RESUMEN
The Tick-borne encephalitis virus (TBEV) causes different disease symptoms varying from asymptomatic infection to severe encephalitis and meningitis suggesting a crucial role of the human host immune system in determining the fate of the infection. There is a need to understand the mechanisms underpinning TBEV-host interactions leading to protective immunity. To this aim, we studied the response of human peripheral blood mononuclear cells (PBMC) to the whole formaldehyde inactivated TBEV (I-TBEV), the drug substance of Encepur, one of the five commercially available vaccine. Immunophenotyping, transcriptome and cytokine profiling of PBMC revealed that I-TBEV generates differentiation of a sub-population of plasmacytoid dendritic cells (pDC) that is specialized in type I interferon (IFN) production. In contrast, likely due to the presence of aluminum hydroxide, Encepur vaccine was a poor pDC stimulus. We demonstrated I-TBEV-induced type I IFN together with Interleukin 6 and BAFF to be critical for B cell differentiation to plasmablasts as measured by immunophenotyping and immunoglobulin production. Robust type I IFN secretion was induced by pDC with the concerted action of both viral E glycoprotein and RNA mirroring previous data on dual stimulation of pDC by both S. aureus and influenza virus protein and nucleic acid that leads to a type I IFN-mediated sustained immune response. E glycoprotein neutralization or high temperature denaturation and inhibition of Toll-like receptor 7 signalling confirmed the importance of preserving the functional integrity of these key viral molecules during the inactivation procedure and manufacturing process to produce a vaccine able to stimulate strong immune responses.
Asunto(s)
Células Dendríticas/inmunología , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/prevención & control , Interacciones Microbiota-Huesped , Interferón Tipo I/metabolismo , Vacunas Virales/inmunología , Antivirales/inmunología , Diferenciación Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Dendríticas/virología , Encefalitis Transmitida por Garrapatas/virología , Humanos , Leucocitos Mononucleares/inmunología , Activación de Linfocitos , ARN Viral/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Autophagy is a primordial eukaryotic pathway, which provides the immune system with multiple mechanisms for the elimination of invading pathogens including Mycobacterium tuberculosis (Mtb). As a consequence, Mtb has evolved different strategies to hijack the autophagy process. Given the crucial role of human primary dendritic cells (DC) in host immunity control, we characterized Mtb-DC interplay by studying the contribution of cellular microRNAs (miRNAs) in the post-transcriptional regulation of autophagy related genes. From the expression profile of de-regulated miRNAs obtained in Mtb-infected human DC, we identified 7 miRNAs whose expression was previously found to be altered in specimens of TB patients. Among them, gene ontology analysis showed that miR-155, miR-155* and miR-146a target mRNAs with a significant enrichment in biological processes linked to autophagy. Interestingly, miR-155 was significantly stimulated by live and virulent Mtb and enriched in polysome-associated RNA fraction, where actively translated mRNAs reside. The putative pair interaction among the E2 conjugating enzyme involved in LC3-lipidation and autophagosome formation-ATG3-and miR-155 arose by target prediction analysis, was confirmed by both luciferase reporter assay and Atg3 immunoblotting analysis of miR-155-transfected DC, which showed also a consistent Atg3 protein and LC3 lipidated form reduction. Late in infection, when miR-155 expression peaked, both the level of Atg3 and the number of LC3 puncta per cell (autophagosomes) decreased dramatically. In accordance, miR-155 silencing rescued autophagosome number in Mtb infected DC and enhanced autolysosome fusion, thereby supporting a previously unidentified role of the miR-155 as inhibitor of ATG3 expression. Taken together, our findings suggest how Mtb can manipulate cellular miRNA expression to regulate Atg3 for its own survival, and highlight the importance to develop novel therapeutic strategies against tuberculosis that would boost autophagy.
