Mycobacterium bovis Bacille-Calmette-Guérin Infection Aggravates Atherosclerosis

Abstract

Tuberculosis has been associated with increased risk of atherosclerotic cardiovascular disease. To examine whether mycobacterial infection exacerbates atherosclerosis development in experimental conditions, we infected low-density lipoprotein receptor knockout (Ldlr-/-) mice with Mycobacterium bovis Bacille-Calmette-Guérin (BCG), an attenuated strain of the Mycobacterium tuberculosis complex. Twelve-week old male Ldlr-/- mice were infected with BCG (0.3–3.0x106 colony-forming units) via the intranasal route. Mice were subsequently fed a western-type diet containing 21% fat and 0.2% cholesterol for up to 16 weeks. Age-matched uninfected Ldlr-/- mice fed with an identical diet served as controls. Atherosclerotic lesions in aorta were examined using Oil Red O staining. Changes induced by BCG infection on the immunophenotyping profile of circulating T lymphocytes and monocytes were assessed using flow cytometry. BCG infection increased atherosclerotic lesions in en face aorta after 8 weeks (plaque ratio; 0.021±0.01 vs. 0.013±0.01; p = 0.011) and 16 weeks (plaque ratio, 0.15±0.13 vs. 0.06±0.02; p = 0.003). No significant differences in plasma cholesterol or triglyceride levels were observed between infected and uninfected mice. Compared to uninfected mice, BCG infection increased systemic CD4/CD8 T cell ratio and the proportion of Ly6Clow non-classical monocytes at weeks 8 and 16. Aortic plaque ratios correlated with CD4/CD8 T cell ratios (Spearman’s rho = 0.498; p = 0.001) and the proportion of Ly6Clow non-classical monocytes (Spearman’s rho = 0.629; p < 0.001) at week 16. In conclusion, BCG infection expanded the proportion of CD4+ T cell and Ly6Clow monocytes, and aggravated atherosclerosis formation in the aortas of hyperlipidemic Ldlr-/- mice. Our results indicate that mycobacterial infection is capable of enhancing atherosclerosis development.

Neonatal Subcutaneous BCG Vaccination Decreases Atherosclerotic Plaque Number and Plaque Macrophage Content in ApoE−/− Mice

“…Results from animal studies on the effects of BCG on atherosclerosis are inconsistent. Experimental approaches vary markedly by model species [ 9 , 11 , 12 ], live versus killed [ 8 , 10 ] BCG inoculum, age [ 10 ], and route of administration [ 9 , 10 , 14 ]. Some reports indicate that BCG vaccination may have proatherogenic effects [ 10 ], albeit under experimental conditions that differ substantially from BCG vaccination in humans, particularly with respect to dose, schedule, route of administration and age at immunization (See Supplementary Table S2 for an overview).…”

“…Experimental approaches vary markedly by model species [ 9 , 11 , 12 ], live versus killed [ 8 , 10 ] BCG inoculum, age [ 10 ], and route of administration [ 9 , 10 , 14 ]. Some reports indicate that BCG vaccination may have proatherogenic effects [ 10 ], albeit under experimental conditions that differ substantially from BCG vaccination in humans, particularly with respect to dose, schedule, route of administration and age at immunization (See Supplementary Table S2 for an overview). For example, in juvenile rabbits, two subcutaneous injections of BCG at 10 weeks (approximating to a full human BCG dose) and 14 weeks of age (half human dose), followed by a high cholesterol diet, resulted in systemic immune activation and increased aortic monocyte recruitment and atherogenesis, without differences in plasma lipids [ 12 ].…”

“…In a study investigating the association between active mycobacterial infection and atherosclerotic CVD, rather than BCG vaccination per se, intra-nasal live BCG inoculation (0.3–3 × 10 6 CFU) of 12-week-old C57BL/6J Ldlr −/− mice fed a high-fat diet induced a persistent systemic BCG infection in lung and spleen [ 10 ]. Compared to uninfected controls, BCG-infected mice had more extensive aortic atherosclerosis and immune activation (non-classical monocyte proportions and increased CD4/CD8 T cell ratio) at 8- or 16-weeks post-vaccination, with no differences in plasma lipid profile between the groups [ 10 ]. These observations are analogous to both active tuberculosis disease and latent tuberculosis infection in humans, which are associated with an increased likelihood of developing subclinical obstructive coronary artery disease [ 22 ].…”

“…Studies of BCG and atherosclerosis are limited to animal models, in which BCG administration differs substantially from that in human infants in terms of age at immunization, BCG preparation [ 8 ], dose [ 9 ], schedule, and anatomical vaccination site [ 10 ]. Moreover, these experimental animal models also vary considerably in other aspects of their methodology.…”

“…Moreover, these experimental animal models also vary considerably in other aspects of their methodology. Studies have used: chickens [ 11 ], rabbits [ 12 ], and atherosclerosis-prone genetically modified mouse strains [ 13 ]; live or freeze-dried BCG [ 8 ]; single and repeated [ 12 ] immunizations; immunizations at varying ages following weaning; weight-equivalent doses that differ by approximately 10-fold; and intranasal [ 10 ], intraperitoneal [ 14 ], and intravenous [ 9 ] administration. The findings from studies using these diverse experimental conditions have been inconsistent, and it is difficult to extrapolate these animal data to humans, in whom BCG immunization is given once, subcutaneously with a live inoculum in the early neonatal period.…”