Oxidized LDL in the Guinea Pig Tuberculosis Model Figure 5. Lung lesions in M. tuberculosis infected guinea pigs have increased expression of CD36. A. The overall expression of CD36 in the lungs increases with time in guinea pigs sham-vaccinated with saline or BCG as determined by scoring of immunostained sections. The bars represent median values plus range (when present) for each group (n = 5). The asterisks denote statistically significant increase compared to the naive animals (* = p,0.05 and ** = p,0.01). The photomicrographs B and C represent immunostaining of CD36 predominantly within lung granulomas at 30 and 60 days of infection respectively (406). The inserts (10006) show intracellular staining within macrophages. doi:10.1371/journal.pone.0034148.g005 predominantly within the inflammatory zone of primary granu- lomas (Figure 6B) with minimal staining of the cellular debris within the necrotic center. The staining was comparatively unchanged within the PLF lung areas at different time points (Figure 6C). The pattern of staining for LOX1 was significantly different than for CD36. Whereas CD36 showed a diffuse staining pattern, macrophages expressing LOX1 had discrete cytoplasmic stippling that was most prominent by day 60 (Figure 6E and F). Similar to OxLDL and CD36, BCG vaccination prior to virulent challenge reduced the extent of LOX1 expression but increases at days 30 and 60 of infection were significantly higher than days 5, 15 and 20. Enhanced bacterial survival in OxLDL-loaded macrophages To determine whether OxLDL accumulation in alveolar macrophages influenced the growth of M. tuberculosis, we treated primary guinea pig alveolar macrophages with purified OxLDL prior to in vitro infection. Macrophages were either treated with non-oxidized low-density lipoprotein or copper-oxidized low- density lipoprotein prior to in vitro infection. One group of macrophages did not receive any treatment prior to infection. Our results showed an approximately 10 fold (p,0.005) increase in bacilli in OxLDL-treated macrophages at days 3 and 7 after the initial infection (Figure 7) compared to non-treated macrophages or those pre-treated with non-oxidized lipoproteins. All bacterial counts were normalized to 105 cells for all time points and groups. No statistically significant differences in the viability of macro- phages were noted between different treatment groups at all time points examined (data not shown) suggesting that the observed differences in bacterial numbers were not related to the differences in the viability of macrophages. To confirm the uptake and accumulation of OxLDL by the primary alveolar macrophages, cells were stained with OxLDL by immunofluorescence. Our results showed significant cytoplasmic accumulation of OxLDL in the treatment groups (Figure 8B, 8D). In M. tuberculosis infected group, bacteria were present within the cytoplasm of cells co-expressing OxLDL (Figure 8D). No noticeable expression of OxLDL was present in the untreated groups including the group that received LDL despite the presence of intracellular bacilli (Figures 8A, 8C). Discussion Our data builds on our previous study that showed that systemic and pulmonary oxidative stress is a prominent feature of experimental tuberculosis in guinea pigs [4]. The major findings in this study was that not only does OxLDL accumulate in the serum and within granulomas of M. tuberculosis-infected guinea pigs but the expression of the scavenger receptors CD36 and LOX1 were also increased with similar kinetics and relative magnitude as PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e34148