Adaptive Immune Responses Extracellular Traps Links Innate and T

T Lymphocyte Priming by Neutrophil Extracellular Traps Links Innate and Adaptive Immune Responses

This information is current as Kati Tillack, Petra Breiden, Roland Martin and Mireia of September 27, 2021. Sospedra J Immunol published online 20 February 2012 http://www.jimmunol.org/content/early/2012/02/20/jimmun ol.1103414 Downloaded from

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 20, 2012, doi:10.4049/jimmunol.1103414 The Journal of Immunology

T Lymphocyte Priming by Neutrophil Extracellular Traps Links Innate and Adaptive Immune Responses

Kati Tillack,* Petra Breiden,* Roland Martin,*,† and Mireia Sospedra*,†

Polymorphonuclear neutrophils constitute the first line of defense against infections. Among their strategies to eliminate pathogens they release neutrophil extracellular traps (NETs), being chromatin fibers decorated with antimicrobial proteins. NETs trap and kill pathogens very efficiently, thereby minimizing tissue damage. Furthermore, NETs modulate inflammatory responses by activating plasmacytoid dendritic cells. In this study, we show that NETs released by human neutrophils can directly prime T cells by reducing their activation threshold. NETs-mediated priming increases T cell responses to specific Ags and even to suboptimal stimuli, which would not induce a response in resting T cells. T cell priming mediated by NETs requires NETs/cell contact and TCR signaling, but unexpectedly we could not demonstrate a role of TLR9 in this mechanism. NETs-mediated T cell activation adds to the list of

neutrophil functions and demonstrates a novel link between innate and adaptive immune responses. The Journal of Immunology, Downloaded from 2012, 188: 000–000.

hen fighting bacterial infections, cells of the innate suppress T cell activation and proliferation. Despite this evolving immune system recognize pathogen-associated mo- evidence, a complete picture about PMN/T cell interactions re- W lecular patterns that are not displayed by host tissue. quires further investigation. Innate immune cells then secrete cytokines and chemokines that PMNs are endowed with a variety of weapons that enable them to http://www.jimmunol.org/ signal danger to other cells and induce inflammation at the site efficiently contain and clear infectious organisms. These include of infection. Polymorphonuclear neutrophils (PMNs) are the first the engulfment and intracellular degradation of microbes (12, 13), immune cells to be recruited to inflamed tissue (1), to contain and production of reactive oxygen species and granule proteins (14), clear infectious organisms, and to direct the extravasation of adap- and the recently described release of extracellular chromatin fibers tive immune cells and their activation (2–4). Adaptive immune decorated with antimicrobial proteins called neutrophil extracel- cells subsequently participate in the elimination of the pathogen lular traps (NETs) (15). NETs are the most efficient means to and set up memory for the case of reinfection. contain and eliminate pathogens (16), since they not only trap and The participation of PMNs in adaptive immune responses has not kill microbes, but also prevent collateral tissue damage by local- been considered relevant until recently, when it has been recog- izing toxic proteases and reducing their proteolytic activity (17). by guest on September 27, 2021 nized that PMNs and T cells may engage in multiple interactions Furthermore, NETs can modulate immune responses by activating and mutual activation (5). PMNs release chemokines that attract plasmacytoid dendritic cells (pDCs), an APC population special- T cells to the site of inflammation (5–7) and also cytokines that ized in sensing infections. NETs activate pDCs through TLR9, influence T differentiation (8). Several cytokines and granule an intracellular receptor that recognizes viral/bacterial DNA (18). proteins secreted by PMNs such as IFN-g, TNF-a, cathepsin G, Under physiological conditions, pDCs do not respond to self- and neutrophil elastase are able to increase T cell proliferation and DNA, most likely because self-DNA lacks CpG motifs found in cytokine production (9, 10), which together enhance adaptive viral/bacterial DNA and also because self-DNA is rapidly de- immune responses. Paradoxically, PMNs also secrete mediators graded in the extracellular environment and therefore fails to such as oxygen species, arginase (11), IL-10, and TGF-b that may access intracellular TLR9. However, damaged cells can release factors such as the neutrophil antimicrobial peptide LL37 and the high-mobility group box protein 1, which can protect self-DNA *Institute for Neuroimmunology and Clinical Multiple Sclerosis Research, Center for Molecular Neurobiology, University Medical Center Hamburg–Eppendorf, 20251 from DNase degradation and deliver it to the intracellular com- Hamburg, Germany; and †Department of Clinical Neuroimmunology and Multiple partment containing TLR9 in pDCs with the consequence of Sclerosis Research, Neurology Clinic, University Hospital Zu¨rich, 8091 Zu¨rich, Swit- TLR9-mediated activation (19–21). LL37 and high-mobility zerland group box protein 1 are also contained in NETs and hence con- Received for publication November 28, 2011. Accepted for publication January 21, 2012. fer to these structures the potential to activate pDCs via TLR9 (22, 23). NETs activation of pDCs seems to play an important role in This work was supported by Deutsche Forschungsgemeinschaft Grant SO 1029/1-1. The Institute for Neuroimmunology and Clinical Multiple Sclerosis Research was the pathogenesis of some autoimmune diseases such as psoriasis supported by the Gemeinnu¨tzige Hertie Stiftung. (19) and systemic lupus erythematosus (22–25), for which it has Address correspondence and reprint requests to Dr. Mireia Sospedra, Department of been suggested that the large amounts of IFN-g produced by Neuroimmunology and Multiple Sclerosis Research, Neurology Clinic, University NETs-activated pDCs can lead to the maturation of myeloid DCs Hospital Zu¨rich, Frauenklinikstrasse 26, 8091 Zu¨rich, Switzerland. E-mail address: [email protected] (mDCs) and exert an effect on T cell function. If such an indirect The online version of this article contains supplemental material. interaction between NETs and T cells were confirmed, it would Abbreviations used in this article: DC, dendritic cell; DPI, diphenyleneiodonium; represent a new NETs-mediated mechanism of communication mDC, myeloid dendritic cell; NET, neutrophil extracellular trap; ODN, oligodeox- between PMNs and T cells. Furthermore, TLR9 is expressed in ynucleotide; pDC, plasmacytoid dendritic cell; PFA, paraformaldehyde; PMN, poly- T lymphocytes, and CpG-containing oligodeoxynucleotides morphonuclear neutrophil. (CpG-ODN) can modulate T cell activation (26, 27). NETs may Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 therefore also be able to directly activate T cells through their

