RESUMO
Inflammatory vasculopathy is critical in sickle cell disease (SCD)-associated organ damage. An imbalance between pro-inflammatory and pro-resolving mechanisms in response to different triggers such as hypoxia/reoxygenation or infections has been proposed to contribute to the progression of SCD. Administration of specialized pro-resolving lipid mediators may provide an effective therapeutic strategy to target inflammatory vasculopathy and to modulate inflammatory response. Epeleuton (15 hydroxy eicosapentaenoic acid ethyl ester) is a novel, orally administered, second-generation ω-3 fatty acid with a favorable clinical safety profile. In this study we show that epeleuton re-programs the lipidomic pattern of target organs for SCD towards a pro-resolving pattern. This protects against systemic and local inflammatory responses and improves red cell features, resulting in reduced hemolysis and sickling compared with that in vehicle-treated SCD mice. In addition, epeleuton prevents hypoxia/reoxygenation-induced activation of nuclear factor-κB with downregulation of the NLRP3 inflammasome in lung, kidney, and liver. This was associated with downregulation of markers of vascular activation in epeleuton-treated SCD mice when compared to vehicle-treated animals. Collectively our data support the potential therapeutic utility of epeleuton and provide the rationale for the design of clinical trials to evaluate the efficacy of epeleuton in patients with SCD.
Assuntos
Anemia Falciforme , Modelos Animais de Doenças , Traumatismo por Reperfusão , Animais , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Anemia Falciforme/complicações , Camundongos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Humanos , Masculino , Hipóxia/metabolismo , Hipóxia/tratamento farmacológicoRESUMO
Mucus plugging and non-resolving inflammation are inherent features of cystic fibrosis (CF) that may lead to progressive lung disease and exercise intolerance, which are the main causes of morbidity and mortality for people with CF. Therefore, understanding the influence of mucus on basic mechanisms underlying the inflammatory response and identifying strategies to resolve mucus-driven airway inflammation and consequent morbidity in CF are of wide interest. Here, we investigated the effects of the proresolving lipid mediator resolvin (Rv) D1 on mucus-related inflammation as a proof-of-concept to alleviate the burden of lung disease and restore exercise intolerance in CF. We tested the effects of RvD1 on inflammatory responses of human organotypic airways and leukocytes to CF mucus and of humanized mice expressing the epithelial Na + channel (ßENaC-Tg) having CF-like mucus obstruction, lung disease, and physical exercise intolerance. RvD1 reduced pathogenic phenotypes of CF-airway supernatant (ASN)-stimulated human neutrophils, including loss of L-selectin shedding and CD16. RNASeq analysis identified select transcripts and pathways regulated by RvD1 in ASN-stimulated CF bronchial epithelial cells that are involved in sugar metabolism, NF-κB activation and inflammation, and response to stress. In in vivo inflammation using ßENaC TG mice, RvD1 reduced total leukocytes, PMN, and interstitial Siglec-MΦ when given at 6-8 weeks of age, and in older mice at 10-12 weeks of age, along with the decrease of pro-inflammatory chemokines and increase of anti-inflammatory IL-10. Furthermore, RvD1 treatment promoted the resolution of pulmonary exacerbation caused by Pseudomonas aeruginosa infection and significantly enhanced physical activity and energy expenditure associated with mucus obstruction, which was impaired in ßENaC-Tg mice compared with wild-type. These results demonstrate that RvD1 can rectify features of CF and offer proof-of-concept for its therapeutic application in this and other muco-obstructive lung diseases.
