Bacterial Ligands Generated in a Phagosome Are Targets of the Cytosolic Innate Immune System

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Abstract
Macrophages are permissive hosts to intracellular pathogens, but upon activation become microbiocidal effectors of innate and cell-mediated immunity. How the fate of internalized microorganisms is monitored by macrophages, and how that information is integrated to stimulate specific immune responses is not understood. Activation of macrophages with interferon (IFN)–γ leads to rapid killing and degradation of Listeria monocytogenes in a phagosome, thus preventing escape of bacteria to the cytosol. Here, we show that activated macrophages induce a specific gene expression program to L. monocytogenes degraded in the phago-lysosome. In addition to activation of Toll-like receptor (TLR) signaling pathways, degraded bacteria also activated a TLR-independent transcriptional response that was similar to the response induced by cytosolic L. monocytogenes. More specifically, degraded bacteria induced a TLR-independent IFN-β response that was previously shown to be specific to cytosolic bacteria and not to intact bacteria localized to the phagosome. This response required the generation of bacterial ligands in the phago-lysosome and was largely dependent on nucleotide-binding oligomerization domain 2 (NOD2), a cytosolic receptor known to respond to bacterial peptidoglycan fragments. The NOD2-dependent response to degraded bacteria required the phagosomal membrane potential and the activity of lysosomal proteases. The NOD2-dependent IFN-β production resulted from synergism with other cytosolic microbial sensors. This study supports the hypothesis that in activated macrophages, cytosolic innate immune receptors are activated by bacterial ligands generated in the phagosome and transported to the cytosol. Innate immune recognition of microorganisms has a direct impact on the type and the magnitude of the immune response elicited. While recognition of microorganisms relies on receptors that sense pathogen-associated molecular patterns, (PAMPs), it was reasonable to suspect that immune cells could discriminate between live and dead bacteria. Listeria monocytogenes is an intracellular pathogenic bacterium used extensively as a model system for studying basic aspects of innate and acquired immunity. L. monocytogenes is internalized by macrophages, escapes from a vacuole, multiplies within the cytosol, and spreads from cell to cell without lysing the cells. We used wild-type and bacterial mutants of L. monocytogenes to demonstrate that macrophages not only respond differently to bacteria that are growing in the cytosol and to non-growing bacteria that are trapped in a vacuole, but that they also can discriminate between intact or degraded bacteria in the vacuole. We showed that macrophages induce specific immune response when bacteria are killed and degraded. This response was directly correlated to the ability of macrophages to degrade bacteria and involved receptors that were located in the host cell cytosol. These observations led us to suggest that bacterial degradation products may serve as messengers that inform immune cells that bacteria were killed and degraded. This information might affect directly the immune response, for example, by down-regulating inflammatory responses that can be deleterious. We call these bacterial degradation products PAMP-PM (PAMP–post-mortem).