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103414 2 T CELL PRIMING BY NETs

TLR9, and this would represent a second and, in this case, direct blocking Ab (provided by Dr. H.G. Rammensee, Department of Immu- mechanism of a NETs-mediated communication between PMNs nology, University of Tu¨bingen, Tu¨bingen, Germany) or 60 mg/ml corre- and T cells. sponding isotype control (BioLegend, San Diego, CA) was added into the culture. As positive control, 1 mg/ml soluble anti-CD3 Ab (OKT3; Ortho As outlined above, there are at least two possibilities of how Biotech Products, Raritan, NJ) or anti-hCD2/anti-hCD3/anti-hCD28 beads PMNs could interact with T cells via NETs production. In this study (cell/bead ratio of 2:1) (T cell activation/expansion kit; Miltenyi Biotec) we have examined whether NETs released by human PMNs are were used (data not shown). + able to exert direct or indirect effects on T cell activation. We found To study the threshold for activation, NETs-primed CD4 T cells were stimulated with 0.025 mg/ml soluble OKT3 (Ortho Biotech Products) that NETs were able to directly prime T cells by reducing their (48 h). NETs-primed TCC36 was seeded in quadruplicate in 96-well plates activation threshold, which increased T cell responses to specific (25,000 cells/well) together with autologous irradiated PBMCs (1 3 105 Ags and even to suboptimal stimuli. Priming by NETs required cells/well) (3000 rad) with or without different concentrations of the NETs/cell contact and TCR signaling, but unexpectedly we failed specific stimulatory peptide (72 h). to show a role of TLR9 in this mechanism. Our results demonstrate Proliferation assays a novel strategy how PMNs are capable of activating adaptive immune responses via NETs. Proliferation in the cocultures was measured after 48 h using a Click-iT EdU Alexa Fluor 647 flow cytometry assay kit (Molecular Probes/Invitrogen) following the manufacturer’s instructions. Cells were stained with anti- Materials and Methods CD3 (PE-Cy7; eBioscience, San Diego, CA), anti-CD4 (allophycocyanin; Cell purification and TCC36 eBioscience), and anti-CD8 (Pacific Blue; Dako) and analyzed by flow cytometry. Sample acquisition was done with a LSRII (BD Biosciences)

Cells were isolated from healthy donors at the Department of Transfusion ﬂow cytometer and data were analyzed with FACSDiva (BD Biosciences) Downloaded from Medicine, University Medical Center Hamburg–Eppendorf, after informed and FlowJo (Tree Star) softwares. consent was obtained. PMNs were isolated from blood or buffy coats using Proliferation of TCC36 was measured by [3H]thymidine (Hartmann dextran-Ficoll, as described previously (28). The purity and viability of Analytic, Braunschweig, Germany) incorporation using a scintillation beta PMNs was $97 and $95%, respectively, as assessed by expression of the counter (Wallac 1450; PerkinElmer, Rodgau-Ju¨rgesheim, Germany). neutrophil-speciﬁc marker CD16b, annexin V expression, and trypan blue exclusion (Supplemental Fig. 1). PBMCs were isolated by density gradi- Cytokine production ent. CD4+ T cells were enriched using the BD IMag human CD4

