Exposure to particulate crystals can induce oxidative stress in phagocytes, which triggers NLRP3 inflammasome-mediated interleukin 1β (IL-1β) secretion to initiate undesirable inflammatory responses that are associated with both autoinflammatory and metabolic diseases. Although mitochondrial reactive oxygen species (ROS) play a central role in NLRP3 inflammasome activation, how ROS signal assembly of the NLRP3 inflammasome remains elusive. Here, we identify liposomes as novel activators of NLRP3 inflammasome and further demonstrate that liposome-induced inflammasome activation also requires mitochondrial ROS. Moreover, we found that stimulation with liposomes/crystals induced ROS-dependent calcium influx via the TRPM2 channel and that macrophages deficient in TRPM2 displayed drastically impaired NLRP3 inflammasome activation and IL-1β secretion. Consistently, Trpm2−/− mice were resistant to crystal-/liposome-induced IL-1β-mediated peritonitis in vivo. Together, these results identify TRPM2 as a key player that links oxidative stress to the NLRP3 inflammasome activation. Therefore, targeting TRPM2 may be effective for the treatment of NLRP3 inflamamsome-associated inflammatory disorders.
Pseudomonas aeruginosa (P. aeruginosa) infections are associated with considerable morbidity and mortality in immunocompromised patients due to antibiotic resistance. Therefore, we investigated the efficacy of the anti-P. aeruginosa serotype O11 lipopolysaccharide monoclonal antibody Panobacumab in a clinically relevant murine model of neutropenia induced by cyclophosphamide and in combination with meropenem in susceptible and meropenem resistant P. aeruginosa induced pneumonia. We observed that P. aeruginosa induced pneumonia was dramatically increased in neutropenic mice compared to immunocompetent mice. First, Panobacumab significantly reduced lung inflammation and enhanced bacterial clearance from the lung of neutropenic host. Secondly, combination of Panobacumab and meropenem had an additive effect. Third, Panobacumab retained activity on a meropenem resistant P. aeruginosa strain. In conclusion, the present data established that Panobacumab contributes to the clearance of P. aeruginosa in neutropenic hosts as well as in combination with antibiotics in immunocompetent hosts. This suggests beneficial effects of co-treatment even in immunocompromised individuals, suffering most of the morbidity and mortality of P. aeruginosa infections.
NLRPs (Nucleotide-binding domain, leucine-rich repeat and pyrin domain containing proteins) are a family of pattern-recognition receptors (PRRs) that sense intracellular microbial components and endogenous stress signals. NLRP10 (also known as PYNOD) is a unique NLRP member characterized by a lack of the putative ligand-binding leucine-rich repeat domain. Recently, human NLRP10 has been shown to inhibit the self-association of ASC into aggregates and ASC-mediated procaspase-1 processing. However, such activities are not found in mouse NLRP10. Here we report the solution structure and dynamics of human NLRP10 pyrin domain (PYD), whose helix H3 and loop H2–H3 adopt a conformation distinct from those of mouse NLRP10. Docking studies show that human and mouse NLRP10 PYDs may interact differently with ASC PYD. These results provide a possible structural explanation for the contrasting effect of NLRP10 on ASC aggregation in human cells versus mouse models. Finally, we also provide evidence that in human NLRP10 the PYD domain may not interact with the NOD domain to regulate its intrinsic nucleotide hydrolysis activity.
Francisella tularensis is a Gram-negative bacterium and the causative agent of the disease tularemia. Escape of F. tularensis from the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1β (IL-1β) and IL-18, which play a crucial role in innate immune responses to F. tularensis. We have identified the 5-formyltetrahydrofolate cycloligase gene (FTL_0724) as being important for F. tularensis live vaccine strain (LVS) virulence. Infection of mice in vivo with a F. tularensis LVS FTL_0724 mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. The FTL_0724 mutant also induced increased inflammasome-dependent IL-1β and IL-18 secretion and cytotoxicity in macrophages in vitro. In contrast, infection of macrophages with a F. tularensis LVS rluD pseudouridine synthase (FTL_0699) mutant resulted in diminished IL-1β and IL-18 secretion from macrophages in vitro compared to infection of macrophages with wild-type LVS. In addition, the FTL_0699 mutant was not attenuated in vivo. These findings further illustrate that F. tularensis LVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogen in vivo.
