B6. MRL-Faslpr/lpr mice (#000482, referred to as Faslpr/lpr mice) and Cd19-Cre mice (#006785) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Sh3bp2-floxed mice have been generated by inGenious Targeting Laboratory, as reported previously . In the Sh3bp2-floxed mice, Sh3bp2 exon3 is flanked by loxP sites. To generate B cell-specific SH3BP2-deficient mice, the Sh3bp2-floxed mice were crossed with the Cd19-Cre mice. To generate SH3BP2 systemic knockout mice, the Sh3bp2-floxed mice were crossed with EIIa-Cre mice (#003724, Jackson Laboratory), in which Cre recombinase is expressed in the germ cells . Elimination of the EIIa-Cre allele was achieved by crossing with mice without the EIIa-Cre allele, resulting in SH3BP2 systemic knockout (Sh3bp2Δ/Δ) mice. All mouse studies were performed using male and female mice of the C57BL/6J background. All mutant mice were maintained in the animal facility of Kawasaki Medical School (Okayama, Japan), and were housed in a group (2–5 mice/cage) and maintained at 22℃ under a 12h:12h light/dark cycle with free access to water and standard laboratory food (MF diet, Oriental Yeast Co., Tokyo, Japan). All animal experiments were approved by the Safety Committee for Recombinant DNA Experiments (Nos.14-32, 15-24, and 20-28) and the Institutional Animal Care and Use Committee of Kawasaki Medical School (Nos. 17-109, 18-105, 18-131, and 18-132). All experimental procedures were conducted by the institutional and National Instituted of Health guidelines for the humane use of animals.
Animal Study: analysis of the lupus-prone mice
To generate SH3BP2-deficient lupus-prone mice, Faslpr/lpr mice were crossed with Sh3bp2Δ/Δ mice (C57BL/6J background) to yield double-mutant mice. Sh3bp2+/+ (n = 10), Sh3bp2Δ/Δ (n = 10), Faslpr/lpr (n = 16), and Sh3bp2Δ/ΔFaslpr/lpr (n = 14) mice were monitored until 35 weeks of age. At the end of the observation period, samples of blood, spleen, and kidney were collected and used for subsequent analyses.
To generate B cell-specific SH3BP2-deficient lupus-prone mice, Faslpr/lpr mice were crossed with Cd19Cre/+Sh3bp2fl/fl mice (C57BL/6J background). Cd19Cre/+Sh3bp2fl/flFaslpr/lpr (n = 14), and Cd19+/+Sh3bp2fl/flFaslpr/lpr (n = 14) were monitored until 35 weeks of age. At the end of the observation period, samples of blood, spleen, and kidney were collected and used for subsequent analyses.
Both male and female mice were included in the mouse studies; no sex-differences were observed in the clinical and immunological phenotypes.
Western blot analysis
Protein expression in the lymph nodes, spleen, and B cells was determined by western blot, as described previously [27, 28]. For the preparation of protein samples, tissues were harvested from 35-week-old mice immediately after euthanasia and soaked in the RIPA lysis buffer (Sigma-Aldrich, St. Lois, MO, USA) containing a protease inhibitor cocktail (P8340, Sigma-Aldrich). The tissues were minced using homogenizers. After centrifugation (15,000×g at 4℃ for 20 min), supernatants were collected, and protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% skim milk in Tris-buffered saline with 0.1% Tween-20, the membranes were incubated with the indicated primary antibodies, followed by incubation with the appropriate horseradish peroxidase (HRP)-conjugated species-specific secondary antibodies. The bands were detected using SuperSignal West Dura or Femto chemiluminescent substrate (Thermo Fisher Scientific) and visualized using an ImageQuant LAS-4000 (GE Healthcare, Little Chalfont, UK). Actin was used as a loading control to normalize the amount of protein. The antibodies used in this study were as follows: anti-SH3BP2 (clone1E9; Abnova, Taipei City, Taiwan) and anti-Actin (A2066; Sigma-Aldrich).
