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1.
J Immunol ; 194(11): 5520-8, 2015 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-25926674

RESUMEN

Bacteria colonize cystic fibrosis (CF) airways, and although T cells with appropriate Ag specificity are present in draining lymph nodes, they are conspicuously absent from the lumen. To account for this absence, we hypothesized that polymorphonuclear neutrophils (PMNs), recruited massively into the CF airway lumen and actively exocytosing primary granules, also suppress T cell function therein. Programmed death-ligand 1 (PD-L1), which exerts T cell suppression at a late step, was expressed bimodally on CF airway PMNs, delineating PD-L1(hi) and PD-L1(lo) subsets, whereas healthy control (HC) airway PMNs were uniformly PD-L1(hi). Blood PMNs incubated in CF airway fluid lost PD-L1 over time; in coculture, Ab blockade of PD-L1 failed to inhibit the suppression of T cell proliferation by CF airway PMNs. In contrast with PD-L1, arginase 1 (Arg1), which exerts T cell suppression at an early step, was uniformly high on CF and HC airway PMNs. However, arginase activity was high in CF airway fluid and minimal in HC airway fluid, consistent with the fact that Arg1 activation requires primary granule exocytosis, which occurs in CF, but not HC, airway PMNs. In addition, Arg1 expression on CF airway PMNs correlated negatively with lung function and positively with arginase activity in CF airway fluid. Finally, combined treatment with arginase inhibitor and arginine rescued the suppression of T cell proliferation by CF airway fluid. Thus, Arg1 and PD-L1 are dynamically modulated upon PMN migration into human airways, and, Arg1, but not PD-L1, contributes to early PMN-driven T cell suppression in CF, likely hampering resolution of infection and inflammation.


Asunto(s)
Arginasa/inmunología , Antígeno B7-H1/inmunología , Fibrosis Quística/inmunología , Neutrófilos/inmunología , Linfocitos T/inmunología , Adulto , Apoptosis/inmunología , Arginasa/biosíntesis , Antígeno B7-H1/antagonistas & inhibidores , Proliferación Celular , Exocitosis/inmunología , Femenino , Humanos , Inflamación/inmunología , Inflamación/patología , Pulmón/inmunología , Pulmón/patología , Activación de Linfocitos/inmunología , Masculino , Pruebas de Función Respiratoria , Mucosa Respiratoria/citología , Mucosa Respiratoria/inmunología , Adulto Joven
2.
Immunol Cell Biol ; 94(1): 24-38, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26010746

RESUMEN

Dendritic cells (DCs) are key regulators of host immunity that are capable of inducing either immune tolerance or activation. In addition to their well-characterized role in shaping immune responses to foreign pathogens, DCs are also known to be critical for the induction and maintenance of anti-tumor immune responses. Therefore, it is important to understand how tumors influence the function of DCs and the quality of immune responses they elicit. Although the majority of studies in this field to date have utilized either immortalized DC lines or DC populations that have been generated under artificial conditions from hematopoietic precursors in vitro, we wished to investigate how tumors impact the function of already differentiated, tissue-resident DCs. Therefore, we used both an ex vivo and in vivo model system to assess the influence of melanoma-derived factors on DC maturation and activation. In ex vivo studies with freshly isolated splenic DCs, we demonstrate that the extent to which DC maturation and activation are altered by these factors correlates with melanoma tumorigenicity, and we identify partial roles for tumor-derived transforming growth factor (TGF)ß1 and vascular endothelial growth factor (VEGF)-A in the altered functionality of DCs. In vivo studies using a lung metastasis model of melanoma also demonstrate tumorigenicity-dependent alterations to the function of lung-resident DCs, and skewed production of proinflammatory cytokines and chemokines by these tumor-altered cells is associated with recruitment of an immune infiltrate that may ultimately favor tumor immune escape and outgrowth.


Asunto(s)
Diferenciación Celular , Células Dendríticas/patología , Pulmón/patología , Melanoma Experimental/patología , Bazo/patología , Animales , Linfocitos T CD8-positivos/inmunología , Recuento de Células , Quimiocinas/genética , Quimiocinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Factores Inmunológicos/metabolismo , Activación de Linfocitos/inmunología , Macrófagos/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Ratones Endogámicos C57BL , Factor de Crecimiento Transformador beta1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo
3.
Cell Immunol ; 272(2): 275-82, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22051048

RESUMEN

Dendritic cells play critical roles in both innate and adaptive immunity, and their numerous functions are tightly linked to their maturation and activation status. Here, we characterize the murine dendritic cell line DC2.4 as a model for studying dendritic cell maturation and activation, and we evaluate the influence of melanoma tumor cells on these processes. Exposure of DC2.4 cells to the Toll-like receptor ligand lipopolysaccharide induces both maturation and activation of these cells, characterized by upregulation of costimulatory molecule expression and proinflammatory cytokine/chemokine production. This maturation and activation is suppressed by soluble factors derived from both the highly tumorigenic B16-F1 and the poorly tumorigenic D5.1G4 murine melanoma cell lines. Interestingly, the extent of DC2.4 immunosuppression by these melanomas correlates with their tumorigenicity, suggesting a potentially vital role for dendritic cell/tumor cell interactions in the regulation of anti-tumor immunity and tumor outgrowth.


Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Factores Inmunológicos/inmunología , Melanoma Experimental/inmunología , Proteínas de Neoplasias/inmunología , Animales , Comunicación Celular/inmunología , Línea Celular , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Femenino , Factores Inmunológicos/metabolismo , Terapia de Inmunosupresión/métodos , Lipopolisacáridos/inmunología , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/metabolismo , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Regulación hacia Arriba/inmunología
4.
J Leukoc Biol ; 112(4): 707-716, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35172381

RESUMEN

Cystic fibrosis (CF) airways feature high extracellular levels of the IL-1 family of proinflammatory mediators. These mediators are cleavage products of caspase-1, the final protease in the inflammasome cascade. Due to the proven chronic presence of reprogrammed neutrophils in the CF airway lumen, understanding inflammasome signaling in these cells is of great importance to understand how disease is perpetuated in this milieu. Here, we hypothesized that CF airway neutrophils contribute to chronic inflammation, in part, via the packaging of inflammasome-inducing signals in extracellular vesicles (EVs). We confirmed that CF airway fluid is enriched in IL-1α, IL-1ß, and IL-18, and that CF airway neutrophils up-regulate the activating receptor IL-1R1. Meanwhile, down-modulatory signals such as IL-1R2 and IL-1RA are unchanged. Active caspase-1 itself is present in CF airway fluid EVs, with neutrophil-derived EVs being most enriched. Using a transmigration model of CF airway inflammation, we show that CF airway fluid EVs are necessary and sufficient to induce primary granule exocytosis by naïve neutrophils (hallmark of reprogramming) and concomitantly activate caspase-1 and IL-1ß production by these cells and that the addition of triple-combination highly effective CFTR modulator therapy does not abrogate these effects. Finally, EVs from activated neutrophils can deliver active caspase-1 to primary tracheal epithelial cells and induce their release of IL-1α. These findings support the existence of a feed-forward inflammatory process by which reprogrammed CF airway neutrophils bypass 2-step control of inflammasome activation in neighboring cells (naïve neutrophils and epithelial cells) via the transfer of bioactive EVs.


Asunto(s)
Fibrosis Quística , Vesículas Extracelulares , Caspasas , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Humanos , Inflamasomas , Inflamación , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-18 , Neutrófilos , Péptido Hidrolasas , Receptores Tipo II de Interleucina-1
5.
Cell Rep Med ; 2(4): 100239, 2021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33948572

RESUMEN

Neutrophils are often considered terminally differentiated and poised for bacterial killing. In chronic diseases such as cystic fibrosis (CF), an unexplained paradox pits massive neutrophil presence against prolonged bacterial infections. Here, we show that neutrophils recruited to CF airways in vivo and in an in vitro transmigration model display rapid and broad transcriptional firing, leading to an upregulation of anabolic genes and a downregulation of antimicrobial genes. Newly transcribed RNAs are mirrored by the appearance of corresponding proteins, confirming active translation in these cells. Treatment by the RNA polymerase II and III inhibitor α-amanitin restores the expression of key antimicrobial genes and increases the bactericidal capacity of CF airway neutrophils in vitro and in short-term sputum cultures ex vivo. Broadly, our findings show that neutrophil plasticity is regulated at the site of inflammation via RNA and protein synthesis, leading to adaptations that affect their canonical functions (i.e., bacterial clearance).


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Neutrófilos/metabolismo , Sistema Respiratorio/microbiología , Fibrosis Quística/genética , Fibrosis Quística/microbiología , Trampas Extracelulares/microbiología , Humanos , Neutrófilos/microbiología , Pseudomonas aeruginosa/patogenicidad , Esputo/metabolismo , Esputo/microbiología
6.
J Cyst Fibros ; 18(1): 64-70, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-29937317

RESUMEN

BACKGROUND: Resistin is an immunometabolic mediator that is elevated in several inflammatory disorders. A ligand for Toll-like receptor 4, resistin modulates the recruitment and activation of myeloid cells, notably neutrophils. Neutrophils are major drivers of cystic fibrosis (CF) lung disease, in part due to the release of human neutrophil elastase- and myeloperoxidase-rich primary granules, leading to tissue damage. Here we assessed the relationship of resistin to CF lung disease. METHODS: Resistin levels were measured in plasma and sputum from three retrospective CF cohorts spanning a wide range of disease. We also assessed the ability of neutrophils to secrete resistin upon activation in vitro. Finally, we constructed a multivariate model assessing the relationship between resistin levels and lung function. RESULTS: Plasma resistin levels were only marginally higher in CF than in healthy control subjects. By contrast, sputum resistin levels were very high in CF, reaching 50-100 fold higher levels than in plasma. Among CF patients, higher plasma resistin levels were associated with allergic bronchopulmonary aspergillosis, and higher sputum resistin levels were associated with CF-related diabetes. Mechanistically, in vitro release of neutrophil primary granules was concomitant with resistin secretion. Overall, sputum resistin levels were negatively correlated with CF lung function, independently of other variables (age, sex, and genotype). CONCLUSIONS: Our data establish relationships between resistin levels in the plasma and sputum of CF patients that correlate with disease status, and identify resistin as a novel mechanistic link between neutrophilic inflammation and lung disease in CF.


