Generation of Shp1-transgenic mice
The Shp1 gene is located in a high-density locus on mouse chromosome 6. In order to investigate the effect of Shp1 overexpression, we generated Shp1-transgenic (Tg) mice. Briefly, we purchased BAC clone RP24-297C1 containing the full-length Shp1 gene with its promoter region from BACPAC Resources Center (Children’s Hospital Oakland Research Institute in Oakland, CA). The 21.9 Kbp genomic fragment containing Shp1 (derived from a C57Bl/6 mouse) was released from the BAC clone by digestion with Fspl restriction enzyme and separated from other DNA fragments in 0.5% agarose gel by pulse-field electrophoresis (Bio-Rad, Hercules, CA). The 21.9 Kbp fragment was further purified using GeneClean Spin kit (BIO101 Systems, Carlsbad, CA) and sequenced. The purified fragment, containing the Shp1 gene, its promoter, and 2 Kbp flanking regions, was sent to the Cornell University Core Transgenic Mouse Facility (Ithaca, NY) for pronuclear injection into fertilized FVB mouse egg cells. We identified a simple sequence length polymorphism (SSLP) in the 5′ flanking region of the Shp1 transgene (from the donor C57Bl/6 mouse), which was not present in FVB (origin of the fertilized egg) or BALB/c cells. This SSLP was located at a TaqαI restriction enzyme-sensitive site, which allowed us to use appropriate primers that could discriminate between the Shp1 transgene (TaqαI-resistant) and the endogenous Shp1 gene (TaqαI-sensitive) in FVB or BALB/c mice. We identified tandem inserts of the Shp1 transgene with its promoter in mouse chromosome 7.
Transgenic founders were backcrossed into the BALB/c (PGIA-susceptible) background for 12 generations. Heterozygous Shp1-Tg BALB/c mice were then intercrossed to generate Shp1-Tg+/−, Shp1-Tg+/+ and WT offspring.
Confirmation of Shp1 gene expression
Shp1 gene expression was quantified by RT-qPCR from spleen cells of WT as well as heterozygous (Shp1-Tg+/−) and homozygous (Shp1-Tg+/+) Tg mice. Female BALB/c mice at 8–12 weeks of age of each genotype were used. Total RNA was extracted with Direct-Zol RNA MiniPrep Plus (Zymo Research, Irvine, CA) and reverse transcribed using the iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, Hercules, CA). cDNA was amplified using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) in a CFX Connect Real-Time PCR Detection System (Bio-Rad). Measured Ct values were normalized to the housekeeping hypoxanthine phosphoribosyltransferase 1 (Hprt1) gene. Relative expression was calculated using CFX Manager Software (Bio-Rad). Shp1 primer sequences were as follows:
Forward: 5′-TCT CAG TCA GGG TGG ATG AT-3′
Reverse: 5′-CCT GCT GCT GCG TGT AAT A-3′
Detection of SHP-1 protein expression by Western blot
Cellular proteins were extracted and the protein concentrations were determined using a BCA Protein Assay kit (ThermoFisher, Rockford, IL) and a Synergy 2 ELISA reader (BioTek Instruments, Winooski, VT). Protein extracts were separated on 4–20% SDS-polyacrylamide gels (Bio-Rad), blotted onto PVDF membranes (Bio-Rad), then blocked with Tris-buffered saline + 0.05% Tween-20 containing 3% blotting-grade blocker (Bio-Rad), and incubated with the following primary antibodies at 4 °C overnight: anti-SHP-1, (GTX20658 GeneTex, Irvine, CA), and anti-GAPDH (G9545 Sigma-Aldrich, St Louis, MO). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Pierce ECL Western Blotting Substrate (ThermoFisher, Rockford, IL) was used to generate chemiluminescence signals, which were detected on X-ray films.
Tyrosine phosphatase activity assay
SHP-1 was immunoprecipitated from whole cell lysates using anti-mouse SHP-1 antibody (GeneTex) and Protein A/G Plus-Agarose beads (Santa Cruz Biotechnology) following the manufacturer’s protocol. SHP-1 tyrosine phosphatase activity was measured with RediPlate 96 EnzCheck Tyrosine Phosphatase Assay Kit (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. Fluorescence was measured in an ELISA reader (BioTek Instruments), and tyrosine phosphatase activity was expressed as the percent of phosphatase activity detected in cell lysates of WT mice.
Flow cytometric analysis of B and T cell populations in the spleen
The spleens were harvested from the mice (n = 4 per genotype). Cells were released by pressing the spleens under sterile metal screens and erythrocytes were lysed in cold ammonium chloride-based buffer. Fc receptors were blocked with TruStain FcX anti-mouse CD16/CD32 (cat# 101320; BioLegend, San Diego, CA), and surface antigens were stained with fluorescence-labeled antibodies to mouse CD3, CD4, CD8, B220/CD45, IgM, CD21/35, CD23, CD19, and CD138 (Additional file 2). Data acquisition and analysis were performed as before [28] using a FACSCanto II instrument and BD FACSDiva software (version 5.0).
