Figure 3A). The degree of NET induction by IL-17 in RA neutrophils was comparable to induction observed by positive control (PMA). In control neutrophils, IL-17A by itself did not significantly enhance NETosis. However, upon priming with TNF-α, recombinant IL-17A led to significant increases in NETosis. In contrast, as previously reported(21), control and RA neutrophils exposed to GM-CSF without other priming stimuli, did not display increased NET formation (Figure 3B).

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Figure 3

Proinflammatory cytokines induce NETosis and this is enhanced in RA neutrophils

A. Results are adjusted to levels observed in unstimulated control neutrophils. DPI: diphenyleneiodonium chloride; NAC: N-acetyl cysteine; Cl-amidine: PAD inhibitor. B. Neutralizing Abs to TNF and/or IL-17R decrease NETosis induced by RA sera. Results are expressed as relative fluorescent units (RFUs) of DNA fluorescence using Sytox green assay. Results represent mean ±SEM of 3 independent experiments, performed in triplicate; * p<0.05, **p < 0.005, ***p < 0.0001, using two-tailed (A) and one-tailed (B) unpaired t tests.

While the molecular mechanisms implicated in NETosis remain incompletely characterized, the NADPH oxidase (NOX) pathway has been implicated as a critical step (22). Further, an important role for PAD4 in NETosis has been proposed, potentially due to its role in histone deimination (18). To gain insights into which of these signaling pathways control IL-17A–induced NETosis, we took a pharmacological approach and investigated the effects of inhibiting PAD4 and the generation of reactive oxygen species (ROS). NOX inhibition with diphenylene iodonium (DPI) or scavenging of ROS with N-acetylcysteine (NAC) decreased NETosis in RA and control neutrophils stimulated with IL-17 or with IL-17+TNF. Similarly, the PAD inhibitor Cl-amidine (23) significantly inhibited NETosis (Figure 3A). These results implicate both the ROS/NOX pathways and PAD activity as important players controlling NETosis induced by inflammatory cytokines characteristic of RA. Conversely, incubating RA neutrophils with RA serum in the presence of neutralizing Abs to TNF-α or to IL-17R significantly decreased NETosis, when compared to RA serum alone. A significant effect was also observed in control neutrophils exposed to RA serum in the presence of neutralizing anti-IL-17R Ab. A non-significant similar trend was observed in control neutrophils exposed to RA serum plus neutralizing anti-TNF-α (Figure 3B). When neutralizing anti-TNF and IL-17R Abs were added in tandem, significant NETosis inhibition was observed in control and RA neutrophils, which was comparable to inhibiting IL-17R alone but more significant that inhibiting TNF alone (Figure 3B). Overall, these observations indicate that various factors present in RA serum, including autoAbs and inflammatory cytokines promote NETosis. The results also suggest that, in comparison to control neutrophils, RA neutrophils are primed to undergo NETosis upon exposure to IL-17A and TNF-α.

NET formation may also occur through a rapid (10 min-1 h) non-lytic process independent of oxidant production (24). As significant NET formation occurred by 1 h in culture, particularly in the RA samples positive for ACPA (Figures 1 and S4), we tested whether this phenomenon was accompanied by lysis detection. Significant LDH release was detected in the same samples where NETs were induced in control and RA neutrophils, in response to various RA-related stimuli (Figure S4). This suggests that enhanced NET formation in RA neutrophils and/or upon exposure to RA autoAbs and inflammatory cytokines within 1 h in culture is associated to cell lysis, as described in other conditions(22).

