Supplementary Materials and Methods
Construction of recombinant adenoviruses encoding murine IFNγ
Briefly, the mIFNγ cDNA was subcloned into the multicloning site of AdTrack-CMV shuttle vector that contains green fluorescent protein as reporter gene. The subclonation of mIFNγ cDNA was in position between the cytomegalovirus promoter and polyadenylation site, specifically in the XhoI and HindIII restriction sites. The resultant plasmid was linearized by digestion with PmeI endonuclease restriction enzyme and cotransformed into E.coli BJ5183 strain cells with the AdEasy-1 adenoviral plasmid, which contains all sequences of adenovirus serotype Ad5 except nucleotides encompassing the E1 and E3 genes. Recombinant bacteria were selected by kanamycin resistance, and recombination was confirmed by PacI endonuclease restriction analysis. Finally the linearized recombinant plasmid was transiently transfected into the packaging 293-cells (ATCC # CRL-1573) that supply the E1 proteins necessary to generate adenovirus, using LipofectAMINE (Invitrogen) following manufacturer's recommendations. Green fluorescent protein recombinant adenovirus (AdGFP) was constructed similarly; the procedure was facilitated because the GFP gene was integrated into the pAdTrack-CMV. Recombinant adenoviruses were selected three times from 293-infected cells and tested each time by Western blot, as described below to confirm mIFNγ expression.
Production of high titers of AdIFNγ and AdGFP
Fibra-cel disks are electrostatical support matrices (http://www.nbsc.com/products/spinner.htm), composed of polyester and polypropylene, conforming a three-dimensional network that permits efficient adenovirus-infected cells attachment and active cell growth. Approximately 150x106 293-cells were inoculated into a spinner basket loaded with 5 g of fibra-cel disks, Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5g/L glucose and 10% fetal bovine serum (FBS) (Gibco/BRL, Gran Island, NY, USA.). The spinner basket was incubated at 37°C under continuous stirring during 7 days. The pH of the culture medium was neutral and was constantly monitored during the incubation time. To generate higher titer viral stocks, these 293-cells in spinner baskets were infected with AdIFNγ or AdGFP at a multiplicity of infection (MOI) of 0.1 for 5 days, which in preliminary kinetic studies showed to be the time and MOI for maximal viral production. The efficiency of this infection was easily followed with the detection of GFP by fluorescence microscopy. After 5 days, the supernatant cultures were collected and immediately processed to purify adenoviruses by precipitation using polyethylene glycol at 10% (Sigma) and 0.5 M NaCl (Sigma), followed by exhaustive dialysis against the storage buffer containing 1 M Tris, pH 8.0, 1 M MgCl2, and 5% sucrose. This procedure usually yielded between 1- 3 x 1011 recombinant purified adenoviruses from 500 ml of supernatant.
Stocks of purified adenovirus were titrated by counting plaques-forming units (PFU). Briefly, 293-cell monolayers were infected with serial dilutions of the virus stock, incubated for 40h at 37°C and fixed with methanol:acetone (1:1), and washed twice with phosphate buffered saline (PBS). Viral plaques detection was performed with polyclonal rabbit anti-adenovirus antibodies diluted 1/1000, and incubated overnight at 4°C. After washing, cells were incubated with protein A labeled with peroxidase, diluted 1/1000 (Sigma) during one hour at room temperature. Peroxidase was revealed with 3,3’dimetoxibenzidine (Sigma) and H2O2. Viral plaques (PFU) were counted using an invert microscope.
Determination of IFNγ secretion and bioactivity in the supernatants of Mv1Lu cells infected with recombinant adenovirus
Mv1Lu lung epithelial cells (ATCC # CCL-64) from the American Type Culture Collection (Rockville, MD) suspended in DMEM containing 10% FBS were seeded in 6-well multicluster wells (7 x 105 per well). On the following day, Mv1Lu cells were infected with (MOI=250) AdIFNγ or AdGFP for 1h in medium without FBS. After 72h of incubation, we checked the efficiency of infection through detection of GFP by fluorescence microscopy. Then, supernatants were recovered and stored at -70°C until use.
