were blocked with 5% BSA in PBS or 5% heat-inactivated rabbit serum, respectively, and examined for the surface expression of SR-B1 or SIGNR1 using rabbit polyclonal anti-mouse SR-B1 IgG (NB400-113, Novus Biologicals) or rat monoclonal anti-mouse ERTR9 IgM (BMA Biomedicals) together with biotinylated mouse anti-rat IgM (Serotec), respectively. Rabbit IgG (Chromopure) or rat IgM (BD PharMingen), respectively, were used as isotype controls. Cells were stained with R-Phycoerythrin-conjugated donkey anti-rabbit IgG (Jackson Immuno Research Laboratories, Inc.) or R-Phycoerythrin-conjugated streptavidin (BD PharMin- gen), respectively, and fixed with 1% formaldehyde in PBS. The analysis was performed using a FACSCalibur (Becton Dickinson) together with the software CellQuest version 4. For the detection of ligand binding to SR-B1, 10mg/ml DiI-labelled LDL (Molecular Probes) was added to SR-B1 expressing R6F cells in serum-free DMEM for 2 hr at 37uC. Cells were then washed three times and lifted with 4mg/ml lidocaine hydrochloride (Sigma) in PBS supplemented with 10mM EDTA. Cells were spun down, resuspended in FACS wash solution containing 1% formaldehyde and analysed for ligand binding by FACS. Animals C57BL/6 mice deficient in SR-B1 and its splice variant SR-B2 (Scarb1tm1Kri/Scarb1tm1Kri) were purchased from the Jackson Labo- ratory which were originally derived by Rigotti et al. [50]. Animals were maintained under specific pathogen-free conditions in the University of Cape Town animal facility. All work was approved by the Faculty of Health Sciences Animal Ethics Committee, University of Cape Town. As homozygous Scarb1tm1Kri/Scarb1tm1Kri female mice were not fertile, breeding had to be performed with heterozygous Scarb1tm1Kri/Scarb1+ mice. However, the numbers of mutant offspring was significantly less than the expected Mendelian ratio of 1:2:1 as described earlier [50,51], which significantly limited the number of animals available for experiments. Where indicated, mice were fed either a low cholesterol (LC, 0.15% cholesterol) or a high cholesterol (HC, 1.25% cholesterol) diet (Research Diets, Inc.) two weeks before and throughout the experiment. For these experiments, serum cholesterol levels were measured using the Cholesterol Enzymatic CHOD-PAP kit (KAT Laboratory & Medical (Pty) Ltd.). Isolation of Primary Macrophages Resident or thioglycollate-elicited peritoneal macrophages as well as bone-marrow derived macrophages (BMDmØ) were isolated from 8–12 weeks old SR-B12/2 mice or wild type C57BL/6 littermates by standard procedures. To isolate resident alveolar macrophages, lungs were lavaged with 1ml cold PBS (representing the bronchoalveolar lavage (BAL)), followed by repeated washes with 1ml PBS/10mM EDTA. Cells were maintained for 24 hr in RPMI (Invitrogen) with 10% heat- inactivated fetal calf serum (delta bioproducts), 2mM L-glutamine, 100U/ml penicillin and 100mg/ml streptomycin (Sigma) at 37uC, except for BMDmØ which were cultured in RPMI medium for 5– 7 days supplemented with 20% (v/v) L-cell conditioned medium as a source of M-CSF. Binding experiments with these macrophages were performed in the absence of serum (and References M. tuberculosis and SR-B1 absence of M-CSF, in the case of BMDmØ) as described above. To determine the levels of TNF released into the supernatant, cells were washed after the binding assay in a parallel set of experiments, fresh DMEM containing 10% serum was added, and cells were incubated at 37uC. The amount of TNF was measured after 4 hr and 24 hr using the OptEIA murine TNFa ELISA kit (BD Biosciences) as described by the manufacturers, and normalized to cell number by CFSE staining. Data represent pooled values from at least 3 independent experiments. M. tuberculosis Infections Frozen stocks of Mycobacterium tuberculosis H37Rv were thawed, briefly vortexed, and clumping disrupted by aspirating 30 times through a 29-gauge needle. The concentration was adjusted in sterile saline to deliver ,100 CFU per mouse (low dose infection) or ,1000 CFU per mouse (high dose infection). Appropriate numbers of 10–14 weeks old SR-B12/2 male and female mice or wild type C57BL/6 littermates were then infected via the aerosol route using an inhalation exposure system (Glas-Col, Terre Haute, IN). In each experiment five mice were sacrificed 24 hr post- infection to confirm the infection dose. Animals were monitored throughout the experiments with regard to body weight and signs of disease. After 2 or 4 months, mice were sacrificed and lungs were removed and weighed. Defined tissue aliquots were homogenized in saline/0.04% Tween-80, and 10-fold serial dilutions were plated in duplicates onto Middlebrook 7H10 agar plates containing 10% OADC (Difco). Plates were incubated at 37uC, and colonies were counted after 3 weeks. Data are expressed as CFU per lung. Lung homogenates were further analysed to measure the amounts of TNF, IFNc, IL10, IL12p70 and IL6 by ELISA with appropriate OptEIA kits (BD Biosciences) as described by the manufacturers. Lungs were also prepared for histology by fixing the big lobes in 4% phosphate-buffered formalin and then embedding in paraffin. 5 mm-thick sections were stained with haematoxylin and eosin (H&E) for evaluation of pathologic changes. The sizes of inflammatory lesions in four serial sections per Mtb infected mouse lung were determined by automated morphometry using a Nikon inverted microscope eclipse 90i and the software NIS- Elements BR 3.1 (Nikon). Acknowledgments We thank Nasiema Allie, Faried Abbass and Lizette Fick for excellent technical assistance. We further thank Prof Monty Krieger (MIT, Cambridge, MA) for providing access to the SR-B12/2 mice, Dr William Bourn and Prof Anna-Lise Williamson (IIDMM, Cape Town, South Africa) for providing the BCG-GFP strain, as well as Dr Stephen Lawn (IIDMM, Cape Town, South Africa) for providing the BCG-lux strain. Author Contributions Conceived and designed the experiments: GS GDB. Performed the experiments: GS RG GM. Analyzed the data: GS GDB. Contributed reagents/materials/analysis tools: FB GDB. Wrote the paper: GS GDB. 1. Schafer G, Jacobs M, Wilkinson RJ, Brown GD (2009) Non-opsonic recognition of Mycobacterium tuberculosis by phagocytes. J Innate Immun 1: 231–243. 2. Zimmerli S, Edwards S, Ernst JD (1996) Selective receptor blockade during phagocytosis does not alter the survival and growth of Mycobacterium tuberculosis in human macrophages. Am J Respir Cell Mol Biol 15: 760– 770. 3. Wieland CW, Koppel EA, den Dunnen J, Florquin S, McKenzie AN, et al. (2007) Mice lacking SIGNR1 have stronger T helper 1 responses to Mycobacterium tuberculosis. Microbes Infect 9: 134–141. 4. Hu C, Mayadas-Norton T, Tanaka K, Chan J, Salgame P (2000) Mycobac- terium tuberculosis infection in complement receptor 3-deficient mice. J Immunol 165: 2596–2602. PLoS ONE | www.plosone.org 12 December 2009 | Volume 4 | Issue 12 | e8448