Chemicals and antibodies
RPMI-1640 medium containing 1 mM sodium pyruvate, Dulbecco’s phosphate-buffered saline (D-PBS) and Hanks’ balanced salt solution (HBSS) were purchased from Gibco (Scotland). Middlebrook OADC (oleic acid albumin dextrose catalase) enrichment, Middlebrook 7H9 broth, and Middlebrook 7H10 agar were obtained from Becton Dickinson (USA). IFN-γ, phorbol 12-myristate 13-acetate (PMA), bovine serum albumin (BSA), fluorescein isothiocyanate (FITC), Tween-20, Tween-80, IRAK1/4 inhibitor, 37% formaldehyde solution (FA), horseradish peroxidase (HRP), 2-mercaptoethanol (2-ME) and luminol were purchased from Sigma-Aldrich (USA). Human type AB serum (off-clot) and fetal bovine serum (FBS) were purchased from PAA-The Cell Culture Company (Austria). Mouse IgG2a anti-human TLR2 (sodium azide-free), phycoerythrin (PE)-conjugated mouse anti-TLR2 (IgG2a), and PE-conjugated mouse IgG2aκ isotype control were obtained from Imgenex (USA). FITC-conjugated mouse anti-human CD14 (IgG2aκ) and PE-conjugated anti-human CD11b (IgG1κ) were purchased from BD Pharmingen (USA). Human TNF-α and human IL-10 Quantikine enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (USA).
Bacterial strains and growth conditions
All strains used in this study were based on M. tuberculosis H37Rv (ATCC) and were maintained on Middlebrook 7H10 agar or 7H9 broth supplemented with 10% OADC enrichment and 25 μg/ml kanamycin, as required. For growth on media supplemented with defined carbon sources, strains were grown in minimal medium supplemented with 0.01% cholesterol, as described previously. The engineering of the Mtb strain deficient for the KstD enzyme (ΔkstD), and ΔkstD complemented with an intact kstD gene (ΔkstD-kstD) was described previously. Wild-type, mutant, and complemented bacterial strains were prepared for infection by growing in roller bottles in Middlebrook 7H9 broth containing 10% OADC enrichment and 0.05% Tween-80 for 4–6 days to reach an optical density at 600 nm (OD600) of 1. A portion of the bacterial culture (approximately 1 × 109 bacilli/ml) was suspended in Middlebrook 7H9 broth and labeled with 100 μg/ml of FITC by incubating for 2 hours at room temperature with gentle agitation in the dark. FITC-labeled bacteria were washed once with Middlebrook 7H9 broth supplemented with 4% BSA and then twice with Middlebrook 7H9 broth without BSA. Unlabeled and FITC-labeled bacteria were divided into equal portions and stored at -85°C. After 1 week, a portion of bacteria was thawed and colony-forming assays were used to determine the number of bacterial colony-forming units (CFUs).
The human monocyte-macrophages cell line THP-1 (ATCC TIB-202, USA) was maintained in culture medium (CM) consisting of RPMI-1640 supplemented with 1 mM sodium pyruvate, 10% FBS, 0.05 mM 2-ME, 100 U/ml of penicillin and 100 μg/ml of streptomycin at 37°C in a humidified 5% CO2 environment. THP-1 cells were passaged every 3–4 days.
Undifferentiated THP-1 cells (monocytes) were distributed into 24- and 96-well plates and differentiated into macrophages (resting MØ) by culturing for 24 hours (37°C, 5% CO2) with PMA (20 ng/ml), as described previously by others[14–16]. The macrophage-like phenotype of the cells was confirmed by assessing CD14 expression using flow cytometry (see below). The ability of resting MØ to adhere to plastic dishes was examined under a light microscope. IFN-γ-activated MØ were prepared by incubating resting MØ with 20 ng/ml of IFN-γ in CM for 24 hours (37°C, 5% CO2). Resting MØ and IFN-γ-activated MØ were infected with bacteria and cultured in CM without antibiotics. IFN-γ (20 ng/ml) was added to cultures of IFN-γ-activated MØ.
Flow cytometry analysis
CD14 surface expression on monocytes and resting MØ was assessed by staining the cells (1 × 105) with 10 μg/ml of a FITC-conjugated monoclonal antibody (mAb) against CD14 or isotype control (IgG2a; 10 μg/ml) for 30 minutes at 4°C. Before staining with anti-TLR2 mAb, crystallizable fragment receptors (FcRs) were blocked in D-PBS containing 10% human AB serum for 15 minutes at room temperature to prevent nonspecific antibody binding. Subsequently, cells were washed twice in D-PBS containing 1% FBS. Resting MØ and IFN-γ-activated MØ (1 × 105 cells) were stained with 10 μg/ml of a PE-conjugated anti-TLR2 mAb or isotype control (IgG1; 10 μg/ml). A concentration of anti-TLR2 mAb sufficient to completely block the expression of TLR2 on cells was determined in preliminary experiments by adding different mAb concentrations (10, 25, and 35 μg/ml) to MØ and incubating for 1 hour (37°C/5% CO2). MØ were then stained with PE-conjugated anti-TLR2 mAb or isotype control, as described above.
