institute, were fed a normal chow (Oriental Yeast, Tokyo, Japan) and water ad libitum,

Transcription

1 Supplement Methods Animals ApoE -/- mice (offspring of homozygous ApoE -/- mice, backcrossed onto the C57BL/6 background) 1 were kept in a specific pathogen-free animal facility at Kobe University institute, were fed a normal chow (Oriental Yeast, Tokyo, Japan) and water ad libitum, and were maintained on a 12:12-h light-dark cycle in the animal facility. All animal experiments were conducted according to the Guidelines for Animal Experiments at Kobe University School of Medicine. Experimental Design We fed six-week-old female ApoE -/- mice 20ng or 200ng calcitriol dissolved in carboxymetylcellulose or vehicle alone by gastric intubation with a plastic tube twice a week for 12 weeks. Mice under the same protocol as described above were injected with either 100 g of neutralizing CD25 monoclonal antibody (clone PC61; Bio Express Inc., West Lebanon, NH) to deplete CD4 + CD25 + Tregs or 100 g of isotype-matched control rat IgG (Bio Express Inc.) once every 4 weeks at 6, 10 and 14 week-old. Mice were killed at 18 weeks of age, and atherosclerotic lesions were examined. Assessment of Biochemical and Physiological Parameters Blood was obtained on the day after the last feeding with calcitriol or vehicle under overnight fasting condition and was collected by the cardiac puncture under anesthetic conditions using pentobarbital sodium (80mg/kg intraperitoneal injection). Plasma was obtained through centrifugation and stored at -80 C. Concentrations of plasma

2 total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglyceride, calcium and phosphorus were determined enzymatically by using an automated chemistry analyzer (SRL, Tokyo, Japan). 1,25(OH) 2 -dihydroxyvitamin D 3 and 25(OH)-hydroxyvitamin D 3, the principal circulating form of vitamin D 3, were analyzed by radioimmunoassay (SRL). Plasma intact parathyroid hormone (PTH) was determined using a commercial ELISA kit (Alpco Diagnostics, Salem, NH). Systolic and diastolic blood pressure was measured with the tail cuff method (Softron blood pressure-98a, Tokyo, Japan). Atherosclerotic Lesions Assessments At 18 weeks of age, mice were anesthetized and the aorta was perfused with saline. For aortic root lesion analysis, samples were cut from the ascending aorta, and the proximal samples containing the aortic sinus were embedded in OCT compound (Tissue-Tek; Sakura Finetek, Tokyo, Japan). Five consecutive sections (thickness, 10 m), spanning 550 m of the aortic sinus, were collected from each mouse and stained with Oil Red O (Sigma, St Louis, MO). For quantitative analysis of atherosclerosis, the total lesion area of 5 separate sections from each mouse was obtained and quantitatively analyzed with the use of the Image J software (National Institutes of Health) as previously described. 1,2 We assessed the aortic sinus mean plaque area in comparison among the groups. Lesion analysis was conducted by a single observer blinded to the group of the mice. Immunohistochemical Analyses of Atherosclerotic Lesions Immunohistochemistry was performed on acetone-fixed cryosections (10 m) of mouse

