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BACKGROUND/OBJECTIVES: Proinflammatory cytokines are increased in obese adipose tissue, including inflammasome key masters. Conversely, IL-18 protects against obesity and metabolic dysfunction. We focused on the IL-18 effect in controlling adipose tissue remodeling and metabolism. MATERIALS/SUBJECTS AND METHODS: We used C57BL/6 wild-type (WT) and interleukine-18 deficient (IL-18-/-) male mice fed a chow diet and samples from bariatric surgery patients. RESULTS: IL-18-/- mice showed increased adiposity and proinflammatory cytokine levels in adipose tissue, leading to glucose intolerance. IL-18 was widely secreted by stromal vascular fraction but not adipocytes from mice's fatty tissue. Chimeric model experiments indicated that IL-18 controls adipose tissue expansion through its presence in tissues other than bone marrow. However, IL-18 maintains glucose homeostasis when present in bone marrow cells. In humans with obesity, IL-18 expression in omental tissue was not correlated with BMI or body fat mass but negatively correlated with IRS1, GLUT-4, adiponectin, and PPARy expression. Also, the IL-18RAP receptor was negatively correlated with IL-18 expression. CONCLUSIONS: IL-18 signaling may control adipose tissue expansion and glucose metabolism, as its absence leads to spontaneous obesity and glucose intolerance in mice. We suggest that resistance to IL-18 signaling may be linked with worse glucose metabolism in humans with obesity.
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Tejido Adiposo , Interleucina-18 , Ratones Endogámicos C57BL , Obesidad , Animales , Interleucina-18/metabolismo , Ratones , Masculino , Tejido Adiposo/metabolismo , Humanos , Obesidad/metabolismo , Intolerancia a la Glucosa/metabolismo , Modelos Animales de Enfermedad , Ratones NoqueadosRESUMEN
OBJECTIVE: Angiotensin-(1-7) [Ang-(1-7)] is a pro-resolving mediator. It is not known whether the pro-resolving effects of Ang-(1-7) are sustained and protect the lung from a subsequent inflammatory challenge. This study sought to investigate the impact of treatment in face of a second allergic or lipopolysaccharide (LPS) challenge. METHODS: Mice, sensitized and challenged with ovalbumin (OVA), received a single Ang-(1-7) dose at the peak of eosinophilic inflammation, 24 h after the final OVA challenge. Subsequently, mice were euthanized at 48, 72, 96, and 120 h following the OVA challenge, and cellular infiltrate, inflammatory mediators, lung histopathology, and macrophage-mediated efferocytic activity were evaluated. The secondary inflammatory stimulus (OVA or LPS) was administered 120 h after the last OVA challenge, and subsequent inflammatory analyses were performed. RESULTS: Treatment with Ang-(1-7) resulted in elevated levels of IL-10, CD4+Foxp3+, Mres in the lungs and enhanced macrophage-mediated efferocytic capacity. Moreover, in allergic mice treated with Ang-(1-7) and then subjected to a secondary OVA challenge, inflammation was also reduced. Similarly, in mice exposed to LPS, Ang-(1-7) effectively prevented the lung inflammation. CONCLUSION: A single dose of Ang-(1-7) resolves lung inflammation and protect the lung from a subsequent inflammatory challenge highlighting its potential therapeutic for individuals with asthma.
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Angiotensina I , Lipopolisacáridos , Pulmón , Ovalbúmina , Fragmentos de Péptidos , Animales , Angiotensina I/uso terapéutico , Angiotensina I/farmacología , Angiotensina I/administración & dosificación , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Fragmentos de Péptidos/administración & dosificación , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/inmunología , Ovalbúmina/inmunología , Ratones , Masculino , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Ratones Endogámicos BALB C , Inflamación/tratamiento farmacológico , Eosinofilia/tratamiento farmacológico , Eosinofilia/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/citologíaRESUMEN
OBJECTIVE AND DESIGN: The aim of this study was to investigate the effects of ethanol exposure on epigenetic markers in bone marrow (BM) and their impact on inflammatory response during Aspergillus fumigatus infection. RESULTS: Chronic ethanol exposure decreased H3K27me3 enrichment in the Il6 promoter region while increased H3K4me3 enrichment in Tnf. Chimeric mice were generated by transplanting BM from mice exposed to ethanol or water. Infection of ethanol-chimeric mice culminated in higher clinical scores, although there was no effect on mortality. However, previous chronic exposure to ethanol affects persistently the inflammatory response in lung tissue, demonstrated by increased lung damage, neutrophil accumulation and IL-6, TNF and CXCL2 production in ethanol-chimeric mice, resulting in a decreased neutrophil infiltration into the alveolar space. Neutrophil killing and phagocytosis were also significantly lower. Moreover, BM derived macrophages (BMDM) from ethanol-chimeric mice stimulated with A. fumigatus conidia exhibited higher levels of TNF, CXCL2 and IL-6 release and a higher killing activity. The Il6 promoter of BMDM from ethanol-chimeric mice exhibited a reduction in H3K27me3 enrichment, a finding also observed in BM donors exposed to ethanol. CONCLUSIONS: These evidences demonstrate that prior chronic alcohol exposure of bone-marrow modify immune effector cells functions impairing the inflammatory response during A. fumigatus infection. These findings highlight the persistent impact of chronic ethanol exposure on infectious disease outcomes.
