RESUMEN
Emerging evidence indicates that the immunomodulatory effect of mesenchymal stem cells (MSCs) is primarily attributed to the paracrine pathway. As a key paracrine effector, MSC-derived exosomes are small vesicles that play an important role in cell-to-cell communication by carrying bioactive substances. We previously found that exosomes derived from tonsil-derived mesenchymal stem cells (T-MSCs) were able to effectively attenuate inflammatory responses in mast cells. Here we investigated how T-MSC exosomes impact mast cells in steady state, and how exposure of T-MSCs to Toll-like receptors (TLRs) ligands changes this impact. Transcriptomic analysis of HMC-1 cells, a human mast cell line, using DNA microarrays showed that T-MSC exosomes broadly regulate genes involved in the normal physiology of mast cells. TLR3 or TLR4 primed T-MSC exosomes impacted fewer genes involved in specific functions in mast cells. This distinguishable regulation also was apparent in the analysis of related gene interactions. Our results suggest that MSC exosomes maintain immune homeostasis in normal physiology and impact the inflammatory state by modulating mast cell transcription.
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Exosomas , Células Madre Mesenquimatosas , Humanos , Mastocitos , Exosomas/genética , Exosomas/metabolismo , Comunicación Celular , Células Madre Mesenquimatosas/metabolismo , Expresión GénicaRESUMEN
(1) Background: six mammalian ceramide synthases (CerS1-6) determine the acyl chain length of sphingolipids (SLs). Although ceramide levels are increased in murine allergic asthma models and in asthmatic patients, the precise role of SLs with specific chain lengths is still unclear. The role of CerS2, which mainly synthesizes C22-C24 ceramides, was investigated in immune responses elicited by airway inflammation using CerS2 null mice. (2) Methods: asthma was induced in wild type (WT) and CerS2 null mice with ovalbumin (OVA), and inflammatory cytokines and CD4 (cluster of differentiation 4)+ T helper (Th) cell profiles were analyzed. We also compared the functional capacity of CD4+ T cells isolated from WT and CerS2 null mice. (3) Results: CerS2 null mice exhibited milder symptoms and lower Th2 responses than WT mice after OVA exposure. CerS2 null CD4+ T cells showed impaired Th2 and increased Th17 responses with concomitant higher T cell receptor (TCR) signal strength after TCR stimulation. Notably, increased Th17 responses of CerS2 null CD4+ T cells appeared only in TCR-mediated, but not in TCR-independent, treatment. (4) Conclusions: altered Th2/Th17 immune response with higher TCR signal strength was observed in CerS2 null CD4+ T cells upon TCR stimulation. CerS2 and very-long chain SLs may be therapeutic targets for Th2-related diseases such as asthma.
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Asma/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Esfingosina N-Aciltransferasa/deficiencia , Células Th17/inmunología , Células Th2/inmunología , Animales , Asma/inducido químicamente , Asma/genética , Asma/patología , Ratones , Ratones Noqueados , Ovalbúmina/toxicidad , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal/genética , Esfingosina N-Aciltransferasa/inmunología , Células Th17/patología , Células Th2/patologíaRESUMEN
Lymphatic vessels serve as conduits through which immune cells traffic. Because lymphatic vessels are also involved in lipid transport, their function is vulnerable to abnormal metabolic conditions such as obesity and hyperlipidemia. Exactly how these conditions impact immune cell trafficking, however, is not well understood. Here, we found higher numbers of LYVE-1-positive lymphatic endothelial cells and CD3-positive T cells in the lymph nodes of mice fed high-cholesterol or high-fat diets compared with those of mice fed a normal chow diet. To confirm the effect of fat content on immune cell trafficking, the lymphatic endothelial SVEC4-10 cell line was treated with palmitic acid at a 100 µM concentration. After 24 h, palmitic acid-treated cells exhibited increased expression of podoplanin and vascular growth-associated molecules (VEGFC, VEGFD, VEGFR3, and NRP2) and enhanced tube formation. Microarray analysis showed an increase in pro-inflammatory cytokine and chemokine transcription after palmitic acid treatment. Finally, transwell migration assay confirmed that T cell line moved toward medium previously cultured with palmitic acid-treated SVEC4-10 cells. Together, our results suggest that hyperlipidemia drives lymphatic vessel remodeling and T cell migration toward lymphatic endothelial cells.
