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Fish erythrocytes remain nucleated, unlike mammalian erythrocytes that undergo enucleation during maturation. Besides oxygen transport, fish erythrocytes are capable of several immune defence processes and thus these cells are candidates for carrying out ETotic responses. ETosis is an evolutionarily conserved innate immune defence process found in both vertebrates and invertebrates, which involves the extrusion of DNA studded with antimicrobial effector proteins into the extracellular space that traps and kills microorganisms. In this present report, we demonstrate that erythrocytes from Danio rerio (zebrafish) produce ETotic-like responses when exposed to both chemical and physiological inducers of ETosis. Furthermore, erythrocytes from Salmo salar (Atlantic salmon) behaved in a similar way. We have termed these ET-like formations, as Fish Erythrocyte Extracellular Traps (FEETs). Several inducers of mammalian ETosis, such as the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin, induced FEETs. Moreover, we found that FEETs depend on the activation of PKC and generation of mitochondrial reactive oxygen species (mROS). This present report is the first demonstration that fish erythrocytes can exhibit ETotic-like responses, unveiling a previously unknown function, which sheds new light on the innate immune arsenal of these cells.
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Trampas Extracelulares , Animales , Pez Cebra , Eritrocitos/metabolismo , MamíferosRESUMEN
BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin E2 (PGE2 ). However, the respective roles for the PGE2 receptors EP2 and EP4 (both share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered. METHODS: The roles of PGE2 receptors EP2 and EP4 on ILC2-mediated lung inflammation were investigated using genetically modified mouse lines and pharmacological approaches in IL-33-induced lung allergy model. The effects of PGE2 receptors and downstream signals on ILC2 metabolic activation and effector function were examined using in vitro cell cultures. RESULTS: Deficiency of EP2 rather than EP4 augments IL-33-induced mouse lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 and EP2 or inhibition of phosphodiesterase markedly restricts IL-33-induced lung ILC2 responses. Mechanistically, PGE2 directly suppresses IL-33-dependent ILC2 activation through the EP2/EP4-cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL-33-dependent ILC2 responses in mice where endogenous PG synthesis or EP2 signaling is blocked but not in mice with intact PGE2 -EP2 signaling. CONCLUSION: We have defined a mechanism for optimal suppression of mouse lung ILC2 responses by endogenous PGE2 -EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE2 -EP4-cAMP and energy metabolic pathways may provide novel opportunities for treating the ILC2-initiated lung inflammation in asthma and NERD.
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Asma , Inmunidad Innata , Ratones , Animales , Interleucina-33/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/genética , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Linfocitos/metabolismo , Dinoprostona/metabolismo , Pulmón/metabolismoRESUMEN
Cyclin-dependent kinase (CDK) inhibitor drugs (CDKi), such as R-roscovitine and AT7519, induce neutrophil apoptosis in vitro and enhance the resolution of inflammation in a number of in vivo models. This class of compounds are potential novel therapeutic agents that could promote the resolution of acute and chronic inflammatory conditions where neutrophil activation contributes to tissue damage and aberrant tissue repair. In this study we investigated CDKi effects on macrophage pro-inflammatory mediator production and viability. Treatment of human monocyte-derived macrophages (MDMs) with the CDKi AT7519 and R-roscovitine at concentrations that induce neutrophil apoptosis had no significant effect on control or LPS-activated MDM apoptosis and viability, and did not detrimentally affect MDM efferocytosis of apoptotic cells. In addition, enhanced efferocytosis, induced by the glucocorticoid dexamethasone, was also unaffected after a short time treatment with R-roscovitine. Macrophage cytokine responses to inflammatory stimuli are also of importance during inflammation and resolution. As a key target of CDKi, CDK9, is involved in protein transcription via the RNA polymerase II complex, we investigated the effect of CDKi drugs on cytokine production. Our data show that treatment with AT7519 significantly downregulated expression and release of key MDM cytokines IL-6, TNF, IL-10 and IL-1ß, as well as markers of pro-inflammatory macrophage polarisation. R-Roscovitine was also able to downregulate inflammatory cytokine protein secretion from MDMs. Using siRNA transfection, we demonstrate that genetic knock-down of CDK9 replicates these findings, reducing expression and release of pro-inflammatory cytokines. Furthermore, overexpression of CDK9 in THP-1 cells can promote a pro-inflammatory phenotype in these cells, suggesting that CDK9 plays an important role in the inflammatory phenotype of macrophages. Overall, this study demonstrates that pharmacological and genetic targeting of CDK9 inhibits an inflammatory phenotype in human MDMs. As such these data indicate that CDK9 may be key to therapeutically targeting pro-inflammatory macrophage functions during chronic inflammation.
