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Caspase-12 is a caspase family member for which functions in regulating cell death and inflammation have previously been suggested. In this study, we used caspase-12 lacZ reporter mice to elucidate the expression pattern of caspase-12 in order to obtain an idea about its possible in vivo function. Strikingly, these reporter mice showed that caspase-12 is expressed explicitly in Purkinje neurons of the cerebellum. As this observation suggested a function for caspase-12 in Purkinje neurons, we analyzed the brain and behavior of caspase-12 deficient mice in detail. Extensive histological analyses showed that caspase-12 was not crucial for establishing cerebellum structure or for maintaining Purkinje cell numbers. We then performed behavioral tests to investigate whether caspase-12 deficiency affects memory, motor, and psychiatric functions in mice. Interestingly, while the absence of caspase-12 did not affect memory and motor function, caspase-12 deficient mice showed depression and hyperactivity tendencies, together resembling manic behavior. Next, suggesting a possible molecular mechanistic explanation, we showed that caspase-12 deficient cerebella harbored diminished signaling through the brain-derived neurotrophic factor/tyrosine kinase receptor B/cyclic-AMP response binding protein axis, as well as strongly enhanced expression of the neuronal activity marker c-Fos. Thus, our study establishes caspase-12 expression in mouse Purkinje neurons and opens novel avenues of research to investigate the role of caspase-12 in regulating psychiatric behavior.
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Background: Autoinflammation with infantile enterocolitis (AIFEC) is an often fatal disease caused by gain-of-function mutations in the NLRC4 inflammasome. This inflammasomopathy is characterized by macrophage activation syndrome (MAS)-like episodes as well as neonatal-onset enterocolitis. Although elevated IL-18 levels were suggested to take part in driving AIFEC pathology, the triggers for IL-18 production and its ensuing pathogenic effects in these patients are incompletely understood. Methods: Here, we developed and characterized a novel genetic mouse model expressing a murine version of the AIFEC-associated NLRC4V341A mutation from its endogenous Nlrc4 genomic locus. Results: NLRC4V341A expression in mice recapitulated increased circulating IL-18 levels as observed in AIFEC patients. Housing NLRC4V341A-expressing mice in germfree (GF) conditions showed that these systemic IL-18 levels were independent of the microbiota, and unmasked an additional IL-18-inducing effect of NLRC4V341A expression in the intestines. Remarkably, elevated IL-18 levels did not provoke detectable intestinal pathologies in NLRC4V341A-expressing mice, even not upon genetically ablating IL-18 binding protein (IL-18BP), which is an endogenous IL-18 inhibitor that has been used therapeutically in AIFEC. In addition, NLRC4V341A expression did not alter susceptibility to the NLRC4-activating gastrointestinal pathogens Salmonella Typhimurium and Citrobacter rodentium. Conclusion: As observed in AIFEC patients, mice expressing a murine NLRC4V341A mutant show elevated systemic IL-18 levels, suggesting that the molecular mechanisms by which this NLRC4V341A mutant induces excessive IL-18 production are conserved between humans and mice. However, while our GF and infection experiments argue against a role for commensal or pathogenic bacteria, identifying the triggers and mechanisms that synergize with IL-18 to drive NLRC4V341A-associated pathologies will require further research in this NLRC4V341A mouse model.
