RESUMEN
NK cells are key innate immune cells that play critical roles in host defense. Although NK cells have been shown to regulate immunity to some infectious diseases, their role in immunity to Trypanosoma congolense has not been investigated. NK cells are vital sources of IFN-γ and TNF-α; two key cytokines that are known to play important roles in resistance to African trypanosomes. In this article, we show that infection with T. congolense leads to increased levels of activated and functional NK cells in multiple tissue compartments. Systemic depletion of NK cells with anti-NK1.1 mAb led to increased parasitemia, which was accompanied by significant reduction in IFN-γ production by immune cells in the spleens and liver of infected mice. Strikingly, infected NFIL3-/- mice (which genetically lack NK cell development and function) on the normally resistant background were highly susceptible to T. congolense infection. These mice developed fulminating and uncontrolled parasitemia and died significantly earlier (13 ± 1 d) than their wild-type control mice (106 ± 26 d). The enhanced susceptibility of NFIL3-/- mice to infection was accompanied by significantly impaired cytokine (IFN-γ and TNF-α) response by CD3+ T cells in the spleens and liver. Adoptive transfer of NK cells into NFIL3-/- mice before infection rescued them from acute death in a perforin-dependent manner. Collectively, these studies show that NK cells are critical for optimal resistance to T. congolense, and its deficiency leads to enhanced susceptibility in infected mice.
Asunto(s)
Células Asesinas Naturales/inmunología , Tripanosomiasis Africana/inmunología , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trypanosoma congolense/inmunologíaRESUMEN
Dendritic cells (DC) connect the innate and adaptive arms of the immune system and carry out numerous roles that are significant in the context of viral disease. Their functions include the control of inflammatory responses, the promotion of tolerance, cross-presentation, immune cell recruitment and the production of antiviral cytokines. Based primarily on the available literature that characterizes the behaviour of many DC subsets during Severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19), we speculated possible mechanisms through which DC could contribute to COVID-19 immune responses, such as dissemination of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to lymph nodes, mounting dysfunctional inteferon responses and T cell immunity in patients. We highlighted gaps of knowledge in our understanding of DC in COVID-19 pathogenesis and discussed current pre-clinical development of therapies for COVID-19.
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COVID-19/inmunología , Células Dendríticas/inmunología , SARS-CoV-2/inmunología , Inmunidad Adaptativa , Animales , COVID-19/patología , COVID-19/terapia , Células Dendríticas/patología , Humanos , Inmunidad Innata , Linfocitos T/inmunologíaRESUMEN
c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.
Asunto(s)
Senescencia Celular , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Epitelio Pigmentado de la Retina/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Línea Celular Transformada , Proteínas de Unión al ADN , Células Epiteliales/citología , Humanos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas de Unión al ARN , Epitelio Pigmentado de la Retina/citología , Factores de TiempoRESUMEN
Semaphorin-3E (Sema-3E) is a member of a large family of proteins originally identified as axon guidance cues in neural development. It is expressed in different cell types, such as immune cells, cancer cells, neural cells, and epithelial cells. Subsequently, dys-regulation of Sema-3E expression has been reported in various biological processes that range from cancers to autoimmune and allergic diseases. Recent work in our laboratories revealed a critical immunoregulatory role of Sema-3E in experimental allergic asthma. We further speculate possible immune modulatory function(s) of Sema-3E on natural killer (NK) cells.
