Netting Neutrophils Activate Autoreactive B Cells in Lupus

Lupus erythematosus (LE) is an autoimmune disease characterized by the aberrant activation of autoreactive B cells with the production of autoantibodies directed against nuclear Ags such as nucleic acids and associated proteins. These autoantibodies bind to extracellular DNA and RNA, leading to the formation of circulating immune complexes (ICs) that are deposited in peripheral tissues, leading to inflammation that damages target organs, including skin, joints, lungs, and kidneys (1, 2). A particularly important role in driving inflammation is played by plasmacytoid dendritic cells (pDC) (3). pDC recognize nucleic acids contained in ICs via TLR7 and TLR9 and produce high levels of type I IFNs, which activate a broad range of immune cells (4).

For many years, apoptotic cells have been considered to be the main source of nucleic acids contained in ICs. However, more recently, compelling evidence has accumulated for a role of neutrophils as an important source of extracellular DNA in LE (5). Neutrophils extrude large quantities of DNA in the context of a NETosis, a form of neutrophil cell death associated with the release of neutrophil extracellular traps (NETs) consisting of DNA filaments in complex with granular antimicrobial peptides such as LL37 and the human neutrophil peptides (HNPs) (6, 7). In addition to the potent antimicrobial activity of NETs, these DNA–antimicrobial peptide complexes possess a specialized capacity to activate pDC (5, 8, 9). In fact, within these complexes, the antimicrobial peptide LL37 converts the DNA from being immunologically inert into a potent activator of pDC by protecting it from extracellular degradation, transporting it into endosomal compartments (10), and promoting the formation of grill-like DNA structures that interdigitate TLR9 (11). In LE, circulating ICs contain DNA complexed with LL37 and HNP (5), suggesting that NETs are one origin of ICs. Large quantities of NETs have indeed been found in the kidneys, skin, and blood of lupus patients, and their presence correlated with the disease activity (9, 12). Increased NETs in LE result from a heightened release by lupus neutrophils (5, 9), as well as from an impaired ability to clear NETs in these patients (13–15).

In addition to being potent stimulators of pDC, LL37–DNA complexes in NETs also represent antigenic targets of autoantibodies in LE (5). In fact, lupus patients develop autoantibodies to LL37, which correlate with the presence of anti-DNA Abs and disease activity. The pathogenic function of these anti-NET Abs appears to be multifold: first, they bind NETs to form ICs that are efficiently taken up by pDC via FcγRII (5); second, they bind to LL37 exposed on the surface of LE neutrophils, leading to additional NET formation by neutrophils (5); and finally, they prevent DNases from accessing and degrading NETs (13). However, how B cells are activated to produce anti-NETs autoantibodies or whether LL37–DNA complexes in NETs play a role in this process is unknown.

In this article, we show that LL37–DNA complexes in NETs can directly activate human memory B cells (mBC). This occurs via the ability of AMP/DNA complexes, including LL37–DNA complexes, present in NETs to access endosomal compartments of B cells and trigger TLR9 activation. In LE patients, LL37 present in NETs additionally activates the LL37-specific mBC leading to the production of pathogenic anti-LL37 Abs.

Studies were approved by the institutional review and privacy boards of the Lausanne University Hospital, Switzerland, and the local ethics committee, in accordance with the Helsinki Declaration. Blood buffy coats of healthy donors were obtained from the Interregional Blood Transfusion Center, Bern, Switzerland. Systemic LE (SLE) and cutaneous LE (CLE) patients with a positive antinuclear Ab test from the dermatology department at the Lausanne University Hospital gave informed consent before study inclusion.

Human PBMCs were isolated by Ficoll-Paque centrifugation (GE Healthcare). mBC were isolated by using the human Memory B Cell Isolation Kit II (Miltenyi Biotec) from fresh PBMCs. The purity of mBC was verified by flow cytometry using monoclonal anti-human CD19 PerCP-Cyanine5.5 and anti-human CD27 allophycocyanin Abs (BD Biosciences) and reached 95%. Neutrophils were isolated with anti-human CD15 microbeads (Miltenyi Biotec). Neutrophil purity was assessed by flow cytometry with monoclonal anti-CD15 FITC (BD Biosciences) and reached 95%.

