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

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 CD11clo/−, 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 CD11clo/− 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.

Efferocytosis and extrusion of leukocytes determine the progression of early mycobacterial pathogenesis

“…Although mycobacteria are notorious for their ability to block apoptosis (Lee et al, 2006;Repasy et al, 2013), our correlative light and electron microscopy images show that macrophages with a large bacterial content, most likely acquired by efferocytosis, have a fragmented nucleus. In addition, by using CLSM, clear fragmentation of infected macrophages, another characteristic of apoptotic cell death, was observed ( Fig.…”

“…In our study, macrophage burst events were defined as highly infected macrophages undergoing necrotic cell death, probably due to cell-death-induced rupture of the membranes surrounding these cells, and spreading of the bacteria to freshly recruited leukocytes (Repasy et al, 2013). Subsequently, the spread bacteria were taken up by neutrophils and macrophages.…”

Interaction of Mycobacterium tuberculosis with Host Cell Death Pathways

“…Other reports showed caspase-independent, programmed necrotic cell death induction after infection of cells with high bacterial numbers that did not lead to bactericidal activity . The threshold for host cells to undergo necrosis was determined to be between 20 and 40 bacteria per phagocyte as determined in ex vivo and in vivo studies Repasy et al 2013). This death pathway was independent of Ca 2þ flux and MPT and did not require lysosomal cathepsin protease activity Welin et al 2011;Wong and Jacobs 2011).…”

Interplay of human macrophages and Mycobacterium tuberculosis phenotypes

“…In vivo, efferocytosis mediates clearance of apoptotic cells and could contribute to clearance of bacteria and resolution of inflammation, at least in those lesions that resolve during treatment. The phenomenon of 'dose-dependency' in terms of correlation of bacterial burden and host cell death or clinical outcome was also demonstrated in other models, such as in mice and NHPs [102,203].…”

“…Furthermore, necrosis was shown to depend on ESAT6 and on intracellular bacterial numbers, indicating that Mtb seeks to disseminate only when a certain amount of bacilli is present [202,203]. Several bacterial effectors are involved in inhibition of apoptosis, such as nuoG, a subunit of the NADPH dehydrogenase that suppresses ROS-induced apoptosis.…”