RESUMO
Uncontrolled inflammation leads to tissue damage and it is central for the development of chronic inflammatory diseases and autoimmunity. An acute inflammatory response is finely regulated by the action of anti-inflammatory and pro-resolutive mediators, culminating in the resolution of inflammation and restoration of homeostasis. There are few studies investigating intracellular signaling pathways associated with the resolution of inflammation. Here, we investigate the role of Rho-associated kinase (ROCK), a serine/threonine kinase, in a model of self-resolving neutrophilic inflammatory. We show that ROCK activity, evaluated by P-MYPT-1 kinetics, was higher during the peak of lipopolysaccharide-induced neutrophil influx in the pleural cavity of mice. ROCK inhibition by treatment with Y-27632 decreased the accumulation of neutrophils in the pleural cavity and was associated with an increase in apoptotic events and efferocytosis, as evaluated by an in vivo assay. In a model of gout, treatment with Y-27632 reduced neutrophil accumulation, IL-1ß levels and hypernociception in the joint. These were associated with reduced MYPT and IκBα phosphorylation levels and increased apoptosis. Finally, inhibition of ROCK activity also induced apoptosis in human neutrophils and destabilized cytoskeleton, extending the observed effects to human cells. Taken together, these data show that inhibition of the ROCK pathway might represent a potential therapeutic target for neutrophilic inflammatory diseases.
Assuntos
Amidas/administração & dosagem , Inflamação/tratamento farmacológico , Lipopolissacarídeos/efeitos adversos , Neutrófilos/citologia , Piridinas/administração & dosagem , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas I-kappa B/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Masculino , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Piridinas/farmacologiaRESUMO
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.
Assuntos
Inflamação/imunologia , Lisofosfolipídeos/metabolismo , Neutrófilos/imunologia , Cavidade Pleural/imunologia , Receptores de Esfingosina-1-Fosfato/metabolismo , Esfingosina/análogos & derivados , Animais , Apoptose , Sobrevivência Celular , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ativação de Neutrófilo , Transdução de Sinais , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/antagonistas & inibidoresRESUMO
Defective apoptosis of eosinophils, the main leukocyte in the pathogenesis of asthma, and delay in its removal lead to lung damage and loss of pulmonary function due to failure in the resolution of inflammation. Here, we investigated the ability of angiotensin-(1-7) [Ang-(1-7)], a pivotal peptide of the renin-angiotensin system, to promote resolution of an allergic lung inflammatory response. Balb/c mice were sensitized and challenged with ovalbumin and treated with Ang-(1-7) at the peak of the inflammatory process. Bronchoalveolar lavage (BAL) fluid and lungs were collected 24 h after treatment. Different lung lobes were processed for histology to evaluate inflammatory cell infiltration, airway and pulmonary remodeling, total collagen staining, and measurements of (i) collagen I and III mRNA expression by qRT-PCR; (ii) ERK1/2, IκB-α, and GATA3 protein levels by Western blotting; and (iii) eosinophilic peroxidase activity. Total number of inflammatory cells, proportion of apoptotic eosinophils and immunofluorescence for caspase 3 and NF-κB in leukocytes were evaluated in the BAL. Mas receptor immunostaining was evaluated in mouse and human eosinophils. Engulfment of human polimorphonuclear cells by macrophages, efferocytosis, was evaluated in vivo. Ang-(1-7) reduced eosinophils in the lung and in the BAL, increased the number of apoptotic eosinophils, shown by histology criteria and by increase in caspase 3 immunostaining. Furthermore, Ang-(1-7) decreased NF-kB immunostaining in eosinophils, reduced GATA3, ERK1/2, and IκB-α expression in the lung and decreased pulmonary remodeling and collagen deposition. Importantly, Ang-(1-7) increased efferocytosis. Our results demonstrate, for the first time, Ang-(1-7) activates events that are crucial for resolution of the inflammatory process of asthma and promotion of the return of lung homeostasis, indicating Ang-(1-7) as novel endogenous inflammation-resolving mediator.
