RESUMEN
Demyelination due to autoreactive T cells and inflammation in the central nervous system are principal features of multiple sclerosis (MS), a chronic and highly disabling human disease affecting brain and spinal cord. Here, we show that treatment with apelin, a secreted peptide ligand for the G protein-coupled receptor APJ/Aplnr, is protective in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Apelin reduces immune cell entry into the brain, delays the onset and reduces the severity of EAE. Apelin affects the trafficking of leukocytes through the lung by modulating the expression of cell adhesion molecules that mediate leukocyte recruitment. In addition, apelin induces the internalization and desensitization of its receptor in endothelial cells (ECs). Accordingly, protection against EAE major outcomes of apelin treatment are phenocopied by loss of APJ/Aplnr function, achieved by EC-specific gene inactivation in mice or knockdown experiments in cultured primary endothelial cells. Our findings highlight the importance of the lung-brain axis in neuroinflammation and indicate that apelin targets the transendothelial migration of immune cells into the lung during acute inflammation.
Asunto(s)
Apelina , Encefalomielitis Autoinmune Experimental , Células Endoteliales , Leucocitos , Ratones Endogámicos C57BL , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Animales , Apelina/metabolismo , Ratones , Células Endoteliales/metabolismo , Células Endoteliales/inmunología , Leucocitos/inmunología , Leucocitos/metabolismo , Femenino , Pulmón/inmunología , Pulmón/patología , Inflamación/metabolismo , Inflamación/inmunología , Receptores de Apelina/metabolismo , Receptores de Apelina/genética , Humanos , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/inmunología , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacos , Ratones Noqueados , Modelos Animales de EnfermedadRESUMEN
Transmigration of circulating monocytes from the bloodstream to tissues represents an early hallmark of inflammation. This process plays a pivotal role during viral neuroinvasion, encephalitis, and HIV-associated neurocognitive disorders. How monocytes locally unzip endothelial tight junction-associated proteins (TJAPs), without perturbing impermeability, to reach the central nervous system remains poorly understood. Here, we show that human circulating monocytes express the TJAP Occludin (OCLN) to promote transmigration through endothelial cells. We found that human monocytic OCLN (hmOCLN) clusters at monocyte-endothelium interface, while modulation of hmOCLN expression significantly impacts monocyte transmigration. Furthermore, we designed OCLN-derived peptides targeting its extracellular loops (EL) and show that transmigration of treated monocytes is inhibited in vitro and in zebrafish embryos, while preserving vascular integrity. Monocyte transmigration toward the brain is an important process for HIV neuroinvasion and we found that the OCLN-derived peptides significantly inhibit HIV dissemination to cerebral organoids. In conclusion, our study identifies an important role for monocytic OCLN during transmigration and provides a proof-of-concept for the development of mitigation strategies to prevent monocyte infiltration and viral neuroinvasion.
Asunto(s)
Células Endoteliales , Monocitos , Ocludina , Migración Transendotelial y Transepitelial , Pez Cebra , Ocludina/metabolismo , Ocludina/genética , Humanos , Monocitos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/virología , Animales , Migración Transendotelial y Transepitelial/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/virología , Células Endoteliales/efectos de los fármacos , Infecciones por VIH/virología , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , VIH-1/efectos de los fármacos , Péptidos/farmacología , Péptidos/metabolismo , Encéfalo/metabolismo , Encéfalo/virologíaRESUMEN
Chronic inflammation is characterized by persisting leukocyte infiltration of the affected tissue, which is enabled by activated endothelial cells (ECs). Chronic inflammatory diseases remain a major pharmacotherapeutic challenge, and thus the search for novel drugs and drug targets is an ongoing demand. We have identified the natural product vioprolide A (vioA) to exert anti-inflammatory actions in vivo and in ECs in vitro through inhibition of its cellular target nucleolar protein 14 (NOP14). VioA attenuated the infiltration of microglia and macrophages during laser-induced murine choroidal neovascularization and the leukocyte trafficking through the vascular endothelium in the murine cremaster muscle. Mechanistic studies revealed that vioA downregulates EC adhesion molecules and the tumor necrosis factor receptor (TNFR) 1 by decreasing the de novo protein synthesis in ECs. Most importantly, we found that inhibition of importin-dependent NF-ĸB p65 nuclear translocation is a crucial part of the action of vioA leading to reduced NF-ĸB promotor activity and inflammatory gene expression. Knockdown experiments revealed a causal link between the cellular target NOP14 and the anti-inflammatory action of vioA, classifying the natural product as unique drug lead for anti-inflammatory therapeutics.
