Previous studies identified nuclear localization of PAD4 in neutrophils (43). We used a slightly different fixation and permeabilization method to stain neutrophils from healthy donors with four different anti-PAD4 antibodies, followed by immunofluorescence microscopy. All anti-PAD4 antibodies resulted in a similar mainly cytosolic, granular staining (Figure 1A). Some staining appeared to be associated with the plasma membrane, and some had a perinuclear distribution. Under our staining conditions, less nuclear PAD4 staining was evident than previously reported (43).

Antibodies against PAD2 produced a similar granular staining of neutrophils (Figure 1B), but no PAD2 was detected within the nucleus. The specificity of the antibodies was confirmed by preincubation with recombinant PADs prior to staining. These findings suggest that PAD2 and PAD4 are not freely soluble cytosolic proteins, but are largely associated with internal structures in the neutrophils.

Since some of the cytosolic PAD2 and PAD4 staining appeared very close to the plasma membrane, we tested whether these proteins could be detected on the neutrophil surface in the absence of permeabilization. Indeed, a punctate signal was observed for PAD4 (Figure 2A) but not for PAD2 (data not shown). Some of the staining appeared to be intracellular, suggesting that surface-exposed PAD4 may be internalized during incubation.

We further confirmed the apparent surface exposure of a fraction of cellular PAD4 in flow cytometric analyses of unfixed human neutrophils isolated from fresh blood samples obtained from healthy volunteers. Analysis was gated on live intact neutrophils with DAPI exclusion to avoid any detection of intracellular PAD in dead or broken cells (negative annexin V gating showed similar results). Clear surface staining was seen with the anti-PAD4 antibody, which was unaffected by preincubation with recombinant PAD2 but abolished by preincubation with recombinant PAD4 (Figures 2B,C). These findings were confirmed in cells from five different donors, although there was some variation in surface PAD4 levels between donors. In contrast, negligible staining with anti-PAD2 antibodies (Figures 2D,E) was seen in cells from the same donors. Surface expression of PAD4 on neutrophils was also verified using ImageStream, which captures fluorescence images of individual cells during flow cytometry (Figures S1A,B in Supplementary Material). Confocal imaging of CD16 and PAD4 co-staining further confirmed the cell membrane location of PAD4 (Figure S2 in Supplementary Material).

To evaluate whether soluble PAD4 (e.g., released from dying cells) could bind to the surface of live neutrophils, we incubated neutrophils with 100 µg/ml of recombinant human PAD4, washed the cells several times, and stained them for PAD4. Analysis of these cells by flow cytometry did not reveal any increase in staining compared with cells treated with medium alone (data not shown), indicating that neutrophils do not bind soluble PAD4. Neutrophils could potentially be damaged or activated during the isolation process. To further rule out exposure of the antibody to intracellular PAD4 and test if PAD4 was expressed in other leukocytes, we directly stained whole blood, which circumvents the need to isolate neutrophils. It is noteworthy that the relative increase in mean fluorescence intensity (MFI) with PAD4 staining was similar whether the staining was performed with whole blood or isolated neutrophils from the same donor. By gating on other leukocyte populations within the whole blood stained samples, we observed some PAD4 reactivity on monocytes, but not on NK cells, B cells, T cells, or platelets (Figure S1C in Supplementary Material). Surface staining of PAD2 was not detected on any cells of the leukocyte lineage.

Taken together, these results suggest that PAD4 but not PAD2 was expressed on the surface of viable resting neutrophils and monocytes.

