Mice
Female MRL/MpJ-Faslpr/J (MRL/lpr) mice were purchased from Jackson Laboratory (Bar Harbor, ME). At 12 weeks of age, 10 mice were started on a high Mg diet containing Mg2800 ppm and 10 mice were started on a regular diet (from here on referred as Mg500 ppm) as a control (see below for details). Both experimental and control groups were sacrificed at 21 weeks of age. Experiments were performed under an Institutional Animal Care and Use Committee (IACUC) approved protocol, and mice were maintained in the same room to limit potential effects of microbiome differences.
Diet chow composition and regimens
Dietary chow. The diets were purchased from Teklad-Envigo Laboratories (Somerset, NJ). Mice received identical diets, except for the amount of magnesium. Specifically, the diets were irradiated and had the following contents (g/kg): protein (17.7), carbohydrates (64.4), fat (6.2), casein (200), DL-methionine (3.0), sucrose (415), corn starch (250), soybean oil (60), cellulose (30), vitamin mix (Teklad 40060), ethoxyquin (antioxidant) (0.01), calcium phosphate, dibasic (13.7), potassium citrate (monohydrate) (7.7), calcium carbonate (4.8), sodium chloride (2.6), potassium sulfate (1.82), ferric citrate (0.25), manganous carbonate (0.12), zinc carbonate (0.056), chromium potassium sulfate (dodecahydrate) (0.02), cupric carbonate (0.012), potassium iodate (0.0004), and sodium selenite, (pentahydrate) (0.0004).
The regular Mg diet had Mg oxide 0.822 g/kg of chow (Mg500 ppm) and the high Mg diet had Mg oxide 2.3 g/kg of chow (Mg2800 ppm). Twelve-week-old mice were randomly assigned to receive the normal Mg diet Mg500 or the high Mg diet Mg2800 for nine weeks.
The dose of high magnesium diet was based on our previous studies showing that a dose of magnesium nearly 5-fold the recommended daily requirements for mice was well-tolerated and induced significant reduction in arthritis severity and joint damage scores, while increasing number of FOXP3 + Tregs in arthritic C57BL/6 and DBA1/ mice [11].
Clinical skin severity scoring
Inflammatory skin lesions on the dorsum of the neck, ears and forehead were scored based on a scale of 0–3 as previously described [13]. 0 = no visible skin changes, 1 = minimal hair loss with redness and a few scattered lesions, 2 = redness, scabbing, and hair loss with a small area of involvement, and 3 = ulcerations with an extensive area of involvement.
Arthritis activity and severity scoring
The clinical arthritis score was determined according to a scoring scale ranging from 0 to 16 per mouse per day as previously reported where 1 = swelling and erythema in a single joint, 2 = swelling and erythema in more than one joint, 3 = swelling of the entire paw and 4 = swelling of paw and inability to bear weight [14].
Histological scoring of the skin and kidney lesions
Following euthanasia (100 mg/kg Ketamine/Xylazine by route of IP injection), mice were perfused with 4% paraformaldehyde in PBS at a rate of 8–10 ml/min. Skin and kidneys were paraffin-embedded (10%). Paraffin-embedded tissue Sect. (3 μm) were mounted on glass slides and stained with H&E (skin) or Periodic-Acid Schiff (PAS) (kidney). Light microscopy images were acquired after on a wide-field microscope (Zeiss AxioImager Z2M). The histology slides were scanned and the high-resolution images scored blindly for treatment identify by two experienced pathologists (RK and FS, respectively) using digital imaging (WSI).
Skin histology: Skin histopathological were graded from 0 to 2 for the following parameters: (a) degree of acanthosis, from 0 (none) to marked 2 (thickened dermis); (b) hyperkeratosis, 0 (none) to 2 (markedly increased keratin); inflammation, 0 (sparse) to 2 (heavy lymphocytic infiltrates); (c) fibrosis, dermal collagen, 0 (normal) to 2 (markedly thickened); (d) vessels, 0 (normal) to 2 (diffusely dilated); (e) ulcer, 0 (absent) or 1 (present). [15]
Renal histology
Kidney histopathological changes were quantitated based on previously reported scoring systems [15,16,17]. Glomerular involvement including mesangial proliferation, mesangial matrix hyperplasia and glomerulosclerosis, each was graded from 0 to 3 (0, absent; 1, mild; 2, moderate; 3, severe) with a maximum score of 9. Glomerular crescentic formation was graded from 0 to 3 (0, absent; 1, less than 25%; 2 more than 25%). The tubulo-interstitial compartment changes, including interstitial fibrosis, tubular atrophy and interstitial inflammation were each scored from 0 to 3 (0, absent; 1, in < 25% of the section; 2, in 25–50% of the section; 3, in 50–100% of the section) with a maximum score for 9. Vasculitis was reported if detected.
