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 naive macrophages produced an antimicrobial effect, but only if naive 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 naive macrophages. The antimicrobial effect of naive 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 naive 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 naive 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.

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and has recently been shown to influence IL-1b secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1b in antigen-presenting cells. After treatment of macrophages with Toll-like receptor (TLR) ligands, pro-IL-1b was specifically sequestered into autophagosomes, while further activation of autophagy with rapamycin induced the degradation of pro-IL-1b and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1b by antigen-presenting cells in a NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species (ROS), but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1b in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1b through at least two separate mechanisms; by targeting pro-IL-1 for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.