RESUMEN
Neutrophils release chromatin extracellular traps (ETs) as part of the fish innate immune response to counter the threats posed by microbial pathogens. However, relatively little attention has been paid to this phenomenon in many commercially farmed species, despite the importance of understanding host-pathogen interactions and the potential to influence ET release to reduce disease outbreaks. The aim of this present study was to investigate the release of ETs by Atlantic salmon (Salmo salar L.) immune cells. Extracellular structures resembling ETs of different morphology were observed by fluorescence microscopy in neutrophil suspensions in vitro, as these structures stained positively with Sytox Green and were digestible with DNase I. Immunofluorescence studies confirmed the ET structures to be decorated with histones H1 and H2A and neutrophil elastase, which are characteristic for ETs in mammals and other organisms. Although the ETs were released spontaneously, release in neutrophil suspensions was stimulated most significantly with 5⯵g/ml calcium ionophore (CaI) for 1â¯h, whilst the fish pathogenic bacterium Aeromonas salmonicida (isolates 30411 and Hooke) also exerted a stimulatory effect. Microscopic observations revealed bacteria in association with ETs, and fewer bacterial colonies of A. salmonicida Hooke were recovered at 3â¯h after co-incubation with neutrophils that had been induced to release ETs. Interestingly, spontaneous release of ETs was inversely associated with fish mass (pâ¯<â¯0.05), a surrogate for age. Moreover, suspensions enriched for macrophages and stimulated with 5⯵g/ml CaI released ET-like structures that occasionally led to the formation of large clumps of cells. A deeper understanding for the roles and functions of ETs within innate immunity of fish hosts, and their interaction with microbial pathogens, may open new avenues towards protecting cultured stocks against infectious diseases.
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Aeromonas salmonicida , Trampas Extracelulares , Salmo salar , Animales , Cromatina , Neutrófilos , SuspensionesRESUMEN
The gut microbiota fundamentally regulates intestinal homeostasis and disease partially through mechanisms that involve modulation of regulatory T cells (Tregs), yet how the microbiota-Treg cross-talk is physiologically controlled is incompletely defined. Here, we report that prostaglandin E2 (PGE2), a well-known mediator of inflammation, inhibits mucosal Tregs in a manner depending on the gut microbiota. PGE2 through its receptor EP4 diminishes Treg-favorable commensal microbiota. Transfer of the gut microbiota that was modified by PGE2-EP4 signaling modulates mucosal Treg responses and exacerbates intestinal inflammation. Mechanistically, PGE2-modified microbiota regulates intestinal mononuclear phagocytes and type I interferon signaling. Depletion of mononuclear phagocytes or deficiency of type I interferon receptor diminishes PGE2-dependent Treg inhibition. Together, our findings provide emergent evidence that PGE2-mediated disruption of microbiota-Treg communication fosters intestinal inflammation.
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Microbioma Gastrointestinal , Linfocitos T Reguladores , Dinoprostona/farmacología , Humanos , Inflamación , Subtipo EP2 de Receptores de Prostaglandina ERESUMEN
A highly sensitive optical probe for the detection of activated neutrophils and Neutrophil Extracellular Traps (NETs) is reported. It is based on a triple-quenched, super-silent tri-branched probe that generates >20 fold increase in fluorescence upon cleavage. The probe was highly specific for human neutrophil elastase, a protease that mediates a variety of inflammatory diseases, and detected NETosis and neutrophil activation in in vitro differentiated neutrophils and isolated human neutrophils.
