Patients and controls. We recruited 15 Mexican-mestizo adult patients with active SLE (SLEDAI > 6) according to the ACR criteria (14), who were followed-up in a tertiary care center (Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán). As a control group, ten age and gender matched healthy subjects were included. Exclusion criteria were applied to those patients with any kind of acute or chronic infection, pregnancy, puerperium and neoplasia. All healthy controls and SLE patients signed an informed consent before inclusion, and the protocol was approved by our Institutional Ethics Committee (Ref. 2152) in compliance with the Helsinki declaration. Laboratory features of SLE patients were assessed as follows: Auto antibodies of SLE patients (anti-dsDNA IgG, anti-nucleaosomes, anti-cardiolipin and anti-β2glycoprotein I) were assayed using a commercial ELISA.
In vitro ubiquitylation of MPO. A commercial Ubiquitylation Kit (Abcam™) (HeLa lysate) was used according to the manufacturer’s instructions. The recombinant human MPO (rhMPO 10 µg) was incubated for 4 h at 37 ° in a water bath and stored at -80°. To confirm that the ubiquitylation reaction took place, a 4–15% polyacrylamide gel electrophoresis was performed with 50 µg of ubiquitylated MPO (UbMPO) and rhMPO. Subsequently, the transfer to a PVDF membrane was carried out for 1 h at 100v, then, the non-specific binding sites were blocked with Starting Block solution (Thermo Fisher ™). Overnight incubation was carried out with the primary anti-ubiquitin antibody (P4D1 Santa Cruz™) and followed by the secondary antibody HRP anti-Mouse for 1 h (Thermo Fisher™). Membranes were developed by enhanced chemiluminiscence with ECL Western Blotting Substrate (BioRad™). Samples were acquired with a digital image analyzer (Chemidoc MP™, Biorad™) and quantified by densitometry with ImageLab software (Biorad™). (Figure S1).
T cell responses upon ubiquitylated MPO (UbMPO). To address T cell responses, cocultures of mature in vitro CD4+ T lymphocytes and monocyte derived dendritic cells (MoDCs) were set up and stimulated with either the native MPO (recombinant human MPO, rhMPO) (5 µg/ml) or UbMPO (5 µg/ml).
Monocyte isolation and MoDCs generation: Peripheral blood mononuclear cells (PBMCs) were isolated from SLE patients and healthy controls by density gradients after centrifugation with Ficoll-Paque PLUS (GE Healthcare™). After washing twice with 5% fetal bovine serum (FBS) in PBS, PBMCs were resuspended in RPMI 1640 medium (Gibco™) supplemented with 10% FBS and 100,000 cells were incubated for 30 min at 4 °C in MACS buffer and anti-CD14 antibodies from CD14 MicroBeads Isolation Kit conjugated with magnetic beads (Miltenyi Biotec ™) to obtain the enriched CD14+ population. To induce differentiation to dendritic cells, GM-CSF (R&D™) (50 ng/ml) and IL4 (PeProTech™) (30 ng/ml) were added. CD14+ monocytes were seeded in 6-well plates in 4 ml (1 × 106 per ml of medium). On the third day, medium change was performed (15). After seven days, DCs were harvested and viability was evaluated using the trypan blue method. They were seeded in 24-well plates (1 × 106 cells per ml of medium). Cells were divided into three reactivation conditions for 24 h (16): a) LPS (1 µg/ml) E. coli O111: B4 LPS (Sigma Aldrich™); b) LPS (1 µg/ml) and rhMPO R&D™ (5 µg/ml); and c) LPS (1 µg/ml) and ubiquitylated MPO (5 µg/ml).
CD4+ T cell isolation and CD4+/MoDC cocultures: Both lupus and control CD4+ T cells were also isolated from total PBMCs by magnetic negative selection with microbeads according to the Human CD4+ Isolation Kit manufacturer instructions (Miltenyi Biotec™). CD4+/MoDC cocultures were performed in a 10:1 ratio at 37 °C, and 4% CO2 for 3 days to evaluate proliferation and for 7 days to evaluate reactivation and cytokine production from CD4+ T cells. The cocultures were set up as follows: a) Unstimulated CD4+ cells; b)CD4+ cells plus DCs activated only with LPS; c) CD4+ cells plus DCs activated with LPS and rhMPO; d)CD4+ cells plus DCs activated with LPS and ubiquitylated MPO; and e)CD4+ cells polyclonally stimulated with 50 ng/mL of αCD3 (HIT3a BD™) and 100 ng/mL of αCD28 (NA/LE BD™).