3 aortic roots using antibodies to identify macrophages (MOMA-2 at a dilution of 1:400; BMA Biomedicals, Augst, Switzerland), and T cells (CD4, 1:100; BD Biosciences, San Jose, CA), followed by detection with biotinylated secondary antibodies and streptavidin-horseradish peroxidase. For natural Tregs, acetone-fixed cryosections of mouse aortic roots were incubated with rat anti-foxp3 antibody (clone FJK-16s, 1:100; ebioscience, San Diego, CA), followed by Alexa Fluor 588 anti-rat secondary antibody (1:500; Molecular Probes, Eugene, OR) as described. 2 For mature DCs, slides were stained by using TSA TM - kit (PerkinElmer LAS, Inc., Boston, MA) according to the manufacturer s instructions. In brief, endogenous peroxidase activity was quenched 0.5% H 2 O 2 for 30 minutes. Sections were blocked with TNB buffer (TSA TM - kit) and hamster anti-cd11c antibody (clone N418, 1:200; BioLegend, San Diego, CA) and rat anti-cd86 (clone PO3.1, 1:200; ebioscience, San Diego, CA) of primary antibodies were applied for 1 hour at room temperature. Slides were washed and incubated with HRP-labeled goat-anti-rat IgG (American Qualex, San Clemente, CA) for 30 minutes. CD86 were detected with Cy3-Tyramide. Next, primary HRP was deactivated by treatment with 0.5% H 2 O 2 for 30 minutes, and the sections were then incubated with biotinylated anti-hamster IgG (Vector Laboratories, Inc., Burlingame, CA). Slides were washed and incubated with streptavidine-hrp (TSA TM - kit). Staining by hamster anti-cd11c was visualized by amplification of the signal with FITC-Tyramide. Nuclei were counterstained with DAPI (Molecular Probes). The appropriate fixation reagent depending on the primary antibodies was used. Negative controls were prepared with substitution with an isotype control antibody. Staining with Masson s trichrome was used to delineate the fibrous area as previously described. 2 Sections were observed under an All-in-one Type Fluorescence Microscope (BZ-8000; Keyence,

4 Osaka, Japan) using BZ Analyzer Software (Keyence). Stained sections were digitally captured, and the percentage of the stained area (the stained area per total atherosclerotic lesion area) was calculated. Quantification of CD4 + T cells, Foxp3 + Tregs, CD11c + DCs and CD11c + CD86 + DCs in atherosclerotic lesions was done by counting positively stained cells, which was divided by total plaque area. Cell Isolation and Flow Cytometry Analyses Purified CD4 + T cells and CD11c + DCs were isolated from mesenteric lymph nodes (MLNs) and spleens. CD4 + T cells and CD11c + DCs were positively selected with anti-cd4 + antibody microbeads and with anti-cd11c + microbeads (Miltenyi Biotec, Inc., Auburn, CA) using an AutoMACS separator (Miltenyi Biotec) according to the manufacturer s instructions. For fluorescence-activated cell sorter (FACS) analyses of lymphoid organs, MLN cells and splenocytes were isolated at 18 weeks of age. Cells were stained in PBS containing 2% FCS. FACS analysis was performed with a FACSCalibur using CellQuest Pro software (BD Bioscience). The antibodies used were as follows; anti-cd16/cd32 (clone 2.4G2; BD Bioscience), FITC-conjugated anti-cd4, Peridinin Chlorophyll Protein cyanin 5.5 (PerCP Cy5.5)-conjugated anti-cd4 (clone H129.19; BD Bioscience), PE-conjugated anti-cd8 (clone ; BD Bioscience), phycoerythrin (PE)-conjugated anti-cd25, allophycocyanin (APC)-conjugated anti-cd25 (clone PC61; BD Bioscience), (APC)-conjugated Foxp3 (clone FJK-16s; ebioscience), PE-conjugated anti-il-4 (clone BVD4-1D11; BD Bioscience), PE-conjugated anti-il-10 (clone JES5-16E3; BD Bioscience), PE-conjugated anti-ifn- (clonexmg1.2; BD Bioscience),PE-conjugated streptavidin (BD Bioscience), biotinylated anti-human LAP (BAF 246; R&D Systems),

