Patients with SLE and healthy controls
A total of 57 patients with SLE and 60 healthy controls were recruited from Jeonbuk National University Hospital in Jeonju, South Korea. All patients were between 18 to 75 years of age and fulfilled the 2012 Systemic Lupus International Collaborating Clinics (SLICC) Classification Criteria for SLE (19). The patients receiving over 1mg/kg/day prednisolone at the time of blood sampling were excluded from this study. SLE disease activity was evaluated using the SLE Disease Activity Index 2000 update (SLEDAI-2K) (20). Sixty age and gender-matched healthy control subjects were recruited. Written informed consent was obtained from all participants. This study was conducted consistent with the Declaration of Helsinki and was approved by the Ethics Committee of the Jeonbuk National University Hospital (CUH 2018-08-005-004).
Lupus murine model induction
BALB/C mice (7-9-week-old females) were purchased from Central Lab Animal Inc. All mice were maintained under a 12 h:12 h light/dark cycle, with the temperature at 24°C, and the humidity at 60% on the standard diet in a conventional cage. We adopted toll-like receptor (TLR) 7 agonist-induced mice as SLE murine models for our experiments. This model presents with the phenotypic findings of lupus including an elevated autoantibody levels, immune complex-deposited nephritis, and marked splenomegaly (21), and has been widely used for experiments in research into SLE pathogenesis (22, 23). The lupus murine model was induced through the topical application of a TLR7 agonist. The skin on the back of the mice was treated topically 3 times weekly over 4 weeks with 100 µg of Resiquimod (R848, Enzo Life Sciences, NY, USA) mixed with 100 µL of acetone. The control group was treated with 100 µL of acetone topically in the same manner. At the end of the treatment, mice were euthanized via intraperitoneal administration of ketamine (75 mg/ kg) plus xylazine (10 mg/kg), followed by the collection of a blood sample through cardiac puncture. All experiments were conducted in accordance with National Institutes of Health guidelines and Animal Research: Reporting of In Vivo Experiments guidelines. The study protocol was approved by the Institutional Animal Care and Use Committee of Jeonbuk National University (JBNU 2020 − 0115).
mRNA extraction and Real-Time PCR
Total RNA was extracted using an RNA Extraction Kit (Bioneer, Daejeon, Korea) according to the manufacturer’s protocol, and cDNA was generated by using an Accupower RocketScript RT Premix, Rnase H Minus kit (Bioneer, Daejeon, Korea). Quantitative PCR was performed with SYBR Green Real time PCR Master mix (TOYOBO, Osaka, Japan) and a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Grand Island, NY, USA). Amplification conditions were as follows: 50℃ for 2 min, then 95℃ for 10 min for 1 cycle, followed by 40 cycles of 95℃ for 15 sec and 60℃ for 1 min. The following primers were used (forward and reverse, respectively): for humans, GAPDH, 5’-tggtatcgtggaaggactca-3’ and 5’-gcagggatgatgttctggag-3’; NRP1, 5’-gcctgactcaaatcctccag-3’ and 5’-acctggtgttttctgtccac-3’; ILT-4, 5’-tcagggcaaacactggacat-3’ and 5’-tcacggcagcatagaggttt-3’; for mice, GAPDH, 5’-acttgaagggtggagccaaa-3’ and 5’-gcaggatgcattgctgacaa-3’; NRP1, 5’-5’-gcctgactcaaatcctccag-3’ and 5’-acctggtgttttctgtccac-3’; ILT-4, 5’- tcagggcaaacactggacat-3’ and 5’-tcacggcagcatagaggttt-3’; for mice, GAPDH, 5’-acttgaagggtggagccaaa-3’ and 5’-gcaggatgcattgctgacaa-3’; NRP1, 5’- tgctctggaatgttgggcat-3’ and 5’-tggtcaccagacggatgttt-3’; Nephrin, 5’- aacatccagctcgtcagcat-3’ and 5’- aaagccaggtttccactcca-3’. The relative expression of the target genes was determined by normalizing the expression of each gene to GAPDH using the ΔΔCt method.
Cell isolation and culture
The peripheral blood of each participant was obtained via venipuncture. 10 ml of blood samples were diluted 1:1 with PBS (pH 7.4) and layered on Lymphoprep (Serumwerk Bernburg AG, Oslo, Norway) following centrifugation at 20°C for 30 min. PBMCs were isolated from the interphase between the Ficoll-Paque and serum layers, and washed twice with PBS.