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Multiple sclerosis is an autoimmune disease that affects the central nervous system. Because of its complexity and the difficulty to treat, searching for immunoregulatory responses that reduce the clinical signs of disease by non-aggressive mechanisms and without adverse effects is a scientific challenge. Herein we propose a protocol of oral tolerance induction that prevented and controlled MOG-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. The genetically modified strain HSP65-producing Lactococcus lactis was orally administered for 5 consecutive days either before or during disease development in mice. Both protocols of feeding HSP65 resulted in significant reduction in the clinical score of EAE. Frequencies of LAP+CD4+Foxp3- regulatory T cells were higher in spleens and inguinal lymph nodes of fed mice. In addition, intravital microscopy showed that adherence of leukocytes to venules in the spinal cord was reduced in orally treated mice. Oral treatment with HSP65-producing L.lactis prevented leukocytes to leave the secondary lymphoid organs, therefore they could not reach the central nervous system. Despite the inhibition of pathological immune response that drive EAE development, activated T cells were at normal frequencies suggesting that oral tolerance did not induce general immunosuppression, but it led to specific control of pathogenic T cells. Our results indicate a novel therapeutic strategy to prevent and control autoimmune diseases such as multiple sclerosis.
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Encefalomielitis Autoinmune Experimental , Lactococcus lactis , Esclerosis Múltiple , Ratones , Animales , Ratones Endogámicos C57BL , Médula EspinalRESUMEN
Phosphoinositide-3-kinases (PI3Ks) are enzymes involved in signalling and modification of the function of all mammalian cells. These enzymes phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositol, resulting in lipid products that act as second messengers responsible for coordinating many cellular functions, including activation, chemotaxis, proliferation and survival. The identification of the functions that are mediated by a specific PI3K isoform is complex and depends on the specific cell type and inflammatory context. In this chapter we will focus on the role of PI3K isoforms in the context of innate immunity, focusing on the mechanisms by which PI3K signalling regulates phagocytosis, the activation of immunoglobulin, chemokine and cytokines receptors, production of ROS and cell migration, and how PI3K signalling plays a central role in host defence against infections and tissue injury.
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Inmunidad Innata , Fosfatidilinositol 3-Quinasas , Animales , Quimiocinas , Inositol , Mamíferos/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/metabolismo , Isoformas de Proteínas/metabolismo , Especies Reactivas de Oxígeno , Receptores de CitocinasRESUMEN
Inflammation resolution is an active process that involves cellular events such as apoptosis and efferocytosis, which are key steps in the restoration of tissue homeostasis. Hepatocyte growth factor (HGF) is a growth factor mostly produced by mesenchymal-origin cells and has been described to act via MET receptor tyrosine kinase. The HGF/MET axis is essential for determining the progression and severity of inflammatory and immune-mediated disorders. Here, we investigated the effect of blocking the HGF/MET signalling pathway by PF-04217903 on the resolution of established models of neutrophilic inflammation. In a self-resolving model of gout induced by MSU crystals, HGF expression on periarticular tissue peaked at 12 h, the same time point that neutrophils reach their maximal accumulation in the joints. The HGF/MET axis was activated in this model, as demonstrated by increased levels of MET phosphorylation in neutrophils (Ly6G+ cells). In addition, the number of neutrophils was reduced in the knee exudate after PF-04217903 treatment, an effect accompanied by increased neutrophil apoptosis and efferocytosis and enhanced expression of Annexin A1, a key molecule for inflammation resolution. Reduced MPO activity, IL-1ß and CXCL1 levels were also observed in periarticular tissue. Importantly, PF-04217903 reduced the histopathological score and hypernociceptive response. Similar findings were obtained in LPS-induced neutrophilic pleurisy. In human neutrophils, the combined use of LPS and HGF increased MET phosphorylation and provided a prosurvival signal, whereas blocking MET with PF-04217903 induced caspase-dependent neutrophil apoptosis. Taken together, these data demonstrate that blocking HGF/MET signalling may be a potential therapeutic strategy for inducing the resolution of neutrophilic inflammatory responses.
