Getting rid of the bad apple: inflammasome-induced extrusion of Salmonella-infected enterocytes.

Two reports in this issue of Cell Host & Microbe (Sellin et al., 2014; Knodler et al., 2014) establish the cell-intrinsic inflammasome-induced extrusion of infected enterocytes as a general defense mechanism against acute bacterial infections.

Epithelium-intrinsic NAIP/NLRC4 inflammasome drives infected enterocyte expulsion to restrict Salmonella replication in the intestinal mucosa.

The gut mucosal epithelium separates the host from the microbiota, but enteropathogens such as Salmonella Typhimurium (S.Tm) can invade and breach this barrier. Defenses against such acute insults remain incompletely understood. Using a murine model of Salmonella enterocolitis, we analyzed mechanisms limiting pathogen loads in the epithelium during early infection. Although the epithelium-invading S.Tm replicate initially, this intraepithelial replicative niche is restricted by expulsion of infected enterocytes into the lumen. This mechanism is compromised if inflammasome components (NAIP1-6, NLRC4, caspase-1/-11) are deleted, or ablated specifically in the epithelium, resulting in ∼100-fold higher intraepithelial loads and accelerated lymph node colonization. Interestingly, the cytokines downstream of inflammasome activation, interleukin (IL)-1α/ß and IL-18, appear dispensable for epithelial restriction of early infection. These data establish the role of an epithelium-intrinsic inflammasome, which drives expulsion of infected cells to restrict the pathogen's intraepithelial proliferation. This may represent a general defense mechanism against mucosal infections.

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.

Germs gone wild.

The normally harmless behavior of bacteria in the intestinal tract is maintained by community structure and the integrity of host defenses. When either or both of these are compromised, a few disgruntled outcasts can cause a riot, taking down the whole neighborhood (pages 799-806).

Lethal inflammasome activation by a multidrug-resistant pathobiont upon antibiotic disruption of the microbiota.

The mammalian intestine harbors a complex microbial community that provides numerous benefits to its host. However, the microbiota can also include potentially virulent species, termed pathobiont, which can cause disease when intestinal homeostasis is disrupted. The molecular mechanisms by which pathobionts cause disease remain poorly understood. Here we describe a sepsis-like disease that occurs upon gut injury in antibiotic-treated mice. Sepsis was associated with the systemic spread of a specific multidrug-resistant Escherichia coli pathobiont that expanded markedly in the microbiota of antibiotic-treated mice. Rapid sepsis-like death required a component of the innate immune system, the Naip5-Nlrc4 inflammasome. In accordance with Koch's postulates, we found the E. coli pathobiont was sufficient to activate Naip5-Nlrc4 and cause disease when injected intravenously into unmanipulated mice. These findings reveal how sepsis-like disease can result from recognition of pathobionts by the innate immune system.

Cooperation between multiple microbial pattern recognition systems is important for host protection against the intracellular pathogen Legionella pneumophila.

Multiple pattern recognition systems have been shown to initiate innate immune responses to microbial pathogens. The degree to which these detection systems cooperate with each other to provide host protection is unknown. Here, we investigated the importance of several immune surveillance pathways in protecting mice against lethal infection by the intracellular pathogen Legionella pneumophila, the causative agent of a severe pneumonia called Legionnaires' disease. Rip2 and Naip5/NLRC4 signaling was found to contribute to the innate immune response generated against L. pneumophila in the lung. Elimination of Rip2 or Naip5/NLRC4 signaling in MyD88-deficient mice resulted in increased replication and dissemination of L. pneumophila and higher rates of mortality. Irradiated wild-type mice receiving bone marrow cells from pattern recognition receptor-deficient mice displayed L. pneumophila infection phenotypes similar to those of donor mice. Rip2 and Naip5/NLRC4 signaling provided additive effects in protecting MyD88-deficient mice from lethal infection by L. pneumophila, with the contribution of Naip5/NLRC4 being slightly greater than that of Rip2. Thus, activation of the Rip2, MyD88, and Naip5/NLRC4 signaling pathways triggers a coordinated and synergistic response that protects the host against lethal infection by L. pneumophila. These data provide new insight into how different pattern recognition systems interact functionally to generate innate immune responses that protect the host from lethal infection by activating cellular pathways that restrict intracellular replication of L. pneumophila and by recruiting to the site of infection additional phagocytes that eliminate extracellular bacteria.