CD4 + T cell proliferation and activation markers assessment by flow cytometry: For the assessment of activation markers, after washing twice with 5% FBS in PBS and incubated with FVS700 to evaluate viability, PBMCs were stained with the following fluorescent surface labelled-antibodies: (CD4-APC, CD25-PE, CD83-PE/Cy5, HLA-DR-PerCP) (All Bio Legend™). After that, the cells were fixed with True-Nuclear™ 1X Fix Concentrate, then washed with FoxP3-Perm Buffer™ and finally incubated with labelled anti-FoxP3 BV421. For the proliferation assay, the same steps described above were performed and after fixation and permeabilization, PBMCs were incubated with anti Ki67-APC (Bio Legend™) instead.
For intracellular cytokine staining, PBMCs were stimulated with PMA (Sigma™), ionomycin from Streptomyces conglobatus (Sigma™) and incubated with monensin (GolgiStop™, BD Horizon™) at 37ºC for 5 h. After washing twice with 5% FBS in PBS, PBMCs were stained with anti CD4-AF488 and then fixed and permeabilized with Cytofix/cytoperm (BD™), and finally incubated with intracellular anti IFNγ-APC (Th1), IL4-PE (Th2), IL17A-BV421V (Th17) (Bio Legend™) (Figure S2).
Molecular Dynamics Methods. To assess the initial positions of all-atoms of the backbone of the MPO were taken from the cryogenic crystal structure isoform C, PDB:1CXP, chains A and C (17), while the ubiquitin coordinates were taken from the crystal structure, PDB:1UBQ, chain B (18). To approach the post-translational modifications of MPO, the solvent accessible surface area (SASA) and the root mean square deviation (RMSD) fit of MPO at lysine residues with K6, K48 and K63 of the ubiquitin, were used as criterium to identify exposed lysines. Larger values of SASA were used to identify susceptible residues for ubiquitylation (an enzymatic reaction that its defined by an isopeptide bond among the amino group of substrate lysine chains and the carboxyl terminus G63 of ubiquitin).
To the generation of simulation trajectories, MPO was defined as the center in the simulation box and the water molecules were added via the solvate plugin of VMD 1.9.1 program (19), filling the space around the protein after a 2 Å cutoff. To define the physiologic electrolyte concentration of NaCl at 0.15 M we used the ionized VMD plugin (19). Every system was committed to energy minimization for 10 K steps of conjugate gradient algorithm. Later, the crystallographic structure was relaxed in steps of 200 ps each, with positional constraints setting force constants of 25, 15, 10 5 3, 2 ,1 kcal/mol Å. After pre-equilibration, all simulation trajectories were produced with the NAMD 2.12 program (20). Configurations in the isothermal-isobaric ensemble, at 300K and 1 bar conditions, were generated using Langevin dynamics scheme to maintain constant temperature (20), and a Nose-Hoover Langevin piston for pressure control (21, 22). For computational efficiency, a multiple time-step integration scheme was set, with 1 fs for bond forces, 2 fs for short-range non-bonding interactions, and 4 fs for full electrostatic forces (23, 24). Coulomb interactions were computed using particle-mesh smooth Ewald summation (25, 26) with a tolerance of 10− 6 for the direct part of the Ewald sum, a fourth-order interpolation scheme, and a grid spacing of ~ 1 Å, for each direction of the simulation box. All bonds for hydrogen atoms were constrained using the SHAKE algorithm (27). All-atom force field parameters were used in this study. The CHARMM 36 force field (28–30) with CMAP correction for the protein atoms (31), and the TIP3P water model (32). In house analysis scripts were developed for specific calculations, such as hydration level at the active site, heme group contact maps.
