ASC controls IFN-γ levels in an IL-18-dependent manner in caspase-1-deficient mice infected with Francisella novicida.

The inflammasome is a signaling platform that is central to the innate immune responses to bacterial infections. Francisella tularensis is a bacterium replicating within the host cytosol. During F. tularensis subspecies novicida infection, AIM2, an inflammasome receptor sensing cytosolic DNA, activates caspase-1 in an ASC-dependent manner, leading to both pyroptosis and release of the proinflammatory cytokines IL-1ß and IL-18. Activation of this canonical inflammasome pathway is key to limit F. novicida infection. In this study, by comparing the immune responses of AIM2 knockout (KO), ASC(KO), and Casp1(KO) mice in response to F. novicida infection, we observed that IFN-γ levels in the serum of Casp1(KO) mice were much higher than the levels observed in AIM2(KO) and ASC(KO) mice. This difference in IFN-γ production was due to a large production of IFN-γ by NK cells in Casp1(KO) mice that was not observed in ASC(KO) mice. The deficit in IFN-γ production observed in ASC(KO) mice was not due to a reduced Dock2 expression or to an intrinsic defect of ASC(KO) NK cells. We demonstrate that in infected Casp1(KO) mice, IFN-γ production is due to an ASC-dependent caspase-1-independent pathway generating IL-18. Furthermore, we present in vitro data suggesting that the recently described AIM2/ASC/caspase-8 noncanonical pathway is responsible for the caspase-1-independent IL-18 releasing activity. To our knowledge, this study is the first in vivo evidence of an alternative pathway able to generate in a caspase-1-independent pathway bioactive IL-18 to boost the production of IFN-γ, a cytokine critical for the host antibacterial response.

Inflammasome activation restricts Legionella pneumophila replication in primary microglial cells through flagellin detection.

Microglial cells constitute the first line of defense of the central nervous system (CNS) against microbial invasion. Pathogens are detected thanks to an array of innate immune receptors termed pattern recognition receptors (PRRs). PRRs have been thoroughly characterized in bone marrow-derived macrophages, but the PRRs repertoire and functionality in microglial cells remain largely unknown. Microglial cells express various Toll-like Receptors and the Nod1/2 receptors. Recently, a novel innate immune signalling pathway, the inflammasome pathway has been uncovered. Inflammasome activation leads to caspase-1 activation, release of the proinflammatory cytokines, IL-1ß and IL-18 and cell death in a process termed pyroptosis. One inflammasome receptor, NLRP3, has been characterized in microglial cells and associated with response to infections and in the initiation of neuro-degeneration in an Alzheimer's disease model. Legionella pneumophila (L.pneumophila) is a flagellated bacterium replicating within macrophages. In bone marrow-derived macrophages, L. pneumophila is detected in a flagellin-dependent manner by the Naip5-NLRC4 (Ipaf) inflammasome pathway. In this study, we decided to use L. pneumophila to investigate the presence and the functionality of this inflammasome in primary murine microglial cells. We show that microglial cells detect L. pneumophila infection in a flagellin-dependent manner leading to caspase-1-mediated bacterial growth restriction, infected cell death and secretion of the proinflammatory cytokines IL-1ß and IL18. Overall, our data demonstrate that microglial cells have a functional Naip5-NLRC4 inflammasome likely to be important to monitor and clear CNS infections by flagellated bacteria.

Cross-talk between Staphylococcus aureus leukocidins-intoxicated macrophages and lung epithelial cells triggers chemokine secretion in an inflammasome-dependent manner.

Staphylococcus aureus is a major pathogen responsible for both nosocomial and community-acquired infections. Central to its virulence is its ability to secrete haemolysins, pore-forming toxins and cytolytic peptides. The large number of membrane-damaging toxins and peptides produced during S. aureus infections has hindered a precise understanding of their specific roles in diseases. Here, we used comprehensive libraries of recombinant toxins and synthetic cytolytic peptides, of S. aureus mutants and clinical strains to investigate the role of these virulence factors in targeting human macrophages and triggering IL-1ß release. We found that the Panton Valentine leukocidin (PVL) is the major trigger of IL-1ß release and inflammasome activation in primary human macrophages. The cytolytic peptides, δ-haemolysin and PSMα3; the pore-forming toxins, γ-haemolysin and LukDE; and ß-haemolysin synergize with PVL to amplify IL-1ß release, indicating that these factors cooperate with PVL to trigger inflammation. PVL(+) S. aureus causes necrotizing pneumonia in children and young adults. The severity of this disease is due to the massive recruitment of neutrophils that cause lung damage. Importantly, we demonstrate that PVL triggers IL-1ß release in human alveolar macrophages. Furthermore, IL-1ß released by PVL-intoxicated macrophages stimulates the secretion of the neutrophil attracting chemokines, IL-8 and monocyte chemotactic protein-1, by lung epithelial cells. Finally, we show that PVL-induced IL-8/monocyte chemotactic protein-1 release is abolished by the inclusion of IL-1 receptor antagonist (IL-1Ra) in a mixed culture of lung epithelial cells and macrophages. Together, our results identify PVL as the predominant S. aureus secreted factor for triggering inflammasome activation in human macrophages and demonstrate how PVL-intoxicated macrophages orchestrate inflammation in the lung. Finally, our work suggests that anakinra, a synthetic IL-1Ra, may be an effective therapeutic agent to reduce the massive neutrophils infiltration observed during necrotizing pneumonia and decrease the resulting host-mediated lung injury.