Male DBA/1 LacJ and BALB/c mice (The Jackson Laboratories, Bar Harbor, ME), 5 weeks of age, were kept in filter-top cages in the Animal Care and Veterinary Services Facility at the University of Western Ontario according to the Canadian Council for Animal Care Guidelines. Mice were fed by food and water and allowed to settle for 2 weeks before initiation of experiments, which had ethical approval from the university review board.
DBA/1 LacJ mice, 7 weeks of age, were intradermally immunized (Day 0) at several sites into the base of the tail with 200 μg of bovine type II collagen (CII) (Sigma-Aldrich, St. Louis, MO) dissolved in 100 μl of 0.05 M acetic acid and mixed with an equal volume of complete Freund's adjuvant (CFA) (Sigma). CII was dissolved at a concentration of 2 mg/ml by stirring overnight at 4°C. On day 21 after priming, the mice received an intraperitoneal booster injection with 200 μg of CII in the equal volume (100 μl) of PBS. Mice were examined visually three times per week for the appearance of arthritis in the peripheral joints, and arthritis score index for disease severity was given as follows: 0 - no evidence of erythema and swelling; 1 - erythema and mild swelling confined to the mid-foot (tarsals) or ankle joint; 2 - erythema and mild swelling extending from the ankle to the mid-foot; 3 - erythema and moderate swelling extending from the ankle to the metatarsal joints; 4 - erythema and severe swelling encompass the ankle, foot, and digits. Scoring was done by two independent observers, without knowledge of the experimental and control groups.
At Day 0, bone marrow cells were flushed from the femurs and tibias of DBA/1 LacJ mice, washed and cultured in 6-well plates (Corning, NY) at 4 × 106 cells/well in 4 ml of complete medium (RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg of streptomycin, 50 μM 2-ME, and 10% FCS (all from Life Technologies, Ontario, Canada) supplemented with recombinant GM-CSF (10 ng/ml; PeproTech, Rocky Hill, NJ) and recombinant mouse IL-4 (10 ng/ml; PeproTech). All cultures were incubated at 37°C in 5% humidified CO2. Non-adherent cells were removed after 48 h of culture (Day 2) and fresh medium was added every 48 h.
shRNA expressing vectors and transfection
siRNA sequences were selected according to the method of Elbashir SM et al. [36
]. The siRNA sequence specific for IL-12p35 (AACCUGCUGAAGACCACAGAU) was selected and cloned into Psilencer 3.1 vector (Ambion, Austin, TX) which expresses short hairpin RNA (shRNA) under the control of the mouse U6 promoter, using the method described by the supplier of the vector. IL-12 shRNA was sequenced and prepared in a large scale for in vitro and in vivo study. Gene silencing was examined with DC. DC were generated from bone marrow progenitor cells as previously described [35
]. Transfection of DC was conducted as described previously [36
]. 24 h before transfection (day 4), DC were plated to be 60-90% confluent on the day of transfection. On day 5, 2 μg of IL-12 shRNA and 3 μl of GenePORTER reagent (Gene Therapy Systems, San Diego, CA) were separately diluted with serum-free medium RPMI 1640 using 1/2 of the transfection volume (125 μl). The diluted DNA was added to the diluted GenePORTER reagent, mixed rapidly and incubated in total volume of 250 μl of the medium at room temperature for 45 min. The culture medium from the DC was aspirated, and the DNA-GenePORTER mixture was added carefully to the DC. Mock controls were transfected with 3 μl of the GenePORTER reagent alone. After 4-h incubation, an equal volume of RPMI 1640 (250 μl) supplemented with 20% FCS was added to the cells. 48 h after the start of transfection (day 7), DC were washed and pulsed with 10 μg/ml of CII for 24 h. At day 8, DC were then activated with LPS/TNF-α for additional 24 h. 7 days before and/or 12 days after priming with CII, different groups of mice with 6 animals per group were i.p. injected with shRNA-transfected or control DC at a dose of 5 × 106
cells per mouse.
Total RNA was extracted from cells using Trizol (Invitrogen). 3 μg total RNA was used to synthesize the cDNA with oligdT and reverse transcriptase (Invitrogen) in 20 μl reaction volume. Primers used for the amplification of murine IL-12, IFNγ, IL-2, IL-4, IL-10 and GAPDH were as follows [37
]: IL-12, 5'- CTT GCC CTC CTA AAC CAC CTC AGT-3' (forward) and 5'- CCA CCA GCA TGC CCT TGT CTA-3' (reverse); IFNγ, 5'- CAC GGC ACA GTC ATT GAA AGC CTA-3' (forward) and 5'- TGA GGC TGG ATT CCG GCA ACA GCT-3' (reverse);
IL-2, 5'- ACA TTG ACA CTT GTG CTC CGT GTC-3' (forward) and 5'- TTG AGG GCT TGT TGA GAT GAT GCT-3' (reverse); IL-4, 5'- AGC TAG TTG TCA TCC TGC TCT TCT-3' (forward) and 5'- CGA GTA ATC CAT TTG CAT GAT GCT-3' (reverse); IL-10, 5'- GAA GAC AAT AAC TGC ACC CAC TTC-3' (forward) and 5'- ATG GCC TTG TAG ACA CCT TGG TCT-3' (reverse); GAPDH, 5'-TGA TGA CAT CAA GAA GGT GGT GAA-3' (forward) and 5'-TGG GAT GGA AAT TGT GAG GGA GAT-3' (reverse).