Histopathologic assessment of kidney
The kidneys were fixed in 4% paraformaldehyde for 2 days and then embedded in paraffin. Kidney sections (2 µm) were stained with periodic acid-Schiff (PAS). Glomerular pathology was assessed on 10 glomerular cross-sections per kidney, and the number of nuclei per glomerulus was calculated to determine glomerular cell hyperproliferation .
Enzyme-linked immunosorbent assay (ELISA) for serum anti-dsDNA antibody and IgM-RF
Anti-dsDNA antibody (IgG) and IgM-RF levels in serum samples were measured by using ELISA kits (Shibayagi, Gumma, Japan) . Diluted sera (anti-dsDNA 1:100 or IgM-RF 1:1000) were incubated on dsDNA- or RF-coated ELISA plates at 25℃ for 2 h. After washing, the plates were incubated with HRP-conjugated goat anti-mouse IgG and anti-mouse IgM, respectively, at 25℃ for 2 h. Tetramethylbenzidine was used for detection, and optical density at 450 nm (OD450) was measured using a microplate reader (Varioskan Flash; Thermo Fisher Scientific). Concentrations of anti-dsDNA antibodies (IgG) and IgM-RF were calculated and expressed as mU/ml.
Serum immunoglobulin measurement
Concentrations of isotype-specific immunoglobulins in serum were measured with an ELISA kit, SBA Clonotyping System-C57BL/6-AP (Southern Biotech, Birmingham, AL, USA), as reported . Each well in the 96-well plate was incubated with goat anti-mouse immunoglobulin (10 µg/mL) as a capture reagent at 4℃ overnight. Wells were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at 25℃ for 1 h with gentle shaking. Diluted serum samples were added to the capture antibody-coated wells and incubated at 25℃ for 1 h with gentle shaking. The wells were then incubated with alkaline phosphatase (AP)-labeled detection antibodies at 25℃ for 1 h. After adding p-nitrophenyl phosphate substrate, optical densities were measured at 405 nm by a microplate reader (Varioskan Flash), and the concentrations of isotype-specific immunoglobulins were determined.
Flow cytometry for immune cell subsets
The subsets of immune cells in the spleen were analyzed with a flow cytometer (FACSCanto II; BD Biosciences, Franklin, NJ, USA), as described . To block FcγR, single-cell suspensions were incubated with the anti-CD16/CD32 antibody (2.4G2; BD Biosciences) on ice for 10 min before staining with the indicated monoclonal antibodies. The following monoclonal antibodies were used in this study: anti-CD45R/B220 (RA3-6B2), anti-CD4 (RM4-4), anti-CD8a (53-6.7), anti-CD69 (H1.2F3, all from BioLegend, San Diego, CA, USA), and anti-CD3ε (145-2C11, eBioscience, San Diego, CA, USA); all antibodies were conjugated with fluorochrome. Dead cells were excluded by 7-aminoactinomycyn D (7-AAD; BioLegend) staining. In most samples, a minimum of 3×104 events were evaluated, with all data analyzed using Flowjo software (version 10.6.1; BD Biosciences).
Functional analyses of CD4+ T cells
CD4+ T cells were isolated from the spleen of 12-week-old mice by negative selection using the CD4+ T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Purified T cells were activated by plate-bound anti-mouse CD3 antibody (145-2C11, 5 to 20 µg/mL; TONBO biosciences, San Diego, CA) and/or anti-mouse CD28 antibody (37.51, 2 µg/mL; TONBO biosciences) at 37℃ in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol.
After the activation for 24 h, the cells were collected and stained for activation marker proteins on the cell surface using specific fluorophore-conjugated antibodies; anti-CD4 (RM4-4), anti-CD25 (PC61), anti-CD62L (MEL-14), anti-CD44 (IM7), and anti-CD69 (H1.2F3, all from BioLegend). For proliferation assessment, cells were stained with 1 µM carboxyfluorescein succinimidyl ester (CFSE; Dojindo laboratories, Kumamoto, Japan) in the dark at 25℃ for 10 min and were activated by plate-bound anti-CD3 antibody and/or anti-CD28 antibody for 72 h. Cells were collected at indicated time points and stained with a fluorophore-conjugated anti-CD4 antibody. All samples were analyzed in a flow cytometer (FACSCanto II) and Flowjo software.