All stained cells were washed twice, resuspended in 200 μl of D-PBS containing 1% FBS, 1% FA and sodium azide, and stored at 4°C until FACS (fluorescence-activated cell sorting) analysis. All samples were examined with a FACS LSR II BD flow cytometer (Becton Dickinson, USA) equipped with BD FACS Diva Software. The results were presented as median fluorescence intensity (MFI), which correlates with the surface expression of the target molecule.
Bacteria were thawed, washed twice in RPMI-1640 medium, and then opsonized (or not) by incubating with 20% human serum AB in RPMI-1640 medium for 30 minutes at 37°C with gentle agitation. Thereafter, bacteria were washed once with RPMI-1640 medium. Opsonized and non-opsonized Mtb were suspended in CM, and clumps were disrupted by multiple passages through a 25-gauge needle. Serial dilutions of bacteria were prepared in CM.
Resting MØ and IFN-γ-activated MØ were infected with opsonized or non-opsonized, unlabeled or FITC-labeled Mtb strains (wild-type, mutant [ΔkstD] or complemented [∆kstD- kstD]) at a multiplicity of infection (MOI) of 1 or 10, as indicated, for 2 hours (37°C, 5% CO2). Extracellular bacteria were removed by extensively washing MØ with warm HBSS. Infected MØ were directly used in tests (day 0) or cultured for 1, 2 or 6 days, as indicated in Figures.
Ingestion of bacteria
Resting MØ and IFN-γ-activated MØ (1 × 105 cells/well) were prepared in 8-well Permanox chamber slides (Nunc, Denmark) and then infected with FITC-labeled Mtb strains at an MOI of 10. After infection, MØ were fixed by incubating with 3% FA for 15 minutes (37°C, 5%, CO2) and washed twice with HBSS. The number of infected MØ and the number of bacteria engulfed by one MØ were determined by fluorescence microscopic examination (Nikon ECLIPSE TE 2000 U). In all cases, 200 MØ were counted.
Intracellular growth of bacteria
Resting MØ and IFN-γ-activated MØ (1 × 105 cells/well) were prepared in 24-well plates (Nunc). MØ were then treated with 10 μM IRAK1/4 inhibitor or with a saturating concentration of anti-TLR2 blocking mAbs (35 μg/ml) for 1 hour or left untreated. Afterwards, MØ were infected with Mtb strains at an MOI of 1. After infection, fresh CM and IRAK1/4 inhibitor or anti-TLR2 blocking mAb (when required) were added, and cells were cultured for 6 days. On the day of infection (day 0) and 6 days post-infection, MØ were lysed with 1 ml of 0.2% Triton X-100 and appropriate dilutions of cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC. After 21 days of culture, CFUs were counted. The data were presented as fold-increase in CFUs, calculated as CFUs on day 6 divided by CFUs on day 0.
Resting MØ and IFN-γ-activated MØ (1 × 105 cells/well) were prepared in 96-well plates (Nunc) and treated with IRAK1/4 inhibitor or left untreated (as described above). Next, MØ were infected with Mtb strains at an MOI of 10 and cultured for 2 days with or without IRAK1/4 inhibitor. The presence of nitrite (stable metabolite of NO) in the culture supernatants was determined using the Griess reagent. OD was determined using a Multiscan RC ELISA reader (Labsystem, Finland). Nitrite concentration was calculated from a standard curve prepared using sodium nitrite as a reference.
Resting MØ and IFN-γ-activated MØ (1 × 105 cells/well) were prepared in 96-well plates (Nunc) and then infected with Mtb strains at an MOI of 10. After culturing for 1 day, 1 μg/ml of PMA (to initiate ROS production) as well as 40 U of HRP and 1 mM luminol (to enhance chemiluminescence) were added to the cells. Chemiluminescence (CL) was recorded over 4 hours at 5-minute intervals using Fluoroscan Ascent FL (Labsystem, Finland). Data were acquired as relative light units (RLUs), and the area under the curve of CL versus assay time (total RLUs) was calculated. Data were presented as percent inhibition of ROS production calculated according to the formula, 1 - (total RLUs for cells infected with bacteria and stimulated with PMA/total RLUs for cells stimulated with PMA) × 100.
TNF-α and IL-10 production
Resting MØ and IFN-γ-activated MØ (1 × 106 cells/well) were prepared in 24-well plates (Nunc), infected with Mtb strains (MOI of 10), and cultured for 24 hours. The presence of TNF-α and IL-10 in the culture supernatants was assessed using Quantikine ELISA kits. The sensitivities of TNF-α and IL-10 assays were 1.6 pg/ml and 3.9 pg/ml, respectively.
Data are presented as means ± SEMs. Statistical significance was verified using nonparametric Wilcoxon’s signed-rank or Mann–Whitney U tests. The Statistica 8.0 (StatSoft, Poland) software package was used for statistical calculations. Statistical significance was defined as p ≤ 0.05.