RESUMEN
The involvement of microRNA (miR) in cystic fibrosis (CF) pathobiology is rapidly emerging. We previously documented that miR-181b controls the expression of the ALX/FPR2 receptor, which is recognized by the endogenous proresolution ligand, lipoxin (LX)A4. Here, we examined whether the miR-181b-ALX/FPR2 circuit was altered in CF. We examined human airways epithelial cells, normal (16HBE14o-), carrying the ΔF508 mutation (CFBE41o-) or corrected for this mutation (CFBE41o-/CEP-CFTR wt 6.2 kb), as well as monocyte-derived macrophages (MΦs) from CF patients. CFBE41o- cells exhibited higher miR-181b and reduced ALX/FPR2 levels compared to 16HBE14o- and CFBE41o-/CEP-CFTR wt 6.2 kb cells. An anti-mir-181b significantly enhanced ALX/FPR2 expression (+ 60%) as well as LXA4-induced increase in transepithelial electric resistance (+ 25%) in CFBE41o- cells. MΦs from CF patients also displayed increased miR-181b (+ 100%) and lower ALX/FPR2 levels (- 20%) compared to healthy cells. An anti-mir-181b enhanced ALX/FPR2 expression (+ 40%) and normalized receptor-dependent LXA4-induced phagocytosis of fluorescent-labeled zymosan particles as well as of Pseudomonas aeruginosa by CF-MΦs. These results provide the first evidence that miR-181b is overexpressed in CF cells, impairing some mechanisms of the ALX/FPR2-dependent pathway of inflammation resolution. Thus, targeting miR-181b may represent a strategy to enhance anti-inflammatory and anti-microbial defense mechanisms in CF.
Asunto(s)
Fibrosis Quística/inmunología , MicroARNs/genética , Fagocitosis , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Células Cultivadas , Fibrosis Quística/genética , Femenino , Humanos , Macrófagos/inmunología , Macrófagos/microbiología , MicroARNs/metabolismo , Pseudomonas aeruginosa/patogenicidad , Receptores de Formil Péptido/genética , Receptores de Lipoxina/genética , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/microbiologíaRESUMEN
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.
Asunto(s)
Micropartículas Derivadas de Células/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Lipoxinas/farmacología , MicroARNs/genética , Línea Celular , Micropartículas Derivadas de Células/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Humanos , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
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.
Asunto(s)
Epigénesis Genética , Histonas/genética , Lipoxinas/metabolismo , ARN Mensajero/genética , Receptores de Formil Péptido/genética , Receptores de Lipoxina/genética , Acetilación , Línea Celular , Línea Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Histonas/metabolismo , Humanos , Inflamación , Lipoxinas/farmacología , Metilación , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Especificidad de Órganos , Cultivo Primario de Células , ARN Mensajero/metabolismo , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Macrófagos/metabolismo , MicroARNs/metabolismo , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Transducción de Señal , Regiones no Traducidas 3' , Ácidos Docosahexaenoicos/farmacología , Humanos , Lipoxinas/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , MicroARNs/genética , Fagocitosis , Receptores de Formil Péptido/genética , Receptores de Lipoxina/genéticaRESUMEN
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.