Polyclonal activation of T cells with anti-mouse CD3/CD28 antibodies
For polyclonal activation, T cells were cultured in the presence of antibodies against mouse CD3 and CD28 (LEAF purified anti-mouse CD3ε antibody, Cat#100314, LEAF purified anti-mouse CD28 antibody, Cat#102112, both from BioLegend). Briefly, the day before harvesting spleen cells, 96-well culture microplates were coated with sterile 0.15 M sodium carbonate coating buffer (pH 9.6) containing 1 μg anti-CD3 and 1 μg anti-CD28 antibodies per well in 100 μl volume. Control wells contained only carbonate buffer. Plates were incubated overnight at room temperature. On the day of harvesting spleen cells, wells were washed 3 times with sterile phosphate-buffered saline (PBS, pH 7.2) and blocked with Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) containing antibiotics/antimycotics and supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT). The spleens from n = 3–6 mice were harvested and the spleen cells prepared as described above. “Untouched” T cells were isolated using immunomagnetic negative selection (EasySep Mouse T cell isolation kit, StemCell Technologies, Vancouver, Canada).
For proliferation assays, purified T cells were suspended in DMEM supplemented with 10% FBS and seeded in the culture microplates (pre-coated with anti-mouse CD3 and CD28 or coating buffer only) at a density of 2 × 105 viable cells in 200 μl of medium per well in triplicate wells. The cultures were maintained for 5 days in a culture incubator at 37 °C with 5% CO2 in air. For determination of cell proliferation, 18–20 h before harvest, the cells were pulsed with 0.5 μCi/well of [3H] thymidine (Perkin Elmer, Boston, MA). Isotope incorporation was measured using a scintillation counter (Micro-Beta; Perkin Elmer) and expressed as counts per minute (cpm). Stimulation index (SI) was calculated by dividing the mean cpm values of stimulated wells with the mean cpm values of control wells.
Activation of B cells with anti-mouse IgM antibody
To assess the polyclonal activation of B cells in WT and Shp1-Tg genotypes, we set up B cell cultures from WT, Shp1-Tg+/+, and Shp1-Tg+/− mice and used anti-mouse IgM for activation (anti-mouse IgM F (ab’)2, μ chain-specific, Functional Grade Purified, eBioscience, cat# 16-5092). Spleen cells of n = 3–6 mice per genotype were harvested. B cells were isolated using immunomagnetic negative selection (EasySep mouse pan B cell isolation kit, StemCell Technologies).
For proliferation assays, B cells were suspended in DMEM supplemented with 10% FBS and seeded in 96-well culture microplates at a density of 2 × 105 viable cells in 200 μl of medium per well. Anti-IgM (10 μg/ml) was added to stimulated wells, while control wells contained only medium and the cells (both in triplicates). The culture was maintained for 5 days. [3H] thymidine (Perkin Elmer) incorporation was measured as described for T cells. Stimulation index (SI) was calculated by dividing the mean cpm values of stimulated wells with the mean cpm values of control wells.
Mice, immunization, and visual assessment of PGIA
Human cartilage PG protein was prepared as described [23] to induce autoimmune arthritis. Adult WT (retired breeder) female BALB/c mice were obtained from the National Cancer Institute (NCI; Frederick, MD) or from the NCI colony of Charles River (Wilmington, MA). Shp1-Tg mice (both homozygous and heterozygous for the transgene) were bred and kept in the animal facility of Rush University Medical Center under specific pathogen-free conditions. All animal protocols were approved by the Institutional Animal Care and Use Committee (Rush University, Chicago, IL, IACUC permit #17-039).
To induce PGIA, mice were immunized intraperitoneally (i.p.) with human PG (100 μg protein in 100 μl sterile PBS emulsified with dimethyl-dioctadecyl ammonium bromide adjuvant (DDA; Sigma-Aldrich) three times, 3 weeks apart as described [29] [30]. In one of the experiments, mice received four PG injections. Mice were inspected for signs of arthritis (swelling and redness) twice a week after the second PG immunization. Upon disease onset, the degree of arthritis for each paw was visually scored every other day on a scale of 0 to 4 for each limb, summing the individual paw scores to a maximum visual arthritis (VA) score (arthritis severity) of 16 per animal [25] [29]. Arthritis incidence was expressed as the percentage of mice showing arthritis symptoms (> 0.5 score on any limb) of all mice examined in each group.
