Neutrophil Extracellular Traps Downregulate Lipopolysaccharide-Induced Activation of Monocyte-Derived Dendritic Cells This information is current as of September 28, 2021. Lorena Barrientos, Alexandre Bignon, Claire Gueguen, Luc de Chaisemartin, Roseline Gorges, Catherine Sandré, Laurent Mascarell, Karl Balabanian, Saadia Kerdine-Römer, Marc Pallardy, Viviana Marin-Esteban and Sylvie Chollet-Martin Downloaded from J Immunol 2014; 193:5689-5698; Prepublished online 22 October 2014; doi: 10.4049/jimmunol.1400586 http://www.jimmunol.org/content/193/11/5689 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2014/10/19/jimmunol.140058 Material 6.DCSupplemental References This article cites 64 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/193/11/5689.full#ref-list-1 by guest on September 28, 2021

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Neutrophil Extracellular Traps Downregulate Lipopolysaccharide-Induced Activation of Monocyte-Derived Dendritic Cells

Lorena Barrientos,*,† Alexandre Bignon,*,‡,1 Claire Gueguen,x,1 Luc de Chaisemartin,*,†,{ Roseline Gorges,*,† Catherine Sandre´,*,† Laurent Mascarell,x Karl Balabanian,*,‡ Saadia Kerdine-Ro¨mer,*,† Marc Pallardy,*,† Viviana Marin-Esteban,*,†,2 and Sylvie Chollet-Martin*,†,{,2

Polymorphonuclear neutrophils (PMN) play a central role in inflammation and participate in its control, notably by modulating dendritic cell (DC) functions via soluble mediators or cell–cell contacts. Neutrophil extracellular traps (NETs) released by PMN Downloaded from could play a role in this context. To evaluate NET effects on DC maturation, we developed a model based on monocyte-derived DC (moDC) and calibrated NETs isolated from fresh human PMN. We found that isolated NETs alone had no discernable effect on moDC. In contrast, they downregulated LPS-induced moDC maturation, as shown by decreased surface expression of HLA-DR, CD80, CD83, and CD86, and by downregulated cytokine production (TNF-a, IL-6, IL-12, IL-23), with no increase in the expression of tolerogenic DC genes. Moreover, the presence of NETs during moDC maturation diminished the capacity of these moDC to induce T lymphocyte proliferation in both autologous and allogeneic conditions, and modulated CD4+ T lymphocyte http://www.jimmunol.org/ polarization by promoting the production of Th2 cytokines (IL-5 and IL-13) and reducing that of Th1 and Th17 cytokines (IFN-g and IL-17). Interestingly, the expression and activities of the lymphoid chemokine receptors CCR7 and CXCR4 on moDC were not altered when moDC matured in the presence of NETs. Together, these findings reveal a new role for NETs in adaptive immune responses, modulating some moDC functions and thereby participating in the control of inflammation. The Journal of Immu- nology, 2014, 193: 5689–5698.

olymorphonuclear neutrophils (PMN) are the first line of of inflammation (3, 4) and pharmacological agents such as PMA antimicrobial defenses and are rapidly recruited to sites of and calcium ionophore (1, 3, 5–7). Netosis was initially described as P inflammation. PMN kill pathogens through various strat- a suicide-like mechanism, but a concept of “vital netosis” is also by guest on September 28, 2021 egies, including phagocytosis, degranulation, the oxidative burst, emerging (8). NETs are composed of extracellular chromatin dec- and release of neutrophil extracellular traps (NETs), a process orated with various granule-derived and cytoplasmic proteins. His- called netosis (1, 2). NETs are released by activated PMN in re- tones are the most abundant components [∼70% of total protein sponse to pathogenic bacteria, fungi, viruses, and protozoa (1, 3–5) (9)], but several important mediators of immune responses are also [reviewed in (6, 7)] and also in response to endogenous mediators present, such as elastase, lactoferrin, calprotectin, myeloperoxidase (MPO), and LL-37 [reviewed in (10)]. *INSERM, Unite´ Mixte de Recherche-S 996, “Cytokines, chimiokines et immunopa- The best-known function of NETs is pathogen trapping: this thologie,” Universite´ Paris-Sud, 92296 Chaˆtenay-Malabry and Clamart, France; property limits the dissemination of pathogens and exposes them † Universite´ Paris-Sud, Faculte´ de Pharmacie, 92296 Chaˆtenay-Malabry, France; to high local concentrations of various molecules immobilized on ‡Laboratory of Excellence in Research on Medication and Innovative Therapeutics, 92296 Clamart, France; xStallergenes, Antony Cedex 92183, France; and {Assistance the NET chromatin backbone (1, 7, 11) [reviewed in (12, 13)]. Publique Hoˆpitaux de Paris, Groupe Hospitalier Paris Nord Val de Seine, Hoˆpital However, NET accumulation can also have adverse consequences Bichat, Unite´ d’Immunologie (Auto-immunite´ et Hypersensibilite´s), 75018 Paris, France in in vitro and in vivo, such as epithelial and endothelial cell injury (6, 11, 14–16) and thrombus formation (17, 18) [reviewed in (10, 1A.B. and C.G. contributed equally to this work. 13)]. Moreover, by exposing self-Ags, NETs might be involved 2V.M.-E. and S.C.-M. contributed equally to this work. in autoimmune disorders, including small-vessel vasculitis, Received for publication March 5, 2014. Accepted for publication September 23, 2014. rheumatoid arthritis, type 1 diabetes, and systemic lupus ery- thematosus (SLE) (14, 19–21). In particular, during SLE, the This work was supported by an “Attractivite´” grant from Universite´ Paris-Sud. A.B. was the recipient of a fellowship from the French Ministry of Higher Education and association of self DNA with some PMN proteins (LL-37, ca- Research and from Fondation pour la Recherche Me´dicale (FDT20130928127). thepsin G, elastase, or secretory leukocyte peptidase inhibitor) Address correspondence and reprint requests to Prof. Sylvie Chollet-Martin, Unite´ induces IFN-a production by plasmacytoid dendritic cells (DC) Mixte de Recherche-S 996, Faculte´ de Pharmacie, 5 Rue J.B. Cle´ment, 92296 (22–25). This plasmacytoid DC/NETs/IFN-a axis could thus Chaˆtenay-Malabry Cedex, France. E-mail address: [email protected] represent an amplification loop of major importance in SLE The online version of this article contains supplemental material. pathogenesis (16, 23, 24) and illustrates how NETs can modulate Abbreviations used in this article: DC, dendritic cell; MFI, mean fluorescence inten- sity; MNase, micrococcal nuclease; moDC, monocyte-derived DC; MPO, myeloper- DC functions in vivo. Furthermore, on forming complexes with oxidase; NET, neutrophil extracellular trap; PI, propidium iodide; PMN, poly- RNA, these PMN proteins are able to activate myeloid DCs from morphonuclear neutrophil; rh, recombinant human; SLE, systemic lupus erythe- SLE patients, but not from controls (22, 24). matosus; TT, tetanus toxin C fragment. The role of PMN in DC functions is a growing field of inves- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 tigation [reviewed in (26, 27)] and has been evaluated using blood www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400586 5690 NETs DOWNREGULATE DENDRITIC CELL ACTIVATION

