Netting Neutrophils Induce Endothelial Damage, Infiltrate Tissues, and Expose Immunostimulatory Molecules in Systemic Lupus Erythematosus This information is current as of September 25, 2021. Eneida Villanueva, Srilakshmi Yalavarthi, Celine C. Berthier, Jeffrey B. Hodgin, Ritika Khandpur, Andrew M. Lin, Cory J. Rubin, Wenpu Zhao, Stephen H. Olsen, Matthew Klinker, David Shealy, Michael F. Denny, Joel Plumas, Laurence Chaperot, Matthias Kretzler, Allen T. Bruce and Mariana J. Kaplan Downloaded from J Immunol 2011; 187:538-552; Prepublished online 25 May 2011; doi: 10.4049/jimmunol.1100450

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Netting Neutrophils Induce Endothelial Damage, Infiltrate Tissues, and Expose Immunostimulatory Molecules in Systemic Lupus Erythematosus

Eneida Villanueva,*,1 Srilakshmi Yalavarthi,*,1 Celine C. Berthier,† Jeffrey B. Hodgin,‡ Ritika Khandpur,x Andrew M. Lin,x Cory J. Rubin,x Wenpu Zhao,* Stephen H. Olsen,‡ Matthew Klinker,* David Shealy,{ Michael F. Denny,*,2 Joel Plumas,‖,#,** Laurence Chaperot,‖,#,** Matthias Kretzler,† Allen T. Bruce,x and Mariana J. Kaplan*

An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these low- density granulocytes (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased Downloaded from levels of proinflammatory cytokines and type I IFNs. To directly establish LDGs as a distinct neutrophil subset, their gene array profiles were compared with those of autologous normal-density neutrophils and control neutrophils. LDGs significantly overex- press mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In con- trast, gene profiles of lupus normal-density neutrophils do not differ from those of controls. LDGs have heightened capacity to synthesize neutrophils extracellular traps (NETs), which display increased externalization of bactericidal, immunostimulatory pro- teins, and autoantigens, including LL-37, IL-17, and dsDNA. Through NETosis, LDGs have increased capacity to kill endothelial http://www.jimmunol.org/ cells and to stimulate IFN-a synthesis by plasmacytoid dendritic cells. Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which expose LL-37 and dsDNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and its subsequent responses may play a prominent deleterious role. The Journal of Immunology, 2011, 187: 538–552.

ecent evidence from our group and others indicates that more, lupus patients with higher circulating LDG numbers have neutrophils may play an important role in the induction of increased prevalence of skin involvement and/or vasculitis (1).

R autoimmune responses and organ damage in systemic Although currently no specific LDG surface markers have been by guest on September 25, 2021 lupus erythematosus (SLE) (1–3). Furthermore, microarray data identified that would allow them to be distinguished from normal- indicate that neutrophil-specific genes are highly expressed in density granulocytes, their nuclear morphology suggests that these PBMCs from lupus patients because of the cosegregation of low- cells present a more immature phenotype (1, 3). However, it is still density granulocytes (LDGs) in mononuclear cell fractions (3, 4). unknown whether lupus LDGs play prominent roles, when com- These LDGs represent a distinct neutrophil subset that is present pared with normal-density lupus neutrophils, in other aspects of in the peripheral blood of all adult SLE patients analyzed. Lupus disease pathogenesis, including providing a supply of potential LDGs are likely to be pathogenic, given their heightened capa- autoantigens or inducing or perpetuating autoimmune responses city to induce vascular damage and synthesize type I IFNs upon and tissue damage. exposure to specific stimulants, such as G-CSF and polyinosinic: Neutrophils immobilize and kill invading microbes extracellu- polycytidylic acid, when compared with autologous lupus normal- larly through the formation of neutrophil extracellular traps (NETs). density neutrophils and healthy control neutrophils (1). Further- As a unique type of neutrophil cell death, recently described as

*Division of Rheumatology, Department of Internal Medicine, University of Mich- (Grant P30 CA46592), the Rheumatic Disease Core Center (Grant P30 AR48310), igan Medical School, Ann Arbor, MI 48109; †Division of Nephrology, Department of and the Applied Systems Biology Core in the O’Brien Renal Center (Grant P30 Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109; DK081943). ‡Department of Pathology, University of Michigan Medical School, Ann Arbor, MI x The sequences presented in this article have been submitted to the Gene Expression 48109; Department of Dermatology, University of Michigan Medical School, Ann { ‖ Omnibus under accession number GSE26975. Arbor, MI 48109; Centocor Research and Development, Radnor, PA 19355; Uni- versite´ Joseph Fourier, Grenoble 38041, France; #INSERM U823, Immunobiologie et Address correspondence and reprint requests to Dr. Mariana J. Kaplan, Division of Immunotherapie des Cancers, La Tronche 38706, France; and **Etablissement Fran- Rheumatology, Department of Internal Medicine, University of Michigan Medical cais du Sang Rhone-Alpes, Laboratoire R&D, La Tronche 38701, France School, 5520 MSRBI, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5680. E-mail address: [email protected] 1E.V. and S.Y. contributed equally to the manuscript and share first authorship. The online version of this article contains supplemental material. 2Current address: Division of Rheumatology and Autoimmunity Center, Temple University, Philadelphia, PA. Abbreviations used in this article: ANCA, anti-neutrophilic cytoplasmic Ab; CTSG, cathepsin-G; DEFA4, defensin a-4; EC, endothelial cell; ELANE, elastase-2; IPA, Received for publication February 11, 2011. Accepted for publication April 24, 2011. Ingenuity Pathway Analysis; LCN2, lipocalin-2; LDG, low-density granulocyte; LTF, This work was supported by the National Institutes of Health through Public Health lactotransferrin; MMP-8, matrix metalloproteinase-8; MNase, micrococcal nuclease; Service Grant HL088419 (to M.J.K.), the Arthritis Foundation (to M.J.K.), the Uni- MPO, myeloperoxidase; NET, neutrophil extracellular trap; pDC, plasmacytoid den- versity of Michigan/Centocor Postdoctoral Research Program (to M.J.K. and E.V.), dritic cell; RNP, ribonucleoprotein; SLE, systemic lupus erythematosus; SLEDAI, the Anthony Gramer Fund in Inflammation Research (to M.J.K.), the Babcock Re- systemic lupus erythematosus disease activity index. search Endowment (to A.T.B.), and Training Grant in Cell and Molecular Dermatol- ogy 5T32AR007197 (to C.J.R.). This work was supported in part by the National Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 Institutes of Health through the University of Michigan’s Cancer Center Support www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100450 The Journal of Immunology 539

NETosis, this response is distinct from apoptosis and necrosis recognizing human neutrophil elastase (Abcam, Cambridge, MA), human and characterized by the active release of nuclear chromatin fi- Ro/SSA and human La/SSB (Santa Cruz Biotechnology, Santa Cruz, CA), bers (5, 6). NETosis is triggered by a variety of stimuli including mouse Abs recognizing human Smith (Abcam), human Cathelicidin/ OSX12 (Abcam), and human dsDNA (Millipore, Temecula, CA), and microorganisms, proinflammatory cytokines, activated platelets, goat anti-human IL-17 (R&D Systems, Minneapolis, MN) were used. and endothelial cells (ECs) (6, 7). A variety of putative auto- Secondary detection was performed using goat anti-rabbit–FITC (Southern antigens are present within and attached to NET chromatin fibers, Biotechnology Associates, Birmingham, AL), donkey anti-goat Alexa 568 including citrullinated histones (8) and various bactericidal pro- (Invitrogen, Carlsbad, CA), and/or anti-mouse Cy3 (Sigma-Aldrich, St. Louis, MO). Prolong Gold Antifade and Hoechst 33342 were from In- teins and/or enzymes such as the cathelicidin LL-37, neutrophil vitrogen. Micrococcal nuclease (MNase) was from Thermo Scientific elastase, and myeloperoxidase (MPO) (9). Upon neutrophil acti- (Rockford, IL). vation, elastase migrates from azurophilic granules to the nucleus, where it partially degrades specific histones and promotes chro- Purification of LDGs matin decondensation. MPO synergizes with elastase in driving Lupus LDGs were purified as described by our group (1). In brief, PBMCs this decondensation (9), a phenomenon that is considered key in were isolated by Ficoll/Hypaque gradient, and cell pellets were incubated NET formation. with LDG isolation mixture (equal volumes of biotinylated Abs recog- nizing human CD3, CD7, CD19, CD79b, CD56, MHC class II, CD86, and Due to the potential role of netting neutrophils in externalizing CD235a), followed by anti-biotin MACS beads (Miltenyi Biotec, Auburn, autoantigens and DNA-modifying factors, thereby making these CA). Cells were applied to an MACS-LS column, and nonimmobilized molecules more exposed to the adaptive and innate immune sys- cells were recovered by negative selection. The purity of the LDG fraction + lo + lo tems, a putative link between NETosis and autoimmunity has been was .95%, and cells were identified as CD15 /CD14 or CD10 /CD14 . recently proposed. It has been shown that neutrophils from patients Isolation of neutrophils Downloaded from with anti-neutrophilic cytoplasmic Ab (ANCA)-positive vasculitis release NETs enriched in MPO and LL-37 (10). NETs are also Normal-density neutrophils were isolated by dextran sedimentation of RBC $ present in the kidneys from patients with this disease, where they pellets, as described (15). Purity was 95%. In previous studies, we had compared the activation status of neutrophils obtained by dextran sedi- may provide a source of antigenic nucleosomes and promote mentation versus double gradient and found no differences in their phe- immune complex formation (10). Further, impaired NET degra- notype or function (1).

dation has been identified in a subset of SLE patients, secondary to http://www.jimmunol.org/ DNase1 inhibitors and anti-NET Abs that prevent DNase1 access NETs immunohistochemical staining and quantification to NETs (2). LDGs may represent an additional source of NETs, Neutrophils or LDGs were isolated as above, and 1 to 2 3 105 cells/ml were leading to heightened autoantigen exposure and modification of seeded in poly-L-lysine coverslips and incubated at 37˚C, 5% CO2 for 15 tissue damage. Recent evidence indicates that NETosis may be min. Cells were washed with ice-cold PBS and either fixed right away with a 4% paraformaldehyde and then blocked overnight at 4˚C with 10% FBS/ enhanced in IFN- –primed lupus neutrophils upon exposure to 1% BSA/0.05% Tween 20 and 2 mM EDTA/PBS or incubated for 2 h in anti-ribonucleoprotein (RNP) Abs. This is accompanied by the RPMI 1640/glutamine/2% BSA in the presence or absence of 20 nM PMA release of LL-37 and high-mobility group protein B1, which fa- to induce NET formation, followed by fixation and overnight blocking at cilitate uptake and recognition of mammalian DNA by plasma- 4˚C. NETs were detected by washing the fixed cells with ice-cold 10%

