RESUMEN
BACKGROUND: Innovative therapies to target tumor-associated neutrophils (PMN) are of clinical interest, since these cells are centrally involved in cancer inflammation and tumor progression. Resolvin D1 (RvD1) is a lipid autacoid that promotes resolution of inflammation by regulating the activity of distinct immune and non-immune cells. Here, using human papilloma virus (HPV) tumorigenesis as a model, we investigated whether RvD1 modulates PMN to reduce tumor progression. METHODS: Growth-curve assays with multiple cell lines and in vivo grafting of two distinct HPV-positive cells in syngeneic mice were used to determine if RvD1 reduced cancer growth. To investigate if and how RvD1 modulates PMN activities, RNA sequencing and multiplex cytokine ELISA of human PMN in co-culture with HPV-positive cells, coupled with pharmacological depletion of PMN in vivo, were performed. The mouse intratumoral immune cell composition was evaluated through FACS analysis. Growth-curve assays and in vivo pharmacological depletion were used to evaluate anti-tumor activities of human and mouse monocytes, respectively. Bioinformatic analysis of The Cancer Genome Atlas (TCGA) database was exploited to validate experimental findings in patients. RESULTS: RvD1 decreased in vitro and in vivo proliferation of human and mouse HPV-positive cancer cells through stimulation of PMN anti-tumor activities. In addition, RvD1 stimulated a PMN-dependent recruitment of classical monocytes as key determinant to reduce tumor growth in vivo. In human in vitro systems, exposure of PMN to RvD1 increased the production of the monocyte chemoattractant protein-1 (MCP-1), and enhanced transmigration of classical monocytes, with potent anti-tumor actions, toward HPV-positive cancer cells. Consistently, mining of immune cells infiltration levels in cervical cancer patients from the TCGA database evidenced an enhanced immune reaction and better clinical outcomes in patients with higher intratumoral monocytes as compared to patients with higher PMN infiltration. CONCLUSIONS: RvD1 reduces cancer growth by activating PMN anti-cancer activities and encouraging a protective PMN-dependent recruitment of anti-tumor monocytes. These findings demonstrate efficacy of RvD1 as an innovative therapeutic able to stimulate PMN reprogramming to an anti-cancer phenotype that restrains tumor growth.
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Ácidos Docosahexaenoicos/uso terapéutico , Monocitos/metabolismo , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Neutrófilos/metabolismo , Animales , Ácidos Docosahexaenoicos/farmacología , Humanos , RatonesRESUMEN
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.
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COVID-19 , Fibrosis Quística , Ácidos Docosahexaenoicos/farmacología , Macrófagos , Infecciones por Pseudomonas , Pseudomonas aeruginosa/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , COVID-19/inmunología , COVID-19/microbiología , COVID-19/patología , Fibrosis Quística/inmunología , Fibrosis Quística/microbiología , Fibrosis Quística/patología , Fibrosis Quística/virología , Citocinas/inmunología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Inflamación/inmunología , Inflamación/microbiología , Inflamación/patología , Inflamación/virología , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/patología , Macrófagos/virología , Masculino , MicroARNs/inmunología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/patología , Infecciones por Pseudomonas/virologíaRESUMEN
Despite the high expectations associated with the recent introduction of CFTR modulators, airway inflammation still remains a relevant clinical issue in cystic fibrosis (CF). The classical anti-inflammatory drugs have shown very limited efficacy, when not being harmful, raising the question of whether alternative approaches should be undertaken. Thus, a better knowledge of the mechanisms underlying the aberrant inflammation observed in CF is pivotal to develop more efficacious pharmacology. In this respect, the observation that endogenous proresolving pathways are defective in CF and that proresolving mediators, physiologically generated during an acute inflammatory reaction, do not completely suppress inflammation, but promote resolution, tissue healing and microbial clearance, without compromising immune host defense mechanisms, opens interesting therapeutic scenarios for CF. In this mini-review, we present the current knowledge and perspectives of proresolving pharmacology in CF, focusing on the specialized proresolving lipid mediators and selected peptides.
RESUMEN
Non-resolving lung inflammation and Pseudomonas aeruginosa infections are the underlying cause of morbidity and mortality in cystic fibrosis (CF). The endogenous lipid mediator resolvin (Rv) D1 is a potent regulator of resolution, and its roles, actions, and therapeutic potential in CF are of interest. Here, we investigated actions and efficacy of RvD1 in preclinical models of cystic fibrosis. Cftr knockout mice with chronic P. aeruginosa lung infection were treated with RvD1 to assess differences in lung bacterial load, inflammation, and tissue damage. Cells from volunteers with CF were treated with RvD1 during ex vivo infection with P. aeruginosa, and effects on phagocytosis and inflammatory signaling were determined. In CF mice, RvD1 reduced bacterial burden, neutrophil infiltration, and histological signs of lung pathology, improving clinical scores of diseases. Mechanistically, RvD1 increased macrophage-mediated bacterial and leukocyte clearance in vivo. The clinical significance of these findings is supported by actions in primary leukocytes and epithelial cells from volunteers with CF where RvD1 enhanced P. aeruginosa phagocytosis and reduced genes and proteins associated to NF-κB activation and leukocyte infiltration. Concentration of RvD1 in sputum from patients with CF was also inversely correlated to those of cytokines and chemokines involved in CF lung pathology. These findings demonstrate efficacy of RvD1 in enhancing resolution of lung inflammation and infections and provide proof of concept for its potential as a prototypic novel pro-resolutive therapeutic approach for CF.
