B10.A mice (I-Ek), C57BL/6 mice (H-2b), TCR-Cyt-5CC7-I/Rag1−/− transgenic (PCCF-specific TCR Tg; I-Ek) and C57BL/6-Tg (Tcra2D2,Tcrb2D2)1Kuch/J (MOG35–55 specific TCR) were purchased from Jackson Laboratory (Bar Harbor, ME) and Taconic (Hudson, NY). Transgenic mice were bred and maintained in the Temple University School of Medicine animal facility (Philadelphia, PA) under pathogen-free conditions. All mice used were between 6 and 10 wk of age. Mice were handled and housed in accordance with the guidelines of the Temple University Animal Care and Use Committee.
Recombinant murine GM-CSF, recombinant murine CCL19, IL-12, IL-6, were purchased from Peprotech Inc (Rocky Hill, NJ). Docosahexaenoic Acid was purchased from Cayman Chemical (Ann Arbor, MI). Lipopolysaccharide (LPS) (Escherichia coli O26:B6), pertussis toxin (PTX), streptavidin-peroxidase, phorbol myristate acetate (PMA) and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO). CD4 and CD11c MicroBeads were purchased from Miltenyi Biotec (Bergish-Gladbach, Germany). Recombinant IL-23, capture and biotinylated anti-mouse IL-23 antibody, PE-conjugated anti-mouse PD-L1 and CD25, FITC-conjugated anti-mouse PD-L2, mouse regulatory T cell staining kit, APC-conjugated anti-mouse INFγ were purchased from eBioscience (San Diego, CA). FITC-conjugated anti-mouse CD80, CD86, CD40, MHCII, CD4, CD44; PerCP-Cy™5.5 conjugated anti-mouse CD69, PE-conjugated anti-mouse IL-17, recombinant mouse IL-10, IFNγ and capture and biotinylated anti-mouse IL-2, IL-12p70, IL-6, IL-10, IFNγ; GolgiPlug, annexin V-FITC apoptosis detection kit I, Cytofix/Cytoperm, Perm/Wash buffer, TMB Substrate Reagent Set, and the Cycle TEST PLUS DNA Reagent Kit were purchased from BD PharMingen (San Diego, CA). Pigeon cytochrome c fragment (PCCF), myelin oligodendrocyte glycoprotein (MOG)35–55, proteolipid protein (PLP)139–151, CFSE Cell Proliferation Kit, 1X HBSS and 10X HBSS were purchased from Invitrogen Corporation (Carlsbad, CA). Capture and biotinylated anti-mouse IL-17, recombinant mouse IL-17, recombinant TGFβ, and recombinant mouse IL-2 were purchased from R&D Systems (Minneapolis, MN). DNase I grade II and Liberase TL were purchased from Roche (Indianapolis, IN). Ketamine HCl was purchased from Fort Dodge Animal Health (Fort Dodge, IA). Xylazine was purchased from Butler Animal Health Supply (Dublin, OH). 0.5 M EDTA was purchased from Promega Corporation (Madison, WI). Percoll was purchased from GE Healthcare (Piscataway, NJ). Mycobacterium tuberculosis H37 RA was purchased from Difco (Detroit, MI).
Generation and purification of DC from bone marrow
DC were generated from bone marrow as described previously (Kong et al., 2010
). On day 7, the non-adherent cells were harvested and purified by immunomagnetic sorting with anti-CD11c–coated magnetic beads using the autoMACS system according to the manufacturer’s instructions (Miltenyi Biotec). The purity of the sorted cells was determined by FACS analysis (>96% CD11c+
Isolation of CD4+ T cells
Purified CD4+ T cells were isolated from the spleen of PCCF-specific TCR-Tg mice or MOG35–55 specific TCR-Tg mice by positive immunomagnetic selection using anti-CD4 mAb magnetic beads (Miltenyi Biotec). The purified T cells were 98% CD4+ as determined by FACS analysis.
Cells were subjected to FACS analysis in a 3-color FACS Calibur (BD Biosciences, Mountain View, CA). Data were collected for 10,000 cells and analyzed using Cellquest software from BD Biosciences (San Jose, CA). DC or T cells washed with ice cold PBS and incubated for 30 minutes at 4°C with various FITC/PE/APC/PerCP conjugated antibodies and were analyzed by flow cytometry. For the detection of Foxp3, cells were first stained with anti-CD4 and anti-CD25, fixed with Cytofix/Cytoperm buffer, incubated with anti-Foxp3, and analyzed by FACS. The specificity of the primary Abs was established with appropriate isotype-matched controls.