CD4+ T cells and monocytes were isolated from PBMCs using a MACS isolation kit (BD Biosciences, San Jose, CA, United States) by negative isolation through a CD8, CD11b, CD16, CD19, CD36, CD56, CD123, and CD235a γδ TCR biotinylated antibody cocktail (Human CD4 T Lymphocyte Enrichment Set; BD Biosciences, San Jose, CA, USA) as well as a CD3, CD45RA, CD19, CD56, and CD235a biotinylated antibody cocktail (Human Monocyte Enrichment Set; BD Biosciences, San Jose, CA, USA). CD4+ T cells or monocytes were purified using a magnetic board of over 90% purity, as assessed by the flow cytometry of CD4+ T cells with anti-Human CD4-FITC (Tonbo Biosciences, CA, USA) and monocytes with mouse anti-human CD14-FITC (BD pharmigen, USA).
Cells were cultured in RPMI 1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Life Technologies Limited, UK), 100 U/mL penicillin (Gibco, Life Technologies Limited, UK), 100 mg/mL streptomycin (Gibco, Life Technologies Limited, UK), and 2 mM L-glutamine (Gibco, Life Technologies Limited, UK). Cells were grown at 37°C in a humidified 5% CO₂ atmosphere.
Dendritic cell differentiation
Isolated monocytes from human PBMCs were cultured in RPMI 1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Life Technologies Limited, UK), penicillin (100 U/mL) (Gibco, Life Technologies Limited, UK), streptomycin (100 mg/mL) (Gibco, Life Technologies Limited, UK), 2 mM L-glutamine, rh-granulocyte-macrophage colony-stimulating factor (GM-CSF) (50 ng/mL) (Peprotech, New Jersey Cranbury, USA), and rh-interleukin (IL)-4 (10 ng/mL) (Peprotech, New Jersey Cranbury, USA) over 6 days in a 96-well plate (Corning-falcon, USA) to obtain monocyte-derived dendritic cells (Mo-DC) as previously described (24).
The generation of bone marrow-derived dendritic cells (BDMCs) was performed using a method previously described elsewhere (25). Briefly, bone marrow cells were isolated and prepared from the bone marrow of the femur and tibia in BALB/C mice as single-cell suspension. The aliquots of 1×106 bone marrow cells were placed in 24-well plates and cultured in RPMI 1640 containing 10% heat-inactivated FBS (Gibco, Life Technologies Limited, UK), 100 U/mL penicillin (Gibco, Life Technologies Limited, UK), 100 mg/mL streptomycin (Gibco, Life Technologies Limited, UK), 2 mM L-glutamine in the presence of rm-GM-CSF (50 ng/mL, Peprotech, New Jersey Cranbury, USA), and rm-IL-4 (10 ng/mL, Peprotech, New Jersey Cranbury, USA). On day 3, two-thirds of the medium was replaced. On day 5, the non-adherent cells were transferred into a six-well plate and cultured for two additional days. On day 7, the acquired dendritic cell morphology was confirmed by microscope and checked for CD11c fluorescence (BD Bioscience, San Jose, CA, USA) using flow cytometry. Then the harvested cells were used in the subsequent experiment.
Glomeruli isolation
The kidneys were harvested, and the medulla was removed carefully. The renal cortexes were minced and digested with collagenase type V (Sigma, dissolved in HBSS) in a water bath at 37℃ for 20 minutes with pipetting at 5-min intervals. The digested mixture was transferred with cold Hanks Balanced Salt Solution (HBSS, Sigma-Aldrich) onto 100-µm, 70-µm, and 40-µm cell strainers to remove cell debris and small tubular fragments. New 40-µm cell strainers were then used to remove the remnant debris, and the retained glomeruli were transferred into another clean culture dish to remove the few residual tubular fragments. The floating glomeruli were collected and centrifuged at 290g at 4℃ for 5 minutes. The glomeruli were examined under a microscope and their purity confirmed by the expression of the glomerular marker using Real-time PCR and Western blot analysis.