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Movimiento Celular , Células Endoteliales/patología , Hiperlipidemias/inmunología , Hiperlipidemias/patología , Ganglios Linfáticos/patología , Linfocitos T/patología , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Quimiocinas/metabolismo , Dieta , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Hiperlipidemias/fisiopatología , Masculino , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Ácido Palmítico/toxicidad , Linfocitos T/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacosRESUMEN
Mutations in MEGF10 cause early onset myopathy, areflexia, respiratory distress, and dysphagia (EMARDD), a rare congenital muscle disease, but the pathogenic mechanisms remain largely unknown. We demonstrate that short hairpin RNA (shRNA)-mediated knockdown of Megf10, as well as overexpression of the pathogenic human p.C774R mutation, leads to impaired proliferation and migration of C2C12 cells. Myoblasts from Megf10-/- mice and Megf10-/-/mdx double knockout (dko) mice also show impaired proliferation and migration compared to myoblasts from wild type and mdx mice, whereas the dko mice show histological abnormalities that are not observed in either single mutant mouse. Cell proliferation and migration are known to be regulated by the Notch receptor, which plays an essential role in myogenesis. Reciprocal co-immunoprecipitation studies show that Megf10 and Notch1 interact via their respective intracellular domains. These interactions are impaired by the pathogenic p.C774R mutation. Megf10 regulation of myoblast function appears to be mediated at least in part via interactions with key components of the Notch signaling pathway, and defects in these interactions may contribute to the pathogenesis of EMARDD.
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Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Receptor Notch1/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Ratones , Ratones Endogámicos mdx , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Mioblastos/metabolismo , Mioblastos/fisiología , Receptor Notch1/genética , Transducción de SeñalRESUMEN
BACKGROUND: The skin is a dynamic body organ that can be activated by both central and local hypothalamic-pituitary-adrenal axis systems. This phenomenon might be the crucial explanation why stress can cause relapse of chronic inflammatory skin diseases, such as psoriasis. Here, we determined the effects of mast cells on keratinocyte proliferation under stress hormone stimulation. METHODS: We subcutaneously injected dexamethasone on the shaved back of mice and evaluated histological changes and keratinocyte growth factor (KGF) expression on dermal mast cells. Further, human mast cell line (HMC-1) and keratinocyte cell line (HaCaT) cells were treated with dexamethasone in vitro to observe the extent of proliferation and the expression of KGF. Finally, the supernatants of HMC-1 cells treated with dexamethasone were used for the culture of HaCaT cells to investigate the effect on proliferation. RESULTS: We observed epidermal thickening in dexamethasone-injected mice, accompanied by an increase in the number of KGF-expressing dermal mast cells. Similar to mouse dermal mast cells, KGF was highly expressed in the human mast cell line HMC-1 following stimulation with dexamethasone. Further, dexamethasone-treated mast cells promoted keratinocyte proliferation in vitro. However, the effects of mast cells on keratinocytes were significantly diminished in the presence of anti-KGF-blocking antibodies. CONCLUSION: Taken together, our results show that a stressful environment may disturb skin barrier homeostasis through mast cell-derived KGF expression.