PMN or PMN-derived purified mediators. Activated PMN can negative fraction was kept for allogeneic T cell proliferation assays. Naive + promote DC migration (28) and also modulate DC maturation and CD4 T lymphocytes were also isolated from this fraction, by negative se- activation through cell–cell contacts (29), ectosome release (30), lection with the MACS naive CD4 isolation kit II (Miltenyi Biotec), to be used for Ag-specific T cell proliferation assays and polarization experiments. or mediator secretion. Interestingly, several granule-derived Total and naive CD4+ T lymphocytes were confirmed to have a purity .95%, mediators such as lactoferrin, LL-37, calprotectins, a-defensins, based on flow cytometry of CD4 and CD45RA expression, respectively. elastase, bactericidal/permeability-increasing protein, MPO, and NET production and isolation proteinase 3 have been shown to either downregulate (31–37) or upregulate (33, 38–41) DC functions, depending on the study. NETs were produced and isolated, as previously described (5). Briefly, 3 6 Few recent studies have evaluated the potential effects of NETs freshly isolated PMN were seeded in 12-well culture plates (1.5 10 cells/well) and stimulated with 5 mM A23187 for 3 h at 37˚C with 5% on APCs. Fully activated PMN bearing NETs can stimulate TNF-a CO2. The cells were carefully washed twice with 1 ml PBS and then and IL-8 release by THP-1 cells (42). NETs recovered after mi- treated for 20 min at 37˚C with 20 U/ml restriction enzyme AluI in HBSS crococcal nuclease treatment of activated PMN can enhance IL-1b to recover large soluble NET fragments. In some experiments, the cells and IFN-a production by LPS-activated macrophages (43). In were treated for 20 min at 37˚C with DNase (10 U/ml) or MNase (20 U/ml), a murine model of autoimmune vasculitis, inflammatory PMN two other restriction enzymes. Supernatants were collected and centrifuged at 300 3 g for 5 min at 4˚C to remove contaminating cells and debris. NET recovered from mouse skin exudates spontaneously produce NETs preparations were then pooled, aliquoted, and stored at 220˚C until use. that have been shown to activate bone marrow–derived DC and to DNA was quantified in NET samples by using PicoGreen (Molecular transfer PMN-derived self-Ags to these cells (21). These reports Probes), as previously described (5). tend to support a proinflammatory effect of NETs on APCs. Generation of human moDC However, an in vitro study showed no proinflammatory effect of Downloaded from 3 6 NETs alone on macrophages (44), and aggregated NETs promote Monocytes (2 10 /ml) were cultured for 5 d in RPMI 1640 Glutamax supplemented with 10% decomplemented FCS, 1 mM sodium pyruvate, the resolution of inflammation in a murine gout model (45). The 100 U/ml penicillin, 100 mg/ml streptomycin, 550 IU/ml rhGM-CSF, and conflicting effects reported in the literature might be related to 550 IU/ml rhIL-4 (R&D Systems, France). On day 5, flow cytometry technical variations in NET preparation and APC models. In (FACSCalibur flow cytometer and Cell Quest software [BD Biosciences]) particular, the proinflammatory effects of NETs on APCs were confirmed the generation of nonactivated moDC with the following phe- notype: CD14neg, DC-SIGNhigh, CD1ahigh, CD83neg, and CD86low,as observed using both activated and primed full PMN (21, 42, 43). http://www.jimmunol.org/ previously described (46, 47). We therefore examined the effects of isolated NETs on monocyte-derived DC (moDC) functions. Large functional NET NET pretreatment and moDC activation fragments were prepared from human fresh blood by using the These moDC (106/ml) were washed once with cold RPMI 1640 for 5 min, restriction enzyme AluI, as we have previously described (5). and incubated with or without AluI-derived NETs (10, 100, or 300 ng/ml These isolated NETs downregulated LPS-induced moDC matu- DNA), MNase-derived NETs (100 ng/ml DNA), or DNase-derived NETs ration, as shown by changes in cell surface marker expression and (100 ng/ml DNA) for 30 min, before adding FCS and LPS (25 ng/ml) or not to the medium. Controls were also done using AluI (20 U/ml), DNAse cytokine production, but did not affect the expression or function (10 U/ml), or MNase (20 U/ml) alone, instead of NETs. In some experi- of selected lymphoid chemokine receptors, or migratory capacity. ments, moDC were activated with R848, a TLR7/TLR8 ligand (10 mg/ml), However, NET-treated mature moDC displayed a decreased ca- or PMA (300 nM) instead of LPS. Cells and supernatants were recovered by guest on September 28, 2021 pacity to activate CD4+ T lymphocyte proliferation and reduced after 24 h at 37˚C, unless otherwise specified. MoDC maturation was Th1 and Th17 polarization in favor of Th2 polarization. These evaluated by quantifying both cytokine release (as indicated below) and HLA-DR, CD40, CD80, and CD86 expression. Phenotypic results were results reveal a new and important role for PMN in the regulation expressed as the relative mean fluorescence intensity (MFI), corresponding of adaptive immune responses, via NETs. to the MFI of the specific label/MFI of the isotype control. CCR5, CCR7, and CXCR4 expression was analyzed with a LSR-Fortessa cytometer, and the results were displayed in MFI, as previously described (48). Cell Materials and Methods viability was determined by labeling with annexin V and 7-amino- Reagents and Abs actinomycin D, following the manufacturer’s instructions, followed by Calcium ionophore A23187, LPS from Escherichia coli serotype O55:B5, analysis on a FACSCalibur cytometer. dexamethasone, tetanus toxin C fragment (TT) from Clostridium tetani, RNA isolation and real-time PCR analysis of cytokine RNAs in PMA, and AMD3100 were from Sigma-Aldrich (Saint-Quentin Fallavier, France). Imiquimod (R848) was from Invivogen (Toulouse, France). FITC-, moDC PE-, PE-Cy7–, or allophycocyanin-conjugated mAbs against human CD4, MoDC (106/ml) were washed once with cold RPMI 1640 for 5 min, and CD40, CD80, CD83, CD86, HLA-DR, CD4, CCR5, CCR7, and CXCR4, incubated with AluI-derived NETs (100 ng/ml DNA) or AluI alone for 30 and their respective isotype controls, were from BD Biosciences (Le Pont min, before adding FCS and LPS (25 ng/ml) or not to the medium. After 2, de Claix, France). CFSE was from Molecular Probes; micrococcal nu- 4, 6, or 8 h at 37˚C, cells were washed twice with cold PBS. Total RNA clease (MNase), DNase I, and AluI were from New England Biolabs (Evry, was isolated by using TRIzol reagent (Invitrogen, Grand Island, NY), and France); and annexin V apoptosis detection kit I and recombinant human cDNA was synthesized from 1 mg RNA by using oligo-dT primers and (rh) TNF-a were from BD Pharmingen and BD Biosciences. The che- avian myeloblastosis virus reverse transcriptase (Promega, Lyon, France), mokines CXCL12 and CCL19 were from R&D Systems. following the manufacturer’s protocols. Gene transcripts were amplified Cell isolation using SYBR Green technology on a Bio-Rad CFX96 system. Each reaction was performed with 4 ng cDNA, 0.5 mM each forward and reverse primer, PMN, PBMC, monocytes, and naive CD4+ T lymphocytes were isolated and Sso Advanced SYBR Green Supermix (Bio-Rad, Marnes la Coquette, from healthy donors’ peripheral blood provided by Etablissement Franc¸ais France) in a 10 mL final volume. After 30 s at 95˚C and 44 cycles of du Sang (Rungis, France). Whole-blood centrifugation (20 min at 690 3 g, amplification (95˚C for 5 s, 60˚C for 5 s), a melting-curve reaction was 20˚C) on lymphocyte separation medium (Eurobio, Les Ulis, France) was performed from 65˚C to 95˚C to confirm the presence of a single ampli- used to separate PBMC (supernatant) from PMN and erythrocytes (pellet). fication product. For each target gene, a 3-fold serial dilution of pooled PMN were then isolated by pellet sedimentation on 5% dextran T500 cDNA was analyzed to calculate the amplification efficiency (E). The (Pharmacia, Uppsala, Sweden) in 0.9% saline at a ratio of 4:1. Contami- relative amount of target mRNA was determined with the cycling threshold nating erythrocytes were removed by hypotonic lysis, and PMN (consis- method [(1 + E)2ΔΔCt], were E is the amplification efficiency. The ratio of tently .95% pure) were resuspended in HBSS (Life Technologies, Saint each target gene threshold value to the geometric average of the reference Aubin, France) supplemented with 0.5% FCS (AA, Les Mureaux, France) gene (ACTB and GAPDH) threshold values was calculated for each sam- and 10 nM HEPES (Life Technologies). Monocytes were isolated from ple. Results were expressed as relative gene expression, corresponding to PBMC by magnetic bead sorting of CD14+ cells (MiniMacs separation the ratio of the former calculated value for a given sample to the value columns; Miltenyi Biotec, Bergisch Gladbach, Germany). The CD14- obtained for the nonactivated sample. The primer sequences for IL-1B, IL- The Journal of Immunology 5691