mBC from healthy donors or LE patients were seeded into 96-well round-bottom plates at 2 × 10⁵ cells in 200 μl RPMI 1640 supplemented with 10% FBS (Connectorate), 1% l-glutamine, 1% penicillin/streptomycin (Sigma-Aldrich), 1% HEPES, 1% amino acid (Life Technologies), IL-15 (10 ng/ml), and IL-2 (20 U/ml) (Miltenyi Biotec). LL37–DNA complexes were generated as follows. Human genomic DNA (BioChain) was digested with DNase1 to obtain sizes of DNA fragments between 250 and 1000 bp. This digestion was done to mimic the degradation of DNA in an extracellular environment in vivo. Then, 50 μg of LL37 (ProteoGenix) was premixed with 10 μg of DNA (peptide/DNA mass ratio of 5:1) in 100 μl of culture medium at room temperature for 20 min. LL37–DNA complexes were added to the mBC at 25 and 5 μg/ml respectively. Alternatively, mBC were stimulated with 1 μM CpG 2006 oligodeoxynucleotides or 10 μg/ml LPS (Thermo Fisher Scientific) or with complexes formed between DNA and the enantiomer form of LL37 (D-LL37). In some experiments, 3 μM chloroquine (Sigma-Aldrich) was used to block endosomal TLR9 activation. To prepare NETs, neutrophils were cultured into 24-well flat-bottom plates at 5 × 10⁶ cells/ml in RPMI 1640 without FBS, supplemented with l-Glutamine (1%) and penicillin/streptomycin (1%). NETosis was induced by stimulating neutrophils with 10 μg/ml anti-human LL37 mAbs (clone 8A8, IgG2b) at 37°C. After 3 h of stimulation, cells were spun down and cell-free NET-containing supernatants were collected and used at a 1:1 dilution to stimulate mBC. Activation of mBC was assessed by flow cytometry with monoclonal CD40 FITC and CD86 PE Abs (BD Biosciences). Proliferation of mBC was monitored with the Cell Proliferation Dye eFluor 670 (eBioscience) by flow cytometry according to the manufacturer’s protocol. Survival of mBC was monitored by flow cytometry using staining with 7-amino-actinomycin D and propidium iodide. The production of IgG Abs was measured in the supernatants by ELISA (Mabtech AB). In some experiments, blocking of the FcγRII was achieved by incubating cells with 10 μg/ml anti-human CD32, clone FLI8.26 (Stemcell Technologies) 15 min before stimulation, and RAGE was inhibited with 10 μM S100P-derived peptide–based competitive antagonist Ac-ELKVLMEKEL-NH2 (Calbiochem) 30 min before stimulation.

To assess the uptake of DNA in mBC, DNA was conjugated with Ulysis Nucleic Labeling Kit Alexa 488 as described previously. Briefly, DNA labeled with Alexa 488 was complexed with LL37 peptide as described above. Then, complexes of LL37–DNA were incubated with mBC for 4 h. Next, cells were extensively washed and stained with anti-human CD19 PerCP-Cyanine5.5 and anti-human CD27 allophycocyanin Abs. Internalization of DNA^A488 was monitored by flow cytometry with a FACSCalibur. In some experiments, mBC were pretreated with 1 μg/ml Cytochalasin D (Sigma-Aldrich) to inhibit endocytosis.

To assess the internalization of LL37–DNA^A488 complexes, cells were seeded onto poly-l-lysine–coated coverslips for 4 h and then fixed with 4% paraformaldehyde and permeabilized with PBS/1% BSA/0.1% saponin. Cells were then stained with mouse anti-human CD27 or anti-human TLR9 Abs (eBioscience) followed by A546-labeled goat anti-mouse IgG Abs. For assessing NET components internalization, fixed and permeabilized cells were then stained with rabbit polyclonal Abs (Abcam) against myeloperoxidase (MPO), lysozyme, lactoferrin, or cathelicidin (LL37), followed by staining with A488-labeled goat anti-rabbit Abs. DAPI was used to stain nuclei (Abcam). To create staining controls of the NET proteins, fresh blood-isolated neutrophils were stimulated with anti-LL37 Abs to produce NETs and were further stained as above. Cells were then analyzed by confocal microscopy (Zeiss).

