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Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility

Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying... Introduction: Neutrophil extracellular traps (NETs) have recently been implicated in a number of autoimmune conditions, including rheumatoid arthritis (RA). We examined the underlying signaling pathways triggering enhanced NETosis in RA and ascertained whether the products of NETosis had diagnostic implications or usefulness. Methods: Neutrophils were isolated from RA patients with active disease and from controls. Spontaneous NET formation from RA and control neutrophils was assessed in vitro with microscopy and enzyme-linked immunosorbent assay (ELISA) for NETosis-derived products. The analysis of the signal-transduction cascade included reactive oxygen species (ROS) production, myeloperoxidase (MPO), neutrophil elastase (NE), peptidyl arginine deiminase 4 (PAD4), and citrullinated histone 3 (citH3). NET formation was studied in response to serum and synovial fluid and immunoglobulin G (IgG) depleted and reconstituted serum. Serum was analyzed for NETosis-derived products, for which receiver operator characteristic (ROC) curves were calculated. Results: Neutrophils from RA cases exhibited increased spontaneous NET formation in vitro, associated with elevated ROS production, enhanced NE and MPO expression, nuclear translocation of PAD4, PAD4-mediated citrullination of H3, and altered nuclear morphology. NET formation in both anti-citrullinated peptide antibody (ACPA)-positive and -negative RA was abolished by IgG depletion, but restored only with ACPA-positive IgG. NETosis-derived products in RA serum demonstrated diagnostic potential, the ROC area under the curve for cell-free nucleosomes being >97%, with a sensitivity of 91% and a specificity of 92%. No significant difference was observed between ACPA-positive and -negative cases. Conclusions: Signaling elements associated with the extrusion of NETs are significantly enhanced to promote NETosis in RA compared with healthy controls. NETosis depended on the presence of ACPA in ACPA-positive RA serum. The quantitation of NETosis-derived products, such as cell-free nucleosomes in serum, may be a useful complementary tool to discriminate between healthy controls and RA cases. * Correspondence: [email protected]; [email protected] Equal contributors Department of Biomedicine, University Hospital Basel, Hebelstrasse 20, 4032 Basel, Switzerland Department of Rheumatology, Kantonsspital Aarau, Tellstrasse, 5001 Aarau, Switzerland Full list of author information is available at the end of the article © 2014 Sur Chowdhury et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 2 of 14 http://arthritis-research.com/content/16/3/R122 Introduction general condition, age ≥28 and ≤70 years, and for blood A novel feature of the biology of polymorphonuclear gran- donors fulfilling national criteria for blood donation. ulocytes (PMNs) is their ability to generate neutrophil Exclusion criteria were current or previous systemic extracellular traps (NETs) [1] via a distinct process of cell autoimmune disease, asthma and reconvalescence after death termed NETosis [2]. NETs consist of extruded major illness, surgery, current medication with corti- chromosomal DNA decorated with granular components costeroids, immunosuppressive agents, and malignant that include antimicrobial peptides and proteases. The neoplasia or chemotherapy within 5 years before molecular pathways leading to NETosis encompass cal- recruitment for the study. RA cases had a DAS ≥2.6, cium mobilization, generation of reactive oxygen species were from age ≥27 to ≤70 years, and had no other (ROS), nuclear delobulation involving the enzymatic activ- systemic autoimmune disease, including ankylosing ities of myeloperoxidase (MPO) and neutrophil elastase spondylitis and psoriatic arthritis. (NE), and chromatin modification via the citrullination of Exclusion criteria were corticosteroids ≥40 mg equiva- histones by peptidyl arginine deiminase 4 (PAD4) [2-6]. lent of prednisone daily, and those mentioned earlier for A number of studies have implicated NETs in the healthy controls. Informed, written consent was obtained etiology of autoinflammatory or autoimmune conditions from all subjects in the study, which was approved by such as preeclampsia, Felty syndrome, systemic lupus ery- the Cantonal Ethical Review Boards of Aargau-Solothurn thematosus (SLE), multiple sclerosis, and, most recently, and Basel/Basel-Land, Switzerland. rheumatoid arthritis (RA) [7-13]. In the context of RA, these findings are especially interesting, as NETs have Preparation of plasma and serum been proposed to contribute to the generation of anti- Plasma and serum were collected and processed as citrullinated protein antibody (ACPA) autoantigens, and described previously [15]. Samples were studied immedi- may also be a target for autoantibodies [13,14]. PMNs iso- ately or stored at −80°C until analysis. lated from RA patients showed an increased propensity to undergo spontaneous and LPS-induced NETosis, which Cell isolation was in part mediated by TNF and IL-17 and could be PMNs were isolated with Dextran-Ficoll density centri- inhibited by blocking NADPH oxidase or PAD4. Whereas fugation [8]. Cell viability was 96% to 98%, with a purity the citrullinated autoantigens vimentin and α-enolase of >95% PMNs. Neutrophils seeded in 24-well plates were expressed on NETs from RA PMNs, antibodies to were allowed to settle for 1 hour at 37°C under 5% CO the former were able to induce NET formation by healthy before further experimentation. control PMNs [13,14]. As we had previously detected significantly increased Cell-free DNA isolation and quantification concentrations of cell-free DNA in the sera of RA patients Cell-free DNA extracted from 850 μlplasmaorserum by compared with healthy controls, we were intrigued using the QIAamp Circulating Nucleic Acid Kit (Qiagen, whether the provenance of this material involved NETosis Valencia, CA, USA) was quantified by TaqMan real-time [15]. The premise for the current investigation was that a PCR (StepOne Plus Real-Time PCR System, Applied Bio- link between circulating cell-free DNA levels and NETs systems, Foster City, CA, USA) specific for the glyceral- has previously been made in a number of conditions, dehyde-3-phosphate dehydrogenase (GAPDH) gene [15]. including preeclampsia, sepsis, cancer, thrombosis, or even storage of blood-transfusion products [16-19]. Detection of neutrophil elastase (NE), myeloperoxidase In view of these findings and reports on the complex in- (MPO), and cell-free nucleosomes volvement of neutrophil NETs in autoimmunity, we sought The concentrations of neutrophil elastase (NE) and mye- to investigatethe NEToticresponseofPMNsinRA, with loperoxidase (MPO) were measured with sandwich ELISA particular regard to the underlying signal-transduction (Elastase/a1-PI Complex ELISA Kit, Calbiochem/EMD, cascade, and whether the products of overt NETosis could Gibbstown, NJ, USA) and the human (MPO ELISA Kit, be diagnostically useful. Hycult Biotech, Plymouth Meeting, PA, USA) respectively. Nucleosomes were measured by using the Human Cell PLUS Materials and methods Death Detection ELISA (Roche Diagnostics, Basel, Human subjects Switzerland). Cell-culture supernatants were incubated with All patients fulfilled the American College of Rheuma- DNaseI (10 U for 5 minutes) (Roche Diagnostics) before tology classification criteria for RA, or for systemic analysis [20]. lupus erythematosus, respectively. Healthy volunteers, matched for gender and age, were recruited at the MPO/DNA complex detection hospitals or at the Blood Bank of the Swiss Red Cross, MPO is present on extruded NETs. To detect such Basel. Inclusion criteria for healthy controls were fair structures, NET-associated MPO/DNA complexes were Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 3 of 14 http://arthritis-research.com/content/16/3/R122 quantified by using a modified capture ELISA [21]. In anti-histone H1 + core proteins (EMD Millipore, Billerica, brief, NET-associated MPO in serum or culture super- MA, USA), and rabbit anti-citrullinated histone H3 natant was captured by using the coated 96-well plate of (citH3, Abcam). Secondary antibodies were goat anti- the human MPO ELISA Kit, (Hycult Biotech), after which rabbit IgG AF555, goat anti-rabbit IgG AF488 (Invitrogen the NET-associated DNA backbone was detected by using Life Technologies, San Diego, CA, USA), and goat anti- the detection antibody of the Human Cell Death Detec- mouse IgG AF647. DNA was stained with 4′,6-diamidino- PLUS tion ELISA (Roche Diagnostics). 2-phenylindole (DAPI, Sigma-Aldrich), and NETs were visualized by using a Zeiss Axioplan 2 Imaging fluores- PAD4/DNA-complex detection cence microscope in conjunction with a Zeiss AxioCam To detect the presence of PAD4 on extruded NETs in MRm monochromatic CCD camera and analyzed with culture supernatants after spontaneous NETosis, cell-free Axiovision 4.8.2 software (Carl Zeiss). A minimum of 20 PAD4/DNA complexes were quantified by using a modi- fields (at least 1,000 PMNs) per case was evaluated for fied capture ELISA, akin to that described for MPO MPO/NE and DNA co-staining; nuclear phenotypes and earlier. In brief, cell-free PAD4 was captured by using the NETs were counted and expressed as percentage of the coated 96-well plate of a commercial human PAD4 ELISA total number of cells in the fields. (USCN Life Science, Inc., Wuhan, China), and associated DNA was detected by using Human Cell Death Detection RA serum depletion, IgG purification, and quantification PLUS ELISA kit (Roche Diagnostics). of NETs After three washes with PBS, 200 μl protein G agarose ROS generation analysis (Pierce Biotechnology Inc, Rockford, IL, USA) was incu- ROS was measured by using a 2′,7′-dichloro dihydrofluor- bated with 200 μl ACPA + and ACPA- RA or control escein diacetate (DCFH-DA) assay [22]. The 5 × 10 cells serum diluted in an equal volume of phenol red-free RPMI in a final volume of 500 μl were incubated for 30 minutes 1640 medium overnight at 4°C. The serum/protein G with 25 μM DCFH-DA (Sigma-Aldrich, St. Louis, MO, agarose mixture was centrifuged at 2,500 g for 5 minutes, USA). Fluorescence was measured with flow cytometry and the supernatant (IgG-depleted serum) was carefully (FACSCalibur; BD Biosciences, San Jose, CA, USA). transferred into a new Eppendorf microcentrifuge tube. The protein G agarose pellet was gently washed 3 times Fluorescence and scanning electron microscopy with 500 μlddH O, and the bound antibody was released The 5 × 10 cells isolated PMNs seeded on poly-L-lysine- by the addition of 50 μl0.1 M glycine pH 2–3, and coated coverslips (BD Biosciences) were stimulated with phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) Table 1 Demographics and patient population for 90 minutes and dehydrated with a graded ethanol characteristics versus healthy blood donors series (30%, 50%, 70%, 100%) [8], coated with 2-nm plat- Controls RA Statistics inum, and analyzed with a Nova NanoSEM 230 scanning Age 50.34 ± 1.5 53.03 ± 1.5 P = 0.214 electron microscope (FEI Co., Hillsboro, OR, USA). PMNs Gender (F/M) 24/32 24/8 - were incubated for 10 minutes with 5 μM Sytox Green DAS28 n.a. 3.07 ± 1.12 - dye (Invitrogen Life Technologies, San Diego, CA, USA) Bone erosion (pos/neg) n.a. 22/10 - for assessment of NETs with an Axiovert fluorescence Serum ACPA (pos/neg) n.a. 20/12 - microscope (Carl Zeiss) coupled to a Zeiss AxioCam color CCD camera (Carl Zeiss Microimaging, Oberkoch, Serum RF (pos/neg) n.a. 19/13 - Germany) [8,23]. Serum ANA (pos/neg) n.a. 21/11 - ESR (mm/h) n.a. 16.8 ± 13.1 - Immunohistochemical staining and quantification of NETs CRP (mg/L) n.d. 6.9 ± 5.2 - The 5 × 10 isolated PMNs were seeded on poly-L- PBMC (cells/μl) 1,961 ± 81.69 1513 ± 75.90 P < 0.0001 lysine-coated glass coverslips (BD Biosciences, San Jose, PMN (cells/μl) 3,641 ± 149.7 4575 ± 546.0 P = 0.021 CA, USA) in tissue-culture wells and allowed to settle before stimulation, as described earlier. Coverslips were Therapy (yes/no) n.a. 31/1 - rinsed with ice-cold HBSS and the cells fixed with 4% DMARDs (yes/no) n.u. 27/5 - paraformaldehyde and blocked overnight (HBSS with Biologics (yes/no) n.a. 30/2 - 10% goat serum, 1% BSA, 0.1% Tween20, and 2 mM F, female; M, male; DAS28, disease activity score; n.a., not applicable; pos, EDTA) at 4°C. NETs were detected with rabbit anti-NE positive; neg, negative; ACPA: anti-citrullinated protein antibodies; RF, rheumatoid factor; ANA, antinuclear antibody; ESR, erythrocyte sedimentation rate; CRP, (Abcam, Cambridge, MA, USA), rabbit anti-MPO C-reactive protein; n.d., not determined; PBMC, peripheral blood mononuclear (Dako, Glostrup, Denmark), two different rabbit anti- cell; PMN, polymorphonuclear leukocyte; DMARD, disease-modifying PAD 4 (Abcam), mouse anti-PAD4 (Abcam), mouse antirheumatic drug; n.u., not used. Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 4 of 14 http://arthritis-research.com/content/16/3/R122 immediately equilibrated with 10 μlof 1 M Tris pH 7.5-9. NETs were quantified after IHC staining with mouse All protein concentrations were determined with the MN anti-human MPO antibody (Abcam) and rabbit anti- Protein Quantification Assay (Macherey-Nagel GmbH, human citH3 antibody (Abcam), or the respective isotype Düren, Germany), and isolation of IgG was verified with controls, followed by incubation with goat anti-mouse IgG Coomassie staining of SDS-PAGE. AF555 and goat anti-rabbit IgG AF488 (Invitrogen Life Neutrophils from healthy donors (n =3) were isolated Technologies). DNA was counterstained with 4′,6-diami- and cultured for 2 hours in 96-well culture dishes (Thermo dino-2-phenylindole (DAPI, Sigma-Aldrich). NETs were Fischer Scientific, Waltham, MA, USA), supplemented visualized by using an Olympus IX81 motorized epifluor- with:serum,depletedserum,and purified IgG fromACPA- escence microscope (Olympus America Inc., Center Val- positive RA patients (n = 3), ACPA-negative RA patients ley, PA, USA) in conjunction with an Olympus XM10 (n = 3), and healthy individuals (n = 3) to a final concentra- monochromatic CCD camera (Olympus) and analyzed tion of 100 μg/ml. with the Olympus CellSens Dimension software (Olympus). A C PMN isolation in vitro cell culture Cell-free nucleosomes * *** 1h resting T1 T2 T3 0.8 0.6 Timecourse 0h 1h 3h 0.4 T1 T2 T3 0.2 NE DAPI T1 T2 T3 T1 T2 T3 Control RA Control MPO/Cell-free DNA complexes ***** ** * 0.12 0.10 0.08 RA 0.06 T1 T2 T3 T1 T2 T3 Control RA E F lobulated delobulated/diffused spread * *** * * *** ** **** 60 **** **** 80 70 **** ** *** * lobulated ** ** * delobulated/ 40 30 diffused 10 10 0 0 0 spread NE T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 DAPI Control RA Control RA Control RA Figure 1 RA-derived PMNs exhibited increased spontaneous NETosis and elevated levels of NET-component release. (A) Schematic representation of the time-course design for studying in vitro spontaneous NET release. (B) Detection of in vitro NETosis by immunohistochemistry for neutrophil elastase (NE) (green) and DAPI (blue). Magnification: 20×; scale bar, 50 nm. (C) Concentration of cell-free nucleosomes in PMN culture supernatant by ELISA. (D) Quantification of NET-associated MPO/DNA complexes. These assays indicate that more-rapid and extensive progression of NET formation is observed in RA versus control PMNs. (E) Changes in PMN nuclear morphology during NETosis detected with immunohistochemistry for NE and DAPI. (F) Steady-state (T1) RA-derived PMNs exhibited a greater proportion of delobulated/diffused cells, and progressed rapidly to a NETotic spread phenotype during in vitro culture. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. All data are representative of at least six independent experiments. % neutrophils % neutrophils Absorbance (A -A ) Absorbance (A ) 450 nm 405nm 490nm % neutrophils Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 5 of 14 http://arthritis-research.com/content/16/3/R122 Figure 2 (See legend on next page.) Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 6 of 14 http://arthritis-research.com/content/16/3/R122 (See figure on previous page.) Figure 2 Increased expression of NET-associated signaling elements and nuclear localization of PAD4, citrullination of histone H3 in RA-derived PMN, and possible extrusion of PAD4 on NETs. Baseline ROS levels, measured with flow cytometry, were higher in RA-derived PMNs than in control PMNs (A). Quantitative real-time PCR analysis of NE mRNA (B) and MPO mRNA expression (C), as well as Western blot analysis of MPO protein levels (D), indicated that the levels of these two components required for NETosis were elevated in RA-derived PMNs. Total PAD4 protein (E) or PAD4 mRNA expression levels (F) did not indicate any significant difference between control and RA-derived PMNs. (G) Quantification of PAD4 protein levels in cytoplasmic and nuclear fractions of PMNs from healthy controls and RA patients. Nuclear levels of PAD4 were significantly increased in RA patients, whereas the cytoplasmic levels were lower compared with the healthy control PMNs. (H) Elevated citrullinated histone H3 levels in RA PMN extracts detected with Western blot. (I) Co-localization of citrullinated histone H3 (green) with histone components detected with a pan-histone antibody (red), spread over the entire NET surface (blue). Magnification: upper panel 20×, scale bar, 50 nm; lower panel 63× scale bar, 10 nm. (J) Extracellular localization of PAD4 (red) on extruded NETs by multicolor fluorescent immunohistochemistry. NET DNA is stained blue (DAPI), and histones (panH) are stained green. Magnification, 20×; scale bar, 50 nm (K). Detection of PAD4/cell-free DNA complexes in the culture supernatants of isolated PMN undergoing spontaneous NETosis. Higher levels of these complexes were detected in RA-derived PMN cultures. Time points correspond to those of Figure 1A. Data are represented as mean ± SEM. **P < 0.01; ***P <0.001; ****P < 0.0001; n.s., statistically not significant. All data are representative of at least six independent experiments. A minimum of 20 fields at 10× magnification (at least 500 selection procedure involving six different endogenous to 1,000 PMNs) per case was evaluated for MPO/citH3 and reference genes, as suggested in the MIQE guidelines [26]. −DDCt DNA co-staining through ImageJ analysis software (National Relative values were calculated with 2 analysis [27]. Institutes of Health Image Processing, Bethesda, MD, USA); nuclear phenotypes and NETs were determined, Statistical analysis counted, and expressed as percentage of the total area of All data are presented as mean ± SEM. Descriptive statis- cells in the fields [24]. tics for continuous parameters consisted of median and range, and categoric variables were expressed as percent- Protein isolation and Western blot analysis ages. Comparisons between patients and healthy controls Total protein was isolated with NucleoSpin TriPrep kit were by the Mann–Whitney U test with a Welch posttest (Macherey-Nagel) from 3 × 10 PMNs. Proteins from the correction. Statistical significance in multiple comparisons nuclear and cytoplasmic fractions were isolated by using was by one-way analysis of variance (ANOVA) with a the Nuclear and Cytoplasmic Protein Extraction Kit Dunn posttest correction. P < 0.05 was considered statisti- (Thermo Scientific). Western blotting was performed cally significant. by using AnykD Mini-PROTEAN TGX Gels (Biorad, Receiver operating characteristic (ROC) curves were Hercules, CA, USA) and nylon/nitrocellulose mem- calculated, and the area under the curve (AUC) with branes (Biorad). Primary and secondary antibodies used corresponding standard errors of means was calculated. were: rabbit anti-PAD4 (Abcam), rabbit anti-MPO (Cell Data were processed in GraphPad Prism version 5.0b for Signalling Technologies, Beverly, MA, USA), mouse MacOSX (GraphPad Software Inc., San Diego, CA, anti-β-Actin (Sigma-Aldrich), goat anti-Mouse and/or USA). Analysis of covariance (ANCOVA) was conducted anti-Rabbit, human anti-HRP (Southern Biotech). HRP with SPSS version 21.0 statistical software (IBM SPSS activity was detected by using SuperSignal West Pico Inc., Chicago, IL, USA). Additional professional statis- Chemiluminescent Substrate (Thermo Scientific). Equal tical assistance was provided by A. Schoetzau, Basel. loading was verified by using beta-actin or histone H3, when appropriate. Western blots of citrullinated H3 (citH3) protein were performed according to Shechter Results et al. [25]. Densitometric analysis and protein quantifi- RA-derived PMN exhibit increased spontaneous NETosis cation of the Western blots was performed by using the Details of the RA study group and healthy control group ImageJ software. are described in Table 1 and Additional file 1. As in very recent observations [13], we observed that RNA isolation and quantitative real-time PCR RA-derived PMNs underwent greater degrees of NETosis Total RNA was isolated by using RNeasy Mini Kit (Qiagen). than did control PMNs in vitro (data not shown). To study TaqMan real-time quantitative RT-PCR was performed by this facet in more detail, we examined the kinetics of using the Applied Biosystems StepOne Plus cycler (Applied spontaneous NET extrusion, for which purpose, PMNs Biosystems) and TaqMan Gene Expression Assay primer/ isolated from peripheral blood samples were allowed to probe sets (Applied Biosystems) for ELANE (HS00236952_ settle for 1 hour and then cultured for a period of up to m1), MPO (HS00924296_m1), PADI4 (HS00202612_m1), 3hours in vitro (Figure 1A), NETs being detected by im- and β -microglobulin B2M (HS99999907_m1). Data were munohistochemistry for neutrophil elastase (NE) and DAPI normalized by using the housekeeping gene B2M, after a (4′,6-diamidino-2-phenylindole)(Figure 1B). In addition, Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 7 of 14 http://arthritis-research.com/content/16/3/R122 Control +PMA RA +PMA Control UT Control +PMA RA UT RA +PMA MPO DAPI MPO lobulated delobulated/diffused spread 80 60 100 **** ** **** n.s. **** **** n.s. **** **** **** * **** 40 30 0 0 0 -+-+ PMA -+-+ PMA -+-+ PMA Control RA Control RA Control RA PAD4 protein expression Cell-free nucleosomes ** 3.0 ** * * * ** 4.0 2.0 0.6 3.0 0.5 0.4 2.0 0.3 0.2 1.0 0.1 0.0 0.0 - - - - PMA + + + + PMA - - - - + + + + Control RA Control RA Cl-amidine - - - - + + + + Cytoplasmic Nuclear Control RA Figure 3 (See legend on next page.) Absorbance (A -A ) % neutrophils 405nm 490nm % neutrophils Arbitrary units % neutrophils Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 8 of 14 http://arthritis-research.com/content/16/3/R122 (See figure on previous page.) Figure 3 Increased NETotic response of RA-derived PMNs to PMA. (A) Scanning electron micrographs of NETs induced by PMA (25 nM) indicate the excessive NETotic response of RA-derived PMN. Scale bar, 20 nm. (B) Assessment of NETs induced by PMA treatment by fluorescent immunohistochemistry for MPO (red) and DAPI (blue), indicating the increased response of RA PMNs to PMA (25 nM). Magnification, 20×; scale bar, 50 nm. (C) Analysis of the nuclear phenotype indicated a vast decrease in delobulated/diffused RA PMN nuclei after treatment with PMA and rapid increase in the NETotic-spread phenotype. (D) Release of cell-free nucleosomes after PMA treatment is abrogated by chloramidine, a PAD4 inhibitor. (E) Increased nuclear localization and concomitant decrease in cytoplasmic PAD4 protein levels after PMA treatment, with a clear tendency for an increased responsiveness to the PMA stimulus by RA PMN. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., statistically not significant. All data are representative of at least four independent experiments. we quantitatively assessed the degree of in vitro NETosis in of NE (Figure 2B) and MPO (Figure 2C and D), as deter- these cultures by determining the concentration of cell-free mined with real-time PCR and/or Western blotting. nucleosomes in the respective supernatants (Figure 1C), Surprisingly, neither PAD4 mRNA expression nor specifically their association with myeloperoxidase (MPO), PAD4 levels in total cellular protein showed any discern- indicative of the NETotic origin of this material [2,21] ible difference between RA and control PMNs (Figure 2E (Figure 1D). We also measured NET-associated MPO en- and F, respectively). Because PAD4 translocates to the zymatic activity by using tetramethylbenzidine (TMB) as a nucleus on PMN activation [4,29], where it citrullinates substrate (data not shown). The results clearly indicate histone proteins, such as H3, we examined its presence that RA-derived PMNs generated NETs more rapidly, to a in nuclear and cytoplasmic PMN fractions. Compared greater magnitude, and more extensively than did control with control PMNs, PAD4 was preferentially located in healthy PMNs, which was particularly evident at the 3-hour the nucleus of RA-derived PMNs (Figure 2G). The nu- stage of in vitro culture (Figure 1B to D). Accounting for clear presence of PAD4 was associated with increased variances in PMN counts, the difference between the citrullinated histone H3 (citH3) levels with Western blot healthy control and RA subjects remained highly significant analysis in PMNs from RA cases compared with controls in an analysis of covariance (ANCOVA) of the nucleosome (Figure 2H). Furthermore, citrullinated histone H3 could assay (P <0.001). be readily detected on NET structures (Figure 2I). During NETosis, the morphology of the PMN nucleus changes from the familiar lobulated to a diffused and then Potential extracellular localization of PAD4 on NETs to a spread phenotype (Figure 1E) [28]. By examining and Because we observed elevated nuclear translocation of enumerating these features, it was observed that at base- PAD4 in RA PMNs, we examined whether this enzyme is line (T1), the nuclei from healthy control PMNs were extruded into the extracellular environment during NETo- predominantly lobulated, whereas in this instance, the sis. Unfortunately, the visualization of such an event by majority of RA-derived PMN nuclei exhibited a delobu- fluorescence immunohistochemistry proved to be difficult lated or diffused nuclear phenotype (Figure 1F). In RA- with a variety of commercially available antibodies, and we derived PMNs, this delobulated population decreased obtained only rudimentary evidence for the presence of over time, giving rise to NETotic cells with a spread PAD4 on NETs by this means (refer to Figure 2J). phenotype (Figure 1F). In contrast, in normal PMNs, we We were, however, able to detect PAD4/cell-free DNA noted a steady progression in the proportion of delobu- complexes readily in culture supernatants from isolated lated cells (Figure 1F). The spontaneous progression of PMNs, the levels of which were increased in cases with RA nuclei to the NETotic-spread phenotype was more pro- (Figure 2K). It is, therefore, quite probable that PAD4 is asso- nounced in RA than in normal PMNs, a feature most ciated with NETs structures after aberrant NETosis in RA. evident after 3 hours (T3) (Figure 1F). PMNs from RA patients present an enhanced NETotic RA-derived PMNs demonstrate increased expression of response to PMA, and normal PMNs are strongly affected NET-associated signaling elements, nuclear localization of by RA serum and synovial fluid PAD4, and augmented H3 citrullination In autoinflammatory or malignant conditions, such as NETosis has been shown to depend on a number of bio- SLE or cancer, an elevated NETotic response of PMNs chemical signaling elements, among which are the gen- to an external activation signal has been shown [9,13]. eration of ROS by nicotinamide adenine dinucleotide In our experiments, we noted that when RA-derived phosphate (NADPH) oxidase, the action of NE in com- PMNs were treated with PMA, they responded far more bination with MPO, and histone citrullination by PAD4 vigorously with regard to NETosis than did controls, as [2,3,5]. RA-derived PMNs exhibited increased basal intra- detected by SEM and fluorescence microscopy (Figure 3A cellular ROS levels (Figure 2A), as well as increased levels and B, respectively). Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 9 of 14 http://arthritis-research.com/content/16/3/R122 Figure 4 (See legend on next page.) Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 10 of 14 http://arthritis-research.com/content/16/3/R122 (See figure on previous page.) Figure 4 Influence of RA serum and synovial fluid on normal PMNs. (A) Incubation of healthy donor PMNs with serum (Se) from healthy donors or from RA patients, synovial fluid (SF) from patients with noninflammatory osteoarthritis (OA) or synovial fluid from RA patients. Immunohistochemical analysis of four main components of NETs (NE, MPO, PAD4, and citH3) revealed that RA-derived serum and SF enhanced NETosis in normal PMN compared to healthy control serum or noninflammatory OA SF. PAD4 (white arrowheads) and citH3 (empty arrowheads) colocalize with unmodified histones on NETs. Magnification, 20×; scale bars, 50 nm. (B) Release of cell-free nucleosomes during in vitro culture by PMNs from healthy controls incubated with control serum, RA serum, OA SF or RA SF, or PMA. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01. (C) Increased ROS generation during in vitro culture by PMNs from healthy controls incubated with control serum, RA serum, OA SF or RA SF, or PMA. Data are presented as mean ± SEM. (D) Release of cell-free nucleosomes during in vitro culture by PMNs from healthy controls, ACPA-positive and ACPA-negative RA patients incubated with serum (Se +), IgG-depleted serum (Se -), or serum reconstituted with their respective eluted IgG, or with PMA. Data are presented as mean ± SEM. *P < 0.05; ***P < 0.001; ****P < 0.0001; n.s., statistically not significant. All data are representative of at least four independent experiments in Figures 4A–C, and 3 in 4D. In addition, morphometric assessment indicated that RA- PMNs, and that ACPA and also IgM RF are to a large derived PMNs exhibited a larger decrease in cells with a part responsible for this effect [13,14]. delobulated phenotype and a greater progression toward a NETotic-spread nuclear phenotype than control PMNs (Figure 3C), a feature accompanied by excessive release of Serum from RA patients shows elevated levels of the cell-free nucleosomes in culture supernatants (Figure 3D). principal components of NETs, indicating enhanced NET PMA appears to activate PAD4, as it enhanced transloca- extrusion during clot formation, which has potential tion from the cytoplasm to the nucleus (Figure 3E). The clinical utility stimulatory effect of PMA on the release of nucleosomes As we had previously observed increased levels of cell-free into the supernatant was abrogated by Cl-amidine, a chem- DNA (cfDNA) in RA sera [15], we determined whether ical inhibitor of PAD4, indicating that PAD4 signaling is ne- this resulted from enhanced NETosis, and whether this cessary for NETosis induced by PMA [4,30] (Figure 3D). could have diagnostic applications. cfDNA concentrations ThesedataindicatethatPMNsinRAare susceptibleto were indeed significantly higher in serum samples from increased NETosis after stimulation by a secondary signal, RA cases compared with age-matched healthy control sera such as that mediated by PMA. (Figure 5A). In parallel, the concentrations of cell-free nu- Because SLE sera and RA sera and synovial fluid (SF) cleosomes, NE, and MPO were significantly elevated in have been shown to confer an increased NETotic response RA serum compared with control sera (Figure 5B to D). [9,13], we examined whether RA-derived sera or SF The association of a significant fraction of MPO with exerted similar effects on normal PMNs. As noninflamma- markedly elevated cell-free nucleosomes in RA serum, tory controls, we used healthy serum or osteoarthritis SF. which constitute a main component of NETs (Figure 5E), Both RA sera and SF induced a pronounced increase in clearly suggests that NETosis is indeed the source of nu- NETosis, which was paralleled by an increase in the cleosome material present in RA serum [21]. nucleosome content of the supernatant (Figure 4A,B), as Since there was no significant elevation of these parame- well as in ROS production (Figure 4C) when compared ters in simultaneously obtained plasma samples that were with healthy serum or osteoarthritis SF, respectively. processed immediately, these data demonstrate a propen- To assess whether antibodies participate in the effects sity for RA PMN to undergo increased NETosis during of RA serum on normal PMNs, we depleted IgG from coagulation (Figure 5A to E). serum of ACPA-positive and -negative RA patients and To ascertain whether NET-associated serum compo- healthy controls. Compared with nondepleted sera, IgG nents could be diagnostically useful, we conducted ROC depletion of both ACPA-positive and -negative sera analyses. For serum cell-free nucleosomes, this yielded the markedly reduced NET induction to levels of normal surprisingly high AUC value of 0.97 (see Additional file 2 serum (Figure 4D). Whereas the reconstitution of ACPA- and Figure 5F). Of interest is that no significant difference negative IgG to serum did not increase NET formation was found in this value regardless of whether the RA cases significantly compared with controls, it was practically were ACPA positive or not (Figure 5G), although a slight reversed to the original value in the ACPA-positive cases. trend for serum nucleosome levels was higher in ACPA- This indicates a prominent role for ACPA in the induction positive cases than in ACPA-negative cases (Figure 5H). of NETs in ACPA-positive RA, while suggesting that an The AUC for serum nucleosomes was significantly higher alternative mechanism is responsible for the increased than for any of the other parameters examined (Figure 5I NETosis in ACPA-negative RA patients. to K). With the cut-off set at 0.78, the ROC AUC trans- These data are in accordance with recent findings that lates into a sensitivity of 91%, with a specificity of 92% for RA-derived serum and SF induce NETosis in normal differentiating between RA cases and healthy controls. Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 11 of 14 http://arthritis-research.com/content/16/3/R122 Figure 5 (See legend on next page.) Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 12 of 14 http://arthritis-research.com/content/16/3/R122 (See figure on previous page.) Figure 5 Elevated serum levels of NET components in RA patients have potential clinical utility. (A) Cell-free DNA levels in plasma and serum from healthy matched blood donors (n = 41) and patients with RA (n = 32) determined with real-time PCR. (B) Cell-free nucleosome levels in plasma and serum from healthy donor controls and patients with RA, determined with ELISA. (C) Determination of NE protein concentrations in plasma and serum from healthy donors and patients with RA, as assessed with sandwich ELISA. (D) MPO concentrations in plasma and serum from healthy donors and patients with RA, as determined with sandwich ELISA. (E) NET-associated MPO/DNA complexes quantified by using a modified capture ELISA. In contrast to the serum levels, none of the plasma levels of these NET components attained statistical significance (Figure 5A to E). (F) ROC analysis of cell-free nucleosomes in serum of patients with RA and healthy controls. (G) Detail of cell-free nucleosome ROC curve with groups of ACPA + and ACPA- RA cases and (H) scatterbox and whisker plots with individual values for control, ACPA + and ACPA- groups. The ROC curve analysis of other NET components, cell-free DNA (I),NE (J), and MPO (K), was not as conclusive as that for cell-free nucleosomes. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., statistically not significant. In contrast to RA cases, cell-free nucleosome serum a finding confirmed by analysis of culture supernatants for values of 14 cases with SLE showed a slight, but statisti- the products of NETosis. cally significant increase (Additional file 2). This translated Akin to that observed in an array of other pathologic into an ROC AUC of 0.7639 (see Additional file 3), conditions ranging from preeclampsia and SLE to cancer which is below the clinically useful value for diagnostic and RA [8,9,12,13,17], PMNs from RA patients exhibited purposes. an increased response to further stimulation (for instance, by treatment with IL-8, the phorbol ester PMA, or with LPS). This response is in part mediated via the action of PAD4, as the effect of PMA could be significantly reduced Discussion by treatment with Cl-amidine, an inhibitor of PAD4 [30]. Although PMNs figure prominently in the joint effu- In addition, PMA treatment led to an increased nuclear sions and inflamed synovial tissue of RA patients [31], localization of this enzyme, where it presumably could the potential roles of NETotic events in the pathophysi- catalyze a more-extensive citrullination of histone pro- ology of this disorder have only recently become a focus teins, thereby speeding the induction of NETosis. of attention [13,14]. These studies indicated that RA- Although our data are preliminary, they do suggest that derived PMNs were more prone to undergo NETosis, PAD4 is extruded onto the NETs during NETosis, as and that NETs themselves could contribute to the detected with ELISA technology and, to a lesser extent, by generation of auto-antigens (ACPAs) or be the target of fluorescence microscopy. Such an occurrence would have auto-antibodies [13,14]. important implications for the development of anti-PAD4 Our studies, performed independently at a time similar autoantibodies observed in cases with RA [32]. Because time to these, corroborate that NETosis is enhanced in the presence of such antibodies precedes the develop- RA, confirming a possible fundamental role of this ment of RA, our data provide further support that NETs phenomenon in the underlying etiology of RA. In may contribute to the underlying etiology of RA, and addition, we extended these observations by examining for maybearelativelyearly event. As thepresenceofsuch changes in the underlying signal-transduction cascade anti-PAD4 antibodies has been shown to enhance the required for the induction of NETosis. The results show enzymatic activity of PAD4 in an extracellular environ- that the propensity of circulatory PMNs in RA patients to ment by reducing the calcium requirement [33], their undergo NETosis is associated with elevations in members combination with NETs-associated PAD4 could lead to of this cascade, including increased intracellular ROS pro- prodigious quantities of citrullinated autoantigens. In duction, enhanced expression of NE and MPO, increased addition, the extracellular presence of PAD4 on NETs nuclear translocation of PAD4, and citrullination of his- may further promote the prodigious generation of tones, notably H3. Consequently, these and other key citrullinated antigens, because molecular structures in- NETotic pathway elements [6] could serve as potential volving the attachment of enzymes to DNA lattices have therapeutic targets for interventional strategies. been shown to increase their catalytic activity enor- Furthermore, by examining kinetic changes during ex- mously, and thereby form the basis of nano-machines tended in vitro culture, different nuclear morphometric or nano-factories, generating such autoantigens [34]. characteristics were observed in PMNs from RA cases, Although these findings must be verified, and it remains with a lower proportion of the classic lobulated pheno- to be ascertained whether extracellular NETs-associated type, coupled with a much higher proportion of delobu- PAD4 is active, these data do support and extend recent re- lated cells at the initial time point. Unlike in controls, in ports indicating that NETs can be a source for citrullinated which an increase in this population was noted over time, autoantigens, and that they react with ACPA or anti-PAD4 this latter population decreased during in vitro culture in antibodies [13,14,35]. Taken together, these data provide RA PMNs. RA PMNs also progressed more rapidly and further evidence concerning a key role for PAD4 in the extensively to a NETotic-spread phenotype than controls, Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 13 of 14 http://arthritis-research.com/content/16/3/R122 underlying etiology of RA, and offer a potential explanation lost the ability to induce NETosis on depletion of IgG for the efficacy of PAD4 inhibitor chloramidine in reducing molecules, but reconstitution of NET induction was seen disease symptoms in collagen-induced rat and murine arth- only with IgG molecules obtained from ACPA-positive ritismodelsfor RA [36]. serum. The assessment of NETosis-derived products in It recently was reported that ACPA or IgM RF led to the sera of suspected RA cases may offer a novel comple- potent increases in NET formation compared with control mentary diagnostic tool. IgG [13]. In our IgG-depletion experiments on ACPA- positive and -negative RA cases, we observed a marked Additional files reduction of NET induction to control levels in both cases, Additional file 1: Figure S1. Neutrophil, peripheral blood leukocyte whereas reconstitution of serum with IgG gained from counts, and age distribution in RA cases and control cohorts. depletion almost completely restored NET induction in Additional file 2: Table S1. AUC values with corresponding 95% the ACPA-positive cases. However, in the ACPA-negative confidence intervals, P values and standard errors for serum cell-free cases, no significant increase followed reconstitution. nucleosomes and the three different parameters, which were analyzed individually by logistic regression. These results support the notion that ACPAs are import- Additional file 3: Figure S2. Elevated serum levels of NET components, ant inducers of NET formation in ACPA-positive RA in RA patients, have potential clinical utility. cases, and indicate that other mechanisms, such as IgG complexes similar to those involved in NET induction in Abbreviations SLE [8], are operative in ACPA-negative RA. Both mecha- ACPA: anti-citrullinated peptide antibody; AUC: area under the curve; nisms could lead to a common distal mechanism of induc- citH3: citrullinated histone; DAPI: 4′,6-diamidino-2-phenylindole; DCFH-DA: 2′,7′-dichlorodihydro fluorescein diacetate; ELISA: enzyme-linked tion of arthritis. immunosorbent assay; GAPDH: glyceraldehyde-3-phosphate The observation that the coagulation of blood samples dehydrogenase; HBSS: Hanks balanced salt solution; MPO: myeloperoxidase; from RA patients during serum preparation triggers ex- NADPH: nicotinamide adenine dinucleotide phosphate; NE: neutrophil elastase; NET: neutrophil extracellular trap; PAD4: peptidyl arginine deiminase 4; tensive NETosis, evident by increased concentrations of PMA: phorbol-12-myristate-13-acetate; PMN: polymorphonuclear granulocyte; cell-free DNA, nucleosomes, or nucleosome/MPO com- RA: rheumatoid arthritis; RF: rheumatoid factor; ROC: receiver operator plexes, may have unexpected clinical ramifications. With characteristic; ROS: reactive oxygen species; SLE: systemic lupus erythematosus. a sensitivity of 91% and a specificity of 92%, it is possible Competing interests that the assessment of serum cell-free nucleosomes may A patent filing has been submitted by the University of Basel and the serve to distinguish suspected RA patients from healthy Cantonal Hospital Aarau for tests developed during this research. We have controls with a high degree of specificity. It is of interest no other interests to declare. that this aspect was not significantly influenced by the Authors’ contributions ACPA status of the RA patients. As such, this assay may CSC and SG carried out molecular, cellular studies, immunoassays, and be a useful complementary test to perform in conjunc- assisted with the manuscript draft. SG conducted in vitro depletion experiments, performed immune histochemical analyses, morphometric tion with current ACPA or RF assays, not only to extend analyses, statistical analysis and contributed to writing the manuscript. UW diagnostic accuracy, but also to assist in detecting RA in and AB participated in the design of the study, assisted with stratification of cases that are either ACPA or RF negative. Similar NET patients and healthy donor controls, and assisted with the manuscript draft. SH and PH conceived the study, participated in its design, coordination and induction by ACPA-positive and -negative RA sera and its wrote the manuscript. All authors read and approved the final manuscript. abrogation by IgG depletion, as discussed earlier, supports the functional aspect of the nucleosome measurement in Acknowledgements RA serum. We are grateful to Peter Erb, Ed Palmer and Alan Tyndall for helpful suggestions and comments. We thank Swarna Machineni for performing the In a preliminary series of SLE sera, a small and not ROS analyses, and Maria Stoikou for assistance with the IgG depletion statistically significant increase of cell-free nucleosomes studies. A. Schoetzau, Eudox, Basel, Switzerland provided statistical over normal controls was observed, indicating a slightly supervision. Norman Bandelow, Christoph Hemmeler, Eric Deman, Rheumaklinik, Kantonsspital Aarau, and Robyn Benz, Department of elevated propensity for PMNs from SLE patients to Rheumatology, University Hospital, Basel, provided patient samples. undergo NETosis. This was, however, nowhere near the This project was supported by a grant from the Fonds W & W of the range seen in RA cases, and failed to reach an ROC Cantonal Hospital Aarau. The position of Chanchal Sur Chowdhury was supported by the University Women's Hospital Basel. AUC considered to be clinically relevant. These aspects must be validated in larger multicenter studies. Author details Department of Biomedicine, University Hospital Basel, Hebelstrasse 20, 4032 Basel, Switzerland. Department of Rheumatology, Kantonsspital Aarau, Conclusions Tellstrasse, 5001 Aarau, Switzerland. Department of Rheumatology, In summary, our data reaffirm an intricate relation between University Hospital Basel, Basel, Switzerland. Division of Haematology, NETosisand theetiologyofRA, becausethe signalingele- Department of Internal Medicine, University Hospital Basel, Blood Transfusion Centre, Swiss Red Cross, Basel, Switzerland. ments associated with NET extrusion are significantly en- hanced to promote NETosis in RA patients compared with Received: 12 July 2013 Accepted: 21 May 2014 healthy controls. Both ACPA-positive and -negative serum Published: 13 June 2014 Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 14 of 14 http://arthritis-research.com/content/16/3/R122 References bacterial killing in the presence of hydrogen peroxide. 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Demers M, Krause DS, Schatzberg D, Martinod K, Voorhees JR, Fuchs TA, Scadden DT, Wagner DD: Cancers predispose neutrophils to release • Convenient online submission extracellular DNA traps that contribute to cancer-associated thrombosis. • Thorough peer review Proc Natl Acad Sci U S A 2012, 109:13076–13081. 18. Fuchs TA, Alvarez JJ, Martinod K, Bhandari AA, Kaufman RM, Wagner DD: • No space constraints or color figure charges Neutrophils release extracellular DNA traps during storage of red blood • Immediate publication on acceptance cell units. Transfusion 2013, 53:3210–3216. 19. Fuchs TA, Brill A, Wagner DD: Neutrophil extracellular trap (NET) impact • Inclusion in PubMed, CAS, Scopus and Google Scholar on deep vein thrombosis. Arterioscler Thromb Vasc Biol 2012, 32:1777–1783. • Research which is freely available for redistribution 20. 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Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility

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Copyright © 2014 by Sur Chowdhury et al.; licensee BioMed Central Ltd.