Asunto(s)
Antiinflamatorios/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Inflamación/tratamiento farmacológico , Carioferinas/metabolismo , Leucocitos/efectos de los fármacos , Compuestos Macrocíclicos/farmacología , Proteínas Nucleares/metabolismo , Factor de Transcripción ReIA/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacos , Transporte Activo de Núcleo Celular , Animales , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/inmunología , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Carioferinas/genética , Leucocitos/inmunología , Leucocitos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/inmunología , Microglía/metabolismo , Proteínas Nucleares/genética , Factor de Transcripción ReIA/genéticaRESUMEN
We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity; two of its chemical moieties (tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of IL-1ß, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug candidate for the treatment of ARDS in COVID-19 patients.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Leucocitos/efectos de los fármacos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/antagonistas & inhibidores , Pirimidinas/farmacología , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Migración Transendotelial y Transepitelial/efectos de los fármacos , Animales , COVID-19/patología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Movimiento Celular/efectos de los fármacos , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Leucocitos/inmunología , Lipopolisacáridos/efectos adversos , Ratones , Ratones Endogámicos BALB C , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Pirimidinas/química , Síndrome de Dificultad Respiratoria/inducido químicamente , SARS-CoV-2RESUMEN
Microtubules (MTs) control cell shape and intracellular cargo transport. The role of MT turnover in the migration of slow-moving cells through endothelial barriers remains unclear. To irreversibly interfere with MT disassembly, we have used the MT-stabilizing agent zampanolide (ZMP) in Β16F10 melanoma as amodel of slow-moving cells. ZMP-treated B16 cells failed to follow chemotactic gradients across rigid confinements and could not generate stable sub-endothelial pseudopodia under endothelial monolayers. In vivo, ZMP-treated Β16 cells failed to extravasate though lung capillaries. In contrast to melanoma cells, the chemotaxis and transendothelial migration of ZMP-treated Tcells were largely conserved. This is afirst demonstration that MT disassembly is akey checkpoint in the directional migration of cancer cells but not of lymphocytes.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Macrólidos/farmacología , Microtúbulos/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacos , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Masculino , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Microtúbulos/efectos de los fármacos , Seudópodos/efectos de los fármacos , Linfocitos T/efectos de los fármacosRESUMEN
Tumor progression and metastatic dissemination are driven by cell-intrinsic and biomechanical cues that favor the growth of life-threatening secondary tumors. We recently identified pro-metastatic vascular regions with blood flow profiles that are permissive for the arrest of circulating tumor cells. We have further established that such flow profiles also control endothelial remodeling, which favors extravasation of arrested CTCs. Yet, how shear forces control endothelial remodeling is unknown. In the present work, we aimed at dissecting the cellular and molecular mechanisms driving blood flow-dependent endothelial remodeling. Transcriptomic analysis of endothelial cells revealed that blood flow enhanced VEGFR signaling, among others. Using a combination of in vitro microfluidics and intravital imaging in zebrafish embryos, we now demonstrate that the early flow-driven endothelial response can be prevented upon specific inhibition of VEGFR tyrosine kinase and subsequent signaling. Inhibitory targeting of VEGFRs reduced endothelial remodeling and subsequent metastatic extravasation. These results confirm the importance of VEGFR-dependent endothelial remodeling as a driving force of CTC extravasation and metastatic dissemination. Furthermore, the present work suggests that therapies targeting endothelial remodeling might be a relevant clinical strategy in order to impede metastatic progression.