To simulate conditions that occur during inflammation, infection, and autoimmune conditions, we stimulated neutrophils and looked for changes in the cell surface expression of PAD4. Neutrophils were stimulated for 15 min, and live cells were gated on the basis of DAPI exclusion. PAD4 expression was determined with fluorescently labeled antibodies. Treatment of neutrophils with ribonucleoprotein immune complexes (RNP-IC) resulted in a dose-dependent induction of PAD4 expression on the surface of neutrophils from three different donors (Figure 3A), with a maximum induction associated with an eightfold increase in MFI. Similarly, stimulation with TNF-α induced a significant elevation in surface PAD4 expression (Figure 3B), as did stimulation with PMA (Figure 3C), the TLR5 agonist flagellin (Figure 3D), and the TLR6/2 agonist FSL-1 (Figure 3E). In contrast, IL-8 did not cause any change in surface PAD4 levels (Figure 3F). Additional stimuli, such as heat-killed Listeria monocytogenes (a TLR2 agonist), the TLR7 agonist imiquimod, the TLR8 agonist ss40RNA, immune complexes (IC) without nucleic acids, and GM-CSF, also induced upregulation of surface PAD4 levels, whereas formyl-peptide, PAM3 (TLR1/2 agonist), and lipopolysaccharide (LPS) (TLR4 agonist) caused small increases that were not statistically significant (data not shown). Stimulation with IL-6 did not cause any changes in PAD4 expression on the surface of neutrophils from any of the three donors (data not shown). To rule out the possibility that surface expression of PAD4 only increased as a consequence of the induction of NETosis, the NETosis inhibitor DPI was used in conjunction with various stimuli. DPI had no effect on the upregulation of PAD4 surface expression induced by RNP-IC, PMA, or TNF-α (Figure S3 in Supplementary Material). We conclude that a number of physiologically relevant activators of neutrophils caused a significant increase in surface PAD4 levels on human neutrophils, whereas others did not.

Since several surface PAD4-increasing stimuli are elevated in the serum of patients with RA or SLE (e.g., TNF-α, GM-CSF, IC), we hypothesize that surface PAD4 could be upregulated in those patients. Thus, we examined the surface expression of PAD4 on neutrophils and monocytes in fresh whole blood samples from patients with RA, patients with SLE, and healthy donors. Surface PAD4 expression was detected on neutrophils and monocytes from RA patients at levels very similar to those of the healthy donors (Figures 4A,B). Surface PAD4 was also detected on neutrophils in patients with SLE, but, similar to patients with RA, there was no difference compared with the levels detected from healthy donors (Figure 4C). These results suggested that surface PAD4 expression were similar in peripheral blood leukocytes between patients with RA or SLE and healthy donors.

To determine whether the PAD4 detected on the surface of intact neutrophils was catalytically active, we incubated freshly isolated neutrophils with a well-known substrate for citrullination, fibrinogen, and used an anti-citrullinated fibrinogen monoclonal antibody (mAb) to detect its citrullination. The anti-citrullinated fibrinogen mAb did not react with native fibrinogen, whereas a brief incubation of fibrinogen with active recombinant human PAD2 or PAD4 resulted in a strong recognition of fibrinogen β-chain citrullination (Figure 5A, lane 9). Fibrinogen citrullination by recombinant PAD4 was also confirmed by mass spectrometry analysis (Table S1 in Supplementary Material).

When freshly isolated neutrophils were incubated with fibrinogen, this protein became reactive with the anti-citrullinated fibrinogen mAbs (Figure 5A). Citrullination was time-dependent, detectable within 1 h, and increased through at least 4 h. Fibrinogen citrullination had an absolute requirement for extracellular calcium and was blocked by chelation with either EDTA or EGTA (Figure 5A, lane 11), thus providing further evidence of extracellular fibrinogen citrullination. Neutrophil-conditioned media was also able to citrullinate fibrinogen (Figure 5A, lane 10). Equal loading of fibrinogen was confirmed by Coomassie staining (Figure 5A, lower panel). Citrullination of the fibrinogen β-chain and additional citrullination sites in the α- and γ-chains in the presence of live neutrophils was also confirmed by mass spectrometry analysis (Table S2 in Supplementary Material).

To exclude the possibility that a few dead or broken neutrophils could be the source of PAD activity, we evaluated levels of the intracellular protein GAPDH in the neutrophil-conditioned media by western blotting. Although GAPDH was not detected up to 4 h after initiation of the culture, disruption of neutrophils by treatment with 0.005% Triton X-100 resulted in some GAPDH release at 4 h (Figure 5B). Furthermore, necrotic or apoptotic cells were not detected by DAPI staining until four or more hours after Triton X-100 treatment, suggesting good neutrophil viability and integrity under our experimental conditions.

To confirm the activity of PAD4 on the surface of neutrophils, we developed an alternative PAD activity assay using histone H3 as a substrate. As histones are readily citrullinated by PAD4 both in vitro and in vivo (25), we incubated freshly isolated human neutrophils with recombinant histone H3 and detected histone citrullination with a mAb specific for histone H3 citrullinated on Arg-2. As seen in Figure 5C, histone H3 became citrullinated within minutes of incubation with freshly isolated human neutrophils (Figure 5C, lanes 1–6). As with fibrinogen citrullination, extracellular calcium was required for histone H3 citrullination (Figure 5C, lane 11). Citrullinated histones were not detected when neutrophils were incubated without histone H3 (Figure 5C, lane 7), indicating that endogenous histones were not released under our experimental conditions. Similar to fibrinogen citrullination, neutrophil-conditioned media was able to citrullinate histone H3 (Figure 5C, lane 10). We also noted the appearance of a lower molecular weight band that increased in density with the duration of incubation of samples with live neutrophils. The density of this band was reduced by the inclusion of protease inhibitors, but not the MMP inhibitor, GM-6001, indicating that this band represents a proteolytically processed histone H3 (Figure 5D).