Asunto(s)
Proteínas Relacionadas con la Autofagia/genética , Autofagia/genética , Células Dendríticas/metabolismo , MicroARNs/genética , Mycobacterium tuberculosis/fisiología , Enzimas Ubiquitina-Conjugadoras/genética , Autofagosomas/inmunología , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/antagonistas & inhibidores , Células Cultivadas , Células Dendríticas/microbiología , Regulación de la Expresión Génica , Células HEK293 , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , MicroARNs/fisiología , Mycobacterium tuberculosis/inmunología , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidoresRESUMEN
Alteration in endogenous Interferon (IFN) system may profoundly impact immune cell function in autoimmune diseases. Here, we provide evidence that dysregulation in IFN-regulated genes and pathways are involved in B cell- and monocyte-driven pathogenic contribution to Multiple Sclerosis (MS) development and maintenance. In particular, by using an Interferome-based cell type-specific approach, we characterized an increased susceptibility to an IFN-linked caspase-3 dependent apoptotic cell death in both B cells and monocytes of MS patients that may arise from their chronic activation and persistent stimulation by activated T cells. Ongoing caspase-3 activation functionally impacts on MS monocyte properties influencing the STAT-3/IL-16 axis, thus, driving increased expression and massive release of the bio-active IL-16 triggering and perpetuating CD4+ T cell migration. Importantly, our analysis also identified a previously unknown multi-component defect in type I IFN-mediated signaling and response to virus pathways specific of MS B cells, impacting on induction of anti-viral responses and Epstein-barr virus infection control in patients. Taking advantage of cell type-specific transcriptomics and in-depth functional validation, this study revealed pathogenic contribution of endogenous IFN signaling and IFN-regulated cell processes to MS pathogenesis with implications on fate and functions of B cells and monocytes that may hold therapeutic potential.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Interferón Tipo I/genética , Monocitos/inmunología , Monocitos/metabolismo , Esclerosis Múltiple/etiología , Esclerosis Múltiple/metabolismo , Transcriptoma , Adulto , Apoptosis , Biomarcadores , Estudios de Casos y Controles , Susceptibilidad a Enfermedades , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Interferón Tipo I/metabolismo , Interleucina-16/genética , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/patología , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Regiones Promotoras Genéticas , Transducción de SeñalRESUMEN
BACKGROUND: B cells are key pathogenic effectors in multiple sclerosis (MS) and several therapies have been designed to restrain B cell abnormalities by directly targeting this lymphocyte population. OBJECTIVES: Moving from our data showing a Toll-like receptor (TLR)7-driven dysregulation of B cell response in relapsing-remitting multiple sclerosis (RRMS) and having found a low serum level of Thymosin-α1 (Tα1) in patients, we investigated whether the addition of this molecule to peripheral blood mononuclear cells (PBMCs) would influence the expansion of regulatory B cell subsets, known to dampen autoimmune inflammation. METHODS: Serum Tα1 level was measured by enzyme immunoassay. Cytokine expression was evaluated by Cytometric Bead Array (CBA), enzyme-linked immunosorbent assay (ELISA), and real-time reverse transcription polymerase chain reaction (RT-PCR). B cell subsets were analyzed by flow cytometry. RESULTS: Tα1 pre-treatment induces an anti-inflammatory status in TLR7-stimulated RRMS PBMC cultures, reducing the secretion of pro-inflammatory interleukin (IL)-6, IL-8, and IL-1ß while significantly increasing the regulatory IL-10 and IL-35. Indeed, Tα1 treatment enhanced expansion of CD19+CD24+CD38hi transitional-immature and CD24low/negCD38hi plasmablast-like regulatory B cell subsets, which likely inhibit both interferon (IFN)-γ and IL-17 production. CONCLUSION: Our study reveals a deficient ability of B cells from MS patients to differentiate into regulatory subsets and unveils a novel anti-inflammatory and repurposing potential for Tα1 in MS targeting B cell response.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Linfocitos B Reguladores/efectos de los fármacos , Citocinas/metabolismo , Interleucina-10/metabolismo , Esclerosis Múltiple Recurrente-Remitente/sangre , Timalfasina/sangre , Timalfasina/farmacología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptor Toll-Like 7/agonistas , Adulto JovenRESUMEN
A major challenge in tuberculosis (TB) is to improve current vaccination and therapeutic strategies and this requires a fine understanding of the mechanisms that mediate protection and pathogenesis. We need to discern how the host perceives Mycobacterium tuberculosis (Mtb) infection, what are the danger signals that activate the immune system and, in turn, how the immune response controls the life-cycle of Mtb. Cytokines, because of their nature of soluble mediators, represent key elements in mediating and tuning these complex processes. In this review, we provide an overview of recent studies on cytokines expression and function in active (mainly human) TB. Understanding of the balance between pro- and anti-inflammatory networks is crucial to refine our knowledge on the immune responses directed against Mtb.