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Dexametasona/farmacología , Factor 7 de Crecimiento de Fibroblastos/análisis , Queratinocitos/efectos de los fármacos , Mastocitos/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Queratinocitos/fisiología , Mastocitos/química , Mastocitos/patología , Ratones , Ratones Endogámicos BALB CRESUMEN
Sphingosine-1-phosphate (S1P) is a signalling sphingolipid metabolite that regulates important cell processes, including cell proliferation and apoptosis. Circulating S1P levels have been reported to be increased in patients with psoriasis relative to healthy patients. The aim of this study was to examine the potency of S1P inhibition using an imiquimod-induced psoriasis mouse model. Both topical ceramidase and sphingosine kinase 1/2 inhibition, which blocks S1P generation, alleviated imiquimod-induced skin lesions and reduced the serum interleukin 17-A levels induced by application of imiquimod. These treatments also normalized skin mRNA levels of genes associated with inflammation and keratinocyte differentiation. Inhibition of sphingosine kinase 2, but not sphingosine kinase 1, diminished levels of suppressor of cytokine signalling 1 and blocked T helper type 17 differentiation of naïve CD4+ T cells; imiquimod-induced psoriasis-like skin symptoms were also ameliorated. These results indicate the distinct effects of sphingosine kinase 1 and sphingosine kinase 2 inhibition on T helper type 17 generation and suggest molecules that inhibit S1P formation, including ceramidase and sphingosine kinase 2 inhibitors, as novel therapeutic candidates for psoriasis.
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Linfocitos T CD4-Positivos/fisiología , Inhibidores Enzimáticos/farmacología , Lisofosfolípidos/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Psoriasis/tratamiento farmacológico , Esfingosina/análogos & derivados , Administración Tópica , Animales , Diferenciación Celular/efectos de los fármacos , Ceramidasas/antagonistas & inhibidores , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Imiquimod , Inmunidad/efectos de los fármacos , Inflamación/genética , Interleucina-17/sangre , Masculino , Ratones , Psoriasis/inducido químicamente , Psoriasis/patología , Quinolonas/farmacología , ARN Mensajero/metabolismo , Esfingosina/biosíntesis , Proteína 1 Supresora de la Señalización de Citocinas , Células Th17RESUMEN
Next-generation sequencing is commonly used to screen for pathogenic mutations in families with Mendelian disorders, but due to the pace of discoveries, gaps have widened for some diseases between genetic and pathophysiological knowledge. We recruited and analyzed 16 families with limb-girdle muscular dystrophy (LGMD) of Arab descent from Saudi Arabia and Sudan who did not have confirmed genetic diagnoses. The analysis included both traditional and next-generation sequencing approaches. Cellular and metabolic studies were performed on Pyroxd1 siRNA C2C12 myoblasts and controls. Pathogenic mutations were identified in eight of the 16 families. One Sudanese family of Arab descent residing in Saudi Arabia harbored a homozygous c.464A>G, p.Asn155Ser mutation in PYROXD1, a gene recently reported in association with myofibrillar myopathy and whose protein product reduces thiol residues. Pyroxd1 deficiency in murine C2C12 myoblasts yielded evidence for impairments of cellular proliferation, migration, and differentiation, while CG10721 (Pyroxd1 fly homolog) knockdown in Drosophila yielded a lethal phenotype. Further investigations indicated that Pyroxd1 does not localize to mitochondria, yet Pyroxd1 deficiency is associated with decreased cellular respiration. This study identified pathogenic mutations in half of the LGMD families from the cohort, including one in PYROXD1. Developmental impairments were demonstrated in vitro for Pyroxd1 deficiency and in vivo for CG10721 deficiency, with reduced metabolic activity in vitro for Pyroxd1 deficiency.