Asunto(s)
Células Asesinas Naturales/metabolismo , Semaforina-3A/metabolismo , Animales , Humanos , Semaforina-3A/química , Semaforina-3A/genética , Transducción de SeñalRESUMEN
CD40 expression on the surface of B lymphocytes is essential for their biological function and fate decision. The engagement of CD40 with its cognate ligand, CD154, leads to a sequence of cellular events in B lymphocytes, including CD40 cytoplasmic translocation, a temporal and spatial organization of effector molecules, and a cascade of CD40-induced signal transduction. The JLP scaffold protein was expressed in murine B lymphocytes. Using B lymphocytes from jlp-deficient mice, we observed that JLP deficiency resulted in defective CD40 internalization upon CD154/CD40 engagement. Examination of interactions and co-localization among CD40, JLP, dynein, and Rab5 in B lymphocytes suggested that CD40 internalization is a process of JLP-mediated vesicle transportation that depends on Rab5 and dynein. JLP deficiency also diminished CD40-dependent activation of MAPK and JNK, but not NF-κB. Inhibiting vesicle transportation from the direction of cell periphery to the cell center by a dynein inhibitor (ciliobrevin D) impaired both CD154-induced CD40 internalization and CD40-dependent MAPK activities in B lymphocytes. Collectively, our data demonstrate a novel role of the JLP scaffold protein in the bridging of CD154-triggered CD40 internalization and CD40-dependent signaling in splenic B lymphocytes.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linfocitos B/metabolismo , Antígenos CD40/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Western Blotting , Ligando de CD40/metabolismo , Células Cultivadas , Dineínas/metabolismo , Endocitosis , Femenino , Citometría de Flujo , Células HEK293 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones Noqueados , Microscopía Confocal , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Unión Proteica , Interferencia de ARN , Bazo/citología , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
CD3ζ has emerged as a clinically important immunological marker in head and neck squamous cell carcinoma (HNSCC) with reduced level of expression reported in both tumor infiltrating lymphocytes and peripheral blood lymphocytes. In this prospective study (power = 0.99, α = 0.05), CD3ζ expression was compared in 47 HNSCC patients and 53 controls using standardized flow cytometric method. There was no statistical difference in the percentages of the CD3 ε+ T-cell subset present in the peripheral blood mononuclear cells of the HNSCC patients and the healthy controls; however, T cells from the HNSCC patients produced a significantly weaker IFN-γ response in comparison to the healthy controls, when they were stimulated by the recall viral CEF peptide antigen. All patients were followed up for at least 3 years with a median follow-up of 45 months. Levels of CD3ζ-chain expression were measured at 117 follow-up visits at six-month intervals. Receiver operating characteristic curve identified the optimal cut off as a 12% increase in post treatment CD3ζ-chain expression from the baseline levels to confirm absence of HNSCC with the area under curve of 0.81 (95% CI = 0.68-0.94) for predicting absence of HNSCC. The specificity, sensitivity and positive predictive value were 81.25% 79.21% and 97.56%, respectively. Three-year disease specific survival (DSS) was significantly lower (p = 0.007) at 63.2% for patients who showed <12% increase in CD3ζ-chain level as compared to 96.2% for patients who had ≥12% increase. Our results indicate that the change in CD3ζ-chain expression from the baseline is an independent predictor of residual and recurrent HNSCC.
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Biomarcadores de Tumor/biosíntesis , Complejo CD3/biosíntesis , Carcinoma de Células Escamosas/genética , Neoplasias de Cabeza y Cuello/genética , Adulto , Anciano , Biomarcadores de Tumor/genética , Complejo CD3/genética , Carcinoma de Células Escamosas/patología , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/patología , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Migration and localization of NK cells into peripheral tissues are tightly regulated under normal and pathological conditions. The physiological importance of NK cell-DC crosstalk has been well documented. However, the ways in which DCs regulate the migratory properties of NK cells (such as chemotaxis, chemokinesis, chemo-repulsion) are not fully defined in vitro. Here, we employed a microfluidic platform to examine, at the single-cell level, C57BL/6 NK-cell migrations in a stable chemical gradient. We observed that soluble factors released by the immature and LPS-activated mature DCs induced a high level of chemotactic movement of IL-2-activated NK cells in vitro. We confirmed these findings in a standard trans-well migration assay, and identified CXCR3 as a key receptor on the NK cells that mediated the migration. More interestingly, we revealed a novel function of granulocyte macrophage colony-stimulating factor in repulsing NK-cell migrations. The future uses of such microfluidic device in the systematic evaluations of NK-cell migratory responses in NK cell-DC crosstalk will provide new insights into the development of DC-based NK-cell therapies against tumor and infections.