Asunto(s)
Enterocolitis , Síndrome de Activación Macrofágica , Humanos , Ratones , Recién Nacido , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Mutación , Síndrome de Activación Macrofágica/genética , Enterocolitis/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismoRESUMEN
Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL-1ß secretion and neutrophil cell death remain unclear. Here, we show that neutrophil-targeted expression of the disease-associated gain-of-function Nlrp3A350V mutant suffices for systemic autoinflammatory disease and tissue pathology in vivo. We confirm the activity of the canonical NLRP3 and NLRC4 inflammasomes in neutrophils, and further show that the NLRP1b, Pyrin and AIM2 inflammasomes also promote maturation and secretion of interleukin (IL)-1ß in cultured bone marrow neutrophils. Notably, all tested canonical inflammasomes promote GSDMD cleavage in neutrophils, and canonical inflammasome-induced pyroptosis and secretion of mature IL-1ß are blunted in GSDMD-knockout neutrophils. In contrast, GSDMD is dispensable for PMA-induced NETosis. We also show that Salmonella Typhimurium-induced pyroptosis is markedly increased in Nox2/Gp91Phox -deficient neutrophils that lack NADPH oxidase activity and are defective in PMA-induced NETosis. In conclusion, we establish the canonical inflammasome repertoire in neutrophils and identify differential roles for GSDMD and the NADPH complex in canonical inflammasome-induced neutrophil pyroptosis and mitogen-induced NETosis, respectively.
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Trampas Extracelulares , Inflamasomas , Neutrófilos , Proteínas de Unión a Fosfato , Proteínas Citotóxicas Formadoras de Poros , Piroptosis , Animales , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitógenos/metabolismo , NADP/metabolismo , NADPH Oxidasas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Neutrófilos/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Pirina/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pbio.3000354.].
RESUMEN
The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1ß and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants.
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Síndromes Periódicos Asociados a Criopirina/genética , Furanos/farmacología , Inflamasomas/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Sulfonamidas/farmacología , Animales , Citocinas/antagonistas & inhibidores , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Células HEK293 , Compuestos Heterocíclicos de 4 o más Anillos , Humanos , Indenos , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Dominios Proteicos , SulfonasRESUMEN
The caspase activation and recruitment domain (CARD)-based inflammasome sensors NLRP1b and NLRC4 induce caspase-1-dependent pyroptosis independent of the inflammasome adaptor ASC. Here, we show that NLRP1b and NLRC4 trigger caspase-8-mediated apoptosis as an alternative cell death program in caspase-1-/- macrophages and intestinal epithelial organoids (IECs). The caspase-8 adaptor FADD was recruited to ASC specks, which served as cytosolic platforms for caspase-8 activation and NLRP1b/NLRC4-induced apoptosis. We further found that caspase-1 protease activity dominated over scaffolding functions in suppressing caspase-8 activation and induction of apoptosis of macrophages and IECs. Moreover, TLR-induced c-FLIP expression inhibited caspase-8-mediated apoptosis downstream of ASC speck assembly, but did not affect pyroptosis induction by NLRP1b and NLRC4. Moreover, unlike during pyroptosis, NLRP1b- and NLRC4-elicited apoptosis retained alarmins and the inflammasome-matured cytokines interleukin 1ß (IL-1ß) and IL-18 intracellularly. This work identifies critical mechanisms regulating apoptosis induction by the inflammasome sensors NLRP1b and NLRC4 and suggests converting pyroptosis into apoptosis as a paradigm for suppressing inflammation.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Proteínas de Unión al Calcio/metabolismo , Caspasa 1/metabolismo , Inflamasomas/metabolismo , Piroptosis , Animales , Caspasa 8/metabolismo , Enterocitos/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores Toll-Like/metabolismoRESUMEN
Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1ß and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations.
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Fiebre Mediterránea Familiar/genética , Fiebre Mediterránea Familiar/metabolismo , Inflamasomas/metabolismo , Mutación , Pirina/genética , Pirina/metabolismo , Animales , Toxinas Bacterianas/toxicidad , Proteínas Adaptadoras de Señalización CARD/metabolismo , Infecciones por Clostridium/inmunología , Infecciones por Clostridium/metabolismo , Enterotoxinas/toxicidad , Fiebre Mediterránea Familiar/inmunología , Células HEK293 , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/inmunología , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microtúbulos/efectos de los fármacos , Microtúbulos/inmunología , Microtúbulos/metabolismo , Pirina/inmunología , Tubulina (Proteína)/metabolismoRESUMEN
Intestinal T cells and group 3 innate lymphoid cells (ILC3 cells) control the composition of the microbiota and gut immune responses. Within the gut, ILC3 subsets coexist that either express or lack the natural cytoxicity receptor (NCR) NKp46. We identified here the transcriptional signature associated with the transcription factor T-bet-dependent differentiation of NCR(-) ILC3 cells into NCR(+) ILC3 cells. Contrary to the prevailing view, we found by conditional deletion of the key ILC3 genes Stat3, Il22, Tbx21 and Mcl1 that NCR(+) ILC3 cells were redundant for the control of mouse colonic infection with Citrobacter rodentium in the presence of T cells. However, NCR(+) ILC3 cells were essential for cecal homeostasis. Our data show that interplay between intestinal ILC3 cells and adaptive lymphocytes results in robust complementary failsafe mechanisms that ensure gut homeostasis.