Listeria monocytogenes is a food-borne pathogen which causes mild to life threatening disease in humans. Ingestion of contaminated food delivers the pathogen to the gastrointestinal tract, where it crosses the epithelial barrier and spreads to internal organs. Type I interferons (IFN-I) are produced during infection and decrease host resistance after systemic delivery of L. monocytogenes. Here we show that mice benefit from IFN-I production following infection with L. monocytogenes via the gastrointestinal route. Intragastric infection lead to increased lethality of IFN-I receptor chain 1-deficient (Ifnar1−/−) animals and to higher bacterial numbers in liver and spleen. Compared to infection from the peritoneum, bacteria infecting via the intestinal tract localized more often to periportal and pericentral regions of the liver and less frequently to the margins of liver lobes. Vigorous replication of intestine-borne L. monocytogenes in the livers of Ifnar1−/− mice 48 h post infection was accompanied by the formation of large inflammatory infiltrates in this organ and massive death of surrounding hepatocytes. This was not observed in Ifnar1−/− mice after intraperitoneal infection. The inflammatory response to infection is shaped by alterations in splenic cytokine production, particularly IFNγ, which differs after intragastric versus intraperitoneal infection. Taken together, our data suggest that the adverse or beneficial role of a cytokine may vary with the route of infection and that IFN-I are not harmful when infection with L. monocytogenes occurs via the natural route.
Nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) are cytosolic receptors that initiate immune responses to sterile and infectious insults to the host. Studies have demonstrated that Nlrp3 is critical for the control of Candida albicans infections and in the generation of anti-fungal Th17 responses. Here we show that the NLR family member Nlrp10 also plays a unique role in the control of disseminated C. albicans infection in vivo. Nlrp10-deficient mice had increased susceptibility to disseminated candidiasis as indicated by decreased survival and increased fungal burdens. In contrast to Nlrp3, Nlrp10-deficiency did not affect innate proinflammatory cytokine production from macrophages and dendritic cells challenged with C. albicans. However, Nlrp10-deficient mice displayed a profound defect in Candida-specific Th1 and Th17 responses. These results demonstrate a novel role for Nlrp10 in the generation of adaptive immune responses to fungal infection.
Endosomal functions are contingent on the integrity of the organelle-limiting membrane, whose disruption induces inflammation and cell death. Here we show that phagocytosis of ultrahigh molecular weight polyethylene particles induces damage to the endosomal-limiting membrane and results in the leakage of cathepsins into the cytosol and NLRP3-inflammasome activation. Annexin A2 recruitment to damaged organelles is shown by two-dimensional DIGE protein profiling, endosomal fractionation, confocal analysis of endogenous and annexin A2–GFP transfected cells, and immunogold labelling. Binding experiments, using fluorescent liposomes, confirms annexin A2 recruitment to endosomes containing phagocytosed polyethylene particles. Finally, an increase in cytosolic cathepsins, NLRP3-inflammasome activation, and IL-1 production is seen in dendritic cells from annexin A2-null mice, following exposure to polyethylene particles. Together, the results indicate a functional role of annexin A2 binding to endosomal membranes following organelle destabilization.
Through pattern recognition receptors the innate immune system detects disruption of the normal function of the organism and initiates responses directed at correcting these derangements. Cellular damage from microbial or non-microbial insults causes the activation of nucleotide-binding domain leucine-rich repeat containing receptors (NLR) in multiprotein complexes called inflammasomes. Here we discuss the role of the NLRP3 inflammasome in the recognition of cellular damage and the initiation of sterile inflammatory responses.
caspase-1; necrosis; danger signals
Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum, human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene (lpda1 [APH_0065]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-κB signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.