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Comunicación Celular/inmunología , Movimiento Celular/inmunología , Células Dendríticas/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Animales , Antineoplásicos/farmacología , Comunicación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Dendríticas/citología , Interleucina-2/farmacología , Células Asesinas Naturales/citología , Lipopolisacáridos/farmacología , Técnicas Analíticas MicrofluídicasRESUMEN
Molecular mechanisms of gene regulation underlying the activity-dependent long term changes of cellular electrical properties, such as those during memory, are largely unknown. We have shown that alternative splicing can be dynamically regulated in response to membrane depolarization and Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) activation, through special CaM kinase responsive RNA elements. However, proteins that mediate this regulation and how they are affected by CaMKIV are not known. Here we show that the regulation of the stress axis-regulated exon of the Slo1 potassium channel transcripts by membrane depolarization requires a highly conserved CaMKIV target serine (Ser-513) of the heterogeneous ribonucleoprotein L. Ser-513 phosphorylation within the RNA recognition motif 4 enhanced heterogeneous ribonucleoprotein L interaction with the CaMKIV-responsive RNA element 1 of stress axis-regulated exon and inhibited binding of the large subunit of the U2 auxiliary factor U2AF65. Both of these activities were abolished by a S513A mutation. Thus, through Ser-513, membrane depolarization/calcium signaling controls a critical spliceosomal assembly step to regulate the variant subunit composition of potassium channels.
Asunto(s)
Empalme Alternativo/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo L/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Potenciales de la Membrana/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Señalización del Calcio/fisiología , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Exones/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo L/genética , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fosforilación/fisiología , Hipófisis/citología , Ratas , Ribonucleoproteínas/metabolismo , Serina/metabolismo , Factor de Empalme U2AFRESUMEN
COVID-19 and influenza both cause enormous disease burdens, and vaccines are the primary measures for their control. Since these viral diseases are transmitted through the mucosal surface of the respiratory tract, developing an effective and convenient mucosal vaccine should be a high priority. We previously reported a recombinant vesicular stomatitis virus (rVSV)-based bivalent vaccine (v-EM2/SPΔC1Delta) that protects animals from both SARS-CoV-2 and influenza viruses via intramuscular and intranasal immunization. Here, we further investigated the immune response induced by oral immunization with this vaccine and its protective efficacy in mice. The results demonstrated that the oral delivery, like the intranasal route, elicited strong and protective systemic immune responses against SARS-CoV-2 and influenza A virus. This included high levels of neutralizing antibodies (NAbs) against SARS-CoV-2, as well as strong anti-SARS-CoV-2 spike protein (SP) antibody-dependent cellular cytotoxicity (ADCC) and anti-influenza M2 ADCC responses in mice sera. Furthermore, it provided efficient protection against challenge with influenza H1N1 virus in a mouse model, with a 100% survival rate and a significantly low lung viral load of influenza virus. All these findings provide substantial evidence for the effectiveness of oral immunization with the rVSV bivalent vaccine.
RESUMEN
Statins inhibit 3-hydroxy-3-methyl-glutarylcoenzyme CoA (HMG-CoA) reductase, the proximal enzyme for cholesterol biosynthesis. They exhibit pleiotropic effects and are linked to health benefits for diseases including cancer and lung disease. Understanding their mechanism of action could point to new therapies, thus we investigated the response of primary cultured human airway mesenchymal cells, which play an effector role in asthma and chronic obstructive lung disease (COPD), to simvastatin exposure. Simvastatin induced apoptosis involving caspase-9, -3 and -7, but not caspase-8 in airway smooth muscle cells and fibroblasts. HMG-CoA inhibition did not alter cellular cholesterol content but did abrogate de novo cholesterol synthesis. Pro-apoptotic effects were prevented by exogenous mevalonate, geranylgeranyl pyrophosphate and farnesyl pyrophosphate, downstream products of HMG-CoA. Simvastatin increased expression of Bax, oligomerization of Bax and Bak, and expression of BH3-only p53-dependent genes, PUMA and NOXA. Inhibition of p53 and silencing of p53 unregulated modulator of apoptosis (PUMA) expression partly counteracted simvastatin-induced cell death, suggesting a role for p53-independent mechanisms. Simvastatin did not induce mitochondrial release of cytochrome c, but did promote release of inhibitor of apoptosis (IAP) proteins, Smac and Omi. Simvastatin also inhibited mitochondrial fission with the loss of mitochondrial Drp1, an essential component of mitochondrial fission machinery. Thus, simvastatin activates novel apoptosis pathways in lung mesenchymal cells involving p53, IAP inhibitor release, and disruption of mitochondrial fission.