T lymphocyte enrichment set–DM (BD Biosciences, Franklin Lakes, NJ), After 24 h coculture, supernatants were collected and cytokine levels were http://www.jimmunol.org/ naive CD4+ T cells using the BD IMag human naive CD4 T lymphocyte measured by ELISA using the following kits: human IFN-g (BioLegend); enrichment set–DM (BD Biosciences), memory CD4+ T cells using the BD IL-4, IL-10, and IL-2 CytoSet (BioSource International, Camarillo, CA); IMag human memory CD4 T lymphocyte enrichment set–DM (BD Bio- and IL-17A (eBioscience). Reactions were performed according to the sciences), CD8+ T cells using the BD IMag anti-human CD8 magnetic manufacturers’ instructions. particles–DM (BD Biosciences), DCs using the Blood Dendritic Cell Isolation Kit II (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). Flow cytometry analysis of surface markers Cells were separated according to the manufacturers’ instructions. T cell activation was assessed using the following Abs: anti-CD3 (PE-Cy7; TCC36 was established from cerebrospinal fluid of an untreated multiple eBioscience), anti-CD4 (allophycocyanin; eBioscience), anti-CD8 (Pacific sclerosis patient by limiting dilution as previously described (29). Stimu- Blue; Dako), anti-CD25 (PE; eBioscience), and anti-CD69 (FITC; BD latory peptides were identified using positional scanning combinatorial Biosciences). DC activation was assessed using the following Abs: anti- by guest on September 27, 2021 peptide libraries (29). CD3 (Pacific Blue; eBioscience), anti-CD14 (Pacific Blue; BD Biosci- Cell stimulation ences), anti-CD19 (Pacific Blue; BD Biosciences), anti-CD56 (Pacific Blue; BD Biosciences), anti-CD11c (PerCP-Cy5.5; BioLegend), anti-CD123 (PE- Purified PMNs were resuspended in HBSS+ medium (Invitrogen, Carlsbad, Cy7; BioLegend), anti-CD40 (PE; Miltenyi Biotec), anti-CD83 (allophy- CA) supplemented with 10 mM HEPES (Invitrogen). Then, 5 3 105–106 cocyanin; BD Biosciences), anti-CD80 (PE; eBioscience), anti-CD86 PMNs/ml were seeded into tissue culture plates on glass coverslips (FITC; Dako), and anti-HLA class II (FITC; BD Biosciences). FITC (Menzel-Gla¨ser, Braunschweig, Germany) pretreated with 0.001% poly-L- (BD Biosciences), PE (BD Biosciences), allophycocyanin (eBioscience), lysine (Sigma-Aldrich, Steinheim, Germany). PMNs were stimulated with Pacific Blue (Dako), PerCP-Cy5.5 (BD Biosciences), and PE-Cy7 (eBio- 25 nM PMA (Sigma-Aldrich) (15 min or 3 h) and with 100 nM fMLP science) isotype controls were also used. (Sigma-Aldrich) (3 h). When indicated, PMNs were preincubated with 100 mM diphenyleneiodonium (DPI; Sigma-Aldrich) 30 min before stimulation Microscopy assays with PMA (3 h) (30). Also when indicated, PMNs were fixed with 4% paraformaldehyde (PFA) (Roth, Karlsruhe, Germany). To induce apoptosis NETs formation was visualized using fluorescence microscopy in PMNs and secondary necrosis, PMNs were exposed to UV light (60 min) and fixed with 4% PFA and blocked overnight with PBS containing 5% donkey subsequently incubated (16 h). Purified CD4+ T cells were stimulated with serum (The Jackson Laboratory, Bar Harbor, ME) and 0.6% Triton X-100 25 nM PMA. (Roth), by staining with a primary anti-human myeloperoxidase-specific Ab (AbD Serotec) followed by the secondary goat anti-mouse Cy3 Ab (The Isolation and quantification of NETs Jackson Laboratory) and the DNA dye Hoechst 33258 (Sigma-Aldrich). T cell cluster formation was analyzed in transmission light. Specimens NETs released by activated PMNs were digested with 10 U/ml micrococcal were analyzed with a confocal microscope F1000 (Olympus, Hicksville, nuclease (Worthington Biochemical, Lakewood, NJ) as previously de- NY) and Axio Imager M1 (Zeiss, Go¨ttingen, Germany), respectively. scribed (31). NETs (myeloperoxidase/DNA complexes) in supernatants were quantified using a capture ELISA as previously described (32). Ab- Analysis of T cell signaling sorbance was measured at 405 nm using a mQuant microplate reader (Bio- + Tek, Winooski, VT). Purified CD4 T cells cocultured with NETs-supernatant (10 min) were harvested and TCR signal transduction was analyzed by intracellular Cell cocultures staining using an Alexa Fluor 488 mouse anti-ZAP70 phosphorylated on tyrosine 319 (pY319) (BD Phosflow) according to the manufacturer’s PMNs stimulated or unstimulated were seeded on coverslips and placed into instructions. tissue culture plates. Autologous PBMCs (3 3 106), purified DCs (4 3 105), + 6 + 6 purified CD4 (2 3 10 ), purified naive CD4 (2 3 10 ), purified memory Statistical analysis CD4+ (2 3 106), or purified CD8+ (2 3 106) T cells were added to the wells. When indicated cells were incubated with PMN-free supernatant Statistical analyses were performed with Prism 5.02 (GraphPad Software, containing NETs or were physically separated by a transwell poly- San Diego, CA). Descriptive statistics are reported as means 6 SEM. carbonate permeable membrane (0.4 mm pore size, Costar; Corning, Parametric tests were applied for two-group comparisons using unpaired t Acton, MA). Also when indicated 2.5 mM chloroquine (Sigma-Aldrich), 1 tests with two-tailed p values. Comparisons of three groups and more were mM TLR9 antagonist (ODN TTAGGG) (CAYLA-InvivoGen, Toulouse, assessed by one-way ANOVA with Bonferroni’s correction for multiple France), 3 mM herbimycin A (Sigma-Aldrich), 60 mg/ml anti–HLA-DR comparisons. A p value ,0.05 was considered statistically significant. The Journal of Immunology 3