FBS/PBS and incubating with anti-human elastase (1:100) or isotype by guest on September 25, 2021 cytoid dendritic cells (pDCs) (11). In addition, recent evidence control for 45 min at 4˚C, followed by incubation with secondary indicates that NETs may be harmful to the endothelium and fluorochrome-conjugated Abs for 45 min at 4˚C. Nuclear DNA was promote thrombosis (7, 12). detected by incubating cells with Hoechst 33342 (1:100) for 10 min at To further clarify the origin of LDGs and assess their pathogenic room temperature. Coverslips were mounted in Prolong Antifade Reagent potential and role in the induction of autoimmune responses, we (Invitrogen) and analyzed using an Olympus microscope (IX70, Center Valley, PA). Statistical background, shading subtraction, and image overlay compared the gene array expression profiles of purified LDGs with were performed with Metamorph v7.7 software (Molecular Devices, those of autologous normal-density lupus neutrophils (referred to Sunnyvale, CA). The recorded images were loaded onto Adobe Photoshop in the text as lupus neutrophils) or healthy control normal-density (Adobe Systems) for further analysis, in which NETs were manually neutrophils (referred to in the text as control neutrophils). We quantified by two independent observers. The number of cells positive for both neutrophil elastase and nuclear staining (Hoechst) were considered also compared the capacity of isolated LDGs to form NETs, ex- a NET and digitally recorded to prevent multiple counts. The percentage of ternalize autoantigens and immunostimulatory molecules, stimu- NETs was calculated as the average of five to six fields (340) normalized late pDCs, and induce endothelial cytotoxicity through NETosis. to the total number of cells. For LL-37 (1:100), dsDNA (1:10), Ro (1:20), Finally, we assessed whether netting neutrophils are present in vivo La (1:20), Smith (1:20), and IL-17 (1:10) quantification, the number of in involved lupus tissue from various organs as an additional in- cells positive for either of these markers colocalized with elastase and Hoechst were counted as part of the overlay (RGB) image and recorded dication of their putative pathogenic role. digitally to prevent multiple counts. RNA isolation Materials and Methods Patient selection Total RNA was isolated with Tripure (Roche, Indianapolis, IN), following manufacturer’s recommendations. For microarray analysis, RNA was The University of Michigan institutional review board approved this study. further purified and concentrated using an RNeasy micro kit (Qiagen, Subjects gave informed consent in accordance with the Declaration of Valencia, CA). RNA samples were processed on an Agilent 2100 Bio- Helsinki. Lupus patients fulfilled the revised American College of Rheu- Analyzer (Agilent Technologies, Santa Clara, CA) to assess integrity. matology criteria for SLE (13) and were enrolled from the University of Michigan outpatient rheumatology clinic. Disease activity was assessed by Microarray data processing, analysis, and pathway mapping the SLE disease activity index (SLEDAI) (14). Gender-matched healthy Affymetrix Human U133 Plus 2.0 Genechips (Affymetrix, Santa Clara, CA) controls were recruited by advertisement. Demographic and clinical in- were processed at the University of Michigan Microarray Core Facility. The formation on the lupus patients enrolled in the study (including medi- samples analyzed and compared were: normal-density neutrophils from cations) is included in Table II. healthy individuals (n = 9) and normal- density neutrophils and autologous Reagents LDGs from lupus patients (n = 10 for each group). CEL files were nor- malized in GenePattern pipeline (http://www.GenePattern.org) using the For LDG purification, biotinylated Abs recognizing CD3, CD7, CD19, Robust MultiChip Average method and the Human Entrez Gene custom CD79b, CD56, MHC class II, CD86, and CD235a were obtained from CDF annotation version 10 (http://brainarray.mbni.med.umich.edu/Brain Ancell (Bayport, MN). For immunohistochemical staining, rabbit Abs array/default.asp). As samples were part of two hybridization rounds, the 540 ABERRANT NETosis IN LUPUS GRANULOCYTES two resulting normalized files were corrected for batch effect (16) within Ag retrieval was enhanced by microwaving the slides in citrate buffer (pH GenePattern. Of the 17,527 gene identification numbers (corresponding to 6; Biogenex) for 10 min. Sections were blocked for 30 min in 10% horse the 54,675 Affymetrix probesets), the number of genes expressed above serum prior to 1-h incubation of primary Abs at 4˚C. Simultaneous stain- the Poly-A Affymetrix control expression baseline (negative controls) and ing was performed with mouse monoclonal anti-human MPO (1:1000; used for further analyses was 15,929. The Gene Expression Omnibus ac- Abcam) and rabbit polyclonal anti-human histone H2A (1:500; Abcam) cession number for these arrays is GSE26975 (http://www.ncbi.nlm.nih. followed by 30 min incubation with matched secondary Abs: anti-mouse gov/geo/). IgG-Alexa Fluor 488 (1:300; Invitrogen) or anti-rabbit IgG-Alexa Fluor Statistical paired analyses were performed using Significance Analysis of 555 (1:300; Invitrogen). DAPI was added to the slides prior to mounting Microarrays method implemented in MultiExperiment Viewer application with cover slips (Invitrogen). For NETosis detection, images were acquired (17, 18) for comparing lupus neutrophils with lupus LDGs; unpaired by a Leica DMIRB inverted microscope and an RT slider digital camera analyses were performed in the comparison of healthy control neutrophils (model 2.3.1; Diagnostic Instruments) using DP Controller, DP Manager with lupus neutrophils and healthy control neutrophils with lupus LDGs. (Olympus, Essex, U.K.) software, and National Institutes of Health ImageJ The canonical pathways derived from the significantly regulated genes software. RBCs were excluded. NETs were considered as extracellular between the groups (q , 0.05 depicting the false discovery rate) were structures that costained for MPO, histone H2A, and DAPI. analyzed using the Ingenuity Pathway Analysis (IPA) Software (http:// www.ingenuity.com). Skin tissue immunohistochemistry and quantification of NETosis Real-time quantitative PCR Skin biopsies from 11 patients with cutaneous lupus involvement and 10 Real-time PCR reactions were run on an ABI Prism 7900HT in duplicate gender-matched healthy controls were analyzed for neutrophil infiltration using 23SYBR Green PCR Master Mix (Applied Biosystems, Foster City, and NETosis. In addition, skin biopsies from 10 patients with psoriasis were CA). Oligonucleotide primers (IDT, Coralville, IA) used in the reactions included as positive controls of IL-17+ cells in the skin. Control donors were: lipocalin, 59-CCCAGCCCCACCTCTGA39 (forward), 59-CTTCCC- were identified from respondents to advertisements, had no personal or Downloaded from CTGGAATTGGTTGTC-39 (reverse); matrix metalloproteinase-8 (MMP8), family history of lupus, and were free of inflammatory skin disease at the 59-GCTGAGGTAGAAAGAGCTATCAAGGA-39 (forward), 59-AGCAAT- time of biopsy. Five-micrometer sections of skin were deparaffinized on GTTGATATCTGCCTCTCC-39 (reverse); MPO, 59-TTTGACAACCTGC- a hot plate at 65˚C for 1 h. They were rehydrated by incubation and heat ACGATGAC-39 (forward), 59-CGGTTGTGCTCCCGAAGTAA-39 (reverse); retrieval in Cell Conditioning Solution (CC1; Ventana Med, Tucson, AZ). cathepsin-G (CTSG), 59-AGAAGAGTCAGACGGAATCGA-39 (forward), The sections were blocked with 0.2% horse serum (Invitrogen) for 30 min. 59-CCCTGACGACTTTCCATAGGA-39 (reverse); LL-37, 59-GCAGTCAC- Simultaneous staining was first performed for 30 min with goat anti–IL- CAGAGGATTGTGAC-39 (forward), 59-CACCGCTTCACCAGCCC-39 (re- 17 (100 mg/ml; R&D Systems), mouse anti-human Cathelicidin/OSX12 verse); IL-17A, 59-ACTCCTGGGAAGACCTCATTGG-39 (forward), 59- (Abcam), or anti-dsDNA (Millipore) and another primary Ab, either rabbit http://www.jimmunol.org/ GGCCACATGGTGGACAATCG-39 (reverse); and GAPDH, 59-TTGCCA- anti-MPO (1:1500; DakoCytomation) or rabbit anti-human neutrophil TCAATGACCCCTTCA-39 (forward), 59-CGCCCCACTTGATTTTGGA-39 elastase (10 mg/ml; Abcam). This was followed by 30-min incubation with (reverse). matched secondary Abs: chicken anti-goat IgG-Alexa Fluor 488 (1:300; Invitrogen) or chicken anti-rabbit IgG-Alexa Fluor 594 (1:300; Invitrogen). EC cytotoxicity assay ProLong Gold antifade reagent with DAPI (Invitrogen) was added to the HUVECs were cultured in MCDB131 basal media (Life Technologies, slides prior to mounting with cover slips. Images were captured with Carlsbad, CA) supplemented with EGM-2MV (without hydrocortisone) a fluorescent imaging microscope (BX50; Olympus) using DP Controller bullet kit (Lonza, Walkersville, MD) on 0.2% gelatin-coated 24-well plates and DP Manager (Olympus) software. The recorded images were loaded (Fisher, Pittsburgh, PA). Lupus LDGs, their autologous lupus neutrophils, onto Adobe Photoshop. The number of cells expressing one or both markers of interest in each 3200 field was manually counted by two in- and healthy control neutrophils were incubated with HUVECs at a 2:1 E by guest on September 25, 2021 dependent blinded observers. (neutrophil):T (HUVEC) ratio in MCDB131/EGM-2MV and 0.1 mg/ml PMA in the presence or absence of MNase (10 U/ml) for 16–20 h. Al- Statistical analysis ternatively, cells were also cocultured with 0.1 mg/ml PMA, with or without MNase, for 1 to 2 h, followed by replacement with fresh media For NET quantification, immunofluorescence was performed in neutrophils with or without nuclease. Following the incubation, nuclease reaction was isolated from individual patients (n $ 4 for both SLE and control patients), stopped by adding EDTA to final concentration of 2 mM. Adherent LDGs and counts were averaged, represented as mean 6 SEM, and analyzed by and neutrophils were collected by gentle pipetting, and HUVECs were two-tailed Student t test, for which p # 0.05 was considered significant. harvested with 0.05% trypsin-EDTA (Life Technologies), followed by Comparison of NETosis within different areas of the skin was performed centrifugation at 1600 rpm for 5 min. HUVECs were resuspended in 1% by repeated-measures one-way ANOVA with a Tukey’s multiple compar- horse serum/1% BSA in PBS, and 104 to 105 cells were incubated with ison posttest. For all other studies, paired or unpaired Student t tests were PE–anti-human CD146, allophycocyanin-Annexin V, PE/Cy5–anti-human performed. Microarray statistical analysis is detailed above. CD45 (BD Pharmingen, San Diego, CA), and/or PE/Cy5-anti-human CD10 (Biolegend), followed by fixation with 4% paraformaldehyde. The Results percentage of HUVEC cytotoxicity was measured in cells double-positive Lupus LDGs have a distinct gene expression profile from for CD146 and Annexin V in the CD45- or CD10-negative gate. autologous lupus neutrophils and control neutrophils pDC lines and IFN-a quantification To determine whether LDGs represent a distinct pool of neu- The previously described human pDC cell line GEN 2.2 (19) was cul- trophils, gene expression profiling of purified neutrophil fractions tured in RPMI 1640 Glutamax/1 mM sodium pyruvate/4 mM glutamine/ of lupus LDGs, autologous lupus neutrophils, and gender-matched nonessential amino acids/10% heat-inactivated FCS. Cells were stimulated for 16 h with either CpG (1 mg /ml) or supernatants collected from either control neutrophils was assessed using Affymetrix genechip mi- control or lupus neutrophils or autologous lupus LDGs that had been croarrays. Samples were matched-paired in comparing lupus cultured for 1 h, and supernatants were treated in the presence or absence LDGs with autologous lupus neutrophils. Although there were of MNase (10 U/ml, 10 min). IFN-a mRNA from pDCs was quantified by no genes significantly differentially regulated when comparing real-time PCR as previously described by our group (1). normal-density lupus neutrophils with healthy control neutrophils, Human kidney tissue and immunofluorescence several genes were differentially expressed in LDGs relative to either autologous lupus neutrophils or healthy control neutrophils. Kidney tissue was obtained from nine renal biopsies from subjects with clinical and histological diagnosis of lupus nephritis according to the new In all, 302 genes were differentially expressed in lupus LDGs when SLE nephritis classification (20) and from one patient with fulminant compared with control neutrophils, and 281 genes were identified Henoch-Scho¨nlein purpura with renal involvement. Relevant clinico- as altered by pairwise comparison of each patient’s LDGs to their histological parameters are in Table II. Immunohistochemical analyses autologous lupus neutrophils (q value ,0.01, fold-change $1.5 were carried out at the University of Michigan Histology and Im- # munohistochemistry Core. Three-micrometer sections of formalin-fixed, and 0.7 for the upregulated and downregulated genes, respec- paraffin-embedded tissues were mounted on plus slides, deparaffinized in tively) (Fig. 1A, Supplemental Tables I, II). A total of 224 genes xylene, and then rehydrated with distilled H2O through graded alcohols. were identified as upregulated in both comparisons (Fig. 1A). Con- The Journal of Immunology 541

FIGURE 1. LDGs overexpress defensins and proinflammatory molecules. A, Schematic represen- tation of the sample group and gene list comparisons made using the gene microarray data (q value ,0.01, fold-change $1.5 and #0.7 for the upregulated and downregulated genes, respectively). Samples were Downloaded from matched-paired when comparing lupus neutrophils with autologous lupus LDGs (n = 10 for each group). B, Lupus LDGs express elevated levels of azurophilic granule genes. Log base 2 mRNA mean expression values of five azurophil genes in control neutrophils (n = 9), lupus neutrophils (n = 10), and lupus LDGs http://www.jimmunol.org/ (n = 10): MPO, ELANE, DEFA4, CTSG, and azur- ocidin 1 (AZU1). Data are presented as mean 6 SD. C, Confirmation of enhanced mRNA expression by real-time PCR of various neutrophils genes in lupus LDGs when compared with autologous lupus neu- trophils (n = 7–12) and control neutrophils (n = 7). Bar graph represents fold mRNA expression (mean 6 SEM) after adjusting for housekeeping gene (GAPDH). *p , 0.05 LDGs compared with control and/or autologous lupus neutrophils, ***p , 0.0001. by guest on September 25, 2021

versely, a total of 57 genes were found to be selectively regulated in using IPA included inflammatory response and hematological and lupus LDGs when compared with autologous lupus neutrophils, and cardiovascular diseases. 78 genes were restricted to LDGs alone when compared with control The top upregulated genes in lupus LDGs in both comparisons neutrophils (Fig. 1A, Supplemental Tables I, II). included a number of serine proteases, bactericidal proteins, and The defined transcripts were associated to canonical pathways other molecules involved in neutrophil regulation of the in- using IPA. The pathways significantly regulated in LDGs compared flammatory response (3, 21–25) when compared with normal- with control neutrophils and autologous lupus neutrophils are listed density lupus and control neutrophils (Table I). Among them, in Supplemental Table III. Actin cytoskeleton, macropinocytosis, mRNA of the cathelicidin gene CAMP (LL-37) was 3.5- and clathrin-mediated endocytosis, and integrin signaling pathways 4.6-fold higher in lupus LDGs when compared with control and were among the most significantly regulated pathways in lupus lupus neutrophils, respectively (q value ,0.01). In the same group, LDGs compared with control and lupus neutrophils. Inhibition of defensin a-4 (DEFA4) was highly upregulated in LDGs com- angiogenesis by Tsp1 was a pathway significantly regulated only in pared with control and lupus neutrophils (22.4- and 25.6-fold, lupus LDGs compared with control neutrophils, whereas Ephrin respectively). Lactotransferrin (LTF), lipocalin-2 (LCN2), MPO, receptor signaling was only significantly regulated in lupus LDGs elastase-2 (ELANE), MMP-8, and CTSG were also significantly compared with lupus neutrophils. The top functions identified upregulated in the lupus LDGs when compared with control and 542 ABERRANT NETosis IN LUPUS GRANULOCYTES

Table I. Affymetrix microarray expression data in lupus LDGs of genes corresponding to enzymes, bactericidal, and other molecules known to be implicated in reactive oxygen species generation and/or NET formation

Lupus LDGs Lupus LDGs Compared with Compared with Control Lupus Neutrophils Neutrophils

Entrez Gene Fold q Fold q Identification No. Gene Symbol Gene Name Change Valuea Change Valuea Enzyme and enzyme inhibitor genes 6036 RNASE2 RNase, RNaseA family, 2 (liver, 4.24 0.004* 3.06 0.066 eosinophil-derived neurotoxin) 4317 MMP8 Matrix metallopeptidase 8 38.53 0.000* 41.53 0.000* (neutrophil collagenase) 4318 MMP9 Matrix metallopeptidase 9 (gelatinase B, 1.08 0.999 1.37 0.047* 92 kDa gelatinase, 92 kDa type IV collagenase) 6037 RNASE3 RNase, RNase A family, 3 (eosinophil 12.34 0.000* 12.07 0.000* cationic protein) 1991 ELANE (Elastase 2) Elastase, neutrophil expressed 11.64 0.000* 10.92 0.000* 3934 LCN2 Lipocalin2 24.49 0.000* 19.02 0.000* 5476 CTSA Cathepsin-A 1.69 0.004* 1.54 0.043* Downloaded from 1511 CTSG Cathepsin-G 13.53 0.000* 17.73 0.000* 4353 MPO Myeloperoxidase 12.02 0.000* 14.66 0.000* Bactericidal molecule genes 1669 DEFA4 Defensin, a-4, corticostatin 22.41 0.000* 25.57 0.000* 820 CAMP (LL-37) Cathelicidin antimicrobial peptide 3.44 0.003* 4.62 0.000* 671 BPI Bactericidal/permeability-increasing protein 13.80 0.000* 16.59 0.000*