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Trampas Extracelulares/química , Colorantes Fluorescentes/química , Inflamación/diagnóstico , Elastasa de Leucocito/química , Péptidos/química , Técnicas Biosensibles , Células HL-60 , Humanos , Activación Neutrófila , Imagen Óptica , Fotólisis , Espectrometría de FluorescenciaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the novel coronavirus disease 2019 (COVID-19), a highly pathogenic and sometimes fatal respiratory disease responsible for the current 2020 global pandemic. Presently, there remains no effective vaccine or efficient treatment strategies against COVID-19. Non-steroidal anti-inflammatory drugs (NSAIDs) are medicines very widely used to alleviate fever, pain, and inflammation (common symptoms of COVID-19 patients) through effectively blocking production of prostaglandins (PGs) via inhibition of cyclooxyganase enzymes. PGs can exert either proinflammatory or anti-inflammatory effects depending on the inflammatory scenario. In this review, we survey the potential roles that NSAIDs and PGs may play during SARS-CoV-2 infection and the development and progression of COVID-19. LINKED ARTICLES: This article is part of a themed issue on The Pharmacology of COVID-19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc.
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Betacoronavirus/aislamiento & purificación , Infecciones por Coronavirus/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacología , COVID-19 , Infecciones por Coronavirus/virología , Progresión de la Enfermedad , Humanos , Inflamación/tratamiento farmacológico , Inflamación/virología , Pandemias , Neumonía Viral/virología , Prostaglandinas/administración & dosificación , Prostaglandinas/farmacología , SARS-CoV-2 , Tratamiento Farmacológico de COVID-19RESUMEN
Eosinophils are key effector cells in allergic diseases. Here we investigated Mcl-1 (an anti-apoptotic protein) in experimental allergic airway inflammation using transgenic overexpressing human Mcl-1 mice (hMcl-1) and reducing Mcl-1 by a cyclin-dependent kinase inhibitor. Overexpression of Mcl-1 exacerbated allergic airway inflammation, with increased bronchoalveolar lavage fluid cellularity, eosinophil numbers and total protein, and an increase in airway mucus production. Eosinophil apoptosis was suppressed by Mcl-1 overexpression, with this resistance to apoptosis attenuated by cyclin-dependent kinase inhibition which also rescued Mcl-1-exacerbated allergic airway inflammation. We propose that targeting Mcl-1 may be beneficial in treatment of allergic airway disease.
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Asma/genética , Eosinófilos/patología , Regulación de la Expresión Génica , Hipersensibilidad/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , ARN/genética , Animales , Apoptosis , Asma/metabolismo , Asma/patología , Líquido del Lavado Bronquioalveolar , Modelos Animales de Enfermedad , Eosinófilos/metabolismo , Femenino , Hipersensibilidad/metabolismo , Hipersensibilidad/patología , Recuento de Leucocitos , Ratones , Ratones Transgénicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesisRESUMEN
Inflammatory bowel disease (IBD) is a condition of chronic inflammatory intestinal disorder with increasing prevalence but limited effective therapies. The purine metabolic pathway is involved in various inflammatory processes including IBD. However, the mechanisms through which purine metabolism modulates IBD remain to be established. Here, we found that mucosal expression of genes involved in the purine metabolic pathway is altered in patients with active ulcerative colitis (UC), which is associated with elevated gene expression signatures of the group 3 innate lymphoid cell (ILC3)-interleukin (IL)-22 pathway. In mice, blockade of ectonucleotidases (NTPDases), critical enzymes for purine metabolism by hydrolysis of extracellular adenosine 5'-triphosphate (eATP) into adenosine, exacerbates dextran-sulfate sodium-induced intestinal injury. This exacerbation of colitis is associated with reduction of colonic IL-22-producing ILC3s, which afford essential protection against intestinal inflammation, and is rescued by exogenous IL-22. Mechanistically, activation of ILC3s for IL-22 production is reciprocally mediated by eATP and adenosine. These findings reveal that the NTPDase-mediated balance between eATP and adenosine regulates ILC3 cell function to provide protection against intestinal injury and suggest potential therapeutic strategies for treating IBD by targeting the purine-ILC3 axis.