To rule out the possibility that histone toxicity was responsible for cell death and the release of PADs, we followed live neutrophils cultured in the presence of 1 mg/ml histone H3 (the same concentration that was used in our H3 citrullination assay) and DAPI (to stain dying cells) (Movie S1 in Supplementary Material). The rate of cell death remained negligible for at least 45 min with >90% viability, and all cells maintained an intact morphology. However, histone H3 citrullination was detectable at 5 min and peaked between 30 and 45 min, demonstrating that the citrullination was indeed mediated by viable neutrophils.

Taken together, the results of both fibrinogen and histone H3 citrullination assays indicated that neutrophil surface PAD4 is enzymatically active. Notably, neutrophils were also able to secrete active PAD into the surrounding environment.

Since neutrophil-conditioned media was capable of citrullinating fibrinogen (Figure 5A, lane 10) or histone H3 (Figure 5B, lane 10), we sought to determine whether the soluble PAD activity was mediated by PAD4 released from the cell surface. We performed western blot analysis of concentrated culture supernatants from healthy donor neutrophils using antibodies for the detection of PAD4 and PAD2. Surprisingly, PAD2 was clearly observed in all four samples (Figure 6A), whereas PAD4 was barely detectable even after longer exposures (Figure 6B). Nano-LC-MS/MS mass spectrometry confirmed the presence of PAD2 in the supernatants with 43 to 58 peptide-spectrum matches (between the four donors) covering an average of 38.8% of the protein. PAD4 was also detected but with far fewer peptide-spectrum matches (Figure S4 in Supplementary Material). These results indicate that the PAD activity in the neutrophil-conditioned media in the absence of stimulation is predominantly due to PAD2.

To evaluate the relative contributions of PAD2 and PAD4 in extracellular protein citrullination, we developed monoclonal antibodies for PAD2 and PAD4 capable of specifically inhibiting the catalytic activity of their respective PAD targets. The specificity of each antibody was demonstrated with recombinant PAD2 and PAD4 using histone H3 as substrate (Figure 6C).

When these antibodies were used in histone H3 citrullination assays with intact healthy neutrophils, both antibodies reduced PAD activity, but the anti-PAD4 antibody had a greater effect (Figure 6D, lanes 3 and 4). The combination of both antibodies completely blocked citrullination of histone H3 (Figure 6D, lane 5). In contrast, when these antibodies were added to histone H3 citrullination assays with neutrophil-conditioned media, the anti-PAD2 antibody had a substantial inhibitory effect, whereas the anti-PAD4 antibody had a minimal effect (Figure 6D, lanes 9 and 10). These data are consistent with the notion that PAD activity secreted by neutrophils is predominantly mediated by PAD2, whereas PAD activity associated with intact neutrophils is largely mediated by cell-bound PAD4, with some contribution from newly secreted PAD2.

In contrast to the histone H3 assay, fibrinogen citrullination of intact neutrophils was inhibited with the anti-PAD2 antibody, and the anti-PAD4 antibody had no effect (Figure 6E). Western blot analysis using an anti-modified citrulline antibody (more broadly citrulline reactive) showed a similar pattern (data not shown). Thus, it appears that extracellular fibrinogen was preferably citrullinated by secreted PAD2 rather than cell surface-exposed PAD4. This result may also explain the slower kinetics of fibrinogen citrullination compared to histone H3 citrullination by live neutrophils.

Secretion of PAD2 could provide means for neutrophils to modify extracellular matrix proteins or even cytokines without physically interacting with those substrates. To further characterize the pattern of PAD secretion, we developed quantitative ELISA-based fibrinogen and histone H3 citrullination assays to measure PAD activity. The assay was first validated with recombinant human PAD2 and PAD4, which both produced dose-dependent increases in citrullination (fibrinogen shown in Figure 7A, histone H3 not shown). The kinetics of PAD secretion was investigated by analysis of neutrophil-conditioned media. Neutrophils secreted measurable PAD activity within minutes of initiation of the assay, with the highest activity detected at approximately 1 h (Figure 7B). Similar kinetics was observed in assays of both histone and fibrinogen substrates.