566 AZU1 Azurocidin 1 11.23 0.000* 12.22 0.000* http://www.jimmunol.org/ Other genes 1088 CEACAM8 (CD66b) Carcinoembryonic Ag-related cell adhesion molecule8 16.63 0.000* 18.27 0.000* 10321 CRISP3 Cysteine-rich secretory protein3 18.39 0.000* 12.18 0.000* 634 CEACAM1 (CD66a) Carcinoembryonic Ag-related cell adhesion 1.50 0.099 1.30 0.017* molecule 1 (biliary glycoprotein) 4057 LTF Lactotransferrin 16.91 0.000* 12.01 0.000 1191 CLU Clusterin 21.24 0.000* 17.39 0.000* aA q value ,0.05 was considered significant (in boldface and with asterisk). by guest on September 25, 2021 lupus neutrophils (fold-change range from 10.9–41.5). These 2). Both control and normal-density lupus neutrophils displayed results indicate that lupus LDGs show increased expression of significantly higher NET formation following a 2-h in vitro sti- azurophilic granule genes that is not secondary to a general in- mulation with PMA when compared with baseline levels (Fig. duction of these genes in SLE, as the autologous lupus neutrophils 2). In contrast, the percentage of NETs in lupus LDGs remained expressed levels comparable to control neutrophils (Fig. 1B). unchanged from baseline (Fig. 2), suggesting that LDGs may be These findings were confirmed by real-time PCR for several of the prestimulated in vivo and resistant to further NET induction. molecules mentioned above (Fig. 1C). Thus, microarray analysis When comparing isolation methods, there were no differences in of lupus LDGs confirmed an enhanced bactericidal and activated NET formation when neutrophils were isolated through gradient signature compared with control or normal-density lupus neu- separation or negative selection with magnetic beads (data not trophils. shown). This excluded a potential effect of the isolation technique Altogether, the transcriptional analysis results indicate that in the differences in induction of NETs in vitro. LDGs, as a distinct subset of lupus granulocytes, have a specific There were no significant correlations between the percentage of molecular pattern that differs from autologous lupus neutrophils LDGs undergoing NETosis at baseline and lupus disease activity and control neutrophils, whereas lupus neutrophils display no (as determined by SLEDAI) (14) or presence or titers of auto- significant differences in gene expression compared with control antibodies. Furthermore, there were no associations between use neutrophils. These results support the hypothesis that LDGs are of various lupus medications (antimalarials, corticosteroids, and/ a specific subset of granulocytes with distinct phenotype and func- or immunosuppressive drugs) and percentage of LDGs undergoing tional capabilities. NETosis (data not shown). These results indicate that enhanced NETosis occurs in lupus LDGs independent of the patients’ dis- NET formation is increased in lupus LDGs, leading to ease activity. enhanced externalization of autoantigens and To further characterize the NET composition and functional immunostimulatory molecules relevance of extracellular trap formation, the localization and Serine proteases and cathelicidins released from neutrophils are expression of immunostimulatory and bactericidal proteins was incorporated into NETs, allowing the delivery of high concen- investigated. Control and lupus neutrophil and LDG NETs express trations of molecules that kill bacteria and degrade their virulence the bactericidal proteins elastase and LL-37. The latter was de- factors (8). We tested if NET formation differed in lupus LDGs tected intracellularly in all neutrophils but was predominantly relative to control neutrophils or autologous neutrophils. At found colocalized within the extracellular NETs. Because a higher baseline, peripheral blood LDGs demonstrated significantly en- percentage of LDGs undergo NETosis, there was significantly hanced NET formation right after isolation when compared with enhanced externalization of LL-37 by these cells when compared healthy control neutrophils or autologous lupus neutrophils (Fig. with control and lupus neutrophils (Fig. 3). The Journal of Immunology 543

FIGURE 2. Circulating lupus LDGs undergo in- creased NETosis. A, Representative images of control neutrophils, lupus neutrophils, and lupus LDGs iso- lated from peripheral blood and analyzed at baseline (T0) or after stimulation for 2 h with DMSO or PMA. Top panels show immunofluorescent merged images of NETs, which were detected by neutrophil elastase

(green), and DNA was labeled with Hoechst 33342 Downloaded from (blue). Original magnification 340. Scale bar, 20 mm. B, Quantification of the percentage of NETs (elastase- labeled cells over total number of cells) are plotted as mean 6 SEM (n = 6 patients/group). *p # 0.05. http://www.jimmunol.org/ by guest on September 25, 2021

Because NETs may be sources of autoantigens in other con- Recent evidence indicates that IL-17 may be involved in the ditions (10), we assessed if various targeted autoantigens were pathogenesis of SLE through its capacity to amplify local in- present in LDG NETs. During quantification, it became evident flammation by recruiting cells from the innate immune system and that all NETs expose dsDNA regardless of the neutrophil source. its ability to stimulate B cell-adaptive immune responses (30). However, as a higher percentage of LDGs undergo NETosis than Indeed, elevated levels of IL-17 and increased numbers of Th17 healthy controls or lupus neutrophils, they lead to overall en- cells have been reported in human and murine lupus, and there is hancement in externalization of dsDNA (Fig. 4A,4C). In contrast, evidence that this cytokine is synthesized in target organs from there was no evidence of expression of other common lupus patients with this disease, including skin and kidney (30–32). autoantigens (Ro, La, or Smith) within the NETs structure. Indeed, However, it is unclear if, in addition to T cells, innate cells in- the expression of Ro, La, and Smith was intracellular and equally cluding neutrophils could represent an enhanced source of IL-17 detected in LDGs, lupus, and control neutrophils (data not shown). production in blood and the periphery in SLE. Indeed, IL-17A was These observations were independent of whether the serum of the detected at the mRNA level in control and lupus neutrophils and in patients was positive or negative for Abs against dsDNA, Smith, LDGs, and there were no significant differences in expression Ro, or La (Table II and data not shown). These results indicate among these three cell subsets (data not shown). To determine that netting neutrophils externalize dsDNA and that LDGs may whether NETosis could be a source of enhanced IL-17 external- represent an enhanced source of extracellular dsDNA through ization in SLE, the expression of this cytokine in the NETs of heightened NET formation. peripheral blood neutrophils and LDGs was compared. Overall, between 8 6 4% to 23 6 5% of all neutrophils isolated from NET formation leads to enhanced externalization of IL-17 in peripheral blood externalize IL-17 in vitro during NETosis, and all lupus LDGs NETS from peripheral blood neutrophils express IL-17. Both Innate IL-17–producing cells are considered an integral part of IL- control and lupus neutrophils externalize IL-17 with similar fre- 17–mediated immune responses (26). Neutrophils have previously quency. As NETosis was significantly enhanced in lupus LDGs, been identified as a source of IL-17, particularly in the context a higher proportion of these cells externalized IL-17 (Fig. 4B,4C). of autoimmunity (27), although the signaling pathways by which These results suggest that NET formation by LDGs in situ may these cells synthesize and release this specific cytokine remain potentiate IL-17–dependent responses. uncharacterized. Neutrophils expressing IL-17 have been reported Overall, these results indicate that, through enhanced NETosis, in diseased tissues such as atherosclerotic plaques (28). Further- LDGs externalize various immunostimulatory molecules and au- more, neutrophils can externalize IL-17 through NETosis in skin toantigens that may be crucial in stimulation of adaptive and and peripheral blood from patients with psoriasis (29). innate immunity. 544 ABERRANT NETosis IN LUPUS GRANULOCYTES

FIGURE 3. LL-37 externalization in NETs is increased in lupus LDGs. A, Representative images of control neutrophils, lupus neutrophils, and lupus LDGs after isolation from peripheral blood. Cells were stained for detection of LL-37 (red) and neutrophil elastase (green), and DNA was labeled with Hoechst 33342 (blue). Top pan- els show images of LL-37 and Hoechst (left panels), elastase and Hoechst (middle panels), and merged LL-37, elastase, and Hoechst (right pan- els). Original magnification 340. Scale bar, 20 Downloaded from mm. Arrows represent areas of LL-37 localization within the NETs. B, Quantification of the per- centage of cells containing LL-37 colocalized with elastase over total number of cells are plotted as mean 6 SEM (n $ 3 patients). *p # 0.05. http://www.jimmunol.org/ by guest on September 25, 2021

Netting neutrophils infiltrate lupus kidneys affected by Netting neutrophils infiltrate lupus skin and expose dsDNA and glomerulonephritis LL-37 Previous studies have indicated that impaired NET degradation in In previous studies, we identified that SLE patients with high levels SLE is associated with the development of lupus nephritis (2). of LDGs in the circulation had increased prevalence of skin in- Furthermore, patients with ANCA+ vasculitis have evidence of volvement (1). After identifying increased NETosis in peripheral netting neutrophils in their kidneys (10). Although a potential role blood lupus LDGs, we proceeded to assess if lupus skin lesions for neutrophils in lupus nephritis was proposed decades ago (33), show evidence of infiltration by netting neutrophils. We analyzed it is unclear if NETosis is observed in lupus nephritis and if NETs skin biopsies from 11 patients with several forms of cutaneous play a pathogenic role in associated renal damage. We analyzed lupus including discoid lupus, acute cutaneous lupus, subacute kidney biopsies from nine lupus patients with World Health Or- cutaneous lupus, and lupus panniculitis (Table II). High-power ganization class III or IV glomerulonephritis (Table II). NETs examination of dual-color immunofluorescence slides of SLE visualized as weblike or granular structures costaining with MPO, patient and control skin revealed numerous NETs visualized histone H2A, and DAPI were observed in the majority of lupus as weblike structures costaining with MPO and DAPI (Fig. 6). nephritis cases (67%) (Fig. 5). Overall, 17% of glomeruli exam- Similar distribution was seen when skin biopsies were stained ined displayed netting neutrophils (range 0–50%). Patients with with a neutrophil elastase Ab (not shown). Frequently observed class IV lupus nephritis had a higher percentage of glomeruli in all cutaneous lupus lesions, these NETS were particularly infiltrated by netting neutrophils than patients with class III ne- enriched near the dermal-epidermal junction in the papillary der- phritis (mean 6 SEM, 27.5 6 10% versus 9.84 6 3%, re- mis and around blood vessels and adnexae, including hair fol- spectively; p = 0.05). Renal biopsies with higher activity index, as licles and eccrine glands (Fig. 6A–C). In the two patients with per World Health Organization classification, had a higher per- lupus panniculitis, aggregates of netting neutrophils were fre- centage of glomeruli infiltrated by netting neutrophils (19.5 6 5% quently observed in the lobular adipose tissue with extension into for biopsies with activity index $10 versus 6.6 6 1.5% for bi- adjacent reticular dermis (Fig. 6D–G). Overall, in lupus biopsies, opsies with activity index ,10). In addition, those patients with 7% of all neutrophils in the epidermis, 24.3% of neutrophils in the netting neutrophils in glomeruli had on average higher levels of papillary dermis, and 25.4% of neutrophils in the reticular dermis anti-dsDNA Abs in serum than those without evidence of these and subcutis had formed NETs. NETosis was particularly enriched cells (669 6 380 versus 189 6 150 IU/ml). In contrast, no in areas with large aggregates of neutrophils (Fig. 6). In contrast, NETosis was observed in the kidney biopsy from a patient with neutrophil infiltration and NETosis were not observed in skin bi- fulminant Henoch-Scho¨nlein purpura and no lupus (not shown). opsies from 10 healthy control individuals (data not shown). The Journal of Immunology 545

FIGURE 4. Lupus LDGs externalize dsDNA and IL-17 through NETosis. Representative images of control neutrophils, lupus neutrophils, and lupus LDGs after isolation from peripheral blood. Cells were stained for detection of neutrophil elastase (green), DNA (Hoechst 33342, blue), and either dsDNA (red) or IL-17 (red). Merged images of dsDNA, elastase, and Hoechst (A)andmerged images of IL-17, elastase, and Hoechst (B). C, Quantification of the percentage of cells containing dsDNA or IL-17 colocalized with elastase over total Downloaded from number of cells is plotted as mean 6 SEM (n $ 5 patients). Scale bars, 20 mm. *p # 0.05. http://www.jimmunol.org/

Interestingly, large aggregates of NETs were seen in the skin IL-17 in lupus skin was significantly higher than in healthy control from all five lupus patients who had increased levels of anti-dsDNA skin (1.9%, n =8;p , 0.001) and similar to that seen in skin from Abs in sera (Table II). Because SLE is frequently associated with patients with psoriasis (32%, n = 12) (Fig. 7 and data not shown). by guest on September 25, 2021 the presence of circulating anti-dsDNA Abs, we tested if large Overall, these results demonstrate that enhanced NETosis and aggregates of NETs in skin could be a potential antigenic source exposure of autoantigens and immunostimulatory proteins occurs for formation of these autoantibodies, especially because LL-37– in vivo in affected organs of SLE patients. DNA complexes present in the NETs may activate pDCs (34). Netting neutrophils stimulate IFN-a synthesis by pDCs Confirming the findings from peripheral blood neutrophils, analy- sis of these cells in affected lupus skin revealed that NETs ex- Recent work indicates that antimicrobial products, including pressed dsDNA (data not shown). Furthermore, LL-37 was clearly LL-37, are immunostimulatory when bound to DNA and induce present in the NETs in lupus skin biopsies (Fig. 6H) and was IFN-a synthesis by pDCs (23). Additionally, others have shown most prominent in netting neutrophils from the deep dermis areas. that NETosis stimulates pDCs to synthesize increased levels of These results indicate that NETs extrude dsDNA and LL-37, pos- IFN-a (11). There is also evidence that type I IFNs can induce sibly providing antigenic stimuli for anti-dsDNA Ab formation. NET formation (35). Given the important pathogenic role of IFN-a in SLE and the increased NET formation and LL-37 exter- Skin biopsies from lupus patients display higher numbers of nalization by LDGs, we tested if these cells also induce pDCs IL-17+ neutrophils to synthesize more IFN-a. Supernatants from control neutrophils Because IL-17 was detected in the NETs from peripheral blood did not induce the pDC cell line Gen2.2 (19) to synthesize IFN-a neutrophils, we also assessed if IL-17–positive (IL-17+) neu- mRNA when compared with pDCs alone. In contrast, supernatants trophils were detected in the affected lupus skin. Using dual-color from both lupus neutrophils and LDGs induced significantly en- immunofluorescence for IL-17 and MPO, we observed that 51.5 6 hanced IFN-a mRNA synthesis in pDCs when compared with 6.8% of all IL-17–expressing cells in lupus skin were neutrophils. healthy controls (Fig. 8A). IFN-a induction by neutrophil super- Further, 19.7 6 7.5% of intact neutrophils stained brightly with natants was significantly decreased in lupus neutrophils and LDGs IL-17. These intact neutrophils were predominantly observed in after MNAse treatment, indicating that NETosis is involved in the dermal blood vessels and in the dermal interstitium of lupus pDC activation. The capacity to stimulate IFN-a in pDCs did lesions (Fig. 7). Only a small proportion (1 6 0.8%) of NETs not significantly differ between LDGs and autologous lupus located in lupus skin were IL-17+. Although the temporal events neutrophils. These results confirm that both lupus neutrophils and surrounding neutrophil extravasation, migration in tissue, and LDGs have heightened capacity to induce IFN-a synthesis in NETosis are unclear, our observations are consistent with a model pDCs, at least in part, through a NET-mediated effect. in which intact neutrophils containing IL-17 circulate in blood vessels and then release IL-17 upon extravasation and migration Lupus netting neutrophils induce endothelial cytotoxicity into tissue, with all IL-17 released by the completion of extra- Patients with SLE display evidence of accelerated EC apoptosis cellular trap formation. The percentage of neutrophils expressing (36), which strongly correlates with the development of aberrant Table II. Clinical characteristics of lupus patients included in the study 546