Asunto(s)
Endotelio Vascular/fisiología , Hemorreología , Migración Transendotelial y Transepitelial , Animales , Animales Modificados Genéticamente , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Embrión no Mamífero/irrigación sanguínea , Embrión no Mamífero/fisiología , Regulación Neoplásica de la Expresión Génica , Ontología de Genes , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas In Vitro , Microscopía Intravital , Microfluídica , Microscopía Confocal , Células Neoplásicas Circulantes , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Transducción de Señal/fisiología , Sunitinib/farmacología , Sunitinib/uso terapéutico , Migración Transendotelial y Transepitelial/efectos de los fármacos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Pez Cebra/embriologíaRESUMEN
The binding of amyloid precursor protein (APP) expressed on tumor cells to death receptor 6 (DR6) could initiate the necroptosis pathway, which leads to necroptotic cell death of vascular endothelial cells (ECs) and results in tumor cells (TCs) extravasation and metastasis. This study reports the first inhibitor of DR6/APP interaction as a novel class of anti-hematogenous metastatic agent. By rationally utilizing three combined strategies including selection based on phage display library, d-retro-inverso modification, and multiple conjugation of screened peptidomimetic with 4-arm PEG, the polymer-peptidomimetic conjugate PEG-tAHP-DRI (tetra-(D-retro-inverso isomer of AHP-12) substitued 4-arm PEG5k ) is obtained as the most promising agent with the strongest binding potency (KD = 51.12 × 10-9 m) and excellent pharmacokinetic properties. Importantly, PEG-tAHP-DRI provides efficient protection against TC-induced ECs necroptosis both in vitro and in vivo. Moreover, this ligand exhibits prominent anti-hematogenous metastatic activity in serval different metastatic mouse models (B16F10, 4T1, CT26, and spontaneous lung metastasis of 4T1 orthotopic tumor model) and displays no apparent detrimental effects in preliminary safety evaluation. Collectively, this study demonstrates the feasibility of exploiting DR6/APP interaction to regulate hematogenous tumor cells transendothelial migration and provides PEG-tAHP-DRI as a novel and promising inhibitor of DR6/APP interaction for developments of anti-hematogenous metastatic therapies.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Comunicación Celular/efectos de los fármacos , Neoplasias Hematológicas/tratamiento farmacológico , Peptidomiméticos/farmacología , Receptores del Factor de Necrosis Tumoral/genética , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Neoplasias Hematológicas/sangre , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Humanos , Ligandos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Metástasis de la Neoplasia , Peptidomiméticos/química , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Migración Transendotelial y Transepitelial/efectos de los fármacos , Migración Transendotelial y Transepitelial/genéticaRESUMEN
[Figure: see text].