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Distrofia Muscular de Cinturas/genética , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Adulto , Animales , Animales Modificados Genéticamente , Respiración de la Célula/genética , Células Cultivadas , Drosophila , Proteínas de Drosophila/genética , Femenino , Humanos , Masculino , Ratones , Mitocondrias Musculares/genética , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/patología , Distrofia Muscular de Cinturas/patología , Mioblastos/patología , Linaje , Arabia Saudita , SudánRESUMEN
Saturated free fatty acids (FFAs) act as lipid mediators and induce insulin resistance in skeletal muscle. Specifically, in obesity-related diseases such as type 2 diabetes, FFAs directly reduce insulin sensitivity and glucose uptake in skeletal muscle. However, the knowledge of how FFAs mediate inflammation and subsequent tissue disorders, including fibrosis in skeletal muscle, is limited. FFAs are a natural ligand for toll-like receptor 2 (TLR2) and TLR4, and induce chronic low-grade inflammation that directly stimulates skeletal muscle tissue. However, persistent inflammatory stimulation in tissues could induce pro-fibrogenic processes that ultimately lead to perturbation of the tissue architecture and dysfunction. Therefore, blocking the link between inflammatory primed skeletal muscle tissue and connective tissue might be an efficient therapeutic option for treating obesity-induced muscle inactivity. In this study, we investigated the impact of conditioned medium obtained from human palatine tonsil-derived mesenchymal stem cells (T-MSCs) on the interaction between skeletal muscle cells stimulated with palmitic acid (PA) and fibroblasts. We found that PA-treated skeletal muscle cells actively secreted interleukin-1ß (IL-1ß) and augmented the migration, proliferation and expression of fibronectin in L929 fibroblasts. Furthermore, T-CM inhibited the skeletal muscle cell-derived pro-fibrogenic effect via the production of the interleukin-1 receptor antagonist (IL-1Ra), which is an inhibitor of IL-1 signalling. Taken together, our data provide novel insights into the therapeutic potential of T-MSC-mediated therapy for the treatment of pathophysiological processes that occur in skeletal muscle tissues under chronic inflammatory conditions.
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Medios de Cultivo Condicionados/metabolismo , Fibroblastos/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Células Madre Mesenquimatosas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Tonsila Palatina/metabolismo , Animales , Línea Celular , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Resistencia a la Insulina/fisiología , Interleucina-1beta/metabolismo , Ratones , Músculo Esquelético/metabolismo , Receptor Toll-Like 2/metabolismoRESUMEN
Although mast cells are traditionally thought to function as effector cells in allergic responses, they have increasingly been recognized as important regulators of various immune responses. Mast cells mature locally; thus, tissue-specific influences are important for promoting mast cell accumulation and survival in the skin and the gastrointestinal tract. In this study, we determined the effects of keratinocytes on mast cell accumulation during Th17-mediated skin inflammation. We observed increases in dermal mast cells in imiquimod-induced psoriatic dermatitis in mice accompanied by the expression of epidermal stem cell factor (SCF), a critical mast cell growth factor. Similar to mouse epidermal keratinocytes, SCF was highly expressed in the human HaCaT keratinocyte cell line following stimulation with IL-17. Further, keratinocytes promoted mast cell proliferation following stimulation with IL-17 in vitro. However, the effects of keratinocytes on mast cells were significantly diminished in the presence of anti-CD117 (stem cell factor receptor) blocking antibodies. Taken together, our results revealed that the Th17-mediated inflammatory environment promotes mast cell accumulation through keratinocyte-derived SCF.
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Queratinocitos/metabolismo , Mastocitos/citología , Mastocitos/inmunología , Factor de Células Madre/metabolismo , Células Th17/inmunología , Línea Celular , Proliferación Celular , HumanosRESUMEN
The current study characterizes a cohort of limb-girdle muscular dystrophy (LGMD) in the United States using whole-exome sequencing. Fifty-five families affected by LGMD were recruited using an institutionally approved protocol. Exome sequencing was performed on probands and selected parental samples. Pathogenic mutations and cosegregation patterns were confirmed by Sanger sequencing. Twenty-two families (40%) had novel and previously reported pathogenic mutations, primarily in LGMD genes, and also in genes for Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital myopathy, myofibrillar myopathy, inclusion body myopathy and Pompe disease. One family was diagnosed via clinical testing. Dominant mutations were identified in COL6A1, COL6A3, FLNC, LMNA, RYR1, SMCHD1 and VCP, recessive mutations in ANO5, CAPN3, GAA, LAMA2, SGCA and SGCG, and X-linked mutations in DMD. A previously reported variant in DMD was confirmed to be benign. Exome sequencing is a powerful diagnostic tool for LGMD. Despite careful phenotypic screening, pathogenic mutations were found in other muscle disease genes, largely accounting for the increased sensitivity of exome sequencing. Our experience suggests that broad sequencing panels are useful for these analyses because of the phenotypic overlap of many neuromuscular conditions. The confirmation of a benign DMD variant illustrates the potential of exome sequencing to help determine pathogenicity.