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Citocromos c/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/patología , Proteínas Mitocondriales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Serina Endopeptidasas/metabolismo , Simvastatina/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Western Blotting , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Colesterol/metabolismo , Fibroblastos/efectos de los fármacos , Serina Peptidasa A2 que Requiere Temperaturas Altas , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Pulmón/metabolismo , Mesodermo/citología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Redirecting the tropism of viral vectors enables specific transduction of selected cells by direct administration of vectors. We previously developed targeting lentiviral vectors by pseudotyping with modified Sindbis virus envelope proteins. These modified Sindbis virus envelope proteins have mutations in their original receptor-binding regions to eliminate their natural tropisms, and they are conjugated with targeting proteins, including antibodies and peptides, to confer their tropisms on target cells. We investigated whether our targeting vectors interact with DC-SIGN, which traps many types of viruses and gene therapy vectors by binding to the N-glycans of their envelope proteins. We found that these vectors do not interact with DC-SIGN. When these vectors were produced in the presence of deoxymannojirimycin, which alters the structures of N-glycans from complex to high mannose, these vectors used DC-SIGN as their receptor. Genetic analysis demonstrated that the N-glycans at E2 amino acid (aa) 196 and E1 aa 139 mediate binding to DC-SIGN, which supports the results of a previous report of cryoelectron microscopy analysis. In addition, we investigated whether modification of the N-glycan structures could activate serum complement activity, possibly by the lectin pathway of complement activation. DC-SIGN-targeted transduction occurs in the presence of human serum complement, demonstrating that high-mannose structure N-glycans of the envelope proteins do not activate human serum complement. These results indicate that the strategy of redirecting viral vectors according to alterations of their N-glycan structures would enable the vectors to target specific cells types expressing particular types of lectins.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Vectores Genéticos , Lectinas Tipo C/metabolismo , Lentivirus , Polisacáridos/metabolismo , Receptores de Superficie Celular/metabolismo , Virus Sindbis/metabolismo , Proteínas del Envoltorio Viral , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Moléculas de Adhesión Celular/genética , Línea Celular , Proteínas del Sistema Complemento/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Lectinas Tipo C/genética , Lentivirus/genética , Lentivirus/metabolismo , Datos de Secuencia Molecular , Polisacáridos/química , Polisacáridos/genética , Receptores de Superficie Celular/genética , Virus Sindbis/genética , Transducción Genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Tandem pleckstrin homology domain proteins (TAPPs) are recruited to the plasma membrane via binding to phosphoinositides produced by phosphoinositide 3-kinases (PI3Ks). Whereas PI3Ks are critical for B-cell activation, the functions of TAPP proteins in B cells are unknown. We have identified 40 potential interaction partners of TAPP2 in B cells, including proteins involved in cytoskeletal rearrangement, signal transduction and endocytic trafficking. The association of TAPP2 with the cytoskeletal proteins utrophin and syntrophin was confirmed by Western blotting. We found that TAPP2, syntrophin, and utrophin are coexpressed in normal human B cells and B-chronic lymphocytic leukemia (B-CLL) cells. TAPP2 and syntrophin expression in B-CLL was variable from patient to patient, with significantly higher expression in the more aggressive disease subset identified by zeta-chain-associated protein kinase of 70 kDa (ZAP70) expression and unmutated immunoglobulin heavy chain (IgH) genes. We examined whether TAPP can regulate cell adhesion, a known function of utrophin/syntrophin in other cell types. Expression of membrane-targeted TAPP2 enhanced B-cell adhesion to fibronectin and laminin, whereas PH domain-mutant TAPP2 inhibited adhesion. siRNA knockdown of TAPP2 or utrophin, or treatment with PI3K inhibitors, significantly inhibited adhesion. These findings identify TAPP2 as a novel link between PI3K signaling and the cytoskeleton with potential relevance for leukemia progression.
