Uptake and Accumulation of Oxidized Low-Density Lipoprotein during Mycobacterium tuberculosis Infection in Guinea Pigs Gopinath S. Palanisamy, Natalie M. Kirk, David F. Ackart, Andre´ s Obrego´ n-Henao, Crystal A. Shanley, Ian M. Orme, Randall J. Basaraba* Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America Abstract The typical host response to infection of humans and some animals by M. tuberculosis is the accumulation of reactive oxygen species generating inflammatory cells into discrete granulomas, which frequently develop central caseous necrosis. In previous studies we showed that infection of immunologically na¨ıve guinea pigs with M. tuberculosis leads to localized and systemic oxidative stress that results in a significant depletion of serum total antioxidant capacity and the accumulation of malondialdehyde, a bi-product of lipid peroxidation. Here we show that in addition, the generation of excessive reactive oxygen species in vivo resulted in the accumulation of oxidized low density lipoproteins (OxLDL) in pulmonary and extrapulmonary granulomas, serum and lung macrophages collected by bronchoalveolar lavage. Macrophages from immunologically na¨ıve guinea pigs infected with M. tuberculosis also had increased surface expression of the type 1 scavenger receptors CD36 and LOX1, which facilitate the uptake of oxidized host macromolecules including OxLDL. Vaccination of guinea pigs with Bacillus Calmette Guerin (BCG) prior to aerosol challenge reduced the bacterial burden as well as the intracellular accumulation of OxLDL and the expression of macrophage CD36 and LOX1. In vitro loading of guinea pig lung macrophages with OxLDL resulted in enhanced replication of bacilli compared to macrophages loaded with non-oxidized LDL. Overall, this study provides additional evidence of oxidative stress in M. tuberculosis infected guinea pigs and the potential role OxLDL laden macrophages have in supporting intracellular bacilli survival and persistence. Citation: Palanisamy GS, Kirk NM, Ackart DF, Obrego´ n-Henao A, Shanley CA, et al. (2012) Uptake and Accumulation of Oxidized Low-Density Lipoprotein during Mycobacterium tuberculosis Infection in Guinea Pigs. PLoS ONE 7(3): e34148. doi:10.1371/journal.pone.0034148 Editor: Tanya Parish, Queen Mary University of London, United Kingdom Received August 30, 2011; Accepted February 23, 2012; Published March 30, 2012 Copyright: 2012 Palanisamy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by National Institutes of Health grants AI070456 (RJB) and AI083856 (IMO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Randall.Basaraba@colostate.edu Introduction It is estimated that one out of every three people worldwide is infected with M. tuberculosis the primary causative agent of human tuberculosis [1]. Because tuberculosis is a chronic inflammatory disease, generation of excessive reactive oxygen species (ROS) leading to a depletion of host antioxidant defenses, has been implicated in the disease pathogenesis. Markers of free radical damage such as malondialdehyde have been shown to be elevated in the peripheral circulation of human patients with active tuberculosis along with decreased serum antioxidants [2,3]. In a previous study, we showed that in vivo oxidative stress in guinea pigs experimentally infected with virulent M. tuberculosis was also due in part to a depletion of host antioxidant defenses [4]. We also showed that treatment of M. tuberculosis infected guinea pigs with the antioxidant drug N-acetylcysteine reduced systemic oxidative stress by restoring the serum total antioxidant capacity, which lessened the severity of granuloma necrosis and reduced extrapulmonary dissemination of bacilli. These data suggest that inhibition of excessive ROS generation therapeutically either alone or in combination with standard drug therapy may be beneficial by limiting the progression of pulmonary and extrapul- monary tuberculosis in humans. It has been known for nearly a century that the caseum of necrotic tuberculous granulomas are rich in host lipids [5]. Besides being an important source of energy, lipids in the form of cholesterol and fatty acids have been shown to facilitate mycobacterial entry and growth [6,7,8,9]. Lipid-laden macro- phages are a prominent and consistent feature of granulomatous lesions in animals and in infected with M. tuberculosis [10,11,12]. Recently lipid-laden macrophages have been shown to support the persistence of non-replicating bacilli and to be defective in phagocytic and bactericidal activity [13]. The diminished antimicrobial capacity of infected macrophages may enable M. tuberculosis to exploit this unique intracellular microenvironment for survival and replication in vivo. Lipid-laden macrophages occur in a variety of infectious and non-infectious inflammatory conditions [14,15]. Vacuolated macrophages also referred to as ‘‘foam cells’’ are central to the pathogenesis of atherosclerosis where oxidative stress plays a major role in disease pathogenesis. The morphologic similarities between foam cells in atherosclerosis and tuberculosis led us to hypothesize that low-density lipoproteins (LDL) are among the host macro- molecules that accumulates within macrophages associated with granulomas of M. tuberculosis infected guinea pigs. Similar to humans, the majority of circulating cholesterol in guinea pigs is PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e34148