The Nod-like receptor family member Naip5/Birc1e restricts Legionella pneumophila growth independently of caspase-1 activation.

Similar to Ipaf and caspase-1, the Nod-like receptor protein Naip5 restricts intracellular proliferation of Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires' disease. Thus, Naip5 has been suggested to regulate Legionella replication inside macrophages through the activation of caspase-1. In this study, we show that cytosolic delivery of recombinant flagellin activated caspase-1 in A/J macrophages carrying a mutant Naip5 allele, and in C57BL/6 (B6) macrophages congenic for the mutant Naip5 allele (B6-Naip5(A/J)), but not in Ipaf(-/-) cells. In line with these results, A/J and B6-Naip5(A/J) macrophages induced high levels of caspase-1 activation and IL-1beta secretion when infected with Legionella. In addition, transgenic expression of a functional Naip5 allele in A/J macrophages did not alter Legionella-induced caspase-1 activation and IL-1beta secretion. Notably, defective Naip5 signaling renders B6-Naip5(A/J) macrophages permissive for Legionella proliferation despite normal caspase-1 activation. These results indicate that the restriction of intracellular Legionella replication is more complex than previously appreciated and requires both Ipaf-dependent caspase-1 activation as well as functional Naip5 signaling.

Restriction of Legionella pneumophila growth in macrophages requires the concerted action of cytokine and Naip5/Ipaf signalling pathways.

Macrophages from the C57BL/6 (B6) mouse strain restrict intracellular growth of Legionella pneumophila, whereas A/J macrophages are highly permissive. The mechanism by which B6 macrophages restrict Legionella growth remains poorly understood, but is known to require the cytosolic microbe sensors Naip5 (Birc1e) and Ipaf. We hypothesized that Naip5 and Ipaf may act in partnership with other antimicrobial signalling pathways in macrophages. Indeed, we found that macrophages lacking either tumour necrosis factor (TNF)-alpha or type I interferon (IFN) signalling are permissive for growth of L. pneumophila, even in the presence of functional Naip5 and Ipaf alleles. Similarly, macrophages lacking Naip5 and/or Ipaf signalling were permissive even though we found that Naip5 or Ipaf were not required for induction of TNF-alpha and type I IFN. Therefore, our data suggest that the mechanism by which B6 macrophages restrict intracellular replication of L. pneumophila is more complex than previously appreciated, and involves the concerted action of cytokine and intracellular microbe sensor signalling pathways.

Birc1e/Naip5 rapidly antagonizes modulation of phagosome maturation by Legionella pneumophila.

Legionella survives intracellularly by preventing fusion with lysosomes, due to phagosome escape from the endocytic pathway at an early stage of phagosome maturation, and by creating a replicative organelle that acquires endoplasmic reticulum (ER) characteristics through sustained interactions and fusion with the ER. Intracellular replication of Legionella pneumophila in mouse macrophages is controlled by the Lgn1 locus. Functional complementation in vivo has identified the Birc1e/Naip5 gene as being responsible for the Lgn1 effect. To understand the function and temporal site of action of Birc1e/Naip5 in susceptibility to L. pneumophila, we examined the biogenesis of Legionella-containing vacuoles (LCVs) formed in permissive A/J macrophages and in their Birc1e/Naip5 transgenic non-permissive counterpart. Birc1e/Naip5 effects on acquisition of lysosomal and ER markers were evident within 1-2 h following infection. A significantly higher proportion of LCVs formed in Birc1e/Naip5 transgenic macrophages had acquired the lysosomal markers cathepsin D and Lamp1 by 2 h post infection, whereas a significantly higher proportion of LCVs formed in permissive macrophages were positively stained for the ER markers BAP31 and calnexin, 6 h post infection. Likewise, studies by electron microscopy showed acquisition of lysosomal contents (horseradish peroxidase), within the first hour following phagocytic uptake, by LCVs formed in Birc1e/Naip5 transgenic macrophages and delivery of the ER marker glucose 6-phosphatase (G6Pase) only to the lumen of LCVs formed in A/J macrophages. Finally, a larger proportion of LCVs formed in A/J macrophages were studded with ribosomes 24 h post infection, compared with LCVs formed in Birc1e/Naip5 transgenic macrophages. These results suggest that sensing of L. pneumophila products by Birc1e/Naip5 in macrophages occurs rapidly following phagocytosis, a process that antagonizes the ability of L. pneumophila to remodel its phagosome into a specialized vacuole with ER characteristics.