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Productos de Degradación de Fibrina-Fibrinógeno/farmacología , Leucocitos/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Vena Cava Inferior/metabolismo , Trombosis de la Vena/sangre , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Estudios de Casos y Controles , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Humanos , Leucocitos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Endogámicos BALB C , Fragmentos de Péptidos/sangre , Embolia Pulmonar/sangre , Células THP-1 , Factores de Tiempo , Vena Cava Inferior/patología , Trombosis de la Vena/patologíaRESUMEN
BACKGROUND: Poor infiltration and limited activation of transferred T cells are fundamental factors impeding the development of adoptive cell immunotherapy in solid tumors. A tumor-penetrating peptide iRGD has been widely used to deliver drugs deep into tumor tissues. CD3-targeting bispecific antibodies represent a promising immunotherapy which recruits and activates T cells. METHODS: T-cell penetration was demonstrated in tumor spheroids using confocal microscope, and in xenografted tumors by histology and in vivo real-time fluorescence imaging. Activation and cytotoxicity of T cells were assessed by flow cytometry and confocal microscope. Bioluminescence imaging was used to evaluate in vivo antitumor effects, and transmission electron microscopy was used for mechanistic studies. RESULTS: We generated a novel bifunctional agent iRGD-anti-CD3 which could immobilize iRGD on the surface of T cells through CD3 engaging. We found that iRGD-anti-CD3 modification not only facilitated T-cell infiltration in 3D tumor spheroids and xenografted tumor nodules but also induced T-cell activation and cytotoxicity against target cancer cells. T cells modified with iRGD-anti-CD3 significantly inhibited tumor growth and prolonged survival in several xenograft mouse models, which was further enhanced by the combination of programmed cell death protein 1 (PD-1) blockade. Mechanistic studies revealed that iRGD-anti-CD3 initiated a transport pathway called vesiculovacuolar organelles in the endothelial cytoplasm to promote T-cell extravasation. CONCLUSION: Altogether, we show that iRGD-anti-CD3 modification is an innovative and bifunctional strategy to overcome major bottlenecks in adoptive cell therapy. Moreover, we demonstrate that combination with PD-1 blockade can further improve antitumor efficacy of iRGD-anti-CD3-modified T cells.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antineoplásicos Inmunológicos/farmacología , Complejo CD3/antagonistas & inhibidores , Movimiento Celular/efectos de los fármacos , Inmunoterapia Adoptiva , Activación de Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/trasplante , Oligopéptidos/farmacología , Neoplasias Gástricas/terapia , Linfocitos T/efectos de los fármacos , Linfocitos T/trasplante , Animales , Complejo CD3/inmunología , Complejo CD3/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Citotoxicidad Inmunológica/efectos de los fármacos , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Esferoides Celulares , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacos , Microambiente Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Electronic (e-) cigarettes are growing in popularity despite uncertainties regarding their long-term health implications. The link between cigarette smoking and initiation of chronic lung disease took decades to unpick so in vitro studies mimicking e-cigarette exposure aim to detect early indicators of harm. In response to e-cigarette exposure, alveolar macrophages adopt a proinflammatory phenotype of increased secretion of proinflammatory cytokines, reduction in phagocytosis, and efferocytosis and reactive oxygen species generation. These effects are largely driven by free radical exposure, changes in PI3K/Akt signaling pathways, nicotine-induced reduction in phagocytosis receptors, and impaired lipid homeostasis leading to a foam-like lipid-laden phenotype. Neutrophils exhibit disrupted chemotaxis and transmigration to chemokines, reduced phagocytosis and bacterial killing, and an increase in protease secretion without corresponding antiproteases in response to e-cigarette exposure. This is driven by an altered ability to respond and to polarize toward chemoattractants, an activation of the p38 MAPK signaling pathway and inability to assemble NADPH oxidase. E-cigarettes induce lung epithelial cells to display decreased ciliary beat frequency and ion channel conductance as well as changes in chemokine secretion and surface protein expression. Changes in gene expression, mitochondrial function, and signaling pathways have been demonstrated in lung epithelial cells to explain these changes. Many functional outputs of alveolar macrophages, neutrophils, and lung epithelial cells have not been fully explored in the context of e-cigarette exposure and the underlying driving mechanisms are poorly understood. This review discusses current evidence surrounding the effects of e-cigarettes on alveolar macrophages, neutrophils, and lung epithelial cells with particular focus on the cellular mechanisms of change.
Asunto(s)
Células Epiteliales Alveolares , Sistemas Electrónicos de Liberación de Nicotina , Macrófagos Alveolares , Neutrófilos , Vapeo , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Animales , Quimiocinas/metabolismo , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Neutrófilos/metabolismo , Neutrófilos/patología , Fagocitosis/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Migración Transendotelial y Transepitelial/efectos de los fármacos , Vapeo/efectos adversos , Vapeo/metabolismo , Vapeo/patologíaRESUMEN
The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers. We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.