Asunto(s)
Linfocitos B/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia de Células B/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Utrofina/metabolismo , Western Blotting , Adhesión Celular/fisiología , Línea Celular Tumoral , Citoesqueleto/metabolismo , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , ARN Interferente PequeñoRESUMEN
Regulation of dendritic cell functions is a complex process in which several mediators play diverse roles as a network in a context-dependent manner. The precise mechanisms underlying dendritic cell functions have remained to be addressed. Semaphorins play crucial roles in regulation of various cell functions. We previously revealed that Semaphorin 3E (Sema3E) contributes to regulation of allergen-induced airway pathology partly mediated by controlling recruitment of conventional dendritic cell subsets in vivo, though the underlying mechanism remained elusive. In this study, we investigate the potential regulatory role of Sema3E in dendritic cells. We demonstrated that bone marrow-derived dendritic cells differentiated from Sema3e-/- progenitors have an enhanced migration capacity both at the baseline and in response to CCL21. The enhanced migration ability of Sema3E dendritic cells was associated with an overexpression of the chemokine receptor (CCR7), elevated Rac1 GTPase activity and F-actin polymerization. Using a mouse model of allergic airway sensitization, we observed that genetic deletion of Sema3E leads to a time dependent upregulation of CCR7 on CD11b+ conventional dendritic cells in the lungs and mediastinal lymph nodes. Furthermore, aeroallergen sensitization of Sema3e-/- mice lead to an enhanced expression of PD-L2 and IRF-4 as well as enhanced allergen uptake in pulmonary CD11b+ DC, compared to wild type littermates. Collectively, these data suggest that Sema3E implicates in regulation of dendritic cell functions which could be considered a basis for novel immunotherapeutic strategies for the diseases associated with defective dendritic cells in the future.
Asunto(s)
Alérgenos/inmunología , Células de la Médula Ósea/inmunología , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Neumonía/inmunología , Semaforinas/fisiología , Actinas/metabolismo , Animales , Movimiento Celular , Quimiocina CCL21/metabolismo , Ratones , Ratones Noqueados , Neuropéptidos/metabolismo , Neumonía/metabolismo , Neumonía/patología , Receptores CCR7/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The prolactin inducible protein (PIP) is expressed to varying degrees in more than 90% of breast cancers (BCs). Although high levels of PIP expression in BC has been shown to correlate with better prognosis and patient response to chemotherapy, some studies suggest that PIP may also play a role in metastasis. Here, we investigated the role of PIP in BC using the well-established 4T1 and E0771 mouse BC cell lines. Stable expression of PIP in both cell lines did not significantly alter their proliferation, migration, and response to anticancer drugs in vitro compared to empty vector control. To assess the effect of PIP expression on breast tumorigenesis in vivo, the 4T1 syngeneic transplantable mouse model was utilized. In immunocompetent syngeneic BALB/c mice, PIP-expressing 4T1 primary tumors displayed delayed tumor onset and reduced tumor growth, and this was associated with higher percentages of natural killer cells and reduced percentages of type 2 T-helper cells in the tumor environment. The delayed tumor onset and growth were abrogated in immunodeficient mice, suggesting that PIP-mediated modulation of primary tumor growth involves an intact immune system. Paradoxically, we also observed that PIP expression was associated with a higher number of 4T1 colonies in the lungs in both the immunocompetent and immunodeficient mice. Gene expression analysis of PIP-expressing 4T1 cells (4T1-PIP) revealed that genes associated with tumor metastasis such as CCL7, MMP3 and MMP13, were significantly upregulated in 4T1-PIP cells when compared to the empty vector control (4T1-EV) cells. Collectively, these studies strongly suggest that PIP may possess a double-edge sword effect in BC, enhancing both antitumor immunity as well as metastasis.
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Understanding how NK cells interact with tumor cells under specific microenvironment will be informative in development of NK-cell based immunotherapy. Applications of microfluidic devices in in vitro studies of NK-cell migrations offer unique opportunities to examine NK-cell migrations at single-cell under controlled cellular microenvironments. Novel devices can be created and engineered to present precise configuration that mimics cellular microenvironments for cell migration studies. We established previously the first application of a simple Y-shaped device for imaging and analysis of the abilities of the immature and mature DC to regulate murine IL-2 activated NK cell migrations. Here we reported the application of our recent technical development of a novel microfluidic device, which is also called the triple docking device (i.e., D3-Chip), for the studies of NK-cell migrations in NK-4T1 breast cancer cell interactions in vitro. Key features of this microfluidic device are its pump-free gradient generation, and the three-parallel units design that supports easy setup and parallel comparison of multiple experimental conditions. The cell docking structure enables the prealignment of all NK cells at the same "start" position before their exposures to the test conditions. As a result, quantification of cell displacement toward a chemical gradient can be quantified by enumeration of the number of cells migrated out of the docking structure and their displacements. Such microfluidic devices can be further modified in future to mimic the complex in vivo microenvironments to support more advanced investigations of NK-cell migratory responses in vitro.