To generate B cells that are deficient for TLR9, we transfected the LCL888 B cell line with two different CRISPR/Cas9 ribonucleoproteins (RNPs) targeting the TLR9 gene using the GeneArt CRISPR gRNA Design Tool from Thermo Fisher Scientific. Synthesis of guide RNA (gRNA) was as follows: two gRNAs targeting the first exon of human TLR9, TLR9-gRNA-T1 5′-ACGGCCTGGTGAACTGCAAC-3′ PAM: TGG and TLR9-gRNA-T3 5′-ACTTGAGGTTGAGATGCCGC-3′ PAM: AGG, were synthesized in vitro using the GeneArt Precision gRNA Synthesis Kit (Thermo Fisher) and were purified using the gRNA Clean Up Kit. Transfection of cells with CRISPR/Cas9 RNP–CRISPR RNP complexes were preformed by mixing 1 μg of GeneArt Platinum Cas9 Nuclease (Thermo Fisher) with 240 ng of gRNA in 5 μl of Resuspension Buffer R (Thermo Fisher) for 10 min at room temperature. For a negative control, Cas9 nucleases without gRNA were used. RNPs were then transferred to 5 μl of Resuspension Buffer R containing 1.10⁵ LCL888 cells and electroporated into cells (1550 V, 20 ms, and 1 pulse) using the Neon Transfection System. Cells were then plated in 24-well plates at 5.10³ cells/ml per well for 1 wk. For the validation of TLR9 deficiency, functional disruption of TLR9 was tested by stimulating transfected cells from the different wells with 1 μM CpG-B and assessing the production of IgG. Cells for which the greater reduction of activity was observed compared with control-transfected LCL888 B cells were then amplified for further experiments, and efficient disruption of the TLR9 gene was verified by sequencing TLR9 exon 1 (Fasteris, Geneva, Switzerland), which showed several indels.

The levels of human IgG Abs were measured by the commercial kit from Mabtech AB. To measure the levels of anti-LL37 Abs, LL37 peptides (ProteoGenix) were coated at 2 μg/ml overnight in Nunc MaxiSorp flat-bottom 96-well plates at 4°C. Plates were then washed in PBS plus Tween 0.05% and saturated with PBS/2% BSA for 1 h. Diluted sera and supernatants were then incubated for 2 h. Following washes, plates were incubated with peroxidase-conjugated AffiniPure F(ab′)₂ Fragment Goat anti-Human IgG (H+L) (1:2500 in PBS–2% BSA, Jackson ImmunoResearch). Plates were finally revealed with 3,3′,5,5′-tetramethylbenzidine (BD Biosciences), and reactions were stopped with H₂SO₄ 2N and read on a spectrophotometer at 450 nm.

To assess the presence of anti-neutrophil cytoplasmic Abs (ANCA), 5 × 10⁴ blood-isolated human neutrophils were seeded onto glass coverslips precoated with poly-l-lysine. Then, cells were fixed with 4% paraformaldehyde for 10 min at 37°C and permeabilized with 0.5% Triton X-100 in PBS for 5 min at 37°C. Nonspecific binding was blocked with PBS/10% BSA. Diluted supernatants (1:5 in 10% BSA) from stimulated mBC were then added for 1 h at room temperature. For the control, diluted sera (1:80) from healthy donors or LE patients were used. Cells were then stained with A488-labeled AffiniPure Goat anti-Human IgG (H+L) (Jackson ImmunoResearch) for 1 h at room temperature. DAPI was used to stain nuclei. Stained cells were analyzed by confocal microscopy (Zeiss), and the percentage of ANCA-positive cells was measured.

Comparisons between groups were performed using paired Student t tests. All statistical analyses were carried out with GraphPad Prism.

To investigate whether LL37–DNA complexes can trigger the activation of B cells, we purified blood CD27⁺CD20 mBC from healthy donors and stimulated them in vitro with fragments of purified human DNA (self-DNA), either alone or in complex with LL37. Whereas self-DNA alone was unable to activate B cells, LL37–DNA complexes triggered polyclonal activation of mBC with marked upregulation of CD40 and CD86 expression and IL-6 production and induced mBC proliferation and B cell survival, shown by the eFluor 670 dye dilution and Annexin V/propidium iodide staining, respectively (Fig. 1A, 1B, Supplemental Fig. 1). Furthermore, LL37–DNA complexes induced production of high levels of IgG Abs by mBC (Fig. 1C). B cell activation did not occur with LL37 alone but was increased with the addition of DNA in a dose-dependent manner (data not shown). Thus, LL37 promotes the ability of self-DNA to activate mBC polyclonally, leading to their proliferation and IgG production.