5 FITC-conjugated anti-cd3e (clone 17A2; BD Bioscience), PE-conjugated anti-nk1.1 (clone PK136; ebioscience), APC-conjugated CD80 (clone 16-10A1; ebioscience), PE-conjugated CD86 (clone PO3.1; ebioscience), and isotype matched control antibodies. For CD80 and CD86 staining, CD11c + DCs were positively selected with anti-cd11c + microbeads as described above and were stained. Intracellular staining of Foxp3 or cytokines such as IL-4, IL-10 and IFN- was performed using the Foxp3 staining buffer set (ebioscience) or intracellular cytokine staining kit (BD Bioscience) and APC-conjugated Foxp3, PE-conjugated anti-il-4, PE-conjugated anti-il-10, PE-conjugated anti-ifn- or antibody as described above according to the manufacturer s instructions. All staining procedures were performed after blocking Fc receptor with anti-cd16/cd32 antibody. Surface staining was performed according to standard procedures at a density of 1x10 6 cells per 100 l, and volumes were scaled up accordingly. Cell Proliferation Assays and Cytokine assays In all cell culture experiments, we used RPMI 1640 medium (Sigma) supplemented with 10% FCS, 10mM Hepes, 50 mol/l 2 -mercaptoethanol and antibiotics. For analysis of in vitro suppressive function of CD11c + DCs, CD11c + cells from spleens of calcitriol-treated or control mice were cultured with 1x10 5 of CD4 + T cells from spleens of Balb/c mice at various ratios (total volume, 200 L/well). These cells were cultured at various ratios in flat-bottomed 96-well plates at 37 C with 5% CO 2 for 72 hours. In these experiments, CD11c + DC were irradiation with 18.5 Gy before co-culture. The cells were pulsed with 1 Ci of [ 3 H]-thymidine (GE Healthcare, Buckinghamshire, UK) for the last 16 hours, and thymidine incorporation was assessed

6 with a LS 6500 liquid scintillation counter (Beckman Coulter, Inc, Brea, CA). For measurement of cytokine such as IL-17, IL-10, IFN-, and IL-6, splenocytes were cultured at a concentration of cells/ml for 72 hours with 2 g/ml concanavalin A (Con A; Sigma). Culture supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA) using paired antibodies specific for corresponding cytokines according to the manufacturer s instructions (R&D Systems). RT-PCR Analysis At 18 weeks old, mice were anesthetized and the aortic roots were excised as described above. Total RNA was extracted from CD4 + T cells, CD11c + DCs, and mouse aortic roots after perfusion with RNA-later (Ambion, Austin, TX) using TRIzol reagent (Invitrogen, Carlsbad, CA). Quantitative PCR was performed using One Step SYBR PrimeScript RT-PCR Kit (Takara, Shiga, Japan) and an ABI PRISM 7500 Sequence Detection system (Applied Biosystems, Foster City, CA) according to the manufacturer s protocol as described previously. 2 The following primers were used to amplify renin, CD25, Foxp3, Cytotoxic lymphocyte antigen 4 (CTLA4), CD11c, CD80, CD86, interleukin (IL)-6, IL-10, IL-12p35, IL12p40, interferon (IFN)-, transforming growth factor (TGF)-, intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, monocyte chemoattractant protein (MCP)-1, thymus and CC-chemokine ligand (CCL)17, CCL22, CC-chemokine receptor (CCR)4, CCR8 and GAPDH: Renin, 5 -CTC CTG GCA GAT CAC GAT GAA G-3 and 5 -GGA GCT CGT AGG AGC CGA GAT A-3 ; CD25, 5 -CTG ATC CCA TGT GCC AGG AA-3 and 5 -AGG GCT TTG AAT GTG GCA TTG-3 ; Foxp3, 5 -CTC ATG ATA GTG CCT GTG TCC TCA A-3 and 5 -AGG GCC AGC ATA GGT GCA AG-3 ; CTLA4,