Peripheral Blood

Patient SLEDAI No. Score dsDNA-Ab C3,C4 ENA ANA Clinical Manifestations Medications 1B 2 + Normal Negative 1:160 Skin, joints, CNS MMF, antimalarials 2B 10 + Normal Negative 1:320 Nephritis, arthritis None 3B 8 + Low Sm, RNP 1:2560 Cytopenias, nephritis PDN, antimalarials 4B 0 2 Normal Ro 1:2560 Neuropathy, Raynaud’s Antimalarials 5B 0 2 Normal Negative 1:320 Arthritis, skin, mucositis Antimalarials 6B 2 + Normal Sm, RNP 1:320 Skin, cytopenias, fever PDN, antimalarials 7B 20 + Low Sm, RNP 1:2560 Arthritis, CNS, cytopenias, skin PDN, antimalarials, MMF 8B 2 2 Low Sm, RNP 1:320 Nephritis, synovitis, serositis PDN, antimalarials, MMF 9B 4 + Normal Negative 1:160 Serositis, synovitis, mucositis, skin Antimalarials, MMF 10B 2 2 Normal Negative 1:160 Skin, nephritis, arthritis PDN, antimalarials, MMF 11B 4 + Normal Ro 1:640 Arthritis, cytopenias Antimalarials 12B 0 2 Normal Negative 1:320 Arthritis, eye Antimalarials 13B 10 + Low Ro, La 1:2560 Arthritis, nephritis, Raynaud’s MMF, PDN 14B 12 + Low Ro, Sm, RNP 1:2560 Arthritis, skin, serositis, kidney, CNS, MMF, MTX, PDN 15B 4 + Low Sm, RNP 1:320 Skin, arthritis, serositis, leukopenia Antimalarials 16B 6 2 Normal Sm, RNP, Ro 1:2560 Nephritis, cytopenias, arthritis PDN, antimalarials, AZA 17B 4 2 Normal Negative 1:2560 Serositis, arthritis, skin Antimalarials 18B 0 2 Normal Sm, RNP Negative Arthritis PDN, antimalarials 19B 10 + Low Ro 1:320 Nephritis, arthritis PDN, MMF, antimalarials 20B 6 2 Normal Negative 1:320 Arthritis, skin PDN, antimalarials

Skin

Patient No. Diagnosis dsDNA-Ab C3, C4 ENA ANA Other Clinical Manifestations Medications 1S DL + Low Negative 1:160 CNS, nephritis MMF, PDN

2S DL + Low RNP, Ro 1:2560 CNS, Raynaud’s PDN, antimalarials GRANULOCYTES LUPUS IN NETosis ABERRANT 3S Interface dermatitis + Low SmRNP 1:2560 Nephritis PDN, antimalarials 4S Lupus panniculitis + Low Negative 1:640 – None 5S Lupus panniculitis + Low SmRNP, RNP 1:2560 Arthritis PDN, antimalarials 6S SCLE 2 Normal Ro Negative Sicca Antimalarials 7S DL NA NA Negative Negative – None 8S DL NA NA NA Negative – None 9S DL NA Normal NA Negative – None 10S DL NA Normal Negative 1:640 Arthritis None 11S Interface dermatitis 2 NA Ro, La Negative – None

Kidney

Kidney Patient Diagnosis; Other Clinical No. Activity/Chronicity dsDNA-Ab C3, C4 ENA ANA Manifestations Medications at Time of Biopsy 1N Class IV/S; 12/8 + Low Sm, RNP 1:160 Serositis, arthritis, PDN, AZA 2N Class IV/S; 11/3 + Low Sm 1:320 Skin, serositis, arthritis, cytopenia PDN, antimalarials 3N Class IV/S; 13/2 NA Low NA 1:640 Cytopenia None 4N Class III/V; 13/2 + Low Ro, La, Sm, RNP 1:1280 Skin, joints, serositis PDN, MMF 5N Class IV; 17/11 + Normal Sm 1:2560 CNS PDN 6N Class III/V; 6/8 + Low Ro, Sm, RNP 1:1280 – PDN, AZA

(Table continues)

Downloaded from from Downloaded http://www.jimmunol.org/ by guest on September 25, 2021 25, September on guest by The Journal of Immunology 547 PDN None None Steroids Medications Antimalarials Antimalarials Antimalarials Antimalarials Antimalarials PDN, antimalarials PDN, antimalarials PDN, antimalarials PDN, AZA, antimalarials PDN, MMF, antimalarials

FIGURE 5. Netting neutrophils are present in glomeruli from patients with lupus nephritis. Colocalization of histone H2A (green), MPO (red), and DNA (white) by direct immunofluorescence reveals in vivo evidence of NET formation in a glomerulus from a representative kidney micro- Skin Skin Downloaded from

Arthritis photograph from a patient with class IV lupus nephritis. Yellow arrows and Skin, arthritis Arthritis, lung Skin, arthritis Arthritis, CNS inset boxes highlight intraglomerular NET formation. Original magnifi- Cytopenias, arthritis Arthritis, cytopenias Cytopenias, arthritis Arthritis, skin, fever Clinical Manifestations cation 320. Skin, arthritis, serositis Skin, arthritis, pleuritis Nephritis, skin, arthritis

vascular function and may predispose to the development of atherosclerosis. Our group had previously reported that lupus http://www.jimmunol.org/ LDGs, and less so lupus neutrophils, induce enhanced EC cyto- toxicity using a coculture system with HUVECs (1). However, the NA

ANA mechanisms implicated in this enhanced EC cytotoxicity have not 1:160 1:160 1:160 1:160 1:160 1:640 1:2560 1:2560 1:2560 1:2560 1:2560 1:2560 Negative been identified. Because EC activation may induce NET for- mation, which in turn promotes endothelial cytotoxicity (7), we tested if NET formation by lupus LDGs could enhance EC death. Incubation of HUVEC monolayer with lupus LDGs induces sig- nificantly elevated levels of EC cytotoxicity, relative to control and

lupus neutrophils (Fig. 8B). As previously reported, lupus neu- by guest on September 25, 2021 Ro NA ENA Negative Negative Negative Negative La, RNP Negative Negative Negative trophils also showed enhanced EC cytotoxicity when compared RNP, Sm Microarray Analysis Sm, RNP Sm, RNP Ro, Sm, RNP with healthy control neutrophils, but significantly less when com- pared with lupus LDGs (1). A component of this enhanced cy- totoxicity was mediated by NET formation because disrupting these structures by treating lupus LDGs and neutrophils with MNase (Fig. 8C) significantly downregulated EC apoptosis (Fig. Low Low Low Low Low Low Low Low Low Low

C3, C4 8B). These results indicate that lupus LDGs mediated more ex- Normal Normal Normal Normal tensive EC killing, at least in part, through their enhanced capacity to form NETs.

Discussion + + + + + + + + 2 2 2 2 2 NA Recent work from various groups indicates that NET formation

dsDNA-Ab may be an important phenomenon in autoantigen modification and exposure to the immune system, as well as in the induction of tissue damage (9, 10). As such, aberrant NET formation may play an important role in the development and perpetuation of autoimmune diseases and organ damage observed in chronic in- flammatory disorders. Our work now expands and reinforces this concept by reporting that a distinct subset of neutrophils found in 4 2 4 2 4 4 8 SLE patients (LDGs) have enhanced capacity to form NETs and 10 14 10

Score upregulate expression of various neutrophil proteins and enzymes SLEDAI Class III; 8/1 Class III; 6/2 Class IV; 17/4 implicated in NET formation and autoimmunity induction. These Henoch-Schonlein; NETs also expose dsDNA, an autoantigen considered key in lupus ) crescentic glomerulonephritis pathogenesis. Furthermore, lupus neutrophils and, in particular, LDGs elicit enhanced EC cytotoxicity through NET formation. This phenomenon also appears to play a role in the induction of

Continued IFN-a synthesis by pDCs. We have also identified that enhanced NETosis occurs in vivo in SLE in affected skin and kidney. Fur- No. Patient 9N 10N 8N 7N 1M 2M 3M 4M 5M 6M 8M 7M 10M 9M

The dashes in the+, dsDNA positive; Ab ANA, column anti-nuclear Ab; mean AZA, negative. azathioprine; The DL, dashes discoid in lupus; the ENA, clinical extractable manifestations nuclear column Ags; mean MMF, mycophenolate none. mofetil; MTX, methotrexate; NA, notthermore, available; PDN, prednisone; SCLE, subacute cutaneous lupus; SM, Smith; neutrophils from blood and skin from SLE patients Table II. ( SmRNP, Smith/RNP. frequently externalize IL-17 as part of the NETosis process, which 548 ABERRANT NETosis IN LUPUS GRANULOCYTES

FIGURE 6. Netting neutrophils are present in affected lupus dermis and subcutis. Direct im- munofluorescence staining of DNA (blue) and MPO (red) reveal NETs throughout affected lupus skin. A, Low-power view of a punch biopsy from lesional lupus skin. Scale bar, 200 mm. B, Arrows highlight perifollicular infiltration of NETs. Scale bar, 50 mm. C, NETs within the papillary dermis. Scale bar, 20 mm. Low-power view of lupus re- ticular dermis (D; scale bar, 200 mm) with arrows

highlighting infiltration by NETs (E; scale bar, 50 Downloaded from mm). Low-power view of a large blood vessel in affected lupus subcutis (F; scale bar, 200 mm), with arrows highlighting NETs in an area of panniculitis (G; scale bar, 20 mm). Dotted line delineates the dermal-epidermal junction (in A, C, D) and circumscribes the follicle (“f”) in B.

Epidermis is designated “e” and dermis by “d”. H, http://www.jimmunol.org/ Cathelicidin (LL-37) is present in NETs within inflamed lupus skin lesions. Expression of LL-37 in neutrophils in lupus tissue was examined by dual-color immunofluorescence staining for LL- 37 (green) and MPO (red) with DAPI counterstain (blue). Representative images from one of three sections stained with LL-37 and MPO (original magnification 3600). Scale bar, 100 mm. I, Fre- quency of NETosis in cutaneous lupus lesions. by guest on September 25, 2021 NETs and neutrophils were counted after immu- nofluorescence staining with MPO and DAPI. Percentage of neutrophils undergoing NETosis of all neutrophils was calculated for the epidermis, papillary dermis, reticular dermis, and subcutis. *p , 0.05.

may contribute to tissue damage and immune dysregulation. Over- environmental factors. As it is expected that LDGs and autologous all, these observations further support a pathogenic role for neu- lupus neutrophils were exposed to a very similar cytokine milieu, trophils in organ damage in SLE. these results indicate that LDGs may indeed represent a distinct One of the findings from our study is that no differences in gene subset of proinflammatory and pathogenic cells within the gran- expression were found when normal density lupus neutrophils were ulocyte spectrum. compared with gender-matched healthy control neutrophils. Thus, It is unclear why LDGs upregulate mRNA of various serine previous reports examining alterations in lupus neutrophils may proteases and bactericidal proteins present in azurophilic granules. in part reflect responses specifically elicited in the LDG pool. In One possibility is that these findings are indicative of a more contrast, lupus LDGs obtained from the same patients from whom immature phenotype of the LDGs, further supported by their the normal-density neutrophils were obtained showed significant immature nuclear morphology (1). Indeed, proteins synthesized at differences in gene expression when compared with healthy control the same time during neutrophil differentiation are colocalized in and lupus neutrophils. The pairwise comparison of gene expression the same granules. The levels of expression of the mRNA that in LDGs and autologous neutrophils in SLE patients provides encode the neutrophil serine proteases are greatest at the pro- a control for many potential sources of variability such as medi- myelocytic stage of neutrophil differentiation in the marrow and cations, disease activity, clinical manifestations, and exposure to are downregulated as neutrophils mature (37). This observation The Journal of Immunology 549

trophil elastase), released after encountering immune complexes, may potentiate a positive autocrine feedback on neutrophil acti- vation (38, 39). Further, these molecules have been implicated in the activation of the proforms of proinflammatory cytokines in- cluding TNF and IL-1b (40). Neutrophil elastase can activate TLR4, eventually resulting in IL-8 production (41). IL-8 levels are elevated in SLE, but the exact mechanisms leading to this increase have been unclear (42). One could propose that enhanced expo- sure to extracellular elastase through NET formation in SLE LDGs could promote enhanced synthesis of IL-8. This cytokine could in turn activate neutrophil recruitment and promote damage in various organs. In addition, we previously showed that LDGs synthesize enhanced levels of IL-8 and TNF upon activation when compared with control and normal-density lupus neutrophils (1). The mechanisms by which LDGs are more primed to make NETs are unclear. To date, the exact molecular mechanisms and sub- cellular events leading to NETosis remain elusive. Although a crucial role for elastase, reactive oxygen species, and the cyto-

skeleton has been proposed, recent reports suggest that NETosis is Downloaded from quite complex. The observed higher expression of elastase and MPO in LDGs could play an important role in enhancing extra- cellular trap synthesis, based on what other groups have recently reported (9). There is also evidence that type I and II IFNs can act as priming factors on mature neutrophils, allowing the formation

of NETs upon subsequent stimulation with complement factor 5a http://www.jimmunol.org/ (35). One possibility may be that LDGs are more sensitive to the effects of type I IFNs and/or to IFN signaling than normal-density lupus neutrophils. However, this would go against the hypothesis that LDGs represent a more immature subset of neutrophils, be- cause previous evidence indicates that granulocyte precursors and less mature cells are fairly insensitive to type I IFN effects when compared with fully differentiated neutrophils (35). Further, al- though one possibility is that LDGs represent cells that have been exposed to elevated levels of type I IFNs in the bone marrow, we by guest on September 25, 2021 do not consider this is likely the case because these cells do not FIGURE 7. IL-17–positive neutrophils infiltrate SLE skin. Direct im- display evidence of increased type I IFN gene signature. Future munofluorescence staining of IL-17 (green) and MPO (red) in affected studies need to explore whether the LDGs are derived from normal + lupus dermis. A, Arrows highlight intact IL-17 neutrophils in a blood neutrophils or result from alterations in granulocyte development. m vessel. Scale bar, 200 m. B, IL-17 present in a NET (arrow). Scale bar, 10 Previous studies have shown that the antimicrobial peptide m m. C, Frequency of IL-17 expression in intact neutrophils and NETs in LL-37 is a key factor that mediates pDC activation in psoriasis (23). SLE skin. Percentages of: 1) IL-17+ neutrophils of all IL-17+ cells (d); 2) LL-37 converts inert self-DNA into a potent trigger of type I IFN IL-17+ neutrophils of all intact neutrophils (n); and 3) IL-17+ NETs of all NETs (:) are reported in the graph. production. This occurs by binding the DNA to form condensed structures that are delivered to and retained within early endocytic compartments in pDCs to trigger TLR9 (23). LL-37 also converts could indicate that LDGs are indeed a more immature neutrophil self-RNA into a trigger of TLR7 and TLR8 in human DCs (22). In subset, despite their apparent expression of markers of fully ma- general, LL-37 is involved in a myriad of important immune tured neutrophils, including CD16 and CD10. Indeed, a previous functions, including chemoattraction of immune cells and release study has shown that, among the lupus bone marrow upregulated of inflammatory mediators from epithelial cells (43). Our findings genes (when compared with controls), the highest overexpression support that the enhanced release of LL-37 through NETosis could occurs in granulopoiesis-related genes. These genes include sev- promote enhanced inflammatory responses in SLE organs. Other eral of the early granulopoiesis genes upregulated in the LDG molecules overexpressed in lupus LDGs, including various de- microarray in our study, including MPO, ELA2, CTSG, DEFA4, fensins, may also be immunostimulatory (44). Our findings are and LTF (24). This is also confirmed by a previous study that in agreement with a recent study that reported that SLE patients showed that the PBMC granulocyte signatures observed in pedi- have elevated serum levels of various neutrophil peptides includ- atric SLE patients were for genes preferentially transcribed within ing MPO and defensins (45, 46). It is possible that these are de- the earliest granulocytes (myeloblast and promyelocytes) and with rived from LDGs. Further, increased anti-defensin and CTSG the presence of immature neutrophils in their peripheral blood (3). ANCA Abs are found in lupus patients (47). Given their immu- These observations further support that LDGs could represent an nomodulatory role, overproduction of alarmins might activate the aberrant immature subset originating from lupus bone marrow that adaptive immune and promote autoimmune responses, as is mani- may persist or expand in the blood and/or other tissues from SLE fested in SLE (48, 49). Another overexpressed molecule in LDGs, patients. neutrophil-gelatinase associated lipocalin/LCN2, has recently The functional consequences of high serine protease expression been proposed as a biomarker of and to have a pathogenic role in LDGs may be varied. It has been proposed that all serine in lupus nephritis (50). Finally, the role of MMP-8 overex- proteases of azurophilic granules (CTSG, proteinase 3, and neu- pression in LDGs remains to be determined, but this metallo- 550 ABERRANT NETosis IN LUPUS GRANULOCYTES

