RESUMEN
Leptin is a pleiotropic adipokine that regulates immunometabolism centrally and peripherally. Obese individuals present increased levels of leptin in the blood and develop hypothalamic resistance to this adipokine. Here we investigated whether leptin effects on the periphery are maintained despite the hypothalamic resistance. We previously reported that leptin injection induces in vivo neutrophil migration and peritoneal macrophage activation in lean mice through TNF-α- and CXCL1-dependent mechanisms. However, leptin effects on leukocyte biology during obesity remain unclear. In this study, we investigated the in vivo responsiveness of leukocytes to i.p. injected leptin in mice with diet-induced obesity (DIO). After 14-16 wk, high-sucrose, high-fat diet (HFD)-fed mice showed hyperglycemia, hyperleptinemia, and dyslipidemia compared to normal-sucrose, normal-fat diet (ND). Exogenous leptin did not reduce food intake in DIO mice in contrast to control mice, indicating that DIO mice were centrally resistant to leptin. Regardless of the diet, we found increased levels of TNF-α and CXCL1 in the animals injected with leptin, alongside a pronounced neutrophil migration to the peritoneal cavity and enhanced biogenesis of lipid droplets in peritoneal macrophages. Supporting our in vivo results, data from ex vivo leptin stimulation experiments confirmed hypothalamic resistance in DIO mice, whereas bone marrow cells responded to leptin stimulation through mTOR signaling despite obesity. Altogether, our results show that leukocytes responded equally to leptin in ND- or HFD-fed mice. These results support a role for leptin in the innate immune response also in obesity, contributing to the inflammatory status that leads to the development of metabolic disease.
Asunto(s)
Inmunidad Innata , Leptina/metabolismo , Obesidad/etiología , Obesidad/metabolismo , Transducción de Señal , Animales , Biomarcadores , Citocinas/metabolismo , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Leucocitos/inmunología , Leucocitos/metabolismo , RatonesRESUMEN
Eosinophils are key regulators of adipose tissue homeostasis, thus characterization of adipose tissue-related molecular factors capable of regulating eosinophil activity is of great interest. Leptin is known to directly activate eosinophils in vitro, but leptin ability of inducing in vivo eosinophilic inflammatory response remains elusive. Here, we show that leptin elicits eosinophil influx as well as its activation, characterized by increased lipid body biogenesis and LTC4 synthesis. Such leptin-triggered eosinophilic inflammatory response was shown to be dependent on activation of the mTOR signaling pathway, since it was (i) inhibited by rapamycin pre-treatment and (ii) reduced in PI3K-deficient mice. Local infiltration of activated eosinophils within leptin-driven inflammatory site was preceded by increased levels of classical mast cell-derived molecules, including TNFα, CCL5 (RANTES), and PGD2. Thus, mice were pre-treated with a mast cell degranulating agent compound 48/80 which was capable to impair leptin-induced PGD2 release, as well as eosinophil recruitment and activation. In agreement with an indirect mast cell-driven phenomenon, eosinophil accumulation induced by leptin was abolished in TNFR-1 deficient and also in HQL-79-pretreated mice, but not in mice pretreated with neutralizing antibodies against CCL5, indicating that both typical mast cell-driven signals TNFα and PGD2, but not CCL5, contribute to leptin-induced eosinophil influx. Distinctly, leptin-induced eosinophil lipid body (lipid droplet) assembly and LTC4 synthesis appears to depend on both PGD2 and CCL5, since both HQL-79 and anti-CCL5 treatments were able to inhibit these eosinophil activation markers. Altogether, our data show that leptin triggers eosinophilic inflammation in vivo via an indirect mechanism dependent on activation of resident mast cell secretory activity and mediation by TNFα, CCL5, and specially PGD2.
Asunto(s)
Eosinófilos/efectos de los fármacos , Leptina/farmacología , Mastocitos/fisiología , Prostaglandina D2/fisiología , Animales , Movimiento Celular/efectos de los fármacos , Quimiocina CCL5/fisiología , Eosinófilos/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Leptin is a cytokine, produced mainly by mature adipocytes, that regulates the central nervous system, mainly to suppress appetite and stimulate energy expenditure. Leptin also regulates the immune response by controlling activation of immunomodulatory cells, including eosinophils. While emerging as immune regulatory cells with roles in adipose tissue homeostasis, eosinophils have a well-established ability to synthesize pro-inflammatory molecules such as lipid mediators, a key event in several inflammatory pathologies. Here, we investigated the impact and mechanisms involved in leptin-driven activation of eicosanoid-synthesizing machinery within eosinophils. Direct in vitro activation of human or mouse eosinophils with leptin elicited synthesis of lipoxygenase as well as cyclooxygenase products. Displaying selectivity, leptin triggered synthesis of LTC4 and PGD2, but not PGE2, in parallel to dose-dependent induction of lipid body/lipid droplets biogenesis. While dependent on PI3K activation, leptin-driven eosinophil activation was also sensitive to pertussis toxin, indicating the involvement of G-protein coupled receptors on leptin effects. Leptin-induced lipid body-driven LTC4 synthesis appeared to be mediated through autocrine activation of G-coupled CCR3 receptors by eosinophil-derived CCL5, inasmuch as leptin was able to trigger rapid CCL5 secretion, and neutralizing anti-RANTES or anti-CCR3 antibodies blocked lipid body assembly and LTC4 synthesis induced by leptin. Remarkably, autocrine activation of PGD2 G-coupled receptors DP1 and DP2 also contributes to leptin-elicited lipid body-driven LTC4 synthesis by eosinophils in a PGD2-dependent fashion. Blockade of leptin-induced PGD2 autocrine/paracrine activity by a specific synthesis inhibitor or DP1 and DP2 receptor antagonists, inhibited both lipid body biogenesis and LTC4 synthesis induced by leptin stimulation within eosinophils. In addition, CCL5-driven CCR3 activation appears to precede PGD2 receptor activation within eosinophils, since neutralizing anti-CCL5 or anti-CCR3 antibodies inhibited leptin-induced PGD2 secretion, while it failed to alter PGD2-induced LTC4 synthesis. Altogether, sequential activation of CCR3 and then PGD2 receptors by autocrine ligands in response to leptin stimulation of eosinophils culminates with eosinophil activation, characterized here by assembly of lipidic cytoplasmic platforms synthesis and secretion of the pleiotropic lipid mediators, PGD2, and LTC4.