7 5 - CCT CTG CAA GGT GGA ACT CAT GTA-3 and 5 -AGC TAA CTG CGA CAA GGA TCC AA-3 ; CD11c, 5 -AGA CGT GCC AGT CAG CAT CAA C-3 and 5 -CTA TTC CGA TAG CAT TGG GTG AGT G-3 ; CD80, 5 -AGT TTC CAT GTC CAA GGC TCA TTC-3 and 5 -TTG TAA CGG CAA GGC AGC AAT A-3 ; CD86, 5 -TGG CAT ATG ACC GTT GTGTGT G-3 ; IL-6, 5 -CCA CTT CAC AAG TCG GAG GCT TA-3 and 5 -GCA AGT GCA TCA TCG TTG TTC ATA C-3 ; IL-10, 5 -GAC CAG CTG GAC AAC ATA CTG CTA A-3 and 5 -GAT AAG GCT TGG CAA CCC AAG TAA-3 ; IL-12p35, 5 -TGT CTT AGC CAG TCC CGA AAC C-3 and 5 -TCT TCA TGA TCG ATG TCT TCA GCA G-3 ; IL-12p40, 5 -GCT CGC AGC AAA GCA AGG TAA-3 and 5 -CCA TGA GTG GAG ACA CCA GCA-3 ; IFN-, 5 -CGG CAC AGT CAT TGA AAG CCT A-3 and 5 -GTT GCT GAT GGC CTG ATT GTC-3 ; TGF-, 5 -GTG TGG AGC AAC ATG TGG AAC TCT A-3 and 5 -TTG GTT CAG CCA CTG CCG TA-3 ; ICAM-1, 5 -CAA TTC ACA CTG AAT GCC AGC TC-3 and 5 -CAA GCA AGT CCG TCT CGT CCA-3 ; VCAM-1, 5 -TGC CGG CAT ATA CGA GTG TGA-3 and 5 -CCC GAT GGC AGG TAT TAC CAA G-3 ; MCP-1, 5 -GCA TCC ACG TGT TGG CTC A-3 and 5 -CTC CAG CCT ACT CAT TGG GAT CA-3 ; CCL17, 5 -CCG AGA GTG CTG CCT GGA TTA-3 and 5 -AGC TTG CCC TGG ACA GTC AGA-3 ; CCL22, 5 -GGC ACC TAT CCA GTG CCA CA-3 and 5 -TGG TGG ACC AGC CTG AAA CTC-3 ; CCR4, 5 -TGC TCG CCT TGT TTC AGT CAG-3 and 5 -AGC CAT CTT GCC ATG GTC TTG-3 ; CCR8, 5 -CAG ACC CAC AAC CTG CTG GA-3 and 5 -GAC AGC GTG GAC AAT AGC CAG A-3 ;GAPDH, 5 -TGT GTC CGT CGT GGA TCT GA-3 and 5 -TTG CTG TTG AAG TCG CAG GAG-3. Amplification reactions were performed in duplicate and fluorescence curves were analyzed with included software. GAPDH was used as an

8 endogenous control reference. Statistical Analysis Data were expressed as means±sem. To detect significant differences between 2 groups or among 3 groups, unpaired Student t test, Mann-Whitney U test, or one way ANOVA with post hoc test was used when appropriate. Statistical values of p<0.05 were considered statistically significant. For statistical analysis, GraphPad Prism 4.0 was used. Supplemental References 1. Ozaki M, Kawashima S, Yamashita T, Hirase T, Namiki M, Inoue N, Hirata K, Yasui H, Sakurai H, Yoshida Y, Masada M, Yokoyama M. Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoe-deficient mice. J Clin Invest. 2002;110: Sasaki N, Yamashita T, Takeda M, Shinohara M, Nakajima K, Tawa H, Usui T, Hirata K. Oral anti-cd3 antibody treatment induces regulatory T cells and inhibits the development of atherosclerosis in mice. Circulation. 2009;120:

9 CD25 + cells / CD4 + cells (%) Thymus Base 2W 4W Foxp3 + / CD4 + CD8 - cells (%) Base 2W 4W Base (4 weeks of age) Control D 3 200ng CD25 + cells / CD4 + cells (%) MLNs * CD25 + Foxp3 + cells / CD4 + cells (%) Base 2W 4W Base 2W 4W * CD25 + cells / CD4 + cells (%) Spleen CD25 + Foxp3 + cells / CD4 + cells (%) Base 2W 4W Base 2W 4W Supplemental Figure I