RA autoAbs and cytokines induce distinct protein cargo in the NETs

Previous work identified 24 NET-associated proteins released from control neutrophils in response to PMA (25). However, it is unclear whether different stimuli trigger the release of a different subset of proteins into the NETs. We exposed control neutrophils to: a) IgG fractions isolated from sera of 4 RA patients with high ACPA and/or RF titers (100 mg/mL); b) purified IgM RF from 4 patients with monoclonal IgM cryoglobulinemia (100 mg/mL), or c) recombinant TNF-α (100 ng/mL) for 1 h. NETs induced by these stimuli were isolated and their protein cargo was determined by proteomics analysis. Depending on the experimental condition, the number of individual NET-specific proteins identified ranged between 28–40, and included various proteases and defensins that are typically present in neutrophil granules, as well as histones, cytoskeleton-related proteins and cytosolic proteins (Figure 4 and Table S3). IgM RF-stimulation and RA IgG enriched in ACPA-stimulation led to the highest number of proteins identified in the NETs (n=36 for each), while TNF-α stimulation led to the identification of 28 proteins. While most NET proteins were conserved upon different types of stimuli (Figure S5), some were only detected following specific stimulation. For example, MMP-8, histone 3 and vasodilator-stimulated phosphoprotein were only detected in RA IgG-induced NETs, whereas catalase, moesin, transaldolase, phosphoglycerate mutase and olfactomedin-4 were only found in IgM RF-induced NETs. For the TNF-induced NETs, calmodulin, tropomyosin-3 and actin-related protein complex-3 were uniquely present in these samples (Table S3). Relative quantification of protein abundance was performed utilizing spectral counts as described(26), and statistically significant differences in NET protein levels in response to different types of stimulation were identified (Figure 4). Indeed, defensin-2 levels were significantly higher in RF-induced NETs than in the TNF-α-induced NETs, while MPO levels were significantly higher in RF-induced NETs than in those induced by RA IgG. Neutrophil gelatinase associated lipocalin and protein S-100A9 levels were significantly higher in the RF-induced NETs than in the other conditions. Overall, these results suggest that the protein cargo of the NETs varies depending on the specific stimulus utilized to induce these structures.

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Figure 4

Various stimuli present in RA sera induce distinct protein cargo in control NETs

Bar graphs represent spectral counts from 25 of the proteins identified in the NETs, which were normalized for protein content. RF=rheumatoid factor, RA IgG=IgG isolated from RA patients with high titers of ACPA and/or RF; *p<0.05, ** p<0.01, when comparing proteins expressed in the NETs induced by the 3 conditions. Results represent mean ±SEM of 3–5 independent experiments. Comparisons among groups were done with one-way ANOVA and p values were adjusted for Bonferroni’s multiple comparison test.

Citrullinated autoAgs are externalized during NETosis

The proteomic analysis described above identified several proteins that, when citrullinated, are important RA autoAgs(5, 7), including vimentin and α-enolase. We subsequently confirmed that these two proteins were present in the NETs formed by stimulation of healthy control neutrophils and in spontaneously formed or stimulated RA neutrophils (Figure 5A). Given the association between autoAbs targeting citrullinated vimentin (CV) and RA pathogenesis and inflammation(27, 28), we assessed the citrullination status of the vimentin in these NETs and found that this protein is indeed citrullinated (Figure 5B). These results indicate that NETs externalize citrullinated autoAgs reported to play important roles in RA pathogenesis.

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Figure 5

A. Vimentin and α-enolase decorate control and RA NETs. Red represents vimentin or α-enolase and blue is Hoechst. Total magnification 63X. B. Vimentin externalized in control and RA NETs is citrullinated. Left panel is visualization with Ponceau. Right panel depicts immunoblot using anti-modified citrulline and detection by chemiluminescence (right). Molecular weight markers (kDa) are indicated. Cit Vim=citrullinated vimentin; Hc=heavy chain; Lc=light chain; Vim=vimentin; IP: immunoprecipitate; IB: Immunoblot. Results are representative of 3 independent experiments. Bar is 10 µm.

Abs to CV stimulate NETosis

Given their high specificity for RA, and because CV is present in the RA-derived NETs, we hypothesized that anti-CV autoAbs can directly stimulate NETosis. To test this hypothesis, we purified human ACPAs with specificity to CV and determined whether they could stimulate NETosis. Indeed, anti-CV Abs potently induced NETs in control and RA neutrophils, when compared to control IgG (Figures 6A and S6). Furthermore, anti-CV Abs recognized vimentin externalized in the NETs (Figure 6B). These results indicate that autoAbs directed to specific citrullinated proteins present in the NETs enhance the formation of these lattices.

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Figure 6

A. Anti-CV Abs isolated from RA sera significantly enhance control neutrophil NETosis. Results represent mean+SEM of 3 independent experiments, each performed in duplicate. Units are expressed as RFUs of DNA fluorescence using Sytox green assay; *p<0.05, ***p<0.001, using two-tailed unpaired t test. B. Anti-CV Abs bind to vimentin externalized in the NETs. Original magnification is 63x; Ctrl=control. Results are representative of 2 independent experiments. Bar is 10 µm.