Results were cocultured with NETs-supernatant in the presence or absence NETs induce cluster formation, upregulation of the activation of two TLR9 inhibitors: chloroquine, which blocks TLR9/DNA markers CD25 and CD69, and phosphorylation of the interactions in endosomes (33) and the TLR9 antagonist (ODN TCR-associated signaling kinase ZAP70 but not proliferation TTAGGG), or the antibiotic herbimycin A, which inhibits TCR- in CD4+ T cells mediated signaling (34). The upregulation of CD25 and CD69 on + To examine the NETs-mediated effect on T cells, purified human CD4 T cells cocultured with NETs-supernatant remained un- CD4+ T cells were cocultured with PMNs releasing NETs changed in the presence of chloroquine and TLR9 antagonist (NETting-PMNs) and unstimulated PMNs. We stimulated NET (ODN TTAGGG), whereas it was reduced by herbimycin A (Fig. formation by incubating highly pure PMNs with 25 nM PMA for 1D), suggesting that the effect of NETs is independent of TLR9, 3 h (Fig. 1A). To assure that we examine the role of NETs and not but involves TCR signaling. + To investigate further the TCR-mediated effect of NETs but also of other PMA-induced mediators, we also incubated CD4 T cells to exclude the possibility that this effect was mediated by PMA that with PMNs pretreated with diphenyleneiodonium (DPI) (DPI- was taken up by neutrophils during stimulation and then released PMNs), a NADPH inhibitor of NET formation, before stimula- bound to NETs, we analyzed whether NETs induced recruitment of tion with PMA. Furthermore, we digested NETs from stimulated the TCR-proximal tyrosine kinase ZAP70. ZAP70 plays a critical PMNs with micrococcal nuclease and incubated purified CD4+ role during early steps of TCR-mediated signaling, but is bypassed T cells with these cell-free supernatants containing NETs (NETs- + by PMA that directly activates protein kinase C without involve- supernatant). CD4 T cells cocultured with NETting-PMNs (Fig. ment of ZAP70 (35). Purified CD4+ T cells were cocultured for 1B) and NETs-supernatant (data not shown) formed clusters in- 10 min with unstimulated PMNs, NETting-PMNs, or NETs- Downloaded from dicative of cell activation or proliferation that were absent when supernatant, and ZAP70 (pY319) phosphorylation was measured cells were cocultured with unstimulated PMNs (Fig. 1B) or DPI- by flow cytometry. ZAP70 phosphorylation was upregulated in PMNs (data not shown). As a further sign of activation, CD25 CD4+ T cells cocultured with NETting-PMNs or NETs-super- + and CD69 expression was upregulated on purified CD4 T cells natant compared with unstimulated PMNs (Fig. 1E), showing an cocultured during 24 h with NETting-PMNs and NETs-super- effect of NETs on purified CD4+ T cells that involves TCR sig- natant, but not on T cells cocultured with unstimulated PMNs or naling and is not mediated by PMA. Furthermore, a putative role http://www.jimmunol.org/ DPI-PMNs (Fig. 1C). To examine whether these effects of NETs of PMA bound to NETs was also excluded by the observation that on CD4+ T cells involved TLR9 and TCR signaling, CD4+ T cells purified CD4+ T cells stimulated with PMA for 48 h proliferated by guest on September 27, 2021

FIGURE 1. CD4+ T cell priming induced by NETs. (A) Representative images of unstimulated neutrophils and NETting-PMNs visualized using fluorescence microscopy. DNA is shown in blue (Hoechst 33258) and myeloperoxidase in red. (B) Transmission light images of cluster formation of purified CD4+ T cells cocultured with unstimulated PMNs and NETting-PMNs (NETs). Original magnification (A, B) 340. (C and D) CD25 and CD69 surface expression on purified CD4+ T cells cocultured 24 h with unstimulated PMNs, NETting-PMNs, DPI-PMNs, and NETs-supernatant with or without chloroquine, TLR9 antagonist (ODN TTAGGG), or herbimycin A. Graphs show mean fluorescence intensity (MFI) 6 SEM from five independent experiments. *p , 0.05, **p , 0.01. (E) Flow cytometry analysis of ZAP70 (pY319) phosphorylation in CD4+ T cells cocultured for 10 min with unstimulated PMNs, NETting-PMNs, and NETs-supernatant. Graph represents MFI 6 SEM from three independent experiments. *p , 0.05. (F) CD4+ T cell proliferation assessed by EdU incorporation after culture during 48 h with NETting-PMNs, NETs-supernatant, and PMA. Histograms represent the proliferation from a representative experiment. Graph represents the percentage of EdU-positive cells (mean values 6 SEM) from three or more independent experiments. ***p , 0.001. 4 T CELL PRIMING BY NETs whereas purified CD4+ T cells cocultured with NETting-PMNs or of resting T cells, stimulates a response of NETs-primed CD4+ NETs-supernatant did not (Fig. 1F). The lack of proliferation of T cells. CD4+ T cells were precultured for 24 h with unstimulated purified CD4+ T cells cocultured with NETs indicated that the PMNs (unprimed CD4) or with NETs-supernatant (NETs-primed increase in ZAP70 phosphorylation induced by NETs was ap- CD4) for 24 h. Simultaneously, purified DCs were precultured parently insufficient to fully activate T cells and support their with unstimulated PMNs (resting DCs). After this period, both cell proliferation. types were carefully washed and cocultured for an additional 48 h. NETs-primed purified CD4+ T cells (8.7 6 1.3%) proliferated and T cell priming by NETs lowers the activation threshold released 2092 6 459 pg/ml IFN-g, whereas no activation was The changes that NETs exerted on CD4+ T cells suggested that observed in unprimed CD4+ T cells (Fig. 3A). To confirm that they led to a state of preactivation or priming. We therefore ex- NETs priming is TLR9 independent, but involves TCR signaling, amined whether interaction with NETs reduces the activation CD4+ T cells were primed with NETs-supernatant in the presence threshold of CD4+ T cells. First, we studied the effect of NETs on or absence of the TLR9 inhibitor chloroquine, TLR9 antagonist Ag-specific responses using a previously well-characterized CD4+ (ODN TTAGGG), or herbimycin A. Proliferation was not affected T cell clone (TCC36) (29). TCC36 was precultured for 24 h with in CD4+ T cells primed in the presence of chloroquine or TLR9 unstimulated PMNs or NETs-supernatant and then seeded with antagonist (ODN TTAGGG); however, herbimycin A clearly re- autologous irradiated PBMCs pulsed or not with its specific target duced proliferation (Fig. 3B), confirming that T cell priming by peptide. TCC36 primed by NETs proliferated significantly more NETs is independent of TLR9 but involves TCR signaling. vigorously in response to PBMCs loaded with 10 and 1 mg/ml To better understand the interaction between NETs-primed specific peptide when compared with the unprimed TCC36, that CD4+ T cells and resting DCs, NETs-primed purified CD4+ Downloaded from is, precultured with unstimulated PMNs (Fig. 2A). T cells and resting DCs were cocultured in the presence or ab- Then, we addressed whether priming by NETs reduces the acti- sence of a blocking anti–HLA-DR Ab, the corresponding isotype vation threshold of CD4+ T cells and renders them capable to be control, or herbimycin A. The presence of an anti–HLA-DR Ab activated by suboptimal stimuli. Purified CD4+ T cells were pre- did not induce a significant reduction of primed CD4+ Tcell cultured with unstimulated PMNs, NETting-PMNs, DPI-PMNs, or proliferation (Fig. 3C). However, because class II molecules