ISG15 detection in ex vivo NETs by Western Blot. After density gradients, neutrophils were isolated with dextran sedimentation as previously described (33). We assessed the spontaneous NET formation (without stimuli) and lipopolysaccharide (LPS)-induced NETosis with 1 µg/mL E. coli O111:B4 LPS (Sigma Aldrich ™) for 4 h. Subsequently, 300 µL of RPMI with micrococcal nuclease (0.01U/µL) were added, the supernatant was centrifuged and stored at -20 °C. The protein was quantified using the bicinchoninic acid method at a wavelength of 562 nm. 4–15% polyacrylamide gel electrophoresis was performed on the lysates from the NETs of patients with SLE and healthy controls. Subsequently, the transfer was carried out for one hour at 100v to a PVDF membrane. Nonspecific binding sites were blocked with the Starting Block Thermo Fisher™ solution. The membrane was then incubated overnight with anti-ISG15 (Abcam ™), after 3 washes with TBS-tween, the secondary antibody HRP anti-Rabbit (Thermo Fisher™) was added for 1 h. Membranes were developed by enhanced chemiluminiscence with ECL Western Blotting Substrate (BioRad). Samples were acquired with a digital image analyzer (Chemidoc MP, Biorad) and quantified by densitometry with ImageLab software (Biorad). Data was expressed as normalized expression to MPO.
Assessment of ISG15 in ex vivo NETs by confocal microscopy. Neutrophils were incubated in RPMI without phenol red (Thermo Fisher ™), 1% FBS, and 1% 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. For confocal microscopy, neutrophils were seeded in 0.01% poly-L-Lysine (Sigma-Aldrich ™) coated coverslips at 37ºC during 1.5 h. After fixation with 4% paraformaldehyde (Santa Cruz ™) (33) at 4ºC during 24 h, washing and blocking with 0.2% gelatin from porcine skin (Sigma-Aldrich ™) was performed. For indirect immunofluorescence, we used the following primary and secondary antibodies diluted in 0.2% gelatin from porcine skin: rabbit anti-human ISG15 (Abcam™), mouse anti-human H2B (Abcam™), donkey anti-rabbit Alexa Fluor 555 (Thermo Fisher™) and donkey anti-mouse Alexa Fluor 488 (Thermo Fisher™). Chromatin was stained with Hoechst 33342 (Thermo Fisher™) and coverslips were mounted on slides with ProLong® Gold Antifade Mountant (Thermo Fisher™)(34). The samples were acquired in an Eclipse Ti-E Nikon confocal microscope (Minato, Tokyo, Japan). The number of cells positive for H2B and nuclear staining (Hoechst) were considered producers of NETs. The mean fluorescence intensity and the area percentage of NETs were quantified using Fiji free software (ImageJ ™) using the average of 6 fields at 40x, normalized for the total number of cells analyzed. The colocalization of ISG15 with histone 2B (H2B) was calculated by Pearson correlation coefficient and validated through the Costes method (35).
Flow cytometry assessment of IFNγ production in PBMC's stimulated with NETs enriched in ISG15. To evaluate IFNγ production, 2 × 106 healthy donor’s PBMCs were stimulated with 100 µg/mL of SLE NETs enriched in ISG15, and NETs from healthy controls lacking ISG15 (expression of ISG15 were corroborated by Western Blot and immunofluorescence, as previously described) or PMA Sigma™ (50 ng/mL) and ionomycin from Streptomyces conglobatus Sigma™ (1 µg/mL) as a positive control. Monensin (GolgiStop™ BD Horizon™) were added and incubated at 37ºC for 18 h. After washing twice with 5% FBS in PBS, PBMCs were stained with the following fluorescent surface labelled-antibodies: (CD3-APCH7, CD4-AF488, CD8-PE/Dazzle, CD56-PE, CD335-BV711) (All Bio Legend™). CD4+ cells were defined as CD3+CD4+, CD8+ cells were CD3+CD8+ and NK cells were CD3−CD56+CD335+. After that, the cells were fixed and permeabilized with Cytofix/cytoperm BD™, and finally incubated with intracellular anti IFNγ-APC (Th1) (Bio Legend™). The samples were acquired on a BD LSRFortessa flow cytometer. Data analysis was performed with the support of FlowJo 10.6 software (FlowStar Inc ™).
Statistical analysis. Quantitative variables were expressed as median with interquartile range (IQR) or minimum and maximum (min-max). The homogeneity of variances of each experiment was determined using the Brown-Forsythe and Bartlett tests. The non-parametric data were analyzed with the Kruskall-Wallis test and the adjustment was made using the Dunn multiple comparison test, as well as Mann-Whitney U test (sum of Wilcoxon ranges) to compare medians between groups with free software Past 3 version™ (United States). Pearson's linear correlation coefficient was used (36), and significance was verified using the Costes method (35). In all cases, significant differences were considered fora p value lower than 0.05. Statistical analysis and graph generation was performed with support of the GraphPad Prism 8 version ™ software (United States).