Assuntos
Fibrose Cística , Humanos , Camundongos , Animais , Fibrose Cística/genética , Tolerância ao Exercício , Pulmão/metabolismo , Inflamação/metabolismoRESUMO
Cardiovascular (CV) disease prevention with low-dose aspirin can be less effective in patients with a faster recovery of platelet (PLT) cyclooxygenase (COX)-1 activity during the 24-hour dosing interval. We previously showed that incomplete suppression of TXA2 over 24 hours can be rescued by a twice daily aspirin regimen. Here we show that reduced PLT glycoprotein (GP)Ibα shedding characterizes patients with accelerated COX-1 recovery and may contribute to higher thrombopoietin (TPO) production and higher rates of newly formed PLT, escaping aspirin inhibition over 24 hours. Two hundred aspirin-treated patients with high CV risk (100 with type 2 diabetes mellitus) were stratified according to the kinetics of PLT COX-1 activity recovery during the 10- to 24-hour dosing interval. Whole proteome analysis showed that PLT from patients with accelerated COX-1 recovery were enriched in proteins involved in cell survival, inhibition of apoptosis and cellular protrusion formation. In agreement, we documented increased plasma TPO, megakaryocyte maturation and proplatelet formation, and conversely increased PLT galactose and reduced caspase 3, phosphatidylserine exposure and ADAM17 activation, translating into diminished GPIbα cleavage and glycocalicin (GC) release. Treatment of HepG2 cells with recombinant GC led to a dose-dependent reduction of TPO mRNA in the liver, suggesting that reduced GPIbα ectodomain shedding may unleash thrombopoiesis. A cluster of clinical markers, including younger age, non-alcoholic fatty liver disease, visceral obesity and higher TPO/GC ratio, predicted with significant accuracy the likelihood of faster COX-1 recovery and suboptimal aspirin response. Circulating TPO/GC ratio, reflecting a dysregulation of PLT lifespan and production, may provide a simple tool to identify patients amenable to more frequent aspirin daily dosing.
Assuntos
Diabetes Mellitus Tipo 2 , Trombocitopenia , Humanos , Aspirina/farmacologia , Trombopoese , Diabetes Mellitus Tipo 2/metabolismo , Plaquetas/metabolismo , Trombocitopenia/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismoRESUMO
The COVID-19 pandemics has made sparkly evident the importance of acute inflammation and its timely resolution to protect humans from pathogenic viruses while sparing them from collateral damages due to an uncontrolled immune response. It is clear now that resolution of inflammation is an active process regulated by endogenous specialized proresolving lipid mediators (SPM) biosynthesized from essential polyunsaturated fatty acids. Accruing evidence indicates that SPM are produced during viral infections and play key roles in controlling the magnitude and duration of the inflammatory response and in regulating adaptive immunity. Here, we reviewed biosynthesis and bioactions of SPM in virus-mediated human diseases. Harnessing SPM and their proresolutive actions can help in providing new therapeutic approaches to current and future human viral diseases by controlling infection, stimulating host immunity, and protecting from organ damage.
Assuntos
COVID-19 , Humanos , Inflamação/tratamento farmacológico , Inflamação/patologia , Eicosanoides , Mediadores da Inflamação , Ácidos Docosa-HexaenoicosRESUMO
An excessive, non-resolving inflammatory response underlies severe COVID-19 that may have fatal outcomes. Therefore, the investigation of endogenous pathways leading to resolution of inflammation is of interest to uncover strategies for mitigating inflammation in people with SARS-CoV-2 infection. This becomes particularly urgent in individuals with preexisting pathologies characterized by chronic respiratory inflammation and prone to bacterial infection, such as cystic fibrosis (CF). Here, we analyzed the immune responses to SARS-CoV-2 virion spike 1 glycoprotein (S1) of macrophages (MΦ) from volunteers with and without CF and tested the efficacy of resolvins (Rv) D1 and D2 in regulating the inflammatory and antimicrobial functions of MΦ exposed to S1. S1 significantly increased chemokine release, including interleukin (IL)-8, in CF and non-CF MΦ, while it enhanced IL-6 and tumor necrosis factor (TNF)-α in non-CF MΦ, but not in CF cells. S1 also triggered the biosynthesis of RvD1 and modulated microRNAs miR-16, miR-29a, and miR-103, known to control the inflammatory responses. RvD1 and RvD2 treatment abated S1-induced inflammatory responses in CF and non-CF MΦ, significantly reducing the release of select chemokines and cytokines including IL-8 and TNF-α. RvD1 and RvD2 both restored the expression of miR-16 and miR-29a, while selectively increasing miR-223 and miR-125a, which are involved in NF-κB activation and MΦ inflammatory polarization. During Pseudomonas aeruginosa infection, S1 stimulated the MΦ phagocytic activity that was further enhanced by RvD1 and RvD2. These results provide a map of molecular responses to SARS-CoV-2 in MΦ, key determinants of COVID-19-related inflammation, unveiling some peculiarity in the response of cells from individuals with CF. They also demonstrate beneficial, regulatory actions of RvD1 and RvD2 on SARS-CoV-2-induced inflammation.