NETs-supernatant for 24 h. Next, T cells were carefully washed and other than DR are expressed on DCs, that is, HLA-DQ and HLA- http://www.jimmunol.org/ cultured for another 48 h with low concentrations of soluble anti- DP molecules, we cannot rule out that HLA class II molecules are CD3 Ab (OKT3) (signal 1) in the absence of APC (signal 2), a involved. The presence of herbimycin A completely abrogated the suboptimal stimulus, which alone is not able to induce proliferation proliferation of primed CD4+ T cells, indicating that activation of of resting T cells. Purified CD4+ T cells precultured with unstimu- primed T cells by DCs requires TCR signaling (Fig. 3C). lated PMNs or with DPI-PMNs failed to proliferate (Fig. 2B). In NETs-activated pDCs are not able to activate unprimed T cells contrast, 27.2 6 6.7% of CD4+ T cells precultured with NETting- PMNs and 13.27 6 3.5% precultured with NETs-supernatant pro- Next, we also examined whether DCs, particularly pDCs, pre- liferated (Fig. 2B). cultured with NETs-supernatant (NETs-activated DCs) were able + ∼ by guest on September 27, 2021 + to activate CD4 T cells. Following purification, 40% of DCs Resting DCs are able to activate NETs-primed CD4 T cells in were pDCs and 60% were mDCs. After 24 h coculture with NETs, the absence of specific Ag all mDCs died, whereas pDCs survived and upregulated some Next, we examined whether a more physiologic, but suboptimal maturation and activation markers such as CD40, CD80, CD83 stimulus such as the interaction of T cells with resting DCs in the and CD86, but not HLA class II (Fig. 4A). Unprimed CD4+ T cells absence of specific Ag, which alone does not induce proliferation cocultured with NETs-activated pDCs did not proliferate nor did

FIGURE 2. T cell priming by NETs lowers the activation threshold. (A) T cell proliferation assessed by thymidine incorporation in TCC36 precultured with unstimulated PMNs or NETs-supernatant and stimulated with autologous irradiated PBMCs pulsed with a speciﬁc peptide. Graph represents cpms (mean values 6 SEM) from three or more independent experiments. (B) T cell proliferation assessed by EdU incorporation in puriﬁed CD4+ T cells precultured with unstimulated PMNs, NETting-PMNs, DPI-PMNs, or NETs-supernatant and stimulated or not with low concentrations of anti-CD3 Ab (OKT3). Graph represents the percentage of EdU-positive cells (mean values 6 SEM) from three or more independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 5

A Pre-culture Co-culture CD4 + DC + IFN PMNs PMNs CD4 DC

Unstimulated Unstimulated Unprimed Resting

NETs Counts Unstimulated NETs-primed Resting ** Supernatant ***

24 h 48 h EdU 0 3 6 9 12 0 1000 2000 3000 EdU+ cells (%) pg/ml

BCCo-culture Co-culture CD4 DC CD4 DC

NETs-primed Resting NETs-primed Resting

NETs-primed Resting + Anti-HLA-DR

(TLR9 Antagonist) Downloaded from

NETs-primed + Isotype control Resting (Chloroquine)

NETs-primed + Herbimycin A Resting (Herbimycin A) * ** 0 2 4 6 8 10 12 http://www.jimmunol.org/ 0 2 4 6 8 10 12 14 16 EdU positive cells (%) EdU positive cells (%) FIGURE 3. Resting DCs are able to activate NETs-primed CD4+ T cells in the absence of speciﬁc Ag. (A–C) CD4+ T cell proliferation assessed by EdU incorporation. Histograms represent the proliferation from a representative experiment. Graph represents the percentage of EdU-positive cells and pg/ml IFN-g (mean values 6 SEM) from three or more independent experiments. (A) Puriﬁed CD4+ T cells precultured with unstimulated PMNs (unprimed) or with NETs-supernatant (NETs-primed) were cocultured with DCs previously precultured with unstimulated PMNs (resting). *p , 0.014, **p , 0.003. (B) CD4+ T cells precultured with NETs-supernatant in presence or absence of TLR9 inhibitors chloroquine and TLR9 antagonist (ODN TTAGGG) or TCR signaling inhibitor herbimycin A, and then cocultured with resting DCs. *p , 0.05. (C) NETs-primed CD4+ T cells cocultured with resting DCs in presence or absence of a HLA-DR blocking Ab, the corresponding isotype control, or herbimycin A. **p , 0.01. by guest on September 27, 2021 they produce IFN-g (Fig. 4B). NETs-activated pDCs were able to T cell activation. NETs activation of pDCs seems to play a role in induce proliferation and IFN-g release only in NETs-primed some autoimmune diseases (19, 22–25), and it has been suggested CD4+ T cells (Fig. 4B), suggesting that the NETs-mediated acti- that the large amounts of IFN-a produced by NETs-activated vation of pDCs in our in vitro setting does not exert an effect on pDCs can activate mDCs and increase Ag presentation to