Mesenchymal stem cell (MSC)-based tissue regeneration is a promising therapeutic strategy for treating damaged tissues. However, the inflammatory microenvironment that exists at a local injury site might restrict reconstruction. Low-power laser irradiation (LPLI) has been widely applied to retard the inflammatory reaction. The purpose of this study was to investigate the anti-inflammatory effect of LPLI on human adipose-derived stem cells (hADSCs) in an inflammatory environment. We showed that the hADSCs expressed Toll-like Receptors (TLR) 1, TLR2, TLR3, TLR4, and TLR6 and that lipopolysaccharide (LPS) significantly induced the production of pro-inflammatory cytokines (Cyclooxygenase-2 (Cox-2), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Interleukin-8 (IL-8)). LPLI markedly inhibited LPS-induced, pro-inflammatory cytokine expression at an optimal dose of 8 J/cm2. The inhibitory effect triggered by LPLI might occur through an increase in the intracellular level of cyclic AMP (cAMP), which acts to down-regulate nuclear factor kappa B (NF-κB) transcriptional activity. These data collectively provide insight for further investigations of the potential application of anti-inflammatory treatment followed by stem cell therapy.
NLRs (nucleotide-binding domain leucine-rich repeat containing receptors; NOD-like receptors) are a class of pattern recognition receptor (PRR) that respond to host perturbation from either infectious agents or cellular stress1,2. The function of most NLR family members has not been characterized and their role in instructing adaptive immune responses remains unclear 2,3. NLRP10 (also known as PYNOD, NALP10, PAN5 and NOD8) is the only NLR lacking the putative ligand binding leucine rich repeat domain, and has been postulated to be a negative regulator of other NLR members including NLRP34–6. We did not find evidence that NLRP10 functions through an inflammasome to regulate caspase-1 activity nor that it regulates other inflammasomes. Instead, Nlrp10−/− mice had a profound defect in helper T cell-driven immune responses to a diverse array of adjuvants including lipopolysaccharide (LPS), aluminium hydroxide (alum) and complete Freund’s adjuvant (CFA). Adaptive immunity was impaired in the absence of NLRP10 due to a dendritic cell (DC) intrinsic defect in emigration from inflamed tissues while upregulation of DC costimulatory molecules and chemotaxis to CCR7-dependent and independent ligands remained intact. The loss of antigen transport to the draining LN by this migratory DC subset resulted in an almost absolute loss in naïve CD4+ T cell priming, highlighting the critical link between diverse innate immune stimulation, NLRP10 activity and the immune function of mature DCs.
Adjuvants; Alum; EAE; Multiple Sclerosis; Asthma; Complete Freund’s Adjuvant; NOD-like Receptors; Nalp; Caspase-1; Pattern Recognition Receptor
The NLRP3 inflammasome is activated in response to a variety of signals that are indicative of damage to the host including tissue damage, metabolic stress, and infection. Upon activation, the NLRP3 inflammasome serves as a platform for activation of the cysteine protease caspase-1, which leads to the processing and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Dysregulated NLRP3 inflammasome activation is associated with both heritable and acquired inflammatory diseases. Here we review new insights into the mechanism of NLRP3 inflammasome activation and its role in disease pathogenesis.
NLRP3; inflammasome; caspase-1; sterile inflammation
To investigate the respective contributions of TLR versus IL-1R mediated signals in MyD88 dependent control of Mycobacterium tuberculosis, we compared the outcome of M. tuberculosis infection in MyD88, TRIF/MyD88, IL-1R1, and IL-1β-deficient mice. All four strains displayed acute mortality with highly increased pulmonary bacterial burden suggesting a major role for IL-1β signaling in determining the MyD88 dependent phenotype. Unexpectedly, the infected MyD88 and TRIF/MyD88-deficient mice, rather than being defective in IL-1β expression, displayed increased cytokine levels relative to wild-type animals. Similarly, infected mice deficient in caspase-1 and ASC, which have critical functions in inflammasome-mediated IL-1β maturation, showed unimpaired IL-1β production and importantly, were considerably less susceptible to infection than IL-1β deficient mice. Together our findings reveal a major role for IL-1β in host resistance to M. tuberculosis and indicate that during this infection the cytokine can be generated by a mechanism that does not require TLR signaling or caspase-1. The Journal of Immunology, 2010, 184: 3326–3330.