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Inmunidad Innata , Interleucinas/biosíntesis , Linfocitos/inmunología , Linfocitos/metabolismo , Animales , Citrobacter rodentium/inmunología , Análisis por Conglomerados , Modelos Animales de Enfermedad , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/metabolismo , Infecciones por Enterobacteriaceae/mortalidad , Infecciones por Enterobacteriaceae/patología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Homeostasis , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/deficiencia , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , Transducción de Señal , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Transcriptoma , Interleucina-22RESUMEN
BACKGROUND: NOD-like receptors (Nlrs) are key regulators of immune responses during infection and autoimmunity. A subset of Nlrs assembles inflammasomes, molecular platforms that are activated in response to endogenous danger and microbial ligands and that control release of interleukin (IL)-1ß and IL-18. However, their role in response to injury in the nervous system is less understood. METHODS: In this study, we investigated the expression profile of major inflammasome components in the peripheral nervous system (PNS) and explored the physiological role of different Nlrs upon acute nerve injury in mice. RESULTS: While in basal conditions, predominantly members of NOD-like receptor B (Nlrb) subfamily (NLR family, apoptosis inhibitory proteins (NAIPs)) and Nlrc subfamily (ICE-protease activating factor (IPAF)/NOD) are detected in the sciatic nerve, injury causes a shift towards expression of the Nlrp family. Sterile nerve injury also leads to an increase in expression of the Nlrb subfamily, while bacteria trigger expression of the Nlrc subfamily. Interestingly, loss of Nlrp6 led to strongly impaired nerve function upon nerve crush. Loss of the inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) and effector caspase-1 and caspase-11 did not affect sciatic nerve function, suggesting that Nlrp6 contributed to recovery after peripheral nerve injury independently of inflammasomes. In line with this, we did not detect release of mature IL-1ß upon acute nerve injury despite potent induction of pro-IL-1ß and inflammasome components Nlrp3 and Nlrp1. However, Nlrp6 deficiency was associated with increased pro-inflammatory extracellular regulated MAP kinase (ERK) signaling, suggesting that hyperinflammation in the absence of Nlrp6 exacerbated peripheral nerve injury. CONCLUSIONS: Together, our observations suggest that Nlrp6 contributes to recovery from peripheral nerve injury by dampening inflammatory responses independently of IL-1ß and inflammasomes.
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Inflamasomas/genética , Enfermedades del Sistema Nervioso Periférico/patología , Receptores de Superficie Celular/genética , Animales , Conducta Animal , Interleucina-1beta/genética , Lipopolisacáridos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Compresión Nerviosa , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/psicología , Receptores de Superficie Celular/deficiencia , Recuperación de la Función , Nervio Ciático/patologíaRESUMEN
The rapid rise of multi-drug-resistant bacteria is a global healthcare crisis, and new antibiotics are urgently required, especially those with modes of action that have low-resistance potential. One promising lead is the liposaccharide antibiotic moenomycin that inhibits bacterial glycosyltransferases, which are essential for peptidoglycan polymerization, while displaying a low rate of resistance. Unfortunately, the lipophilicity of moenomycin leads to unfavourable pharmacokinetic properties that render it unsuitable for systemic administration. In this study, we show that using moenomycin and other glycosyltransferase inhibitors as templates, we were able to synthesize compound libraries based on novel pyranose scaffold chemistry, with moenomycin-like activity, but with improved drug-like properties. The novel compounds exhibit in vitro inhibition comparable to moenomycin, with low toxicity and good efficacy in several in vivo models of infection. This approach based on non-planar carbohydrate scaffolds provides a new opportunity to develop new antibiotics with low propensity for resistance induction.