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Colitis/etiología , Colitis/metabolismo , Inmunidad Innata , Linfocitos/inmunología , Linfocitos/metabolismo , Purinas/metabolismo , Animales , Biomarcadores , Colitis/patología , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Citometría de Flujo , Perfilación de la Expresión Génica , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Noqueados , TranscriptomaRESUMEN
Acute lung injury is a neutrophil-dominant, life-threatening disease without effective therapies and better understanding of the pathophysiological mechanisms involved is an urgent need. Here we show that interleukin (IL)-22 is produced from innate lymphoid cells (ILC) and is responsible for suppression of experimental lung neutrophilic inflammation. Blocking prostaglandin E2 (PGE2) synthesis reduces lung ILCs and IL-22 production, resulting in exacerbation of lung neutrophilic inflammation. In contrast, activation of the PGE2 receptor EP4 prevents acute lung inflammation. We thus demonstrate a mechanism for production of innate IL-22 in the lung during acute injury, highlighting potential therapeutic strategies for control of lung neutrophilic inflammation by targeting the PGE2/ILC/IL-22 axis.
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Dinoprostona/farmacología , Inmunidad Innata/efectos de los fármacos , Interleucinas/biosíntesis , Linfocitos/metabolismo , Neumonía/prevención & control , Animales , Modelos Animales de Enfermedad , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Neumonía/inmunología , Neumonía/metabolismo , Interleucina-22RESUMEN
BACKGROUND: Eosinophils play a central role in propagation of allergic diseases, including asthma. Both recruitment and retention of eosinophils regulate pulmonary eosinophilia, but the question of whether alterations in apoptotic cell clearance by phagocytes contributes directly to resolution of allergic airway inflammation remains unexplored. OBJECTIVES: In this study we investigated the role of the receptor tyrosine kinase Mer in mediating apoptotic eosinophil clearance and allergic airway inflammation resolution in vivo to establish whether apoptotic cell clearance directly affects the resolution of allergic airway inflammation. METHODS: Alveolar and bone marrow macrophages were used to study Mer-mediated phagocytosis of apoptotic eosinophils. Allergic airway inflammation resolution was modeled in mice by using ovalbumin. Fluorescently labeled apoptotic cells were administered intratracheally or eosinophil apoptosis was driven by administration of dexamethasone to determine apoptotic cell clearance in vivo. RESULTS: Inhibition or absence of Mer impaired phagocytosis of apoptotic human and mouse eosinophils by macrophages. Mer-deficient mice showed delayed resolution of ovalbumin-induced allergic airway inflammation, together with increased airway responsiveness to aerosolized methacholine, increased bronchoalveolar lavage fluid protein levels, altered cytokine production, and an excess of uncleared dying eosinophils after dexamethasone treatment. Alveolar macrophage phagocytosis was significantly Mer dependent, with the absence of Mer attenuating apoptotic cell clearance in vivo to enhance inflammation in response to apoptotic cells. CONCLUSIONS: We demonstrate that Mer-mediated apoptotic cell clearance by phagocytes contributes to resolution of allergic airway inflammation, suggesting that augmenting apoptotic cell clearance is a potential therapeutic strategy for treating allergic airway inflammation.
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Apoptosis/inmunología , Eosinófilos/inmunología , Macrófagos/inmunología , Hipersensibilidad Respiratoria/inmunología , Tirosina Quinasa c-Mer/inmunología , Alérgenos/inmunología , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/inmunología , Femenino , Humanos , Inflamación/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , Fagocitosis , Tirosina Quinasa c-Mer/genéticaRESUMEN
BACKGROUND: Cystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation. METHODS: Blood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR. RESULTS: CF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF. CONCLUSIONS: CF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.