To elucidate the mechanism by which LL37–DNA complexes trigger polyclonal B cell activation, we used human DNA labeled with the fluorescent dye Alexa 488 to stimulate mBC. Confocal microscopy revealed that the LL37–DNA complexes were internalized by B cells and localized in vesicular structures between the CD27⁺ cell membrane and the DAPI⁺ nucleus of B cells (Fig. 2A), suggesting endocytosis of the complexes. Quantification of internalized DNA using flow cytometry confirmed that DNA in complex with LL37, but not DNA alone, was present in B cells (Fig. 2B), and that this process was completely inhibited by the chemical inhibitor of endocytosis cytochalasin (Fig. 2C). We then asked whether endocytosis of LL37–DNA complexes by B cells involves a specific receptor. To answer this question, we used the enantiomeric form of LL37, which retains the secondary structure of the peptides but not the amino acid sequence required to bind a potential receptor. Like LL37, the LL37 enantiomer was not only able to bind DNA but also able to internalize it into endocytic compartments (Fig. 2C) with consequent polyclonal B cell activation and IgG production (Fig. 2D), demonstrating that endocytosis of the complexes does not involve a scavenger receptor specific for the LL37 amino acid sequence. Because TLR9 is the specific endosomal DNA receptor, known to be expressed by B cells, we next investigated the role of this receptor in the activation of mBC. By using confocal microscopy, we found that endosomal TLR9 colocalizes with internalized LL37–DNA complexes in mBC (Fig. 2E). Next, we used chloroquine to inhibit endosomal TLR signaling, which is shown by its ability to block B cell activation by the TLR9 agonist CpG, but not the TLR4 agonist LPS. Pretreatment of mBC with chloroquine prior to LL37–DNA stimulation completely blocked IgG secretion (Fig. 2F). Furthermore, specific TLR9 disruption in B cells using CRISPR/Cas9 knockdown technologies abrogated IgG induction in response to LL37–DNA (Fig. 2G). Together, these data indicate that LL37–DNA complexes trigger the polyclonal activation of mBC via TLR9.

Because NETs contain LL37–DNA complexes that activate TLR9 in pDC, we next investigated whether NETs could also activate mBC. We generated NETs by stimulating neutrophils with anti-LL37 Abs. Anti-LL37 Abs strongly induced the formation of NETs, similar to LPS stimulation (Fig. 3A) that contained NET-derived LL37–DNA complexes (Fig. 3B). We then used anti-LL37–induced NETs to stimulate mBC. The use of LPS- or PMA-induced NETs to stimulate B cells was excluded, as residues of these compounds can activate B cells directly. Anti-LL37–induced NETs induced strong polyclonal mBC activation with IgG Ab production (Fig. 3C, Supplemental Fig. 2A). B cell activation was not triggered via the low-affinity IgG receptor CD32 by residual Ab present in the supernatant (Supplemental Fig. 2B) but was entirely mediated by NET/DNA, as the IgG production was blocked completely by chloroquine (Fig. 3D) and decreased in TLR9-deficient LCL888 B cells (Fig. 3E). We then tested the requirement of LL37–DNA complexes in this process by using AAPV-CMK, a proteinase 3 inhibitor that blocks the cleavage of LL37 from the inactive precursor protein (5). NET-induced B cell activation was partially decreased by pretreatment of neutrophils with AAPV-CMK (Fig. 3F), suggesting on one hand that LL37 is an important molecule in the NET-mediated activation of B cells but on the other hand that additional neutrophil proteins also contribute to this effect. In fact, other NET-binding proteins such as MPO, lactoferrin, and lysozyme were found to be internalized into B cells along with NET-derived LL37–DNA complexes (Fig. 3G). Importantly, uptake into B cells was independent of the RAGE receptor (Supplemental Fig. 2C), which was previously shown to mediate the uptake of NETs and activation of synovial fibroblasts. Like LL37–DNA complexes, lysozyme/DNA and lactoferrin/DNA, but not MPO/DNA, complexes were also found to activate B cells to produce IgG (Fig. 3H). Together, these data indicate that NETs can activate mBC to produce IgG via LL37–DNA complexes and potentially other DNA complexes involving DNA-binding neutrophil AMPs, such as lysozyme and lactoferrin.