Flow cytometry analysis
Spleens were harvested and individually analyzed. Single cell suspensions (1 to 3 × 106) were stained with fluorescent-labelled monoclonal antibodies for cell surface antigens and incubated for 10 min at 25 °C, or 30 min at 4 °C. For intracellular staining cells were fixed with Cytofix/Cytoperm™ (Cat. 554714, BD Bioscience, San Jose, CA) for 20 min at 4 °C, followed by permeabilization in 1X Perm/Wash™ solution (Cat. 554723, BD Bioscience) for 15 min. In selected experiments, cells were fixed using eBioscience™ FOXP3/Transcription Factor Staining Buffer Set (Cat. 00-5523-00, Thermo Fisher Scientific, Waltham, MA). Fixed cells were stained with fluorochrome-conjugated antibodies for 30 min–1 h at 4 °C in the dark. CD4+FOXP3+ regulatory T cells (Tregs) were stained using anti-CD4-PE-Cy7 (clone GK1.5) (TONBO bioscience, San Diego, CA), anti-CD8a-Pacific Blue (clone 53 − 6.7) (Biolegend, San Diego, CA), and anti-FOXP3-APC (clone FJK-16 S) (Thermo Fisher Scientific). Tr1 cells were identified by anti-CD4-Pacific blue (clone: RM4-5), anti-CD45RA-PE (clone: 14.8), anti-CD49B-PeCy7 (clone: DX-5), anti-LAG3-APC (clone: C9B7W) and anti-IL-10-PerCP/Cy5.5 (clone: JES5-16E3). TFH cells were stained with anti-CD4-Pacific blue (clone: RM4-5), anti-CXCR5-biotin-APC (clone 2G8), anti-PD1-PeCy7 (clone: RMP1-30), anti-BCL6-PE (Clone: IG191E/A8) and anti-IL-10-PerCP/Cy5.5 (clone: JES5-16E3) (antibodies from Biolegend or BD Bioscience). In selected experiment TFH cells were analyzed using biotinylated anti-CXCR5 (clone 2G8) (BD Pharmingen) followed by Pacific Blue streptavidin (Thermo Fisher Scientific), anti-TCRb-PerCP-Cy5.5 (clone H57-597), anti-PD1-PE-Cy7 (clone RMP1-30), anti-CD4-BV510 (clone GK1.5) (BD Pharmingen), and anti-FOXP3-FITC (clone FJK-16 S) (Thermo Fisher Scientific). Germinal Center and Class switched B cells were stained using anti-B220-Pacific Blue (clone RA 3–6 B2), anti-FAS-APC (clone Jo2), and anti-GL7-FITC (clone GL7) (BD Pharmingen); anti-IgD-APC-Cy7 (clone 11-26c), and anti-IgM-PE-Cy7 (clone eB121-15F9) (Thermo Fisher Scientific).
For intracellular cytokine analysis, cells were treated with GolgiPlug (1 µg/ml; BD Biosciences, at 37 °C for 4 h), and stained with ) (BioLegend), anti-CD4-APC-Cy7 (clone GK1.5) (TONBO bioscience), anti-CD8-PacBlue (clone 53 − 6.7) (Biolegend), anti-IL-1B-: PE-Cy7 (clone NJTEN3 ) (company eBioscience), anti-TNF-α-FITC (clone MP6-XT22) (eBioscience), and anti-INF-ɣ-APC (clone XMG1.2) (eBioscience). At least 50,000 cells were acquired per sample. Samples were acquired on a BD LSRII, on a three-laser Canto II (BD Biosciences) flow cytometer, and analyzed with FlowJo (https://www.flowjo.com) software (Ashland, OR).
Anti-dsDNA antibodies
Blood was collected via puncture of the submandibular vein, and serum isolated for ELISA. Antibodies were quantified using a commercially available kit (Mouse anti-dsDNA IgG-specific ELISA Kit, Cat. 5120, Alpha Diagnostic Intl. Inc., San Antonio, TX).
Urinary and serum renal function chemistry measurements
Urine samples were collected from individual mice through gentle restrain in a collection device. Urine creatinine and albumin were quantified using commercial kits (Cayman Chemical, Cat. 500701, Ann Harbor, MI; Bethyl Laboratory Inc., Cat. E99-134, Montgomery, TX, respectively). Albuminuria was expressed as the ratio of urine albumin to urine creatinine. Serum BUN was quantified in serial blood collections using a colorimetric detection kit (Thermo Scientific, Cat. EIABUN) according to the manufacturers’ instructions.
Serum levels of cytokines/chemokines
Blood samples collected after nine weeks on the Mg500 or Mg2800 diets, and serum used for cytokine quantification. BAFF, IFN-ɣ, IL-6, IL-10, IL-16, IL-18, IL-21, IP-10 (CXCL10) and TNF-α were measured using a bead-based multiplex array ProcartaPlex mouse Mix&Match panel (ThemoFisher Scientific). All specimens were assayed in duplicate according to the manufacturer’s instructions. The assay was performed using MAGPIX instrument with xPONENT software (Luminex Corporation, Austin, TX) and analyte concentration calculated from standard curves using 5-parameter logistic curve fit.
Statistics
All variables were normally distributed and therefore means were compared with unpaired t-test or two-way ANOVA (Sidak’s multiple comparison test). P values < 0.05 were considered significant. All statistical analyses were performed using GraphPad Prism (version 8 for Windows, GraphPad Software, Inc.).
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