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Movimiento Celular , Pruebas Inmunológicas de Citotoxicidad/métodos , Células Asesinas Naturales/fisiología , Técnicas Analíticas Microfluídicas/métodos , Animales , Neoplasias de la Mama , Técnicas de Cocultivo , Femenino , Células Asesinas Naturales/inmunología , RatonesRESUMEN
JLP belongs to the JIP family whose members serve as scaffolding proteins that link motor proteins and their cargo for intracellular transport. Although JLP is mainly cytoplasmic, it accumulates as a focus in the perinuclear region when stimulated by extracellular stimuli. Focus formation, which changes the nucleus shape and concentrates the nuclear pores, depends on p38MAPK activation and the dynein retrograde motor protein complex. Extracellular stimuli trigger the tethering of PLK1 to the centrosome by JLP, leading to centrosome maturation and microtubule array formation. The centrosome localization domain of JLP is important for the binding of the centrosome and the formation of the JLP focus and the microtubule array. Furthermore, the formation of the JLP focus and the microtubule array is interdependent and important for the transport of NF-κB p65 to the nucleus and its unloading therein. In conclusion, JLP exhibits multiple functions in the nuclear translocation of NF-κB p65.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Citoplasma/metabolismo , Microtúbulos/metabolismo , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Dineínas/metabolismo , Células HEK293 , Humanos , Cinesinas/metabolismo , Unión Proteica/fisiología , Transporte de Proteínas/fisiología , Transducción de Señal/fisiología , Factor de Transcripción ReIARESUMEN
The importance of the bi-directional natural killer-dendritic cell crosstalk in coordinating anti-tumour and anti-microbial responses in vivo has been well established. However, physical parameters associated with natural killer-dendritic cell interactions have not been fully elucidated. We have previously used a simple "Y" shaped microfluidic device to study natural killer cell-migratory responses toward chemical gradients from a conditioned medium of dendritic cells. There are, however, limitations of the Y-shaped microfluidic devices that could not support higher throughput analyses and studies of cell-cell interactions. Here, we report two novel microfluidic devices (D3-Chip, T2-Chip) we applied in advanced studies of natural killer-cell migrations and their interactions with dendritic cells in vitro. The D3-Chip is an improved version of the previously published Y-shaped device that supports high-throughput analyses and docking of the cells of interest in the migration assay before they are exposed to a chemical gradient. The T2-Chip is created to support analyses of natural killer-dendritic cell cell-cell interactions without the requirement of promoting a natural killer cell to migrate long distances to find a loaded dendritic cell in the device. Using these two microfluidic platforms, we observe quantitative differences in the abilities of the immature and lipopolysaccharide-activated mature dendritic cells to interact with activated natural killer cells. The contact time between the activated natural killer cells and immature dendritic cells is significantly longer than that of the mature dendritic cells. There is a significantly higher frequency of an immature dendritic cell coming into contact with multiple natural killer cells and/or making multiple simultaneous contacts with multiple natural killer cells. To contrast, an activated natural killer cell has a significantly higher frequency of coming into contact with the mature dendritic cells than immature dendritic cells. Collectively, these differences in natural killer-dendritic cell interactions may underlie the differential maturation of immature dendritic cells by activated natural killer cells. Further applications of these microfluidic devices in studying natural killer-dendritic cell crosstalk under defined microenvironments shall enrich our understanding of the functional regulations of natural killer cells and dendritic cells in the natural killer-dendritic cell crosstalk.