10 MLNs CD25 + cells / CD4 + cells (%) * * Spleen CD25 + cells / CD4 + cells (%) * * Supplemental Figure II

11 A IFN γ / CD4 + cells (%) MLN Spleen IL-4 / CD4 + cells (%) MLN Spleen Control D 3 200ng B IL-10 / CD4 + cells (%) MLN Spleen LAP + / CD4 + cells (%) CD25 + LAP + / CD4 + cells (%) CD25 - LAP + / CD4 + cells (%) MLN Spleen MLN Spleen MLN Spleen C (pg/ml) IL-10 (pg/ml) IL-17 (pg/ml) IFN-γ (pg/ml) IL-6 * * D CD3 - NK1.1 + (%) CD3 + NK1.1 + (%) MLN Spleen MLN Spleen Supplemental Figure III

12 Supplemental Figure Legends Supplemental Figure I. Effects of Calcitriol on stimulating the expansion of thymus and peripherally induced Tregs. We fed 4 week-old female ApoE -/- mice 200ng calcitriol dissolved in carboxymetylcellulose or vehicle alone by gastric intubation with a plastic tube twice a week for 4 weeks. For FACS analyses of lymphoid organs, thymus cells, MLN cells and splenocytes were isolated at 4, 6 and 8 weeks of age. Thymus cells were stained with FITC-conjugated anti-cd4, PE-conjugated anti-cd8 and APC-conjugated anti- Foxp3. MLN cells and splenocytes cells were stained with FITC-conjugated anti-cd4, PE-conjugated anti-cd25 and APC-conjugated anti-foxp3. For all FACS analyses, data represent means ± SEM of 3 mice in each group. Horizontal bars represent means. *p<0.05 vs. control. Supplemental Figure II. The dynamics of the alteration of Tregs after a single intraperitoneal injection of 100 g of CD25-depleting PC61 antibody. MLN cells and splenocytes from calcitriol with anti-cd25 antibodies (D 3 200ng+anti- CD25 Ab; closed circle), control with anti-cd25 antibodies (Control+anti-CD25 Ab; open circle), and calcitriol with isotype-matched control antibodies (D 3 200ng+rat-IgG; gray circle) were isolated 1, 2, and 4 weeks after a single intraperitoneal injection of 100 µg of CD25-depleting PC61 antibody. Data represent means ± SEM of 3 mice in each group. *p<0.05 vs. D 3 200ng+anti-CD25. p<0.05 vs. control+anti-cd25 Ab.

13 Supplemental Figure III. Effects of calcitriol on the phenotypes of T lymphocytes and other cells in MLNs and spleen. For FACS analyses of lymphoid organs, MLN cells and splenocytes were isolated at 18 weeks of age. CD4 + T cells were stained with PE-conjugated anti-ifn-γ for Th1 lymphocytes, PE-conjugated anti-il-4 for Th2 lymphocytes, PE-conjugated anti-il-10 for Tr1 cells, APC-conjugated anti-cd25 and PE-conjugated anti-lap for Th3, or FITC-conjugated anti-cd3e and PE-conjugated anti-nk1.1 for Natural Killer (NK) cells or NKT cells (A, B, and D). There was no significant differences in IFN-γ producing CD4 + T cells (Th1) and IL-4 producing CD4 + T cells (Th2) in the calcitrioltreated group and control group (A). Furthermore, there was no obvious induction of IL-10 producing CD4 + T cells (Tr1) and LAP + Tregs (mainly Th3) in calcitriol-treated mice (B). Furthermore, there were no significant difference in NK cells (CD3 - NK1.1 + ) and NKT cells (CD3 + NK1.1 + ) between the two groups (D). Data represent means ± SEM of 3 mice in each group. Horizontal bars represent means. *p<0.05 vs. control. Splenocytes were stimulated with Con A in vitro for 72 hours. IL-17, IL-10, IFN-γ, and IL-6 productions in supernatants were measured by ELISA (C). The productions of IL- 10 and IL-17 from splenocytes were similar between the two groups, suggesting it is unlikely calcitriol affected Tr1 and Th17 in the spleen. Productions of inflammatory cytokine such as IFN-γ and IL-6 from splenocytes were significantly decreased in calcitriol-treated mice. Data represent means±sem of at least 6 mice in each group. Horizontal bars represent means. *p<0.05 vs. control.