Supplementary Methods

Transcription

1 Supplementary Methods Mice injections. C57BL/6 female mice 6-10 weeks of age were purchased from the Jackson Laboratory. Soluble rapamycin (Sigma) was diluted in PBS and administered i.p. to mice at 1.5 mg/kg/day for 3 days. Rapamycin encapsulation in PLGA microparticles was resuspended in PBS, sonicated and administered i.p. to mice at 2 mg/day for 3 days. On day 4, mice were challenged with CpG-A (50 μg/mouse) or YF-17D (1 x 10 6 PFU/mouse). At different time points, mouse serum was obtained via retro-orbital bleed. Mouse was then sacrificed (CO 2 asphyxiation) for spleen removal. pdcs were enriched from spleen using anti-mpdca1 microbeads (Miltenyi Biotec) (purity>80%) and cultured for 40 h. Supernatant was then collected for IFN-α ELISA. Rapamycin encapsulation in PLGA microparticles and characterization. Rapamycin was encapsulated in PLGA microparticles using a standard oil in water (O/W) based single emulsion particle synthesis approach. Briefly, 160 µl of rapamycin (25 mg/ml in DMSO) combined with 2 ml of 10% wt/v PLGA in dicholoromethane was homogenized in a 5% PVA solution at speed setting 4 for 2 minutes using a Fisher Powergen 500 homogenizer (Fisher Scientific). The resulting oil in water emulsion (O/W) was added to 85ml of 5% PVA solution and magnetically stirred for 4 hours to evaporate the organic solvent. Resulting rapamycin encapsulated microparticles were centrifuged at 4000g for 20 min, washed and lyophilized using a Labconco Freezone 2.5 L benchtop lyophilizer. Microparticles were stored at 20 C until further use. Number average size distribution of rapamycin microparticles analyzed using a Brookhaven 90plus particle size analyzer (BIC) indicated a mean size of 1400nm. Encapsulation efficiency of rapamycin was calculated by modifying a technique described to evaluate protein loading in PLGA microspheres. 80% encapsulation efficiency at 16 ug of rapamycin/mg of PLGA formulation was realized in 2 independent microparticle batches synthesized indicating reproducibility. Apoptosis assays for pdcs. pdcs ( /well) were treated with rapamycin (500 nm or 20 nm) in 96-well flat-bottom tissue culture plates for 3 h. The cells were then harvested and stained with APC-Annexin V (Sigma) and propidium iodide to quantitate live cells by flow cytometry. 1

2 Cytokine detection. For the mouse cytokines, the following kits were used: IL-4, IFN-γ, IL-10, IL-12p40, IL-12p70, TNF and IL-6 were obtained from BD Bioscience; IL-13, IL-17 were obtained from ebioscience; and IP-10 was obtained from R&D Systems. Human IL-12p70, IL-6, TNF, IP-10 were all measured using kits obtained from BD Bioscience. Flow cytometry. Isolated mouse pdcs were fixed and permeablized by using Cytofix-Cytoperm and stained with anti-tlr9 polyclonal antibody (Imgenex) following by PE conjugated goat anti-rabbit antibody (Santa Cruz). To detect IRF7 phosphorylation, human pdcs were pretreated with rapamycin or vehicle for 3 h. Cells were then stimulated with CpG-A. After 45 min, cells were fixed for 10 min at 37 C with BD Cytofix buffer and then permeabilized on ice for at least 30 min with BD Phosflow Perm Buffer III. Cells were stained with PE mouse anti-irf7 (BD Biosciences). Flow cytometric analysis was performed on a Becton Dickinson FACSCaliber flow cytometer at the Emory Vaccine Center. For analysis of DC population in knockout mice, cells were stained with anti-cd11c and anti-cd45ra or anti-cd11c and antimpdca-1 and analyzed by flow cytometry. Microarray analysis. Total RNA was extracted from freshly isolated, flow-cytometry-sorted mouse pdcs (CD11c int CD45RA + ) with an RNeasy kit (Qiagen). RNA quality was assessed with an Agilent Bioanalyzer 2100 and only RNA with minimal degradation and distinct 18s and 28s rrna bands was used for analysis. Microarray processing was done by the Vanderbilt Microarray Shared Resource. Fragmented and biotin-labeled cdna was synthesized from 100 ng purified mrna with the Ovation Biotin System (Nugen). The cdna was hybridized to Affymetrix Murine Genome microarray chips (Affymetrix). Hybridized chips were stained and washed and were scanned with a GeneArray scanner (Affymetrix). GeneSpring software (Silicon Genetics) was used for data analysis. Imaging. For imaging on pdcs, purified cells were pretreated with rapamycin following by CpG-A stimulation. After 12 h stimulation, pdcs were fixed with 3.7% formaldehyde for 10 min at room temperature. The fixed cells were seeded on glass cover slips and then permeablized with 0.5% saponin (Sigma). Cells were blocked with 20% goat serum for 30 min and incubated with primary antibody, anti-irf7 (Santa Cruz), at a 1:20 dilution, or normal rabbit IgG, as an 2