Assuntos
COVID-19 , Fibrose Cística , Ácidos Docosa-Hexaenoicos/farmacologia , Macrófagos , Infecções por Pseudomonas , Pseudomonas aeruginosa/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , COVID-19/imunologia , COVID-19/microbiologia , COVID-19/patologia , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Fibrose Cística/virologia , Citocinas/imunologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Inflamação/virologia , Macrófagos/imunologia , Macrófagos/microbiologia , Macrófagos/patologia , Macrófagos/virologia , Masculino , MicroRNAs/imunologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/patologia , Infecções por Pseudomonas/virologiaRESUMO
In human medicine, the progression from early neoplasia development to either complete resolution of tumorigenesis and associated inflammation or chronicity and fatal outcomes remain difficult to predict. Resolution of inflammation is an active process that stimulates the termination of the inflammatory response and promotes return to homeostasis, while failure in resolution contributes to the development of a number of diseases. To understand how resolution pathways contribute to tumorigenesis, we defined and employed a cumulative score based on the expression level of genes involved in synthesis, signaling, and metabolism of the D-series resolvin (RvD). This score was used for comparative analyses of clinical, cellular, and molecular features of tumors, based on RNA-sequencing (RNA-seq) datasets collected within The Cancer Genome Atlas (TCGA) program. Our results indicate that higher RvD scores are associated with better clinical outcome of patients with head and neck squamous cell carcinoma (HNSC), and with molecular and cellular signatures indicative of enhanced anti-tumor immunity and better response to immune-checkpoint inhibitors (ICI), also in human papilloma virus (HPV) negative HNSC subtypes. Thus, higher activity of the RvD pathway identifies patients with improved resolution and a more efficient immune reaction against cancer.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Biomarcadores Tumorais/genética , Carcinogênese , Carcinoma de Células Escamosas/metabolismo , Transformação Celular Neoplásica , Expressão Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Humanos , Inflamação , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Resolvins (Rvs), endogenous lipid mediators, play a key role in the resolution of inflammation. Sickle cell disease (SCD), a genetic disorder of hemoglobin, is characterized by inflammatory and vaso-occlusive pathologies. We document altered proresolving events following hypoxia/reperfusion in humanized SCD mice. We demonstrate novel protective actions of 17R-resolvin D1 (17R-RvD1; 7S, 8R, 17R-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid) in reducing ex vivo human SCD blood leukocyte recruitment by microvascular endothelial cells and in vivo neutrophil adhesion and transmigration. In SCD mice exposed to hypoxia/reoxygenation, oral administration of 17R -RvD1 reduces systemic/local inflammation and vascular dysfunction in lung and kidney. The mechanism of action of 17R-RvD1 involves (1) enhancement of SCD erythrocytes and polymorphonuclear leukocyte efferocytosis, (2) blunting of NF-κB activation, and (3) a reduction in inflammatory cytokines, vascular activation markers, and E-selectin expression. Thus, 17R-RvD1 might represent a new therapeutic strategy for the inflammatory vasculopathy of SCD.
Assuntos
Anemia Falciforme/complicações , Anti-Inflamatórios não Esteroides/administração & dosagem , Ácidos Docosa-Hexaenoicos/administração & dosagem , Nefropatias/prevenção & controle , Pneumonia/prevenção & controle , Animais , Citocinas/metabolismo , Humanos , Nefropatias/etiologia , Nefropatias/patologia , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Pneumonia/etiologia , Pneumonia/patologiaRESUMO
Autophagy is a catabolic pathway that accounts for degradation and recycling of cellular components to extend cell survival under stress conditions. In addition to this prominent role, recent evidence indicates that autophagy is crucially involved in the regulation of the inflammatory response, a tightly controlled process aimed at clearing the inflammatory stimulus and restoring tissue homeostasis. To be efficient and beneficial to the host, inflammation should be controlled by a resolution program, since uncontrolled inflammation is the underlying cause of many pathologies. Resolution of inflammation is an active process mediated by a variety of mediators, including the so-called specialized pro-resolving lipid mediators (SPMs), a family of endogenous lipid autacoids known to regulate leukocyte infiltration and activities, and counterbalance cytokine production. Recently, regulation of autophagic mechanisms by these mediators has emerged, uncovering unappreciated connections between inflammation resolution and autophagy. Here, we summarize mechanisms of autophagy and resolution, focusing on the contribution of autophagy in sustaining paradigmatic examples of chronic inflammatory disorders. Then, we discuss the evidence that SPMs can restore dysregulated autophagy, hypothesizing that resolution of inflammation could represent an innovative approach to modulate autophagy and its impact on the inflammatory response.
