RESUMO
The inflammatory caspases 1 and 11 are activated in response to different agonists and act independently to induce pyroptosis. In the context of IL-1ß/IL-18 secretion, however, in vitro studies indicate that caspase-11 acts upstream of NLRP3 and caspase-1. By contrast, studying infection in vivo by the cytosol-invasive bacterium Burkholderia thailandensis, we find that caspase-1 activity is required upstream of caspase-11 to control infection. Caspase-1-activated IL-18 induces IFN-γ to prime caspase-11 and rapidly clear B. thailandensis infection. In the absence of IL-18, bacterial burdens persist, eventually triggering other signals that induce IFN-γ. Whereas IFN-γ was essential, endogenous type I interferons were insufficient to prime caspase-11. Although mice transgenic for caspase-4, the human ortholog of caspase-11, cleared B. thailandensis in vivo, they did not strictly require IFN-γ priming. Thus, caspase-1 provides priming signals upstream of caspase-11 but not caspase-4 during murine defense against a cytosol-invasive bacterium.
Assuntos
Burkholderia/imunologia , Caspase 1/metabolismo , Caspases/metabolismo , Citosol/microbiologia , Inflamassomos/metabolismo , Interferon gama/metabolismo , Interleucina-18/metabolismo , Animais , Caspases Iniciadoras/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Transdução de SinaisRESUMO
The immunostimulatory properties conferred by vaccine adjuvants require caspase-1 for processing of IL-1ß and IL-18. Caspase-1 is activated in response to a breach of the cytosolic compartment by microbes and the process is initiated by intracellular pattern recognition receptors within inflammasomes. Listeria monocytogenes is detected in the cytosol by the NLRC4, NLRP3 and AIM2 inflammasomes. NLRC4 is activated by flagellin, and L. monocytogenes evades NLRC4 by repressing flagellin expression. We generated an L. monocytogenes strain that was forced to express flagellin in the host cell cytosol. This strain hyperactivated caspase-1 and was preferentially cleared via NLRC4 detection in an IL-1ß/IL-18 independent manner. We also created a strain of L. monocytogenes with forced expression of another NLRC4 agonist, PrgJ, from the Type III secretion system of Salmonella typhimurium. Forced expression of flagellin or PrgJ resulted in attenuation, yet both strains conferred protective immunity in mice against lethal challenge with L. monocytogenes. This work is the first demonstration of specific targeting of the caspase-1 activation pathway to generate a safe and potent L. monocytogenes-based vaccine. Moreover, the attenuated strains with embedded flagellin or PrgJ adjuvants represent attractive vectors for vaccines aimed at eliciting T-cell responses.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Vacinas Bacterianas , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 1/metabolismo , Listeria monocytogenes/imunologia , Listeria monocytogenes/metabolismo , Adjuvantes Imunológicos , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sistemas de Secreção Bacterianos/genética , Vacinas Bacterianas/imunologia , Células Cultivadas , Flagelina/imunologia , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Salmonella typhimurium/imunologiaRESUMO
Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of interleukin 1ß (IL-1ß) and IL-18. Although infection with wild-type Salmonella typhimurium is lethal to mice, we show here that a strain that persistently expresses flagellin was cleared by the cytosolic flagellin-detection pathway through the activation of caspase-1 by the NLRC4 inflammasome; however, this clearance was independent of IL-1ß and IL-18. Instead, caspase-1-induced pyroptotic cell death released bacteria from macrophages and exposed the bacteria to uptake and killing by reactive oxygen species in neutrophils. Similarly, activation of caspase-1 cleared unmanipulated Legionella pneumophila and Burkholderia thailandensis by cytokine-independent mechanisms. This demonstrates that activation of caspase-1 clears intracellular bacteria in vivo independently of IL-1ß and IL-18 and establishes pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.
Assuntos
Apoptose/imunologia , Caspase 1/imunologia , Imunidade Inata/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Animais , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imuno-Histoquímica , Inflamassomos/imunologia , Inflamassomos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Pathogens are detected by pattern recognition receptors that, upon activation, orchestrate an appropriate immune response. The TLRs and the nucleotide-binding oligomerization domain-like receptors (NLRs) are prototypic pattern recognition receptors that detect extracellular and cytosolic pathogens, respectively. Listeria monocytogenes has both extracellular and cytosolic phases and is detected in the cytosol by members of the NLR family. These include two NLR members, NLRC4 and NLRP3, that, upon detection of cytosolic L. monocytogenes, induce the assembly of the inflammasome. Inflammasomes serve as platforms for the activation of the protease caspase 1, which mediates the processing and secretion of pro-IL-1beta and pro-IL-18. We previously provided evidence that L. monocytogenes is also detected by a third inflammasome. We now use biochemical and genetic approaches to demonstrate that the third detector senses bacterial DNA and identify it as Aim2, a receptor that has previously been shown to detect viral DNA.