T cell proliferation assay
DC-CD4+ T cell co-cultures or splenocytes were cultured in 96-well flat bottom plates. On day 3 of co-culture, [3H]-thymidine (1µCi per well) was added and incorporation was measured after 16h. Cells were harvested on fiberglass filters, and [3H]-thymidine incorporation was measured in a liquid scintillation counter.
Proliferation suppressive assays were performed as follows: 1×105 MOG35–55 specific CD4+ T cells were activated with MOG-pulsed DC or DC-DHA in the presence or absence of 2 ng/ml TGFβ and 50 U/ml IL-2 for 3 days, rested for 2 days in the presence of IL-2 and re-cultured with CFSE-labeled (5 µM, according to the manufacturer’s Iprotocol) naïve MOG Tg-CD4+ T cells (0.1× 105) and 0.1× 104 MOG-pulsed DC, in 200 µl medium in 96-well plates. Three days later, proliferation was assessed by CFSE dilution using FACS. The proliferation of naïve CD4+ T cells was based on gated CFSE labeled cells.
Apoptosis assay with annexin V and propidium iodide (PI) staining
After treatment, DC or T cells were washed and adjusted to 1 × 106 cells/ml in staining buffer. Annexin V and PI staining was performed according to the protocol provided by BD Pharmingen, and the cells were analyzed immediately by flow cytometry.
Cell cycle analysis
DC or DC treated with DHA (DC-DHA) were pulsed with 50µg/ml MOG35–55 and stimulated with 0.1 µg/ml LPS for 24 h. After extensive washing, DC or DC-DHA were co-cultured with MOG35–55 specific CD4+ T cells at a 1/20 ratio. Three days later, cell cycle analysis was performed on activated T cells using the Cycle TEST PLUS DNA Reagent Kit according to the manufacturer's instructions. Samples were analyzed by FACS.
Supernatants from DC, DC-CD4+ T cell co-cultures or splenocyte cultures were harvested and subjected to sandwich ELISA for IL-12p70, IL-23, IL-6, IL-10, IL-2, IFNγ and IL-17. The detection limits were: 15 pg/ml for IL-6, IL-17, IL-2 and IL-10, 30 pg/ml for IL-23, IFNγ and IL-12p70.
Purified DC were preincubated with 50µM DHA for 24h, followed by an additional 24h treatment with 0.1 µg/ml LPS and assayed for migration in response to the chemokine CCL19 (100 ng/ml). The lower chambers of Transwell plates (8.0µm pore size; Corning, Acton, MA) were filled with 600µl serum-free medium with or without CCL19. DC (1×105 cells in 0.1 ml) resuspended in serum-free medium were deposited in the upper chambers of the Transwell plates and allowed to migrate for 4h at 37°C in 5% CO2. The numbers of migrated DC harvested from the lower chambers were counted by FACS (60-second counts).