Asunto(s)
Mucosa Intestinal/citología , Mucosa Intestinal/fisiología , Modelos Biológicos , Migración Transendotelial y Transepitelial , Animales , Línea Celular , Quimiocinas/farmacología , Citometría de Flujo , Mucosa Intestinal/efectos de los fármacos , Ratones Endogámicos C57BL , Neutrófilos/citología , Permeabilidad , Estándares de Referencia , Migración Transendotelial y Transepitelial/efectos de los fármacosRESUMEN
The role of glycosaminoglycans on the surface of immune cells has so far been less studied compared to their participation in inflammatory responses as members of the endothelium and the extracellular matrix. In this study we have therefore investigated if glycosaminoglycans on immune cells act in concert with GPC receptors (i.e. both being cis-located on leukocytes) in chemokine-induced leukocyte mobilisation. For this purpose, freshly-prepared human neutrophils and monocytes were treated with heparinase III or chondroitinase ABC to digest heparan sulfate -chains or chondroitin sulfate-chains, respectively, from the leukocyte surfaces. Subsequent analysis of CXCL8- and CCL2-induced chemotaxis revealed that leukocyte migration was strongly reduced after eliminating heparan sulfate from the surface of neutrophils and monocytes. In the case of monocytes, an additional dependence of CCL2-induced chemotaxis on chondroitin sulfate was observed. We compared these results with the effect on chemotaxis of a heparan sulfate masking antibody and obtained similarly reduced migration. Following our findings, we postulate that glycosaminoglycans located on target leukocytes act synergistically with GPC receptors on immune cell migration, which is further influenced by glycosaminoglycans located on the inflamed tissue (i.e. trans with respect to the immune cell/GPC receptor). Both glycosaminoglycan localization sites seem to be important during inflammatory processes and could potentially be tackled in chemokine-related diseases.
Asunto(s)
Movimiento Celular , Quimiocina CCL2/farmacología , Glicosaminoglicanos/metabolismo , Interleucina-8/farmacología , Monocitos/metabolismo , Neutrófilos/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Condroitinasas y Condroitín Liasas/metabolismo , Femenino , Glipicanos/genética , Glipicanos/metabolismo , Liasa de Heparina/metabolismo , Humanos , Monocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Porcinos , Sindecanos/genética , Sindecanos/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacosRESUMEN
Intestinal injury is observed in cancer patients after radiotherapy and in individuals exposed to radiation after a nuclear accident. Radiation disrupts normal vascular homeostasis in the gastrointestinal system by inducing endothelial damage and senescence. Despite advances in medical technology, the toxicity of radiation to healthy tissue remains an issue. To address this issue, we investigated the effect of atorvastatin, a commonly prescribed hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor of cholesterol synthesis, on radiation-induced enteropathy and inflammatory responses. We selected atorvastatin based on its pleiotropic anti-fibrotic and anti-inflammatory effects. We found that atorvastatin mitigated radiation-induced endothelial damage by regulating plasminogen activator inhibitor-1 (PAI-1) using human umbilical vein endothelial cells (HUVECs) and mouse model. PAI-1 secreted by HUVECs contributed to endothelial dysfunction and trans-endothelial monocyte migration after radiation exposure. We observed that PAI-1 production and secretion was inhibited by atorvastatin in irradiated HUVECs and radiation-induced enteropathy mouse model. More specifically, atorvastatin inhibited PAI-1 production following radiation through the JNK/c-Jun signaling pathway. Together, our findings suggest that atorvastatin alleviates radiation-induced enteropathy and supports the investigation of atorvastatin as a radio-mitigator in patients receiving radiotherapy.