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Exoma/genética , Pruebas Genéticas/métodos , Distrofia Muscular de Cinturas/diagnóstico , Distrofia Muscular de Cinturas/genética , Secuencia de Bases , Miopatías Distales/diagnóstico , Miopatías Distales/genética , Femenino , Enfermedad del Almacenamiento de Glucógeno Tipo II/diagnóstico , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Humanos , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular Facioescapulohumeral/diagnóstico , Distrofia Muscular Facioescapulohumeral/genética , Mutación/genética , Miopatías Estructurales Congénitas/diagnóstico , Miopatías Estructurales Congénitas/genética , Análisis de Secuencia de ADN/métodos , Estados UnidosRESUMEN
BACKGROUND AND PURPOSE: Most patients with cerebral infarction die of brain edema because of the breakdown of the blood-brain barrier (BBB) in ischemic tissue. Caveolins (a group of proteins) are key modulators of vascular permeability; however, a direct role of caveolin-1 (Cav-1) in the regulation of BBB permeability during ischemic injury has yet to be identified. METHODS: Cav-1 expression was measured by immunoblotting after photothrombotic ischemia. A direct functional role of Cav-1 in cerebral edema and BBB permeability during cerebral ischemia was investigated by genetic manipulation (gene disruption and re-expression) of Cav-1 protein expression in mice. RESULTS: There was a significant correlation between the extent of BBB disruption and the Cav-1 expression. In Cav-1-deficient (Cav-1(-/-)) mice, the extent of BBB disruption after cerebral ischemia was increased compared with wild-type (Cav-1(+/+)) mice, whereas the increase in cerebral edema volume was ameliorated by lentiviral-mediated re-expression of Cav-1. Furthermore, Cav-1(-/-) mice had significantly higher degradation of tight junction proteins and proteolytic activity of matrix metalloproteinase than Cav-1(+/+) mice. Conversely, re-expression of Cav-1 in Cav-1(-/-) mice restored tight junction protein expression and reduced matrix metalloproteinase proteolytic activity. CONCLUSIONS: These results indicate that Cav-1 is a critical determinant of BBB permeability. Strategies for regulating Cav-1 represent a novel therapeutic approach to controlling BBB disruption and subsequent neurological deterioration during cerebral ischemia.
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Barrera Hematoencefálica/metabolismo , Edema Encefálico/metabolismo , Isquemia Encefálica/metabolismo , Caveolina 1/metabolismo , Animales , Caveolina 1/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Ratones , Ratones NoqueadosRESUMEN
INTRODUCTION: The genetic causes of limb-girdle muscular dystrophy (LGMD) have been studied in numerous countries, but such investigations have been limited in Egypt. METHODS: A cohort of 30 families with suspected LGMD from Assiut, Egypt, was studied using immunohistochemistry, homozygosity mapping, Sanger sequencing, and whole exome sequencing. RESULTS: Six families were confirmed to have pathogenic mutations, 4 in SGCA and 2 in DMD. Of these, 3 families harbored a single nonsense mutation in SGCA, suggesting that this may be a common mutation in Assiut, Egypt, originating from a founder effect. CONCLUSIONS: The Assiut region in Egypt appears to share at least several of the common LGMD genes found in other parts of the world. It is notable that 4 of the 6 mutations were ascertained by means of whole exome sequencing, even though it was the last approach adopted. This illustrates the power of this technique for identifying causative mutations for muscular dystrophies. Muscle Nerve 54: 690-695, 2016.
