RESUMEN
Multiple sclerosis (MS) is a demyelinating inflammatory disease of the central nervous system (CNS), which is a chronic progressive disease and is caused by uncontrolled activation of myelin antigen specific T cells. It has high unmet medical needs due to the difficulty of efficient drug delivery into the CNS to control tissue inflammation. In this study, we demonstrate that a fusion protein of NOD-like receptor family member X1 (NLRX1) and blood brain barrier (BBB)-permeable peptide, dNP2 ameliorates experimental autoimmune encephalomyelitis (EAE). Methods: We purified recombinant LRR or NBD regions of NLRX1 protein conjugated with dNP2. To examine intracellular delivery efficiency of the recombinant protein, we incubated the proteins with Jurkat T cells or murine splenic T cells and their delivery efficiency was analyzed by flow cytometry. To investigate the therapeutic efficacy in an EAE model, we injected the recombinant protein into mice with 3 different treatment schemes e.g., prevention, semi-therapeutic, and therapeutic. To analyze their functional roles in T cells, we treated MACS-sorted naïve CD4 T cells with the proteins during their activation and differentiation into Th1, Th17, and Treg cells. Results: dNP2-LRR protein treatment showed significantly higher delivery efficiency than TAT-LRR or LRR alone in Jurkat T cells and mouse splenic T cells. In all three treatment schemes of EAE experiments, dNP2-LRR administration showed ameliorated tissue inflammation and disease severity with reduced number of infiltrating T cells producing inflammatory cytokines such as IFNγ. In addition, dNP2-LRR inhibited T cell activation, cytokine production, and Th1 differentiation. Conclusion: These results suggest that dNP2-LRR is a novel agent, which regulates effector T cell functions and could be a promising molecule for the treatment of CNS autoimmune diseases such as multiple sclerosis.
Asunto(s)
Péptidos de Penetración Celular/administración & dosificación , Portadores de Fármacos/administración & dosificación , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Proteínas Mitocondriales/química , Linfocitos T/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Barrera Hematoencefálica , Péptidos de Penetración Celular/farmacocinética , Portadores de Fármacos/farmacocinética , Evaluación Preclínica de Medicamentos , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Humanos , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Linfocinas/biosíntesis , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/farmacocinética , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Dominios Proteicos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/uso terapéutico , Organismos Libres de Patógenos Específicos , Médula Espinal/metabolismo , Médula Espinal/patología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
BACKGROUND: Blood-brain barrier (BBB) disruption and neuroinflammation are considered key mechanisms of pathogenic Escherichia coli invasion of the brain. However, the specific molecules involved in meningitic E. coli-induced BBB breakdown and neuroinflammatory response remain unclear. Our previous RNA-sequencing data from human brain microvascular endothelial cells (hBMECs) revealed two important host factors: platelet-derived growth factor-B (PDGF-B) and intercellular adhesion molecule-1 (ICAM-1), which were significantly upregulated in hBMECs after meningitic E. coli infection. Whether and how PDGF-B and ICAM-1 contribute to the development of E. coli meningitis are still unclear. METHODS: The western blot, real-time PCR, enzyme-linked immunosorbent assay, immunohistochemistry, and immunofluorescence were applied to verify the significant induction of PDGF-B and ICAM-1 by meningitic E. coli in vivo and in vitro. Evan's blue assay and electric cell-substrate impedance sensing assay were combined to identify the effects of PDGF-B on BBB permeability. The CRISPR/Cas9 technology, cell-cell adhesion assay, and electrochemiluminescence assay were used to investigate the role of ICAM-1 in neuroinflammation subversion. RESULTS: We verified the significant induction of PDGF-B and ICAM-1 by meningitic E. coli in mouse as well as monolayer hBMECs models. Functionally, we showed that the increase of PDGF-B may directly enhance the BBB permeability by decreasing the expression of tight junction proteins, and the upregulation of ICAM-1 contributed to neutrophils or monocytes recruitment as well as neuroinflammation subversion in response to meningitic E. coli infection. CONCLUSIONS: Our findings demonstrated the roles of PDGF-B and ICAM-1 in mediating bacterial-induced BBB damage as well as neuroinflammation, providing new concepts and potential targets for future prevention and treatment of bacterial meningitis.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Infecciones por Escherichia coli/metabolismo , Mediadores de Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/biosíntesis , Linfocinas/biosíntesis , Meningitis Bacterianas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Animales , Barrera Hematoencefálica/microbiología , Barrera Hematoencefálica/patología , Células Cultivadas , Escherichia coli , Infecciones por Escherichia coli/patología , Femenino , Meningitis Bacterianas/patología , Ratones , Uniones Estrechas/metabolismo , Uniones Estrechas/microbiología , Regulación hacia Arriba/fisiologíaRESUMEN
Bile duct cancer is known to contain numerous fibroblasts, and reported to recruit cancer- associated fibroblasts by secreting platelet-derived growth factor-D (PDGF-D) which needs serine proteases, such as matriptase, to behave as a ligand. However, their expression pattern, and prognostic value have not been clarified. In this study, we investigated the clinicopathological significance of PDGF-D and matriptase expression in patients with extrahepatic bile duct cancer. The samples were obtained from 256 patients who underwent the surgical resection between 1991 and 2015, and the expression levels of PDGF-D and matriptase were evaluated immunohistochemically. Staining intensities and distribution were scored, and finally classified into low and high expression groups in cancer cells and stroma respectively. High expression of matriptase in the cancer stroma was detected in 91 tumors (40%). The high stromal matriptase expression was significantly associated with shorter recurrence-free survival (RFS) and overall survival (OS) (P = 0.0027 and 0.0023, respectively). Multivariate analyses also demonstrated that the stromal matriptase expression level was an independent influential factor in RFS (P = 0.0050) and OS (P = 0.0093). Our findings suggest that the high stromal matriptase expression was strongly associated with tumor progression, recurrence and poor outcomes in patients with extrahepatic bile duct cancer.