Renal histopathologic assessment and immunofluorescence assay
Kidneys were harvested after perfusion with saline, and were fixed in 4% formaldehyde for 24 hr, embedded in paraffin, and separated into fixed sections of 10-µm thickness. After being stained with hematoxylin and eosin (H&E), and periodic acid–Schiff (PAS), renal pathology was evaluated according to a previously described scoring scale (26). Briefly, we assessed glomerular pathology in 20 glomeruli per kidney and interstitial/tubular pathology in randomly selected 10 high-power fields, semi-quantitatively on a scale of 0–3. For the immunofluorescence assay, the 10-µm-thick acetone fixed sections were stained with rabbit anti-mouse IgG-heavy and light chain antibody, FITC-conjugated (Bethyl Laboratories Inc., USA). Slides were then incubated at room temperature for 1 hr. Slides were stained with rat-anti mouse C3 (Abcam, Cambridge, United Kingdom) and then were incubated at 4℃ overnight. The slides were stained with goat anti-rat IgG-heavy and light chain antibody FITC-conjugated and then incubated for 1 hr at room temperature in a dark humid box. They were then mounted with mounting medium using DAPI (Abcam, Cambridge, United Kingdom). Staining for IgG and NRP1 was performed in the same manner, and the antibodies used are listed below. Slides were observed with a confocal microscope (Zeiss LSM 880, Oberkochen, Germany) and analyzed by ZEN 3.2 Zeiss Microscopy GmbH software (version 3.2.0.0000) (Zeiss LSM 880, Oberkochen, Germany). The fluorescence intensity (MFI) of the glomeruli in different groups was calculated using ImageJ software (National Institutes of Health, USA) for each section with 10 glomeruli. 1) For IgG staining: rabbit anti-mouse IgG-heavy and light chain antibody FITC-conjugated (Bethyl Laboratories Inc., USA), 2). For NRP1 staining: NRP1 antibody (eBioscience, Carlsbad, CA, USA) (1:200), with goat anti-rat IgG-heavy and light chain antibody FITC-conjugated (Bethyl Laboratories Inc., USA)
Immunocytochemistry assay
BMDCs were placed in a 35 mm confocal dish (SPL Life Sciences Co., Korea) at a number of 0.5×106 cells and incubated at 37°C for 2 hrs. The cells were fixed using 3.7% paraformaldehyde in PBS pH 7.4 for 10 minutes at room temperature and washed three times with cold PBS. The cells were incubated for 10 min with PBS containing 0.1% Triton X-100 at room temperature for permeabilization and washed in PBS three times. The washed cells were incubated with 1% BSA in PBST (PBS + 0.1% Tween 20) for 30 minutes to block the unspecific binding of the antibodies. The cells were stained in the diluted antibody in 1% BSA in PBST overnight at 4°C. After washing the cells in PBS, they were stained with the secondary antibody in 1% BSA in PBS for 1 hr at room temperature in the dark and washed in PBS. The cells were mounted with mounting medium with DAPI (Abcam). The following antibodies were used in the immunocytochemical analysis: Neuropilin-1 antibody (eBioscience, Carlsbad, CA, USA), Goat anti-rabbit IgG, highly Cross-Adsorbed Secondary Antibody, goat anti-rat IgG-heavy, and light chain antibody FITC-conjugated (Bethyl Laboratories Inc., USA). The slides were observed with a confocal microscope (Zeiss LSM 880, Oberkochen, Germany) and analyzed by ZEN 3.2 Zeiss Microscopy GmbH software (version 3.2.0.0000) (Zeiss, Oberkochen, Germany). Fluorescence intensity was calculated using ImageJ software (National Institutes of Health, USA) and the scores were evaluated through methods described previously (27).
Cell cytotoxicity and proliferation assay
BMDCs were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine at 37°C under 5% CO2. BMDCs were seeded at 5×104 cells/well in a 96-well plate and treated with various doses of R848 (0, 1, 2.5, 5, and 10 µg/mL) (Enzo Life Sciences, NY, USA) and EG00229 (Tocris Bioscience, United Kingdom) that had been dissolved in 0.2M NaOH (0, 10, 30, 100 and 300 µM) (Tocris Bioscience, United Kingdom) for 24 hrs. The 10 µL/well of the Lysis solution was added to the control group as a positive control. The cytotoxicity of the cells was evaluated using a LDH Cell Cytotoxicity Assay Kit (DoGenBio, Seoul, Korea). To assess the cell proliferation, BMDCs were seeded (5×104 cells/mL) in a 96-well plate. The BMDCs were pretreated with EG00229, an NRP1 antagonist (Tocris Bioscience, United Kingdom) (100 µM), for 2 hrs, followed by treatment with R848 for 24 hrs and 48 hrs. Cell proliferation was measured using a Cell Counting Kit-8 (Enzo Life Sciences, NY, USA). Absorbance was measured using a microplate reader (Bio-Rad) at 450nm.