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Factor de Crecimiento de Hepatocito , Neutrófilos , Humanos , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Factor de Crecimiento de Hepatocito/uso terapéutico , Lipopolisacáridos/farmacología , Inflamación/metabolismo , Apoptosis , Proteínas Proto-Oncogénicas c-met/metabolismo , HomeostasisRESUMEN
BACKGROUND/OBJECTIVES: Platelet-activating factor receptor (PAFR) activation controls adipose tissue (AT) expansion in animal models. Our objective was twofold: (i) to check whether PAFR signaling is involved in human obesity and (ii) investigate the PAF pathway role in hematopoietic or non-hematopoietic cells to control adipocyte size. MATERIALS/SUBJECTS AND METHODS: Clinical parameters and adipose tissue gene expression were evaluated in subjects with obesity. Bone marrow (BM) transplantation from wild-type (WT) or PAFR-/- mice was performed to obtain chimeric PAFR-deficient mice predominantly in hematopoietic or non-hematopoietic-derived cells. A high carbohydrate diet (HC) was used to induce AT remodeling and evaluate in which cell compartment PAFR signaling modulates it. Also, 3T3-L1 cells were treated with PAF to evaluate fat accumulation and the expression of genes related to it. RESULTS: PAFR expression in omental AT from humans with obesity was negatively correlated to different corpulence parameters and more expressed in the stromal vascular fraction than adipocytes. Total PAFR-/- increased adiposity compared with WT independent of diet-induced obesity. Differently, WT mice receiving PAFR-/--BM exhibited similar adiposity gain as WT chimeras. PAFR-/- mice receiving WT-BM showed comparable augmentation in adiposity as total PAFR-/- mice, demonstrating that PAFR signaling modulates adipose tissue expansion through non-hematopoietic cells. Indeed, the PAF treatment in 3T3-L1 adipocytes reduced fat accumulation and expression of adipogenic genes. CONCLUSIONS: Therefore, decreased PAFR signaling may favor an AT accumulation in humans and animal models. Importantly, PAFR signaling, mainly in non-hematopoietic cells, especially in adipocytes, appears to play a significant role in regulating diet-induced AT expansion.
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Tejido Adiposo/fisiopatología , Obesidad/complicaciones , Glicoproteínas de Membrana Plaquetaria/farmacología , Tejido Adiposo/metabolismo , Adulto , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Obesidad/fisiopatología , Paris , Receptores Acoplados a Proteínas G , Transducción de Señal/fisiologíaRESUMEN
Uncontrolled inflammation and failure to resolve the inflammatory response are crucial factors involved in the progress of inflammatory diseases. Current therapeutic strategies aimed at controlling excessive inflammation are effective in some cases, though they may be accompanied by severe side effects, such as immunosuppression. Phytochemicals as a therapeutic alternative can have a fundamental impact on the different stages of inflammation and its resolution. Biochanin A (BCA) is an isoflavone known for its wide range of pharmacological properties, especially its marked anti-inflammatory effects. Recent studies have provided evidence of BCA's abilities to activate events essential for resolving inflammation. In this review, we summarize the most recent findings from pre-clinical studies of the pharmacological effects of BCA on the complex signaling network associated with the onset and resolution of inflammation and BCA's potential protective functionality in several models of inflammatory diseases, such as arthritis, pulmonary disease, neuroinflammation, and metabolic disease.