To examine the respective contributions of PAD2 and PAD4 to citrullination in this ELISA assay format, conditioned media were incubated with blocking antibodies prior to the assay. These experiments demonstrated that most of the activity was mediated by PAD2 with a minor or negligible contribution by PAD4 (Figures 7C,D). This result is consistent with the detection of PAD2 in neutrophil supernatants by mass spectrometry and western blotting, and the inhibition of PAD activity observed in western blot assays. Taken together, these data confirm that PAD2 is secreted spontaneously by neutrophils and is predominately responsible for the PAD activity found in the neutrophil-conditioned media.

Surface PAD4 levels were found to change in response to neutrophil activators, thus we evaluated the impact of these stimuli on PAD2 secretion. Interestingly, PAD2 secretion from neutrophils was significantly reduced by stimulation with PMA or ICs (Figure 8). LPS also reduced PAD2 secretion, but to a lesser extent (Figure 8). IL-6 reduced PAD2 secretion in some but not all donors. Other stimuli (IL-8, GM-CSF, TNF-α, fMLP, flagellin, FSL-1, PAM3, heat-killed L. monocytogenes, imiquimod, and ss40RNA) had no detectable effects on PAD2 secretion (data not shown). This result demonstrated that PAD2 secretion was reduced by some inflammatory stimuli, and it was regulated differently from PAD4 surface expression.

Blood from healthy volunteers was obtained with informed consent under MedImmune, LLC’s blood donation program, and studies using human cells were performed in accordance with the Institutional Review Board guidelines. Neutrophils were isolated from heparin anti-coagulated blood on a discontinuous Ficoll gradient as previously described (72).

Blood samples from patients with RA and systemic lupus erythematosus were collected from Johns Hopkins University Hospital Arthritis Center (Baltimore, MD, USA) and the National Institutes of Health (Bethesda, MD, USA) lupus clinic, respectively, with informed consent and IRB approval. All patients met the American College of Rheumatology classification criteria (73, 74).

Antibodies for detection of PAD4 (Ab128086 and Ab50247), PAD2 (Ab16478), citrullinated histone H3 (R2 citrullination, Ab176843), and total histone H3 (Ab24834) were from Abcam (Cambridge, MA, USA). Antibodies for detection of PAD4 (MABE254) and modified citrulline were from EMD Millipore (Billerica, MA, USA). Antibodies for flow cytometric analyses (FITC-CD45, PE-CD15, BV421-CD3, APC-CY7-CD56, and PE-CY7-CD14) and CD16 (clone 3G8) were from BioLegend (San Diego, CA, USA) and BUV395-CD19 was from BD Bioscience (San Jose, CA, USA).

Recombinant human PAD2, recombinant human PAD4, and human IgG1 clone NIP228 were made in house. Alexa-647-labeled PAD2 and PAD4 staining antibodies and PAD2 and PAD4 blocking antibodies were generated in house and were human IgG1isotype. Control antibodies, PAD blocking antibodies, and Alexa-647 fluorochrome-labeled anti-PAD monoclonal antibodies were engineered with mutant Fc regions to subvert complement and Fcγ receptor binding (75) to minimize non-specific interactions with neutrophils. Recombinant human histone H3, human fibrinogen, and the anti-citrullinated fibrinogen western blot antibody (clone 10E9.2) were from Cayman Chemicals (Ann Arbor, MI, USA). Anti-GAPDH (D16H11) was from Cell Signaling (Danvers, MA, USA). The anti-citrullinated fibrinogen ELISA antibody (clone 20B2) was from Modiquest (Molenstraat, Netherlands).

DPI, TNF-α, fMLP, PMA, HRP-, or FITC-labeled anti-mouse IgG and anti-rabbit IgG secondary antibodies were from Invitrogen (Carlsbad, CA, USA). Recombinant GM-CSF, IL-6, and IL-8 were from R&D system (Minneapolis, MN, USA). The human TLR1-9 agonist kit was from InvivoGen (San Diego, CA, USA). The MMP inhibitor GM-6001 was from Enzo Life Sciences (Farmingdale, NY, USA). The protease inhibitor cocktail was from Thermo Fisher (Waltham, MA, USA).