Lupus patients have circulating ANCA including anti-LL37 Abs (5). To investigate whether, in addition to the polyclonal B cell stimulation, NETs would more specifically activate lupus B cells to produce anti-neutrophilic Abs in an Ag-specific manner, we selected six LE patients with circulating anti-neutrophilic Abs and anti-LL37 Abs and compared them with six healthy donors whose sera did not contain these Abs (Fig. 4A, Supplemental Fig. 3A–C, Supplemental Table I). mBC of both cohorts were then stimulated with LL37–DNA complexes or NETs, and production of anti-neutrophilic Abs and anti-LL37 Abs was determined (Fig. 4B). LL37–DNA complexes and NETs induced the production of high levels of total IgG in B cells of both lupus patients and healthy donors (Supplemental Fig. 3D); however, they only induced the production of anti-neutrophilic Abs and anti-LL37 Abs in lupus B cells (Fig. 4C). This finding is consistent with the presence of autoreactive B cells against neutrophil Ags in lupus patients but not in healthy donors. We then asked whether activation of these autoreactive lupus B cells by NETs involved Ag-specific recognition in addition to TLR9 activation. To address this question, we used the LL37 enantiomer, which retains TLR9 activation capacity when complexed with DNA but is unable to engage the LL37-specific BCR because of its inverted amino acid sequence. Both the original LL37 peptide and the LL37 enantiomer stimulated polyclonal B cell activation via TLR9 (Fig. 2D), but only the original LL37 peptide was able to induce the production of detectable levels of anti-LL37 Abs (Fig. 4C). This finding indicates that activation of autoreactive B cells requires Ag-specific recognition of LL37. The expansion of LL37-specific B cells was entirely abrogated if B cells were pretreated with chloroquine (data not shown), indicating that both a concomitant BCR and TLR9 activation are required for an efficient stimulation of NET-specific B cells in lupus.

Anti-LL37 Abs have been linked to disease activity in LE patients (5). Although increased NETosis of lupus neutrophils has been described (5), its link to the presence of circulating anti-LL37 Abs has not been established. Because in vitro NETs can directly restimulate lupus B cells to produce anti-LL37 Abs, we sought to investigate whether the link exists in vivo. We purified and stimulated peripheral blood neutrophils of six SLE and four CLE patients for 3 h before measuring NET production in the supernatant by the fluorescent PicoGreen staining. In parallel, we collected sera of these 10 patients to measure the presence of anti-LL37 Abs. We found a highly significant correlation between the NETing activity of neutrophils and the presence of anti-LL37 Abs in the circulation of these patients (Fig. 5). Thus, the increased NETting activity of lupus neutrophils directly correlates with the induction of pathogenic anti-NETs Ab in lupus patients.

Our study identifies a link between neutrophils, NETs, and the activation of self-reactive B cells. We show that neutrophils undergoing NETosis expose their DNA in complex with the granular antimicrobial peptides. These structures are highly immunogenic and trigger polyclonal B cell activation through TLR9 but also stimulate NET-specific self-reactive B cells by simultaneously engaging the BCR. In fact, via this mechanism, the increased NET formation in lupus patients triggers the activation of NET-specific self-reactive B cells that produce pathogenic anti-LL37 Abs and, potentially, anti-DNA Abs.

Normally, self-reactive B cells are eliminated in the bone marrow by clonal deletion. However, self-reactive B cells with DNA specificity can be detected even in healthy individuals as part of the normal human peripheral repertoire (16). These self-reactive B cells are unresponsive to extracellular DNA, as the DNA fails to access endosomal TLR9-contaning compartments even when internalized via the BCR (10, 17). Our study now shows that B cell unresponsiveness is broken when the DNA originates from NETs. In fact, DNA in NETs forms complexes with granular antimicrobial peptides, allowing protection from degradation and internalization into endosomal compartments. As a result, NET/DNA is able to trigger a polyclonal TLR9 activation of B cells with IgG production. In lupus, NETs also engage the specific BCR leading to the activation of self-reactive B cells that produce Abs against LL37. Potentially, NETs may also induce Abs against DNA contained in NETs, as there is a significant correlation between the anti-LL37 and the anti-DNA Ab titers in lupus patients (5).

Our studies demonstrate that NETs reactivate NET-specific mBC of lupus patients, most likely because of the increased frequency of NET-specific B cells in the B cell memory pool of these patients compared with healthy individuals. Our in vitro experiments were unable to prime NET-specific B cells from the naive B cells. This is most likely due to the lower precursor frequency in the naive B cell pool but is also due to the lower levels of TLR expression on naive B cells (18) and the requirement that T cells help with their activation (19).