Asunto(s)
Neoplasias de los Conductos Biliares/patología , Biomarcadores de Tumor/análisis , Colangiocarcinoma/patología , Serina Endopeptidasas/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de los Conductos Biliares/enzimología , Neoplasias de los Conductos Biliares/mortalidad , Conductos Biliares Extrahepáticos/patología , Colangiocarcinoma/enzimología , Colangiocarcinoma/mortalidad , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Linfocinas/biosíntesis , Masculino , Persona de Mediana Edad , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , PronósticoRESUMEN
BACKGROUND: Studies implicate that angiotensin 1-7 (Ang1-7) imparts protective effects in the kidney. However, its relevance in hypertensive kidney disease is not fully understood. The purpose of this study was to explore the role of Ang1-7 on renal damage/remodeling during hypertension and its potential underlying molecular-cellular mechanisms. METHODS: Hypertension was induced in adult Sprague-Dawley rats by infusion of aldosterone (ALDO; 0.75 µg/hour) for 4 weeks with or without co-treatment of Ang1-7 (1 mg/kg/day). Untreated rats served as controls. Systolic blood pressure was monitored by tail-cuff technique. Renal fibrosis was evaluated by picrosirius red staining and renal collagen volume fraction was quantitated using imaging analyzing system. The expression of profibrotic factors [transforming growth factor-ß1 (TGF-ß1), platelet-derived growth factor-D (PDGF-D), fibroblast growth factor-1 (FGF-1), vascular endothelial growth factor-D (VEGF-D), and tissue inhibitors of metalloproteinases (TIMPs)] and free radical producing enzymes (inducible nitric oxide synthase and nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) in the kidney were examined by reverse transcription-polymerase chain reaction and western blot. Renal oxidative stress was assessed by malondialdehyde (MDA) measurement. RESULTS: Chronic ALDO infusion caused hypertension and hypertensive renal disease represented as glomerular damage/sclerosis. Ang1-7 co-treatment did not affect blood pressure in ALDO-treated rats, but significantly attenuated the glomerular damage/fibrosis. ALDO treatment significantly elevated renal expression of profibrogenic factors, including TGF-ß1, TIMP-1/TIMP-2, FGF-1, PDGF-D, and VEGF-D, whereas Ang1-7 co-treatment significantly reduced renal TGF-ß1, TIMP-1/TIMP-2, and FGF-1, but not PDGF-D and VEGF-D. Furthermore, ALDO infusion elevated NADPH oxidase (gp91phox) and MDA in the kidney, which was attenuated by Ang1-7 co-treatment. CONCLUSIONS: Ang1-7 plays a protective role in the hypertensive kidney disease independent of blood pressure. The beneficial effects of Ang1-7 are likely mediated via suppressing TGF-ß/FGF-1 pathways and oxidative stress.
Asunto(s)
Angiotensina I/farmacología , Hipertensión Renal/tratamiento farmacológico , Riñón/metabolismo , Nefritis/tratamiento farmacológico , Estrés Oxidativo , Fragmentos de Péptidos/farmacología , Animales , Antihipertensivos/farmacología , Presión Sanguínea/fisiología , Western Blotting , Modelos Animales de Enfermedad , Fibrosis/genética , Fibrosis/metabolismo , Fibrosis/patología , Regulación de la Expresión Génica , Hipertensión Renal/metabolismo , Hipertensión Renal/patología , Riñón/efectos de los fármacos , Riñón/patología , Linfocinas/biosíntesis , Linfocinas/genética , Masculino , Nefritis/metabolismo , Nefritis/patología , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/genética , ARN/genética , Ratas , Ratas Sprague-Dawley , Inhibidores Tisulares de Metaloproteinasas/biosíntesis , Inhibidores Tisulares de Metaloproteinasas/genética , Factor D de Crecimiento Endotelial Vascular/biosíntesis , Factor D de Crecimiento Endotelial Vascular/genéticaRESUMEN
T cell expression of TIM-3 following Ag encounter has been associated with a continuum of functional states ranging from effector memory T cells to exhaustion. We have designed an in vitro culture system to specifically address the impact of anti-TIM-3/TIM-3 engagement on human Ag-specific CD8 T cells during a normal response to Ag and found that anti-TIM-3 treatment enhances T cell function. In our in vitro T cell culture system, MART1-specific CD8 T cells were expanded from healthy donors using artificial APCs. To ensure that the T cells were the only source of TIM-3, cells were rechallenged with peptide-loaded artificial APCs in the presence of anti-TIM-3 Ab. In these conditions, anti-TIM-3 treatment promotes generation of effector T cells as shown by acquisition of an activated phenotype, increased cytokine production, enhanced proliferation, and a transcription program associated with T cell differentiation. Activation of mTORC1 has been previously demonstrated to enhance CD8 T cell effector function and differentiation. Anti-TIM-3 drives CD8 T cell differentiation through activation of the mTORC1 as evidenced by increased levels of phosphorylated S6 protein and rhebl1 transcript. Altogether these findings suggest that anti-TIM-3, together with Ag, drives differentiation in favor of effector T cells via the activation of mTOR pathway. To our knowledge, this is the first report demonstrating that TIM-3 engagement during Ag stimulation directly influences T cell differentiation through mTORC1.