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Genisteína , Isoflavonas , Genisteína/farmacología , Genisteína/uso terapéutico , Humanos , Inflamación/tratamiento farmacológico , Fitoquímicos/farmacología , FitoterapiaRESUMEN
Cannabidiol (CBD) is a highly lipidic phytocannabinoid with remarkable anti-inflammatory effects. The aim of this study was to evaluate CBD's effects and mechanisms of action in the treatment of mice subjected to acute graft-versus-host disease (aGVHD). aGVHD was induced by the transplantation of bone marrow cells and splenocytes from C57BL-6j to Balb-c mice. The recipient mice were treated daily with CBD, and the treatment reduced mouse mortality by decreasing inflammation and injury and promoting immune regulation in the jejunum, ileum, and liver. Analysis of the jejunum and ileum showed that CBD treatment reduced the levels of C-C motif chemokine ligand (CCL) 2, CCL3, CCL5, tumor necrosis factor α, and interferon γ (IFNγ). CCL3 and IFNγ levels were also decreased in the liver. Mechanistically, CBD also increased the number of cannabinoid receptor type 2 (CB2) receptors on CD4+ and forkhead box P3+ cells in the intestine, which may explain the reduction in proinflammatory cytokines and chemokines. Antagonists of the CB2 receptor reduced the survival rates of CBD-treated mice, suggesting the participation of this receptor in the effects of CBD. Furthermore, treatment with CBD did not interfere with the graft-versus-leukemia response. CBD treatment appears to protect aGVHD mice by anti-inflammatory and immunomodulatory effects partially mediated by CB2 receptor interaction. Altogether, our study suggests that CBD represents an interesting approach in the treatment of aGVHD, with potential therapeutic applications in patients undergoing bone marrow transplantation. SIGNIFICANCE STATEMENT: This study provides for the first time a mechanism by which cannabidiol, a phytocannabinoid with no psychoactive effect, induces immunomodulation in the graft-versus-host disease. Enhancing intestinal cannabinoid receptor type 2 (CB2) receptor expression on CD4+ and forkhead box P3+ cells and increasing the number of these regulatory cells, cannabidiol decreases proinflammatory cytokines and increases graft-versus-host disease mice survival. This effect is dependent of CB2 receptor activation. Besides, cannabidiol did not interfere with graft-versus-leukemia response, a central response to avoid primary disease relapse.
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Cannabidiol/uso terapéutico , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Mucosa Intestinal/metabolismo , Leucemia/terapia , Receptor Cannabinoide CB2/metabolismo , Animales , Trasplante de Médula Ósea/efectos adversos , Trasplante de Médula Ósea/métodos , Cannabidiol/farmacología , Células Cultivadas , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Mucosa Intestinal/efectos de los fármacos , Leucemia/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/genética , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Resolution failure of exacerbated inflammation triggered by Influenza A virus (IAV) prevents return of pulmonary homeostasis and survival, especially when associated with secondary pneumococcal infection. Therapeutic strategies based on pro-resolving molecules have great potential against acute inflammatory diseases. Angiotensin-(1-7) [Ang-(1-7)] is a pro-resolving mediator that acts on its Mas receptor (MasR) to promote resolution of inflammation. We investigated the effects of Ang-(1-7) and the role of MasR in the context of primary IAV infection and secondary pneumococcal infection and evaluated pulmonary inflammation, virus titers and bacteria counts, and pulmonary damage. Therapeutic treatment with Ang-(1-7) decreased neutrophil recruitment, lung injury, viral load and morbidity after a primary IAV infection. Ang-(1-7) induced apoptosis of neutrophils and efferocytosis of these cells by alveolar macrophages, but had no direct effect on IAV replication in vitro. MasR-deficient (MasR-/-) mice were highly susceptible to IAV infection, displaying uncontrolled inflammation, increased viral load and greater lethality rate, as compared to WT animals. Ang-(1-7) was not protective in MasR-/- mice. Interestingly, Ang-(1-7) given during a sublethal dose of IAV infection greatly reduced morbidity associated with a subsequent S. pneumoniae infection, as seen by decrease in the magnitude of neutrophil influx, number of bacteria in the blood leading to a lower lethality. Altogether, these results show that Ang-(1-7) is highly protective against severe primary IAV infection and protects against secondary bacterial infection of the lung. These effects are MasR-dependent. Mediators of resolution of inflammation, such as Ang-(1-7), should be considered for the treatment of pulmonary viral infections.
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Angiotensina I/uso terapéutico , Antiinflamatorios/uso terapéutico , Fragmentos de Péptidos/uso terapéutico , Infecciones Neumocócicas/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , Proteínas Proto-Oncogénicas/inmunología , Receptores Acoplados a Proteínas G/inmunología , Células A549 , Angiotensina I/farmacología , Animales , Antiinflamatorios/farmacología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/inmunología , Perros , Humanos , Virus de la Influenza A , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Células de Riñón Canino Madin Darby , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Fragmentos de Péptidos/farmacología , Peroxidasa/inmunología , Fagocitosis/efectos de los fármacos , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/patología , Neumonía Viral/inmunología , Neumonía Viral/patología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Receptores Acoplados a Proteínas G/genética , Streptococcus pneumoniaeRESUMEN
Sphingosine-1-phosphate (S1P) is an important sphingolipid derived from plasma membrane and has a known role in productive phase of inflammation, but its role in neutrophil survival and resolution phase of inflammation is unknown. Here, we investigated the effects of inhibition of S1P receptors and the blockade of S1P synthesis in BALB/c mice and human neutrophils. S1P and S1PR1-3 receptors expression were increased in cells from the pleural cavity stimulated with LPS. Using different antagonists of S1PRs and inhibitors of different steps of the metabolic pathway of S1P production, we show that S1P and its receptors are involved in regulating neutrophil survival and resolution of inflammation in the pleural cavity. Given the role of the S1P-S1PR axis in resolution of inflammation, we sought to identify whether blockade at different levels of the sphingosine-1-phosphate synthesis pathway could affect neutrophil survival in vitro. Inhibitors of the S1P pathway were also able to induce human neutrophil apoptosis. In addition, blockade of S1P synthesis or its receptor facilitated the efferocytosis of apoptotic neutrophil. Taken together, our data demonstrate a fundamental role for S1P in regulating the outcome of inflammatory responses, and position S1P-S1PR axis as a potential target for treatment of neutrophilic inflammation.
