Two isolates of Mbv from animals with tuberculosis were used in this study. The strain B2 was isolated from buffalo and gently provided by Dr. Eliana Roxo (Biological Institute, USP, São Paulo, Brazil). The bovine strain MP287/03 was kindly provided by Dr. José Soares Ferreira Neto (Institute for Veterinary Medicine, USP, São Paulo, Brazil). M. tuberculosis strain H37Rv (ATCC) was kindly provided by Dr. Philip Suffys (Oswaldo Cruz Foundation, FIOCRUZ, Rio de Janeiro, Brazil).
Mycobacterial strains were grown in suspension in complete 7H9 Middlebrook broth (Difco, Detroit, MI), containing 10% albumin dextrose complex, ADC (BD, Sparks, MD), 0.5% glycerol and 0.05% Tween-80 at 37°C under Biosecurity level 3 containment conditions. Additionally, sodium pyruvate 0.4% was added to the cultures of Mbv. Bacterial cultures were grown to mid-logarithmic phase, aliquoted, and stored at −70°C. Before experiments, the aliquots were thawed, resuspended in complete 7H9 medium and cultured for 5 days. Bacterial suspensions were ultrasonicated in water bath to disrupt small clumps and obtain single cell suspensions. The resulted dispersion of bacteria was tested by microscopic examination of the suspension samples stained by the acid-fast staining procedure. The densities of the suspensions were measured by spectrophotometry, and corresponding concentrations were determined by serial dilution plating of each strain on Middlebrook 7H10 agar (Difco, Detroit, MI) plates supplemented with 0.5% glycerol, 10% oleic acid–albumin-dextrose–catalase enrichment, OADC (BD, Sparks, MD), and, additionally, with 0.4% sodium pyruvate in the case of Mbv cultures. After 21 days, total CFU were determined.
Quantification of mycobacterial growth in 7 H9 broth
The bacterial capacity to grow in 7H9 broth was measured by spectrophotometry. Bacterial suspensions adjusted to OD600 = 0.1 were cultured at 37°C for twelve days with daily agitation. Bacterial tubes were then vortexed, ultrasonicated in a water bath, and the OD of suspension was measured. To confirm the lack of significant alteration in OD600 readings, colony forming units (CFU) were determined for each culture on day 0 through the plating of appropriate bacterial dilution onto the 7H10 agar.
Generation of bone marrow- derived macrophages
Bone marrow- derived macrophages (BMDM) were obtained as previously described  with some modifications. Briefly, bone marrow cells were flushed from the femur of eight- to ten- week-old specific pathogen-free C57BL/6 mice. The cells were dispersed in Dulbecco’s modified Eagle’s medium, DMEM (Sigma, St Louis, MO), supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 0.05 M 2-mercaptoethanol (Gibco BRL, Grand Island, NY), 10% heat-inactivated FBS (Hyclone, Road Logan, UT), 50 μg/ml gentamicin (Gibco BRL, Grand Island, NY), and cultured at 37°C in 5% CO2 atmosphere. Nonadherent cells were collected after 18 h, resuspended in the complete DMEM, supplemented with 20% L929 cell-conditioned medium as a source of M-CSF, and cultured for 7 days, replacing the medium on day 3. The monolayer cells were scraped, resuspended in DMEM, supplemented with 2% FBS, without antibiotics, and plated at a concentration of 5 x 105 cell/ml in a 96-well plates, 100 μl/well.
Treatment with cytokines and infection of cell cultures
The BMDM cultures were incubated overnight, pretreated, or not, with murine recombinant IFN-γ, 100 U/ml (Bioscience, Camarillo, CA), or IL-10, 20 ηg/ml (Bioscience, Camarillo, CA) for 2 h, and infected with single-cell suspensions of mycobacterial strains at MOI 1:1 and 5:1. After 3 h of incubation at 37°C, infected monolayers were washed and incubated for additional 6 d in new aliquots of culture medium. In the pretreated cultures, the cytokines were renewed and were present throughout the incubation period. Cell viability of infected MΦ was monitored by trypan blue exclusion and was over 90% in all experiments.
