Oxidized LDL in the Guinea Pig Tuberculosis Model Figure 3. OxLDL is concentrated and accumulates in macrophages within lung granulomas of M. tuberculosis infected guinea pigs. A. The expression of OxLDL in the lungs lesions increased with the progression of disease in guinea pigs sham vaccinated with saline and BCG vaccinated animals as determine 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 OxLDL predominantly within primary granulomas at 30 and 60 days of infection respectively (406). The inserts (10006) show intracellular staining within vascular endothelial cells (VE), epithelioid macrophages (EM), airway epithelial cells (AE) and foamy macrophages (FM). doi:10.1371/journal.pone.0034148.g003 lesion scores. Increases in CD36 expression were statistically significant on days 30 and 60 of infection (p,0.05 and 0.01 respectively). Non-infected guinea pigs expressed minimal to no CD36 staining. The increase in the CD36 expression was predominantly within the inflammatory zone of the primary granulomas (Figure 5B) similar to the pattern seen for OxLDL. In Figure 4. OxLDL accumulates in pulmonary alveolar macro- phages of M. tuberculosis-infected guinea pigs. A and B represent OxLDL immunostaining in BAL cells collected from M. tuberculosis infected guinea pigs at day 30 and day 60 after infection respectively (10006 magnification). Predominantly macrophages (arrows) and occasionally granulocytes (arrowhead) show intracellular staining. doi:10.1371/journal.pone.0034148.g004 contrast, the expression within the secondary lesions and non- affected lung was unchanged even out to 60 days of infection (Figure 5.5C). Despite the reduced lesion burden seen in BCG vaccinated guinea pigs, there was an increase in CD36 expression by day 60 of infection. The cell types that showed the most intense CD36 expression were macrophages including those with vacuolated cytoplasm, airway epithelial cells, endothelial cells and granulocytes (Figure 5E and F). LOX1 expression in lungs LOX1 is another major receptor for oxidized LDL and is known to be transcriptionally up-regulated during various inflammatory diseases that have oxidative stress as a central pathogenesis [26]. Similar to CD36, we sought to evaluate the LOX1 receptor expression levels in the lungs M. tuberculosis infection in guinea pigs using immunohistochemistry. Similar to OxLDL and C36, overall LOX1 protein expression levels increased significantly as the infection progressed (Figure 6A), a rate that correlated (r = 0.99) with the increase in the lung lesion scores. Lungs from non-infected guinea pigs had minimal to no LOX1 expression by immunohistochemistry. This increase in LOX1 expression was statistically significant on days 30 and 60 after infection (p,0.05 and 0.01 respectively). Similar to CD36 expression, the increase in the LOX1 immunostaining occurred PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e34148