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Antibacterianos/síntesis química , Glicosiltransferasas/antagonistas & inhibidores , Oligosacáridos/química , Animales , Antibacterianos/uso terapéutico , Femenino , Humanos , Mastitis/tratamiento farmacológico , Ratones , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Staphylococcus aureusRESUMEN
Over recent years, inflammasomes have emerged as key regulators of immune and inflammatory responses. They induce programmed cell death and direct the release of danger signals and the inflammatory cytokines interleukin (IL)-1ß and IL-18. The concerted actions of inflammasomes are of utmost importance for responding adequately to harmful environmental agents and infections. However, deregulated inflammasome signaling is increasingly linked to a diversity of human pathologies, including rheumatoid arthritis, inflammatory bowel disease, and rare, hereditary periodic fever syndromes. In this review, we discuss recent insight in the protective and detrimental roles of inflammasomes in selected infectious, autoinflammatory and autoimmune diseases, and cover clinically approved therapies that interfere with inflammasome signaling. These findings highlight the importance of fine-balancing the Ying and Yang activities of inflammasomes for sustained homeostasis and suggest that further understanding of inflammasome mechanisms may offer new cures for human diseases.
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Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinflamatorias Hereditarias/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Animales , Enfermedades Autoinmunes/etiología , Interacciones Huésped-Patógeno/inmunología , Humanos , Inflamación/etiologíaRESUMEN
The detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined with regard to the molecular and cellular components contributing to this defense mechanism in mammals and importance during infection. Here, we reveal the central role of the IL-1ß signaling axis and Gr1+ cells in controlling the Escherichia coli burden in the blood in response to the sensing of the Rho GTPase-activating toxin CNF1. Consistently, this innate immune response is abrogated in caspase-1/11-impaired mice or following the treatment of infected mice with an IL-1ß antagonist. In vitro experiments further revealed the synergistic effects of CNF1 and LPS in promoting the maturation/secretion of IL-1ß and establishing the roles of Rac, ASC and caspase-1 in this pathway. Furthermore, we found that the α-hemolysin toxin inhibits IL-1ß secretion without affecting the recruitment of Gr1+ cells. Here, we report the first example of anti-virulence-triggered immunity counteracted by a pore-forming toxin during bacteremia.
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Toxinas Bacterianas/inmunología , Infecciones por Escherichia coli/inmunología , Proteínas de Escherichia coli/inmunología , Proteínas Hemolisinas/inmunología , Inmunidad Innata/inmunología , Transducción de Señal/inmunología , Animales , Bacteriemia/inmunología , Modelos Animales de Enfermedad , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Femenino , Interacciones Huésped-Patógeno/inmunología , Interleucina-1beta/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Virulencia , Factores de Virulencia/inmunologíaRESUMEN
Caspase-11 contributes to host defense against Gram-negative bacterial pathogens by inducing an inflammatory form of programmed cell death in infected cells. Lipopolysaccharides (LPS) have been identified as the microbial agents that stimulate caspase-11 activation; however, the mechanism of LPS detection has been unknown. In a recent study, Shao and colleagues demonstrate that caspase-11 and its human homologues, caspases -4 and -5, unexpectedly act as direct sensors of cytosolic LPS.
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Bacterias Gramnegativas/inmunología , Interacciones Huésped-Patógeno , Macrófagos/inmunología , Modelos Inmunológicos , Animales , Bacterias Gramnegativas/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologíaRESUMEN
Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1.