Assuntos
Angiotensina I/metabolismo , Asma/imunologia , Asma/metabolismo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Fragmentos de Peptídeos/metabolismo , Angiotensina I/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Líquido da Lavagem Broncoalveolar , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Eosinófilos/efeitos dos fármacos , Imunofluorescência , Fator de Transcrição GATA3/metabolismo , Contagem de Leucócitos , Masculino , Camundongos , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Fragmentos de Peptídeos/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Leukotriene B4 (LTB4), a proinflammatory mediator produced by the enzyme 5-lipoxygenase (5-LO), is associated with the development of many inflammatory diseases. In this study, we evaluated the participation of the 5-LO/LTB4 axis in graft-versus-host disease (GVHD) pathogenesis by transplanting 5-LO-deficient leukocytes and investigated the effect of pharmacologic 5-LO inhibition by zileuton and LTB4 inhibition by CP-105,696. Mice that received allogeneic transplant showed an increase in nuclear 5-LO expression in splenocytes, indicating enzyme activation after GVHD. Mice receiving 5-LO-deficient cell transplant or zileuton treatment had prolonged survival, reduced GVHD clinical scores, reduced intestinal and liver injury, and decreased levels of serum and hepatic LTB4 These results were associated with inhibition of leukocyte recruitment and decreased production of cytokines and chemokines. Treatment with CP-105,696 achieved similar effects. The chimerism or the beneficial graft-versus-leukemia response remained unaffected. Our data provide evidence that the 5-LO/LTB4 axis orchestrates GVHD development and suggest it could be a target for the development of novel therapeutic strategies for GVHD treatment.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Transplante de Células/métodos , Doença Enxerto-Hospedeiro/metabolismo , Leucotrieno B4/metabolismo , Animais , Araquidonato 5-Lipoxigenase/genética , Benzopiranos/farmacologia , Ácidos Carboxílicos/farmacologia , Transplante de Células/efeitos adversos , Quimiocinas/metabolismo , Citocinas/metabolismo , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/etiologia , Hidroxiureia/análogos & derivados , Hidroxiureia/farmacologia , Leucócitos/citologia , Leucócitos/enzimologia , Leucócitos/metabolismo , Antagonistas de Leucotrienos/farmacologia , Leucotrieno B4/antagonistas & inibidores , Inibidores de Lipoxigenase/farmacologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Confocal , Transplante HomólogoRESUMO
Platelet-activating factor (PAF) is known to be an important mediator of anaphylaxis. However, there is a lack of information in the literature about the role of PAF in food allergy. The aim of this work was to elucidate the participation of PAF during food allergy development and the consequent adipose tissue inflammation along with its alterations. Our data demonstrated that, both before oral challenge and after 7 days receiving ovalbumin (OVA) diet, OVA-sensitized mice lacking the PAF receptor (PAFR) showed a decreased level of anti-OVA IgE associated with attenuated allergic markers in comparison to wild type (WT) mice. Moreover, there was less body weight and adipose tissue loss in PAFR-deficient mice. However, some features of inflamed adipose tissue presented by sensitized PAFR-deficient and WT mice after oral challenge were similar, such as a higher rate of rolling leukocytes in this tissue and lower circulating levels of adipokines (resistin and adiponectin) in comparison to nonsensitized mice. Therefore, PAF signaling through PAFR is important for the allergic response to OVA but not for the adipokine alterations caused by this inflammatory process. Our work clarifies some effects of PAF during food allergy along with its role on the metabolic consequences of this inflammatory process.
Assuntos
Adipocinas/sangue , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade Alimentar/metabolismo , Glicoproteínas da Membrana de Plaquetas/imunologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Ração Animal , Animais , Biomarcadores/sangue , Peso Corporal/imunologia , Dieta , Hipersensibilidade Alimentar/sangue , Imunoglobulina E/imunologia , Inflamação/sangue , Inflamação/imunologia , Inflamação/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Ovalbumina/imunologiaRESUMO
OBJECTIVE: Allergic mice show a reduction in body weight and adiposity with a higher inflammatory response in the adipose tissue similar to obese fat tissue. This study aimed to evaluate whether the low-grade inflammatory milieu of mice with diet-induced mild obesity interferes with the allergic response induced by ovalbumin (OVA). METHODS: BALB/c mice were divided into four groups: 1) non-allergic (OVA-) mice fed chow diet, 2) allergic (OVA+) mice fed chow diet, 3) OVA- mice fed high-refined carbohydrate-containing (HC) diet, and 4) OVA+ mice fed HC diet. After 5 wk, allergic groups were sensitized with OVA and received a booster 14 d later. All groups received an oral OVA challenge 7 d after the booster. RESULTS: Allergic groups showed increased serum levels of total IgE, anti-OVA IgE, and IgG1; a high disease activity index score; aversion to OVA; and increased intestinal eosinophil infiltration. Non-allergic mild-obese mice also showed aversion to OVA and an increased number of eosinophils in the proximal jejunum. After the allergic challenge, OVA+ mice fed chow diet showed weight loss and lower adiposity in several adipose tissue depots. OVA+ mice fed HC diet showed a loss of fat mass only in the mesenteric adipose tissue. Furthermore, increased levels of TNF, IL-6, and IL-10 were observed in this tissue. CONCLUSIONS: Our data show that mild-obese allergic mice do not present severe pathologic features of food allergy similar to those exhibited by lean allergic mice. Mild obesity promoted by HC diet ingestion causes important intestinal disorders that appear to modulate the inflammatory response during the antigen challenge.