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Apoptosis/fisiología , Fibrosis Quística/patología , Trampas Extracelulares , Neutrófilos/fisiología , Adulto , Animales , Estudios de Casos y Controles , Supervivencia Celular , Fibrosis Quística/sangre , Fibrosis Quística/inmunología , Humanos , Inflamación , Porcinos , Factores de TiempoRESUMEN
BACKGROUND: Atopic dermatitis (AD) and allergic contact dermatitis (ACD) are both forms of eczema and are common inflammatory skin diseases with a central role of T cell-derived IL-22 in their pathogenesis. Although prostaglandin (PG) E2 is known to promote inflammation, little is known about its role in processes related to AD and ACD development, including IL-22 upregulation. OBJECTIVES: We sought to investigate whether PGE2 has a role in IL-22 induction and development of ACD, which has increased prevalence in patients with AD. METHODS: T-cell cultures and in vivo sensitization of mice with haptens were used to assess the role of PGE2 in IL-22 production. The involvement of PGE2 receptors and their downstream signals was also examined. The effects of PGE2 were evaluated by using the oxazolone-induced ACD mouse model. The relationship of PGE2 and IL-22 signaling pathways in skin inflammation were also investigated by using genomic profiling in human lesional AD skin. RESULTS: PGE2 induces IL-22 from T cells through its receptors, E prostanoid receptor (EP) 2 and EP4, and involves cyclic AMP signaling. Selective deletion of EP4 in T cells prevents hapten-induced IL-22 production in vivo, and limits atopic-like skin inflammation in the oxazolone-induced ACD model. Moreover, both PGE2 and IL-22 pathway genes were coordinately upregulated in human AD lesional skin but were at less than significant detection levels after corticosteroid or UVB treatments. CONCLUSIONS: Our results define a crucial role for PGE2 in promoting ACD by facilitating IL-22 production from T cells.
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Dermatitis Alérgica por Contacto/inmunología , Dinoprostona/inmunología , Interleucinas/inmunología , Piel/inmunología , Linfocitos T/inmunología , Animales , Dermatitis Alérgica por Contacto/genética , Dermatitis Alérgica por Contacto/patología , Dinoprostona/genética , Humanos , Interleucinas/genética , Ratones , Ratones Noqueados , Piel/patología , Linfocitos T/patología , Interleucina-22RESUMEN
BACKGROUND: Acute respiratory distress syndrome (ARDS) is an often fatal neutrophil-dominant lung disease. Although influenced by multiple proinflammatory mediators, identification of suitable therapeutic candidates remains elusive. We aimed to delineate the presence of mitochondrial formylated peptides in ARDS and characterise the functional importance of formyl peptide receptor 1 (FPR1) signalling in sterile lung inflammation. METHODS: Mitochondrial formylated peptides were identified in bronchoalveolar lavage fluid (BALF) and serum of patients with ARDS by liquid chromatography-tandem mass spectrometry. In vitro, human neutrophils were stimulated with mitochondrial formylated peptides and their effects assessed by flow cytometry and chemotaxis assay. Mouse lung injury was induced by mitochondrial formylated peptides or hydrochloric acid. Bone marrow chimeras determined the contribution of myeloid and parenchymal FPR1 to sterile lung inflammation. RESULTS: Mitochondrial formylated peptides were elevated in BALF and serum from patients with ARDS. These peptides drove neutrophil activation and chemotaxis through FPR1-dependent mechanisms in vitro and in vivo. In mouse lung injury, inflammation was attenuated in Fpr1-/- mice, effects recapitulated by a pharmacological FPR1 antagonist even when administered after the onset of injury. FPR1 expression was present in alveolar epithelium and chimeric mice demonstrated that both myeloid and parenchymal FPR1 contributed to lung inflammation. CONCLUSIONS: We provide the first definitive evidence of mitochondrial formylated peptides in human disease and demonstrate them to be elevated in ARDS and important in a mouse model of lung injury. This work reveals mitochondrial formylated peptide FPR1 signalling as a key driver of sterile acute lung injury and a potential therapeutic target in ARDS.