Asunto(s)
Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Memoria Inmunológica/inmunología , Diana Mecanicista del Complejo 1 de la Rapamicina/inmunología , Anticuerpos Monoclonales/farmacología , División Celular , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Humanos , Activación de Linfocitos , Linfocinas/biosíntesis , Linfocinas/genética , Antígeno MART-1/inmunología , Fosforilación , Procesamiento Proteico-Postraduccional , Especificidad del Receptor de Antígeno de Linfocitos T , Proteínas ras/biosíntesis , Proteínas ras/genéticaRESUMEN
Introducción. El caseinato de sodio, una sal de la caseína utilizada como agente proinflamatorio en ratones, es capaz de inducir granulopoyesis en vivo e incrementar la producción de citocinas esenciales en dicho evento.Objetivo. Evaluar si el caseinato de sodio es capaz de inducir un efecto biológico en células de origen linfoide y la producción de citocinas involucradas con este linaje.Materiales y métodos: Se utilizaron ratones hembra BALB/c de 8 a 12 semanas de edad. Los animales se inyectaron cuatro veces, con intervalos de 48 horas, por vía intraperitoneal con 1 ml de caseinato de sodio (10 % de SFB p/v). La población de linfocitos B y la incorporación de bromodesoxiuridina (BrdU) se analizaron mediante citometría de flujo. La detección de la interleucina 7 se evaluó mediante la técnica de ELISA.Resultados. Tras la inyección por vía intraperitoneal, el número de linfocitos B 220+ provenientes del bazo de ratones tratados con caseinato de sodio aumentó comparados con los que solo recibieron el vehículo como tratamiento (89,01±1,03 Vs. 75,66±2,08), así como la incorporación de BrdU en células B220+ (38,59±4,48 Vs. 11,82±1,04). Se evidenció, asimismo, el incremento en la concentración de la interleucina 7 (IL-7) en el suero de los ratones tratados con caseinato de sodio, comparados con los que solo recibieron el vehículo (62,1±17,5 Vs. 26,9±4,4 pg/ml).Conclusión. El caseinato de sodio fue capaz de aumentar el número de linfocitos B en bazo de ratones, así como inducir la producción de IL-7, citocina clave para la linfopoyesis B.
Asunto(s)
Linfocitos B/efectos de los fármacos , Caseínas/farmacología , Linfopoyesis/efectos de los fármacos , Animales , Médula Ósea/efectos de los fármacos , Caseínas/administración & dosificación , Caseínas/toxicidad , División Celular , Femenino , Inyecciones Intraperitoneales , Interleucina-7/biosíntesis , Interleucina-7/sangre , Interleucina-7/genética , Recuento de Linfocitos , Linfocinas/biosíntesis , Linfocinas/genética , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Anti-VEGF-A therapy has proven to be effective for many neovascular diseases. However, drug resistance to anti-VEGF-A treatment can develop. Also, not all patients with neovascular diseases are responsive to anti-VEGF-A treatment. The mechanisms underlying these important issues remain unclear. In this study, using different model systems, we found that inhibition of VEGF-A directly upregulated PDGF-CC and its receptors in multiple cell types in pathological angiogenesis in vitro and in vivo. Importantly, we further revealed that combinatorial targeting of VEGF-A and PDGF-CC suppressed pathological angiogenesis more efficiently than monotherapy. Given the potent angiogenic activity of PDGF-CC, our findings suggest that the development of resistance to anti-VEGF-A treatment may be caused by the compensatory upregulation of PDGF-CC, and combined inhibition of VEGF-A and PDGF-CC may have therapeutic advantages in treating neovascular diseases.
