Patients and healthy controls
The study included consecutive 70 new-onset and treatment naïve patients with SLE (including 60 females and 10 males) who met the 1997 American College of Rheumatology (ACR) classification criteria for the diagnosis of SLE confirmed by two qualified rheumatologists (Jialin Teng and Chengde Yang) [21]. Demographic data, clinical characteristics, and laboratory findings such as anti-dsDNA IgG levels, erythrocyte sedimentation rate (ESR), white blood cell counts in blood (WBC), haemoglobin (Hb), platelets (PLT), C-reactive protein (CRP), immunoglobulin G (IgG), complement 3 (C3) and complement 4 (C4) of SLE patients were collected. In addition, samples from 70 sex- and age-matched healthy donors with neither autoimmune nor infectious diseases were collected as healthy controls (HCs) (including 58 females and 12 males). All of the sera samples were stored at -80 °C until use. Disease activity was measured using SLEDAI score [22]. The study was performed in accordance with the Declaration of Helsinki and the Principles of Good Clinical Practice. Biological samples were obtained under a protocol approved by the Institutional Research Ethics Committee of Ruijin Hospital (ID: 2016-62), Shanghai, China.
Detection of anti-Tyro3 autoantibody
Recombinant human Tyro3 (Abnova, Taiwan) was prepared by wheat germ expression system. The protein was fused with a GST-tag at N-terminal and purified by glutathione sepharose 4 fast flow. Antibody against human Tyro3 receptor in the sera of SLE patients and HCs was determined by an enzyme-linked immunosorbent assay (ELISA). Ninety-six-well high binding plates (Corning, New York, USA) were coated with recombinant human Tyro3 protein in 0.05 mol/L carbonate buffer sodium (pH=9.6) overnight at 4 °C. The antigen-coated wells were washed three times with PBST (PBS plus 0.05% Tween-20) and blocked with PBST containing 5% bovine serum albumin (BSA) for 2 h at 37 °C. The blocking buffer was removed, and the plates were washed as described above before the addition of 100 μl of serum sample (1:100 diluted in 1% BSA). The human sera were incubated for 2 h at room temperature followed by incubation with HRP-conjugated goat anti-human IgG (Abcam, Cambridge, UK) for another 1 h at room temperature. Then, the plates were washed, and 100 μl of tetramethylbenzidine substrate solution was added. The color development was stopped by the addition of 50 μl of 0.5 M H2SO4. The absorbance was measured at a wavelength of 450 nm in a microplate reader (Bio-Rad Laboratories, Richmond, USA).
IgG purification
Total IgG was isolated from the serum of new-onset SLE patients by Thiophilic Adsorbent reagent (Pierce, Thermo Scientific, Rockford, USA) according to the manufacturer’s instructions and concentrated in a centrifuge tube (Amicon, Millipore, Eschborn, Germany). The total protein content was estimated using a spectrophotometer (NanoDrop, Thermo Scientific, USA).
Purification of anti-Tyro3 IgG
The purification of the specific antibody was performed using AminoLink Plus Coupling Resin (MicroLink, Thermo Scientific, USA). We purchased recombinant human Tyro3 protein (Abnova, Taiwan) and coupled the protein to the resin. Then, we used the purified total IgG from SLE patients to form the resin-bound complex and incubated them with gentle end-over-end mixing. The eluted antibody was neutralized with 1 M Tris (pH=9.0). After sterile filtration, the autoantibody was stored as small aliquots at -80 °C.
Preparation of macrophages
Peripheral blood mononuclear monocytes (PBMCs) were isolated from the blood of healthy volunteers using Ficoll density gradient centrifugation (GE Healthcare, Madison, USA). The CD14+ monocytes were isolated by positive selection using CD14 microbeads (Miltenyi Biotec, Auburn, USA) according to the manufacturer’s instructions. The selected cells were cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS; Gibco, NY, USA), 100 units/ml penicillin, and 100 μg/ml streptomycin that was supplemented with 100 ng/ml of macrophage colony stimulating factor (M-CSF) (R&D Systems, Minneapolis, USA) in a humidified 5% CO2 incubator. On day 4, the medium was replaced with RPMI 1640 medium containing M-CSF, and on day 7, the mature macrophages were harvested.
Preparation of apoptotic cells
To generate apoptotic cells, Jurkat cells (purchased from ATCC, Manassas, USA) were cultured in RPMI 1640 medium without FCS, and apoptosis was induced with 0.5 μg/mL staurosporine (BBI Life Science, Shanghai, China) for 3 h, as previously reported that the inhibitor of protein kinases staurosporine showed remarkable activity in inducing apoptosis in a wide variety of mammalian cells [23]. Afterwards, the cells were washed three times with PBS and resuspended in RPMI 1640 medium. Staurosporine treatment yielded a population of 90% apoptotic cells, which was verified by staining with annexin V and 7-amino-actinomycin D (7-AAD, Tianjin Sungene Biotech Co., China). Before being fed to the macrophages, the apoptotic cells were labeled with iFL Green dye (pHrodo, Invitrogen, Thermo Scientific, USA), which could be detected with FITC (fluorescein), protected from light at room temperature for 20 minutes.
Efferocytosis assays
Human CD14 positive monocyte derived macrophages were incubated with fresh medium containing 60 μg/ml purified human Tyro3 antibody from SLE patients or normal human IgG (BBI Life Science, Shanghai) for 1 h at 37 °C in an incubator.
Then, the macrophages were incubated with staurosporine induced apoptotic Jurkat cells labeled with iFL Green dye (ratio of macrophages : apoptotic cells = 1:5) in RPMI 1640 medium without FCS in an incubator for 30 minutes. After washed with PBS, the macrophages were collected using trypsin. Efferocytosis was determined according to the percentage of macrophages that phagocytosed apoptotic cells labeled with FITC using a FACS Canto II cytometer (BD Biosciences, San Jose, USA). The data were analyzed using FlowJo software (Tree Star Inc., Ashland, USA).
For immunofluorescence, the apoptotic cells were labeled with 5 μM 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) (eBioscience, Invitrogen, Thermo Scientific, USA), protected from light at room temperature for 10 minutes and washed with PBS. Then, the macrophages were incubated with induced apoptotic Jurkat cells for 30 minutes. After washing with PBS, macrophages were fixed with 4% formaldehyde for 20 minutes. After three rinses with PBS, the cells were permeabilized with Triton X-100 (Beyotime, Shanghai, China) for 5 minutes. The cytoskeleton was dyed with phalloidin (Servicebio, Shanghai, China), and incubated for 1 h protected from light. DNA was stained with 10 µg/ml 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (hoechst 33324) (Thermo Scientific, USA) for 5 minutes protected from light. After rinsing 3 times with PBS, the cells were viewed by confocal fluorescence microscope LSM 800 (ZEISS, Oberkochen, Germany).
Statistics
Continuous variables were tested for normality by One-Sample Kolmogorov-Smirnov Test. Continuous variables were expressed as mean ± SD or median (interquartile range) as per distribution type, and categorical data were expressed as frequency and percentages. Receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC) were used to assess the sensitivity and specificity of anti-Tyro3 IgG for the diagnosis of SLE. Statistical analysis was performed using independent samples t-test for normal data and Mann-Whitney U-test for non-normal data. The analyses were carried out under the two-sided principle. Correlations between groups were evaluated by the Spearman correlation analysis. A P-value less than 0.05 was considered statistically significant. Graphs were drawn using Graphpad Prism (version 7, GraphPad Software Inc, San Diego, USA) and data were analyzed using the SPSS software for Windows (Version 23; SPSS Inc., Chicago USA).