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Codón sin Sentido/genética , Homocigoto , Distrofia Muscular de Cinturas/epidemiología , Distrofia Muscular de Cinturas/genética , Sarcoglicanos/genética , Egipto/epidemiología , Femenino , Humanos , Masculino , Distrofia Muscular de Cinturas/diagnóstico , LinajeRESUMEN
In a previous study, we reported that intrathecal injection of mesenchymal stem cells (MSCs) slowed disease progression in G93A mutant superoxide dismutase1 transgenic mice. In this study, we found that intrathecal MSC administration vastly increased the infiltration of peripheral immune cells into the spinal cord of Amyotrophic lateral sclerosis (ALS) mice (G93A mutant superoxide dismutase1 transgenic). Thus, we investigated the immunomodulatory effect of MSCs on peripheral blood mononuclear cells (PBMCs) in ALS patients, focusing on regulatory T lymphocytes (Treg ; CD4(+) /CD25(high) /FoxP3(+) ) and the mRNA expression of several cytokines (IFN-γ, TNF-α, IL-17, IL-4, IL-10, IL-13, and TGF-ß). Peripheral blood samples were obtained from nine healthy controls (HC) and sixteen patients who were diagnosed with definite or probable ALS. Isolated PBMCs from the blood samples of all subjects were co-cultured with MSCs for 24 or 72 h. Based on a fluorescence-activated cell sorting analysis, we found that co-culture with MSCs increased the Treg /total T-lymphocyte ratio in the PBMCs from both groups according to the co-culture duration. Co-culture of PBMCs with MSCs for 24 h led to elevated mRNA levels of IFN-γ and IL-10 in the PBMCs from both groups. However, after co-culturing for 72 h, although the IFN-γ mRNA level had returned to the basal level in co-cultured HC PBMCs, the IFN-γ mRNA level in co-cultured ALS PBMCs remained elevated. Additionally, the levels of IL-4 and TGF-ß were markedly elevated, along with Gata3 mRNA, a Th2 transcription factor mRNA, in both HC and ALS PBMCs co-cultured for 72 h. The elevated expression of these cytokines in the co-culture supernatant was confirmed via ELISA. Furthermore, we found that the increased mRNA level of indoleamine 2,3-dioxygenase (IDO) in the co-cultured MSCs was correlated with the increase in Treg induction. These findings of Treg induction and increased anti-inflammatory cytokine expression in co-cultured ALS PBMCs provide indirect evidence that MSCs may play a role in the immunomodulation of inflammatory responses when MSC therapy is targeted to ALS patients. We propose the following mechanism for the effect of mesenchymal stem cells (MSCs) administered intrathecally in amyotrophic lateral sclerosis (ALS): MSCs increase infiltration of peripheral immune cells into CNS and skew the infiltrated immune cells toward regulatory T lymphocytes (Treg ) and Th2 lymphocytes. Treg and Th2 secret anti-inflammatory cytokines such as IL-4, IL-10, and TGF-ß. A series of immunomodulatory mechanism provides a new strategy for ALS treatment.
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Esclerosis Amiotrófica Lateral/inmunología , Esclerosis Amiotrófica Lateral/terapia , Inmunomodulación/inmunología , Leucocitos Mononucleares/inmunología , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/inmunología , Adulto , Animales , Técnicas de Cocultivo , Femenino , Humanos , Inyecciones Espinales , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Linfocitos T Reguladores/inmunología , Adulto JovenRESUMEN
BACKGROUND: The pathogenesis of inflammatory skin disease involves the release of cytokines from keratinocytes, and one of these, IL-1ß, has been previously implicated in inflammatory skin disease. T(h)17 cells, a subset of T(h) cells involved in autoimmunity and inflammation, possess IL-1ß receptors and secrete cytokines such as IL-17 and IL-22 in response to IL-1ß stimulation. A mutation in the inflammasome protein NLRP3 (NACHT, LRR and PYD domains-containing protein 3) causes excess production of IL-1ß, resulting in an augmentation of T(h)17-dominant pathology. METHODS: To determine the feedback effect, if any, of IL-17 and/or IL-22 on the secretion of IL-1ß from keratinocytes, we stimulated the human keratinocyte cell line HaCaT, as well as caspase-1-deficient mice, with IL-17 or IL-22. RESULTS: We found that treatment with IL-17 and IL-22 causes an increase in IL-1ß via the activation of NLRP3 by a process that involves the generation of reactive oxygen species. Moreover, skin inflammation induced by IL-17 and IL-22 was lower in caspase-1 knockout (KO) mice relative to that induced by IL-1ß treatment. Additionally, skin inflammation induced by the drug imiquimod was lower in caspase-1 KO mice than in wild-type mice. CONCLUSION: These results indicate that cytokines from T(h)17 cells may potentiate IL-1ß-mediated skin inflammation and result in phenotypic alterations of keratinocytes via a feedback mechanism.