6, IL-10, and IL-23A have been published elsewhere (49). The sequences and 1:30). On day 6, cells were stained with PI and analyzed by flow of the other primers were as follows: TNF (59-GCCCAGGCAGTCA- cytometry. Proliferation of CD4+ T lymphocytes was expressed as the GATCATCT-39 and 59-TTGAGGGTTTGCTACAACATGG-39), IL-8 (59- percentage of CFSElow PI-negative cells present in the sample. TCTCTTGGCAGCCTTCCATGA-39 and 59-TGGGGTGGAAAGGTTT- To assess T lymphocyte polarization, moDC pretreated with NETs (100 GGAG-39), ACTB (59-GGCATCCTCACCCTGAAGTA-39 and 59-GCA- ng/ml DNA) were cocultured with allogeneic naive CD4+ T lymphocytes CACGCAGCTCATTGTAG-39), and GAPDH (59-CAGCCTCAAGATC- (moDC:T lymphocyte ratio 1:10) for 5 d, as previously described (50). ATCAGCA-39 and 59-TGTGGTCATGAGTCCTTCCA-39). Supernatants were then analyzed for cytokine release, as described below. Quantification of effector and tolerogenic moDC gene Cytokine quantification in moDC and moDC/T lymphocyte expression culture supernatants MoDC (106/ml) were washed once with cold RPMI 1640 for 5 min and MoDC supernatants were assayed for IL-6, IL-8, IL-10, IL-4, and TNF-a by incubated with or without AluI-derived NETs (100 ng/ml DNA) for 30 using a cytometric bead array (BD Biosciences) with a detection limit of min, before adding FCS and LPS (25 ng/ml) to the medium. After 24 h at 7.2 pg/ml. IL-23 was quantified by ELISA with the ready-SET-Go kit from 37˚C, cells were washed twice with cold PBS, and effector and tolerogenic eBioscience (San Diego, CA), with a detection limit of 15 pg/ml. In some moDC genes were analyzed, as previously described (47). Total RNA was experiments, TNF-a was quantified by using the optEIA set from BD extracted from moDC with RNeasy minikits and the Qiacube robot Biosciences (detection limit 7.8 pg/ml). IL-12p70 was quantified with the (Qiagen, Courtaboeuf, France); cDNAs were obtained by using TaqMan Quantikine reagent kit (R&D Systems), with a detection limit of 5 pg/ml. reverse-transcription reagents (Applied Biosystems). mRNA expression Supernatants of moDC/naive CD4+ T lymphocyte cocultures were ana- was evaluated by quantitative PCR on a 7900HT real-time PCR system lyzed for IFN-g, IL-4, IL-5, IL-9, IL-10, IL-13, and IL-17A by using (Applied Biosystems) using predesigned TaqMan gene expression assays a human cytokine bead kit (Merck Millipore, Darmstadt, Germany) and the and reagents, according to the manufacturer’s instructions. The expression Magpix system (Luminex, Austin, TX).