Fresh isolated neutrophils were resuspended in Hanks’ balanced salt solution (HBSS) without calcium and magnesium (Invitrogen) with 2% heat-inactivated human AB serum (Sigma-Aldrich, St. Louis, MO, USA) at a density of 500,000 cells/ml and seeded for adherence on l-lysine-coated glass coverslips. Subsequently, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. For permeabilization, fixed cells were treated with 1% Triton X-100 for 1 min and blocked in 1% BSA in PBS for 1 h at 37°C. All primary antibodies (Alexa-647-labeled anti-PAD4 staining antibody, Ab128086, MABE254 and Ab50247 for PAD4; Alexa-647-labeled anti-PAD2 staining antibody for PAD2, Alexa-488-labeled CD16) were used at a concentration of 10 µg/ml in PBS with 1% BSA. Cells were incubated with antibodies for 1 h at 37°C. For recombinant PAD competition, the primary antibody was first incubated with 100 µg/ml recombinant human PAD2 or PAD4 for 15 min at room temperature. A FITC-conjugated secondary antibody (Invitrogen) was used at 1:5,000 dilutions for detection of unlabeled primary antibodies. DAPI was used as a counterstain in permeabilized cells. Images were acquired using a TCS SP2 Leica laser-scanning confocal microscope with 63× objective.

Isolated neutrophils were resuspended in HBSS without calcium and magnesium at a density of 2 × 10⁶ cells/ml. The Alexa-647 fluorochrome-labeled anti-PAD2 staining antibody and anti-PAD4 staining antibody were added at 5 µg/ml. For recombinant PAD competition, antibodies were preincubated with 50 µg/ml recombinant human PAD2 or PAD4 for 15 min at room temperature. Cells were incubated with antibodies on ice for 30 min. Flow cytometry was performed using LSRII and ImageStream X. The results were analyzed using the FlowJo software package (Tree Star) or ImageStream analysis software (EMD Millipore). Neutrophils were gated based on forward and side scatter patterns.

Fresh blood was obtained from healthy donors, RA patients, and lupus patients. Aliquots of 50 µl blood were stained on ice for 30 min with an Ab cocktail (FITC-CD45, PE-CD15, BV421-CD3, APC-CY7-CD56, PE-CY7-CD14, BUV395-CD19, and Medi-PAD2/PAD4 staining antibody, all at 5 µg/ml). Flow cytometry was performed using an LSRII Instrument. After acquisition, the flow cytometry data were analyzed using the FlowJo software package (Tree Star). The following populations were gated for analysis: neutrophils (CD45+ CD15+), monocytes (CD45+ CD14+), T cells (CD45+ CD3+), B cells (CD45+ CD19+), and NK cells (CD45+ CD56+).

Neutrophils were resuspended in HBSS at a density of 10⁷ cells/ml. For surface PAD activity assays, one million neutrophils were incubated with 100 µg human fibrinogen or recombinant human histone H3 for various durations at 37°C in HBSS with 1 mM DTT and 2 mM CaCl₂. After incubation, cells were removed by 10-min 300 g centrifugation, and the supernatant was collected for western blot analysis. Supernatants from neutrophils alone, naïve fibrinogen or histone H3, and fibrinogen histone H3 citrullinated by recombinant human PAD4 were used as controls.

For secreted PAD activity assays, one million neutrophils were incubated in HBSS buffer for 1 h at 37°C. Subsequently, cells were removed by 10-min 300 g centrifugation, and supernatants were collected, which were then incubated for 1 h at 37°C with 100 µg fibrinogen or histone H3 in HBSS with 1 mM DTT and 2 mM CaCl₂. Western blot analysis was performed afterward to detect citrullinated substrates.

Freshly isolated neutrophils were resuspended in HBSS at a density of 2 × 10⁷ cells/ml and incubated at 37°C for 1 h. Cells were removed by 10-min 300 g centrifugation, and supernatants were concentrated by filtering through an Amicon ultra-0.5 ml 30 kDa protein concentration column (EMD Millipore). Concentrated neutrophil-conditioned media was analyzed by western blotting and mass spectrometry.

Recombinant human PAD2 or PAD4 (5 ng) was preincubated with a NIP228 control antibody, PAD2 blocking antibody, and PAD4 blocking antibody for 10 min at room temperature. Histone H3 (5 µg) was then added with 2 mM calcium and incubated for another hour at 37°C.