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Interleucina-17/inmunología , Interleucina-1beta/metabolismo , Interleucinas/inmunología , Queratinocitos/metabolismo , Transducción de Señal , Piel/inmunología , Animales , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Caspasa 1/inmunología , Caspasa 1/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Immunoblotting , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-17/metabolismo , Interleucinas/metabolismo , Queratinocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Piel/metabolismo , Piel/patología , Células Th17/inmunología , Interleucina-22RESUMEN
One of the most affected aspects of the aging process is immunity, with age-related immune system decline being responsible for an increase in susceptibility to infectious diseases and cancer risk. On the other hand, the aging process is accompanied with low-grade pro-inflammatory status. This condition involves a persistent rise in cytokine levels that can activate both innate and adaptive immune systems. Finally, despite the fact that immunological responses to antigenic stimulations decrease with age, the incidence and prevalence of many common autoimmune diseases increase in the elderly population. Overall, the co-existence of a prolonged, low-grade inflammatory status and declining immune activity appears to be a paradoxical phenomenon. This study characterized skin inflammation in mouse dermatitis model of various ages to monitor possible changes of inflammatory responses during aging.
RESUMEN
BACKGROUND: To achieve optimal bone marrow engraftment during bone marrow transplantation, migration of donor bone marrow cells (BMCs) toward the recipient's bone marrow is critical. Despite the enhanced engraftment of BMCs by co-administration of mesenchymal stem cells (MSCs), the efficiency can be variable depending on MSC donor. The purpose of this study is to examine the functional heterogeneity of tonsil-derived MSCs (TMSCs) and to identify a marker to evaluate efficacy for the enhancement of BMC migration. METHODS: To examine the donor-to-donor variation of TMSCs in potentiating BMC migration, we isolated TMSCs from 25 independent donors. Transcriptome of TMSCs and proteome of conditioned medium derived from TMSC were analyzed. RESULTS: Enhanced BMC migration by conditioned medium derived from TMSCs was variable depending on TMSC donor. The TMSCs derived from 25 donors showed distinct expression profiles compared with other cells, including fibroblasts, adipose-derived MSCs and bone marrow-derived MSCs. TMSCs were distributed in two categories: high- and low-efficacy groups for potentiating BMC migration. Transcriptome analysis of TMSCs and proteome profiles of conditioned medium derived from TMSCs revealed higher expression and secretion of matrix metalloproteinase (MMP) 1 in the high-efficacy group. MMP1 knockdown in TMSCs abrogated the supportive efficacy of conditioned medium derived from TMSC cultures in BMC migration. CONCLUSION: These data suggest that secreted MMP1 can be used as a marker to evaluate the efficacy of TMSCs in enhancing BMC migration. Furthermore, the strategy of analyzing transcriptomes and proteomes of the MSCs may be useful to set the standard for donor variation.
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Células Madre Mesenquimatosas , Tonsila Palatina , Células de la Médula Ósea , Medios de Cultivo Condicionados/farmacología , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteoma/metabolismo , HumanosRESUMEN
Allergic asthma is characterized by infiltration of eosinophils, elevated Th2 cytokine levels, airway hyperresponsiveness, and IgE. In addition to eosinophils, mast cells, and basophils, a variety of cytokines are also involved in the development of allergic asthma. The pivotal role of eosinophils in the progression of the disease has been a subject of controversy. To determine the role of eosinophils in the progression of airway inflammation, we sensitized and challenged BALB/c wild-type (WT) mice and eosinophil-deficient ΔdblGATA mice with ovalbumin (OVA) and analyzed different aspects of inflammation. We observed increased eosinophil levels and a Th2-dominant response in OVA-challenged WT mice. In contrast, eosinophil-deficient ΔdblGATA mice displayed an increased proportion of mast cells and a Th17-biased response following OVA inhalation. Notably, the levels of IL-33, an important cytokine responsible for Th2 immune deviation, were not different between WT and eosinophil-deficient mice. We also demonstrated that mast cells induced Th17-differentiation via IL-33/ST2 stimulation in vitro. These results indicate that eosinophils are not essential for the development of allergic asthma and that mast cells can skew the immune reaction predominantly toward Th17 responses via IL-33 stimulation.