FIGURE 8. Netting lupus LDGs stimulate IFN-a synthesis by pDCs and kill ECs. A, Gen2.2 pDCs were stimulated for 16 h with supernatants from control neutrophils (n = 4), lupus neutrophils (n = 6), and autologous lupus LDGs (n = 6) that had been previously cultured in the presence or absence of MNase for 1 h. Results represent mean 6 SEM IFN-a mRNA expression in Gen2.2 cells. *p , 0.05 for control neutrophils compared with lupus neutrophils and with LDGs and for lupus and LDGs compared with lupus or LDGs+MNAse. B, Bar graph represents mean 6 SEM percentage of apoptotic HUVECs after exposure to activated LDGs, autologous neutrophils,

and control neutrophils (n = 4 to 5/group) in the Downloaded from presence or absence of MNase. *p , 0.05. C, Rep- resentative images of LDGs isolated from peripheral blood cultured for 2 h followed by either no MNase treatment (T2) or with MNase (T2+MNase) for 10 min at room temperature and processed for immu- nofluorescence staining of elastase (green) and DNA

(Hoechst 33342, blue). Original magnification 340. http://www.jimmunol.org/ Scale bar, 20 mm. by guest on September 25, 2021 proteinase may play a key role in vascular damage in other con- ous involvement. Although mechanisms of skin damage in SLE ditions (51). are likely multifactorial (54), recent evidence has identified an Lupus patients develop accelerated atherosclerosis, leading to enhanced type I interferogenic signature and IFN-a–producing premature cardiovascular disease. We had previously found that pDCs in lupus skin (55). The role of pDCs in the inflammation ECs from lupus patients undergo accelerated apoptosis in vivo and observed in autoimmune skin disease in animal models has re- that this phenomenon may be pathogenic, as it correlates with cently supported the notion that IFN-a may be crucial in cuta- endothelial dysfunction development (36). LDGs are capable of neous involvement in SLE (56). In a lupus murine model, tape killing ECs, but the mechanism involved was unclear (1). The stripping led to an influx into skin of neutrophils forming NETs, current study shows that, through enhanced NET formation, LDGs which contain DNA and RNA associated with LL-37 (56). Our acquire a heightened capability to damage the endothelium, as data confirm a similar pattern in human lupus skin biopsies, in cytotoxicity was abrogated with MNAse treatment. As such, ac- which abundant dsDNA+, LL-37+ NETs are seen in multiple celerated NETosis may represent an important mechanism of layers of affected skin. Interestingly, these findings are associated premature vascular damage in SLE. with the presence of elevated anti-dsDNA Abs in the circulation, Death by NETosis may also represent an important immunos- supporting the notion that autoantigen exposure in the NETs could timulatory event with regards to activation of innate immunity in promote autoantibody formation in vivo. SLE, given the enhanced capacity of netting lupus neutrophils to Similar findings were seen in the kidneys of lupus patients af- activate pDCs. This could promote chronic activation of the im- fected by class III and IV glomerulonephritis. This supports recent mune system and perpetuation of type I IFN synthesis characteristic findings that impaired NET degradation in SLE is associated with of this disease (52). Enhanced NETosis in SLE may be one of the renal involvement in this disease (2). Indeed, IgG deposition on mechanisms explaining why infections may trigger flares in this NETs in tubuli and glomeruli in the kidney of an SLE patient who disease (53), with NET induction by microorganisms that in turn degraded NETs poorly was reported in that study (2). We have leads to autoantigen externalization and immune system activa- now expanded this observation by studying a larger number of tion. The observation that both lupus neutrophils and LDGs in- patients with lupus nephritis, and the presence of netting neu- duced comparable induction of IFN-a synthesis by pDCs may trophils in glomeruli was observed in a majority of them. In- indicate that this process cannot be fully explained by pure en- terestingly, patients with a higher proportion of glomeruli in- hancement of NETosis in LDGs and that other variables are in- filtrated by netting neutrophils had higher levels of circulating volved in this phenomenon. Future studies are needed to better anti-dsDNA Abs and higher activity index in renal biopsies. It has understand how this process occurs. been previously proposed that anti-NET Abs and persistent NETs The skin is an important target organ of the immune system in could form NET immune complexes that could be relevant in SLE, and a significant proportion of lupus patients have cutane- lupus disease severity (2). It is possible that the presence of in- The Journal of Immunology 551

filtrating netting neutrophils in lupus tissue samples represented Note added in proof. While this manuscript was in the process of a combination of enhanced NETosis and impaired NET degrada- being accepted, two articles that also demonstrate the putative tion. We could then propose a model of profound imbalance in importance of NETs in lupus have been published (65, 66). NET production and degradation. On the one hand, NETosis would be enhanced in lupus LDGs. On the other hand, the activity Acknowledgments of DNase1 (an enzyme important in NET degradation) is decreased We thank Dr. Michelle Kahlenberg for critical review of the manuscript. in a subset of SLE patients (2). This may provide the conditions by which NETs may persist and constitute a prolonged source Disclosures of autoantigen exposure in an immunostimulatory context, leading D.S. is an employee of Centocor Research & Development and owns stock to enhanced formation of immune complexes and induction of in Johnson & Johnson. The other authors have no financial conflicts of autoantibodies that could further contribute to tissue damage. interest. Whether the infiltrating neutrophils in the skin and kidney cor- respond primarily to LDGs is unclear at this point, as no specific cell marker has to this date been identified to distinguish these cells References 1. Denny, M. F., S. Yalavarthi, W. Zhao, S. G. Thacker, M. Anderson, A. R. Sandy, from normal-density neutrophils. A better understanding of the W. J. McCune, and M. J. Kaplan. 2010. 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Interferon and granulopoiesis signatures in systemic lupus could also support that this cell subset is pathogenic to cutaneous erythematosus blood. J. Exp. Med. 197: 711–723. 4. Hacbarth, E., and A. Kajdacsy-Balla. 1986. Low density neutrophils in patients tissue (1). Future studies in a larger number of patients are re- with systemic lupus erythematosus, rheumatoid arthritis, and acute rheumatic quired to assess the specific role that these cells play in tissue fever. Arthritis Rheum. 29: 1334–1342. 5. Brinkmann, V., U. Reichard, C. Goosmann, B. Fauler, Y. Uhlemann, D. S. Weiss, damage and progression of disease in SLE. http://www.jimmunol.org/ Y. Weinrauch, and A. Zychlinsky. 2004. Neutrophil extracellular traps kill IL-17 has recently been linked to the pathogenesis of SLE (31) bacteria. Science 303: 1532–1535. by participation in the amplification of autoimmune responses by 6. Fuchs, T. A., U. Abed, C. Goosmann, R. Hurwitz, I. Schulze, V. 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Zychlinsky. 2010. by guest on September 25, 2021 been shown in other conditions (28) (Lin et al., submitted for Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil publication), neutrophils expressing IL-17 are seen at significantly extracellular traps. J. Cell Biol. 191: 677–691. enhanced levels in blood and affected skin from lupus patients. 10. Kessenbrock, K., M. Krumbholz, U. Schonermarck, W. Back, W. L. Gross, Z. Werb, H. J. Grone, V. Brinkmann, and D. E. Jenne. 2009. Netting neutrophils This phenomenon could initiate a cycle in which IL-17–secreting in autoimmune small-vessel vasculitis. Nat. Med. 191: 677–691. cells in lupus skin lesions (including innate and adaptive im- 11. Caielli, S., G. Garcia.-Romo, B. Vega, J. Connolly, F. Xu, Z. Allantaz, M. Punaro, J. Baisch, C. Guiducci, F. Barrat, et al. 2010. Netting neutrophils are mune cells) would recruit additional neutrophils. 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Entrez Gene Gene name Fold- q-value Gene ID symbol change

proteinkinase,cAMP- 5577 PRKAR2B dependent,regulatory,typeII,beta 64.90 0.000

guaninenucleotidebindingprotein(Gprotein),gamma 2791 GNG11 11 41.86 0.000

4317 MMP8 matrixmetallopeptidase8(neutrophilcollagenase) 38.53 0.000

83699 SH3BGRL2 SH3domainbindingglutamicacid-richproteinlike2 36.00 0.000

ELOVLfamilymember7,elongationoflongchainfattyaci 79993 ELOVL7 ds(yeast) 34.10 0.000

pro-plateletbasicprotein(chemokine(C-X- 5473 PPBP Cmotif)ligand7) 31.69 0.000

81027 TUBB1 tubulin,beta1 30.66 0.000

5196 PF4 plateletfactor4 29.74 0.000

guaninenucleotidebindingprotein(Gprotein),alphazp 2781 GNAZ olypeptide 26.48 0.000

3934 LCN2 lipocalin2 24.49 0.000

9886 RHOBTB1 Rho-relatedBTBdomaincontaining1 22.61 0.000

1669 DEFA4 defensin,alpha4,corticostatin 22.41 0.000

2162 F13A1 coagulationfactorXIII,A1polypeptide 21.62 0.000

1191 CLU clusterin 21.24 0.000

10562 OLFM4 olfactomedin4 20.89 0.000

139105 BEND2 BENdomaincontaining2 20.02 0.000

8436 SDPR 19.35 0.000 serumdeprivationresponse(phosphatidylserinebindi ngprotein)

10321 CRISP3 cysteine-richsecretoryprotein3 18.39 0.000

4638 MYLK myosinlightchainkinase 17.59 0.000

56911 C21orf7 chromosome21openreadingframe7 17.58 0.000

4057 LTF lactotransferrin 16.91 0.000

3693 ITGB5 integrin,beta5 16.80 0.000

carcinoembryonicantigen- 1088 CEACAM8 relatedcelladhesionmolecule8 16.63 0.000

membrane-spanning4- domains,subfamilyA,member3(hematopoieticcell- 932 MS4A3 specific) 15.49 0.000

8969 HIST1H2AG histonecluster1,H2ag 15.41 0.000

1950 EGF epidermalgrowthfactor(beta-urogastrone) 15.39 0.000

10826 C5orf4 chromosome5openreadingframe4 15.38 0.000

51266 CLEC1B C-typelectindomainfamily1,memberB 14.63 0.000

239 ALOX12 arachidonate12-lipoxygenase 14.34 0.000

6678 SPARC secretedprotein,acidic,cysteine-rich(osteonectin) 13.93 0.000

carcinoembryonicantigen- relatedcelladhesionmolecule6(non- 4680 CEACAM6 specificcrossreactingantigen) 13.92 0.000

671 BPI bactericidal/permeability-increasingprotein 13.80 0.000

1511 CTSG cathepsinG 13.53 0.000

2017 CTTN cortactin 13.33 0.000

4900 NRGN neurogranin(proteinkinaseCsubstrate,RC3) 13.00 0.000

ribonuclease,RNaseAfamily,3(eosinophilcationicprot 6037 RNASE3 ein) 12.34 0.000

4353 MPO myeloperoxidase 12.02 0.000 10158 PDZK1IP1 PDZK1interactingprotein1 11.78 0.000

1991 ELANE elastase,neutrophilexpressed 11.64 0.000

ArfGAPwithSH3domain,ankyrinrepeatandPHdomain 8853 ASAP2 2 11.59 0.000

375033 PEAR1 plateletendothelialaggregationreceptor1 11.46 0.000

7292 TNFSF4 tumornecrosisfactor(ligand)superfamily,member4 11.45 0.000

90952 ESAM endothelialcelladhesionmolecule 11.45 0.000

84886 C1orf198 chromosome1openreadingframe198 11.42 0.000

84281 C2orf88 chromosome2openreadingframe88 11.32 0.000

566 AZU1 azurocidin1 11.23 0.000

10398 MYL9 myosin,lightchain9,regulatory 11.20 0.000

7504 XK X-linkedKxbloodgroup(McLeodsyndrome) 10.97 0.000

5627 PROS1 proteinS(alpha) 10.57 0.000

400360 C15orf54 chromosome15openreadingframe54 10.42 0.000

340348 TSPAN33 tetraspanin33 10.28 0.000

94121 SYTL4 synaptotagmin-like4 9.74 0.000

116173 CMTM5 CKLF-likeMARVELtransmembranedomaincontaining5 9.74 0.000

2982 GUCY1A3 guanylatecyclase1,soluble,alpha3 9.63 0.000

25927 CNRIP1 cannabinoidreceptorinteractingprotein1 9.54 0.000

340205 TREML1 triggeringreceptorexpressedonmyeloidcells-like1 9.36 0.000

8848 TSC22D1 TSC22domainfamily,member1 9.22 0.000

disabledhomolog2,mitogen- 1601 DAB2 responsivephosphoprotein(Drosophila) 9.17 0.000

23531 MMD monocytetomacrophagedifferentiation-associated 8.97 0.000

934 CD24 CD24 molecule 8.93 0.000 integrin,alpha2b(plateletglycoproteinIIbofIIb/IIIacom 3674 ITGA2B plex,antigenCD41) 8.63 0.000

4211 MEIS1 Meishomeobox1 8.31 0.000

10634 GAS2L1 growtharrest-specific2like1 8.06 0.000

4052 LTBP1 latenttransforminggrowthfactorbetabindingprotein1 7.76 0.000

22885 ABLIM3 actinbindingLIMproteinfamily,member3 7.72 0.000

91624 NEXN nexilin(Factinbindingprotein) 7.63 0.000

728926 LOC728926 truncated alpha-1,3-galactosyltransferase 7.04 0.000

25893 TRIM58 tripartitemotif-containing58 6.95 0.000

10810 WASF3 WASproteinfamily,member3 6.83 0.000

5197 PF4V1 plateletfactor4variant1 6.72 0.000

30845 EHD3 EH-domaincontaining3 6.71 0.000

219670 ENKUR enkurin,TRPCchannelinteractingprotein 6.59 0.000

92597 MOBKL1A MOB1,MpsOneBinderkinaseactivator-like1A(yeast) 6.59 0.000

6915 TBXA2R thromboxaneA2receptor 6.52 0.000

84519 ACRBP acrosinbindingprotein 6.45 0.000

11167 FSTL1 follistatin-like1 6.42 0.000

4602 MYB v-mybmyeloblastosisviraloncogenehomolog(avian) 6.40 0.000

prostaglandin- endoperoxidesynthase1(prostaglandinG/Hsynthasea 5742 PTGS1 ndcyclooxygenase) 6.33 0.000