of the following genes was assessed, as previously described (47, 50): Downloaded from glucocorticoid-induced leucine zipper (GILZ) (Hs00608272_m1), com- Statistical analysis C1QA CATC plement component C1q ( ) (Hs00381122_m1), cathepsin C ( ) 6 (Hs00175188_m1), myxovirus resistance 1 (MX1) (Hs00895608_m1), Data are expressed as means SEM. Differences between groups were NMES1 evaluated with the Wilcoxon signed rank test or the Mann–Whitney U test normal mucosa of esophagus-specific 1 ( ) (Hs00260902_m1), , stabilin 1 (STAB1) (Hs01109068_m1), and retinaldehyde dehydrogenase 1 (Prism software; GraphPad, La Jolla, CA). The p values 0.05 were (RALDH1) (Hs00167445_m1). Data were interpreted for each target gene considered to denote statistical significance. by comparison with ACTB (Hs99999903_m1) as an endogenous control. The relative amount of each target gene in each sample was calculated by http://www.jimmunol.org/ comparison with the calibrator sample (untreated sample), using the cy- Results cling threshold method described above. NETs prevent full moDC maturation TNF-a degradation by NETs Recent studies suggest that PMN having undergone netosis (21, 42), and supernatants of activated PMN containing NETs (23, 43), A fixed amount of TNF-a (rhTNF-a or pooled LPS-activated moDC su- can modulate APC functions. In this study, we evaluated the pernatant, final volume 40 mL) was incubated with increasing concen- trations of NETs (0, 10, 30, 100, or 300 ng/ml DNA) for 24 h, and then effects of isolated NETs on moDC. We sheared NETs from TNF-a was measured by ELISA, as described above. A23187-activated PMN by using the restriction enzyme AluI, MNase, or DNase, thereby recovering NET fragments in fresh

MoDC chemotaxis assay by guest on September 28, 2021 HBSS medium that could be quantified by DNA assay, as we have Chemotaxis upon CCL19 or CXCL12 induction was measured in a previously described (5). 5 Transwell assay, as previously described (48). Briefly, 1 3 10 moDC pre- We first examined whether these isolated NETs could modify m treated with NETs (100 ng/ml DNA) were resuspended in 150 l RPMI moDC maturation, either directly or in response to LPS. MoDC 1640 medium supplemented with 20 mM HEPES/0.2% AB human serum (Sigma-Aldrich) and were added to the upper chamber of a 6.5-mm–diam- were preincubated with NETs for 30 min in the absence of FCS to eter, 5-mm pore-size polycarbonate Transwell culture insert (Corning Costar, avoid NET dismantling by serum DNases (51). Then the culture Brumath, France). The same medium (600 ml), with or without CCL19 (1, medium was supplemented with FCS, and LPS was added. After 10, or 50 nM) or CXCL12 (50 nM), was placed in the lower chamber. 24 h, the expression of several moDC maturation markers was AMD3100 was used at 10 mM to inhibit CXCR4-dependent signaling. MoDC that migrated to the lower chamber after 3 h of incubation at 37˚C in quantified by flow cytometry, and cytokines were assayed in the humidified air with 5% CO2 were collected and counted by flow cytometry supernatants. A first set of experiments showed that the effect of (LSR-Fortessa). Cells were gated on forward and side scatter to eliminate NETs on LPS-induced moDC maturation (in terms of HLA-DR debris. Then forward scatter-W/A and side scatter-W/A were selected to gate expression and TNF-a release) was only observed when NETs single cells. The fraction of moDC that migrated across the polycarbonate were recovered after AluI digestion, as MNase- and DNase- membrane was calculated, as previously described (48): [(number of cells migrating to the lower chamber in response to chemokine or medium)/ derived NETs did not modify these moDC markers (Fig. 1A). (number of cells added to the upper chamber at the start of the assay)] 3 100. Moreover, the effect of AluI-derived NETs was concentration T lymphocyte proliferation and polarization in response to dependent and led us to choose a NET concentration of 100 ng/ml moDC DNA for all subsequent experiments (Supplemental Fig. 1). Fig. 1B shows that the expression of HLA-DR, CD40, CD80, + The capacity of moDC to activate CD4 lymphocytes was evaluated with and CD86 was not modified by NETs alone. As expected, LPS two different approaches: activation of allogeneic CD14neg PBMCs and activation of autologous TT-specific CD4+ T lymphocytes. In both ap- alone induced moDC maturation, as shown by increased expression proaches, CD14neg PBMCs and CD4+ T lymphocytes, respectively, were of all the chosen markers. However, this marker upregulation was first labeled with 5 mM CFSE, following the manufacturer’s instructions. significantly attenuated when moDC were pretreated with NETs, In the first approach, moDC pretreated with NETs (100 ng/ml DNA) were with the following percentage reductions: HLA-DR (29.3 6 6.6%), 3 5 neg incubated with 1.5 10 CD14 PBMC from allogeneic or autologous CD40 (28.9 6 2.0%), CD80 (18.9 6 1.3%),andCD86(23.86 donors at a moDC:PBMC ratio of 1:3 or 1:9, in RPMI 1640 Glutamax supplemented with 10% AB human serum, in round-bottom 96-well plates, 2.5%) (Fig. 1B). in triplicate. On day 6, cells were labeled with propidium iodide (PI) and In the same experiments, NETs alone had no effect on cyto- with an allophycocyanin-labeled anti-CD4 Ab. Proliferation of CD4+ kine release in moDC supernatants (data not shown). In contrast, + neg T lymphocytes was analyzed by flow cytometry (CD4 PI cells). The NETs significantly downregulated LPS-induced cytokine release by second approach evaluated the activation of Ag-specific lymphocytes. 6 Autologous CD4+ T lymphocytes (1 3 105) were coincubated with moDC moDC, with the following percentage reductions: TNF-a (60.0 pretreated with NETs (100 ng/ml DNA) and preloaded with TT (0.2 mg/ 7.7%), IL-6 (58.9 6 7.4%), IL-8 (26.4 6 10.8%), IL-10 (52.8 6 ml) during the LPS activation process (moDC:lymphocyte ratios of 1:10 11.5%), IL-12p70 (81.1 6 9.2%), and IL-23 (62.5 6 8.0%) 5692 NETs DOWNREGULATE DENDRITIC CELL ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. AluI-derived NETs downregulate moDC maturation, as reflected by cell surface marker expression and cytokine release. (A) NETs recovered by AluI digestion (N-A), by MNase digestion (N-M), or by DNase digestion (N-D) were added to immature moDCs for 30 min before adding LPS. As a control, cells were treated with 20 U/ml AluI (A), 20 U/ml MNase (M), or 10 U/ml DNase (D) alone or in the presence of LPS. After 24 h of incubation, the expression of HLA-DR was analyzed by flow cytometry, and TNF-a was quantified in the culture supernatants by ELISA (n = 4). *p , 0.05 as compared with LPS-treated moDC (Wilcoxon signed rank test). Immature moDC (NS) were then incubated with AluI-derived NETs (100 ng/ml DNA), LPS (25 ng/ml), or NETs plus LPS (NET/LPS). After 24 h, (B) the expression of cell surface markers was quantified by flow cytometry, and (C) cytokines were assayed in the supernatants by cytometric bead array and ELISA. Data represent the results of six independent experiments. *p , 0.05, **p , 0.01, as determined by Wilcoxon signed rank test.