Asunto(s)
Atorvastatina/farmacología , Rayos gamma/efectos adversos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Enfermedades Intestinales/metabolismo , Monocitos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Traumatismos Experimentales por Radiación/metabolismo , Migración Transendotelial y Transepitelial , Animales , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Enfermedades Intestinales/patología , Ratones , Monocitos/patología , Traumatismos Experimentales por Radiación/patología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Migración Transendotelial y Transepitelial/efectos de la radiaciónRESUMEN
BACKGROUND: Environmental enteropathy (EE) is associated with stunting, impairment of responses to oral vaccines, and other adverse health consequences in young children throughout the developing world. EE is characterized by chronic low-grade intestinal inflammation and disrupted epithelial barrier integrity, partly resulting from dysregulation of tight junction proteins, observed in other enteropathies such as celiac disease. During EE, this dysregulation of tight junction expression amplifies translocation of pathogenic bacteria across the intestinal mucosa. AIMS: The aim was to determine whether enteropathogen-mediated epithelial barrier failure can be ameliorated using contra-pathogenicity therapies. METHODS: Intestinal epithelial barrier damage was assessed in Caco-2 cells incubated with three important enteropathogens identified in EE patients: Enteropathogenic Escherichia coli (EPEC), Citrobacter rodentium (C. rodentium), and Cryptosporidium parvum (C. parvum). Potential therapeutic molecules were tested to detect effects on transepithelial resistance (TER), bacterial translocation (BT), claudin-4 expression, and regulation of the inflammatory cytokine response. RESULTS: All three enteropathogens compared to uninfected cells, reduced TER (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0007), reduced claudin-4 expression, and permitted BT (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0003) through the monolayer. Zinc, colostrum, epidermal growth factor, trefoil factor 3, resistin-like molecule-ß, hydrocortisone, and the myosin light chain kinase inhibitor ML7 (Hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine hydrochloride); ML7) improved TER (up to 70%) and decreased BT (as much as 96%). Only zinc demonstrated modest antimicrobial activity. CONCLUSION: The enteropathogens impaired intestinal-epithelial barrier integrity with dysregulation of claudin-4 and increased bacterial translocation. Enteropathogen-mediated damage was reduced using contra-pathogenicity agents which mitigated the effects of pathogens without direct antimicrobial activity.
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Traslocación Bacteriana/fisiología , Citrobacter rodentium/metabolismo , Cryptosporidium parvum/metabolismo , Escherichia coli Enteropatógena/metabolismo , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Traslocación Bacteriana/efectos de los fármacos , Células CACO-2 , Citrobacter rodentium/efectos de los fármacos , Cryptosporidium parvum/efectos de los fármacos , Escherichia coli Enteropatógena/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento Epidérmico/uso terapéutico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Humanos , Hidrocortisona/farmacología , Hidrocortisona/uso terapéutico , Enfermedades Intestinales/tratamiento farmacológico , Enfermedades Intestinales/metabolismo , Enfermedades Intestinales/microbiología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Migración Transendotelial y Transepitelial/fisiologíaRESUMEN
Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg.
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Colforsina/farmacología , Endotelio Vascular , Neoplasias Pulmonares/patología , Células Neoplásicas Circulantes , Niclosamida/farmacología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Animales , Comunicación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales/métodos , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Quininógenos/análisis , Masculino , Metabolómica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Invasividad Neoplásica , Proteómica , Pez CebraRESUMEN
Multiple sclerosis (MS) is an autoimmune disorder where auto-aggressive T cells target the central nervous system (CNS), causing demyelination. The trans-endothelial migration of leucocytes across the blood-brain barrier (BBB) is one of the earliest CNS events in MS pathogenesis. We examined the effect of the disease state and treatment with fingolimod on the transmigration of peripheral blood mononuclear cells (PBMCs) in an in vitro BBB model. Patients' leucocyte numbers, subsets and phenotypes were assessed by flow cytometry. As expected, fingolimod treatment induced a significant reduction in T cell and B cell numbers compared to untreated MS patients and healthy controls. Interestingly fingolimod led to a marked reduction of CD4+ and a significant increase in CD8+ cell numbers. In migrated cells, only CD3+ cell numbers were reduced in fingolimod-treated, compared to untreated patients; it had no effect on B cell or monocyte transmigration. T cells were then differentiated into naïve, effector and memory subsets based on their expression of CCR7. This showed that MS patients had increased numbers of effector memory CD4+ cells re-expressing CD45RA (TEMRA) and a decrease in central memory (CM) CD8+ cells. The former was corrected by fingolimod, while the latter was not. CM CD4+ and CD8+ cells migrated across BBB more efficiently in fingolimod-treated patients. We found that while fingolimod reduced the proportions of naïve CD19+ B cells, it significantly increased the proportions of these cells which migrated. When B cells were further stratified based on CD24, CD27 and CD38 expression, the only effect of fingolimod was an enhancement of CD24hiCD27+ B cell migration, compared to untreated MS patients. The migratory capacities of CD8hi Natural Killer (NK), CD8dim NK and NK-T cells were also reduced by fingolimod. While the disease-modifying effects of fingolimod are currently explained by its effect on reducing circulating auto-aggressive lymphocytes, our data suggests that fingolimod may also have a direct though differential effect on the trans-endothelial migration of circulating lymphocyte populations.