Assuntos
Autofagia , Ácidos Docosa-Hexaenoicos/metabolismo , Eicosanoides/metabolismo , Inflamação/metabolismo , Animais , Doença Crônica , HumanosRESUMO
The proresolution lipid mediator lipoxin (LX)A4 bestows protective bioactions on endothelial cells. We examined the impact of LXA4 on transcellular endothelial signaling via microRNA (miR)-containing microvesicles. We report LXA4 inhibition of MV release by TNF-α-treated HUVECs, associated with the down-regulation of 18 miR in endothelial microvesicles (EMVs) and the up-regulation of miR-126-5p, both in HUVECs and in EMVs. LXA4 up-regulated miR-126-5p by â¼5-fold in HUVECs and promoted a release of microvesicles (LXA4-EMVs) that enhanced miR-126-5p by â¼7-fold in recipient HUVECs. In these cells, LXA4-EMVs abrogated the up-regulation of VCAM-1, induced in recipient HUVECs by EMVs released by untreated or TNF-α-treated HUVECs. LXA4-EMVs also reduced by â¼40% the expression of SPRED1, which we validated as an miR-126-5p target, whereas they stimulated monolayer repair in an in vitro wound assay. This effect was lost when the EMVs were depleted of miR-126-5p. These results provide evidence that changes in miR expression and microvesicle packaging and transfer represent a mechanism of action of LXA4, which may be relevant in vascular biology and inflammation.-Codagnone, M., Recchiuti, A., Lanuti, P., Pierdomenico, A. M., Cianci, E., Patruno, S., Mari, V. C., Simiele, F., Di Tomo, P., Pandolfi, A., Romano, M. Lipoxin A4 stimulates endothelial miR-126-5p expression and its transfer via microvesicles.
Assuntos
Micropartículas Derivadas de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Lipoxinas/farmacologia , MicroRNAs/genética , Linhagem Celular , Micropartículas Derivadas de Células/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Lipoxin (LX) A4, a main stop signal of inflammation, exerts potent bioactions by activating a specific G protein-coupled receptor, termed formyl peptide receptor 2 and recently renamed ALX/FPR2. Knowledge of the regulatory mechanisms that drive ALX/FPR2 gene expression is key for the development of innovative anti-inflammatory pharmacology. Here, we examined chromatin patterns of the ALX/FPR2 gene. We report that in MDA-MB231 breast cancer cells, the ALX/FPR2 gene undergoes epigenetic silencing characterized by low acetylation at lysine 27 and trimethylation at lysine 4, associated with high methylation at lysine 27 of histone 3. This pattern, which is consistent with transcriptionally inaccessible chromatin leading to low ALX/FPR2 mRNA and protein expression, is reversed in polymorphonuclear leukocytes that express high ALX/FPR2 levels. Activation of p300 histone acetyltransferase and inhibition of DNA methyltransferase restored chromatin accessibility and significantly increased ALX/FPR2 mRNA transcription and protein levels in MDA-MB231 cells, as well as in pulmonary artery endothelial cells. In both cells types, changes in the histone acetylation/methylation status enhanced ALX/FPR2 signaling in response to LXA4. Collectively, these results uncover unappreciated epigenetic regulation of ALX/FPR2 expression that can be exploited for innovative approaches to inflammatory disorders.