(Fig. 1C). NETs did not modify the very low capacity of IL-4 limited to LPS induction (TLR4 agonist), as TNF-a production production that could be quantified in LPS-treated moDC from two was also significantly reduced when moDC were preincubated donors upon seven tested (50 and 57 pg/ml, respectively). We chose with NETs before being stimulated with Resiquimod R-848 to focus on TNF-a production in some subsequent experiments, as (TLR7/8 agonist) or PMA (protein kinase C activator) (Fig. 2A). TNF-a is one of the earliest cytokines induced by inflammation. Together, these observations suggest that isolated NETs prevent As the restriction enzyme AluI cannot be removed from NET full moDC maturation in various conditions, in terms of both preparations, we confirmed that AluI alone did not modify LPS- surface marker expression and cytokine release. induced TNF-a production nor HLA-DR expression (Fig. 1A). a Moreover, to rule out NET cytotoxicity for moDC, we quantified NET-induced downregulation of TNF- release is not related a annexin V and 7-aminoactinomycin D staining of LPS-activated to a direct effect on LPS or TNF- moDC preincubated with increasing NET concentrations (from 10 To detect any indirect effect of NETs on moDC activation, control to 300 ng/ml DNA), and found that NETs were neither proap- experiments were performed with TNF-a release as the readout. optotic nor cytotoxic (Supplemental Fig. 2). Interestingly, the First, moDC were activated with LPS for 5, 30, or 120 min. After downregulating effect of NETs on moDC maturation was not one washing step to remove LPS, NETs were added and TNF-a The Journal of Immunology 5693

FIGURE 2. The NET-induced decrease in TNF-a production by moDC is not due to in- direct effects. (A) MoDC were activated with R848 (100 ng/ml) or with PMA (300 nM) in the absence or presence of NETs, and TNF-a was quantified by ELISA in the culture supernatants (n =4)(*p , 0.05, as determined by Wilcoxon signed rank test). (B) MoDC were first activated with LPS for the indicated times, and then washed and incubated for 24 h in the absence or presence of NETs. TNF-a was quantified by ELISA in the culture supernatants (n =4)(*p , 0.05, as determined by Wilcoxon signed rank test). A fixed concentration of TNF-a, consist- ing of either rhTNF-a (C) or culture supernatant of LPS-activated moDC (D), was incubated with increasing concentrations of NETs. After 24 h, the remaining TNF-a was quantified by Downloaded from ELISA (n = 4).

was assayed in 24-h culture supernatants. As expected, a time- 20.1%), IL-6 (41.7 6 4.6%), IL-8 (22.1 6 12.4%), IL-10 (28.2 6 http://www.jimmunol.org/ dependent effect of LPS was observed (Fig. 2B). The same time 18.3%), and IL-23A (52.3 6 19.1%) (Fig. 3B). As AluI cannot be course was maintained when NETs were added after LPS stimu- removed from NET preparations, we confirmed that AluI alone did lation, but TNF-a levels were always significantly lower (Fig. 2B). not induce TNF, IL-1B, IL-6,orIL-23A gene transcription and did These results suggest that NET-induced downregulation of TNF-a not modify their mRNA accumulation at 2 h in response to LPS release is not due to physical sequestration of LPS by NETs or to (Supplemental Fig. 3). TLR4 blockade by NETs, hindering LPS ligation. NETs thus These results show that NETs negatively impact LPS-induced appear to negatively modulate ongoing moDC activation by LPS. cytokine production by moDC at both the protein and transcrip- Second, we examined whether the decreased TNF-a levels in tional levels. NET-treated moDC supernatants might be due to TNF-a inter- by guest on September 28, 2021 nalization or binding to membrane receptors. TNF-a immuno- The immunoregulatory effect of NETs on moDC is not staining of permeabilized cells and flow cytometry showed very associated with a tolerogenic gene signature low and similar TNF-a expression (both membrane associated and We then investigated whether NETs drive LPS-activated moDC intracellular) by LPS-activated moDC in the presence and absence toward a tolerogenic phenotype. We thus quantified the expression of of NETs (data not shown). NMES1 and MX, two effector-related genes, and also GILZ, STAB1, Finally, we examined whether NET-associated proteases might C1QA, CATC,andRALDH, genes recently shown by Zimmer et al. directly degrade TNF-a during its production. For this purpose, (47) to be associated with tolerogenic DC. NETs alone induced no 400 pg rhTNF-a (Fig. 2C) or LPS-activated moDC supernatants significant change in effector-associated gene expression, which, as containing ∼300 pg native TNF-a (Fig. 2D) were incubated for expected, was upregulated by LPS. The presence of NETs during 24 h in the presence or absence of increasing amounts of NETs LPS activation tended to reinforce the effector phenotype, with (from 10 to 300 pg/ml DNA), and TNF-a was assayed. TNF-a a trend toward increased NMES1 and MX1 expression (Fig. 4). levels in both conditions were unaffected by NETs, whatever their In addition, tolerogenic markers were downregulated in LPS- concentration. Therefore, in our experimental conditions, NETs, activated moDC even when NETs were present during the activa- and particularly NET-associated proteases, do not seem to directly tion process (Fig. 4), whereas they were significantly upregulated in degrade TNF-a released by activated moDC. dexamethasone-treated control moDC, as expected for this positive Together, these results suggest that NET-induced downregula- control (p , 0.05). Together, these results suggest that NETs do not tion of cytokine release by LPS-activated moDC is not due to LPS induce a tolerogenic DC gene profile, but might rather enhance the sequestration, TLR4 hindrance, intracellular or membrane cytokine expression of some effector genes in LPS-activated moDC. sequestration, or extracellular cytokine degradation by NETs. NETs do not affect the migratory capacity of mature moDC NETs downregulate cytokine gene transcription in in vitro LPS-stimulated moDC We then analyzed the impact of NETs on the rapid and coordinated To better understand these effects of NETs on cytokine production, switch in chemokine receptor expression during DC maturation, we evaluated their impact on TNF, IL1B, IL6, IL8, IL10,andIL23A and on DC migratory capacity. Using flow cytometry, we first gene transcription. In response to LPS, mRNA accumulation was quantified the membrane expression of CCR5, CCR7, and CXCR4, maximal at 2 h for TNF, IL1B, IL6,andIL23A, and at 4 h for IL8 which are involved in DC maturation and homing to lymph nodes. and IL10 (Fig. 3A). The kinetics were similar in the presence of As expected, CCR5 expression was high on immature moDC and NETs (Fig. 3A), but the relative amounts of the mRNAs at their was strongly downregulated by LPS activation (Fig. 5A). In respective transcription peaks were significantly reduced, with the contrast, CCR7 and CXCR4 were barely detectable on immature following percentage reductions: TNF (42.1 6 8.6%), IL-1B (45.0 6 moDC and were upregulated on mature moDC. Interestingly, the 5694 NETs DOWNREGULATE DENDRITIC CELL ACTIVATION Downloaded from http://www.jimmunol.org/