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Inflamación/inmunología , Lisofosfolípidos/metabolismo , Neutrófilos/inmunología , Cavidad Pleural/inmunología , Receptores de Esfingosina-1-Fosfato/metabolismo , Esfingosina/análogos & derivados , Animales , Apoptosis , Supervivencia Celular , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Activación Neutrófila , Transducción de Señal , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/antagonistas & inhibidoresRESUMEN
Patients with rheumatoid arthritis experience chronic pain, depression and fatigue, even when inflammation of the joints is well controlled. To study the relationship between arthritis, depression, and sustained pain when articular inflammation is no longer observed, we tested the hypothesis that brain TNF drives post-inflammation depression-like behavior and persistent pain in experimental arthritis. The murine model of antigen-induced arthritis (AIA) was used to evaluate the effects of knee inflammation on sustained pain and depression-like behavior. We measured joint pain using an automated dynamic plantar algesiometer and depression-like behavior with the tail suspension test. Cytokines were measured by Luminex assay and ELISA. TNF in the brain was blocked by intracerebroventricular injection of anti-TNF antibodies. Histological damage and elevated levels of cytokines were observed in the knee 24 h after antigen treatment, but not at 13 days. Reduced pain thresholds were seen 24 h and 13 days after treatment. Depression-like behavior was observed on day 13. Treatment with the antidepressant imipramine reduced both depression-like behavior and persistent pain. However, blocking joint pain with the analgesic dipyrone did not alter depression-like behavior. Elevated levels of TNF, CCL2, and CXCL-1 were observed in the hippocampus 24 h after treatment, with TNF remaining elevated at day 13. Intracerebroventricular infusion of an anti-TNF antibody blocked depression-like behavior and reduced persistent pain. We have demonstrated that depression-like behavior and pain is sustained in AIA mice after the resolution of inflammation. These changes are associated with elevated levels of TNF in the hippocampus and are dependent upon brain TNF. The findings reveal an important mechanistic link between the expression of chronic pain and depression in experimental arthritis. Furthermore, they suggest treating depression in rheumatoid arthritis may positively impact other debilitating features of this condition.
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Artritis Experimental , Factor de Necrosis Tumoral alfa , Animales , Artritis Experimental/complicaciones , Encéfalo/metabolismo , Depresión , Humanos , Inflamación , Ratones , Dolor , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A complex intracellular signaling governs different cellular responses in inflammation. Extracellular stimuli are sensed, amplified, and transduced through a dynamic cellular network of messengers converting the first signal into a proper response: production of specific mediators, cell activation, survival, or death. Several overlapping pathways are coordinated to ensure specific and timely induction of inflammation to neutralize potential harms to the tissue. Ideally, the inflammatory response must be controlled and self-limited. Resolution of inflammation is an active process that culminates with termination of inflammation and restoration of tissue homeostasis. Comparably to the onset of inflammation, resolution responses are triggered by coordinated intracellular signaling pathways that transduce the message to the nucleus. However, the key messengers and pathways involved in signaling transduction for resolution are still poorly understood in comparison to the inflammatory network. cAMP has long been recognized as an inducer of anti-inflammatory responses and cAMP-dependent pathways have been extensively exploited pharmacologically to treat inflammatory diseases. Recently, cAMP has been pointed out as coordinator of key steps of resolution of inflammation. Here, we summarize the evidence for the role of cAMP at inducing important features of resolution of inflammation.
