carried as LDL whereas in other rodent species, less than 50% of circulating cholesterol is in the form of LDL. Guinea pigs and humans share other similarities in cholesterol and lipoprotein metabolism, which makes them an ideal model to study lipid metabolism during M. tuberculosis infection. Unlike LDL which is taken up by specific macrophage LDL receptors (LDL-R), the oxidized form of LDL (OxLDL) is taken up by the scavenger receptors SR-A, SR-B1, CD36 and lectin-like oxidized low-density lipoprotein receptor-1 (LOX1) during foam cell formation in atherosclerotic lesions [16]. Scavenger receptors recognize and facilitate the uptake of macromolecules with negative charges including modified LDL. Among the multiple OxLDL receptors that have been characterized, CD36 and LOX1 are the most dominant with both being involved in the pathogenesis of cardiovascular disease and atherosclerosis [17,18,19,20]. More- over, CD36 has been shown to be involved in the uptake of M. tuberculosis by macrophages and non-phagocytic cells [21]. In this study, we investigated whether oxidative stress associated with experimental M. tuberculosis infection in guinea pigs results in the accumulation of OxLDL and increased expression of the OxLDL receptors, CD36 and LOX1. Because BCG vaccination of guinea pigs prior to challenge delays granuloma formation and reduces disease severity, we investigated whether BCG vaccination abrogates OxLDL accumulation and CD36 and LOX1 receptor expression in guinea pigs infected with the H37Rv strain of M. tuberculosis. Finally, we determined whether in vitro OxLDL loading of guinea pig alveolar macrophages altered the growth and intracellular survival of M. tuberculosis. Methods Ethics Statement All experimental protocols were in accordance with the National Research Councils Guide for the Care and Use of Laboratory Animals and was approved by the Animal Care and Usage Committee of Colorado State University under protocol number 08-212A-02. Aerosol infection M. tuberculosis H37Rv strain (TMC#102; Trudeau Institute, Saranac Lake, NY) collected and frozen at mid-log phase of growth in Proskauer-Beck liquid medium containing 0.05% Tween 80 was used for infection. A thawed aliquot of M. tuberculosis was diluted in sterile water to 106 CFU/ml for a total working stock volume of 20 ml. Guinea pigs, approximately 9 months old, were purchased from Charles River Laboratories (North Wilmington, MA, USA) were randomly assigned to treatment groups and were exposed to aerosolized bacilli using a Madison aerosol generating chamber (University of Wisconsin Machine Shop, Madison, WI) with a starting volume of 15 ml of working stock. BCG vaccination Guinea pigs were vaccinated with 16104 Mycobacterium bovis (BCG, strain Pasteur) or mock vaccinated with saline, intra- dermally 4 weeks prior to aerosol exposure to M. tuberculosis. M. bovis BCG was grown in Proskauer-Beck similar to M. tuberculosis and frozen at 280uC until used for infection [22]. Euthanasia and sample collection At 5, 15, 20, 30, or 90 days after infection, guinea pigs were euthanized by an overdose (1 ml per 0.75 kg body weight) of sodium pentobarbital (Sleepaway; Fort Dodge Laboratories Inc.) by intraperitoneal injection. Following euthanasia, the left Oxidized LDL in the Guinea Pig Tuberculosis Model pulmonary lobes were infused in situ with 5 ml of 4% paraformaldehyde and were allowed to fix for 48 hours and then transferred to and stored in 70% ethanol. Tissues were routinely processed by embedding in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin (H&E) [23]. Evaluation of serum OxLDL levels Systemic oxidized LDL levels were measured in serum collected from BCG or saline vaccinated guinea pigs at 15, 30, and 60 days after infection using a commercially available mouse anti-human copper oxidized LDL (mAb-4E6)-based competitive ELISA (Mercodia, Winston Salem NC). The specificity of mAb-4E6 (kindly provided by Mercodia) for guinea pig OxLDL was confirmed by the loss of immunoreactivity on lung tissue sections in the presence of excess antigen (human copper-oxidized LDL). Immunohistochemistry Approximately 5 mm thick paraffin lung sections from infected guinea pigs were collected on positively charged glass slides, deparaffinized, rehydrated and antigen retrieval enhanced by incubating in Target Retrieval solution, pH 6.0 (DAKO, Carpen- taria, CA) for 25 min at 90uC, followed by a 20 min cooling period at room temperature. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide treatment for 15 min. The slides were subjected to two blocking steps after rinsing once in Tris buffered saline with 1% Tween-20 (TTBS): (i) 15 min incubation with 0.15 mM glycine in PBS, and (ii) 30 min incubation with 1% normal horse serum with a rinse in TTBS in between. The slides were then incubated with rabbit polyclonal antibody to human CD36, LOX1 (Santa Cruz Biotechnology, Santa Cruz, CA) and copper-oxidized LDL (Abcam, Cambridge, MA), or non-immune rabbit serum at a 1:100 dilution in blocking buffer followed by several rinses in TTBS and incubated for 30 min. with biotinylated goat-anti rabbit-IgG (Vector Laborato- ries). Bound antibody was visualized using the Avidin-Biotin system (Vectastain; Vector Laboratories) and diaminobenzidine substrate (Dako; Carpentaria, CA). Sections were counterstained with Meyer’s hematoxylin (Scytek Laboratories; Logan, Utah), mounted, cover slipped and examined by light microscopy. For ex vivo OxLDL immunocytochemistry, alveolar macrophages were collected at necropsy from M. tuberculosis-infected guinea pigs at by bronchioalveolar lavage as described below and cytospin prepa- rations prepared (Thermo Scientific, Waltham, MA) on charged glass slides. Slides were air dried and fixed with 100% ice cold methanol and processed as described earlier starting with the primary antibody incubation. The specificity of anti-human CD36 and LOX1 antibodies on guinea pig tissues was confirmed by blocking the primary antibodies with the respective purified antigens (CD36 - Affinity Bioreagents, Golden CO; LOX1 - Santa Cruz Biotechnology, Santa Cruz, CA; copper oxidized LDL - Biomedical Technologies, Stoughton, MA) at an antibody to antigen ratio of 1:5. Photomicrographs were acquired with an Olympus DP70 camera and associated computer software. Microscopic lesions stained with H&E and sections stained by immunohistochemistry, were scored by the reviewer blinded to the treatment groups. Lesion analysis To evaluate the progression of disease over time, a previously described histological grading system was used [24]. Lung sections were scored based on the following six criteria: (i) percent of lung affected were ranked at low magnification based on the percent of the lung affected by lesion as follows: 0-no lesions, 1, #25% of lung involved, 2, 25–50% of lung involved, 3, 50–75% of lung PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e34148