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Receptores de Formil Péptido/inmunología , Síndrome de Dificultad Respiratoria/inmunología , Animales , Líquido del Lavado Bronquioalveolar/química , Quimiotaxis de Leucocito/inmunología , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Ratones , Mitocondrias/inmunología , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Espectrometría de Masas en TándemRESUMEN
Acute respiratory distress syndrome (ARDS) is a neutrophil-dominant disorder with no effective pharmacological therapies. While the cyclin-dependent kinase inhibitor AT7519 induces neutrophil apoptosis to promote inflammation resolution in preclinical models of lung inflammation, its potential efficacy in ARDS has not been examined. Untreated peripheral blood sepsis-related ARDS neutrophils demonstrated prolonged survival after 20â hours in vitro culture. AT7519 was able to override this phenotype to induce apoptosis in ARDS neutrophils with reduced expression of the pro-survival protein Mcl-1. We demonstrate the first pharmacological compound to induce neutrophil apoptosis in sepsis-related ARDS, highlighting cyclin-dependent kinase inhibitors as potential novel therapeutic agents.
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Apoptosis/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Piperidinas/uso terapéutico , Pirazoles/uso terapéutico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/etiología , Sepsis/complicaciones , Adulto , Anciano , Biomarcadores/sangre , Estudios de Casos y Controles , Citocinas/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Tasa de SupervivenciaRESUMEN
Systemic inflammation, which results from the massive release of proinflammatory molecules into the circulatory system, is a major risk factor for severe illness, but the precise mechanisms underlying its control are not fully understood. We observed that prostaglandin E2 (PGE2), through its receptor EP4, is down-regulated in human systemic inflammatory disease. Mice with reduced PGE2 synthesis develop systemic inflammation, associated with translocation of gut bacteria, which can be prevented by treatment with EP4 agonists. Mechanistically, we demonstrate that PGE2-EP4 signaling acts directly on type 3 innate lymphoid cells (ILCs), promoting their homeostasis and driving them to produce interleukin-22 (IL-22). Disruption of the ILC-IL-22 axis impairs PGE2-mediated inhibition of systemic inflammation. Hence, the ILC-IL-22 axis is essential in protecting against gut barrier dysfunction, enabling PGE2-EP4 signaling to impede systemic inflammation.
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Dinoprostona/inmunología , Inflamación/inmunología , Interleucinas/inmunología , Intestinos/inmunología , Linfocitos/inmunología , Subtipo EP4 de Receptores de Prostaglandina E/inmunología , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Expresión Génica , Humanos , Inmunidad Innata , Inflamación/tratamiento farmacológico , Inflamación/microbiología , Intestinos/microbiología , Ratones , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/genética , Transducción de Señal , Interleucina-22RESUMEN
The fundamental role played by neutrophils for an efficient, acute inflammatory response has long been appreciated, with the underlying molecular and cellular mechanisms largely elucidated over the past decades. However, more recent work suggests that the biological functions exerted by this fascinating leucocyte are somewhat more extensive than previously acknowledged. Here we discuss how extravasated neutrophils govern the initiation of the resolution phase of inflammation by enabling activation of pro-resolving circuits to ensure the safe conclusion of the inflammatory response. The neutrophil 'alarm bell' on resolution is effected through release of soluble mediators as well as apoptotic bodies and other vesicles, which, in turn, can inform and modify the microenvironment ultimately leading to termination of the inflammatory response coinciding with re-establishment of tissue homeostasis and functionality.