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BACKGROUND: Hepatitis C virus (HCV) infection has a high rate of chronicity, attributable to its capacity to alter host immunity, including natural killer (NK) cell function. In this study, the interaction between NK cell activity and HCV viral load was investigated. METHODS: Peripheral blood NK cells were examined for cytotoxicity and interferon (IFN)-γ expression in HCV infected low (LVL, < 800,000 IU/mL, n = 10) and high (HVL, > 800,000 IU/mL, n = 13) viral load patient cohorts. RESULTS: Spontaneous NK cell cytotoxicity was more robust in the LVL cohort resulting in a negative correlation with viral loads (spontaneous, r = -0.437, P = 0.037; IFN-α activated, r = -0.372, P = 0.081). Although the percent of IFN-γ+ NK cells did not associate with viral load, within the LVL cohort there was a marked increase in IFN-γ+ NK cells upon IFN-α activation relative to medium alone (P < 0.01). To examine the inability of NK cells derived from HVL patients to be further activated, the expression of the exhaustion marker programmed cell death protein (PD)-1 was evaluated. PD-1 expression upon NK cell activation correlated with viral load (r = 0.649, P = 0.009). In addition, HCV proteins upregulated PD-1 expression in vitro (P < 0.05), suggesting that HCV can directly promote NK cell exhaustion. Cells from HVL patients were also more likely to produce IFN-γ in response to HCV core protein. The finding that NK cell PD-1 and IFN-γ expression are linked (r = 0.542, P < 0.05) suggests that increased IFN-γ levels may induce PD-1 as a negative feedback mechanism. CONCLUSIONS: High HCV loads appear to promote NK exhaustion in chronic HCV infection.
RESUMEN
Natural killer (NK) cells are lymphocytes that provide an important line of defense against many types of microorganisms, viruses and tumors. The development of an efficient gene transfer system for genetically modifying primary murine NK cells will facilitate the studies of NK cell differentiation, acquisition of self-tolerance, and induction of anti-tumor responses. In this study we used an enhanced green fluorescent protein (EGFP)-expressing vector to carry out a systematic evaluation of the efficiency of lentiviral transduction of primary murine NK cells with or without prior interleukin-2 (IL-2) activation. In a single-step transduction protocol, we demonstrated that human immunodeficiency virus type 1-based lentiviral vectors support an average of 40% transduction efficiency on primary NK cells. These genetically modified NK cells are found to maintain stable EGFP transgene expression in vitro, and can be further expanded in IL-2 supplemented culture medium. Lentiviral transduction does not affect NK surface phenotypes or functions (apoptosis, cytokine production and cytotoxicity). We further demonstrated efficient gene transfer into differentiating NK cells derived from the lentiviral-transduced murine hematopoietic progenitor cells in vitro. This study therefore establishes a simple and efficient approach to the genetic engineering of primary murine NK cells, and will prove useful in studying basic NK cell biology and in exploring the therapeutic potential of NK cells in inbred and transgenic mouse models.
Asunto(s)
Vectores Genéticos/genética , Células Asesinas Naturales/metabolismo , Lentivirus/genética , Transgenes/genética , Animales , Células Cultivadas , Citocinas/inmunología , Citotoxicidad Inmunológica/inmunología , Expresión Génica , Genes Reporteros/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Natural killer (NK) cells and dendritic cells (DCs) are two innate immune cells that are critical in regulating innate and adaptive immunity. Cellular functions and migratory responses of NK or DC can be further regulated in NK-DC crosstalk that involves multiple cytokine signals and/or direct cell-cell contacts. Semaphorin-3E (Sema-3E) is a member of a large family of Semaphorin proteins that play diverse regulatory functions in different biological systems upon its binding to the cognate receptors. However, possible role(s) of Sema-3E on the regulation of NK-cell functions has not been elucidated. Here, we first demonstrated that DC and NK cells expressed Sema-3E and its receptors, respectively. To formally address the importance of DC-derived Sema-3E in regulating NK-cell migration, we compared in vitro migratory responses of activated NK cells (aNKs) toward different conditioned media of DCs (immature, lipopolysaccharide- or Poly I:C-stimulated) derived from Sema-3E+/+ or Sema-3E-/- mice. We observed that aNKs exhibited enhanced migrations toward the conditioned medium of the immature Sema-3E-/- DC, when compared with that of the immature Sema-3E+/+ DC. Addition of exogenous recombinant Sema-3E to the conditioned medium of the Sema-3E-/- immature DC (iDC) abrogated such enhanced NK-cell migration. Our current work revealed a novel role of Sema-3E in limiting NK-cell migrations toward iDC in NK-DC crosstalk.