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Medicine & Public Health; Rheumatology; Orthopedics
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Abstract

Introduction: Neutrophil extracellular traps (NETs) have recently been implicated in a number of autoimmune conditions, including rheumatoid arthritis (RA). We examined the underlying signaling pathways triggering enhanced NETosis in RA and ascertained whether the products of NETosis had diagnostic implications or usefulness. Methods: Neutrophils were isolated from RA patients with active disease and from controls. Spontaneous NET formation from RA and control neutrophils was assessed in vitro with microscopy and enzyme-linked immunosorbent assay (ELISA) for NETosis-derived products. The analysis of the signal-transduction cascade included reactive oxygen species (ROS) production, myeloperoxidase (MPO), neutrophil elastase (NE), peptidyl arginine deiminase 4 (PAD4), and citrullinated histone 3 (citH3). NET formation was studied in response to serum and synovial fluid and immunoglobulin G (IgG) depleted and reconstituted serum. Serum was analyzed for NETosis-derived products, for which receiver operator characteristic (ROC) curves were calculated. Results: Neutrophils from RA cases exhibited increased spontaneous NET formation in vitro, associated with elevated ROS production, enhanced NE and MPO expression, nuclear translocation of PAD4, PAD4-mediated citrullination of H3, and altered nuclear morphology. NET formation in both anti-citrullinated peptide antibody (ACPA)-positive and -negative RA was abolished by IgG depletion, but restored only with ACPA-positive IgG. NETosis-derived products in RA serum demonstrated diagnostic potential, the ROC area under the curve for cell-free nucleosomes being >97%, with a sensitivity of 91% and a specificity of 92%. No significant difference was observed between ACPA-positive and -negative cases. Conclusions: Signaling elements associated with the extrusion of NETs are significantly enhanced to promote NETosis in RA compared with healthy controls. NETosis depended on the presence of ACPA in ACPA-positive RA serum. The quantitation of NETosis-derived products, such as cell-free nucleosomes in serum, may be a useful complementary tool to discriminate between healthy controls and RA cases. * Correspondence: [email protected]; [email protected] Equal contributors Department of Biomedicine, University Hospital Basel, Hebelstrasse 20, 4032 Basel, Switzerland Department of Rheumatology, Kantonsspital Aarau, Tellstrasse, 5001 Aarau, Switzerland Full list of author information is available at the end of the article © 2014 Sur Chowdhury et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 2 of 14 http://arthritis-research.com/content/16/3/R122 Introduction general condition, age ≥28 and ≤70 years, and for blood A novel feature of the biology of polymorphonuclear gran- donors fulfilling national criteria for blood donation. ulocytes (PMNs) is their ability to generate neutrophil Exclusion criteria were current or previous systemic extracellular traps (NETs) [1] via a distinct process of cell autoimmune disease, asthma and reconvalescence after death termed NETosis [2]. NETs consist of extruded major illness, surgery, current medication with corti- chromosomal DNA decorated with granular components costeroids, immunosuppressive agents, and malignant that include antimicrobial peptides and proteases. The neoplasia or chemotherapy within 5 years before molecular pathways leading to NETosis encompass cal- recruitment for the study. RA cases had a DAS ≥2.6, cium mobilization, generation of reactive oxygen species were from age ≥27 to ≤70 years, and had no other (ROS), nuclear delobulation involving the enzymatic activ- systemic autoimmune disease, including ankylosing ities of myeloperoxidase (MPO) and neutrophil elastase spondylitis and psoriatic arthritis. (NE), and chromatin modification via the citrullination of Exclusion criteria were corticosteroids ≥40 mg equiva- histones by peptidyl arginine deiminase 4 (PAD4) [2-6]. lent of prednisone daily, and those mentioned earlier for A number of studies have implicated NETs in the healthy controls. Informed, written consent was obtained etiology of autoinflammatory or autoimmune conditions from all subjects in the study, which was approved by such as preeclampsia, Felty syndrome, systemic lupus ery- the Cantonal Ethical Review Boards of Aargau-Solothurn thematosus (SLE), multiple sclerosis, and, most recently, and Basel/Basel-Land, Switzerland. rheumatoid arthritis (RA) [7-13]. In the context of RA, these findings are especially interesting, as NETs have Preparation of plasma and serum been proposed to contribute to the generation of anti- Plasma and serum were collected and processed as citrullinated protein antibody (ACPA) autoantigens, and described previously [15]. Samples were studied immedi- may also be a target for autoantibodies [13,14]. PMNs iso- ately or stored at −80°C until analysis. lated from RA patients showed an increased propensity to undergo spontaneous and LPS-induced NETosis, which Cell isolation was in part mediated by TNF and IL-17 and could be PMNs were isolated with Dextran-Ficoll density centri- inhibited by blocking NADPH oxidase or PAD4. Whereas fugation [8]. Cell viability was 96% to 98%, with a purity the citrullinated autoantigens vimentin and α-enolase of >95% PMNs. Neutrophils seeded in 24-well plates were expressed on NETs from RA PMNs, antibodies to were allowed to settle for 1 hour at 37°C under 5% CO the former were able to induce NET formation by healthy before further experimentation. control PMNs [13,14]. As we had previously detected significantly increased Cell-free DNA isolation and quantification concentrations of cell-free DNA in the sera of RA patients Cell-free DNA extracted from 850 μlplasmaorserum by compared with healthy controls, we were intrigued using the QIAamp Circulating Nucleic Acid Kit (Qiagen, whether the provenance of this material involved NETosis Valencia, CA, USA) was quantified by TaqMan real-time [15]. The premise for the current investigation was that a PCR (StepOne Plus Real-Time PCR System, Applied Bio- link between circulating cell-free DNA levels and NETs systems, Foster City, CA, USA) specific for the glyceral- has previously been made in a number of conditions, dehyde-3-phosphate dehydrogenase (GAPDH) gene [15]. including preeclampsia, sepsis, cancer, thrombosis, or even storage of blood-transfusion products [16-19]. Detection of neutrophil elastase (NE), myeloperoxidase In view of these findings and reports on the complex in- (MPO), and cell-free nucleosomes volvement of neutrophil NETs in autoimmunity, we sought The concentrations of neutrophil elastase (NE) and mye- to investigatethe NEToticresponseofPMNsinRA, with loperoxidase (MPO) were measured with sandwich ELISA particular regard to the underlying signal-transduction (Elastase/a1-PI Complex ELISA Kit, Calbiochem/EMD, cascade, and whether the products of overt NETosis could Gibbstown, NJ, USA) and the human (MPO ELISA Kit, be diagnostically useful. Hycult Biotech, Plymouth Meeting, PA, USA) respectively. Nucleosomes were measured by using the Human Cell PLUS Materials and methods Death Detection ELISA (Roche Diagnostics, Basel, Human subjects Switzerland). Cell-culture supernatants were incubated with All patients fulfilled the American College of Rheuma- DNaseI (10 U for 5 minutes) (Roche Diagnostics) before tology classification criteria for RA, or for systemic analysis [20]. lupus erythematosus, respectively. Healthy volunteers, matched for gender and age, were recruited at the MPO/DNA complex detection hospitals or at the Blood Bank of the Swiss Red Cross, MPO is present on extruded NETs. To detect such Basel. Inclusion criteria for healthy controls were fair structures, NET-associated MPO/DNA complexes were Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 3 of 14 http://arthritis-research.com/content/16/3/R122 quantified by using a modified capture ELISA [21]. In anti-histone H1 + core proteins (EMD Millipore, Billerica, brief, NET-associated MPO in serum or culture super- MA, USA), and rabbit anti-citrullinated histone H3 natant was captured by using the coated 96-well plate of (citH3, Abcam). Secondary antibodies were goat anti- the human MPO ELISA Kit, (Hycult Biotech), after which rabbit IgG AF555, goat anti-rabbit IgG AF488 (Invitrogen the NET-associated DNA backbone was detected by using Life Technologies, San Diego, CA, USA), and goat anti- the detection antibody of the Human Cell Death Detec- mouse IgG AF647. DNA was stained with 4′,6-diamidino- PLUS tion ELISA (Roche Diagnostics). 2-phenylindole (DAPI, Sigma-Aldrich), and NETs were visualized by using a Zeiss Axioplan 2 Imaging fluores- PAD4/DNA-complex detection cence microscope in conjunction with a Zeiss AxioCam To detect the presence of PAD4 on extruded NETs in MRm monochromatic CCD camera and analyzed with culture supernatants after spontaneous NETosis, cell-free Axiovision 4.8.2 software (Carl Zeiss). A minimum of 20 PAD4/DNA complexes were quantified by using a modi- fields (at least 1,000 PMNs) per case was evaluated for fied capture ELISA, akin to that described for MPO MPO/NE and DNA co-staining; nuclear phenotypes and earlier. In brief, cell-free PAD4 was captured by using the NETs were counted and expressed as percentage of the coated 96-well plate of a commercial human PAD4 ELISA total number of cells in the fields. (USCN Life Science, Inc., Wuhan, China), and associated DNA was detected by using Human Cell Death Detection RA serum depletion, IgG purification, and quantification PLUS ELISA kit (Roche Diagnostics). of NETs After three washes with PBS, 200 μl protein G agarose ROS generation analysis (Pierce Biotechnology Inc, Rockford, IL, USA) was incu- ROS was measured by using a 2′,7′-dichloro dihydrofluor- bated with 200 μl ACPA + and ACPA- RA or control escein diacetate (DCFH-DA) assay [22]. The 5 × 10 cells serum diluted in an equal volume of phenol red-free RPMI in a final volume of 500 μl were incubated for 30 minutes 1640 medium overnight at 4°C. The serum/protein G with 25 μM DCFH-DA (Sigma-Aldrich, St. Louis, MO, agarose mixture was centrifuged at 2,500 g for 5 minutes, USA). Fluorescence was measured with flow cytometry and the supernatant (IgG-depleted serum) was carefully (FACSCalibur; BD Biosciences, San Jose, CA, USA). transferred into a new Eppendorf microcentrifuge tube. The protein G agarose pellet was gently washed 3 times Fluorescence and scanning electron microscopy with 500 μlddH O, and the bound antibody was released The 5 × 10 cells isolated PMNs seeded on poly-L-lysine- by the addition of 50 μl0.1 M glycine pH 2–3, and coated coverslips (BD Biosciences) were stimulated with phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) Table 1 Demographics and patient population for 90 minutes and dehydrated with a graded ethanol characteristics versus healthy blood donors series (30%, 50%, 70%, 100%) [8], coated with 2-nm plat- Controls RA Statistics inum, and analyzed with a Nova NanoSEM 230 scanning Age 50.34 ± 1.5 53.03 ± 1.5 P = 0.214 electron microscope (FEI Co., Hillsboro, OR, USA). PMNs Gender (F/M) 24/32 24/8 - were incubated for 10 minutes with 5 μM Sytox Green DAS28 n.a. 3.07 ± 1.12 - dye (Invitrogen Life Technologies, San Diego, CA, USA) Bone erosion (pos/neg) n.a. 22/10 - for assessment of NETs with an Axiovert fluorescence Serum ACPA (pos/neg) n.a. 20/12 - microscope (Carl Zeiss) coupled to a Zeiss AxioCam color CCD camera (Carl Zeiss Microimaging, Oberkoch, Serum RF (pos/neg) n.a. 19/13 - Germany) [8,23]. Serum ANA (pos/neg) n.a. 21/11 - ESR (mm/h) n.a. 16.8 ± 13.1 - Immunohistochemical staining and quantification of NETs CRP (mg/L) n.d. 6.9 ± 5.2 - The 5 × 10 isolated PMNs were seeded on poly-L- PBMC (cells/μl) 1,961 ± 81.69 1513 ± 75.90 P < 0.0001 lysine-coated glass coverslips (BD Biosciences, San Jose, PMN (cells/μl) 3,641 ± 149.7 4575 ± 546.0 P = 0.021 CA, USA) in tissue-culture wells and allowed to settle before stimulation, as described earlier. Coverslips were Therapy (yes/no) n.a. 31/1 - rinsed with ice-cold HBSS and the cells fixed with 4% DMARDs (yes/no) n.u. 27/5 - paraformaldehyde and blocked overnight (HBSS with Biologics (yes/no) n.a. 30/2 - 10% goat serum, 1% BSA, 0.1% Tween20, and 2 mM F, female; M, male; DAS28, disease activity score; n.a., not applicable; pos, EDTA) at 4°C. NETs were detected with rabbit anti-NE positive; neg, negative; ACPA: anti-citrullinated protein antibodies; RF, rheumatoid factor; ANA, antinuclear antibody; ESR, erythrocyte sedimentation rate; CRP, (Abcam, Cambridge, MA, USA), rabbit anti-MPO C-reactive protein; n.d., not determined; PBMC, peripheral blood mononuclear (Dako, Glostrup, Denmark), two different rabbit anti- cell; PMN, polymorphonuclear leukocyte; DMARD, disease-modifying PAD 4 (Abcam), mouse anti-PAD4 (Abcam), mouse antirheumatic drug; n.u., not used. Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 4 of 14 http://arthritis-research.com/content/16/3/R122 immediately equilibrated with 10 μlof 1 M Tris pH 7.5-9. NETs were quantified after IHC staining with mouse All protein concentrations were determined with the MN anti-human MPO antibody (Abcam) and rabbit anti- Protein Quantification Assay (Macherey-Nagel GmbH, human citH3 antibody (Abcam), or the respective isotype Düren, Germany), and isolation of IgG was verified with controls, followed by incubation with goat anti-mouse IgG Coomassie staining of SDS-PAGE. AF555 and goat anti-rabbit IgG AF488 (Invitrogen Life Neutrophils from healthy donors (n =3) were isolated Technologies). DNA was counterstained with 4′,6-diami- and cultured for 2 hours in 96-well culture dishes (Thermo dino-2-phenylindole (DAPI, Sigma-Aldrich). NETs were Fischer Scientific, Waltham, MA, USA), supplemented visualized by using an Olympus IX81 motorized epifluor- with:serum,depletedserum,and purified IgG fromACPA- escence microscope (Olympus America Inc., Center Val- positive RA patients (n = 3), ACPA-negative RA patients ley, PA, USA) in conjunction with an Olympus XM10 (n = 3), and healthy individuals (n = 3) to a final concentra- monochromatic CCD camera (Olympus) and analyzed tion of 100 μg/ml. with the Olympus CellSens Dimension software (Olympus). A C PMN isolation in vitro cell culture Cell-free nucleosomes * *** 1h resting T1 T2 T3 0.8 0.6 Timecourse 0h 1h 3h 0.4 T1 T2 T3 0.2 NE DAPI T1 T2 T3 T1 T2 T3 Control RA Control MPO/Cell-free DNA complexes ***** ** * 0.12 0.10 0.08 RA 0.06 T1 T2 T3 T1 T2 T3 Control RA E F lobulated delobulated/diffused spread * *** * * *** ** **** 60 **** **** 80 70 **** ** *** * lobulated ** ** * delobulated/ 40 30 diffused 10 10 0 0 0 spread NE T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 DAPI Control RA Control RA Control RA Figure 1 RA-derived PMNs exhibited increased spontaneous NETosis and elevated levels of NET-component release. (A) Schematic representation of the time-course design for studying in vitro spontaneous NET release. (B) Detection of in vitro NETosis by immunohistochemistry for neutrophil elastase (NE) (green) and DAPI (blue). Magnification: 20×; scale bar, 50 nm. (C) Concentration of cell-free nucleosomes in PMN culture supernatant by ELISA. (D) Quantification of NET-associated MPO/DNA complexes. These assays indicate that more-rapid and extensive progression of NET formation is observed in RA versus control PMNs. (E) Changes in PMN nuclear morphology during NETosis detected with immunohistochemistry for NE and DAPI. (F) Steady-state (T1) RA-derived PMNs exhibited a greater proportion of delobulated/diffused cells, and progressed rapidly to a NETotic spread phenotype during in vitro culture. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. All data are representative of at least six independent experiments. % neutrophils % neutrophils Absorbance (A -A ) Absorbance (A ) 450 nm 405nm 490nm % neutrophils Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 5 of 14 http://arthritis-research.com/content/16/3/R122 Figure 2 (See legend on next page.) Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 6 of 14 http://arthritis-research.com/content/16/3/R122 (See figure on previous page.) Figure 2 Increased expression of NET-associated signaling elements and nuclear localization of PAD4, citrullination of histone H3 in RA-derived PMN, and possible extrusion of PAD4 on NETs. Baseline ROS levels, measured with flow cytometry, were higher in RA-derived PMNs than in control PMNs (A). Quantitative real-time PCR analysis of NE mRNA (B) and MPO mRNA expression (C), as well as Western blot analysis of MPO protein levels (D), indicated that the levels of these two components required for NETosis were elevated in RA-derived PMNs. Total PAD4 protein (E) or PAD4 mRNA expression levels (F) did not indicate any significant difference between control and RA-derived PMNs. (G) Quantification of PAD4 protein levels in cytoplasmic and nuclear fractions of PMNs from healthy controls and RA patients. Nuclear levels of PAD4 were significantly increased in RA patients, whereas the cytoplasmic levels were lower compared with the healthy control PMNs. (H) Elevated citrullinated histone H3 levels in RA PMN extracts detected with Western blot. (I) Co-localization of citrullinated histone H3 (green) with histone components detected with a pan-histone antibody (red), spread over the entire NET surface (blue). Magnification: upper panel 20×, scale bar, 50 nm; lower panel 63× scale bar, 10 nm. (J) Extracellular localization of PAD4 (red) on extruded NETs by multicolor fluorescent immunohistochemistry. NET DNA is stained blue (DAPI), and histones (panH) are stained green. Magnification, 20×; scale bar, 50 nm (K). Detection of PAD4/cell-free DNA complexes in the culture supernatants of isolated PMN undergoing spontaneous NETosis. Higher levels of these complexes were detected in RA-derived PMN cultures. Time points correspond to those of Figure 1A. Data are represented as mean ± SEM. **P < 0.01; ***P <0.001; ****P < 0.0001; n.s., statistically not significant. All data are representative of at least six independent experiments. A minimum of 20 fields at 10× magnification (at least 500 selection procedure involving six different endogenous to 1,000 PMNs) per case was evaluated for MPO/citH3 and reference genes, as suggested in the MIQE guidelines [26]. −DDCt DNA co-staining through ImageJ analysis software (National Relative values were calculated with 2 analysis [27]. Institutes of Health Image Processing, Bethesda, MD, USA); nuclear phenotypes and NETs were determined, Statistical analysis counted, and expressed as percentage of the total area of All data are presented as mean ± SEM. Descriptive statis- cells in the fields [24]. tics for continuous parameters consisted of median and range, and categoric variables were expressed as percent- Protein isolation and Western blot analysis ages. Comparisons between patients and healthy controls Total protein was isolated with NucleoSpin TriPrep kit were by the Mann–Whitney U test with a Welch posttest (Macherey-Nagel) from 3 × 10 PMNs. Proteins from the correction. Statistical significance in multiple comparisons nuclear and cytoplasmic fractions were isolated by using was by one-way analysis of variance (ANOVA) with a the Nuclear and Cytoplasmic Protein Extraction Kit Dunn posttest correction. P < 0.05 was considered statisti- (Thermo Scientific). Western blotting was performed cally significant. by using AnykD Mini-PROTEAN TGX Gels (Biorad, Receiver operating characteristic (ROC) curves were Hercules, CA, USA) and nylon/nitrocellulose mem- calculated, and the area under the curve (AUC) with branes (Biorad). Primary and secondary antibodies used corresponding standard errors of means was calculated. were: rabbit anti-PAD4 (Abcam), rabbit anti-MPO (Cell Data were processed in GraphPad Prism version 5.0b for Signalling Technologies, Beverly, MA, USA), mouse MacOSX (GraphPad Software Inc., San Diego, CA, anti-β-Actin (Sigma-Aldrich), goat anti-Mouse and/or USA). Analysis of covariance (ANCOVA) was conducted anti-Rabbit, human anti-HRP (Southern Biotech). HRP with SPSS version 21.0 statistical software (IBM SPSS activity was detected by using SuperSignal West Pico Inc., Chicago, IL, USA). Additional professional statis- Chemiluminescent Substrate (Thermo Scientific). Equal tical assistance was provided by A. Schoetzau, Basel. loading was verified by using beta-actin or histone H3, when appropriate. Western blots of citrullinated H3 (citH3) protein were performed according to Shechter Results et al. [25]. Densitometric analysis and protein quantifi- RA-derived PMN exhibit increased spontaneous NETosis cation of the Western blots was performed by using the Details of the RA study group and healthy control group ImageJ software. are described in Table 1 and Additional file 1. As in very recent observations [13], we observed that RNA isolation and quantitative real-time PCR RA-derived PMNs underwent greater degrees of NETosis Total RNA was isolated by using RNeasy Mini Kit (Qiagen). than did control PMNs in vitro (data not shown). To study TaqMan real-time quantitative RT-PCR was performed by this facet in more detail, we examined the kinetics of using the Applied Biosystems StepOne Plus cycler (Applied spontaneous NET extrusion, for which purpose, PMNs Biosystems) and TaqMan Gene Expression Assay primer/ isolated from peripheral blood samples were allowed to probe sets (Applied Biosystems) for ELANE (HS00236952_ settle for 1 hour and then cultured for a period of up to m1), MPO (HS00924296_m1), PADI4 (HS00202612_m1), 3hours in vitro (Figure 1A), NETs being detected by im- and β -microglobulin B2M (HS99999907_m1). Data were munohistochemistry for neutrophil elastase (NE) and DAPI normalized by using the housekeeping gene B2M, after a (4′,6-diamidino-2-phenylindole)(Figure 1B). In addition, Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 7 of 14 http://arthritis-research.com/content/16/3/R122 Control +PMA RA +PMA Control UT Control +PMA RA UT RA +PMA MPO DAPI MPO lobulated delobulated/diffused spread 80 60 100 **** ** **** n.s. **** **** n.s. **** **** **** * **** 40 30 0 0 0 -+-+ PMA -+-+ PMA -+-+ PMA Control RA Control RA Control RA PAD4 protein expression Cell-free nucleosomes ** 3.0 ** * * * ** 4.0 2.0 0.6 3.0 0.5 0.4 2.0 0.3 0.2 1.0 0.1 0.0 0.0 - - - - PMA + + + + PMA - - - - + + + + Control RA Control RA Cl-amidine - - - - + + + + Cytoplasmic Nuclear Control RA Figure 3 (See legend on next page.) Absorbance (A -A ) % neutrophils 405nm 490nm % neutrophils Arbitrary units % neutrophils Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 8 of 14 http://arthritis-research.com/content/16/3/R122 (See figure on previous page.) Figure 3 Increased NETotic response of RA-derived PMNs to PMA. (A) Scanning electron micrographs of NETs induced by PMA (25 nM) indicate the excessive NETotic response of RA-derived PMN. Scale bar, 20 nm. (B) Assessment of NETs induced by PMA treatment by fluorescent immunohistochemistry for MPO (red) and DAPI (blue), indicating the increased response of RA PMNs to PMA (25 nM). Magnification, 20×; scale bar, 50 nm. (C) Analysis of the nuclear phenotype indicated a vast decrease in delobulated/diffused RA PMN nuclei after treatment with PMA and rapid increase in the NETotic-spread phenotype. (D) Release of cell-free nucleosomes after PMA treatment is abrogated by chloramidine, a PAD4 inhibitor. (E) Increased nuclear localization and concomitant decrease in cytoplasmic PAD4 protein levels after PMA treatment, with a clear tendency for an increased responsiveness to the PMA stimulus by RA PMN. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., statistically not significant. All data are representative of at least four independent experiments. we quantitatively assessed the degree of in vitro NETosis in of NE (Figure 2B) and MPO (Figure 2C and D), as deter- these cultures by determining the concentration of cell-free mined with real-time PCR and/or Western blotting. nucleosomes in the respective supernatants (Figure 1C), Surprisingly, neither PAD4 mRNA expression nor specifically their association with myeloperoxidase (MPO), PAD4 levels in total cellular protein showed any discern- indicative of the NETotic origin of this material [2,21] ible difference between RA and control PMNs (Figure 2E (Figure 1D). We also measured NET-associated MPO en- and F, respectively). Because PAD4 translocates to the zymatic activity by using tetramethylbenzidine (TMB) as a nucleus on PMN activation [4,29], where it citrullinates substrate (data not shown). The results clearly indicate histone proteins, such as H3, we examined its presence that RA-derived PMNs generated NETs more rapidly, to a in nuclear and cytoplasmic PMN fractions. Compared greater magnitude, and more extensively than did control with control PMNs, PAD4 was preferentially located in healthy PMNs, which was particularly evident at the 3-hour the nucleus of RA-derived PMNs (Figure 2G). The nu- stage of in vitro culture (Figure 1B to D). Accounting for clear presence of PAD4 was associated with increased variances in PMN counts, the difference between the citrullinated histone H3 (citH3) levels with Western blot healthy control and RA subjects remained highly significant analysis in PMNs from RA cases compared with controls in an analysis of covariance (ANCOVA) of the nucleosome (Figure 2H). Furthermore, citrullinated histone H3 could assay (P <0.001). be readily detected on NET structures (Figure 2I). During NETosis, the morphology of the PMN nucleus changes from the familiar lobulated to a diffused and then Potential extracellular localization of PAD4 on NETs to a spread phenotype (Figure 1E) [28]. By examining and Because we observed elevated nuclear translocation of enumerating these features, it was observed that at base- PAD4 in RA PMNs, we examined whether this enzyme is line (T1), the nuclei from healthy control PMNs were extruded into the extracellular environment during NETo- predominantly lobulated, whereas in this instance, the sis. Unfortunately, the visualization of such an event by majority of RA-derived PMN nuclei exhibited a delobu- fluorescence immunohistochemistry proved to be difficult lated or diffused nuclear phenotype (Figure 1F). In RA- with a variety of commercially available antibodies, and we derived PMNs, this delobulated population decreased obtained only rudimentary evidence for the presence of over time, giving rise to NETotic cells with a spread PAD4 on NETs by this means (refer to Figure 2J). phenotype (Figure 1F). In contrast, in normal PMNs, we We were, however, able to detect PAD4/cell-free DNA noted a steady progression in the proportion of delobu- complexes readily in culture supernatants from isolated lated cells (Figure 1F). The spontaneous progression of PMNs, the levels of which were increased in cases with RA nuclei to the NETotic-spread phenotype was more pro- (Figure 2K). It is, therefore, quite probable that PAD4 is asso- nounced in RA than in normal PMNs, a feature most ciated with NETs structures after aberrant NETosis in RA. evident after 3 hours (T3) (Figure 1F). PMNs from RA patients present an enhanced NETotic RA-derived PMNs demonstrate increased expression of response to PMA, and normal PMNs are strongly affected NET-associated signaling elements, nuclear localization of by RA serum and synovial fluid PAD4, and augmented H3 citrullination In autoinflammatory or malignant conditions, such as NETosis has been shown to depend on a number of bio- SLE or cancer, an elevated NETotic response of PMNs chemical signaling elements, among which are the gen- to an external activation signal has been shown [9,13]. eration of ROS by nicotinamide adenine dinucleotide In our experiments, we noted that when RA-derived phosphate (NADPH) oxidase, the action of NE in com- PMNs were treated with PMA, they responded far more bination with MPO, and histone citrullination by PAD4 vigorously with regard to NETosis than did controls, as [2,3,5]. RA-derived PMNs exhibited increased basal intra- detected by SEM and fluorescence microscopy (Figure 3A cellular ROS levels (Figure 2A), as well as increased levels and B, respectively). Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 9 of 14 http://arthritis-research.com/content/16/3/R122 Figure 4 (See legend on next page.) Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 10 of 14 http://arthritis-research.com/content/16/3/R122 (See figure on previous page.) Figure 4 Influence of RA serum and synovial fluid on normal PMNs. (A) Incubation of healthy donor PMNs with serum (Se) from healthy donors or from RA patients, synovial fluid (SF) from patients with noninflammatory osteoarthritis (OA) or synovial fluid from RA patients. Immunohistochemical analysis of four main components of NETs (NE, MPO, PAD4, and citH3) revealed that RA-derived serum and SF enhanced NETosis in normal PMN compared to healthy control serum or noninflammatory OA SF. PAD4 (white arrowheads) and citH3 (empty arrowheads) colocalize with unmodified histones on NETs. Magnification, 20×; scale bars, 50 nm. (B) Release of cell-free nucleosomes during in vitro culture by PMNs from healthy controls incubated with control serum, RA serum, OA SF or RA SF, or PMA. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01. (C) Increased ROS generation during in vitro culture by PMNs from healthy controls incubated with control serum, RA serum, OA SF or RA SF, or PMA. Data are presented as mean ± SEM. (D) Release of cell-free nucleosomes during in vitro culture by PMNs from healthy controls, ACPA-positive and ACPA-negative RA patients incubated with serum (Se +), IgG-depleted serum (Se -), or serum reconstituted with their respective eluted IgG, or with PMA. Data are presented as mean ± SEM. *P < 0.05; ***P < 0.001; ****P < 0.0001; n.s., statistically not significant. All data are representative of at least four independent experiments in Figures 4A–C, and 3 in 4D. In addition, morphometric assessment indicated that RA- PMNs, and that ACPA and also IgM RF are to a large derived PMNs exhibited a larger decrease in cells with a part responsible for this effect [13,14]. delobulated phenotype and a greater progression toward a NETotic-spread nuclear phenotype than control PMNs (Figure 3C), a feature accompanied by excessive release of Serum from RA patients shows elevated levels of the cell-free nucleosomes in culture supernatants (Figure 3D). principal components of NETs, indicating enhanced NET PMA appears to activate PAD4, as it enhanced transloca- extrusion during clot formation, which has potential tion from the cytoplasm to the nucleus (Figure 3E). The clinical utility stimulatory effect of PMA on the release of nucleosomes As we had previously observed increased levels of cell-free into the supernatant was abrogated by Cl-amidine, a chem- DNA (cfDNA) in RA sera [15], we determined whether ical inhibitor of PAD4, indicating that PAD4 signaling is ne- this resulted from enhanced NETosis, and whether this cessary for NETosis induced by PMA [4,30] (Figure 3D). could have diagnostic applications. cfDNA concentrations ThesedataindicatethatPMNsinRAare susceptibleto were indeed significantly higher in serum samples from increased NETosis after stimulation by a secondary signal, RA cases compared with age-matched healthy control sera such as that mediated by PMA. (Figure 5A). In parallel, the concentrations of cell-free nu- Because SLE sera and RA sera and synovial fluid (SF) cleosomes, NE, and MPO were significantly elevated in have been shown to confer an increased NETotic response RA serum compared with control sera (Figure 5B to D). [9,13], we examined whether RA-derived sera or SF The association of a significant fraction of MPO with exerted similar effects on normal PMNs. As noninflamma- markedly elevated cell-free nucleosomes in RA serum, tory controls, we used healthy serum or osteoarthritis SF. which constitute a main component of NETs (Figure 5E), Both RA sera and SF induced a pronounced increase in clearly suggests that NETosis is indeed the source of nu- NETosis, which was paralleled by an increase in the cleosome material present in RA serum [21]. nucleosome content of the supernatant (Figure 4A,B), as Since there was no significant elevation of these parame- well as in ROS production (Figure 4C) when compared ters in simultaneously obtained plasma samples that were with healthy serum or osteoarthritis SF, respectively. processed immediately, these data demonstrate a propen- To assess whether antibodies participate in the effects sity for RA PMN to undergo increased NETosis during of RA serum on normal PMNs, we depleted IgG from coagulation (Figure 5A to E). serum of ACPA-positive and -negative RA patients and To ascertain whether NET-associated serum compo- healthy controls. Compared with nondepleted sera, IgG nents could be diagnostically useful, we conducted ROC depletion of both ACPA-positive and -negative sera analyses. For serum cell-free nucleosomes, this yielded the markedly reduced NET induction to levels of normal surprisingly high AUC value of 0.97 (see Additional file 2 serum (Figure 4D). Whereas the reconstitution of ACPA- and Figure 5F). Of interest is that no significant difference negative IgG to serum did not increase NET formation was found in this value regardless of whether the RA cases significantly compared with controls, it was practically were ACPA positive or not (Figure 5G), although a slight reversed to the original value in the ACPA-positive cases. trend for serum nucleosome levels was higher in ACPA- This indicates a prominent role for ACPA in the induction positive cases than in ACPA-negative cases (Figure 5H). of NETs in ACPA-positive RA, while suggesting that an The AUC for serum nucleosomes was significantly higher alternative mechanism is responsible for the increased than for any of the other parameters examined (Figure 5I NETosis in ACPA-negative RA patients. to K). With the cut-off set at 0.78, the ROC AUC trans- These data are in accordance with recent findings that lates into a sensitivity of 91%, with a specificity of 92% for RA-derived serum and SF induce NETosis in normal differentiating between RA cases and healthy controls. Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 11 of 14 http://arthritis-research.com/content/16/3/R122 Figure 5 (See legend on next page.) Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 12 of 14 http://arthritis-research.com/content/16/3/R122 (See figure on previous page.) Figure 5 Elevated serum levels of NET components in RA patients have potential clinical utility. (A) Cell-free DNA levels in plasma and serum from healthy matched blood donors (n = 41) and patients with RA (n = 32) determined with real-time PCR. (B) Cell-free nucleosome levels in plasma and serum from healthy donor controls and patients with RA, determined with ELISA. (C) Determination of NE protein concentrations in plasma and serum from healthy donors and patients with RA, as assessed with sandwich ELISA. (D) MPO concentrations in plasma and serum from healthy donors and patients with RA, as determined with sandwich ELISA. (E) NET-associated MPO/DNA complexes quantified by using a modified capture ELISA. In contrast to the serum levels, none of the plasma levels of these NET components attained statistical significance (Figure 5A to E). (F) ROC analysis of cell-free nucleosomes in serum of patients with RA and healthy controls. (G) Detail of cell-free nucleosome ROC curve with groups of ACPA + and ACPA- RA cases and (H) scatterbox and whisker plots with individual values for control, ACPA + and ACPA- groups. The ROC curve analysis of other NET components, cell-free DNA (I),NE (J), and MPO (K), was not as conclusive as that for cell-free nucleosomes. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., statistically not significant. In contrast to RA cases, cell-free nucleosome serum a finding confirmed by analysis of culture supernatants for values of 14 cases with SLE showed a slight, but statisti- the products of NETosis. cally significant increase (Additional file 2). This translated Akin to that observed in an array of other pathologic into an ROC AUC of 0.7639 (see Additional file 3), conditions ranging from preeclampsia and SLE to cancer which is below the clinically useful value for diagnostic and RA [8,9,12,13,17], PMNs from RA patients exhibited purposes. an increased response to further stimulation (for instance, by treatment with IL-8, the phorbol ester PMA, or with LPS). This response is in part mediated via the action of PAD4, as the effect of PMA could be significantly reduced Discussion by treatment with Cl-amidine, an inhibitor of PAD4 [30]. Although PMNs figure prominently in the joint effu- In addition, PMA treatment led to an increased nuclear sions and inflamed synovial tissue of RA patients [31], localization of this enzyme, where it presumably could the potential roles of NETotic events in the pathophysi- catalyze a more-extensive citrullination of histone pro- ology of this disorder have only recently become a focus teins, thereby speeding the induction of NETosis. of attention [13,14]. These studies indicated that RA- Although our data are preliminary, they do suggest that derived PMNs were more prone to undergo NETosis, PAD4 is extruded onto the NETs during NETosis, as and that NETs themselves could contribute to the detected with ELISA technology and, to a lesser extent, by generation of auto-antigens (ACPAs) or be the target of fluorescence microscopy. Such an occurrence would have auto-antibodies [13,14]. important implications for the development of anti-PAD4 Our studies, performed independently at a time similar autoantibodies observed in cases with RA [32]. Because time to these, corroborate that NETosis is enhanced in the presence of such antibodies precedes the develop- RA, confirming a possible fundamental role of this ment of RA, our data provide further support that NETs phenomenon in the underlying etiology of RA. In may contribute to the underlying etiology of RA, and addition, we extended these observations by examining for maybearelativelyearly event. As thepresenceofsuch changes in the underlying signal-transduction cascade anti-PAD4 antibodies has been shown to enhance the required for the induction of NETosis. The results show enzymatic activity of PAD4 in an extracellular environ- that the propensity of circulatory PMNs in RA patients to ment by reducing the calcium requirement [33], their undergo NETosis is associated with elevations in members combination with NETs-associated PAD4 could lead to of this cascade, including increased intracellular ROS pro- prodigious quantities of citrullinated autoantigens. In duction, enhanced expression of NE and MPO, increased addition, the extracellular presence of PAD4 on NETs nuclear translocation of PAD4, and citrullination of his- may further promote the prodigious generation of tones, notably H3. Consequently, these and other key citrullinated antigens, because molecular structures in- NETotic pathway elements [6] could serve as potential volving the attachment of enzymes to DNA lattices have therapeutic targets for interventional strategies. been shown to increase their catalytic activity enor- Furthermore, by examining kinetic changes during ex- mously, and thereby form the basis of nano-machines tended in vitro culture, different nuclear morphometric or nano-factories, generating such autoantigens [34]. characteristics were observed in PMNs from RA cases, Although these findings must be verified, and it remains with a lower proportion of the classic lobulated pheno- to be ascertained whether extracellular NETs-associated type, coupled with a much higher proportion of delobu- PAD4 is active, these data do support and extend recent re- lated cells at the initial time point. Unlike in controls, in ports indicating that NETs can be a source for citrullinated which an increase in this population was noted over time, autoantigens, and that they react with ACPA or anti-PAD4 this latter population decreased during in vitro culture in antibodies [13,14,35]. Taken together, these data provide RA PMNs. RA PMNs also progressed more rapidly and further evidence concerning a key role for PAD4 in the extensively to a NETotic-spread phenotype than controls, Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 13 of 14 http://arthritis-research.com/content/16/3/R122 underlying etiology of RA, and offer a potential explanation lost the ability to induce NETosis on depletion of IgG for the efficacy of PAD4 inhibitor chloramidine in reducing molecules, but reconstitution of NET induction was seen disease symptoms in collagen-induced rat and murine arth- only with IgG molecules obtained from ACPA-positive ritismodelsfor RA [36]. serum. The assessment of NETosis-derived products in It recently was reported that ACPA or IgM RF led to the sera of suspected RA cases may offer a novel comple- potent increases in NET formation compared with control mentary diagnostic tool. IgG [13]. In our IgG-depletion experiments on ACPA- positive and -negative RA cases, we observed a marked Additional files reduction of NET induction to control levels in both cases, Additional file 1: Figure S1. Neutrophil, peripheral blood leukocyte whereas reconstitution of serum with IgG gained from counts, and age distribution in RA cases and control cohorts. depletion almost completely restored NET induction in Additional file 2: Table S1. AUC values with corresponding 95% the ACPA-positive cases. However, in the ACPA-negative confidence intervals, P values and standard errors for serum cell-free cases, no significant increase followed reconstitution. nucleosomes and the three different parameters, which were analyzed individually by logistic regression. These results support the notion that ACPAs are import- Additional file 3: Figure S2. Elevated serum levels of NET components, ant inducers of NET formation in ACPA-positive RA in RA patients, have potential clinical utility. cases, and indicate that other mechanisms, such as IgG complexes similar to those involved in NET induction in Abbreviations SLE [8], are operative in ACPA-negative RA. Both mecha- ACPA: anti-citrullinated peptide antibody; AUC: area under the curve; nisms could lead to a common distal mechanism of induc- citH3: citrullinated histone; DAPI: 4′,6-diamidino-2-phenylindole; DCFH-DA: 2′,7′-dichlorodihydro fluorescein diacetate; ELISA: enzyme-linked tion of arthritis. immunosorbent assay; GAPDH: glyceraldehyde-3-phosphate The observation that the coagulation of blood samples dehydrogenase; HBSS: Hanks balanced salt solution; MPO: myeloperoxidase; from RA patients during serum preparation triggers ex- NADPH: nicotinamide adenine dinucleotide phosphate; NE: neutrophil elastase; NET: neutrophil extracellular trap; PAD4: peptidyl arginine deiminase 4; tensive NETosis, evident by increased concentrations of PMA: phorbol-12-myristate-13-acetate; PMN: polymorphonuclear granulocyte; cell-free DNA, nucleosomes, or nucleosome/MPO com- RA: rheumatoid arthritis; RF: rheumatoid factor; ROC: receiver operator plexes, may have unexpected clinical ramifications. With characteristic; ROS: reactive oxygen species; SLE: systemic lupus erythematosus. a sensitivity of 91% and a specificity of 92%, it is possible Competing interests that the assessment of serum cell-free nucleosomes may A patent filing has been submitted by the University of Basel and the serve to distinguish suspected RA patients from healthy Cantonal Hospital Aarau for tests developed during this research. We have controls with a high degree of specificity. It is of interest no other interests to declare. that this aspect was not significantly influenced by the Authors’ contributions ACPA status of the RA patients. As such, this assay may CSC and SG carried out molecular, cellular studies, immunoassays, and be a useful complementary test to perform in conjunc- assisted with the manuscript draft. SG conducted in vitro depletion experiments, performed immune histochemical analyses, morphometric tion with current ACPA or RF assays, not only to extend analyses, statistical analysis and contributed to writing the manuscript. UW diagnostic accuracy, but also to assist in detecting RA in and AB participated in the design of the study, assisted with stratification of cases that are either ACPA or RF negative. Similar NET patients and healthy donor controls, and assisted with the manuscript draft. SH and PH conceived the study, participated in its design, coordination and induction by ACPA-positive and -negative RA sera and its wrote the manuscript. All authors read and approved the final manuscript. abrogation by IgG depletion, as discussed earlier, supports the functional aspect of the nucleosome measurement in Acknowledgements RA serum. We are grateful to Peter Erb, Ed Palmer and Alan Tyndall for helpful suggestions and comments. We thank Swarna Machineni for performing the In a preliminary series of SLE sera, a small and not ROS analyses, and Maria Stoikou for assistance with the IgG depletion statistically significant increase of cell-free nucleosomes studies. A. Schoetzau, Eudox, Basel, Switzerland provided statistical over normal controls was observed, indicating a slightly supervision. Norman Bandelow, Christoph Hemmeler, Eric Deman, Rheumaklinik, Kantonsspital Aarau, and Robyn Benz, Department of elevated propensity for PMNs from SLE patients to Rheumatology, University Hospital, Basel, provided patient samples. undergo NETosis. This was, however, nowhere near the This project was supported by a grant from the Fonds W & W of the range seen in RA cases, and failed to reach an ROC Cantonal Hospital Aarau. The position of Chanchal Sur Chowdhury was supported by the University Women's Hospital Basel. AUC considered to be clinically relevant. These aspects must be validated in larger multicenter studies. Author details Department of Biomedicine, University Hospital Basel, Hebelstrasse 20, 4032 Basel, Switzerland. Department of Rheumatology, Kantonsspital Aarau, Conclusions Tellstrasse, 5001 Aarau, Switzerland. Department of Rheumatology, In summary, our data reaffirm an intricate relation between University Hospital Basel, Basel, Switzerland. Division of Haematology, NETosisand theetiologyofRA, becausethe signalingele- Department of Internal Medicine, University Hospital Basel, Blood Transfusion Centre, Swiss Red Cross, Basel, Switzerland. ments associated with NET extrusion are significantly en- hanced to promote NETosis in RA patients compared with Received: 12 July 2013 Accepted: 21 May 2014 healthy controls. Both ACPA-positive and -negative serum Published: 13 June 2014 Sur Chowdhury et al. Arthritis Research & Therapy 2014, 16:R122 Page 14 of 14 http://arthritis-research.com/content/16/3/R122 References bacterial killing in the presence of hydrogen peroxide. 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