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Inflamación/etiología , Inflamación/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Animales , Biomarcadores , Citocinas/metabolismo , Humanos , Inmunidad Innata , Inflamación/patología , Mediadores de Inflamación , Activación Neutrófila/inmunología , Infiltración Neutrófila/inmunología , Neutrófilos/patología , Transducción de Señal , Cicatrización de Heridas/inmunologíaRESUMEN
Cyclin-dependent kinases (CDKs) have been traditionally associated with the cell cycle. However, it is now known that CDK7 and CDK9 regulate transcriptional activity via phosphorylation of RNA polymerase II and subsequent synthesis of, for example, inflammatory mediators and factors that influence the apoptotic process; including apoptosis of granulocytes such as neutrophils and eosinophils. Successful resolution of inflammation and restoration of normal tissue homeostasis requires apoptosis of these inflammatory cells and subsequent clearance of apoptotic bodies by phagocytes such as macrophages. It is believed that CDK7 and CDK9 influence resolution of inflammation since they are involved in the transcription of anti-apoptotic proteins such as Mcl-1 which is especially important in granulocyte survival.This chapter describes various in vitro and in vivo models used to investigate CDKs and their inhibitors in granulocytes and particularly the role of CDKs in the apoptosis pathway. This can be performed in vitro by isolation and use of primary granulocytes and in vivo using animal models of inflammatory disease in rodents and zebrafish. Some of the methods described here to assess the role of CDKs in inflammation and apoptosis include flow cytometry and western blotting, together with imaging and quantification of apoptosis in fixed tissue, as well as in vivo models of inflammation.
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Quinasas Ciclina-Dependientes/metabolismo , Inflamación/metabolismo , Biología Molecular/métodos , Animales , Apoptosis , Bleomicina/química , Lavado Broncoalveolar , Ciclo Celular , Eosinófilos/metabolismo , Citometría de Flujo , Granulocitos/citología , Granulocitos/metabolismo , Humanos , Leucocitos/metabolismo , Pulmón/patología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Mitocondrias/metabolismo , Neutrófilos/metabolismo , Permeabilidad , Fagocitosis , Fosforilación , ARN Interferente Pequeño/metabolismo , Pez CebraRESUMEN
The effects of nanomaterials (NMs) on biological systems, especially their ability to stimulate inflammatory responses requires urgent investigation. We evaluated the response of the human differentiated HL60 neutrophil-like cell line to NMs. It was hypothesised that NM physico-chemical characteristics would influence cell responsiveness by altering intracellular Ca2+ concentration [Ca2+]i and reactive oxygen species production. Cells were exposed (1.95-125 µg/ml, 24 h) to silver (Ag), zinc oxide (ZnO), titanium dioxide (TiO2), multi-walled carbon nanotubes (MWCNTs) or ultrafine carbon black (ufCB) and cytotoxicity assessed (alamar blue assay). Relatively low (TiO2, MWCNTs, ufCB) or high (Ag, ZnO) cytotoxicity NMs were identified. Sub-lethal impacts of NMs on cell function were investigated for selected NMs only, namely TiO2, Ag and ufCB. Only Ag stimulated cell activation. Within minutes, Ag stimulated an increase in [Ca2+]i (in Fura-2 loaded cells), and a prominent inward ion current (assessed by electrophysiology). Within 2-4 h, Ag increased superoxide anion release and stimulated cytokine production (MCP-1, IL-8) that was diminished by Ca2+ inhibitors or trolox. Light microscopy demonstrated that cells had an activated phenotype. In conclusion NM toxicity was ranked; Ag>ufCB>TiO2, and the battery of tests used provided insight into the mechanism of action of NM toxicity to guide future testing strategies.
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Nanoestructuras/toxicidad , Activación Neutrófila/efectos de los fármacos , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Células HL-60 , Humanos , Nanotubos de Carbono/toxicidad , Plata/toxicidad , Hollín/toxicidad , Superóxidos/metabolismo , Titanio/toxicidad , Óxido de Zinc/toxicidadRESUMEN
Controlled release of chromatin from the nuclei of inflammatory cells is a process that entraps and kills microorganisms in the extracellular environment. Now termed ETosis, it is important for innate immunity in vertebrates. Paradoxically, however, in mammals, it can also contribute to certain pathologies. Here we show that ETosis occurs in several invertebrate species, including, remarkably, an acoelomate. Our findings reveal that the phenomenon is primordial and predates the evolution of the coelom. In invertebrates, the released chromatin participates in defence not only by ensnaring microorganisms and externalizing antibacterial histones together with other haemocyte-derived defence factors, but crucially, also provides the scaffold on which intact haemocytes assemble during encapsulation; a response that sequesters and kills potential pathogens infecting the body cavity. This insight into the early origin of ETosis identifies it as a very ancient process that helps explain some of its detrimental effects in mammals.