The expression of p27(kip1), Tbet, GATA3, RORC, Foxp3, CCR5, CCR7, IL-10 and TGFβ were detected by SYBR Green-based real-time RT-PCR. RNA was prepared from 4×106 T cells activated by DC or DC-DHA using an Ultraspec RNA isolation system according to the manufacturer’s instructions (Biotecx Laboratories, Houston, TX). RNA (1 µg) was reversed transcribed to cDNA and subjected to real time PCR. The PCR mixture (20 µl), consists of 4 µl diluted cDNA, 16 µl of SYBR Green containing the PCR master mix and 150 nM of each primer. Real-time PCR was performed using the Stratagene Mx3005P. The following primers were used: p27(kip1) sense, 5'-CGGCGGCAAGGTTTGGAGAGG-3' and antisense, 5'- GGAGGAGGCAGGAGGAGGTGG-3'; Tbet sense, 5'-CGGTA CCAGAGCGGCAAGT-3', and antisense, 5'-CATGCTGCCTTCTGCCTTTC-3'; GATA3 sense, 5'-TACTTGCGTTTTTCGCAGGA-3', and antisense, 5'-GATCTGTCGCTTTCGGGCCT-3'; RORC sense, 5'-GCGGAGCAGACACACTTACA-3', and antisense, 5'-TCCACCACCACAGCTGAGAGG-3'; Foxp3 sense, 5'- CAGCTGCCTACAGTGCCCCTA-3', and antisense 5'-CATTTGCCAGCAGTGGGTAG-3'; CCR5 sense, 5′-CATCGATTATGGTATGTCAGC ACC-3′ and antisense, 5′-CAGAATGGTAGTGTGAGCAGGAA-3′; CCR7 sense 5′- CCAGGAAAAACGTGCTGGTG-3′ and antisense 5′-GGCCAGGTTGAGCAGGTAG G-3′; IL-10 sense, 5'-ACCTGCTCCACTGCCTTGCT-3', and antisense 5'-GGTTGCCAAGCCTTATCGGA-3'; TGFβ sense, 5'-GACCTGGGTTGGAAGTGGATC-3', and antisense 5'-GAAGTT GGCATGGTAGCCCTT-3'; β-actin sense, 5’-TCCACCA CCACAGCTGAGAGG-3’ and antisense, 5’-CAGCTTCTCTTTGATGTCACG-3’. The cycling conditions were 95°C for 15 s, 75°C for 1 min, 57°C for 30 sec, for 40 cycles, followed by a melting point determination or dissociation curves. The expression level of each gene is indicated by the number of cycles needed for the cDNA amplification to reach a threshold. The amount of DNA is calculated from the number of cycles by using standard curves and the results are normalized to β-actin.
C57BL/6 mice (3 wks old) were fed with control or DHA diet for 5 weeks as described before (Kong et al. 2010
). Mice were injected with 200µg MOG33–55
peptide emulsified in complete Freund’s adjuvant containing Mycobacterium tuberculosis
H37 RA (final concentration 2mg/ml) s.c
. on day 0 and 100 ng pertussis toxin (PTX) i.p
. on day 0 and on day 2. Clinical scores were as follows: 0, normal mouse, no overt signs of disease; 1, limp tail or hind limb weakness; 2, limp tail and hind limb weakness; 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, moribund state. At stage 5, animals were euthanized and removed from the calculation for the clinical score. Both clinical scores and weight were followed for 60 days.
Isolation of inflammatory CD4+ T cells from central nervous system (CNS)
C57BL/6 mice were immunized as described before. Isolation of mononuclear cells was performed at peak of clinical disease (day 18). Mice were anesthetized with 20 µl of mix of ketamine HCl and xylazine and perfused through the left cardiac ventricle with 30 ml of HBSS containing 2mM EDTA. The brain was dissected and spinal cord was flushed out with HBSS. CNS tissue was digested with 10 ml HBSS containing DNAse I (0.1 mg/ml for brain and 0.05 mg/ml for spinal cord) and Liberase (0.05 mg/ml for brain and 0.025 mg/ml for spinal cord) for 45 min at 37°C with shaking, followed by blocking solution (10% FCS, 10 mM EDTA in HBSS). The tissue was pelleted and resuspended in 10 ml of 30% isotonic Percoll (diluted with 10x HBSS and distilled water), underlaid with 5 ml of 70% isotonic Percoll. Mononuclear cells were isolated from the 30/70 interphase after gradient centrifugation. Cells were washed with RPMI 1640 medium. Mononuclear cells were cultured in the presence of PMA (50 ng/ml), ionomycin (500ng/ml) and GolgiPlug (1µl/ml) for 4h. Cells were stained with FITC anti-CD4 for 30 min, fixed and permeabilized using Cytofix/Cytoperm and Perm/Wash buffer according to the manufacturer’s instructions. Cells were stained with APC anti-IFNγ and PE anti-IL-17 for 30 min. FACS analysis was performed. T cells were identified by gating on CD4+ cells. The number of cytokine producing cells was calculated from the percentage of cytokine-positive CD4+ T cells. We determined the number of CD4+ T cells in the CNS by multiplying the percentage of positive cells by the total number of mononuclear cells isolated from the CNS.
Results are described as mean +/− SD. Comparisons between two groups were done using Student t test, whereas comparisons among multiple groups were done by one way ANOVA. Bonferroni test was used for post hoc comparisons among multiple groups where appropriate. Statistical significance was determined as p values less than 0.05. All statistical analyses were performed using SPSS 12.0 software.