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Public Library of Science, PLoS Pathogens, 2(9), p. e1003190, 2013

DOI: 10.1371/journal.ppat.1003190

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Intracellular Bacillary Burden Reflects a Burst Size for Mycobacterium tuberculosis In Vivo

This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Data provided by SHERPA/RoMEO

Abstract

We previously reported that Mycobacterium tuberculosis triggers macrophage necrosis in vitro at a threshold intracellular load of ~25 bacilli. This suggests a model for tuberculosis where bacilli invading lung macrophages at low multiplicity of infection proliferate to burst size and spread to naïve phagocytes for repeated cycles of replication and cytolysis. The current study evaluated that model in vivo, an environment significantly more complex than in vitro culture. In the lungs of mice infected with M. tuberculosis by aerosol we observed three distinct mononuclear leukocyte populations (CD11b(-) CD11c(+/hi), CD11b(+/lo) CD11c(lo/-), CD11b(+/hi) CD11c(+/hi)) and neutrophils hosting bacilli. Four weeks after aerosol challenge, CD11b(+/hi) CD11c(+/hi) mononuclear cells and neutrophils were the predominant hosts for M. tuberculosis while CD11b(+/lo) CD11c(lo/-) cells assumed that role by ten weeks. Alveolar macrophages (CD11b(-) CD11c(+/hi)) were a minority infected cell type at both time points. The burst size model predicts that individual lung phagocytes would harbor a range of bacillary loads with most containing few bacilli, a smaller proportion containing many bacilli, and few or none exceeding a burst size load. Bacterial load per cell was enumerated in lung monocytic cells and neutrophils at time points after aerosol challenge of wild type and interferon-γ null mice. The resulting data fulfilled those predictions, suggesting a median in vivo burst size in the range of 20 to 40 bacilli for monocytic cells. Most heavily burdened monocytic cells were nonviable, with morphological features similar to those observed after high multiplicity challenge in vitro: nuclear condensation without fragmentation and disintegration of cell membranes without apoptotic vesicle formation. Neutrophils had a narrow range and lower peak bacillary burden than monocytic cells and some exhibited cell death with release of extracellular neutrophil traps. Our studies suggest that burst size cytolysis is a major cause of infection-induced mononuclear cell death in tuberculosis.