FIGURE 4. NETs-activated pDCs do not exert an effect on unprimed CD4+ T cells. (A) HLA class II, CD40, CD80, CD83, and CD86 expression on gated NETs-activated pDCs. Values show mean ﬂuorescence intensity (MFI) 6 SEM. *p = 0.0263, **p = 0.0041, ***p = 0.0004. (B) Puriﬁed CD4+ T cells precultured with unstimulated PMNs (unprimed) or with NETs-supernatant (NETs-primed) were cocultured with DCs previously precultured with NETs-supernatant (NETs-activated). CD4+ T cell proliferation was assessed by EdU incorporation. Histograms represent the proliferation from a representative experiment. Graph represents the percentage of EdU-positive cells and pg/ml IFN-g (mean values 6 SEM) from three or more independent experiments. *p , 0.04. 6 T CELL PRIMING BY NETs

T cells. The loss of mDCs after culture with NETs most likely T cells and 11.6 6 1.3% of CD8+ T cells proliferated (Fig. 5A, prevented this indirect effect of NETs-activated pDCs on T cells in 5B). Regarding cytokine production, purified CD4+ T cells our in vitro system. cocultured with NETting-PMNs and DCs secreted 901 6 233 pg/ml IFN-g and 289 6 99 pg/ml IL-2. Purified CD8+ T cells Coculture of T cells, NETting-PMNs, and DCs results in T cell secreted only IFN-g (981.6 6 293 pg/ml) (Fig. 5B). Direct contact activation of T cells with DCs and NETting-PMNs was required for T cell We then asked whether coculture of T cells, DCs, and NETting- activation. When CD4+ or CD8+ T cells were physically separated PMNs results in T cell activation and whether NETs exert the from DCs and NETting-PMNs by a transwell, no activation was same effect on CD4+ and CD8+ T cells. We used NETting-PMNs detected (Fig. 5A, 5B). The proliferation of CD4+ T cells cocul- instead of NETs-supernatant as the more physiologic condition. tured with NETting-PMNs and DCs remained unchanged when Purified CD4+ or CD8+ T cells were cocultured with unstimulated CD8+ T cells were added (18.5 6 6.5%); conversely, the prolif- or NETting-PMNs in the absence or presence of purified DCs. eration of purified CD8+ T cells cocultured with NETting-PMNs Proliferation and secretion of IFN-g, IL-17A, IL-4, IL-10, and and DCs increased when purified CD4+ cells were added to the IL-2 were measured as indicators of T cell activation (Fig. 5). coculture (27 6 6.7%) (Fig. 5A). Purified CD4+ and CD8+ T cells cocultured with unstimulated We also addressed whether the ability of NETs to induce T cell PMNs alone or in the presence of purified DCs did not proliferate proliferation was comparable between naive and memory CD4+ or produce cytokines (Fig. 5A, 5B). Similarly, no proliferation or T cells. Purified naive or memory CD4+ T cells were cocultured cytokine release was observed upon coculture with NETting- with unstimulated PMNs or NETting-PMNs in the presence of pu-

PMNs in the absence of DCs (Fig. 5A, 5B). In contrast, we ob- rified DCs. We found that 14.7 6 6.8% of naive T cells and 11.1 6 Downloaded from served both proliferation and cytokine release when purified DCs 5% of memory CD4+ T cells cocultured with NETting-PMNs were added to the cocultures. We found that 16.2 6 2.8% of CD4+ and purified DCs proliferated, whereas neither naive nor memory http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. Coculture of T cells with NETting-PMNs and DCs results in T cell activation. (A) T cell proliferation assessed by EdU incorporation in purified CD4+ (red) and purified CD8+ (blue) T cells cocultured with unstimulated PMNs in the presence or absence of DCs, NETting-PMNs (NETs) in the presence or absence of DCs and separated or not by a transwell, and in the presence or absence of additional purified CD8+ or CD4+ T cells, respectively. Histograms represent the proliferation from a representative experiment. Graphs represent the percentage of EdU-positive cells (mean values 6 SEM) from three or more independent experiments. **p , 0.01, ***p , 0.001. (B) IFN-g, IL-17A, IL-4, IL-10, and IL-2 produced by purified CD4+ (in red), CD8+ (in blue), or both T cells together (in gray) cocultured as indicated in (A). Values show mean pg/ml 6 SEM. *p , 0.05, ***p , 0.001. (C) T cell proliferation assessed by EdU incorporation in purified naive (open bars) and memory (filled bars) CD4+ T cells cocultured with unstimulated PMNs or NETting-PMNs in presence of DCs. Graph represents the percentage of EdU-positive cells (mean values 6 SEM) from three independent experiments. The Journal of Immunology 7

CD4+ T cells proliferated upon coculture with unstimulated PMNs activation, whereas the coculture with NETs-supernatants induced and DCs (Fig. 5C). proliferation and upregulation of activation markers but not cytokine production (Fig. 7). Proteases released by neutrophils that Characterization of NETs-mediated T cell activation in are eliminated during the careful washing of NETting-PMNs are PBMCs not eliminated, but concentrated in NETs-supernatants and most Finally, we characterized the activation of CD4+ and CD8+ T cells likely degradated cytokines (36). mediated by NETs in bulk PBMCs, a condition that reflects better the circumstances under which T cells will encounter NETting- Discussion PMNs in vivo. PBMCs include, in addition to T cells and DCs, The main function of the innate immune system during infection is monocytes and B cells that could both influence T cell activation to rapidly sense microbial pathogens, limit their spread, and elim- mediated by NETs. PBMCs cocultured with NETting-PMNs inate them with minimum collateral tissue damage. The com- formed large clusters that were absent when cells were cocul- position of NETs, being fibers of chromatin decorated with anti- tured with unstimulated PMNs (Fig. 6A) and strongly upregulated microbial proteins, turns them into optimal structures to perform CD25 and CD69 expression on both CD4+ and CD8+ T cells (Fig. this task (16). The main advantages of NETs are the following: 1) 6B). Additionally, within the PBMCs, 10.5 6 1.8% of CD4+ NET fibers trap pathogens and act as physical barriers, preventing T cells and 32.3 6 3.1% of CD8+ proliferated after coculture with microbial spread; 2) NETs render antimicrobial proteins more NETting-PMNs, whereas no proliferation was observed when efficient by concentrating them on the DNA/protein fibers, which PBMCs were cocultured with unstimulated PMNs (Fig. 6C). The keeps them together and allows them to act synergistically; 3) the ability of NETting-PMNs to mediate proliferation in CD4+ and collateral tissue damage is reduced since proteases do not diffuse Downloaded from CD8+ T cells showed great interindividual variability (Fig. 6D). into the tissue, and, additionally, binding to NETs reduces the PBMCs cocultured with NETting-PMNs also secreted 1777 6 272 toxic activity of some of these proteases (17); and 5) NETs also pg/ml IFN-g and 93.5 6 25 pg/ml IL-17A, but IL-4 and IL-10 did allow colocalization of adjuvants and danger signals that can not (Fig. 6D). No cytokine secretion was detected when PBMCs modulate inflammation, for example of self-DNA and LL-37, were cocultured with unstimulated PMNs. which are able to activate pDCs. In this study, we describe as