Serologic analysis and Urinalysis
Anti-dsDNA antibodies (FUJIFILM Wako Shibayagi Corporation, Ishihara, Japan), urine albumin and creatinine (Exocell, Philadelphia, USA), and Human Neuropilin-1 Quantikine (CUSABIO, Houston, TX, USA) were quantified by ELISA consistent with the manufacturer’s instructions.
NRP1 inhibition and co-culture condition
CD4+ CD25− T cells and CD11c+ dendritic cells were isolated from the PBMCs of participants by BD FACSAria ІІІ flow cytometer sorting (> 95%). The CD4+ CD25− T cells from the spleen of the BALB/C mice were sorted by BD FACSAria ІІІ (> 95%). Dendritic cells were pre-treated with EG00229 (100 µM) for 2 hrs and then stimulated by R848 (5 ug/mL) for 0.5 hr. CD4+ CD25− T cells were incubated in the presence of anti-CD3 and anti-CD28 antibody coated beads (eBioscience, Carlsbad, CA, USA) as described previously (28). After 24 hrs of incubation, the T cells were labeled with 2.5 µM CFSE using a Cell Trace Cell Proliferation Kit (Molecular probes) and then co-cultured for 5 days with dendritic cells with target cells: the dendritic cell to target cell ratio was 1:1. The stained cells were detected using an FACSCalibur flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo software (TreeStar Inc., Ashland, OR, United States).
Western blot analysis
Proteins were extracted from isolated glomeruli and BMDCs using RIPA buffer containing protease inhibitors. (Thermo scientific, USA). Proteins were separated on SDS-PAGE gels (10%) and then transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon, Millipore). Membranes were blocked in 5% non-fat dry milk in TBST for 1 hr at room temperature with shaking and then probed overnight at 4℃ with primary antibodies (1:1,000) against Nephrin (Invitrogen, USA), Neuropilin-1 (Cell Signaling, Danvers, MA, USA), p-STAT1 (Cell Signaling, Danvers, MA, USA), STAT1 (Cell Signaling, Danvers, MA, USA), p-STAT3 (Cell Signaling, Danvers, MA, USA), STAT3 (Cell Signaling, Danvers, MA, USA), p-ERK 1/2 (Cell Signaling, Danvers, MA, USA), ERK 1/2 (Cell Signaling, Danvers, MA, USA), p-JNK (Cell Signaling, Danvers, MA, USA), JNK (Cell Signaling, Danvers, MA, USA), p-P38 (Cell Signaling, Danvers, MA, USA), P38 (Cell Signaling, Danvers, MA, USA), p-NF-κB, NF-κB (Cell Signaling, Danvers, MA, USA), p-IκB (Cell Signaling, Danvers, MA, USA), IκB (Cell Signaling, Danvers, MA, USA), and β-actin (Bioworld Technology Inc, USA). After three washes, the membranes were incubated with HRP-conjugated rabbit or mouse secondary antibody (1:3,000) at room temperature for 2 hrs. The reactive proteins were detected using an ECL (GE healthcare, USA) and the intensity of the bands was quantified using a Vilber Lourmat Fusion Fx7 system (Vilber Lourmat, Collégien, France).
Statistical analysis
The Mann-Whitney test with Bonferroni’s correction was used to compare two groups and the Kruskal-Wallis test was used to compare three or more groups for the analysis of pathology scores and IF scoring. All other parametrically distributed data, including flow cytometry, immunoblotting, and cell proliferation assay, were analyzed by a one-way ANOVA with a Turkey post hoc test. Spearmans’ rho test was applied to the analysis of correlation. SPSS 22.0 software (SPSS Inc., Chicago, Illinois, U.S.A.) was used for statistical analysis. P values of < 0.05 were considered statistically significant and results were expressed as mean ± SEM.