Asunto(s)
Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/metabolismo , Linfocinas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Células Cultivadas , Neovascularización Coroidal/patología , Resistencia a Medicamentos , Femenino , Humanos , Linfocinas/biosíntesis , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Células RAW 264.7 , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We previously demonstrated that treatment of diabetic peripheral neuropathy with the short (4 hours) half-life phosphodiesterase 5 (PDE5) inhibitor, sildenafil, improved functional outcome in diabetic db/db mice. To further examine the effect of PDE5 inhibition on diabetic peripheral neuropathy, we investigated the effect of another potent PDE5 inhibitor, tadalafil, on diabetic peripheral neuropathy. Tadalafil is pharmacokinetically distinct from sildenafil and has a longer half-life (17+hours) than sildenafil. Diabetic mice (BKS.Cg-m+/+Leprdb/J, db/db) at age 20 weeks were treated with tadalafil every 48 hours for 8 consecutive weeks. Compared with diabetic mice treated with saline, tadalafil treatment significantly improved motor and sensory conduction velocities in the sciatic nerve and peripheral thermal sensitivity. Tadalafil treatment also markedly increased local blood flow and the density of FITC-dextran perfused vessels in the sciatic nerve concomitantly with increased intraepidermal nerve fiber density. Moreover, tadalafil reversed the diabetes-induced reductions of axon diameter and myelin thickness and reversed the diabetes-induced increased g-ratio in the sciatic nerve. Furthermore, tadalafil enhanced diabetes-reduced nerve growth factor (NGF) and platelet-derived growth factor-C (PDGF-C) protein levels in diabetic sciatic nerve tissue. The present study demonstrates that tadalafil increases regional blood flow in the sciatic nerve tissue, which may contribute to the improvement of peripheral nerve function and the amelioration of diabetic peripheral neuropathy.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Nervio Ciático/efectos de los fármacos , Tadalafilo/administración & dosificación , Animales , Glucemia , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatología , Neuropatías Diabéticas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Linfocinas/biosíntesis , Linfocinas/genética , Ratones , Ratones Endogámicos NOD/genética , Actividad Motora/efectos de los fármacos , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/patología , Factor de Crecimiento Nervioso/biosíntesis , Factor de Crecimiento Nervioso/genética , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/genética , Nervio Ciático/irrigación sanguínea , Nervio Ciático/fisiopatologíaRESUMEN
Our previous work has shown that cerebellar interposed nucleus (IN) modulates immune function. Herein, we reveal mechanism underlying the immunomodulation. Treatment of bilateral cerebellar IN of rats with 3-mercaptopropionic acid (3-MP), a glutamic acid decarboxylase antagonist that reduces γ-aminobutyric acid (GABA) synthesis, enhanced cellular and humoral immune responses to bovine serum albumin, whereas injection of vigabatrin, a GABA-transaminase inhibitor that inhibits GABA degradation, in bilateral cerebellar IN attenuated the immune responses. The 3-MP or vigabatrin administrations in the cerebellar IN decreased or increased hypothalamic GABA content and lymphoid tissues' norepinephrine content, respectively, but did not alter adrenocortical or thyroid hormone levels in serum. In addition, a direct GABAergic projection from cerebellar IN to hypothalamus was found. These findings suggest that GABAergic neurons in cerebellar IN regulate immune system via hypothalamic and sympathetic pathways.
Asunto(s)
Núcleos Cerebelosos/inmunología , Neuronas GABAérgicas/inmunología , Hipotálamo/inmunología , Inmunidad Celular/fisiología , Inmunidad Humoral/fisiología , Sistema Nervioso Simpático/inmunología , Corticoesteroides/sangre , Animales , Bovinos , Núcleos Cerebelosos/efectos de los fármacos , Agonistas del GABA/farmacología , Hipotálamo/metabolismo , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/genética , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Linfocinas/biosíntesis , Linfocinas/genética , Vías Nerviosas/fisiología , Norepinefrina/fisiología , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Albúmina Sérica Bovina/inmunología , Hormonas Tiroideas/sangre , Ácido gamma-Aminobutírico/metabolismoRESUMEN
DCs are the first immune cells to be exposed to allergens, including chemical sensitizers, such as nickel, a human TLR4 agonist that induces DC maturation. In ACD, DCs can interact with PMNs that are recruited and activated, leading, in particular, to ectosome release. The objective of this work was to characterize the effects of PMN-Ect on DC functions in an ACD context. We first developed a standardized protocol to produce, characterize, and quantify ectosomes by use of human PLB-985 cells, differentiated into mature PMN (PLB-Ect). We then studied the in vitro effects of these purified ectosomes on human moDC functions in response to NiSO4 and to LPS, another TLR4 agonist. Confocal fluorescence microscopy showed that PLB-Ect was internalized by moDCs and localized in the lysosomal compartment. We then showed that PLB-Ect down-regulated NiSO4-induced moDC maturation, as witnessed by decreased expression of CD40, CD80, CD83, CD86, PDL-1, and HLA-DR and by decreased levels of IL-1ß, IL-6, TNF-α, and IL-12p40 mRNAs. These effects were related to p38MAPK and NF-κB down-regulation. However, no increase in pan-regulatory DC marker genes (GILZ, CATC, C1QA) was observed; rather, levels of effector DC markers (Mx1, NMES1) were increased. Finally, when these PLB-Ect + NiSO4-treated moDCs were cocultured with CD4(+) T cells, a Th2 cytokine profile seemed to be induced, as shown, in particular, by enhanced IL-13 production. Together, these results suggest that the PMN-Ect can modulate DC maturation in response to nickel, a common chemical sensitizer responsible for ADC.