a novel function of NETs their ability to directly prime T cells by http://www.jimmunol.org/ NETs and not other factors mediate T cell activation in PBMCs reducing their activation threshold and in consequence mediate To confirm that NETs and not other factors mediated the substantial T cell activation. This previously unknown property of NETs T cell activation in bulk PBMCs, we included a broad range of demonstrates that their role is not limited to innate immune additional controls. First, to discard that residual contaminating mechanisms, but is also involved in activating the adaptive im- PMA activates PBMCs despite careful washing of NETting-PMNs mune system. after PMA stimulation, PBMCs were cocultured with PMNs treated CD4+ T cell activation mediated by NETs unfolds as a two-step with PMA for only 15 min, a period too short to induce substantial process. In the first step, NETs prime CD4+ T cells by direct NET production. No upregulation of CD25 and CD69 expression contact, which reduces their threshold of activation. CD4+ T cells (Fig. 7A), T cell proliferation (Fig. 7B), or production of cytokines primed by NETs showed increased Ag-specific responses and can by guest on September 27, 2021 (Fig. 7C) was observed under this condition. PMNs activated with be activated by suboptimal stimuli such as soluble anti-CD3 Ab fMLP, a stimulus less efficient than PMA in inducing NETs, also (signal 1) in the absence of APCs (signal 2) or resting DCs in the failed to mediate T cell activation (Fig. 7). NETting-PMNs, that is, absence of specific Ag, which are both not sufficient to induce PMNs undergoing NETosis, are dying cells. To exclude an effect a response of resting CD4+ T cells. We observed that NETs/T cell of dead cells on T cell activation, we cocultured PBMCs with contact induced the formation of cell clusters, upregulation of the PMNs dying by other mechanisms than NETosis. We induced activation markers CD25 and CD69, as well as some phosphor- apoptosis in PMNs by exposure to UV light for 60 min. Cells were ylation of ZAP70 in CD4+ T cells. These changes were insuffi- subsequently incubated for additional 16 h to increase the number cient to fully activate T cells and support T cell proliferation, but of dying cells and then cocultured with PBMCs. No activation was they lowered their activation threshold. Unexpectedly, we could detected under these coculture conditions (Fig. 7). not demonstrate a role of TLR9 in this NETs-mediated T cell To exclude that PMA-induced mediators other than NETs were priming because neither of the two TLR9 inhibitors, the TLR9 responsible for T cell activation, we stimulated PMNs with PMA inhibitor chloroquine and the TLR9 antagonist (ODN TTAGGG), for 3 h and then fixed them with PFA prior to coculture with had an effect. Whether T cell priming mediated by NETs is a PBMCs. After fixation, PMNs are not able to produce any medi- DNA activation pathway that is TLR9 independent, such as those ators, and only NETs remained on cells. PBMCs cocultured with induced by CpG in monocytes (37) or nucleosomes in neutrophils NETting, PFA-fixed PMNs showed upregulation of CD25 and (38), requires further investigation. Although TLR9 does not seem CD69 expression (Fig. 7A), T cell proliferation (Fig. 7B), and to play a role, T cell priming by NETs apparently involves TCR cytokine release (Fig. 7C) similar to those observed with unfixed engagement and signaling since they induced phosphorylation of cells. As an additional control, PBMCs were cocultured with the TCR-associated signaling kinase ZAP70, and herbimycin A, NETting-PMNs, but physically separated by a transwell to avoid a well-known inhibitor of TCR-mediated signal transduction, cell/cell or NETs/cell contact. No upregulation of CD25 expres- strongly reduced T cell priming. Further studies need to determine sion, T cell proliferation, or release of cytokines (Fig. 7) was which of the many components of NETs engage the TCR and detected under this condition, excluding that soluble mediators induce TCR signaling. produced by PMNs are responsible for T cell activation. Contact The lower activation threshold of NETs-primed T cells has been between NETs and PBMCs was required for T cell activation, demonstrated only for CD4+ T cells. The survival of CD8+ T cells as already observed with purified cells (Fig. 5). Interestingly, precultured with NETs and subsequently incubated with DCs was some upregulation of CD69 was observed in this condition, sug- very low and prevented us from performing priming experiments gesting that the expression of this molecule is partially mediated with CD8+ T cells. It has been reported that the antimicrobial by PMA-induced soluble factors (Fig. 7A). Finally, as expected, peptide LL-37, which is present in NETs, induces granzyme- the coculture of PBMCs with DPI-PMNs did not induce T cell mediated apoptosis of cytotoxic T lymphocytes (39), which 8 T CELL PRIMING BY NETs Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. NETs-mediated T cell activation in PBMCs. (A) Transmission light images of cluster formation of PBMCs cocultured with unstimulated PMNs and NETting-PMNs. Original magnification 340. (B) CD25 and CD69 surface expression after gating on CD4+ (red) and CD8+ (blue) T cells. Histograms represent the expression from a representative experiment. Dotted line indicates PBMCs cocultured with unstimulated PMNs; solid line indicates PBMCs cocultured with NETting-PMNs (NETs). Graphs represent mean fluorescence intensity (MFI) 6 SEM from five independent experiments. ***p , 0.001. (C) T cell proliferation in PBMCs cocultured with unstimulated PMNs and NETting-PMNs. T cell proliferation was assessed by EdU incorporation after gating on CD4+ (red) and CD8+ (blue) T cells. Graphs represent the percentage of EdU-positive cells (mean values 6 SEM) from 21 independent experiments. **p , 0.01, ***p , 0.001. Histograms represent the proliferation from a representative experiment. (D) Scatter plot showing T cell proliferation, in which each dot represents one individual donor. (E) IFN-g, IL-17A, IL-4, and IL-10 produced by PBMCs cocultured with unstimulated PMNs and NETting-PMNs (NETs). Values show mean pg/ml 6 SEM from five independent experiments. **p , 0.01, ***p , 0.001. could explain the lower survival of CD8+ T cells pre-exposed to in NETs-primed CD4+ T cells (Supplemental Fig. 2), which NETs, but we did not address this possibility in this study. CD8+ suggests a similar behavior of both cell types after NETs priming. T cells primed with NETs showed cluster formation and upregu- Furthermore, only minor differences were found in proliferation lation of CD25 and CD69 expression comparable to that observed and release of IFN-g between CD8+ and CD4+ T cells cocultured The Journal of Immunology 9

