Figure 7. OxLDL loading of guinea pig pulmonary alveolar macrophages results in increased mycobacterial survival. Normal guinea pig alveolar macrophages were obtained by broncho- alveolar lavage and loaded with oxidized or non-oxidized LDL prior to in vitro infection with the H37Rv strain of M. tuberculosis. The data represents the mean CFU (6 SD) recovered from infected cultures from 4 hours to 7 days of infection from 4 separate experiments (n = 4). The asterisks denote statistically significant increase compared to the control group at respective time points (* = p,0.05 and *** = p,0.001). doi:10.1371/journal.pone.0034148.g007 discrepancy could reflect the degree and duration of the lipid accumulation over the course of the disease with foamy macrophages representing mature or more chronic accumulations [36]. OxLDL staining was also observed in airway epithelial and vascular endothelial cells especially in the early time points. This observation may have important implications in ROS-mediated Figure 8. OxLDL loaded guinea pig pulmonary alveolar macrophages support intracellular growth of M. tuberculosis in vitro. A. Immunofluorescence of normal guinea pig alveolar macrophages show no evidence of OxLDL cytoplasmic staining. B. Uninfected alveolar macrophages loaded with OxLDL show strong cytoplasmic immunofluorescence staining after 3 days of treatment. C. Alveolar macrophages loaded with LDL for 3 days and infected with M. tuberculosis (stained with rhodamine/auramine) show minimal to no positive staining for OxLDL. D. Co-localization of M. tuberculosis stained with rhodamine/auramine within alveolar macrophages loaded with OxLDL for 3 days in vitro. doi:10.1371/journal.pone.0034148.g008 Oxidized LDL in the Guinea Pig Tuberculosis Model damage to other cell types during the early phase of disease. Endothelial cells are particularly susceptible to oxidative injury. Ongoing studies in our laboratory indicate that in the early stages of granuloma formation, there is loss of vascular integrity within the inflammatory zone of primary granulomas, which may contribute to the pathogenesis of caseous necrosis. Besides the accumulation of OxLDL, we also show that two OxLDL receptors, CD36 and LOX1 show increased expression over the course of M. tuberculosis infection. The scavenger receptor CD36 may have several important functions in the pathogenesis of tuberculosis. Previous studies have shown that CD36 functions in the uptake of M. tuberculosis by macrophages [21]. Increased CD36 receptor expression by platelets has been shown to be pro- thrombotic in several mouse models of microvascular thrombosis [37]. Because CD36 is an important receptor for the uptake of OxLDL in other diseases, the increased expression of CD36 and OxLDL in M. tuberculosis infected guinea pigs may reflect their importance in the pathogenesis of tuberculosis as well. Data from these studies suggest that the parallel expression of CD36 and LOX1 may function to bind and facilitate the uptake of OxLDL generated during the oxidative stress conditions associated with M. tuberculosis infections as is described for other chronic inflammatory diseases [18,25]. In addition, both CD36 and LOX1 have been shown to serve as the receptor for advanced glycation end products (AGEs) which form following the non-enzymatic glycation of proteins, lipids and nucleic acids [38,39]. The formation of AGEs is promoted during oxidative stress conditions and have been shown to accumulate in human tuberculosis lesions [40]. Recent studies in our laboratory have shown that AGEs accumulate within necrotic lesions and within the peripheral circulation of guinea pigs infected with M. tuberculosis (unpublished data). Collectively, these data suggest a number of pathways in which OxLDL and scavenger receptor expression on macrophages and other cells might be involved in tuberculosis granuloma pathogenesis [41,42]. Besides OxLDL, oxidation of cellular debris containing various fatty acids may also contribute to foam cell formation. However, the contribution from oxidized fatty acids to foam cell formation is unclear. While the scavenger receptor pathway has been shown to result in uptake and stable accumulation of lipid components of OxLDL, the uptake pathway and subsequent stable accumulation of oxidized fatty acids has not been well established. Vaccination of guinea pigs with BCG prior to infection significantly delayed the increase in serum and lesion OxLDL levels compared to animals that were mock-vaccinated with saline. BCG vaccinated guinea pigs maintained serum OxLDL levels within the range of normal, uninfected controls. However, BCG was less effective at preventing the accumulation of OxLDL as well as CD36 and LOX1 receptor expression within lung granulomas. In all cases, BCG vaccinated animals had a progressive accumulation of OxLDL, CD36 and LOX1 by day 60 that was significantly increased above values at day 5 of infection. These data are consistent with the ability of BCG vaccination to delay but not prevent the progression of pulmonary and extra- pulmonary disease [43,44]. Our in-vitro study using alveolar macrophages confirms the ability of guinea pig alveolar macrophages to bind and accumulate OxLDL. The reason OxLDL treated macrophages favored bacilli growth is unknown but may be related to the utilization of intracellular host lipids especially cholesterol as an energy source [7]. Increased, albeit seemingly counter-intuitive, replication of mycobacteria noted in guinea pig broncho-alveolar macrophages laden with OxLDL conflicts with the proposed dynamic hypothesis of latent tuberculosis. Our in-vitro system likely PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e34148