Asunto(s)
Alérgenos/inmunología , Antígenos CD/biosíntesis , Micropartículas Derivadas de Células/fisiología , Células Dendríticas/inmunología , Dermatitis Alérgica por Contacto/inmunología , Regulación de la Expresión Génica/inmunología , Linfocinas/biosíntesis , Células Mieloides/inmunología , Neutrófilos/inmunología , Níquel/inmunología , Células Th2/citología , Alérgenos/farmacología , Antígenos CD/genética , Antígeno B7-H1/biosíntesis , Antígeno B7-H1/genética , Diferenciación Celular , Técnicas de Cocultivo , Células Dendríticas/efectos de los fármacos , Dermatitis Alérgica por Contacto/etiología , Antígenos HLA-DR/biosíntesis , Antígenos HLA-DR/genética , Humanos , Leucemia Mieloide Aguda/patología , Liposomas , Linfocinas/genética , Monocitos/citología , Células Mieloides/ultraestructura , Neutrófilos/ultraestructura , Níquel/farmacología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/inmunologíaRESUMEN
Platelet-derived growth factor-D (PDGF-D) was recently identified, and acts as potent mitogen for mesenchymal cells. PDGF-D also induces cellular transformation and promotes tumor growth. However, the functional role of PDGF-D in adipose-derived stem cells (ASCs) has not been identified. Therefore, we primarily investigated the autocrine and paracrine roles of PDGF-D in this study. Furthermore, we identified the signaling pathways and the molecular mechanisms involved in PDGF-D-induced stimulation of ASCs. It is of interest that PDGF-B is not expressed, but PDGF-D and PDGF receptor-ß are expressed in ASCs. PDGF-D showed the strongest mitogenic effect on ASCs, and PDGF-D regulates the proliferation and migration of ASCs through the PI3K/Akt pathways. PDGF-D also increases the proliferation and migration of ASCs through generation of mitochondrial reactive oxygen species (mtROS) and mitochondrial fission. mtROS generation and fission were mediated by p66Shc phosphorylation, and BCL2-related protein A1 and Serpine peptidase inhibitor, clade E, member 1 mediated the proliferation and migration of ASCs. In addition, PDGF-D upregulated the mRNA expression of diverse growth factors such as vascular endothelial growth factor A, fibroblast growth factor 1 (FGF1), FGF5, leukemia inhibitory factor, inhibin, beta A, interleukin 11, and heparin-binding EGF-like growth factor. Therefore, the preconditioning of PDGF-D enhanced the hair-regenerative potential of ASCs. PDGF-D-induced growth factor expression was attenuated by a pharmacological inhibitor of mitogen-activated protein kinase pathway. In summary, PDGF-D is highly expressed by ASCs, where it acts as a potent mitogenic factor. PDGF-D also upregulates growth factor expression in ASCs. Therefore, PDGF-D can be considered a novel ASC stimulator, and used as a preconditioning agent before ASC transplantation.
Asunto(s)
Tejido Adiposo/metabolismo , Regulación de la Expresión Génica/fisiología , Linfocinas/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Células Madre/metabolismo , Tejido Adiposo/citología , Células Cultivadas , Citocinas/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Linfocinas/farmacología , Mitocondrias/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Dinámicas Mitocondriales/fisiología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Células Madre/citologíaRESUMEN
Multipotent mesenchymal stromal cells (MSCs) possess reparative and immunoregulatory properties, making them attractive candidates for cellular therapy. However, the majority of MSCs administered i.v. encounter a pulmonary impasse and soon disappear from the lungs, raising the question of how they induce such durable immunosuppressive effects. Using a mouse model of allergic asthma, we show that administration of MSCs isolated from human bone marrow, umbilical cord, or adipose tissue provoked a pronounced increase in alveolar macrophages and inhibited hallmark features of asthma, including airway hyperresponsiveness, eosinophilic accumulation, and Th2 cytokine production. Importantly, selective depletion of this macrophage compartment reversed the therapeutic benefit of MSC treatment on airway hyperresponsiveness. Our data demonstrate that human MSCs exert cross-species immunosuppressive activity, which is mediated by alveolar macrophages in allergic asthma. As alveolar macrophages are the predominant immune effector cells at the air-tissue interface in the lungs, this study provides a compelling mechanism for durable MSC effects in the absence of sustained engraftment.
Asunto(s)
Asma/terapia , Terapia de Inmunosupresión/métodos , Macrófagos Alveolares/fisiología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Tejido Adiposo/citología , Animales , Asma/etiología , Asma/inmunología , Asma/patología , Asma/fisiopatología , Células de la Médula Ósea/citología , Hiperreactividad Bronquial/inducido químicamente , Hiperreactividad Bronquial/etiología , Líquido del Lavado Bronquioalveolar , Ácido Clodrónico/farmacología , Eosinofilia/etiología , Eosinofilia/inmunología , Femenino , Genes Reporteros , Supervivencia de Injerto , Xenoinjertos , Humanos , Inmunización , Interleucina-10/biosíntesis , Interleucina-10/genética , Pulmón/patología , Linfocinas/biosíntesis , Linfocinas/genética , Macrófagos Alveolares/efectos de los fármacos , Cloruro de Metacolina , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , Ovalbúmina/inmunología , Ovalbúmina/toxicidad , Especificidad de la Especie , Organismos Libres de Patógenos Específicos , Células Th2/metabolismo , Transducción Genética , Cordón Umbilical/citologíaRESUMEN
After antigen encounter by CD4(+) T cells, polarizing cytokines induce the expression of master regulators that control differentiation. Inactivation of the histone methyltransferase Ezh2 was found to specifically enhance T helper 1 (Th1) and Th2 cell differentiation and plasticity. Ezh2 directly bound and facilitated correct expression of Tbx21 and Gata3 in differentiating Th1 and Th2 cells, accompanied by substantial trimethylation at lysine 27 of histone 3 (H3K27me3). In addition, Ezh2 deficiency resulted in spontaneous generation of discrete IFN-γ and Th2 cytokine-producing populations in nonpolarizing cultures, and under these conditions IFN-γ expression was largely dependent on enhanced expression of the transcription factor Eomesodermin. In vivo, loss of Ezh2 caused increased pathology in a model of allergic asthma and resulted in progressive accumulation of memory phenotype Th2 cells. This study establishes a functional link between Ezh2 and transcriptional regulation of lineage-specifying genes in terminally differentiated CD4(+) T cells.