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AMP Cíclico/metabolismo , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/metabolismo , Sistemas de Mensajero Secundario , Animales , Apoptosis , Quimiotaxis de Leucocito , Granulocitos/inmunología , Granulocitos/metabolismo , Granulocitos/patología , Humanos , Inflamación/inmunología , Inflamación/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Fagocitosis , FenotipoRESUMEN
OBJECTIVE AND DESIGN: Biochanin A (BCA), a phytoestrogen, has various pharmacological properties. This study was conducted to compare BCA's therapeutic property against 17-ß estradiol replacement therapy in zymosan-induced arthritis (ZIA) in mice. Additionally, we further investigated in vitro the anti-inflammatory action on neutrophils. TREATMENT: Ovariectomized (OVX) and non-OVX mice were pretreated with BCA (1, 3 and 9 mg/kg) or estrogen (50 µg/kg) for 14 days prior to ZIA. Neutrophils were pretreated with BCA (1, 10 and 100 µM) for 1 h prior to phorbol 12-myristate 13-acetate. METHODS: Anti-inflammatory effects of BCA were evaluated by cellular infiltrate, paw edema and cytokine measurement. In vitro, apoptosis was assessed by morphology and flow cytometry. Neutrophil extracellular traps (NET) were determined by fluorescent microscopy and DNA release. Statistical differences were determined by one- or two-way ANOVA. RESULTS: BCA inhibited neutrophil accumulation, paw edema and proinflammatory cytokine (TNF-α and IFN-γ) and increased anti-inflammatory cytokines (IL-4 and IL-10) in OVX and non-OVX mice, similar to 17-ß estradiol replacement therapy. In vitro, BCA increased apoptosis and consequently reduced NETs. CONCLUSION: BCA has a notable anti-inflammatory effect, similar to 17-ß estradiol, and is especially effective for treatment of ZIA. These results suggest that BCA may be promising for the treatment of postmenopausal arthritis.
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Antiinflamatorios no Esteroideos/uso terapéutico , Artritis/tratamiento farmacológico , Estradiol/uso terapéutico , Terapia de Reemplazo de Estrógeno , Genisteína/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Artritis/inducido químicamente , Citocinas/metabolismo , ADN/metabolismo , Edema/inducido químicamente , Edema/tratamiento farmacológico , Femenino , Ratones , Neutrófilos/efectos de los fármacos , Ovariectomía , Acetato de Tetradecanoilforbol , ZimosanRESUMEN
Inflammation resolution is an active process that functions to restore tissue homeostasis. The participation of the plasminogen (Plg)/plasmin (Pla) system in the productive phase of inflammation is well known, but its involvement in the resolution phase remains unclear. Therefore, we aimed to investigate the potential role of Plg/Pla in key events during the resolution of acute inflammation and its underlying mechanisms. Plg/Pla injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that were primarily macrophages of anti-inflammatory (M2 [F4/80high Gr1- CD11bhigh]) and proresolving (Mres [F4/80med CD11blow]) phenotypes, without changing the number of macrophages with a proinflammatory profile (M1 [F4/80low Gr1+ CD11bmed]). Pleural injection of Plg/Pla also increased M2 markers (CD206 and arginase-1) and secretory products (transforming growth factor ß and interleukin-6) and decreased the expression of inducible nitric oxide synthase (M1 marker). During the resolving phase of lipopolysaccharide (LPS)-induced inflammation when resolving macrophages predominate, we found increased Plg expression and Pla activity, further supporting a link between the Plg/Pla system and key cellular events in resolution. Indeed, Plg or Pla given at the peak of inflammation promoted resolution by decreasing neutrophil numbers and increasing neutrophil apoptosis and efferocytosis in a serine-protease inhibitor-sensitive manner. Next, we confirmed the ability of Plg/Pla to both promote efferocytosis and override the prosurvival effect of LPS via annexin A1. These findings suggest that Plg and Pla regulate several key steps in inflammation resolution, namely, neutrophil apoptosis, macrophage reprogramming, and efferocytosis, which have a major impact on the establishment of an efficient resolution process.
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Anexina A1/metabolismo , Reprogramación Celular , Fibrinolisina/metabolismo , Macrófagos/metabolismo , Plasminógeno/metabolismo , Enfermedad Aguda , Animales , Anexina A1/genética , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Fibrinolisina/genética , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Lipopolisacáridos/toxicidad , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neutrófilos/metabolismo , Neutrófilos/patología , Plasminógeno/genética , Células RAW 264.7RESUMEN
Annexin A1 (AnxA1) is a glucocorticoid-regulated protein known for its anti-inflammatory and pro-resolving effects. We have shown previously that the cAMP-enhancing compounds rolipram (ROL; a PDE4 inhibitor) and Bt2cAMP (a cAMP mimetic) drive caspase-dependent resolution of neutrophilic inflammation. In this follow-up study, we investigated whether AnxA1 could be involved in the pro-resolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or Bt2cAMP at the peak of inflammation shortened resolution intervals, improved resolution indices, and increased AnxA1 expression. In vitro studies showed that ROL and Bt2cAMP induced AnxA1 expression and phosphorylation, and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA in ROL-induced AnxA1 expression. Akin to these in vitro findings, H89 prevented ROL- and Bt2cAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway (by using N-t-Boc-Met-Leu-Phe, a nonselective AnxA1 receptor antagonist, or by using an anti-AnxA1 neutralizing antiserum) prevented ROL- and Bt2cAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or Bt2cAMP to induce neutrophil apoptosis was impaired in AnxA-knock-out mice. Finally, in in vitro settings, ROL and Bt2cAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1-dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the pro-resolving properties of cAMP-elevating agents and cAMP-mimetic drugs.