NETs activate RA and OA fibroblast-like synoviocytes (FLS)

To expand our understanding of the putative pathogenic roles of NETosis in the joints, we investigated whether NETs can stimulate FLS, which are the cells that invade cartilage in RA. Based on established kinetics of cytokine upregulation at the mRNA and protein level in these cells(29), we exposed FLS obtained from RA or OA patients to purified RA NETs for 24–48 h, and quantified the synthesis of IL-6, IL-8, CCL20 and ICAM-1 by ELISA and/or real-time quantitative PCR. Treatment of RA and OA FLS with RA NETs significantly upregulated IL-6 and IL-18 mRNA and protein levels, with responses being more dramatic in RA FLS (Figure 7A–B and S7). Neither IL-6 nor IL-8 were detected in the NETs alone condition. RA NETs also significantly upregulated mRNA levels of CCL20 and/or ICAM-1 in RA and OA FLS (Figure 7B). In additional experiments, when the NETs’ architecture was disrupted with DNase, their stimulatory effect on FLS decreased (42–44% reduction in induction of IL-6 secretion by OA and RA FLS when stimulated with nuclease-treated versus untreated NETs; 50–74% and 43–58% decrease in IL-8 and CCL20 mRNA fold-induction, respectively in OA and RA FLS; p<0.05 when comparing NET-exposed versus unstimulated FLS and p=NS when comparing FLS with FLS exposed to DNase-treated NETs). These results indicate that NETs possess a robust capacity as strong stimulants of FLS expression of proinflammatory genes, a phenomenon that may amplify deleterious inflammatory responses in the RA synovium.

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Figure 7

A. RA and OA FLS upregulate inflammatory cytokine synthesis upon exposure to RA NETs for 48 h. Results represent mean±SEM of 2 independent experiments, each performed in triplicate-quadruplicate; *p<0.05, ** p<0.01, ***p<0.001, using two-tailed unpaired t tests. B. RA and OA FLS upregulate mRNA synthesis of inflammatory cytokines, chemokines and adhesion molecules upon exposure to RA NETs. Results are expressed as fold induction of the specific gene at 48 h, when compared to untreated FLS at 24 h of culture, adjusted for housekeeping gene (GAPDH). Error bars represent maximum RQ value for each sample and represent mean±SEM of 2–3 independent experiments. *p<0.05 comparing change in mean DCt compared with untreated samples at same time-point, using two-tailed unpaired t tests. For untreated OA FLS, there was no amplification of CCL20, suggesting the basal expression was below the level of detection.

Discussion

Experimental evidence suggests that NETosis plays an important role in modification of autoAgs, their exposure to the immune system, and the induction of tissue damage in conditions like SLE, SVV and psoriasis(12–14). As activated neutrophils and anti-granulocyte Abs occur in RA, and there is a prominence of neutrophil recruitment in arthritis animal models (10, 11), we hypothesized that NETs may also play a pathogenic role in RA. Here we show that the RA pro-inflammatory milieu, characterized by specific autoAbs and increased proinflammatory cytokines, is highly conducive for the induction of NETosis in the absence of microbial stimuli. In turn, NETs externalize various immunostimulatory molecules and citrullinated autoAgs that, in predisposed individuals, may perpetuate a vicious cycle leading to generation of specific autoAbs and inflammatory responses (Figure 8).

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Figure 8

Proposed model of the role of NETosis in RA

RA NETs may provide a source of autoAgs and activate FLS and B cells. AutoAbs (RF, ACPA) and inflammatory cytokines (IL-17A, TNF-α, and IL-8) are all potential stimuli for enhanced NETosis in RA. In turn, RA NETs are a source of citrullinated (Cit-) autoAgs, including vimentin, further triggering production of ACPA. RA NETs also promote activation and cytokine release by FLS, with implications for joint damage and further propagation of a vicious cycle of NET induction and autoAb formation.

NETs may provide the immune system with access to enhanced sources of citrullinated proteins, and thereby represent an early event preceding epitope spreading. In addition, RA NETs induce proinflammatory responses in FLS, revealing an additional amplifying mechanism of joint damage. Indeed, the secretion of proinflammatory cytokines, such as IL-8, by FLS upon exposure to extracellular traps, may further enhance NETosis (30), amplify citrullinated autoAg exposure and promote autoAb generation in predisposed hosts. Recent observations indicate that CV, one of the autoAgs we identified in the NETs, induces proinflammatory cytokine secretion, PAD4 and RANKL expression in RA FLS (31). This supports that CV and other autoAgs present in NETs may be crucial in inflammatory response activation in the joint.

The increase in NET formation in RA neutrophils and/or upon exposure to RA-associated autoAbs and cytokines was observed as early as 1 h and was accompanied by evidence of cell lysis. The observation that RA autoAbs (including anti-CV and RF) potently induce NETosis is similar to what has been reported for other autoAbs (ANCA for SVV and anti-RNP for SLE) (12, 19). We also found that RA sera, but not control, can bind to NETs. Circulating autoAbs present in serum of patients with Felty's syndrome, a severe variant of RA, can bind to deiminated histones and NETs (32). While in that study only a small percentage of RA serum samples showed preferential binding to deiminated histones(32), it is possible that RA serum or autoAbs can recognize other citrullinated epitopes present in netting neutrophils. In support of this hypothesis, anti-CV Abs bound to vimentin present in the NETs.