Assuntos
Epigênese Genética , Histonas/genética , Lipoxinas/metabolismo , RNA Mensageiro/genética , Receptores de Formil Peptídeo/genética , Receptores de Lipoxinas/genética , Acetilação , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Histonas/metabolismo , Humanos , Inflamação , Lipoxinas/farmacologia , Metilação , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Especificidade de Órgãos , Cultura Primária de Células , RNA Mensageiro/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Transdução de SinaisRESUMO
Endothelial cell (EC) dysfunction has been reported in cystic fibrosis (CF) patients. Thus, the availability of CF EC is paramount to uncover mechanisms of endothelial dysfunction in CF. Using collagenase digestion, we isolated cells from small fragments of pulmonary artery dissected from non-CF lobes or explanted CF lungs. These cells were a heterogeneous mixture, containing variable percentages of EC. To obtain virtually pure pulmonary artery endothelial cells (PAEC), we developed an easy, inexpensive, and reliable method, based on the differential adhesion time of pulmonary artery cells collected after collagenase digestion. With this method, we obtained up to 95% pure non-CF and CF-PAEC. Moreover, we also succeed at immortalizing both PAEC and CF-PAEC, which remained viable and with unchanged phenotype and proliferation rate over the 30th passage. These cells recapitulated cystic fibrosis transmembrane conductance regulator expression and functions of the parental cells. Thus, we isolated for the first time endothelial cells from CF patients, providing a valuable tool to define the emerging role of EC in CF lung and vascular disease.
Assuntos
Fibrose Cística/patologia , Endotélio Vascular/patologia , Pulmão/patologia , Artéria Pulmonar/patologia , Substituição de Aminoácidos , Biomarcadores/metabolismo , Adesão Celular , Linhagem Celular Transformada , Proliferação de Células , Separação Celular , Sobrevivência Celular , Células Cultivadas , Colagenases/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/cirurgia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Impedância Elétrica , Endotélio Vascular/metabolismo , Humanos , Imunofenotipagem , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/cirurgia , Mutação , Pneumonectomia , Artéria Pulmonar/metabolismo , Técnicas de Cultura de TecidosRESUMO
Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced transendothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a ß2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/patologia , Células Endoteliais/patologia , Antígenos CD/metabolismo , Caderinas/metabolismo , Proliferação de Células/fisiologia , AMP Cíclico/metabolismo , Fibrose Cística/metabolismo , Citocinas/metabolismo , Células Endoteliais/metabolismo , Homeostase/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Insulina/farmacologia , Interleucina-8/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxidos de Nitrogênio/metabolismo , Fosforilação , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , beta-Arrestina 2/metabolismoRESUMO
Regulatory mechanisms of ALX/FPR2, the lipoxin A4 receptor, expression have considerable relevance in inflammation resolution. Because microRNAs (miRs) are emerging as key players in inflammation resolution, here we examined microRNA-mediated regulation of ALX/FPR2 (lipoxin A4 receptor/formyl peptide receptor 2) expression. By matching data from bioinformatic algorithms, we found 27 miRs predicted to bind the 3'-UTR of ALX/FPR2. Among these, we selected miR-181b because of its link with inflammation. Using a luciferase reporter system, we assessed miR-181b binding to ALX/FPR2 3'-UTR. Consistent with this, miR-181b overexpression in human macrophages significantly down-regulated ALX/FPR2 protein levels (-25%), whereas miR-181b knockdown gave a significant increase in ALX/FPR2 (+60%). miR-181b levels decreased during monocyte to macrophage differentiation (-50%), whereas ALX/FPR2 expression increased significantly (+60%). miR-181b overexpression blunted lipoxin A4 (0.1-10 nm)- and resolvin D1 (0.01-10 nm)-stimulated phagocytic activity of macrophages. These results unravel novel regulatory mechanisms of ALX/FPR2 expression and ligand-evoked macrophages proresolution responses mediated by miR-181b, thus uncovering novel components of the endogenous inflammation resolution circuits.