Interleukin-1β (IL-1β) is important for host resistance against Mycobacterium tuberculosis (Mtb) infections. The response of the dendritic cell inflammasome during Mtb infections has not been investigated in detail.
Here we show that Mtb infection of bone marrow-derived dendritic cells (BMDCs) induces IL-1β secretion and that this induction is dependent upon the presence of functional ASC and NLRP3 but not NLRC4 or NOD2. The analysis of cell death induction in BMDCs derived from these knock-out mice revealed the important induction of host cell apoptosis but not necrosis, pyroptosis or pyronecrosis. Furthermore, NLRP3 inflammasome activation and apoptosis induction were both reduced in BMDCs infected with the esxA deletion mutant of Mtb demonstrating the importance of a functional ESX-1 secretion system. Surprisingly, caspase-1/11-deficient BMDCs still secreted residual levels of IL-1βand IL-18 upon Mtb infection which was abolished in cells infected with the esxA Mtb mutant.
Altogether we demonstrate the partially caspase-1/11-independent, but NLRP3- and ASC- dependent IL-1β secretion in Mtb-infected BMDCs. These findings point towards a potential role of DCs in the host innate immune response to mycobacterial infections via their capacity to induce IL-1β and IL-18 secretion.
The mechanisms by which the intracellular pathogen Francisella tularensis evades innate immunity are not well defined. We have identified a gene with homology to Escherichia coli mviN, a putative lipid II flippase, which F. tularensis uses to evade activation of innate immune pathways. Infection of mice with a F. tularensis mviN mutant resulted in improved survival and decreased bacterial burdens compared to infection with wild-type F. tularensis. The mviN mutant also induced increased AIM2 inflammasome-dependent IL-1β secretion and cytotoxicity in macrophages. The compromised in vivo virulence of the mviN mutant depended upon inflammasome activation, as caspase-1- and ASC-deficient mice did not exhibit preferential survival following infection. This study demonstrates that mviN limits F. tularensis-induced AIM2 inflammasome activation which is critical for its virulence in vivo.
Pattern recognition receptors (PRRs) detect conserved microbial structures generally absent from eukaryotes. Bacterial pathogens commonly utilize pore-forming toxins or specialized secretion systems to deliver virulence factors that promote bacterial replication by modulating host cell physiology. Detection of these secretion systems or toxins by nucleotide-binding oligomerization domain leucine-rich-repeat proteins (NLRs) triggers the assembly of multiprotein complexes, termed inflammasomes, necessary for caspase-1 activation. Here we demonstrate that caspase-1 activation in response to the Yersinia type III secretion system (T3SS) requires the adapter ASC, and involves both NLRP3 and NLRC4 inflammasomes. We further identify a Yersinia type III secreted effector protein, YopK, which prevents inflammasome activation by preventing cellular recognition of the T3SS. Inflammasome-mediated sensing of the T3SS promotes bacterial clearance from infected tissues in vivo. These data demonstrate that a class of bacterial proteins interferes with cellular recognition of bacterial secretion systems, which contributes to bacterial survival within host tissues.
The proinflammatory cytokine IL-1β plays an important role in antifungal immunity; however the mechanisms by which fungal pathogens trigger IL-1β secretion are unclear. Here we show that infection with C. albicans is sensed by the Nlrp3 inflammasome resulting in the subsequent release of IL-1β. The ability of C. albicans to switch from a unicellular yeast form into a filamentous form is essential for activation of the Nlrp3 inflammasome as C. albicans mutants incapable of forming hyphae were defective in their ability to induce macrophage IL-1β secretion. Nlrp3-deficient mice also demonstrated increased susceptibility to infection with C. albicans consistent with a key role for Nlrp3 in innate immune responses to the pathogen C. albicans.