Asunto(s)
Asma/patología , Inflamación/patología , Interleucinas/fisiología , Mastocitos/metabolismo , Células Th17/metabolismo , Animales , Asma/inducido químicamente , Asma/inmunología , Asma/metabolismo , Líquido del Lavado Bronquioalveolar , Broncoconstrictores/farmacología , Recuento de Células , Diferenciación Celular , Citocinas/genética , Citocinas/metabolismo , Eosinófilos/metabolismo , Femenino , Expresión Génica , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/metabolismo , Interleucinas/farmacología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Pulmón/fisiopatología , Mastocitos/inmunología , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ovalbúmina , Receptores de Interleucina/metabolismo , Células Th17/inmunología , Células Th2/metabolismoRESUMEN
Allogenic bone marrow transplantation (BMT), an important treatment for hematological malignancies, is often complicated by graft-versus-host disease (GVHD). Suppression of GVHD is associated with the unwanted diminishment of the graft-versus-leukemia (GVL) response. The aim of this study was to maintain the benefits of GVL during GVHD suppression through isolated blockade of T-cell migration factors. To this end, we developed a murine model of B-cell leukemia, which was treated with BMT to induce GVHD. Within this model, functional blockade of MIP-2/CXCR2 was analyzed by observing proteomic, histologic and clinical variables of GVHD manifestation. Luminex assay of collected tissue identified several cytokines [granulocyte colony-stimulating factor (G-CSF), keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), and interleukin-23 (IL-23)] that were upregulated during GHVD, but reduced by neutralizing the MIP-2/CXCR2 axis. In addition, donor T-cell blockade of CXCR2 combined with recipient administration of anti-MIP-2 caused a significant decrease in GVHD while preserving the GVL response. We propose that blocking the MIP-2/CXCR2 axis represents a novel strategy to separate the toxicity of GVHD from the beneficial effects of GVL after allogenic BMT.
Asunto(s)
Quimiocina CXCL2/antagonistas & inhibidores , Enfermedad Injerto contra Huésped/inmunología , Receptores de Interleucina-8B/antagonistas & inhibidores , Linfocitos T/inmunología , Animales , Trasplante de Médula Ósea , Línea Celular Tumoral , Movimiento Celular/inmunología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-ß in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-α, G-CSF, and TGF-ß. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.
Asunto(s)
Técnicas de Cocultivo , Células Madre Mesenquimatosas/fisiología , Neutrófilos/inmunología , Tejido Adiposo/citología , Diferenciación Celular , Supervivencia Celular/genética , Expresión Génica , Factor Estimulante de Colonias de Granulocitos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Células HL-60 , Humanos , Interferón-alfa/inmunología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Neutropenia/terapia , Neutrófilos/citología , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Crecimiento Transformadores/inmunologíaRESUMEN
Tonsils comprise part of the mucosal immune system and contain lymphocytes, macrophages, and follicular dendritic cells (FDCs). FDCs are located in the B cell area of the follicles of secondary lymphoid organs, such as the spleen, tonsils, or lymph nodes, and they trap and retain immune complexes on their surfaces to regulate B cell activation and maturation. Stromal cells from the palatine tonsils are often used for FDC in vitro studies, and it has been reported that human palatine tonsils may be a good source of multipotent mesenchymal cells. Therefore, we assessed whether tonsil-derived mesenchymal stromal cells could differentiate into a FDC-like phenotype. We discovered that stromal cells isolated from human tonsils not only had the potential to differentiate into various cell types of mesenchymal origin, but they also could differentiate into FDC-like cells under cytokine stimulation in vitro.