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Clorhidrato de Fingolimod/uso terapéutico , Inmunosupresores/uso terapéutico , Subgrupos Linfocitarios/efectos de los fármacos , Esclerosis Múltiple/sangre , Esclerosis Múltiple/tratamiento farmacológico , Migración Transendotelial y Transepitelial/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Femenino , Clorhidrato de Fingolimod/farmacología , Humanos , Inmunosupresores/farmacología , Subgrupos Linfocitarios/metabolismo , Masculino , Migración Transendotelial y Transepitelial/fisiología , Resultado del TratamientoRESUMEN
Herpes simplex virus (HSV)-1 encephalitis has significant morbidity partly because of an over-exuberant immune response characterized by leukocyte infiltration into the brain and increased blood-brain barrier (BBB) permeability. Determining the role of specific leukocyte subsets and the factors that mediate their recruitment into the brain is critical to developing targeted immune therapies. In a murine model, we find that the chemokines CXCL1 and CCL2 are induced in the brain following HSV-1 infection. Ccr2 (CCL2 receptor)-deficient mice have reduced monocyte recruitment, uncontrolled viral replication, and increased morbidity. Contrastingly, Cxcr2 (CXCL1 receptor)-deficient mice exhibit markedly reduced neutrophil recruitment, BBB permeability, and morbidity, without influencing viral load. CXCL1 is produced by astrocytes in response to HSV-1 and by astrocytes and neurons in response to IL-1α, and it is the critical ligand required for neutrophil transendothelial migration, which correlates with BBB breakdown. Thus, the CXCL1-CXCR2 axis represents an attractive therapeutic target to limit neutrophil-mediated morbidity in HSV-1 encephalitis.
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Astrocitos/metabolismo , Barrera Hematoencefálica/patología , Quimiocina CXCL1/metabolismo , Encefalitis Viral/patología , Neuronas/metabolismo , Neutrófilos/patología , Migración Transendotelial y Transepitelial , Aciclovir/farmacología , Animales , Astrocitos/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Femenino , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/fisiología , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Permeabilidad , Receptores de Interleucina-8B/deficiencia , Receptores de Interleucina-8B/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacosRESUMEN
Inflammatory cell infiltration contributes to the pathogenesis of acute respiratory distress syndrome (ARDS). Protectin DX (PDX), an endogenous lipid mediator, shows anti-inflammatory and proresolution bioactions. In vivo, the mice were intraperitoneally injected with PDX (0.1 µg/mouse) after intratracheal (1 mg/kg) or intraperitoneal (10 mg/kg) LPS administration. Flow cytometry was used to measure inflammatory cell numbers. Clodronate liposomes were used to deplete resident macrophages. RT-PCR, and ELISA was used to measure MIP-2, MCP-1, TNF-α and MMP9 levels. In vitro, sorted neutrophils, resident and recruited macrophages (1 × 106 ) were cultured with 1 µg/mL LPS and/or 100 nmol/L PDX to assess the chemokine receptor expression. PDX attenuated LPS-induced lung injury via inhibiting recruited macrophage and neutrophil recruitment through repressing resident macrophage MCP-1, MIP-2 expression and release, respectively. Finally, PDX inhibition of neutrophil infiltration and transmembrane was associated with TNF-α/MIP-2/MMP9 signalling pathway. These data suggest that PDX attenuates LPS-stimulated lung injury via reduction of the inflammatory cell recruitment mediated via resident macrophages.
