Interactions between Naïve and Infected Macrophages Reduce Mycobacterium tuberculosis Viability

Abstract

A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with naïve macrophages produced an antimicrobial effect, but only if naïve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the naïve macrophages. The antimicrobial effect of naïve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the naïve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of naïve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which promotes nitric oxide-dependent antimicrobial activity against bacilli in autolysosomes of heavily infected macrophages. This cooperative, innate antimicrobial interaction may limit the maximal growth rate of M. tuberculosis prior to the expression of adaptive immunity in pulmonary tuberculosis.

Efferocytosis Is an Innate Antibacterial Mechanism

“…The assay system developed here will be an important tool to study how efferocytosis is regulated (or subverted) in the context of infection. Our data does not exclude the possibility of other cell contact and non-contact mechanisms of bacterial control exerted by uninfected macrophages seen in other high-dose Mtb-induced cytolysis models (Hartman and Kornfeld, 2011). In fact, in the recent study by Hartman and Kornfeld, efferocytosis appeared to be inhibited and, in the context of our results, may indicate that high-MOI non-apoptotic cell death inhibits efferocytosis or prevents the expression of relevant ‘eat me’ signals on the dying cell.…”

Programmed cell death as a defence against infection

“…This efferocytic phagosome matures, acidifies and kills the bacteria via ROS. Notably, pathogens that resist apoptosis, such as Mycobacterium , also interfere with efferocytosis, which increases their survival 136 . L. monocytogenes , on the other hand, exploits efferocytosis receptors in order to spread from cell to cell 137 .…”

Mycobacterium tuberculosis and the human macrophage : shifting the balance through inflammasome activation

“…Inversely, efferocytosis of dead macrophages by recruited neutrophils in M. marinum-infected zebrafish leads to ROS-mediated killing by the neutrophils, showing that efferocytosis is not a specific mechanism unique for macrophages (238). Efferocytosis of apoptotic macrophages by non-infected macrophages also controls bacterial growth both in vitro and in vivo (230), while efferocytosis of infected macrophages that display PS but have undergone more necrotic-like cell death does not have any bactericidal effects (239). Furthermore, the phagosome containing the M. tuberculosis-carrying apoptotic body is more efficiently acidified and spacious than the phagosome containing directly phagocytosed M. tuberculosis (230).…”

“…IL-1β released from infected macrophages can also stimulate other cells like lung epithelial cells or uninfected macrophages through IL-1RI to express antimicrobial peptides. These peptides are then secreted, leading to decreased bacterial numbers and increased survival of infected macrophages (239,358). Addition of IL-1β to murine macrophages also increases killing of virulent M. tuberculosis by inducing autophagy (359) and leads to increased maturation of mycobacterial phagosomes (167).…”