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Fibrosis Quística/inmunología , Fibrosis Quística/microbiología , Ácidos Docosahexaenoicos/farmacología , Neumonía/inmunología , Infecciones por Pseudomonas , Animales , Fibrosis Quística/patología , Humanos , Ratones , Ratones Noqueados , Infiltración Neutrófila/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Neumonía/microbiología , Neumonía/patología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosaRESUMEN
Accumulating evidence indicates that stem cells (SCs) possess immunomodulatory, anti-inflammatory, and prohealing properties. The mechanisms underlying these functions are being investigated with the final goal to set a solid background for the clinical use of SCs and/or their derivatives. Specialized proresolving lipid mediators (SPMs) are small lipids formed by the enzymatic metabolism of polyunsaturated fatty acids. They represent a leading class of molecules that actively and timely regulate the resolution of inflammation and promote tissue/organ repair. SC formation of these mediators as well as expression of their receptors has been recently reported, suggesting that SPMs may be involved in the immunomodulatory, proresolving functions of SCs. In the present review, we summarize the current knowledge on SPMs in SCs, focusing on biosynthetic pathways, receptors, and bioactions, with the intent to provide an integrated view of SPM impact on SC biology. Stem Cells Translational Medicine 2019;8:992-998.
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Mediadores de Inflamación/metabolismo , Metabolismo de los Lípidos/genética , Células Madre/metabolismo , HumanosRESUMEN
Resolution Pharmacology identifies drugs developed on the biology of the resolution phase of inflammation, the complex molecular and cellular network of events that ensure the tight temporal and spatial control on the inflammatory response. As such, new anti-inflammatory and pro-resolving drugs could derive from pro-resolving mediators and receptors. To implement faithful screening programs, however, it is important to rely on predictive signaling pathway relevant for the ultimate bio-action of interest. Herein we performed an analysis with four prototypical melanocortin receptor (MC1,3,4,5) agonists. The choice fell on the natural agonist αMSH, the small molecule BMS-470539, and the synthetic derivatives [D-Trp8]-γMSH and [Nle4,D-Phe7]-αMSH. We used human macrophages and quantified the effect of the four agonists on inhibition of cytokine release and promotion of efferocytosis. All agonists (1-10 µM) significantly inhibited cytokine release by LPS-stimulated cells whereas [D-Trp8]-γMSH was the most effective in inducing efferocytosis (â¼60% increase). To study the signaling profile, we monitored cAMP accumulation and ERK1/2 phosphorylation, and constructed biased plots that revealed a marked biased profile of [D-Trp8]-γMSH toward phospho-ERK1/2. Correlation matrix analysis of all data pointed at phospho-ERK1/2 at any receptor as the most prominent pathway to attain pro-phagocytic actions, and MC1 receptor as the most relevant to drive anti-cytokine effects. In conclusion, the present study highlights the need to associate single-target signaling data with relevant functional outcomes. In this manner, we would increase our chances to optimize drug discovery programs during the early target validation and hit-to-lead phases.
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.
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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
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.
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Fibrosis Quística/patología , Endotelio Vascular/patología , Pulmón/patología , Arteria Pulmonar/patología , Sustitución de Aminoácidos , Biomarcadores/metabolismo , Adhesión Celular , Línea Celular Transformada , Proliferación Celular , Separación Celular , Supervivencia Celular , Células Cultivadas , Colagenasas/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/cirugía , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Impedancia Eléctrica , Endotelio Vascular/metabolismo , Humanos , Inmunofenotipificación , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Pulmón/cirugía , Mutación , Neumonectomía , Arteria Pulmonar/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
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.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Células Endoteliales/patología , Antígenos CD/metabolismo , Cadherinas/metabolismo , Proliferación Celular/fisiología , AMP Cíclico/metabolismo , Fibrosis Quística/metabolismo , Citocinas/metabolismo , Células Endoteliales/metabolismo , Homeostasis/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Insulina/farmacología , Interleucina-8/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxidos de Nitrógeno/metabolismo , Fosforilación , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Arrestina beta 2/metabolismoRESUMEN
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.
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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.