Measurement of cytokines in cell culture supernatants
In order to determine net cytokine secretion (stimulated minus unstimulated wells) spleen cells of naïve and PG-immunized mice were harvested, suspended in DMEM supplemented with 10% FBS, and dispensed into 48-well culture plates (2 × 106 viable cells in 900 μl medium per well). Cells were cultured in the absence or presence of human PG (25 μg/ml). On day 4 of culture, the supernatants were collected. Soluble IFNγ, IL-17, IL-10, and IL-4 concentrations were measured using murine cytokine ELISA kits (R & D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Briefly, 96-well Maxisorp ELISA plates (Thermo Scientific, Rochester, NY) were coated with the purified anti-cytokine capture antibodies at the recommended concentration overnight. After repeated washing, free binding sites were blocked with a blocking buffer containing 1% bovine serum albumin (BSA; Sigma-Aldrich). Undiluted cell culture supernatants (100 μl/well) and serially diluted cytokine standards were then incubated in duplicate wells with the immobilized capture antibody for 2 h. After repeated washing, HRP-conjugated anti-cytokine detection antibody was applied to the wells. The color reaction was developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (BD Biosciences). The optical density (OD) values at 450 nm and corresponding cytokine concentrations were determined. Net cytokine secretion was expressed as pg/ml in PG-stimulated minus non-stimulated cells’ supernatants.
Anti-human PG antibody ELISA
For the measurement of serum anti-human PG antibodies, wells of 96-well ELISA plates (Nunc) were coated overnight with human PG (1 μg/well each) in 100 μl/well of sodium carbonate buffer at room temperature [31]. Unbound antigen was removed by washing with 0.05% Tween 20 in PBS. Wells were blocked with 1.5% non-fat milk in 250 μl/well PBS for 1 h at room temperature on a shaker platform. Serum samples were diluted to 1:100 in blocking buffer and incubated with the antigen-coated wells (100 μl/well, duplicate wells) for 2 h at room temperature with shaking. Bound IgG was detected by incubation with 100 μl/well of HRP-conjugated anti-mouse IgG (BD Biosciences) at a 1:2000 dilution for 2 h at room temperature with shaking. Unbound material was removed with wash buffer between each of these steps. The color reaction was developed with 100 μl/well of O-phenylene-diamine solution (Sigma-Aldrich) dissolved freshly in PBS with H2O2, and incubation for 30 min in the dark. The reaction was stopped with 25 μl/well stop solution (4 N H2SO4). Absorbance at 450 nm was read in an ELISA reader.
Phosphorylation-specific protein microarray analysis
To determine which proteins were tyrosine-phosphorylated differentially in the CD4+ T cells of WT versus Shp1-Tg mice, Tyrosine Phosphorylation ProArray was performed by the Full Moon Biosystems (Sunnyvale, CA). This array uses site-specific phospho-tyrosine antibodies allowing determination of tyrosine phosphorylation of proteins at 228 specific sites. The array contained 6 technical replicates for each phosphorylation site. Spleen cells were harvested from PG-immunized WT and Shp1-Tg+/− mice in 3 independent experiments. Cells were pooled from 5 mice per genotype (a total of 15 WT and 15 Shp1-Tg+/− mice were used). Spleen cells were separated using EasySep magnetic separation kits to obtain CD4+/CD25− cell populations. Cell pellets were snap frozen and sent on dry ice to the Full Moon Biosystems for analysis. For data analysis, the intensity of protein phosphorylation in the array was measured using GenePix Pro 6.0 software (Axon Instruments, Molecular Devices, San Jose, CA). The normalized data for each array were computed as follows: normalized data = (Average Signal Intensity of Replicate Spots)/(Median Signal of the Average Signal Intensity) for all antibodies on the array. The normalized data were then used to determine the differences (fold change) between the control (WT) and Shp1-Tg samples. Proteins of CD4+ cells showing the highest degree of decrease in Tyr phosphorylation were selected. Statistical overrepresentation test was performed using the Panther Classification System v14.0 (http://www.pantherdb.org/) to annotate signaling pathways to the most underphosphorylated proteins. The reference set contained the genes of proteins included in the microarray.
In vivo SHP-1 activation
In order to investigate if the SHP-1 activator regorafenib (Selleckchem, Houston, TX) has an effect on PGIA development, we administered it preventively to WT BALB/c mice via oral gavage, daily at 10 and 20 mg/kg doses starting at the day of the 3rd PG injection. Regorafenib doses were chosen based on literature provided by the manufacturer, reporting beneficial effects on tumor growth in mice [32]. Vehicle (2% DMSO, 30% PEG300, and 5% Tween 80 in water) served as control. Drug administration and visual arthritis scoring was continued for 24 days after the last immunization. PGIA incidence and severity, in vitro cytokine secretion, and the presence of anti-human PG antibodies (in serum) were evaluated. Shp1-Tg+/− mice immunized with PG but without activator treatment were included for reference. In a second setting, 15 mg/kg regorafenib was administered therapeutically, 5 times a week via oral gavage after the initial signs of arthritis developed. Treatment was continued until day 27, and arthritis severity and body weight were monitored.
Statistical analysis
Data were analyzed and graphs created using Prism 8.0.2 software (GraphPad, USA). Results are expressed as the mean ± SEM unless specified otherwise. Groups were compared using one-way analysis of variance (ANOVA) or Student’s t test, and groups affected by two factors were compared by two-way ANOVA followed by Dunn’s or Tukey’s multiple comparison test as appropriate. For time-course experiments, area under the curve (AUC) calculations were also used. In each case, p < 0.05 was set as threshold for statistical significance.