Asunto(s)
Regulación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/fisiología , Complejo Represivo Polycomb 2/fisiología , Subgrupos de Linfocitos T/citología , Células TH1/citología , Células Th2/citología , Animales , Asma/genética , Asma/inmunología , Asma/patología , Diferenciación Celular , Células Cultivadas/citología , Células Cultivadas/inmunología , Células Cultivadas/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Factor de Transcripción GATA3/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/deficiencia , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Memoria Inmunológica , Ensayos de Liberación de Interferón gamma , Linfocinas/biosíntesis , Linfocinas/genética , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/deficiencia , Complejo Represivo Polycomb 2/genética , Procesamiento Proteico-Postraduccional , Eliminación de Secuencia , Proteínas de Dominio T Box/biosíntesis , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
The molecular mechanisms responsible for the elevated metastatic potential of malignant melanoma are still not fully understood. In order to shed light on the molecules involved in the acquisition by melanoma of a highly aggressive phenotype, we compared the gene expression profiles of two cell clones derived from the human cutaneous metastatic melanoma cell line M14: a highly invasive clone (M14C2/MK18) and a clone (M14C2/C4) with low ability to invade the extracellular matrix (ECM). The highly invasive phenotype of M14C2/MK18 cells was correlated with overexpression of neuropilin-1, activation of a vascular endothelial growth factor (VEGF)-A/VEGFR-2 autocrine loop and secretion of matrix metalloprotease-2. Moreover, in an in vivo murine model, M14C2/MK18 cells displayed a higher growth rate as compared with M14C2/C4 cells, even though in vitro both clones possessed comparable proliferative potential. Microarray analysis in M14C2/MK18 cells showed a strong upregulation of platelet-derived growth factor (PDGF)-C, a cytokine that contributes to angiogenesis, and downregulation of calpain-3, a calcium-dependent thiol-protease that regulates specific signalling cascade components. Inhibition of PDGF-C with a specific antibody resulted in a significant decrease in ECM invasion by M14C2/MK18 cells, confirming the involvement of PDGF-C in melanoma cell invasiveness. Moreover, the PDGF-C transcript was found to be upregulated in a high percentage of human melanoma cell lines (17/20), whereas only low PDGF-C levels were detected in a few melanocytic cultures (2/6). By contrast, inhibition of calpain-3 activity in M14C2/C4 control cells, using a specific chemical inhibitor, markedly increased ECM invasion, strongly suggesting that downregulation of calpain-3 plays a role in the acquisition of a highly invasive phenotype. The results indicate that PDGF-C upregulation and calpain-3 downregulation are involved in the aggressiveness of malignant melanoma and suggest that modulators of these proteins or their downstream effectors may synergise with VEGFA therapies in combating tumour-associated angiogenesis and melanoma spread.
Asunto(s)
Calpaína/genética , Linfocinas/genética , Melanoma/genética , Proteínas Musculares/genética , Invasividad Neoplásica/genética , Factor de Crecimiento Derivado de Plaquetas/genética , Animales , Calpaína/biosíntesis , Línea Celular Tumoral , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Linfocinas/antagonistas & inhibidores , Linfocinas/biosíntesis , Metaloproteinasa 2 de la Matriz/genética , Melanoma/patología , Ratones , Proteínas Musculares/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Transducción de Señal/genética , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chronic beryllium disease (CBD) is an occupational lung disorder characterized by granulomatous inflammation and the accumulation of beryllium-responsive CD4(+) T cells in the lung. These differentiated effector memory T cells secrete IL-2, IFN-γ, and TNF-α upon in vitro activation. Beryllium-responsive CD4(+) T cells in the lung are CD28 independent and have increased expression of the coinhibitory receptor, programmed death 1, resulting in Ag-specific T cells that proliferate poorly yet retain the ability to express Th1-type cytokines. To further investigate the role of coinhibitory receptors in the beryllium-induced immune response, we examined the expression of CTLA-4 in blood and bronchoalveolar lavage cells from subjects with CBD. CTLA-4 expression was elevated on CD4(+) T cells from the lungs of study subjects compared with blood. Furthermore, CTLA-4 expression was greatest in the beryllium-responsive subset of CD4(+) T cells that retained the ability to proliferate and express IL-2. Functional assays show that the induction of CTLA-4 signaling in blood cells inhibited beryllium-induced T cell proliferation while having no effect on the proliferative capacity of beryllium-responsive CD4(+) T cells in the lung. Collectively, our findings suggest a dysfunctional CTLA-4 pathway in the lung and its potential contribution to the persistent inflammatory response that characterizes CBD.