FIGURE 4. NETs do not induce the expression of tolerogenic-related genes in activated moDC. MoDC were incubated with dexamethasone (DEX) (1 mg/ml), NETs (100 ng/ml DNA), LPS (25 ng/ml), or NETs plus LPS, for 24 h. Relative expression of genes associated with a tolerogenic by guest on September 28, 2021 profile (GILZ, STAB1, C1QA, CATC, and RALDH1) or an effector profile (MX1 and NMES) was evaluated by quantitative PCR (n = 3).

tagonist AMD3100. These findings suggest that moDC migratory capacity, notably in response to lymphoid chemokines, is maintained in the presence of NETs. FIGURE 3. NETs reduce moDC cytokine production at the transcrip- NETs undermine the capacity of LPS-treated moDC to activate tional level. After preincubation with NETs for 30 min, moDC were ac- CD4+ T lymphocyte proliferation tivated with LPS for 2, 4, or 8 h. (A) Representative kinetic curves for To further document the effects of NETs on moDC functions, we cytokine gene expression (TNF, IL-23A, IL-6, IL-1B, IL-8, and IL-10), + indicating the relative amount of mRNA at each time point in DC/LPS evaluated the capacity of these cells to activate CD4 T lymphocyte conditions (dark gray squares) and DC/NET-LPS conditions (light gray proliferation. In a first set of experiments, LPS-activated moDC squares), as evaluated by quantitative PCR. The curves are representative treated with NETs were coincubated with autologous or allogeneic of four independent experiments. (B) Relative amount of each cytokine monocyte-depleted PBMC loaded with CFSE. Proliferation was mRNA recovered at the transcriptional peak (2 h for TNF, IL-23A, IL-6, evaluated on day 6 as the percentage of CFSElow CD4+ T cells. and IL-1B, and 4 h for IL-8 and IL-10) is presented (n = 4). *p , 0.05, as As expected, PBMC activation with PHA resulted in .50% of determined by Wilcoxon signed rank test. CFSElow cells, whereas, in the absence of stimulus, ,2% of PBMC were CFSElow (Fig. 6A). Preincubation with NETs had no impact on presence of NETs during moDC maturation did not modulate the LPS concentration-dependent moDC-induced proliferation of autol- expression of CCR5, CCR7, or CXCR4 (Fig. 5A). We then compared ogous CD4+ T lymphocytes. In allogeneic conditions, LPS-activated CXCL12 (CXCR4 ligand)- and CCL19 (CCR7 ligand)-promoted moDC induced strong proliferation of CD4+ T lymphocytes, with migration of unstimulated or LPS-activated moDC in vitro, in the 67.6 6 9.2% and 46.6 6 6.3% of CFSElow cells at moDC:PBMC presence and absence of NETs, using a Transwell-based chemotaxis ratios of 1:3 and 1:9, respectively (Fig. 6A). This proliferation was assay. In line with the pattern of receptor expression, the addition of significantly diminished by NET preincubation (35.2 6 4.7% and CXCL12 or CCL19 stimulated LPS-activated moDC migration 26.1 6 3.5% CFSElow cells, respectively). across the Transwell membrane, whereas immature moDC migrated We also measured the proliferation of Ag-specific autologous inefficiently (Fig. 5B). When moDC were treated with NETs prior to T lymphocytes. Purified CD4+ T lymphocytes were cocultured LPS activation, moDC migration in response to the two homeostatic with LPS-activated moDC loaded with TT and pretreated with chemokines was unaffected. As expected, moDC migration upon NETs. On day 6 of coculture, in the absence of Ag and inde- CXCL12 exposure was totally inhibited by the specific CXCR4 an- pendently of NET pretreatment, ,20% of total cells were The Journal of Immunology 5695

FIGURE 5. NETs do not modify CXCR4 or CXCR7 expression and function on mature moDC. (A) CCR5, CCR7, and CXCR4 expression at the surface of unstimulated or LPS-activated moDC in the presence or absence of NETs was determined by flow cytometry using con- jugated anti-CCR5, anti-CCR7, and anti- CXCR4 mAbs. Background fluorescence is shown (shaded area). Histograms are representative of three independent experiments. (B)MoDCweretestedfor their ability to migrate in response to the indicated chemokine concentrations. Note the inhibition of CXCR4-mediated che- motaxis by AMD3100 (10 mM) added to both chambers. Transmigrated cells re- Downloaded from coveredinthelowerchamberwere counted by flow cytometry. Data are the percentage of input moDC that migrated to the lower chamber (n = 3). *p , 0.05, **p , 0.005, ***p , 0.0005, as deter- mined by Mann–Whitney U test. http://www.jimmunol.org/