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Infecciones por Escherichia coli/inmunología , Inmunidad Innata , Interleucina-1beta/inmunología , Glándulas Mamarias Animales/inmunología , Mastitis/inmunología , Infecciones Estafilocócicas/inmunología , Animales , Caspasa 1/deficiencia , Caspasa 1/genética , Caspasa 1/inmunología , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Escherichia coli/inmunología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Femenino , Regulación de la Expresión Génica , Interleucina-1alfa/genética , Interleucina-1alfa/inmunología , Interleucina-1beta/deficiencia , Interleucina-1beta/genética , Glándulas Mamarias Animales/microbiología , Glándulas Mamarias Animales/patología , Mastitis/genética , Mastitis/microbiología , Mastitis/patología , Ratones , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/inmunología , Transducción de Señal , Especificidad de la Especie , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , Staphylococcus aureus/inmunologíaRESUMEN
RATIONALE: Sepsis is one of the leading causes of death around the world. The failure of clinical trials to treat sepsis demonstrates that the molecular mechanisms are multiple and are still insufficiently understood. OBJECTIVES: To clarify the long disputed hierarchical contribution of several central inflammatory mediators (IL-1ß, IL-18, caspase [CASP] 7, CASP1, and CASP11) in septic shock and to explore their therapeutic potential. METHODS: LPS- and tumor necrosis factor (TNF)-induced lethal shock, and cecal ligation and puncture (CLP) were performed in genetically or pharmacologically targeted mice. Body temperature and survival were monitored closely, and plasma was analyzed for several markers of cellular disintegration and inflammation. MEASUREMENTS AND MAIN RESULTS: Interestingly, deficiency of both IL-1ß and IL-18 additively prevented LPS-induced mortality. The detrimental role of IL-1ß and IL-18 was confirmed in mice subjected to a lethal dose of TNF, or to a lethal CLP procedure. Although their upstream activator, CASP1, and its amplifier, CASP11, are considered potential therapeutic targets because of their crucial involvement in endotoxin-induced toxicity, CASP11- or CASP1/11-deficient mice were not, or hardly, protected against a lethal TNF or CLP challenge. In line with our results obtained in genetically deficient mice, only the combined neutralization of IL-1 and IL-18, using the IL-1 receptor antagonist anakinra and anti-IL-18 antibodies, conferred complete protection against endotoxin-induced lethality. CONCLUSIONS: Our data point toward the therapeutic potential of neutralizing IL-1 and IL-18 simultaneously in sepsis, rather than inhibiting the upstream inflammatory caspases.
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Antiinflamatorios/uso terapéutico , Autoanticuerpos/uso terapéutico , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Interleucina-18/deficiencia , Interleucina-1beta/deficiencia , Choque Séptico/prevención & control , Animales , Biomarcadores/sangre , Caspasa 1/sangre , Caspasa 1/deficiencia , Caspasa 7/sangre , Caspasa 7/deficiencia , Caspasas/sangre , Caspasas/deficiencia , Caspasas Iniciadoras , Ciego/cirugía , Quimioterapia Combinada , Interleucina-18/antagonistas & inhibidores , Interleucina-18/sangre , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/sangre , Lipopolisacáridos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Choque Séptico/sangre , Choque Séptico/etiología , Factor de Necrosis Tumoral alfaRESUMEN
Bovine mastitis undermines udder health, jeopardizes milk production, and entails prohibitive costs, estimated at $2 billion per year in the dairy industry of the United States. Despite intensive research, the dairy industry has not managed to eradicate the 3 major bovine mastitis-inducing pathogens: Staphylococcus aureus, Streptococcus uberis, and Escherichia coli. In this study, the antimicrobial efficacy of a newly formulated biphenomycin compound (AIC102827) was assessed against intramammary Staph. aureus, Strep. uberis, and E. coli infections, using an experimental mouse mastitis model. Based on its effective and protective doses, AIC102827 applied into the mammary gland was most efficient to treat Staph. aureus, but also adequately reduced growth of Strep. uberis or E. coli, indicating its potential as a broad-spectrum candidate to treat staphylococcal, streptococcal, and coliform mastitis in dairy cattle.