Neutrophils or neutrophil-conditioned media were preincubated with the NIP228 control antibody, PAD2 blocking antibody and PAD4 blocking antibody for 15 min at room temperature. For fibrinogen citrullination, all antibodies were used at 1 µg/ml final concentration. For histone H3 citrullination, the antibody as used at a final concentration of 5 µg/ml. Fibrinogen and histone H3 were added after preincubation. Fibrinogen was incubated for an additional 4 h at 37°C. Histone H3 was incubated for 30 min. Western blot analysis was performed afterward to determine substrate citrullination level.

96-well ELISA plates were coated with either 1 µg/ml human fibrinogen or histone H3 overnight at 4°C. Freshly isolated neutrophils were resuspended in HBSS at a density of 10⁷ cells/ml and incubated at 37°C from 5 min to 2 h. Cells were removed by 10-min 300 g centrifugation, and supernatants were diluted 1:10 in Tris–HCl buffer with 1 mM DTT and 5 mM calcium.

Diluted supernatants were either added directly to coated plates or preincubated with blocking antibodies at a concentration of 10 µg/ml for 15 min before adding to plates.

Citrullination assays were performed at 37°C for 90 min. The reaction was stopped by washing the plates with PBS + 0.05% tween20. Anti-citrullinated fibrinogen (20B2) and anti-citrullinated histone H3 (Ab176843) antibodies were used for detection. The OD450 was measured.

10× RNP-IC was prepared by incubating equal volumes of 20 µg/ml RNP and 40% lupus serum containing a high titer of anti-RNP IgG for 1 h at room temperature. 10× Ig-IC was prepared by incubating equal volumes of 1 mg/ml biotinylated R347 IgG and 3 µM streptavidin for 1 h at room temperature. TNF-α, fMLP, PMA, GM-CSF, IL-6, IL-8, and a human TLR1–9 agonist kit were used according to the manufacturers’ protocols. For NETosis inhibition, cells were incubated with 10 µM DPI for 10 min at room temperature prior to adding stimuli.

Isolated neutrophils were resuspended in HBSS at a density of 2 × 10⁶ cells/ml. All stimuli were used with a 1:3:9:27:81 dilution series. Activation was performed at 37°C for 15 min. Cells were washed and resuspended after stimulation before FACS analysis.

Isolated neutrophils were treated separately with the highest dose of the stimuli in HBSS for 30 min. Cells were then removed by 10-min 300 g centrifugation, and supernatants were collected for ELISA analysis.

Concentrated neutrophil-conditioned media samples were processed using the filter-aided sample preparation method (76). Resulting peptides were analyzed using nanoflow LC-MS/MS (Dionex RSLCnano interfaced with Orbitrap Fusion Tribrid mass spectrometer). Mass spectrometry data were analyzed using the Mascot search engine with Proteome Discoverer software (Thermo Fisher) and quantified using Scaffold software. The normalized total spectra counts for PAD4 and PAD2 were compared to estimate the expression levels of PAD4 and PAD2.

Recombinant PAD4 or neutrophil-citrullinated fibrinogen samples (5 µg) were reduced and alkylated prior to automated on-column trypsin digestion using a Perfinity™ workstation. Peptide samples from each experiment were analyzed using a nanoflow LC-MS/MS system. Mass spectrometry data were searched using Mascot software, with the following search parameters: nine missed trypsin cleavages, oxidation of methionine, and deamidation of N and Q residues. Arginine citrullination was confirmed using the Mascot (v2.5.1) search engine by including neutral loss of 43.006 Da in addition to 0.984-Da mass shift for deamidation.

Equivalent amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis (4–12% gel) and then transferred to a nitrocellulose membrane. Membranes were then probed using anti-citrullinated fibrinogen (clone 10E9.2), anti-citrullinated histone H3 (Ab176843), anti-total histone H3 (Ab24834), anti-GAPDH (clone D16H11), anti-PAD2 (Ab16478), and anti-PAD4 (MABE254) antibodies using the iBind (Invitrogen) system. An HRP-conjugated anti-mouse IgG and anti-rabbit IgG were used as the secondary detection antibodies before visualization of immunoreactive bands with an ECL reagent (Thermo Fisher).

Data analysis was performed with GraphPad Prism (GraphPad, La Jolla, CA, USA). Unpaired t-test was used in PAD4 expression comparison between patients with RA or SLE and healthy donors. Paired t-test was used in other statistical analysis.