Assuntos
Macrófagos/metabolismo , MicroRNAs/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Transdução de Sinais , Regiões 3' não Traduzidas , Ácidos Docosa-Hexaenoicos/farmacologia , Humanos , Lipoxinas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , MicroRNAs/genética , Fagocitose , Receptores de Formil Peptídeo/genética , Receptores de Lipoxinas/genéticaRESUMO
Resolvin D1 (RvD1; 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) is an endogenous immunoresolvent that regulates acute inflammation and orchestrates resolution. Here, we investigated anti-inflammatory and proresolving actions of RvD1 after oral administration. RvD1 rapidly accumulated in the mouse plasma after oral delivery and dose-dependently (1-100 ng/mouse) reduced leukocyte infiltration in zymosan A-induced acute peritonitis. Using mathematical resolution indices, RvD1 reduced Ψmax by â¼50%, shortened the resolution interval by 3 h, and significantly reduced total leukocyte (by â¼30-45%) and polymorphonuclear neutrophil (by â¼40-55%) accumulation when administered at the peak of peritonitis. RvD1 also improved course and outcome of severe peritonitis, shifting it toward resolution. In peritoneal macrophages (MΦs) from the resolution phase of peritonitis, RvD1 down-regulated (by 2- to 3-fold) select genes that control gene transcription, namely coactivator-associated arginine methyltransferase 1 (CARM1), and downstream genes, such as colony-stimulating factor 3, intercellular adhesion molecule 1, and monocyte inflammatory protein 2, which promote neutrophil infiltration and reduce MΦ phagocytosis. Congruently, CARM1 knockdown in human and murine MΦs induced a proresolving phenotype, recapitulating in vivo actions of RvD1. These results establish novel properties of RvD1 and demonstrate that RvD1 modifies the transcription control machinery in MΦs, as part of its mechanisms of action during the resolution of acute inflammation.-Recchiuti, A., Codagnone, M., Pierdomenico, A. M., Rossi, C., Mari, V. C., Cianci, E., Simiele, F., Gatta, V., Romano, M. Immunoresolving actions of oral resolvin D1 include selective regulation of the transcription machinery in resolution-phase mouse macrophages.
Assuntos
Ácidos Docosa-Hexaenoicos/imunologia , Ácidos Docosa-Hexaenoicos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologia , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Leucócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Peritonite/tratamento farmacológico , Peritonite/genética , Peritonite/imunologia , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Fagocitose/imunologia , Transcrição Gênica/genéticaRESUMO
Histone deacetylases (HDACs) regulate inflammatory gene expression, as indicated by the potent antiinflammatory activity of pan-HDAC inhibitors. However, the specific contribution of each of the 11 HDAC proteins to the inflammatory gene expression program is unknown. Using an integrated genomic approach, we found that Hdac3-deficient macrophages were unable to activate almost half of the inflammatory gene expression program when stimulated with LPS. A large part of the activation defect was attributable to loss of basal and LPS-inducible expression of IFN-ß, which maintains Stat1 protein levels in unstimulated cells and acts in an autocrine/paracrine manner after stimulation to promote a secondary wave of Stat1-dependent gene expression. Loss of Hdac3-mediated repression of nuclear receptors led to hyperacetylation of thousands of genomic sites and associated gene derepression. The up-regulation of the constitutively expressed prostaglandin endoperoxide synthase, Ptgs1 (Cox-1), a nuclear receptor target, had a causative role in the phenotype because its chemical inhibition reverted, albeit partially, the Ifn-ß activation defect. These data indicate a central role for Hdac3 in inflammation and may have relevance for the use of selective Hdac inhibitors as antiinflammatory agents.
Assuntos
Regulação da Expressão Gênica/genética , Histona Desacetilases/metabolismo , Macrófagos/metabolismo , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , Ciclo-Oxigenase 1/metabolismo , Citocinas/análise , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Genômica , Histona Desacetilases/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Aging is characterized by increased oxidation and reduced efficiency of cytoprotective mechanisms. Nuclear factor erythroid-2-related factor (Nrf2) is a key transcription factor, controlling the expression of multiple antioxidant proteins. Here, we show that Nrf2-/- mice displayed an age-dependent anemia, due to the combined contributions of reduced red cell lifespan and ineffective erythropoiesis, suggesting a role of Nrf2 in erythroid biology during aging. Mechanistically, we found that the expression of antioxidants during aging is mediated by activation of Nrf2 function by peroxiredoxin-2. The absence of Nrf2 resulted in persistent oxidation and overactivation of adaptive systems such as the unfolded protein response (UPR) system and autophagy in Nrf2-/- mouse erythroblasts. As Nrf2 is involved in the expression of autophagy-related proteins such as autophagy-related protein (Atg) 4-5 and p62, we found impairment of late phase of autophagy in Nrf2-/- mouse erythroblasts. The overactivation of the UPR system and impaired autophagy drove apoptosis of Nrf2-/- mouse erythroblasts via caspase-3 activation. As a proof of concept for the role of oxidation, we treated Nrf2-/- mice with astaxanthin, an antioxidant, in the form of poly (lactic-co-glycolic acid) (PLGA)-loaded nanoparticles (ATS-NPs) to improve its bioavailability. ATS-NPs ameliorated the age-dependent anemia and decreased ineffective erythropoiesis in Nrf2-/- mice. In summary, we propose that Nrf2 plays a key role in limiting age-related oxidation, ensuring erythroid maturation and growth during aging.