Asunto(s)
Fibrosis Quística/metabolismo , Flujo Espiratorio Forzado/fisiología , Resistina/metabolismo , Esputo/metabolismo , Adolescente , Adulto , Biomarcadores/metabolismo , Fibrosis Quística/diagnóstico , Fibrosis Quística/fisiopatología , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Pulmón/fisiopatología , Masculino , Persona de Mediana Edad , Pronóstico , Pruebas de Función Respiratoria , Estudios Retrospectivos , Adulto Joven
7.
JCI Insight ; 4(23)2019 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-31661469

RESUMEN

Pulmonary drug delivery presents a unique opportunity to target lower airway inflammation, which is often characterized by the massive recruitment of neutrophils from blood. However, specific therapies are lacking modulation of airway neutrophil function, and difficult challenges must be overcome to achieve therapeutic efficacy against pulmonary inflammation, notably drug hydrophobicity, mucociliary and macrophage-dependent clearance, and high extracellular protease burden. Here, we present a multistage, aerodynamically favorable delivery platform that uses extracellular proteolysis to its advantage to deliver nanoparticle-embedded hydrophobic drugs to neutrophils within the lower airways. Our design consists of a self-regulated nanoparticle-in-microgel system, in which microgel activation is triggered by extracellular elastase (degranulated by inflammatory neutrophils), and nanoparticles are loaded with Nexinhib20, a potent neutrophil degranulation inhibitor. Successful in vivo delivery of Nexinhib20 to the airways and into neutrophils promoted resolution of the inflammatory response by dampening neutrophil recruitment and degranulation, proinflammatory cytokine production in both airway and systemic compartments, as well as the presence of neutrophil-derived pathological extracellular vesicles in the lung fluid. Our findings showcase a new platform that overcomes challenges in pulmonary drug delivery and allows customization to match the proteolytic footprint of given diseases.


Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Infiltración Neutrófila , Neutrófilos/metabolismo , Neumonía/tratamiento farmacológico , Animales , Líquido del Lavado Bronquioalveolar , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Inflamación/tratamiento farmacológico , Pulmón/efectos de los fármacos , Pulmón/patología , Macrófagos , Ratones , Ratones Endogámicos C57BL , Microgeles , Nanopartículas , Activación Neutrófila/efectos de los fármacos , Elastasa Pancreática , Neumonía/patología
8.
J Leukoc Biol ; 104(4): 665-675, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29741792

RESUMEN

Recruitment of neutrophils to the airways, and their pathological conditioning therein, drive tissue damage and coincide with the loss of lung function in patients with cystic fibrosis (CF). So far, these key processes have not been adequately recapitulated in models, hampering drug development. Here, we hypothesized that the migration of naïve blood neutrophils into CF airway fluid in vitro would induce similar functional adaptation to that observed in vivo, and provide a model to identify new therapies. We used multiple platforms (flow cytometry, bacteria-killing, and metabolic assays) to characterize functional properties of blood neutrophils recruited in a transepithelial migration model using airway milieu from CF subjects as an apical chemoattractant. Similarly to neutrophils recruited to CF airways in vivo, neutrophils migrated into CF airway milieu in vitro display depressed phagocytic receptor expression and bacterial killing, but enhanced granule release, immunoregulatory function (arginase-1 activation), and metabolic activities, including high Glut1 expression, glycolysis, and oxidant production. We also identify enhanced pinocytic activity as a novel feature of these cells. In vitro treatment with the leukotriene pathway inhibitor acebilustat reduces the number of transmigrating neutrophils, while the metabolic modulator metformin decreases metabolism and oxidant production, but fails to restore bacterial killing. Interestingly, we describe similar pathological conditioning of neutrophils in other inflammatory airway diseases. We successfully tested the hypothesis that recruitment of neutrophils into airway milieu from patients with CF in vitro induces similar pathological conditioning to that observed in vivo, opening new avenues for targeted therapeutic intervention.


Asunto(s)
Fibrosis Quística/inmunología , Neutrófilos/inmunología , Animales , Compuestos de Azabiciclo/farmacología , Benzoatos/farmacología , Células Sanguíneas , Células de la Médula Ósea , Células Cultivadas , Quimiotaxis de Leucocito , Medios de Cultivo Condicionados/farmacología , Fibrosis Quística/patología , Exocitosis/efectos de los fármacos , Citometría de Flujo , Glucólisis , Humanos , Elastasa de Leucocito/metabolismo , Leucotrieno B4/farmacología , Lipopolisacáridos/farmacología , Metformina/farmacología , Ratones , Activación Neutrófila , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/patología , Consumo de Oxígeno , Pinocitosis , Pseudomonas aeruginosa , Sistema Respiratorio/inmunología , Sistema Respiratorio/patología , Esputo/inmunología , Migración Transendotelial y Transepitelial/efectos de los fármacos
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