Quantification of mycobacterial growth in macrophages
Mycobacterial ability to grow intracellularly was evaluated by colony-forming units (CFU) test in the MΦ cultures infected at a MOI of 1:1. After 0, 3 and 6 d of incubation, cells were lysed with 1% saponin to release intracellular bacteria. Lysates of infected cells were resuspended, transferred into screw caps, vortexed and sonicated in a preheated waterbath sonicator (Unique 800, Brazil) at 37°C for 2 min. Aliquots of the sonicate were diluted 10-fold in PBS, plated in quadriplicates on 7 H10 agar plates and incubated at 37°C for 21 days.
To study cytokines secreted by infected MΦ, the cell cultures were infected at a MOI 5:1 in the presence or absence of recombinant IFN-γ and IL-10, as indicated above. The infected monolayers were washed and incubated for additional 48 h. After incubation, the culture supernatants were collected, filtered through 0.22 μm Spin-X centrifuge tube filters (Corning, NY), and the supernatant aliquots were stocked at −70°C for posterior cytokine determination. The cells left untreated and cells stimulated with LPS of Escherichia coli 011B (Sigma Aldrich, MO), 1 μg/ml, and r-IFN-γ for 48 h were used as a negative and positive control of proinflammatory macrophage activation, respectively. The frozen samples of culture supernatants of the infected BMDM were then thawed and immediately analyzed using Bio-Plex Pro Mouse Cytokine Assay (BioRad Laboratories, Hercules, CA), following the manufacturers protocol. Standard curves for each cytokine were generated using reference cytokine concentrations supplied by the manufacturer.
Nitric oxide determination
Nitric oxide (NO) generation in the culture supernatants was assessed by the Griess method to measure nitrites, which are stable breakdown products of NO. Briefly, culture supernatant was incubated with the Griess reagents I (1% sulfanilamide in 2.5% phosphoric acid) and II (0.1% naphthylenediamine in 2.5% phosphoric acid). The absorbency was read within 5 min at 550 nm and actual concentration calculated using a standard curve with serial dilutions of sodium nitrite.
Detection of iNOS, ARG-1 and MR by Western blot
The infected adherent cells were resuspended in lysis buffer (10% SDS, 20% glycerol, 5% 2-mercaptoethanol, 2% bromphenol blue and 1 M Tris HCl, pH 6.8) for western blotting analysis. Cell samples in the lysis buffer were harvested and equal amounts of proteins were electrophoresed in a 10% or 8% sodium SDS-PAGE gel under nonreducing conditions. The proteins were then transferred to nitrocellulose membrane (Amersham Hybond-ECL GE) using standard procedures. After overnight blocking with 0.5% non-fatty milk in PBS, the blots were incubated for 1 hr at room temperature with Ab against iNOS, 1:1000 (Santa Cruz Biotechnology, CA), Arg-1, 1:1000 (BD Bioscience), or MR/CD206, 1:100 (Santa Cruz Biotechnology, CA), dissolved in 0.5% non-fatty milk in PBS. The blots were then washed and incubated with peroxidase-conjugated secondary Ab, 1:8000, for 1 hr at room temperature, and the resulting membranes were developed using diaminobenzidine/H2O2 as a substrate for peroxidase. Densitometric analysis of the protein bands was performed using the software ImageJ for Windows (NIH, Bethesda, MD). The value for the control condition (untreated cells) was set as 1 and other conditions were recalculated correspondingly to allow ratio comparisons.
Statistical analysis was performed using the unpaired Student’s t test, one-way analysis of variance (ANOVA) and Bonferroni procedure for multiple range tests, employing Prism 4 software (GraphPad, San Diego, CA) to assess statistical significance between groups of data defining different error probabilities. A value of p < 0.05 was considered to be significant.