RESUMO
Resolution of acute inflammation is an active process that involves the biosynthesis of specialized proresolving lipid mediators. Among them, resolvin D1 (RvD1) actions are mediated by two G protein-coupled receptors (GPCRs), ALX/FPR2 and GPR32, that also regulate specific microRNAs (miRNAs) and their target genes in novel resolution circuits. We report the ligand selectivity of RvD1 activation of ALX/FPR2 and GPR32. In addition to RvD1, its aspirin-triggered epimer and RvD1 analogs each dose dependently and effectively activated ALX/FPR2 and GPR32 in GPCR-overexpressing ß-arrestin systems using luminescence and electric cell-substrate impedance sensing. To corroborate these findings in vivo, neutrophil infiltration in self-limited peritonitis was reduced in human ALX/FPR2-overexpressing transgenic mice that was further limited to 50% by RvD1 treatment with as little as 10 ng of RvD1 per mouse. Analysis of miRNA expression revealed that RvD1 administration significantly up-regulated miR-208a and miR-219 in exudates isolated from ALX/FPR2 transgenic mice compared with littermates. Overexpression of miR-208a in human macrophages up-regulated IL-10. In comparison, in ALX/FPR2 knockout mice, RvD1 neither significantly reduced leukocyte infiltration in zymosan-induced peritonitis nor regulated miR-208a and IL-10 in these mice. Together, these results demonstrate the selectivity of RvD1 interactions with receptors ALX/FPR2 and GPR32. Moreover, they establish a new molecular circuit that is operative in the resolution of acute inflammation activated by the proresolving mediator RvD1 involving specific GPCRs and miRNAs.
Assuntos
Ácidos Docosa-Hexaenoicos/fisiologia , Inflamação/metabolismo , MicroRNAs/genética , Receptores Acoplados a Proteínas G/fisiologia , Doença Aguda , Animais , Arrestinas/metabolismo , Células Cultivadas , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Relação Dose-Resposta a Droga , Impedância Elétrica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Inflamação/genética , Interleucina-10/biossíntese , Interleucina-10/genética , Ligantes , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/fisiologia , Peritonite/tratamento farmacológico , Peritonite/metabolismo , Peritonite/patologia , Receptores de Formil Peptídeo/fisiologia , Receptores de Lipoxinas/fisiologia , Regulação para Cima/efeitos dos fármacos , beta-ArrestinasRESUMO
Lipoxin (LX) A(4,) a main endogenous stop-signal of inflammation, activates the G-protein-coupled receptor FPR2/ALX, which triggers potent anti-inflammatory signaling in vivo. Thus, the regulation of FPR2/ALX expression may have pathophysiological and therapeutic relevance. Here, we mapped a nucleotide sequence with strong FPR2/ALX promoter activity. Chromatin immunoprecipitation revealed specificity protein 1 (Sp1) binding to the core promoter. Site-directed mutagenesis of the Sp1 cis-acting element and Sp1 overexpression established that this transcription factor is key for maximal promoter activity, which is instead suppressed by DNA methylation. LXA(4) enhanced FPR2/ALX promoter activity (+74%) and mRNA expression (+87.5%) in MDA-MB231 cells. A single nucleotide mutation (A/G) was detected in the core promoter of one subject with history of cardiovascular disease and of his two daughters. This mutation reduced by â¼35-90% the promoter activity in vitro. Moreover, neutrophils from individuals carrying the A/G variant displayed â¼10- and 3-fold reduction in FPR2/ALX mRNA and protein, respectively, compared with cells from their relatives or healthy volunteers expressing the wild-type allele. These results uncover FPR2/ALX transcriptional regulation and provide the first evidence of mutations that affect FPR2/ALX transcription, thus opening new opportunities for the understanding of the LXA(4)-FPR2/ALX axis in human disease.