145567 TTC7B tetratricopeptiderepeatdomain7B 6.33 0.000

147199 SCGB1C1 secretoglobin,family1C,member1 6.08 0.000

9124 PDLIM1 PDZandLIMdomain1 5.97 0.000

257019 FRMD3 FERMdomaincontaining3 5.95 0.000

9717 SEC14L5 SEC14-like5(S.cerevisiae) 5.82 0.000 8339 HIST1H2BG histonecluster1,H2bg 5.73 0.000

150 ADRA2A adrenergic,alpha-2A-,receptor 5.70 0.002

81579 PLA2G12A phospholipaseA2,groupXIIA 5.68 0.000

11343 MGLL monoglyceridelipase 5.67 0.000

10857 PGRMC1 progesteronereceptormembranecomponent1 5.65 0.000

643008 LOC643008 hypotheticalproteinLOC643008 5.63 0.000

7180 CRISP2 cysteine-richsecretoryprotein2 5.59 0.000

27030 MLH3 mutLhomolog3(E.coli) 5.55 0.000

3690 ITGB3 integrin,beta3(plateletglycoproteinIIIa,antigenCD61) 5.54 0.000

nervegrowthfactorreceptor(TNFRSF16)associatedpro 27018 NGFRAP1 tein1 5.52 0.000

5747 PTK2 PTK2proteintyrosinekinase2 5.28 0.000

2811 GP1BA glycoproteinIb(platelet),alphapolypeptide 5.19 0.000

51206 GP6 glycoproteinVI(platelet) 4.99 0.000

6374 CXCL5 chemokine(C-X-Cmotif)ligand5 4.98 0.000

55076 TMEM45A transmembraneprotein45A 4.95 0.000

3667 IRS1 insulinreceptorsubstrate1 4.95 0.000

145781 GCOM1 GRINL1Acomplexlocus 4.93 0.000

v- etserythroblastosisvirusE26oncogenehomolog(avian 2078 ERG ) 4.86 0.000

154664 ABCA13 ATP-bindingcassette,sub-familyA(ABC1),member13 4.85 0.000

64805 P2RY12 purinergicreceptorP2Y,G-proteincoupled,12 4.85 0.000

727722 LOC727722 homogentisate 1,2-dioxygenase like 4.76 0.000

5154 PDGFA platelet-derivedgrowthfactoralphapolypeptide 4.65 0.000

55859 BEX1 brainexpressed,X-linked1 4.64 0.000 3012 HIST1H2AE histonecluster1,H2ae 4.62 0.000

54874 FNBP1L forminbindingprotein1-like 4.60 0.000

57121 LPAR5 lysophosphatidicacidreceptor5 4.56 0.000

8555 CDC14B CDC14celldivisioncycle14homologB(S.cerevisiae) 4.52 0.000

26577 PCOLCE2 procollagenC-endopeptidaseenhancer2 4.51 0.003

55536 CDCA7L celldivisioncycleassociated7-like 4.44 0.000

64759 TNS3 tensin3 4.37 0.000

transcobalaminI(vitaminB12bindingprotein,Rbinderf 6947 TCN1 amily) 4.37 0.004

7041 TGFB1I1 transforminggrowthfactorbeta1inducedtranscript1 4.33 0.002

56952 PRTFDC1 phosphoribosyltransferasedomaincontaining1 4.25 0.000

polycystickidneyandhepaticdisease1(autosomalreces 93035 PKHD1L1 sive)-like1 4.25 0.000

ribonuclease,RNaseAfamily,2(liver,eosinophil- 6036 RNASE2 derivedneurotoxin) 4.24 0.004

440712 C1orf186 chromosome1openreadingframe186 4.21 0.000

1359 CPA3 carboxypeptidaseA3(mastcell) 4.20 0.008

3005 H1F0 H1histonefamily,member0 4.14 0.000

196527 ANO6 anoctamin6 4.07 0.000

10638 SPHAR S-phaseresponse(cyclinrelated) 4.06 0.000

9402 GRAP2 GRB2-relatedadaptorprotein2 4.04 0.000

23584 VSIG2 V-setandimmunoglobulindomaincontaining2 4.00 0.000

340146 SLC35D3 solutecarrierfamily35,memberD3 3.96 0.000

23052 ENDOD1 endonucleasedomaincontaining1 3.94 0.000

25907 TMEM158 transmembraneprotein158 3.87 0.000

56729 RETN resistin 3.82 0.000 ATP-bindingcassette,sub- 8714 ABCC3 familyC(CFTR/MRP),member3 3.81 0.000

79895 ATP8B4 ATPase,classI,type8B,member4 3.80 0.000

63933 CCDC90A coiled-coildomaincontaining90A 3.78 0.007

153643 FAM81B familywithsequencesimilarity81,memberB 3.76 0.004

7168 TPM1 tropomyosin1(alpha) 3.75 0.000

6241 RRM2 ribonucleotidereductaseM2 3.71 0.000

84632 AFAP1L2 actinfilamentassociatedprotein1-like2 3.70 0.000

254427 C10orf47 chromosome10openreadingframe47 3.67 0.000

FcfragmentofIgE,highaffinityI,receptorfor;alphapolyp 2205 FCER1A eptide 3.63 0.008

N-acetyltransferase8B(GCN5- 51471 NAT8B related,putative,gene/pseudogene) 3.58 0.000

solutecarrierfamily24(sodium/potassium/calciumexc 57419 SLC24A3 hanger),member3 3.56 0.000

23205 ACSBG1 acyl-CoAsynthetasebubblegumfamilymember1 3.56 0.008

351 APP amyloidbeta(A4)precursorprotein 3.54 0.000

29780 PARVB parvin,beta 3.54 0.006

55287 TMEM40 transmembraneprotein40 3.53 0.000

2766 GMPR guanosinemonophosphatereductase 3.53 0.000

395 ARHGAP6 RhoGTPaseactivatingprotein6 3.49 0.000

10787 NCKAP1 NCK-associatedprotein1 3.46 0.003

4286 MITF microphthalmia-associatedtranscriptionfactor 3.44 0.008

51316 PLAC8 placenta-specific8 3.44 0.000

2876 GPX1 glutathioneperoxidase1 3.44 0.000

820 CAMP cathelicidinantimicrobialpeptide 3.44 0.003 9240 PNMA1 paraneoplasticantigenMA1 3.34 0.000

10079 ATP9A ATPase,classII,type9A 3.31 0.004

serpinpeptidaseinhibitor,cladeB(ovalbumin),membe 5273 SERPINB10 r10 3.30 0.000

6001 RGS10 regulatorofG-proteinsignaling10 3.27 0.003

1118 CHIT1 chitinase1(chitotriosidase) 3.25 0.004

5087 PBX1 pre-B-cellleukemiahomeobox1 3.22 0.000

5352 PLOD2 procollagen-lysine,2-oxoglutarate5-dioxygenase2 3.21 0.000

23601 CLEC5A C-typelectindomainfamily5,memberA 3.16 0.006

selectinP(granulemembraneprotein140kDa,antigenC 6403 SELP D62) 3.15 0.000

solutecarrierfamily2(facilitatedglucose/fructosetrans 6518 SLC2A5 porter),member5 3.15 0.000

56034 PDGFC plateletderivedgrowthfactorC 3.11 0.000

9768 KIAA0101 KIAA0101 3.11 0.008

55363 HEMGN hemogen 3.10 0.000

8328 GFI1B growthfactorindependent1Btranscriptionrepressor 3.09 0.000

124790 HEXIM2 hexamthylenebis-acetamideinducible2 3.09 0.000

928 CD9 CD9molecule 3.05 0.000

3832 KIF11 kinesinfamilymember11 3.04 0.004

654 BMP6 bonemorphogeneticprotein6 3.01 0.003

2039 EPB49 erythrocytemembraneproteinband4.9(dematin) 2.98 0.000

synuclein,alpha(nonA4componentofamyloidprecurs 6622 SNCA or) 2.98 0.003

10184 LHFPL2 lipomaHMGICfusionpartner-like2 2.97 0.000

26052 DNM3 dynamin3 2.96 0.000 CTD(carboxy- terminaldomain,RNApolymeraseII,polypeptideA)sma 10217 CTDSPL llphosphatase-like 2.95 0.000

51109 RDH11 retinoldehydrogenase11(all-trans/9-cis/11-cis) 2.92 0.000

23002 DAAM1 dishevelledassociatedactivatorofmorphogenesis1 2.89 0.006

4086 SMAD1 SMADfamilymember1 2.88 0.000

2040 STOM stomatin 2.88 0.003

10282 BET1 blockedearlyintransport1homolog(S.cerevisiae) 2.83 0.007

8655 DYNLL1 dynein,lightchain,LC8-type1 2.82 0.000

23365 ARHGEF12 Rhoguaninenucleotideexchangefactor(GEF)12 2.81 0.003

79628 SH3TC2 SH3domainandtetratricopeptiderepeats2 2.77 0.007

7122 CLDN5 claudin5 2.77 0.000

57699 CPNE5 copineV 2.77 0.003

93663 ARHGAP18 RhoGTPaseactivatingprotein18 2.75 0.000

2983 GUCY1B3 guanylatecyclase1,soluble,beta3 2.74 0.000

solutecarrierfamily22(organiccation/carnitinetransp 85413 SLC22A16 orter),member16 2.72 0.003

55211 DPPA4 developmentalpluripotencyassociated4 2.72 0.008

858 CAV2 caveolin2 2.67 0.000

7429 VIL1 villin1 2.67 0.000

284422 LOC284422 similartoHSPC323 2.64 0.003

200230 LOC200230 similar to KIAA0386 2.64 0.003

60485 SAV1 salvadorhomolog1(Drosophila) 2.63 0.003

55353 LAPTM4B lysosomalproteintransmembrane4beta 2.62 0.000

10643 IGF2BP3 insulin-likegrowthfactor2mRNAbindingprotein3 2.61 0.006

11142 PKIG 2.55 0.007 proteinkinase(cAMP- dependent,catalytic)inhibitorgamma

147947 ZNF542 zincfingerprotein542 2.53 0.007

7357 UGCG UDP-glucoseceramideglucosyltransferase 2.52 0.000

8936 WASF1 WASproteinfamily,member1 2.49 0.004

platelet- 5156 PDGFRA derivedgrowthfactorreceptor,alphapolypeptide 2.49 0.006

81691 LOC81691 exonucleaseNEF-sp 2.49 0.004

259215 LY6G6F lymphocyteantigen6complex,locusG6F 2.46 0.000

5874 RAB27B RAB27B,memberRASoncogenefamily 2.45 0.003

90355 C5orf30 chromosome5openreadingframe30 2.45 0.000

ectonucleotidepyrophosphatase/phosphodiesterase 5168 ENPP2 2 2.43 0.007

57584 ARHGAP21 RhoGTPaseactivatingprotein21 2.42 0.003

2624 GATA2 GATAbindingprotein2 2.42 0.000

7485 WRB tryptophanrichbasicprotein 2.41 0.002

7057 THBS1 thrombospondin1 2.40 0.000

9848 MFAP3L microfibrillar-associatedprotein3-like 2.39 0.003

117178 SSX2IP synovialsarcoma,Xbreakpoint2interactingprotein 2.38 0.000

1536 CYBB cytochromeb-245,betapolypeptide 2.38 0.000

4860 NP nucleosidephosphorylase 2.36 0.008

2274 FHL2 fourandahalfLIMdomains2 2.36 0.003

2281 FKBP1B FK506bindingprotein1B,12.6kDa 2.33 0.003

5657 PRTN3 proteinase3 2.33 0.006

51203 NUSAP1 nucleolarandspindleassociatedprotein1 2.33 0.000

5028 P2RY1 purinergicreceptorP2Y,G-proteincoupled,1 2.30 0.006 222166 C7orf41 chromosome7openreadingframe41 2.30 0.000

55281 TMEM140 transmembraneprotein140 2.29 0.003

388115 C15orf52 chromosome15openreadingframe52 2.23 0.000

megalencephalicleukoencephalopathywithsubcortic 23209 MLC1 alcysts1 2.23 0.008

647115 FLJ36848 hypotheticalLOC647115 2.22 0.000

9315 C5orf13 chromosome5openreadingframe13 2.21 0.000

8987 STBD1 starchbindingdomain1 2.20 0.000

120939 C12orf59 chromosome12openreadingframe59 2.19 0.006

7298 TYMS thymidylatesynthetase 2.17 0.003

27102 EIF2AK1 eukaryotictranslationinitiationfactor2-alphakinase1 2.16 0.008

1182 CLCN3 chloridechannel3 2.15 0.000

solutecarrierfamily27(fattyacidtransporter),member 11001 SLC27A2 2 2.14 0.003

3903 LAIR1 leukocyte-associatedimmunoglobulin-likereceptor1 2.10 0.000

NADHdehydrogenase(ubiquinone)1alphasubcomple 25915 NDUFAF3 x,assemblyfactor3 2.10 0.003

253650 ANKRD18A ankyrinrepeatdomain18A 2.04 0.006

buddinguninhibitedbybenzimidazoles1homologbeta( 701 BUB1B yeast) 2.04 0.008

64420 SUSD1 sushidomaincontaining1 2.03 0.004

29841 GRHL1 grainyhead-like1(Drosophila) 2.01 0.007

64407 RGS18 regulatorofG-proteinsignaling18 1.99 0.000

7414 VCL vinculin 1.99 0.000

25932 CLIC4 chlorideintracellularchannel4 1.99 0.004

2184 FAH 1.96 0.000 fumarylacetoacetatehydrolase(fumarylacetoacetase )

80230 RUFY1 RUNandFYVEdomaincontaining1 1.96 0.007

1164 CKS2 CDC28proteinkinaseregulatorysubunit2 1.96 0.004

tyrosine3-monooxygenase/tryptophan5- 7533 YWHAH monooxygenaseactivationprotein,etapolypeptide 1.95 0.006