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Antibacterianos/administración & dosificación , Escherichia coli/efectos de los fármacos , Mastitis/veterinaria , Péptidos Cíclicos/administración & dosificación , Staphylococcus aureus/efectos de los fármacos , Streptococcus/efectos de los fármacos , Animales , Bovinos , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/veterinaria , Femenino , Glándulas Mamarias Animales/efectos de los fármacos , Mastitis/tratamiento farmacológico , Mastitis/microbiología , Mastitis Bovina/tratamiento farmacológico , Ratones , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/veterinaria , Infecciones Estreptocócicas/tratamiento farmacológico , Infecciones Estreptocócicas/veterinariaAsunto(s)
Caspasas/genética , Mutación/genética , Sepsis/genética , Animales , Caspasas Iniciadoras , Ratones , Proyectos de InvestigaciónRESUMEN
Neutrophils are essential for the innate immune response against bacterial pathogens and play a key role during the early phases of infection, including mastitis and endometritis in cows. When directly challenged with bacteria, neutrophils undergo phagocytosis induced cell death (PICD). The molecular mechanisms of this cell death modality are poorly understood, especially for bovine neutrophils. Therefore, this study aimed to determine the mechanisms and hallmarks of PICD in bovine neutrophils after in vitro challenge with Escherichia coli (E. coli). Our data show that various apoptotic hallmarks such as blebbing, chromatin condensation and executioner caspase (C)-3/-7 activity are only observed during constitutive bovine neutrophil apoptosis. In contrast, bovine neutrophil PICD is characterized by production of reactive oxygen species (ROS), pro-inflammatory C-1 activation, nuclear factor (NF)-κB activation, and interleukin (IL)-1ß and IL-6 secretion. Nevertheless, under both conditions these phagocytes undergo cell death with the exposure of phosphatidylserine (PS). Although PS exposure is generally attributed to the anti-inflammatory features of executioner caspase-dependent apoptosis, it surprisingly preceded plasma membrane rupture during bovine neutrophil PICD. Moreover, C-1 inhibition strongly affected IL-1ß production but not the PICD kinetics. This indicates that the secretion of the latter pro-inflammatory cytokine is a bystander effect rather than a regulator of PICD in bovine neutrophils, in marked contrast to the IL-1ß-dependent pyroptosis reported for macrophages.
Asunto(s)
Caspasas/fisiología , Escherichia coli/patogenicidad , Neutrófilos/inmunología , Fosfatidilserinas/fisiología , Animales , Apoptosis , Caspasa 1/fisiología , Caspasa 3/fisiología , Caspasa 7/fisiología , Bovinos , Interleucina-1beta/biosíntesis , Interleucina-6/biosíntesis , FN-kappa B/metabolismo , Activación Neutrófila , Neutrófilos/fisiología , Fagocitosis , Poli(ADP-Ribosa) Polimerasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Enteric pathogens represent a major cause of morbidity and mortality worldwide. Toll-like receptor (TLR) and inflammasome signaling are critical for host responses against these pathogens, but how these pathways are integrated remains unclear. Here, we show that TLR4 and the TLR adaptor TRIF are required for inflammasome activation in macrophages infected with the enteric pathogens Escherichia coli and Citrobacter rodentium. In contrast, TLR4 and TRIF were dispensable for Salmonella typhimurium-induced caspase-1 activation. TRIF regulated expression of caspase-11, a caspase-1-related protease that is critical for E. coli- and C. rodentium-induced inflammasome activation, but dispensable for inflammasome activation by S. typhimurium. Thus, TLR4- and TRIF-induced caspase-11 synthesis is critical for noncanonical Nlrp3 inflammasome activation in macrophages infected with enteric pathogens.