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Anexina A1/agonistas , Bucladesina/uso terapéutico , AMP Cíclico/agonistas , Infiltración Neutrófila/efectos de los fármacos , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Pleuresia/tratamiento farmacológico , Rolipram/uso terapéutico , Animales , Anexina A1/antagonistas & inhibidores , Anexina A1/genética , Anexina A1/metabolismo , Apoptosis/efectos de los fármacos , Bucladesina/antagonistas & inhibidores , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Inhibidores de Fosfodiesterasa 4/química , Fosforilación/efectos de los fármacos , Pleuresia/inmunología , Pleuresia/metabolismo , Pleuresia/patología , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Células RAW 264.7 , Rolipram/antagonistas & inhibidoresRESUMEN
Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties. Intact AnxA1 is a 37-kDa protein that may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3, generating the 33-kDa isoform that is largely inactive. In this study, we investigated the dynamics of AnxA1 expression and the effects of synthetic (sivelestat [SIV]; Eglin) and natural (secretory leukocyte protease inhibitor [SLPI]; Elafin) protease inhibitors on the resolution of LPS-induced inflammation. During the settings of LPS inflammation AnxA1 cleavage associated closely with the peak of neutrophil and elastase expression and activity. SLPI expression increased during resolving phase of the pleurisy. Therapeutic treatment of LPS-challenge mice with recombinant human SLPI or Elafin accelerated resolution, an effect associated with increased numbers of apoptotic neutrophils in the pleural exudates, inhibition of elastase, and modulation of the survival-controlling proteins NF-κB and Mcl-1. Similar effects were observed with SIV, which dose-dependently inhibited neutrophil elastase and shortened resolution intervals. Mechanistically, SIV-induced resolution was caspase-dependent, associated to increased levels of intact AnxA1 and decreased expression of NF-κB and Mcl-1. The proresolving effect of antiproteases was also observed in a model of monosodium urate crystals-induced inflammation. SIV skewed macrophages toward resolving phenotypes and enhanced efferocytosis of apoptotic neutrophils. A neutralizing antiserum against AnxA1 and a nonselective antagonist of AnxA1 receptor abolished the accelerated resolution promoted by SIV. Collectively, these results show that elastase inhibition not only inhibits inflammation but actually promotes resolution, and this response is mediated by protection of endogenous intact AnxA1 with ensuing augmentation of neutrophil apoptosis.
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Anexina A1/inmunología , Inflamación/inmunología , Inhibidores de Proteasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Citometría de Flujo , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Inflamación/metabolismo , Elastasa de Leucocito/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Neutrófilos/inmunología , Inhibidores de Proteasas/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/farmacología , Sulfonamidas/farmacologíaRESUMEN
BACKGROUND & AIMS: Resident macrophages are derived from yolk sac precursors and seed the liver during embryogenesis. Native cells may be replaced by bone marrow precursors during extensive injuries, irradiation, and infections. We investigated the liver populations of myeloid immune cells and their location, as well as the dynamics of phagocyte repopulation after full depletion. The effects on liver function due to the substitution of original phagocytes by bone marrow-derived surrogates were also examined. METHODS: We collected and analyzed liver tissues from C57BL/6 (control), LysM-EGFP, B6 ACTb-EGFP, CCR2-/-, CD11c-EYFP, CD11c-EYFP-DTR, germ-free mice, CX3CR1gfp/gfp, CX3CR1gpf/wt, and CX3CR1-DTR-EYFP. Liver nonparenchymal cells were immunophenotyped using mass cytometry and gene expression analyses. Kupffer and dendritic cells were depleted from mice by administration of clodronate, and their location and phenotype were examined using intravital microscopy and time-of-flight mass cytometry. Mice were given acetaminophen gavage or intravenous injections of fluorescently labeled Escherichia coli, blood samples were collected and analyzed, and liver function was evaluated. We assessed cytokine profiles of liver tissues using a multiplexed array. RESULTS: Using mass cytometry and gene expression analyses, we identified 2 populations of hepatic macrophages and 2 populations of monocytes. We also identified 4 populations of dendritic cells and 1 population of basophils. After selective depletion of liver phagocytes, intravascular myeloid precursors began to differentiate into macrophages and dendritic cells; dendritic cells migrated out of sinusoids, after a delay, via the chemokine CX3CL1. The cell distribution returned to normal in 2 weeks, but the repopulated livers were unable to fully respond to drug-induced injury or clear bacteria for at least 1 month. This defect was associated with increased levels of inflammatory cytokines, and dexamethasone accelerated the repopulation of liver phagocytes. CONCLUSIONS: In studies of hepatic phagocyte depletion in mice, we found that myeloid precursors can differentiate into liver macrophages and dendritic cells, which each localize to distinct tissue compartments. During replenishment, macrophages acquire the ability to respond appropriately to hepatic injury and to remove bacteria from the blood stream.