3 isotype control at 4 C overnight. After washing, cells were incubated with Biotin-conjugated anti-rabbit IgG 1:100 (Santa Cruz) and then Strepavidin-FITC (BD Bioscience). The cells were washed and mounted using ProLong Gold anti-fade reagent with DAPI (Invitrogen). Immunofluorescence for IRF7 staining was detected using a LSM510 confocal microscope and images were captured and analyzed using the Zesis LSM Image Browser. For imaging on RAW cells, cells were cultured on cover slips in 24 well plate and transfected with TLR9-YFP plasmids. 40 h later, cells were pretreated with rapamycin (100 nm) or vehicle. Cells were then stimulated with 5 μg/ml CpG-A-Cy5-DOTAP for 90 min. After washing, cells were mounted using Fluorsave (Calbiocam) before analysis using deconvolution microscopy. Luciferase reporter assay. For NF-κB assay, HEK293 cells were co-transfected with NF-κB-luciferase, Renilla- luciferase and TLR9 constructs. For IFN-β assay, the cells were transfected with constitutively active IRF7 plasmid (or empty vector), together with IFN-βluciferase, Renilla-luciferase, TLR9, wild type IRF7 and MyD h after transfection, cells were stimulated with CpG-A with or without rapamycin pretreatment. NF-κB or IFN-β luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega) according to the manufacture s instructions. Data are normalized for transfection efficiency by dividing firefly luciferase activity with that of Renilla luciferase. 3

4 Supplementary Figures Supplementary Figure 1. Rapamycin inhibits human pdcs secretion of IFN-β and other cytokines. ELISA assay on isolated human pdcs pretreated with different dosage of rapamycin for 3 h and then stimulated with CpG-A (10 μg/ml). Supernatants were collected 24 h later and IFN-β, TNF, IL-6 and IP-10 production was measured. Data are representative of three independent experiments. 4

5 Supplementary Figure 2. Rapamycin, CsA and FK506 differentially inhibit mouse BMDCs and human MoDCs cytokine production. ELISA assay on in vitro cultured mouse BMDCs and human MoDCs pretreated with rapamycin, CsA or FK506 and then stimulated with LPS (1 μg/ml). Supernatants were collected 24 h later and IL-12, TNF, IL-6 and IL-10 production was measured. Data are representative of at least three independent experiments. 5

6 Supplementary Figure 3. Analysis of pdcs and conventional DCs (cdcs) in S6K1,2-KO mice. Flow cytometry of splenocytes were prepared from wild-type and S6K1-S6K2 double deficient mice (S6K1,2-KO) and stained with the indicated combination of antibodies. The numbers represent the percentage of cells and absolute cell numbers were also shown in parentheses. Data are representative of two independent experiments. 6

7 Supplementary Figure 4. Knock down of S6K1,2 inhibits TLR9-MyD88 interaction. Immunoprecipitation and immunoblot analysis of lysates of RAW cells (4 x 10 6 ) transfected with MyD88-HA and TLR9-GFP plasmids; 24h later, the cells were transfected with sirnas against S6K1,2 or control sirna. After an additional 24h, the cells were stimulated with CpG-A- DOTAP for 90 min. Cell extracts were immunoprecipitated (IP) with anti-gfp and immunoblot (IB) was performed to detect co-precipitated MyD88. Rapamcyin treatment was used as a control for TLR9-MyD88 dissociation. Data are representative of two independent experiments. 7

8 Supplementary Figure 5. Total IRF7 protein expression was not affected by rapamycin. Immunoblot analysis of cell lysates from medium cultured, CpG-A activated pdcs (with or without rapamycin pretreatment) analyzed for total IRF-7 protein expression. Data are representative of three independent experiments. 8

9 Supplementary Figure 6. Regulation of anti-viral and other anti-inflammatory gene expression by rapamycin in pdcs. Heat-map of gene expression in flow cytometry-sorted mouse pdcs activated with CpG-A for 4 or 12 h in the presence or absence of rapamycin. Total RNAs were extracted and subjected to microarray analysis. Expression of anti-viral and other anti-inflammatory genes is presented. 9

10 Supplementary Figure 7. In vivo administration of rapamycin inhibits CpG-A-induced IFN-α production. ELISA assay of pdcs from 57BL/6 mice were treated daily with rapamycin microparticle (2 mg/mouse/day) for 3 days or left untreated. On day 4, mice were vaccinated subcutaneously with CpG-A (50 μg/mouse). At indicated time points, mice were bled and sacrificed for spleen removal. pdcs were enriched from spleen using anti-mpdca-1 microbeads. IFN-α production in serum as well as in pdc culture supernatant was measured. Data are representative of three independent experiments. 10

11 Supplementary Figure 8 Knock-down of S6K1,2 inhibits YF-17D induced IFN-α secretion in pdcs. Immunoassays of mouse pdcs transfected with S6K1,2 sirna pools for 5 h, followed by YF-17D stimulation for 24 h. S6K expression was determined by immunoblot and IFN-α production in supernatant was determined by ELISA assay. Rapamcyin treatment was used as a control for inhibition of IFN-α production. Data are representative of two independent experiments. 11