The innate immune system senses danger signals via evolutionary conserved receptors. The nucleotide-binding domain leucine-rich repeat containing receptors (NLR) family is a group of intracellular receptors that drive a wide variety of inflammatory responses. A number of the NLR family members can form inflammasomes, which are multiprotein complexes that can activate caspase-1 and ultimately lead to the processing and secretion of interleukin (IL)-1β, IL-18 and IL-33. One of the best-studied members of the NLR family is NLRP3 for which a number of divergent activators have recently been described. These and other studies examining the NLRP3 inflammasome will be discussed in this review.
Inflammasome; NLRP3; Caspase-1; Interleukin-1β
Anthrax lethal toxin (LT) contributes to the immune evasion strategy of B. anthracis by impairing the function of cells of the immune system, such as macrophages and dendritic cells (DCs). Macrophages from certain inbred mice strains undergo rapid death upon LT treatment mediated by caspase-1 activation dependent on Nalp1b, an inflammasome component. Rapid LT-induced death is however not observed in macrophages from human and many mouse strains. Here, we focused on the responses of various murine DCs to LT. Using a variety of knock-out mice, we found that depending on the mouse strain, death of bone marrow derived DCs and macrophages was mediated either by a fast Nalp1b and caspase-1 dependent, or by a slow caspase-1 independent pathway that was triggered by the impairment of MEK1/2 pathways. Caspase-1 independent death was observed in cells of different genetic backgrounds and interestingly occurred only in immature DCs. Maturation, triggered by different types of stimuli, led to full protection of DCs. These studies illustrate that the cellular damage inflicted by LT depends not only on the innate responses but also on the maturation stage of the cell, which modulates the more general caspase-1 independent responses.
The NLR (nucleotide-binding domain leucine-rich repeat containing) family of intracellular sensors is a critical component of the innate immune system. A number of NLR family members can form multiprotein complexes, called inflammasomes, and are capable of activating the cysteine protease caspase-1 in response to a wide range of stimuli including both microbial and self molecules. Caspase-1 activation leads to processing and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18, which play crucial roles in host defense to infectious insults. Dysregulation of the inflammasome has also been linked to a number of autoinflammatory and autoimmune disorders. Recent advances in the inflammasome field will be discussed in this review.
The intraerythrocytic parasite Plasmodium—the causative agent of malaria—produces an inorganic crystal called hemozoin (Hz) during the heme detoxification process, which is released into the circulation during erythrocyte lysis. Hz is rapidly ingested by phagocytes and induces the production of several pro-inflammatory mediators such as interleukin-1β (IL-1β). However, the mechanism regulating Hz recognition and IL-1β maturation has not been identified. Here, we show that Hz induces IL-1β production. Using knockout mice, we showed that Hz-induced IL-1β and inflammation are dependent on NOD-like receptor containing pyrin domain 3 (NLRP3), ASC and caspase-1, but not NLRC4 (NLR containing CARD domain). Furthermore, the absence of NLRP3 or IL-1β augmented survival to malaria caused by P. chabaudi adami DS. Although much has been discovered regarding the NLRP3 inflammasome induction, the mechanism whereby this intracellular multimolecular complex is activated remains unclear. We further demonstrate, using pharmacological and genetic intervention, that the tyrosine kinases Syk and Lyn play a critical role in activation of this inflammasome. These findings not only identify one way by which the immune system is alerted to malarial infection but also are one of the first to suggest a role for tyrosine kinase signaling pathways in regulation of the NLRP3 inflammasome.