Isolation of B cells
B cells were isolated from the spleen of mice by negative selection using the B Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.
Generation of dendritic cells (DCs) from bone marrow cells
DCs were generated in vitro from bone marrow cells of 16-week-old mice, as described previously , with some modifications. Briefly, bone marrow cells were flushed from the tibia and femur of the mice; the collected cells were plated at a density of 1×106/mL and cultured for 10 days in 5% CO2 at 37℃ in RPMI1640 medium containing 10% heat-inactivated FBS, 10 ng/mL recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech, Rocky Hill, NJ, USA), and 5 ng/mL mouse interleukin (IL)-4 (PeproTech). The yielded DCs were used in subsequent experiments.
Flow cytometric analyses of DCs
After the culture for 10 days with GM-CSF and IL-4, the DCs were collected and stained for marker proteins on the cell surface using specific fluorophore-conjugated antibodies; anti-CD11c (N418), anti-CD80 (16-10A1), anti-CD86 (GL-1), anti-CD54/ICAM1 (YN1/1.7.4, all from BioLegend), and anti-MHC Class II (I-A/I-E; M5/114.15.2, eBioscience). Splenic cells isolated from 30-week-old mice were stained with anti-CD11c (N418) and anti-MHC Class II (I-A/I-E; M5/114.15.2, eBioscience). Dead cells were excluded by 7-AAD (BioLegend) staining. All samples were analyzed in a flow cytometer (FACSCanto II) and Flowjo software.
Culture of bone marrow-derived macrophages
Isolation and culture of primary bone marrow cells were performed, as previously described [28, 30]. Briefly, bone marrow cells were isolated from the long bones of 16-week-old female mice and cultured on Petri dishes for 2 h at 37℃ under 5% CO2. Non-adherent bone marrow cells were re-seeded on culture plates at a density of 1×106 cells/mL and then incubated for 2 days in α-minimum essential medium (α-MEM) containing 10% heat-inactivated FBS and 25 ng/mL recombinant mouse macrophage colony-stimulating factor (M-CSF; PeproTech). After the 2-day pre-culture, the yielded bone marrow-derived macrophages were stimulated with Toll-like receptors ligands, such as lipopolysaccharide (LPS, Sigma-Aldrich), polyinosinic-polycytidylic acid sodium salt (Poly(I:C)), single-strand RNA (ssRNA), and CpG oligodeoxynucleotides (ODN, all from InvivoGen, San Diego, CA, USA), for 6 h in the presence of M-CSF. RNA samples were isolated from bone marrow-derived macrophages at the indicated time points and subjected to gene expression analysis.
Real-time quantitative polymerase chain reaction (qPCR)
Total RNA was extracted from culture cells by using RNAiso Plus (Takara Bio, Shiga, Japan) and solubilized in RNase-free water as previously described [23, 27]. cDNA was synthesized by using Prime Script RT reagent Kit (Takara Bio). qPCR reactions were performed by using TB Green PCR Master Mix (Takara Bio) with StepOne Plus System (Thermo Fisher Scientific). Gene expression levels relative to Hprt were calculated by ΔΔCt method and normalized to control samples indicated in each experiment. The qPCR analysis used following primers; 5’-tcgtctttcacaagtgtcttcag-3’ and 5’-ttgccagtagattcggtcttc-3’ for Cd80, 5’-gaagccgaatcagcctagc-3’ and 5’-cagcgttactatcccgctct-3’ for Cd86, 5’-catcttctcaaaattcgagtgaca-3’ and 5’-tgggagtagacaaggtacaaccc-3’ for Tnf, 5’-tcctcctcagaccgctttt-3’ and 5’-cctggttcatcatcgctaatc-3’ for Hprt, respectively. All qPCR reactions yielded products with single peak dissociation curves.
Individual values are presented as dots and the means or the means ± standard deviation. Statistical analysis was performed by the two-tailed unpaired Student’s t-test to compare two groups and by one-way ANOVA (Tukey post-hoc test) to compare three or more groups using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). P values less than 0.05 were considered statistically significant.