Assuntos
DNA Bacteriano/imunologia , Listeria monocytogenes/imunologia , Macrófagos/imunologia , Proteínas Nucleares/imunologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Citosol/imunologia , Citosol/microbiologia , Proteínas de Ligação a DNA , Ensaio de Imunoadsorção Enzimática , Interações Hospedeiro-Patógeno , Interleucina-1beta/metabolismo , Células L , Listeria monocytogenes/genética , Listeria monocytogenes/fisiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Nucleares/genética , Fagossomos/imunologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Interferência de RNARESUMO
The mammalian innate immune system uses Toll-like receptors (TLRs) and Nod-LRRs (NLRs) to detect microbial components during infection. Often these molecules work in concert; for example, the TLRs can stimulate the production of the proforms of the cytokines IL-1beta and IL-18, whereas certain NLRs trigger their subsequent proteolytic processing via caspase 1. Gram-negative bacteria use type III secretion systems (T3SS) to deliver virulence factors to the cytosol of host cells, where they modulate cell physiology to favor the pathogen. We show here that NLRC4/Ipaf detects the basal body rod component of the T3SS apparatus (rod protein) from S. typhimurium (PrgJ), Burkholderia pseudomallei (BsaK), Escherichia coli (EprJ and EscI), Shigella flexneri (MxiI), and Pseudomonas aeruginosa (PscI). These rod proteins share a sequence motif that is essential for detection by NLRC4; a similar motif is found in flagellin that is also detected by NLRC4. S. typhimurium has two T3SS: Salmonella pathogenicity island-1 (SPI1), which encodes the rod protein PrgJ, and SPI2, which encodes the rod protein SsaI. Although PrgJ is detected by NLRC4, SsaI is not, and this evasion is required for virulence in mice. The detection of a conserved component of the T3SS apparatus enables innate immune responses to virulent bacteria through a single pathway, a strategy that is divergent from that used by plants in which multiple NB-LRR proteins are used to detect T3SS effectors or their effects on cells. Furthermore, the specific detection of the virulence machinery permits the discrimination between pathogenic and nonpathogenic bacteria.
Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Infecções Bacterianas/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Caspase 1/imunologia , Imunidade Inata/imunologia , Proteínas de Membrana/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação ao Cálcio/genética , Caspase 1/metabolismo , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Conformação Proteica , TransfecçãoRESUMO
Listeria monocytogenes escapes from the phagosome of macrophages and replicates within the cytosolic compartment. The macrophage responds to L. monocytogenes through detection pathways located on the cell surface (TLRs) and within the cytosol (Nod-like receptors) to promote inflammatory processes aimed at clearing the pathogen. Cytosolic L. monocytogenes activates caspase 1, resulting in post-translational processing of the cytokines IL-1beta and IL-18 as well as caspase 1-dependent cell death (pyroptosis). We demonstrate that the presence of L. monocytogenes within the cytosolic compartment induces caspase 1 activation through multiple Nod-like receptors, including Ipaf and Nalp3. Flagellin expression by cytosolic L. monocytogenes was detected through Ipaf in a dose-dependent manner. Concordantly, detection of flagellin promoted bacterial clearance in a murine infection model. Finally, we provide evidence that suggests cytosolic L. monocytogenes activates caspase 1 through a third pathway, which signals through the adaptor protein ASC. Thus, L. monocytogenes activates caspase 1 in macrophages via multiple pathways, all of which detect the presence of bacteria within the cytosol.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Proteínas do Citoesqueleto/metabolismo , Listeria monocytogenes/imunologia , Listeriose/enzimologia , Listeriose/imunologia , Animais , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Transporte/imunologia , Proteínas do Citoesqueleto/imunologia , Citosol/microbiologia , Ativação Enzimática , Flagelina/imunologia , Flagelina/metabolismo , Interleucina-18/biossíntese , Interleucina-18/imunologia , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Listeria monocytogenes/metabolismo , Listeriose/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fagossomos/imunologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismoRESUMO
The innate immune system encodes cytosolic Nod-like receptors (NLRs), several of which activate caspase 1 processing and IL-1beta and IL-18 secretion. Macrophages respond to Salmonella typhimurium infection by activating caspase 1 through the NLR Ipaf. This activation is mediated by cytosolic flagellin through the activity of the virulence-associated type III secretion system (T3SS). We demonstrate here that Pseudomonas aeruginosa activates caspase 1 and induces IL-1beta secretion in infected macrophages. While live, virulent P. aeruginosa activate IL-1beta secretion through caspase 1 and Ipaf, strains that have mutations in the T3SS or in flagellin did not. Ipaf-dependent caspase 1 activation could be recapitulated by delivering P. aeruginosa flagellin to the macrophage cytosol. We examined the role of Naip5 in P. aeruginosa-induced caspase 1 activation by using A/J (Naip5-deficient) compared with C57BL/6 and BALB/c (Naip5-sufficient) macrophages and observed that A/J macrophages secrete IL-1beta in response to P. aeruginosa, S. typhimurium, and Listeria monocytogenes infection, as well as in response to cytosolic flagellin, but at slightly reduced levels. Thus, Ipaf-dependent detection of cytosolic flagellin is a conserved mechanism by which macrophages detect the presence of pathogens that use T3SS.