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Lesión Pulmonar Aguda/patología , Ácidos Docosahexaenoicos/uso terapéutico , Macrófagos/efectos de los fármacos , Lesión Pulmonar Aguda/inducido químicamente , Administración Intranasal , Animales , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Quimiocina CXCL2/biosíntesis , Quimiocina CXCL2/genética , Quimiocina CXCL2/fisiología , Quimiotaxis de Leucocito/efectos de los fármacos , Ácido Clodrónico/administración & dosificación , Ácido Clodrónico/farmacología , Ácidos Docosahexaenoicos/farmacología , Ácidos Docosahexaenoicos/fisiología , Inflamación , Inyecciones Intraperitoneales , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/toxicidad , Liposomas , Macrófagos/fisiología , Metaloproteinasa 9 de la Matriz/fisiología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Receptores CCR2/antagonistas & inhibidores , Receptores de Interleucina-8B/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Migración Transendotelial y Transepitelial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Granulocyte-monocyte colony stimulating factor (GM-CSF) is used as an adjuvant in various clinical and preclinical studies with contradictory results. These were attributed to opposing effects of GM-CSF on the immune or myeloid systems of the treated patients or to lack of optimal dosing regimens. The results of the present study point to inter-tumor heterogeneity as a possible mechanism accounting for the contrasting responses to GM-CSF incorporating therapies. Employing xenograft models of human melanomas in nude mice developed in our lab, we detected differential functional responses of melanomas from different patients to GM-CSF both in vitro as well as in vivo. Whereas cells of one melanoma acquired pro metastatic features following exposure to GM-CSF, cells from another melanoma either did not respond or became less malignant. We propose that inter-melanoma heterogeneity as manifested by differential responses of melanoma cells (and perhaps also of other tumor) to GM-CSF may be developed into a predictive marker providing a tool to segregate melanoma patients who will benefit from GM-CSF therapy from those who will not.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Melanoma/patología , Neoplasias Cutáneas/patología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Microambiente Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Interleucina-1alfa/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Solubilidad , Migración Transendotelial y Transepitelial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
CONTEXT: Palmatine, a biologically active isoquinoline alkaloid, possesses multiple pharmaceutical activities against mucosal infection and inflammation. OBJECTIVE: There are no reports about the influence of palmatine on uterine mucosal epithelial cells. MATERIALS AND METHODS: We used proteomics to analyse differentially expressed proteins (DEPs) in goat endometrial epithelial cells (EECs) stimulated by lipopolysaccharide (LPS, 5 µg/mL, the dosage can induce inflammatory response, according to our previous study) for 12 h and then treated with palmatine (80 µg/mL) for 8 h; the dosage was selected based on MTT assay. The EECs without any treatment were used as controls. Every group was treated in triplicate. RESULTS: A total of 428 DEPs in LPS-stimulated group and 486 DEPs in the palmatine-treated group were identified. Functional annotation analysis showed that palmatine mainly regulated the protein expression of structural molecules involved in the response to stimuli. Pathway analysis showed that cell adhesion molecule (CaM) pathways were most significant enriched due to palmatine treatment. Junction adhesion molecule 1 (JAM1), nectin 1 (NECT1) and cadherin 5 (CDH5), which play important roles in the transepithelial migration (TEpM) of leukocytes, were significantly downregulated by palmatine. Meanwhile, other proteins essential to the maintenance of cell adhesion and those that facilitate leukocyte migration were upregulated after palmatine treatment. Discussion and conclusions: The results suggested that palmatine regulates the expression of CaMs to affect TEpM during uterine mucosal inflammation and provides novel insight to understanding and developing palmatine pharmacology. Palmatine is a promising drug for treatment of mucosal inflammation.