Asunto(s)
Beriliosis/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígeno CTLA-4/inmunología , Pulmón/inmunología , Subgrupos de Linfocitos T/inmunología , Antígeno B7-1/biosíntesis , Antígeno B7-1/genética , Antígeno B7-2/biosíntesis , Antígeno B7-2/genética , Beriliosis/sangre , Beriliosis/patología , Líquido del Lavado Bronquioalveolar/inmunología , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/patología , Antígeno CTLA-4/biosíntesis , Antígeno CTLA-4/genética , División Celular , Enfermedad Crónica , Regulación de la Expresión Génica/inmunología , Humanos , Memoria Inmunológica , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-2/metabolismo , Pulmón/patología , Activación de Linfocitos , Linfocinas/biosíntesis , Linfocinas/genética , Modelos Inmunológicos , Receptor de Muerte Celular Programada 1/análisis , Subgrupos de Linfocitos T/patología , Células TH1/inmunología , Células TH1/patologíaRESUMEN
The severity of schistosome egg-induced hepatic granulomatous pathology depends markedly on the nature of the host immune responses. In this study, we used LMM and microarray analysis to compare gene expression profiles of histologically distinct zones within, and directly proximal to, hepatic granulomas that developed in C57BL/6 mice infected with Schistosoma japonicum. There was significant up-regulation of type-1, type-2, and type-17 immune-associated genes within the granuloma core (adjacent to eggs), followed by increased expression of type-2 and fibrotic genes at the outer zones of granulomas. Neutrophil-associated genes were also found to be expressed differentially in the core and at the peripheral zone of granulomas, present at 7 weeks p.i., demonstrating a significant role of neutrophils in S. japonicum granulomatous pathology. The release of NETs was observed microscopically in granulomas obtained from the livers of infected mice and when human neutrophils were incubated in vitro in the presence of S. japonicum eggs. These finding are the first to suggest a novel, dual role for neutrophils in the mediation of tissue damage and repair in S. japonicum egg-induced hepatic granulomatous lesions. Together, these results provide an overview of the local events occurring within the granuloma microenvironment.
Asunto(s)
Proteínas de la Matriz Extracelular/biosíntesis , Perfilación de la Expresión Génica , Granuloma/genética , Interacciones Huésped-Parásitos/genética , Hepatopatías/genética , Linfocinas/biosíntesis , Neutrófilos/fisiología , Schistosoma japonicum/fisiología , Esquistosomiasis Japónica/genética , Transcriptoma , Animales , Quimiocinas/biosíntesis , Quimiocinas/genética , Matriz Extracelular/ultraestructura , Proteínas de la Matriz Extracelular/genética , Femenino , Granuloma/inmunología , Granuloma/metabolismo , Granuloma/parasitología , Granuloma/patología , Interacciones Huésped-Parásitos/inmunología , Humanos , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Hepatopatías/inmunología , Hepatopatías/metabolismo , Hepatopatías/parasitología , Hepatopatías/patología , Linfocinas/genética , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Neutrófilos/ultraestructura , Óvulo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Esquistosomiasis Japónica/inmunología , Esquistosomiasis Japónica/metabolismo , Esquistosomiasis Japónica/patología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Regulación hacia ArribaRESUMEN
Tuberculosis (TB) remains a serious threat to public health, causing 2 million deaths annually world-wide. The control of TB has been hindered by the requirement of long duration of treatment involving multiple chemotherapeutic agents, the increased susceptibility to Mycobacterium tuberculosis infection in the HIV-infected population, and the development of multi-drug resistant and extensively resistant strains of tubercle bacilli. An efficacious and cost-efficient way to control TB is the development of effective anti-TB vaccines. This measure requires thorough understanding of the immune response to M. tuberculosis. While the role of cell-mediated immunity in the development of protective immune response to the tubercle bacillus has been well established, the role of B cells in this process is not clearly understood. Emerging evidence suggests that B cells and humoral immunity can modulate the immune response to various intracellular pathogens, including M. tuberculosis. These lymphocytes form conspicuous aggregates in the lungs of tuberculous humans, non-human primates, and mice, which display features of germinal center B cells. In murine TB, it has been shown that B cells can regulate the level of granulomatous reaction, cytokine production, and the T cell response. This chapter discusses the potential mechanisms by which specific functions of B cells and humoral immunity can shape the immune response to intracellular pathogens in general, and to M. tuberculosis in particular. Knowledge of the B cell-mediated immune response to M. tuberculosis may lead to the design of novel strategies, including the development of effective vaccines, to better control TB.
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Anticuerpos Antibacterianos/inmunología , Subgrupos de Linfocitos B/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Células Presentadoras de Antígenos/inmunología , Centro Germinal/inmunología , Humanos , Inmunidad Celular , Tuberculosis Latente/inmunología , Activación de Linfocitos , Cooperación Linfocítica , Linfocinas/biosíntesis , Linfocinas/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Modelos Inmunológicos , Primates , Receptores de IgG/inmunología , Especificidad de la Especie , Subgrupos de Linfocitos T/inmunología , Tuberculoma/inmunología , Tuberculoma/patología , Vacunas contra la Tuberculosis/inmunologíaRESUMEN
Proteoliposomes purified from the Outer Membrane of Neisseria meningitidis B, have been successfully used as core for adjuvants and vaccine formulations. We have tried to increase their structural definition and to conserve their efficacy and stability avoiding the addition of the aluminum hydroxide to the final formulation. Liposomal particle systems were prepared from components of defined molecular structure, such as a Neisseria meningitidis B protein complex, extracted and purified without forming vesicle structures. Liposomes were prepared from a mixture of dioleoyl phosphatidyl serine and cholesterol, using the classical dehydration-rehydration method. Transmission Electron Microscopy (TEM) was used to characterize the liposomes. BALB/c mice were used for animal testing procedures. Analysis of specific IgG response, serum bactericidal activity as well as DTH reaction was carried out. Isolation and purification of mRNA and real-time PCR, was performed to determine the dominating Th lymphokine pattern. The new antimeningococcal formulation without aluminum hydroxide prepared with components of defined molecular structure assembled itself into Neoproteoliposomes (NPL) ranging from 50 to 70 nm in diameter. The extraction and purification of selected membrane proteins to provide the antigen for this new formulation (PD-Tp), as well as the NPL-formulation favors a Th1 response pattern, suggested by the higher percentages of DTH, increased expression of proinflamatory lymphokine mRNAs when administered by intramuscular and intranasal routes. It stimulates a systemic bactericidal antibody response against Neisseria meningitidis B and immunologic memory similar to the Cuban VA-MENGOC-BC vaccine, even at lower dosages and is less reactogenic at the injection site in comparison with the formulation with aluminum hydroxide. This new adjuvant formulation could be applicable to the development of new and improved vaccines against meningococcal disease, and eventually as modulators of the immune response against other diseases.