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Antígenos CD/análisis , Células de la Médula Ósea/fisiología , Diferenciación Celular , Hígado/citología , Hígado/fisiopatología , Células Mieloides/fisiología , Acetaminofén , Animales , Células de la Médula Ósea/citología , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Quimiocina CX3CL1/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/química , Inmunofenotipificación/métodos , Microscopía Intravital , Lectinas/genética , Hígado/inmunología , Hígado/metabolismo , Macrófagos/química , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microvasos/metabolismo , Monocitos/química , Células Mieloides/química , Fenotipo , TranscriptomaRESUMEN
Glucocorticoid (GC)-induced leucine zipper (GILZ) has been shown to mediate or mimic several actions of GC. This study assessed the role of GILZ in self-resolving and GC-induced resolution of neutrophilic inflammation induced by LPS in mice. GILZ expression was increased during the resolution phase of LPS-induced pleurisy, especially in macrophages with resolving phenotypes. Pretreating LPS-injected mice with trans-activator of transcription peptide (TAT)-GILZ, a cell-permeable GILZ fusion protein, shortened resolution intervals and improved resolution indices. Therapeutic administration of TAT-GILZ induced inflammation resolution, decreased cytokine levels, and promoted caspase-dependent neutrophil apoptosis. TAT-GILZ also modulated the activation of the survival-controlling proteins ERK1/2, NF-κB and Mcl-1. GILZ deficiency was associated with an early increase of annexin A1 (AnxA1) and did not modify the course of neutrophil influx induced by LPS. Dexamethasone treatment resolved inflammation and induced GILZ expression that was dependent on AnxA1. Dexamethasone-induced resolution was not altered in GILZ(-/-) mice due to compensatory expression and action of AnxA1. Our results show that therapeutic administration of GILZ efficiently induces a proapoptotic program that promotes resolution of neutrophilic inflammation induced by LPS. Alternatively, a lack of endogenous GILZ during the resolution of inflammation is compensated by AnxA1 overexpression.
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Inflamación/inmunología , Macrófagos/inmunología , Pleuresia/inmunología , Factores de Transcripción/inmunología , Animales , Anexina A1/inmunología , Apoptosis/inmunología , Western Blotting , Movimiento Celular , Modelos Animales de Enfermedad , Citometría de Flujo , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Irinotecan is a useful chemotherapeutic agent for the treatment of several solid tumors. However, this therapy is associated with side effects, including leukopenia and mucositis. Reactive oxygen species (ROS) activate inflammatory pathways and contribute to Irinotecan-induced mucositis. Fullerol is a nanocomposite with anti-oxidant properties that may reduce tissue damage after inflammatory stimuli. In this paper, the effects of Fullerol and mechanisms of protection were investigated in a model of Irinotecan-induced mucositis. Mucositis was induced by an injection of Irinotecan per 4 days in C57BL/6. Fullerol or a vehicle was injected every 12h. On day 7, the intestines were removed to evaluate histological changes, leukocyte influx, and the production of cytokines and ROS. Irinotecan therapy resulted in weight loss, an increased clinical score and intestinal injury. Treatment with Fullerol attenuated weight loss, decreased clinical score and intestinal damage. Irinotecan also induced increased ROS production in enterocytes, oxidative stress, IL-1ß production, neutrophil and eosinophil influx in the ileum. Fullerol treatment decreased production of ROS in the enterocytes, oxidative stress, IL-1ß production, neutrophil and eosinophil influx in the ileum. Irinotecan therapy also induced leukopenia in an ROS-dependent manner because leukopenia reverted in WT mice treated with Fullerol or Apocynin or in Gp91phox(-/-) mice. Mice treated with Irinotecan presented less melanoma tumor growth compared to the control group. Fullerol does not interfere in the anti-tumor action of Irinotecan. Fullerol has a great pharmacology potential to decreases the severity of mucositis and of leukopenia during chemotherapy treatment.