Asunto(s)
Eosinófilos/inmunología , Leptina/metabolismo , Leucotrieno C4/biosíntesis , Receptores CCR3/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Animales , Células Cultivadas , Quimiocina CCL5/antagonistas & inhibidores , Quimiocina CCL5/metabolismo , Eosinófilos/citología , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Femenino , Humanos , Hidantoínas/farmacología , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/metabolismo , Leptina/inmunología , Leucotrieno C4/inmunología , Gotas Lipídicas/inmunología , Gotas Lipídicas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Piperidinas/farmacología , Cultivo Primario de Células , Prostaglandina D2/metabolismo , Receptores CCR3/antagonistas & inhibidores , Receptores CCR3/inmunología , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/inmunología , Receptores de Prostaglandina/antagonistas & inhibidores , Receptores de Prostaglandina/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
Leptin directly activates macrophages and lymphocytes, but the role of leptin in neutrophil activation and migration is still controversial. Here, we investigate the in vivo mechanisms of neutrophil migration induced by leptin. The intraperitoneal injection of leptin (1 mg/kg) induces a time- and concentration-dependent neutrophil influx. We did not observe the enhancement of lipid bodies/droplets in neutrophils, after leptin treatment, as we had observed previously in peritoneal macrophages. The participation of leukotriene B4 (LTB4) in neutrophil recruitment triggered by leptin was investigated using different strategies. Leptin-induced neutrophil recruitment occurs both in the absence of 5-lipoxygenase activity in 5-lipoxygenase (5-LO)-/- mice and after the administration of either 5-LO inhibitor (Zileuton) or the LTB4 receptor antagonist (U-75302). Moreover, no direct induction of LTB4 by leptin could be observed. Neutrophil influx could not be prevented by the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, contrasting with the leptin-induced signaling for lipid body formation in macrophage that is mTOR-dependent. Leptin administration led to tumor necrosis factor-alpha (TNFα) production by the peritoneal cells both in vivo and in vitro. In addition, neutrophil recruitment was inhibited in tumor necrosis factor receptor 1 (TNFR1-/-) mice, indicating a role for TNF in leptin-induced neutrophil recruitment to the peritoneal cavity. Leptin-induced neutrophil influx was PI3Kγ-dependent, as it was absent in PI3Kγ-/- mice. Accordingly, leptin induced the peritoneal cells to produce CXCL1, both in vivo and in vitro, and the neutrophil influx was ablated after using an antibody against CXCL1. Our results establish TNFα/TNFR1- and CXCL1-dependent signaling as important pathways for leptin-induced neutrophil migration in vivo.
Asunto(s)
Quimiocina CXCL1/fisiología , Leptina/fisiología , Neutrófilos/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Araquidonato 5-Lipooxigenasa/genética , Movimiento Celular , Quimiocina CCL3/genética , Macrófagos Peritoneales/inmunología , Masculino , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila , Fosfatidilinositol 3-Quinasas/genéticaRESUMEN
Chikungunya fever is an emerging disease and a significant public health problem in tropical countries. Recently reported outbreaks in Brazil in 2015 drew attention to the need to develop prevention and treatment options, as no antiviral chemotherapy or vaccines are currently available for this disease. Two strategies have been proved to accelerate the discovery of new anti-infectives: phenotypic screening and drug repurposing. Phenotypic screening can support the fast interrogation of compounds without the need for a pre-validated drug target, which is not available for the chikungunya virus (CHIKV) and has the additional advantage of facilitating the discovery of antiviral with novel mechanism of action. Drug repurposing can save time and resources in drug development by enabling secondary uses for drugs that are already approved for human treatment, thus precluding the need for several of the mandatory preclinical and clinical studies necessary for drug approval. A phenotypic screening assay was developed by infecting the human hepatoma Huh-7 cells with CHIKV 181/25 and quantifying infection through indirect immunofluorescence. The compound 6-azauridine was used as a positive control drug. The screening assay was validated by testing a commercial library of 1,280 compounds, including FDA-approved drugs, and used to screen a panel of broad-spectrum antiviral compounds for anti-CHIKV activity. A high content assay was set up in Huh-7 cells-infected with CHIKV. The maximum rate of infection peaked at 48 hours post-infection, after which the host cell number was greatly reduced due to a strong cytopathic effect. Assay robustness was confirmed with Z’-factor values >0.8 and high correlation coefficient between independent runs, demonstrating that the assay is reliable, consistent and reproducible. Among tested compounds, sofosbuvir, an anti-hepatitis C virus drug, exhibited good selectivity against CHIKV with an EC50 of 11 µM, suggesting it is a promising candidate for repurposing.