Malaria is widespread in the tropical and sub-tropical regions of the world, and is responsible for 2–3 million deaths annually. This disease is caused by parasites of the Plasmodium genus. The parasite feeds on the hemoglobin of red blood cells and generates a metabolic waste called hemozoin (Hz). Hz is released into the blood circulation during the rupture of red blood cells, which coincides with the production of many cytokines such as interleukin-1β (IL-1β), responsible in part for the periodic fever that is characteristic of the malaria disease. Here, we investigated how Hz activates macrophages (cells that engulf foreign material) to produce IL-1β. We found that Hz is taken up by macrophages initiating signals such as the tyrosine kinases Syk and Lyn that communicate to intracellular receptors. We also showed that Hz-induced IL-1β production is dependent on activation of the intracellular receptor NLRP3, the adaptor protein ASC and a protease called caspase-1 that cleaves IL-1β, therefore allowing it to be released from the cells. These findings not only identify one way in which the immune system is alerted to malarial infection but also dissect some of the signaling events triggered by Hz in the NLRP3 inflammasome pathway.
The NOD-like receptor (NLR) family of proteins is involved in the regulation of innate immune responses and cell death pathways. Recent findings show that the NLR family member NLRC4 (also known as IPAF) has important roles in innate immune responses to Gram-negative bacteria. Macrophages infected with Legionella pneumophila, Salmonella typhimurium, Shigella flexneri, or Pseudomonas aeruginosa activate caspase-1 in an NLRC4-dependent manner leading to macrophage cell death and the release of proinflammatory cytokines. This review will discuss these findings as well as the role of bacterial type III and type IV secretion systems and flagellin in NLRC4-mediated caspase-1 activation.
Innate Immunity; NOD-like receptors; NLRC4; Caspase-1; Inflammasome; Macrophage; Flagellin; Type 3 Secretion System; type 4 Secretion System
Hepatocyte death results in a sterile inflammatory response that amplifies the initial insult and increases overall tissue injury. One important example of this type of injury is acetaminophen-induced liver injury, in which the initial toxic injury is followed by innate immune activation. Using mice deficient in Tlr9 and the inflammasome components Nalp3 (NACHT, LRR, and pyrin domain–containing protein 3), ASC (apoptosis-associated speck-like protein containing a CARD), and caspase-1, we have identified a nonredundant role for Tlr9 and the Nalp3 inflammasome in acetaminophen-induced liver injury. We have shown that acetaminophen treatment results in hepatocyte death and that free DNA released from apoptotic hepatocytes activates Tlr9. This triggers a signaling cascade that increases transcription of the genes encoding pro–IL-1β and pro–IL-18 in sinusoidal endothelial cells. By activating caspase-1, the enzyme responsible for generating mature IL-1β and IL-18 from pro–IL-1β and pro–IL-18, respectively, the Nalp3 inflammasome plays a crucial role in the second step of proinflammatory cytokine activation following acetaminophen-induced liver injury. Tlr9 antagonists and aspirin reduced mortality from acetaminophen hepatotoxicity. The protective effect of aspirin on acetaminophen-induced liver injury was due to downregulation of proinflammatory cytokines, rather than inhibition of platelet degranulation or COX-1 inhibition. In summary, we have identified a 2-signal requirement (Tlr9 and the Nalp3 inflammasome) for acetaminophen-induced hepatotoxicity and some potential therapeutic approaches.
Pseudomonas aeruginosa is a Gram-negative bacterium that causes opportunistic infections in immunocompromised individuals. P. aeruginosa employs a type III secretion system to inject effector molecules into the cytoplasm of the host cell. This interaction with the host cell leads to inflammatory responses that eventually result in cell death. We show that infection of macrophages with P. aeruginosa results in activation of caspase-1 in an IPAF-dependent, but flagellin-independent, manner. Macrophages deficient in IPAF or caspase-1 were markedly resistant to P. aeruginosa–induced cell death and release of the proinflammatory cytokine interleukin (IL)-1β. A subset of P. aeruginosa isolates express the effector molecule exoenzyme U (ExoU), which we demonstrate is capable of inhibiting caspase-1–driven proinflammatory cytokine production. This study shows a key role for IPAF and capase-1 in innate immune responses to the pathogen P. aeruginosa, and also demonstrates that virulent ExoU-expressing strains of P. aeruginosa can circumvent this innate immune response.