Macrophage Apoptosis in Response to High Intracellular Burden of Mycobacterium tuberculosis Is Mediated by a Novel Caspase-Independent Pathway

Abstract

We previously reported that macrophage exposure to attenuated strains of pathogenic mycobacteria at multiplicities of infection (MOI) ≤ 10 triggers TNF-α-mediated apoptosis which reduces the viability of intracellular bacilli. Virulent strains were found to suppress macrophage apoptosis, and it was proposed that apoptosis is an innate defense against intracellular Mycobacterium tuberculosis analogous to apoptosis of virus-infected cells. The potential similarity of host cell responses to intracellular infection with mycobacteria and viruses suggests that M. tuberculosis might lyse infected macrophage when that niche is no longer needed. To investigate this question, we challenged murine macrophages with high intracellular bacillary loads. A sharp increase in cytolysis within 24 h was observed at MOI ≥ 25. The primary death mode was apoptosis, based on nuclear morphology and phosphatidyl serine exposure, although the apoptotic cells progressed rapidly to necrosis. Apoptosis at high MOI differs markedly from low MOI apoptosis: it is potently induced by virulent M. tuberculosis, it is TNF-α-independent, and it does not reduce mycobacterial viability. Caspase inhibitors failed to prevent high MOI apoptosis, and macrophages deficient in caspase-3, MyD88, or TLR4 were equally susceptible as wild type. Apoptosis was reduced in the presence of cathepsin inhibitors, suggesting the involvement of lysosomal proteases in this novel death response. We conclude that the presence of high numbers of intracellular M. tuberculosis bacilli triggers a macrophage cell death pathway that could promote extracellular spread of infection and contribute to the formation of necrotic lesions in tuberculosis.

A Novel Mechanism Underlying the Basic Defensive Response of Macrophages against Mycobacterium Infection

“…The ligation of TLR2 during phagocytic uptake of the microorganism might be important for BCG infection-induced apoptosis under our experimental conditions. Meanwhile, several reports have also mentioned the importance of TLR4 (19,20,23), despite the fact that M. tuberculosis is a Gram-positive microorganism. Thus, controversy remains over which TLRs contribute predominantly to the M. tuberculosis infection-induced apoptosis of macrophages.…”

“…The mammalian innate immune system recognizes invading microorganisms by phagocytic receptors, such as TLRs and b 2 integrin (14)(15)(16)(17)(18). In fact, a role for TLRs in M. tuberculosis infection-induced macrophage death has been reported (19)(20)(21)(22)(23)(24)(25). However, the signaling pathway transmitting TLR activation to macrophage death remains unclear.…”

Interaction of Mycobacterium tuberculosis with Host Cell Death Pathways

“…Instead Mtb, after infection and multiplication, induces programmed necrosis to escape the cell (Chen et al 2006;Lee et al 2006;Behar et al 2010).…”

In vitro infection of human cells with Mycobacterium tuberculosis

“…Large MOI (5-100 bacteria per cell) induce necrosis and apoptosis 60,61 , which will greatly interfere with the intracellular multiplication of M. tuberculosis. For this reason, infected cells are incubated for a short period of time (1 hour to overnight) and non-phagocyted bacteria are removed by extensive washing.…”