FIGURE 7. NETs and not other factors mediate T cell activation. (A) CD25 and CD69 surface expression after gating on CD4+ (red) and CD8+ (blue) T cells in PBMCs cocultured with unstimulated PMNs, NETting- PMNs (NETs), PMNs stimulated with PMA for 15 min, PMNs stimulated with fMLP, dying PMNs exposed to UV light, NETting-PMNs fixed with PFA, NETting-PMNs physically separated by a transwell, DPI-PMNs, and NETs-supernatant. Graphs represent mean fluorescence intensity (MFI) 6 SEM from five independent experiments. (B) T cell Downloaded from proliferation assessed by EdU incorporation after gating on CD4+ (red) and CD8+ (blue) T cells in PBMCs cocultured as indicated in (A). Graphs represent the percentage of EdU-positive cells (mean values 6 SEM) from five C independent experiments. ( ) IFN-g, http://www.jimmunol.org/ IL-17A, IL-4, and IL-10 produced by PBMCs cocultured as indicated in (A). Values show mean pg/ml 6 SEM from five independent experiments. *p , 0.5, **p , 0.01, ***p , 0.001. by guest on September 27, 2021

with NETting-PMNs and DCs. However, higher proliferation and and psoriasis (19). Because of the central role of T cells in most IFN-g release were found in CD8+ T cells cocultured with NETs of these disorders, our finding that NETs are able to mediate T and DCs in the presence of CD4+ T cells. These data suggest that, cell proliferation and secretion of Th1 proinflammatory cytokines although CD8+ T cells were activated by NETs, CD4+ T cells represents a novel mechanism of how NETs may contribute to most likely enhanced CD8+ T cell activation via IL-2 production T cell-mediated autoimmune diseases or certain aspects of their when both cell types were present. Unexpectedly, NETs mediated pathogenesis. the activation of both CD4+ naive and memory T cells. It is well In conclusion, we describe in this study for the first time, to our known that memory CD4+ T cells have less stringent activation knowledge, the ability of NETs to directly prime T cells by re- requirements than do naive CD4+ cells and are able to respond ducing their activation threshold and consequently to enhance faster and stronger to lower doses of Ag and to lower levels of adaptive immune responses. This novel feature adds to the list of costimulation than do naive CD4+ T cells (40–42). In this context, interesting properties of NETs and the functions of neutrophils in our results suggest that priming of naive CD4+ T cells by NETs general. NETs as a highly specialized structure of PMNs are, may induce changes in these cells similar to those that accompany according to our observations, not only important for the first-line memory T cell generation. innate immune defense, but they also participate in rendering Despite the well-documented importance of NETs as effective adaptive immune responses more efficient. Our data on the in- antimicrobial first-line defense mechanism, there is increasing evi- volvement of NETs in adaptive immunity still leaves many im- dence that NETs occur in various clinical settings in the absence of portant questions, and, among them, it will be very interesting to microbial infections and that they are probably also associated with examine the role of NETs in pathological immune responses and pathophysiological conditions (43). Abnormally high production autoimmune diseases and explore whether a better understanding and/or low degradation of NETs can lead to tissue damage, and may lead to new therapeutic strategies. the activation of pDCs and simultaneous exposure of the immune system to autoantigens may result in the generation of anti-self Acknowledgments Abs, which in addition can stimulate NET formation, creating We thank all healthy donors for blood donations and the staff of the Depart- a vicious cycle. Aberrant NETs formation can therefore not only ment for Transfusion Medicine, University Medical Centre Hamburg– participate in the organ damage observed in chronic inflammatory Eppendorf, for providing the samples. We also thank Nancy Martinez disorders (44–47), but furthermore contribute to the development (Max Planck Institute for Molecular Physiology, Dortmund, Germany) and perpetuation of autoimmune diseases such as systemic lupus for technical advice, and Raquel Planas and Benjamin Schattling for crit- erythematosus (19, 22, 23, 48, 49), small-vessel vasculitis (32), ical reading of the manuscript. 10 T CELL PRIMING BY NETs

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