CFSElow. Conversely, in the presence of TT, and with moDC: study, we show that NETs influence moDC maturation in vitro: T lymphocyte ratios of 1:10 and 1:30, respectively, 37.9 6 5.3% surface markers and cytokine production were both downregu- and 23.8 6 2.4% of recovered cells were CFSElow. In keeping lated by NETs, whereas tolerogenic gene expression was not with the results obtained in allogenic conditions, moDC matura- upregulated and chemokine receptor expression and moDC mi- by guest on September 28, 2021 tion in the presence of NETs led to significantly lower prolifera- gratory capacity were unaffected. NET-treated moDC displayed tion of TT-specific CD4+ T lymphocytes, with 21.8 6 4.4% and a diminished capacity to induce Ag-specific and allogeneic CD4+ 14.1 6 2.8% of CFSElow cells, respectively, at moDC:T lymphocyte T lymphocyte proliferation, and promoted Th2 CD4+ T lymphocytes. ratios of 1:10 and 1:30 (Fig. 6B). Although PMN are well known for their proinflammatory Collectively, these findings reveal that NETs downregulate the properties, evidence has emerged that PMN also participate in capacity of mature moDC to activate autologous and allogeneic regulating inflammation. Indeed, after target eradication, PMN CD4+ T cell proliferation. metabolism shifts toward a proresolutive state, thus damping inflammation and facilitating tissue repair [reviewed in (52)]. In NETs affect the capacity of LPS-activated moDC to polarize particular, PMN can control DC activation via several mecha- CD4+ T cells nisms [reviewed in (26, 27)]. The effects of NETs on APC To identify the type of effector moDC induced by NETs, we an- maturation are unclear (21, 42–44). We thus took advantage of + alyzed the polarization of naive allogeneic CD4 T cells cocultured our previously described method for preparing and quantifying with moDC. MoDC were first incubated for 24 h with medium isolated functional NETs (5) to study the impact of calibrated alone, NETs, LPS, or NETs plus LPS, and then washed and NETs on moDC maturation and its functional consequences. + cocultured with allogeneic naive CD4 T lymphocytes. The ef- We chose to use LPS, a major TLR4 ligand, to induce moDC fector phenotype of activated T lymphocytes was evaluated after maturation and assess the effect of NET preincubation on this 5 d by quantifying cytokines in the culture supernatants (Fig. 7). process. NETs issued from AluI-, MNase-, and DNase-treated NETs alone or AluI alone (data not shown) did not induce more PMN were compared, and we chose to use the large NETs from cytokine production than medium alone. As expected, LPS treat- AluI preparations, as smaller MNase- and DNase-derived NETS ment of moDC enhanced the secretion of the different cytokines. did not modulate moDC maturation. We can hypothesize that When moDC were pretreated with NETs, IFN-g, IL-17A, and IL- long-size isolated NETs, close to in vivo physiological NETs, 10 were significantly downregulated. Moreover, a significant in- better maintain a high local concentration of NET-associated crease in IL-5 and IL-13 production was observed. Together, these molecules (1) and even enhance their activities. Indeed, it was results suggest that NETs could favor the capacity of LPS- recently shown that DNA binding increases elastase or cathepsin activated moDC to promote effector Th2 responses and hinder G function (53). We found that NETs alone or AluI alone had no polarization toward Th1 or Th17 phenotypes. effect, but that they attenuated LPS-induced upregulation of HLA-DR and some costimulatory molecules (CD40, CD80, and Discussion CD86) in a concentration-dependent manner. In addition, TNF-a, The biological properties of NETs, and especially their immuno- IL-6, IL-8, IL-10, and IL-23 production was downregulated in the modulatory functions, are drawing increasing attention. In this presence of NETs at both the mRNA and protein levels. 5696 NETs DOWNREGULATE DENDRITIC CELL ACTIVATION Downloaded from