TLRS in the gut. II. Flagellin-induced inflammation and antiapoptosis.

Flagellin is bacterial protein that serves as a danger signal across a wide variety of eukaryotes and is a potent inducer of inflammatory effector responses in the mammalian gut. Recent findings utilizing purified flagellin and flagellate/aflagellate bacteria in in vitro and in vivo systems have revealed the important roles played by flagellin in the initial encounter between mucosa and flagellate bacteria, specifically in the modulation of apoptotic responses.

Unique anti-apoptotic activity of EAAC1 in injured motor neurons.

Injured motor neurons of the adult rat can survive, whereas similar axotomy causes gradual motor neuron death in the adult mouse. We report that the decreased expression of the neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1) following nerve injury is associated with motor neuron death in the mouse. Glutamate transporters play a crucial role in prevention of neuronal death by suppressing glutamate toxicity. However, the possible functional role of EAAC1 in preventing neuron death has not been resolved as compared with glial glutamate transporters such as GLT-1. Here, we have revealed a unique 'rescue' function of EAAC1, which is independent of removal of extracellular glutamate. During apoptotic stimuli, a mitochondrial protein, holocytochrome c synthetase (HCCS), translocates to outside the mitochondria, binds to and suppresses the X-linked inhibitor of apoptosis protein (XIAP), leading to activation of caspase-3. The N-terminus of EAAC1 can bind to HCCS, which interferes with the HCCS-XIAP association, and thereby maintain XIAP activity. This unique anti-apoptotic mechanism of EAAC1 functions in rescuing PC12 cells and motor neurons from NGF deprivation and nerve injury, respectively.

Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection.

To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1beta secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.

Flagellin-deficient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity.

Macrophages from C57BL/6J (B6) mice restrict growth of the intracellular bacterial pathogen Legionella pneumophila. Restriction of bacterial growth requires caspase-1 and the leucine-rich repeat-containing protein Naip5 (Birc1e). We identified mutants of L. pneumophila that evade macrophage innate immunity. All mutants were deficient in expression of flagellin, the primary flagellar subunit, and failed to induce caspase-1-mediated macrophage death. Interestingly, a previously isolated flagellar mutant (fliI) that expresses, but does not assemble, flagellin did not replicate in macrophages, and induced macrophage death. Thus, flagellin itself, not flagella or motility, is required to initiate macrophage innate immunity. Immunity to Legionella did not require MyD88, an essential adaptor for toll-like receptor 5 (TLR5) signaling. Moreover, flagellin of Legionella and Salmonella induced cytotoxicity when delivered to the macrophage cytosol using Escherichia coli as a heterologous host. It thus appears that macrophages sense cytosolic flagellin via a TLR5-independent pathway that leads to rapid caspase-1-dependent cell death and provides defense against intracellular bacterial pathogens.

CATERPILLERs, pyrin and hereditary immunological disorders.

The newly described CATERPILLER family (also known as NOD-LRR or NACHT-LRR) is comprised of proteins with a nucleotide-binding domain and a leucine-rich region. This family has gained rapid prominence because of its demonstrated and anticipated roles in immunity, cell death and growth, and diseases. CATERPILLER proteins are structurally similar to a subgroup of plant-disease-resistance (R) proteins and to the apoptotic protease activating factor 1 (APAF1). They provide positive and negative signals for the control of immune and inflammatory responses, and might represent intracellular sensors of pathogen products. Most importantly, they are genetically linked to several human immunological disorders.

Autophagy and inflammatory cell death, partners of innate immunity.

By law in the evolutionary jungle, any host defense mechanism that efficiently kills microbes also exerts a strong selective pressure for tolerant variants to emerge. As a consequence, pathogens can be exploited as powerful tools to examine host defense mechanisms. Recent studies of the confrontation between macrophages and the opportunistic pathogen Legionella pneumophila have revealed a regulatory mechanism that may link autophagy to pyroptosis, a type of programmed cell death. Building from the extensive literature on autophagy, cell death, and innate immunity, we propose here a testable model in which the NOD-LRR protein Naip5 dictates whether murine macrophages elevate autophagy or pyroptosis as a barrier to infection.