8349 HIST2H2BE histonecluster2,H2be 1.95 0.002

1053 CEBPE CCAAT/enhancerbindingprotein(C/EBP),epsilon 1.95 0.003

387103 C6orf173 chromosome6openreadingframe173 1.89 0.003

3624 INHBA inhibin,betaA 1.88 0.003

93129 ORAI3 ORAIcalciumrelease-activatedcalciummodulator3 1.88 0.007

85027 MST150 MSTP150 1.88 0.000

3987 LIMS1 LIMandsenescentcellantigen-likedomains1 1.87 0.003

51566 ARMCX3 armadillorepeatcontaining,X-linked3 1.86 0.004

339541 C1orf228 chromosome1openreadingframe228 1.86 0.004

55852 TEX2 testisexpressed2 1.85 0.000

vesicleaminetransportprotein1homolog(T.californica 10493 VAT1 ) 1.85 0.004

163259 DENND2C DENN/MADDdomaincontaining2C 1.85 0.000

367 AR androgenreceptor 1.83 0.000

4130 MAP1A microtubule-associatedprotein1A 1.83 0.000

54443 ANLN anillin,actinbindingprotein 1.83 0.004

825 CAPN3 calpain3,(p94) 1.82 0.000

8645 KCNK5 potassiumchannel,subfamilyK,member5 1.82 0.000

55840 EAF2 ELLassociatedfactor2 1.81 0.000

8345 HIST1H2BH histonecluster1,H2bh 1.80 0.004 255631 COL24A1 collagen,typeXXIV,alpha1 1.80 0.008

9468 PCYT1B phosphatecytidylyltransferase1,choline,beta 1.79 0.002

25934 NIPSNAP3A nipsnaphomolog3A(C.elegans) 1.78 0.000

54498 SMOX spermineoxidase 1.78 0.000

80177 MYCT1 myctarget1 1.75 0.003

977 CD151 CD151molecule(Raphbloodgroup) 1.74 0.003

6335 SCN9A sodiumchannel,voltage-gated,typeIX,alphasubunit 1.74 0.002

9446 GSTO1 glutathioneS-transferaseomega1 1.74 0.000

332 BIRC5 baculoviralIAPrepeat-containing5 1.73 0.006

136 ADORA2B adenosineA2breceptor 1.72 0.006

116028 C16orf75 chromosome16openreadingframe75 1.71 0.007

400073 C12orf76 chromosome12openreadingframe76 1.71 0.004

2273 FHL1 fourandahalfLIMdomains1 1.71 0.009

5476 CTSA cathepsinA 1.69 0.004

79641 ROGDI rogdihomolog(Drosophila) 1.68 0.000

51701 NLK nemo-likekinase 1.67 0.008

23446 SLC44A1 solutecarrierfamily44,member1 1.66 0.007

3295 HSD17B4 hydroxysteroid(17-beta)dehydrogenase4 1.62 0.004

6120 RPE ribulose-5-phosphate-3-epimerase 1.62 0.003

81888 HYI hydroxypyruvateisomerasehomolog(E.coli) 1.62 0.000

25840 METTL7A methyltransferaselike7A 1.61 0.004

2997 GYS1 glycogensynthase1(muscle) 1.60 0.003

v- mafmusculoaponeuroticfibrosarcomaoncogenehom 4097 MAFG ologG(avian) 1.59 0.007 eukaryotictranslationinitiationfactor2B,subunit2beta 8892 EIF2B2 ,39kDa 1.59 0.004

199221 DZIP1L DAZinteractingprotein1-like 1.59 0.006

9425 CDYL chromodomainprotein,Y-like 1.59 0.003

170591 S100Z S100calciumbindingproteinZ 1.58 0.000

253827 MSRB3 methioninesulfoxidereductaseB3 1.56 0.000

9811 KIAA0427 KIAA0427 1.56 0.000

85411 C6orf114 chromosome6openreadingframe114 1.55 0.003

4000 LMNA laminA/C 1.55 0.006

8372 HYAL3 hyaluronoglucosaminidase3 1.55 0.000

896 CCND3 cyclinD3 1.53 0.004

23286 WWC1 WWandC2domaincontaining1 1.52 0.003

3875 KRT18 keratin18 1.52 0.008

8343 HIST1H2BF histonecluster1,H2bf 1.52 0.008

3820 KLRB1 killercelllectin-likereceptorsubfamilyB,member1 0.12 0.003

3003 GZMK granzymeK(granzyme3;tryptaseII) 0.10 0.004

3040 HBA2 hemoglobin,alpha2 0.03 0.004

3043 HBB hemoglobin,beta 0.03 0.004

Supplementary Table II: List and regulation of the 281 genes significantly differentially regulated in lupus LDGs compared to lupus neutrophils (q-value<0.01 fold-change ≥ 1.5 and ≤ 0.7 for the up-regulated and down-regulated genes respectively).

Entrez Gene Gene name Fold- q- Gene ID symbol change value

4317 MMP8 matrixmetallopeptidase8(neutrophilcollagenase) 41.53 0.000

ELOVLfamilymember7,elongationoflongchainfattyacids(yea 79993 ELOVL7 st) 31.87 0.000

PRKAR2 5577 B proteinkinase,cAMP-dependent,regulatory,typeII,beta 31.44 0.000

10562 OLFM4 olfactomedin4 29.41 0.000

1669 DEFA4 defensin,alpha4,corticostatin 25.57 0.000

SH3BGR 83699 L2 SH3domainbindingglutamicacid-richproteinlike2 23.51 0.000

RHOBTB 9886 1 Rho-relatedBTBdomaincontaining1 23.12 0.000

2791 GNG11 guaninenucleotidebindingprotein(Gprotein),gamma11 22.78 0.000

4638 MYLK myosinlightchainkinase 20.18 0.000

5473 PPBP pro-plateletbasicprotein(chemokine(C-X-Cmotif)ligand7) 20.04 0.000

guaninenucleotidebindingprotein(Gprotein),alphazpolypept 2781 GNAZ ide 19.94 0.000

81027 TUBB1 tubulin,beta1 19.46 0.000

3934 LCN2 lipocalin2 19.02 0.000

CEACA 1088 M8 carcinoembryonicantigen-relatedcelladhesionmolecule8 18.27 0.000

139105 BEND2 BENdomaincontaining2 17.90 0.000

932 MS4A3 17.85 0.000 membrane-spanning4-

1

domains,subfamilyA,member3(hematopoieticcell-specific)

1511 CTSG cathepsinG 17.73 0.000

carcinoembryonicantigen- CEACA relatedcelladhesionmolecule6(non- 4680 M6 specificcrossreactingantigen) 17.40 0.000

1950 EGF epidermalgrowthfactor(beta-urogastrone) 17.40 0.000

1191 CLU clusterin 17.39 0.000

3693 ITGB5 integrin,beta5 17.29 0.000

671 BPI bactericidal/permeability-increasingprotein 16.59 0.000

2162 F13A1 coagulationfactorXIII,A1polypeptide 15.16 0.000

5196 PF4 plateletfactor4 14.76 0.000

4353 MPO myeloperoxidase 14.66 0.000

10826 C5orf4 chromosome5openreadingframe4 14.62 0.000

serumdeprivationresponse(phosphatidylserinebindingprote 8436 SDPR in) 13.74 0.000

5627 PROS1 proteinS(alpha) 13.43 0.000

239 ALOX12 arachidonate12-lipoxygenase 13.25 0.000

2017 CTTN cortactin 12.68 0.000

HIST1H2 8969 AG histonecluster1,H2ag 12.58 0.000

566 AZU1 azurocidin1 12.22 0.000

10321 CRISP3 cysteine-richsecretoryprotein3 12.18 0.000

6037 RNASE3 ribonuclease,RNaseAfamily,3(eosinophilcationicprotein) 12.07 0.000

4057 LTF lactotransferrin 12.01 0.000

8853 ASAP2 ArfGAPwithSH3domain,ankyrinrepeatandPHdomain2 11.42 0.000

375033 PEAR1 plateletendothelialaggregationreceptor1 11.26 0.000

2

6678 SPARC secretedprotein,acidic,cysteine-rich(osteonectin) 11.23 0.000

C15orf5 400360 4 chromosome15openreadingframe54 11.11 0.000

1991 ELANE elastase,neutrophilexpressed 10.92 0.000

TSPAN3 340348 3 tetraspanin33 10.91 0.000

90952 ESAM endothelialcelladhesionmolecule 10.88 0.000

116173 CMTM5 CKLF-likeMARVELtransmembranedomaincontaining5 10.73 0.000

10398 MYL9 myosin,lightchain9,regulatory 10.31 0.000

84281 C2orf88 chromosome2openreadingframe88 10.27 0.000

51266 CLEC1B C-typelectindomainfamily1,memberB 10.22 0.000

PDZK1IP 10158 1 PDZK1interactingprotein1 10.10 0.003

4900 NRGN neurogranin(proteinkinaseCsubstrate,RC3) 10.00 0.000

7292 TNFSF4 tumornecrosisfactor(ligand)superfamily,member4 9.88 0.000

94121 SYTL4 synaptotagmin-like4 9.81 0.000

56911 C21orf7 chromosome21openreadingframe7 9.51 0.000

GUCY1A 2982 3 guanylatecyclase1,soluble,alpha3 9.46 0.000

disabledhomolog2,mitogen- 1601 DAB2 responsivephosphoprotein(Drosophila) 9.32 0.000

25927 CNRIP1 cannabinoidreceptorinteractingprotein1 9.16 0.004

C1orf19 84886 8 chromosome1openreadingframe198 9.13 0.000

integrin,alpha2b(plateletglycoproteinIIbofIIb/IIIacomplex,a 3674 ITGA2B ntigenCD41) 9.00 0.000

340205 TREML1 triggeringreceptorexpressedonmyeloidcells-like1 8.89 0.000

3

LOC728 728926 926 truncated alpha-1,3-galactosyltransferase 8.57 0.000

7504 XK X-linkedKxbloodgroup(McLeodsyndrome) 8.56 0.000

4052 LTBP1 latenttransforminggrowthfactorbetabindingprotein1 8.52 0.000

934 CD24 CD24 molecule 8.43 0.000

10810 WASF3 WASproteinfamily,member3 7.95 0.000

TSC22D 8848 1 TSC22domainfamily,member1 7.69 0.000

25893 TRIM58 tripartitemotif-containing58 7.42 0.000

4211 MEIS1 Meishomeobox1 7.26 0.000

5197 PF4V1 plateletfactor4variant1 7.19 0.000

22885 ABLIM3 actinbindingLIMproteinfamily,member3 7.11 0.000

23531 MMD monocytetomacrophagedifferentiation-associated 7.06 0.000

11167 FSTL1 follistatin-like1 6.83 0.000

91624 NEXN nexilin(Factinbindingprotein) 6.79 0.000

6915 TBXA2R thromboxaneA2receptor 6.56 0.000

4602 MYB v-mybmyeloblastosisviraloncogenehomolog(avian) 6.51 0.000

10634 GAS2L1 growtharrest-specific2like1 6.44 0.000

30845 EHD3 EH-domaincontaining3 6.38 0.000

84519 ACRBP acrosinbindingprotein 6.36 0.000

9124 PDLIM1 PDZandLIMdomain1 6.25 0.000

219670 ENKUR enkurin,TRPCchannelinteractingprotein 6.24 0.008

150 ADRA2A adrenergic,alpha-2A-,receptor 6.23 0.009

9717 SEC14L5 SEC14-like5(S.cerevisiae) 6.11 0.000

147199 secretoglobin,family1C,member1 6.10 0.004 SCGB1C 4

1

7180 CRISP2 cysteine-richsecretoryprotein2 6.04 0.004

3690 ITGB3 integrin,beta3(plateletglycoproteinIIIa,antigenCD61) 6.03 0.000

51206 GP6 glycoproteinVI(platelet) 6.00 0.000

TMEM4 55076 5A transmembraneprotein45A 5.99 0.000

3240 HP haptoglobin 5.80 0.007

prostaglandin- endoperoxidesynthase1(prostaglandinG/Hsynthaseandcycl 5742 PTGS1 ooxygenase) 5.68 0.000

145781 GCOM1 GRINL1Acomplexlocus 5.57 0.000

2811 GP1BA glycoproteinIb(platelet),alphapolypeptide 5.50 0.000

27030 MLH3 mutLhomolog3(E.coli) 5.39 0.000

PLA2G1 81579 2A phospholipaseA2,groupXIIA 5.38 0.003

1359 CPA3 carboxypeptidaseA3(mastcell) 5.36 0.005

8555 CDC14B CDC14celldivisioncycle14homologB(S.cerevisiae) 5.35 0.000

64805 P2RY12 purinergicreceptorP2Y,G-proteincoupled,12 5.32 0.000

2078 ERG v-etserythroblastosisvirusE26oncogenehomolog(avian) 5.02 0.000

PGRMC 10857 1 progesteronereceptormembranecomponent1 5.00 0.000

SLC35D 340146 3 solutecarrierfamily35,memberD3 4.93 0.000

6374 CXCL5 chemokine(C-X-Cmotif)ligand5 4.93 0.004

11343 MGLL monoglyceridelipase 4.90 0.003

57121 LPAR5 lysophosphatidicacidreceptor5 4.88 0.006

5

145567 TTC7B tetratricopeptiderepeatdomain7B 4.83 0.000

PRTFDC 56952 1 phosphoribosyltransferasedomaincontaining1 4.74 0.000

10079 ATP9A ATPase,classII,type9A 4.68 0.000

PCOLCE 26577 2 procollagenC-endopeptidaseenhancer2 4.65 0.009

54874 FNBP1L forminbindingprotein1-like 4.63 0.000

3667 IRS1 insulinreceptorsubstrate1 4.62 0.000

820 CAMP cathelicidinantimicrobialpeptide 4.62 0.000

9402 GRAP2 GRB2-relatedadaptorprotein2 4.59 0.003

154664 ABCA13 ATP-bindingcassette,sub-familyA(ABC1),member13 4.54 0.000

LOC727 727722 722 homogentisate 1,2-dioxygenase like 4.54 0.004

5747 PTK2 PTK2proteintyrosinekinase2 4.52 0.000

LOC643 643008 008 hypotheticalproteinLOC643008 4.52 0.000

10666 CD226 CD226molecule 4.51 0.008

5154 PDGFA platelet-derivedgrowthfactoralphapolypeptide 4.51 0.000

MOBKL1 92597 A MOB1,MpsOneBinderkinaseactivator-like1A(yeast) 4.43 0.004

6947 TCN1 transcobalaminI(vitaminB12bindingprotein,Rbinderfamily) 4.41 0.000

ENDOD 23052 1 endonucleasedomaincontaining1 4.39 0.000

257019 FRMD3 FERMdomaincontaining3 4.37 0.000

23584 VSIG2 V-setandimmunoglobulindomaincontaining2 4.33 0.000

5352 PLOD2 procollagen-lysine,2-oxoglutarate5-dioxygenase2 4.30 0.000

6

2205 FCER1A FcfragmentofIgE,highaffinityI,receptorfor;alphapolypeptide 4.26 0.008

NGFRAP 27018 1 nervegrowthfactorreceptor(TNFRSF16)associatedprotein1 4.24 0.004

1118 CHIT1 chitinase1(chitotriosidase) 4.20 0.000

C1orf18 440712 6 chromosome1openreadingframe186 4.20 0.006

PKHD1L polycystickidneyandhepaticdisease1(autosomalrecessive)- 93035 1 like1 4.19 0.004

TMEM4 55287 0 transmembraneprotein40 4.16 0.000

AFAP1L 84632 2 actinfilamentassociatedprotein1-like2 4.15 0.000

TMEM1 200728 7 transmembraneprotein17 4.15 0.004

55859 BEX1 brainexpressed,X-linked1 4.06 0.000

56729 RETN resistin 4.03 0.003

C10orf4 254427 7 chromosome10openreadingframe47 4.00 0.007

7168 TPM1 tropomyosin1(alpha) 3.98 0.000

CCDC90 63933 A coiled-coildomaincontaining90A 3.96 0.009

6241 RRM2 ribonucleotidereductaseM2 3.95 0.004

KIAA010 9768 1 KIAA0101 3.87 0.005

10643 IGF2BP3 insulin-likegrowthfactor2mRNAbindingprotein3 3.83 0.000

4973 OLR1 oxidizedlowdensitylipoprotein(lectin-like)receptor1 3.76 0.003

79895 ATP8B4 ATPase,classI,type8B,member4 3.75 0.000

5087 PBX1 pre-B-cellleukemiahomeobox1 3.74 0.004

7

HIST1H2 3012 AE histonecluster1,H2ae 3.74 0.000

ARHGAP 395 6 RhoGTPaseactivatingprotein6 3.68 0.000

8714 ABCC3 ATP-bindingcassette,sub-familyC(CFTR/MRP),member3 3.66 0.003

56344 CABP5 calciumbindingprotein5 3.62 0.008

196527 ANO6 anoctamin6 3.61 0.003

N-acetyltransferase8B(GCN5- 51471 NAT8B related,putative,gene/pseudogene) 3.58 0.003