Assuntos
Dieta , Carboidratos da Dieta/administração & dosagem , Hipersensibilidade Alimentar/imunologia , Tecido Adiposo/metabolismo , Adiposidade , Animais , Peso Corporal , Hipersensibilidade Alimentar/sangue , Teste de Tolerância a Glucose , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Inflamação/sangue , Inflamação/imunologia , Resistência à Insulina , Interleucina-10/sangue , Interleucina-6/sangue , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Obesidade/metabolismo , Ovalbumina/imunologiaRESUMO
BACKGROUND: Ethanol (EtOH) consumption is able to disturb the ovalbumin (OVA)-oral tolerance induction by interfering on the function of antigen presenting cells (APC), down-regulating dendritic cells (DCs) and macrophages and up-regulating B-lymphocytes and their function, which results in an overall allergic-type immune status. In this study, the potential of a priori administration of Lactococcus lactis (LL) in avoiding loss of oral tolerance in EtOH-treated mice was investigated. METHODS: Female C57BL/6 mice received, by oral route, ad libitum wild-type (WT) LL or heat-shock protein producer (Hsp65) LL for 4 consecutive days. Seven days later, mice were submitted to short-term high-dose EtOH treatment. After 24 hours, stomach, intestine, spleen, mesenteric lymph nodes (mLN) specimens were collected for biomarkers analysis. Following EtOH-treatment protocol, a group of animals underwent single-gavage OVA-tolerance protocol and sera samples collected for antibody analysis. RESULTS: The ingestion of WT LL or Hsp65 LL is able to restore oral tolerance to OVA in EtOH-treated mice, by reducing local and systemic allergic outcomes such as gastric mast cells and gut-interleukin-4, as well as serum IgE. WT LL treatment prevents the decrease of mLN regulatory T cells induced by the EtOH treatment. Moreover, LL treatment preserves APC hierarchy and antigen presentation commitment in EtOH-treated mice, with conserved DC and macrophage activity over B lymphocytes in mLN and preserved macrophage activity over DC and B-cell subsets in the spleen. CONCLUSIONS: The present findings suggest that a priori ingestion of LL preserves essential mechanisms associated with oral tolerance induction that are disturbed by EtOH ingestion. Maintenance of mucosal homeostasis by preserving APC hierarchy and antigen presentation commitment could be associated with T-regulatory subset activities in the gastrointestinal tract.
Assuntos
Apresentação de Antígeno/imunologia , Etanol/administração & dosagem , Trato Gastrointestinal/imunologia , Tolerância Imunológica/imunologia , Lactococcus lactis , Administração Oral , Animais , Apresentação de Antígeno/efeitos dos fármacos , Feminino , Trato Gastrointestinal/efeitos dos fármacos , Tolerância Imunológica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
We investigated the effect of dietary supplementation with n-3 PUFA (fish oil source) in an experimental model of food allergy. Mice were sensitized (allergic group) or not (nonallergic group) with OVA and were fed with OVA diet to induce allergy signals. Mice were fed with regular diet in which 7% of lipid content was provided by soybean (5% of n-3 PUFA) or fish (25% of n-3 PUFA) oil. Allergic group mice had increased serum levels of antiovalbumin IgE and IgG1 and changes in small intestine, characterized by an increased edema, number of rolling leukocytes in microcirculation, eosinophil infiltration, mucus production, and Paneth cell degranulation, in comparison to non-allergic group. All these inflammatory parameters were reduced in mice fed high-n-3-PUFA diet. Our data together suggest that diet supplementation with n-3 PUFA from fish oil may consist of a valid adjuvant in food allergy treatment.
Assuntos
Ácidos Graxos Ômega-3/farmacologia , Óleos de Peixe/farmacologia , Hipersensibilidade Alimentar/metabolismo , Ovalbumina/imunologia , Animais , Suplementos Nutricionais , Ácidos Graxos Ômega-3/administração & dosagem , Feminino , Óleos de Peixe/administração & dosagem , Hipersensibilidade Alimentar/imunologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
To investigate the consequences of food allergy in adipose tissue and metabolism, we used a murine model in which mice have been sensitized subcutaneously with ovalbumin and further received antigen-containing diet. Allergic mice presented a significant weight loss 7 days after oral challenge with a concomitant decrease in epididymal adipose tissue mass. This decrease was associated with increased lipolysis and local inflammation. In adipose tissue of allergic mice there were increased leukocyte rolling and adhesion in the microvasculature, increased number of leukocytes in the tissue, especially macrophages (F4/80(+) cells) and increased pro-inflammatory cytokines levels, including TNF-α, IL-6 and CCL2. In addition, we observed low serum concentrations of triglyceride, glucose, total cholesterol and free fatty acids in the allergic mice. Our results suggest that the induction of food allergy in mice leads to adipose tissue inflammation and systemic metabolic alterations that contribute to the weight loss observed.