Periodontitis has emerged as a risk factor for RA(33). Infection with P. gingivalis (Pg), the anaerobic pathogen primarily responsible for periodontal disease and also associated with chronic bacteremia(34), may play a central role in the early loss of tolerance to self-antigens in RA(35). Indeed, this microbe is the only identified prokaryote that expresses a PAD orthologue, and immunization with Pg enolase induces autoimmunity to mammalian α-enolase and arthritis in DR4-IE-transgenic mice (36). Importantly, increased NETosis occurs in gingival crevicular fluid from patients with periodontitis (37) and Pg can induce NET formation(38). One could speculate that enhanced citrullination and NETosis induced in the oral cavity in patients with Pg-induced periodontitis could be an initial event leading to generation of citrullinated autoAgs and autoAbs that could further promote NET enhancement and exacerbate autoimmune responses.

Smoking is also considered to be an important risk factor for RA. One of the proposed mechanisms is that tobacco smoke enhances pulmonary PAD expression, leading to generation of citrullinated proteins in this organ (39). Neutrophil derived PADs, present in the airways of smokers and those suffering other inflammatory conditions, can locally citrullinate the immunostimulatory peptide LL37. This renders the protein more chemotactic and alters its overall activity (40). Since LL37 is externalized during NETosis(14, 17), it is possible that its citrullination (as well as the citrullination of other targets in the lung parenchyma), could further promote pathogenic responses in RA and other autoimmune diseases.

IL-17 and TNF are increased in RA sera and SF and their elevated levels predict joint damage progression. IL-17 has widespread inflammatory effects on the joint, orchestrates bone and cartilage damage and induces recruitment of proinflammatory mediators to the synovium(41). While IL-17 is known to potently recruit neutrophils, a role for this cytokine in the induction of NETs had not been previously described.

Our study also supports the concept that not all NETs are created equal, as NET protein content of healthy control neutrophils varied depending on the source of stimulation. This supports the need to better understand how NET composition is regulated in the healthy individual and in disease states, and may allow the development of therapeutic compounds to selectively target some of the deleterious aspects triggered by these traps. Indeed, it will be important to further explore the relevance of the differential protein expression in the NETs triggered by autoAbs, cytokines and other “sterile” conditions.

One limitation of this study pertains to the use of samples from patients that were already receiving DMARDs and/or biologics. While we did not observe any correlation between the use of these medications and the ability of RA neutrophils to form NETs, studies in therapy-naïve patients and longitudinal assessments of the impact of medications on the regulated development and severity of NETosis will be needed. In addition, the proteomic analysis might have failed to comprehensively identify all proteins with potential relevance to induction of pro inflammatory responses in RA, revealing a need to focus efforts on the identification of molecules of low abundance but high biological activity present in the NETs. As an example, LL37 has not been identified in the NETs by proteomic analyses by us and other groups (25), while it is readily identified in these structures by other techniques(14).

Identifying the role of aberrant NET formation in animal models of RA, as well as further characterizing the in vivo responses of FLS in response to netting neutrophils in the specific joint milieu, will clarify the rationale for testing NETosis inhibitors in future clinical trials in this and, potentially, other chronic inflammatory conditions. The observation that the PAD inhibitor Cl-amidine is effective in CIA(23) further supports this hypothesis, as this compound decreased IL-17A–induced NET formation in neutrophils. It will also be important to investigate the role that NETs play in the development of extra-articular manifestations of RA, including lung involvement and cardiovascular disease. Indeed, various studies in other patient populations and murine systems are supporting a role for NETs in both vascular damage and inflammatory conditions of the lung (14, 42).

We have shown that NETosis is enhanced in the PB and the synovium of patients with RA and that these structures contain targeted citrullinated autoAgs. Furthermore, this phenomenon correlates with presence and levels of ACPA Abs and with systemic inflammation. ACPAs and RF may perpetuate a vicious circle of NET production that maintains the delivery of modified autoAgs to the immune system. The propensity of neutrophils to form NETs in individuals with RA may be further enhanced by microbes or their products. Indeed, RA is known to frequently flare following bacterial or viral infections(43). Identifying the role of NETosis in RA pathogenesis could provide new potential targets for the treatment of this disease and its associated complications.

Materials and Methods

Supplementary Material

Acknowledgments

Footnotes

References and Notes