SERPINB 5273 10 serpinpeptidaseinhibitor,cladeB(ovalbumin),member10 3.58 0.005

HIST1H2 8339 BG histonecluster1,H2bg 3.42 0.003

55363 HEMGN hemogen 3.39 0.000

ARHGAP 93663 18 RhoGTPaseactivatingprotein18 3.39 0.000

26052 DNM3 dynamin3 3.39 0.000

6622 SNCA synuclein,alpha(nonA4componentofamyloidprecursor) 3.37 0.003

2766 GMPR guanosinemonophosphatereductase 3.33 0.000

25925 ZNF521 zincfingerprotein521 3.32 0.000

55536 CDCA7L celldivisioncycleassociated7-like 3.22 0.000

solutecarrierfamily24(sodium/potassium/calciumexchanger 57419 SLC24A3 ),member3 3.20 0.003

7122 CLDN5 claudin5 3.20 0.000

124790 HEXIM2 hexamthylenebis-acetamideinducible2 3.19 0.004

3832 KIF11 kinesinfamilymember11 3.16 0.008

55211 DPPA4 developmentalpluripotencyassociated4 3.13 0.003

8

10638 SPHAR S-phaseresponse(cyclinrelated) 3.13 0.007

8842 PROM1 prominin1 3.12 0.000

8328 GFI1B growthfactorindependent1Btranscriptionrepressor 3.09 0.000

351 APP amyloidbeta(A4)precursorprotein 3.09 0.000

2039 EPB49 erythrocytemembraneproteinband4.9(dematin) 3.07 0.004

3005 H1F0 H1histonefamily,member0 3.06 0.000

PLEKHA pleckstrinhomologydomaincontaining,familyA(phosphoinos 51054 9 itidebindingspecific)member9 3.04 0.007

TCTEX1 200132 D1 Tctex1domaincontaining1 3.00 0.009

2040 STOM stomatin 2.99 0.003

10282 BET1 blockedearlyintransport1homolog(S.cerevisiae) 2.99 0.004

solutecarrierfamily2(facilitatedglucose/fructosetransporter) 6518 SLC2A5 ,member5 2.98 0.003

ARHGAP 57584 21 RhoGTPaseactivatingprotein21 2.97 0.000

56034 PDGFC plateletderivedgrowthfactorC 2.96 0.000

GUCY1B 2983 3 guanylatecyclase1,soluble,beta3 2.95 0.000

57699 CPNE5 copineV 2.95 0.007

2624 GATA2 GATAbindingprotein2 2.88 0.000

60485 SAV1 salvadorhomolog1(Drosophila) 2.87 0.006

5156 PDGFRA platelet-derivedgrowthfactorreceptor,alphapolypeptide 2.87 0.008

9240 PNMA1 paraneoplasticantigenMA1 2.86 0.008

ARHGEF 23365 12 Rhoguaninenucleotideexchangefactor(GEF)12 2.85 0.008

9

858 CAV2 caveolin2 2.84 0.003

654 BMP6 bonemorphogeneticprotein6 2.81 0.007

LOC284 284422 422 similartoHSPC323 2.79 0.005

TMEM1 25907 58 transmembraneprotein158 2.79 0.004

8936 WASF1 WASproteinfamily,member1 2.74 0.000

7429 VIL1 villin1 2.74 0.004

800 CALD1 caldesmon1 2.70 0.000

928 CD9 CD9molecule 2.68 0.008

CTD(carboxy- terminaldomain,RNApolymeraseII,polypeptideA)smallphos 10217 CTDSPL phatase-like 2.67 0.003

9848 MFAP3L microfibrillar-associatedprotein3-like 2.63 0.000

51109 RDH11 retinoldehydrogenase11(all-trans/9-cis/11-cis) 2.62 0.004

259215 LY6G6F lymphocyteantigen6complex,locusG6F 2.60 0.000

6403 SELP selectinP(granulemembraneprotein140kDa,antigenCD62) 2.58 0.009

5874 RAB27B RAB27B,memberRASoncogenefamily 2.56 0.007

51097 SCCPDH saccharopinedehydrogenase(putative) 2.56 0.009

3673 ITGA2 integrin,alpha2(CD49B,alpha2subunitofVLA-2receptor) 2.54 0.004

LOC816 81691 91 exonucleaseNEF-sp 2.53 0.009

3248 HPGD hydroxyprostaglandindehydrogenase15-(NAD) 2.47 0.003

DCLRE1 9937 A DNAcross-linkrepair1A(PSO2homolog,S.cerevisiae) 2.47 0.003

FLJ3684 647115 8 hypotheticalLOC647115 2.41 0.000

10

2281 FKBP1B FK506bindingprotein1B,12.6kDa 2.36 0.009

383 ARG1 arginase,liver 2.35 0.000

2876 GPX1 glutathioneperoxidase1 2.31 0.003

286319 TUSC1 tumorsuppressorcandidate1 2.31 0.008

3987 LIMS1 LIMandsenescentcellantigen-likedomains1 2.28 0.000

11001 SLC27A2 solutecarrierfamily27(fattyacidtransporter),member2 2.27 0.009

tyrosine3-monooxygenase/tryptophan5- 7533 YWHAH monooxygenaseactivationprotein,etapolypeptide 2.27 0.000

proteintyrosinephosphatase,receptortype,fpolypeptide(PTP 8497 PPFIA4 RF),interactingprotein(liprin),alpha4 2.26 0.000

117178 SSX2IP synovialsarcoma,Xbreakpoint2interactingprotein 2.24 0.007

6925 TCF4 transcriptionfactor4 2.22 0.000

SHROO 57477 M4 shroomfamilymember4 2.21 0.006

8266 UBL4A ubiquitin-like4A 2.20 0.008

8987 STBD1 starchbindingdomain1 2.18 0.000

56670 SUCNR1 succinatereceptor1 2.16 0.007

9315 C5orf13 chromosome5openreadingframe13 2.16 0.000

8655 DYNLL1 dynein,lightchain,LC8-type1 2.14 0.005

51203 NUSAP1 nucleolarandspindleassociatedprotein1 2.13 0.007

222166 C7orf41 chromosome7openreadingframe41 2.12 0.000

7424 VEGFC vascularendothelialgrowthfactorC 2.08 0.009

10644 IGF2BP2 insulin-likegrowthfactor2mRNAbindingprotein2 2.08 0.006

3624 INHBA inhibin,betaA 2.05 0.004

DENND2 163259 C DENN/MADDdomaincontaining2C 2.04 0.000

11

1053 CEBPE CCAAT/enhancerbindingprotein(C/EBP),epsilon 2.04 0.005

29970 SCHIP1 schwannomininteractingprotein1 2.04 0.003

54443 ANLN anillin,actinbindingprotein 2.03 0.004

29841 GRHL1 grainyhead-like1(Drosophila) 2.03 0.005

4130 MAP1A microtubule-associatedprotein1A 2.02 0.000

1182 CLCN3 chloridechannel3 2.02 0.000

C15orf5 388115 2 chromosome15openreadingframe52 2.02 0.000

7357 UGCG UDP-glucoseceramideglucosyltransferase 1.99 0.004

7485 WRB tryptophanrichbasicprotein 1.97 0.000

55852 TEX2 testisexpressed2 1.96 0.004

51701 NLK nemo-likekinase 1.96 0.000

2184 FAH fumarylacetoacetatehydrolase(fumarylacetoacetase) 1.94 0.005

8895 CPNE3 copineIII 1.93 0.007

284415 VSTM1 V-setandtransmembranedomaincontaining1 1.93 0.006

COL24A 255631 1 collagen,typeXXIV,alpha1 1.91 0.000

7033 TFF3 trefoilfactor3(intestinal) 1.90 0.000

367 AR androgenreceptor 1.88 0.000

7414 VCL vinculin 1.88 0.000

90355 C5orf30 chromosome5openreadingframe30 1.87 0.005

56953 NT5M 5',3'-nucleotidase,mitochondrial 1.85 0.000

9468 PCYT1B phosphatecytidylyltransferase1,choline,beta 1.83 0.007

977 CD151 CD151molecule(Raphbloodgroup) 1.83 0.003

30850 CDR2L cerebellardegeneration-relatedprotein2-like 1.82 0.004

12

6319 SCD stearoyl-CoAdesaturase(delta-9-desaturase) 1.82 0.005

guaninenucleotidebindingprotein(Gprotein),alphainhibiting 2770 GNAI1 activitypolypeptide1 1.82 0.008

COL17A 1308 1 collagen,typeXVII,alpha1 1.81 0.009

1178 CLC Charcot-Leydencrystalprotein 1.81 0.003

PGLYRP 8993 1 peptidoglycanrecognitionprotein1 1.80 0.000

6335 SCN9A sodiumchannel,voltage-gated,typeIX,alphasubunit 1.78 0.003

solutecarrierfamily28(sodium- 64078 SLC28A3 couplednucleosidetransporter),member3 1.77 0.008

1428 CRYM crystallin,mu 1.76 0.000

7060 THBS4 thrombospondin4 1.74 0.009

ARMCX 51566 3 armadillorepeatcontaining,X-linked3 1.74 0.007

54498 SMOX spermineoxidase 1.73 0.003

ANKRD1 253650 8A ankyrinrepeatdomain18A 1.72 0.008

80177 MYCT1 myctarget1 1.71 0.003

HIST2H2 8349 BE histonecluster2,H2be 1.71 0.009

55331 ACER3 alkalineceramidase3 1.71 0.003

23286 WWC1 WWandC2domaincontaining1 1.71 0.000

23446 SLC44A1 solutecarrierfamily44,member1 1.69 0.005

57126 CD177 CD177molecule 1.68 0.003

C9orf15 286343 0 chromosome9openreadingframe150 1.68 0.003

13

5746 PTH2R parathyroidhormone2receptor 1.68 0.003

C16orf7 116028 5 chromosome16openreadingframe75 1.67 0.000

5867 RAB4A RAB4A,memberRASoncogenefamily 1.66 0.007

1071 CETP cholesterylestertransferprotein,plasma 1.65 0.009

23314 SATB2 SATBhomeobox2 1.63 0.009

5360 PLTP phospholipidtransferprotein 1.63 0.000

55695 NSUN5 NOL1/NOP2/Sundomainfamily,member5 1.62 0.000

389119 C3orf54 chromosome3openreadingframe54 1.62 0.000

TACSTD 4070 2 tumor-associatedcalciumsignaltransducer2 1.61 0.004

9911 TMCC2 transmembraneandcoiled-coildomainfamily2 1.60 0.000

proteintyrosinephosphatase- 9200 PTPLA like(prolineinsteadofcatalyticarginine),memberA 1.55 0.003

2815 GP9 glycoproteinIX(platelet) 1.55 0.004

C6orf14 221749 5 chromosome6openreadingframe145 1.54 0.008

64407 RGS18 regulatorofG-proteinsignaling18 1.54 0.003

transientreceptorpotentialcationchannel,subfamilyC,memb 7225 TRPC6 er6 1.52 0.003

3915 LAMC1 laminin,gamma1(formerlyLAMB2) 1.52 0.000

SERPINE serpinpeptidaseinhibitor,cladeE(nexin,plasminogenactivato 5054 1 rinhibitortype1),member1 1.51 0.003

7739 ZNF185 zincfingerprotein185(LIMdomain) 1.51 0.007

30846 EHD2 EH-domaincontaining2 1.51 0.007

14

Supplementary Table III: Canonical pathways regulated with a p-value <0.05, as assessed by IPA (Ingenuity Pathway Analysis).

From the 302 genes regulated in lupus LDGs compared to control neutrophils (q-value <0.01, fold-change ≥ 1.5 and ≤ 0.7 for the up-regulated and down-regulated genes respectively)

Canonical pathways (number of genes in the p-value Number of regulated genes pathway)

1 RhoA Signaling (110) 0.003 7

2 Actin Cytoskeleton Signaling (238) 0.005 10

3 ILK Signaling (191) 0.006 9

4 PAK Signaling (107) 0.006 6

5 Macropinocytosis Signaling (76) 0.009 5

6 Integrin Signaling (205) 0.009 9

7 Clathrin-mediated Endocytosis Signaling (171) 0.009 8

8 IL-8 Signaling (193) 0.014 8

9 Inhibition of Angiogenesis by TSP1 (37) 0.019 3

10 P2Y Purigenic Receptor Signaling Pathway (138) 0.023 6

11 Thrombin Signaling (208) 0.025 8

12 Eicosanoid Signaling (84) 0.028 4

13 Atherosclerosis Signaling (115) 0.034 5

14 Extrinsic Prothrombin Activation Pathway (20) 0.035 2

15 cAMP-mediated Signaling (217) 0.040 8 16 Caveolar-mediated Endocytosis Signaling (85) 0.042 4

From the 281 genes regulated in lupus LDGs compared to lupus neutrophils (q-value <0.01, fold-change ≥ 1.5 and ≤ 0.7 for the up-regulated and down-regulated genes respectively)

Canonical pathways (number of genes in the p-value Number of regulated genes pathway)

1 Clathrin-mediated Endocytosis Signaling (171) 0.000 10

2 PAK Signaling (107) 0.001 7

3 RhoA Signaling (110) 0.002 7

4 Ephrin Receptor Signaling (199) 0.003 9

5 Actin Cytoskeleton Signaling (238) 0.003 10

6 Axonal Guidance Signaling (410) 0.004 14

7 Thrombin Signaling (208) 0.006 9

8 Agrin Interactions at Neuromuscular Junction (69) 0.006 5

9 Macropinocytosis Signaling (76) 0.006 5

10 Caveolar-mediated Endocytosis Signaling (85) 0.007 5

11 IL-8 Signaling (193) 0.010 8

Nitric Oxide Signaling in the Cardiovascular 12 0.012 5 System (103)

13 P2Y Purigenic Receptor Signaling Pathway (138) 0.017 6

14 Integrin Signaling (205) 0.017 8

15 G Beta Gamma Signaling (120) 0.021 5

16 Coagulation System (38) 0.022 3 17 Eicosanoid Signaling (84) 0.022 4

18 Atherosclerosis Signaling (115) 0.026 5

19 Ovarian Cancer Signaling (145) 0.027 6

20 Relaxin Signaling (161) 0.029 6

21 Extrinsic Prothrombin Activation Pathway (20) 0.031 2

Hepatic Fibrosis / Hepatic Stellate Cell Activation 22 0.032 6 (147)

23 ILK Signaling (191) 0.033 7

24 Sphingosine-1-phosphate Signaling (121) 0.037 5

25 Leukocyte Extravasation Signaling (199) 0.041 7

26 Androgen Signaling (145) 0.046 5

27 Reelin Signaling in Neurons (82) 0.047 4