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Adyuvantes Inmunológicos , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Proteolípidos/inmunología , Células TH1/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Hidróxido de Aluminio , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Hipersensibilidad Tardía/inmunología , Inmunoglobulina G/inmunología , Linfocinas/biosíntesis , Linfocinas/inmunología , Vacunas Meningococicas/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Proteolípidos/administración & dosificación , ARN Mensajero/análisisRESUMEN
Platelet-derived growth factors (PDGFs) and their tyrosine kinase receptors play instrumental roles in embryonic organogenesis and diseases of adult organs. In particular, platelet-derived growth factor receptor-alpha (PDGFRα) is expressed by multipotent cardiovascular progenitors in mouse and human embryonic stem cell systems. Although cardiac PDGFRα expression has been studied in multiple species, little is known about its expression in the human heart. Using immunofluorescence, we analyzed PDGFRα expression in both human fetal and diseased adult hearts, finding strong expression in the interstitial cells of the epicardium, myocardium, and endocardium, as well as the coronary smooth muscle. Only rare endothelial cells and cardiomyocytes expressed PDGFRα. This pattern was consistent for both the fetal and adult diseased hearts, although more PDGFRα+ cardiomyocytes were noted in the latter. In vitro differentiation assays were then performed on the PDGFRα+ cell fraction isolated from the cardiomyocyte-depleted human fetal hearts. Protocols previously reported to direct differentiation to a cardiomyocyte (5-azacytidine), smooth muscle (PDGF-BB), or endothelial cell fates (vascular endothelial growth factor [VEGF]) were used. Although no significant cardiomyocyte differentiation was observed, PDGFRα+ cells generated significant numbers of smooth muscle cells (smooth muscle-α-actin+ and smooth muscle myosin+) and endothelial cells (CD31+). These data suggest that a subfraction of the cardiac PDGFRα+ populations are progenitors contributing predominantly to the vascular and mesenchymal compartments of the human heart. It may be possible to control the fate of these progenitors to promote vascularization or limit fibrosis in the injured heart.
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Linfocinas/biosíntesis , Miocardio/metabolismo , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Células Madre/metabolismo , Animales , Azacitidina/farmacología , Diferenciación Celular/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Lesiones Cardíacas/metabolismo , Lesiones Cardíacas/terapia , Humanos , Linfocinas/metabolismo , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/crecimiento & desarrollo , Miocardio/patología , Miocitos Cardíacos/citología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
In refractory asthma, neutrophils, rather than eosinophils, often predominate in the airways. Neutrophilic airway inflammation appears to be resistant to steroids and may be related to the Th17, rather than the Th2, cytokine milieu. However, the role of GATA-3 and RORγt, transcription factors for Th2 and Th17 cell differentiation, respectively, in the pathogenesis of steroid-insensitive asthma remains unclear. To examine the effect of GATA-3- and RORγt-overexpression backgrounds on airway inflammation and steroid sensitivity, we generated two strains of transgenic mice overexpressing GATA-3 or RORγt. Mice were sensitized and challenged with OVA. Some OVA-sensitized/challenged mice were treated with dexamethasone, anti-IL-17 Ab, CXCR2 antagonist, or anti-IL-6R Ab to demonstrate their therapeutic effects on airway inflammation. Although Ag-specific airway inflammation and hyperresponsiveness were induced in each mouse, the phenotype of inflammation showed a distinct difference that was dependent upon the genotype. GATA-3-overexpressing mice exhibited steroid-sensitive eosinophilic inflammation with goblet cell hyperplasia and mucus hyperproduction under Th2-biased conditions, and RORγt-overexpressing mice developed steroid-insensitive neutrophilic inflammation under Th17-biased conditions. The levels of keratinocyte-derived chemokine, MIP-2, IL-6, and other neutrophil chemotaxis-related mediators were significantly elevated in OVA-exposed RORγt-overexpressing mice compared with wild-type mice. Interestingly, airway hyperresponsiveness accompanied by neutrophilic airway inflammation in RORγt-overexpressing mice was effectively suppressed by anti-IL-17 Ab, CXCR2 antagonist, or anti-IL-6R Ab administration. In conclusion, our results suggest that the expression levels of GATA-3 and RORγt may be important for determining the phenotype of asthmatic airway inflammation. Furthermore, blockade of the Th17-signaling pathway may be a treatment option for steroid-insensitive asthma.