+ FIGURE 6. NETs diminish mature moDC-induced CD4 T lymphocyte http://www.jimmunol.org/ proliferation. (A) CFSE-loaded CD14-negative PBMC were cultured with PHA or with autologous or allogeneic moDC preactivated with LPS in the absence or presence of NETs, at two DC/PBMC ratios. (B) In other + experiments, CFSE-loaded CD4 T lymphocytes were incubated with FIGURE 7. NETs modify mature moDC-induced CD4+ T lymphocyte autologous immature moDC, or with moDC preactivated with LPS and polarization. Naive CD4+ T lymphocytes were coincubated with moDC NETs, and loaded with TT. Two ratios of DC/PBMC were tested. On day low (both immature and LPS treated) in the absence or presence of NETs. On 6, the percentage of CFSE cells was determined by flow cytometry, by day 5, cytokines were quantified in the coculture supernatants (n = 5). + , gating in CD4 PI-negative cells (n = 5). *p 0.05, as determined by *p , 0.05, as determined by Wilcoxon signed rank test. Wilcoxon signed rank test. by guest on September 28, 2021 proteins in NETs, might also be involved in the observed regulatory We then examined the mechanisms underlying these regulatory actions (9). The effect of histones on DC activation has not yet been effects on LPS-induced maturation. We first showed that NETs reported, even in their citrullinated form largely present in NETs. themselves did not have cytotoxic or proapoptotic effects on moDC NET-associated histones are known to be cytotoxic for endothelia or degrade TNF-a. We also ruled out a role of indirect effects such and epithelia (6, 15, 16), but we observed no such cytotoxic effect as LPS trapping or TLR4 blockade, as previously reported for on moDC in our experimental conditions. Another NET-associated some PMN proteins present on NETs. Indeed, lactoferrin and protein candidate is high-mobility group box 1, an important alar- bactericidal/permeability-increasing protein can sequester LPS min (23) able to modulate DC functions and induce tolerance (58). (35, 36), and lactoferrin, cathepsin G, and elastase have been re- The NET components involved in downregulating moDC activation ported to alter LPS binding to TLR4, either by competition or by are currently under investigation in our model. partial proteolysis of CD14 or TLR4 (39, 54, 55). Interestingly, we We then documented the functional consequences of NET found that the NET regulatory effect on moDC was not limited to downregulation of moDC maturation. We first examined the mo- TLR4 activation, but also occurred when a TLR7/TLR8 ligand or lecular signature of effector and regulatory DCs recently described PMA was used. by Zimmer et al. (50). Although NETs downregulated moDC The active NET component(s) responsible for these regulatory maturation in response to LPS, the resulting cells exhibited no effects on moDC maturation might be one or several NET- increase in their expression of pan-regulatory DC markers (C1QA, associated granular proteins. Indeed, antimicrobial molecules CATC, and GILZ genes), but rather tended toward DC effector such as LL-37, lactoferrin, and a-defensins can inhibit DC acti- marker expressions (MX1 and NMES1 genes). We then found that vation (33, 54, 56, 57). In addition, elastase can induce TGF-b NETs did not modify the LPS-induced chemokine receptor ex- (32) and reduce proinflammatory cytokine production by moDC, pression pattern at the moDC surface (CCR7 and CXCR4) (59) or thus promoting CD4+ lymphocyte T regulatory polarization (31). the migratory capacity of these cells toward the lymphoid che- Recently, Schauer et al. (45) evidenced that aggregated NETs pro- mokines CCL19 and CXCL12 in vitro. This suggests that, even mote the resolution of inflammation in a murine model of gout, via after interaction with NETs at inflammatory sites, DC would in particular elastase-induced cytokine and chemokine degradation; maintain their capacity to migrate to lymph nodes. as we did not evidence such a proteolytic effect of purified NETs on Finally, we studied the effects of NET-treated moDC on TNF-a in our study, we can assume that other mechanisms are T lymphocyte proliferation and polarization. We observed reduced involved. Finally, MPO has been identified as an inhibitor of DC CD4+ T lymphocyte proliferation when these cells were exposed activation and function both in vivo and in vitro, inhibiting adaptive to moDC preincubated with NETs during their maturation, in both immune responses (37). Besides these granular candidates, NET- allogeneic and Ag-specific conditions. Conversely, Tillack et al. associated nuclear proteins such as histones, the most abundant (60) recently reported that NETs alone can interact directly with The Journal of Immunology 5697 the TCR and thereby enhance Ag-specific T cell proliferation. 11. McDonald, B., R. Urrutia, B. G. Yipp, C. N. Jenne, and P. Kubes. 2012. Intra- vascular neutrophil extracellular traps capture bacteria from the bloodstream Together, these results suggest that NETs might differentially during sepsis. Cell Host Microbe 12: 324–333. modulate T lymphocyte proliferation, through either a direct effect 12. Papayannopoulos, V., and A. Zychlinsky. 2009. NETs: a new strategy for using on TCR or an indirect effect via moDC modulation. To investi- old weapons. Trends Immunol. 30: 513–521. + 13. Brinkmann, V., and A. Zychlinsky. 2012. Neutrophil extracellular traps: is im- gate the effect of these moDC on CD4 T cell polarization, we munity the second function of chromatin? J. Cell Biol. 198: 773–783. + cocultured CD4 T cells with moDC matured in the presence of 14. Kessenbrock, K., M. Krumbholz, U. Scho¨nermarck, W. Back, W. L. Gross, NETs and then measured T cell–derived cytokines. A significant Z. Werb, H. J. Gro¨ne, V. Brinkmann, and D. E. Jenne. 2009. Netting neutrophils in autoimmune small-vessel vasculitis. Nat. Med. 15: 623–625. decreased IFN-g, IL-10, and IL-17 production was found, in 15. Saffarzadeh, M., C. Juenemann, M. A. Queisser, G. Lochnit, G. Barreto, keeping with the observed decrease in IL-12p70 and IL-23 pro- S. P. Galuska, J. Lohmeyer, and K. T. Preissner. 2012. Neutrophil extracellular duction by moDC [reviewed in (61)]. These results are also in traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS One 7: e32366. keeping with those of Doz et al. (62), who recently reported that 16. Villanueva, E., S. Yalavarthi, C. C. Berthier, J. B. Hodgin, R. Khandpur, mycobacteria-infected murine DC inhibited IL-17A production by A. M. Lin, C. J. Rubin, W. Zhao, S. H. Olsen, M. Klinker, et al. 2011. Netting + neutrophils induce endothelial damage, infiltrate tissues, and expose immunos- Th17 CD4 T cells via neutrophil-derived IL-10. Moreover, Th2 timulatory molecules in systemic lupus erythematosus. J. Immunol. 187: 538– polarization was enhanced in our cocultures, as reflected by sig- 552. nificant higher levels of IL-5 and IL-13. MoDC-derived IL-4 was 17. de Boer, O. J., X. Li, P. Teeling, C. Mackaay, H. J. Ploegmakers, C. M. van der Loos, M. J. Daemen, R. J. de Winter, and A. C. van der Wal. 2013. Neutrophils, low or undetectable, suggesting that this cytokine did not play neutrophil extracellular traps and interleukin-17 associate with the organisation a role in driving Th2 polarization in our model. In some cir- of thrombi in acute myocardial infarction. Thromb. Haemost. 109: 290–297. cumstances, PMN are known to favor Th2 polarization in vitro 18. Demers, M., D. S. Krause, D. Schatzberg, K. Martinod, J. R. Voorhees, T. A. Fuchs, D. T. Scadden, and D. D. Wagner. 2012. Cancers predispose neu- (57); moreover, in vivo, in a bacterial infection model, neutrophil- trophils to release extracellular DNA traps that contribute to cancer-associated Downloaded from deficient mice exhibit enhanced Th1 responses and diminished thrombosis. Proc. Natl. Acad. Sci. USA 109: 13076–13081. Th2 responses (63). Proteases are candidate NET constituents that 19. Diana, J., Y. Simoni, L. Furio, L. Beaudoin, B. Agerberth, F. Barrat, and A. Lehuen. 2013. Crosstalk between neutrophils, B-1a cells and plasmacytoid might be involved in inducing Th2 responses, as already described dendritic cells initiates autoimmune diabetes. Nat. Med. 19: 65–73. for several allergen serine proteases such as dust mite (61). High- 20. Pratesi, F., I. Dioni, C. Tommasi, M. C. Alcaro, I. Paolini, F. Barbetti, F. Boscaro, mobility group box 1 present on NETs might also play a role, as F. Panza, I. Puxeddu, P. Rovero, and P. Migliorini. 2014. Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in this protein can also induce Th2 responses (64). Finally, DNA has neutrophils extracellular traps. Ann. Rheum. Dis. 73: 1414–1422. http://www.jimmunol.org/ been reported to trigger Th2 responses in a mouse model, through 21. Sangaletti, S., C. Tripodo, C. Chiodoni, C. Guarnotta, B. Cappetti, P. Casalini, S. Piconese, M. Parenza, C. Guiducci, C. Vitali, and M. P. Colombo. 2012. an IFN regulatory factor 3-dependent mechanism (65). Neutrophil extracellular traps mediate transfer of cytoplasmic neutrophil anti- In summary, we describe a new role for NETs. Indeed, NETs gens to myeloid dendritic cells toward ANCA induction and associated auto- prevent full maturation of moDC in vitro, leading to decreased immunity. Blood 120: 3007–3018. + 22. 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Supplementary Figure 1. Concentration-effect of NETs on the moDC cell-surface phenotype and TNF release. Mo-DC were incubated with increasing concentrations of NETs (10, 100 and 300 ng/mL DNA) in FCS-free RPMI medium for 30 min before adding LPS (25 ng/mL) and FCS. After 24 hours of incubation, the expression of cell-surface markers was analysed by flow cytometry (A) and TNF was quantified in the culture supernatants by ELISA (B) (n=4). *p<0.05 as compared with LPS treated moDC (Wilcoxon signed rank test)

Supplementary Figure 2. MoDC viability in the presence of NETs. Mo-DC were incubated with increasing concentrations of NETs (10, 100 and 300 ng/mL DNA) in FCS-free RPMI medium for 30 min before adding LPS (25 ng/mL) and FCS. After 24 hours of incubation, the cells were stained with annexin V-PE and 7-AAD. Results of flow cytometric analysis of viable cells (annexin V-negative, 7-AAD-negative), apoptotic cells (annexin V-positive, 7 AAD- negative) and necrotic cells (annexin V-positive, 7-AAD-positive) are shown. (n=3)

Supplementary figure 3. Alu I is not implicated in the NET-induced decreased cytokine gene expression in response to LPS. The effect of Alu I alone was documented, in the absence of LPS or NETs. After pre-incubation with NETs or Alu I for 30 minutes, moDC were activated with LPS for 2 hours. Relative amount of mRNA of TNF, IL-1B, IL-6 and IL-23A at each condition were evaluated by qPCR. (n=4). *p<0.05 as compared with LPS treated moDC (Wilcoxon signed rank test).