Supernatants were used to quantify levels of secreted IFNγ using ELISA kit (Pharmigen, San Jose CA, USA). Briefly, 96-well plates were precoated with 0.5 μg/ml of monoclonal anti-mouse IFNγ dissolved in 100 μl of 0.05 M carbonate buffer, pH 9.5, incubated overnight at 4°C. After washing with PBS-Tween 20 at 0.05%, wells were blocked with 3% bovine serum albumin (BSA) (Sigma) in PBS for 2h at 37°C. Diluted supernatants and the standard curve of recombinant IFNγ were incubated per duplicate for 2h at 37°C. After washing, biotinylated polyclonal rabbit anti-mouse IFNγ diluted 1/250 in PBS-3% BSA were incubated with streptavidin peroxidase diluted 1/250 in PBS-3% BSA for 1 h at room temperature. To reveal, tetramethylbenzidine and H2O2 (Pharmigen, San Diego CA.US) were used, and the optical density measured at 450 nm.
For Western blotting, equal amounts of protein from supernatants were electrophoresed in polyacrylamide gels (8%) and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were probed using 1/500 of the anti-mouse IFNγ antibody (R&D systems) and 1/10000 of the peroxidase anti-IgG goat (R&D systems). Immunoblots were revealed using the enhanced chemiluminescence kit (Amersham Biosciences, New Jersey, USA ).
To verify the biological activity of the secreted IFNγ, we determined nitrite production by mouse macrophages stimulated during 18h with supernatants from Mv1Lu cells infected with either AdIFNγ or AdGFP. To obtain peritoneal macrophages, Balb/c mice were intraperitoneally injected with 1 ml of sterile light mineral oil (Sigma), and after 5 days peritoneal macrophages were collected from 10 animals in sterile conditions. Collected cells were washed twice with Alsever solution (0.1M glucose, 27 mM sodium citrate, 2 mM citric acid, 71 mM sodium chloride, pH 6.1) to remove oil excess.
Then, cells were centrifuged and red cells were eliminated with lysis buffer (0.2% sodium chloride, stirring and adding 1.6% sodium chloride). After washing with Alsever solution, recovered macrophages were resuspended in DMEM medium with 10% FBS, and seeded in 96-well multicluster wells (1 x 106 per well). After three hours, the cultured medium was substituted by different dilutions of supernatants from Mv1Lu cells infected with AdIFNγ or AdGFP, and incubated at 37°C with 5% CO2 for 18h. To determine nitrites production, 50 μl of supernatant from each well was transferred to another plate and incubated with 150 μl of 1:1 solution of 0.1% sulfanilamide dissolved in water, and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5% phosphoric acid (Griess reactant). After 10 min, nitrite concentration was determined by spectrophotometry at 540 nm on a microtiter plate reader using a standard curve solution with known concentrations of sodium nitrite.
Experimental model of progressive pulmonary tuberculosis.
Male Balb/c mice 6-8 weeks age were anesthetized with sevofluorane and infected using a rigid stainless steel cannula as described above, administering 2.5 x 105 viable bacteria suspended in 100 μl of PBS. Infected mice were maintained in groups of five in cages fitted with microisolators connected to negative pressure. All procedures were performed in a biological security cabinet, P3. The protocol was approved by the Ethics Committee for Experimentation in Animals of the National Institute of Medical Sciences and Nutrition in México.
Immunohistochemistry.
The same paraffin embedded tissue used for histology was used for this method. Lung sections were mounted in silane covered slides, deparaffinized, and the endogenous activity of peroxidase was quenched with 0.03% H2O2 in absolute methanol. Lung sections were incubated over night at room temperature with rabbit polyclonal specific antibodies against murine IFNγ (Santa Cruz, Biotechnology, USA) or rabbit polyclonal specific antibodies against adenovirus antigens. After incubation with goat anti-rabbit antibodies labeled with biotin, bound antibodies were detected with the system avidin-biotin peroxidase, and revealed according to manufacturer’s instructions (Vectastain, Vector laboratories , CA, USA).
Primers to real time PCR analysis
Specific primers were designed using the program Primer Express (Applied Biosystems, USA) for the following targets: glyceraldehyde-3-phosphate dehydrogenase (G3PDH): 5´-ggcgctcaccaaaacatca-3´, 5´-ccggaatgccattcctgtta -3´, iNOS: 5´-agcgaggagcaggtggaag-3´, 5´-catttcgctgtctccccaa-3´, TNFα: 5´-tgtggcttcgacctctacctc-3´, 5´-gccgagaaaggctgcttg-3´, IFNγ: 5´-ggtgacatgaaaatcctgcag-3´, 5´-cctcaaacttggcaatactcatga-3´, CCL2 5’-ctctcttcctccaccaccatg-3’, 5’-ttaactgcatctgcctgagcc-3’ and CCL3 5’ –ccagccaggtgtcattttcc’, 5’ttggagtcagcgcagatgtg-3’.