Polymerase chain reaction (PCR) was performed in a 25 μl of reaction volume containing 0.2 μmol/L primers, 1 U Taq DNA polymerase under the following conditions: 95°C for 30 s, 58°C for 30 s, and then 72°C for 30 s (30 cycles). PCR products were visualized with ethidium bromide on 1.5% agarose gel.
Mixed leukocyte reaction (MLR)
At day 5 of culture, bone marrow-derived DC from DBA/1 LacJ mice were transfected with IL-12 and scrambled siRNAs or mock-transfected followed by activation with LPS/TNF-α. Activated DC were irradiated (3,000 rad) and seeded in triplicate in a flat-bottom 96-well plate (Corning) for use as stimulator cells. Spleen T cells from BALB/c mice were isolated by gradient centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech, Quebec) and added as responders (5 × 105 cells/well). The mixed lymphocytes were cultured at 37°C for 72 h in 200 μl of RPMI 1640 supplemented with 10% FCS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin and pulsed with 1 μCi/well of 3H-labelled thymidine (Amersham Pharmacia Biotech) for the last 16 h of culture. Finally, cells were harvested onto glass fiber filters, and the radioactivity incorporated was quantitated using a Wallac Betaplate liquid scintillation counter (Beckman, Fullerton, CA). Results were expressed as the mean counts per min of triplicate cultures ± SEM.
T cell proliferative responses to CII in subsequent groups of mice were measured with a standard microtiter assay. Following CII immunization, the proliferative responses could be detected for several weeks. Immune cells from either draining lymph node or spleen T cells collected from the mice treated with CII and IL-12 silenced DC or control DC, at 5 × 105/well were seeded to a 96-well flat-bottom microtiter plate (Corning) in triplicates and mixed with serial dilutions of CII with concentrations ranging from 5 to 50 μg/well. Following a 72 h incubation, 1 μCi of [3H] thymidine (Amersham) was added to each well for 16 h. Using an automated cell harvester, the cells were collected onto glass microfiber filter, and the radioactive labeling incorporation was measured by a Wallac Betaplate liquid scintillation counter.
Anti-CII antibody measurement
CII-specific Abs were evaluated using a standard indirect ELISA in which 500 ng of CII was absorbed to each well of a 96-well microtitre plate. Following blocking and washing steps, serial dilutions of immune mouse serum were added to the appropriate wells in duplicates and incubated overnight at 4°C. Dilutions of serum were 1:100-1:100,000. To develop the ELISA, horseradish peroxidase-conjugated goat anti-mouse IgG Fc and ortho-phenylenediamine dihydrochloride substrate buffer (Sigma) were used. The OD of each well was measured at a wavelength of 490 nm in an ELISA plate reader.
Mock or shRNA-transfected DC of DBA/1 LacJ origin were cultured with the allogeneic (BALB/c) T cells or alone for 48 h. The supernatants were collected and assessed for DC cytokines (IL-10 and IL-12) and T cell cytokines (IFN-γ and IL-4) by ELISA. Cytokine-specific ELISA (Endogen, Rockford, IL) was used for detecting cytokine concentrations in culture supernatants according to the manufacturer's instructions using a Benchmark Microplate Reader (Bio-Rad, Hercules, CA).
Paws from experimental and control groups of freshly dissected mice were removed and joint tissues were immersion-fixed for 4 day in 10% (wt/vol) neutral buffered formalin in 0.15 M PBS (pH 7.4). After decalcification by immersing in Decalcifier I solution (Winnipeg, Canada) overnight and subsequent dehydration in a gradient of alcohols, tissues were rinsed in running water. The specimens were processed for paraffin embedding in paraplast (BDH, Dorset, UK) as routine procedure. Serial paraffin sections throughout the joint were cut at 5-μm thickness on a microtome, heated at 60°C for 30 min, and deparaffinized. Hydration was done by transferring the sections through the following solutions: triple to xylene for 6 min, and then for 2 min to 100% ethanol twice, 95% ethanol, and 70% ethanol. Sections were stained with H&E and mounted on glass slides.
Intracellular cytokine staining and flow cytometry
Transfected DCs were treated with 20 ng/ml phorbol myristate acetate and stained with FITC-conjugated IL-12. Ig of the same isotype was used as controls. Flow cytometry analysis was performed in a FACScan II (Becton Dickinson, San Jose, CA- BD) system using FACSDiva software (BD).
Data are expressed as mean ± SEM. Differences between different groups of mice were compared using the Mann-Whitney U test for nonparametric data. A P value less than 0.05 was considered significant.