A possible explanation for the fact that NETs lead to the activation of self-reactive B cells that produce Abs against NETs in LE patients but not in healthy donors is that LE patients have a genetically determined tendency for increased B cell activation. In fact, susceptibility genes involving B cell function and signaling, including BANK, BLK, and LYN, have been found in LE patients (20). Other susceptibility genes include genes involved in type I IFN signaling, such as TLR7, IRF5, TYK2, STAT4, IFIH1, TREX, and STING (20), which may decrease the threshold for B cell activation in LE. As a consequence, the break of B cell tolerance leads to the production of anti-NET Abs that further promote NET formation (5) and decrease NET degradation in LE (13–15), providing a feedback loop that sustains the inflammatory process.

Whether, in vivo, B cell activation requires a direct interaction with NETting neutrophils remains to be determined. Recent studies indicate that neutrophils can localize in the vicinity of B cells in the lymph nodes, where they fuel Ab production by secreting BAFF (21). Although such an interaction may also occur in LE, it is likely that NETs are released by circulating neutrophils and subsequently deposited in B cell areas of lymphoid organs. In favor of this hypothesis, blood neutrophils have an activated phenotype (22, 23) and NETs are detectable in the circulation of lupus patients as cell-free DNA or within ICs (5, 12).

Recently, a new form of NET resulting from the expulsion of mitochondrial DNA has been described (24, 25). Mitochondrial NETs result from a defective mitophagy in neutrophils, which leads to an abnormal disposal of mitochondrial DNA. As a result, mitochondrial DNA accumulates as oxidized DNA that is highly interferogenic upon expulsion (24). In LE, mitochondrial NETs are induced by exposure to type I IFN or RNP-ICs (24) and are abundantly released by a form of immature neutrophils called low-density granulocytes (25). Based on the potent capacity of mitochondrial NETs to trigger type I IFNs in pDC, it has been proposed that a high ratio of mitochondrial NETs versus classical NETs is pathogenic in lupus. Although classical and mitochondrial NETs are indistinguishable on a purely morphological basis, one major difference is that only classical NETs contain granule-derived proteins such as LL37. Because we find that classical NETs can activate lupus B cells and induce the production of pathogenic anti-LL37 Abs, our data provide evidence for an additional role of classical NETs in lupus autoimmunity.

An autoantigenic role of NETs and their associated granular antimicrobial proteins has also been described in other B cell–driven autoimmune diseases. In LE, the antimicrobial peptides LL37 and HNP represent the relevant NET autoantigens, whereas other granular antimicrobial proteins such as MPO and PR3 appear to be more important in ANCA-associated vasculitis (26, 27). Unlike LL37, MPO and PR3 are unable to promote TLR9 activation of B cells, suggesting they become autoantigens by epitope spreading in the context of NET-induced TLR9 activation of B cells. We found that antimicrobial proteins lysozyme and lactoferrin can, like LL37, promote TLR9 activation of B cells in complex with DNA. Interestingly, pathogenic anti-lysozyme abs have been described in Behçet’s disease (28), whereas antilactoferrin abs have been described in autoimmune hepatitis (29), suggesting a mechanism of dual TLR9/BCR engagement for autoantibody induction that is similar to that described for LL37.

In rheumatoid arthritis, NET-derived peptides, including LL37, become autoantigenic because they are citrullinated and recognized by anticitrullinated peptide Abs (30, 31). Future studies will have to determine why specific autoantigens in NETs are related to certain autoimmune diseases and what role citrullination in NETs plays for their antigenicity.

NETs or LL37–DNA complexes should be targeted for therapy of LE. Potential strategies include the use of N-acetylcysteine, an antioxidant that reduces reactive oxygen species and NET formation (32); DNase1, an endonuclease to digest NETs (33); PAD4 inhibitors, which deaminate histones and inhibit classical NETosis (34); or eculizumab, an anti-C5 Ab that reduces NET formation and favors NET degradation by preventing complement deposits in NETs (35). Additional therapeutic strategies may include direct targeting of LL37–DNA complexes and inhibiting their ability to activate B cells. In conclusion, we find a direct role of NETs in activation of self-reactive B cells via TLR9 and specific BCR, providing evidence for NETosis as a key pathogenic event in lupus that should be targeted therapeutically.

We thank Ana Joncic for technical assistance.

The authors have no financial conflicts of interest.