Asunto(s)
Citocinas/inmunología , Pulmón/inmunología , Ganglios Linfáticos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Células Epiteliales Alveolares/inmunología , Células Epiteliales Alveolares/microbiología , Células Epiteliales Alveolares/patología , Citocinas/genética , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Células Dendríticas/patología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Inflamación/genética , Inflamación/inmunología , Inflamación/microbiología , Inflamación/patología , Pulmón/microbiología , Pulmón/patología , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/patología , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/microbiología , Linfocitos T/patología , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patologíaRESUMEN
Growing evidences put B lymphocytes on a central stage in multiple sclerosis (MS) immunopathology. While investigating the effects of interferon-ß (IFN-ß) therapy, one of the most used first-line disease-modifying drugs for the treatment of relapsing-remitting MS, in circulating B-cell sub-populations, we found a specific and marked decrease of CD27+ memory B cells. Interestingly, memory B cells are considered a population with a great disease-driving relevance in MS and resulted to be also target of B-cell depleting therapies. In addition, Epstein-Barr virus (EBV), associated with MS etiopathogenesis, harbors in this cell type and an IFN-ß-induced reduction of the memory B-cell compartment, in turn, resulted in a decreased expression of the EBV gene latent membrane protein 2A in treated patients. We found that in vivo IFN-ß therapy specifically and highly induced apoptosis in memory B cells, in accordance with a strong increase of the apoptotic markers Annexin-V and active caspase-3, via a mechanism requiring the FAS-receptor/TACI (transmembrane activator and CAML interactor) signaling. Thus, efficacy of IFN-ß therapy in MS may rely not only on its recognized anti-inflammatory activities but also on the specific depletion of memory B cells, considered to be a pathogenic cell subset, reducing their inflammatory impact in target organs.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos B/inmunología , Memoria Inmunológica/efectos de los fármacos , Interferón beta/farmacología , Interferón beta/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Receptor fas/metabolismo , Adulto , Linfocitos B/efectos de los fármacos , Proteína Ligando Fas/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/patología , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/inmunología , Esclerosis Múltiple Recurrente-Remitente/patología , Proteína Activadora Transmembrana y Interactiva del CAML/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Adulto JovenRESUMEN
The implication of B lymphocytes in the immunopathology of multiple sclerosis (MS) is increasingly recognized. Here we investigated the response of B cells to IFN-ß, a first-line therapy for relapsing-remitting MS patients, upon stimulation with TLR. IFN-ß restored the frequency of TLR7-induced IgM and IgG-secreting cells in MS patients to the levels found in healthy donors, showing a specific deficiency in the TLR7 pathway. However, no difference was observed in the TLR9 response. Furthermore, in MS-derived PBMCs, TLR7-mediated production of IL-6 and the ex vivo expression of B-cell-activating factor of the TNF family, two crucial cytokines for B-cell differentiation and survival, were induced by IFN-ß. Depletion of monocytes, which are key producers of both IL-6 and B-cell-activating factor of the TNF family, showed that TLR7-mediated B-cell differentiation into Ig-secreting cells is strongly dependent on the cross-talk between B cells and monocytes. Accordingly, impaired expression of TLR7 mRNA was observed in PBMCs and monocytes isolated from MS-affected individuals as compared with those from healthy donors, which was rescued by IFN-ß therapy. Collectively, our data unveil a novel TLR7-regulated mechanism in in vivo IFN-ß-stimulated whole leukocytes that could be exploited to define new TLR7-based strategies for the treatment of MS.
Asunto(s)
Linfocitos B/efectos de los fármacos , Interferón beta/uso terapéutico , Monocitos/efectos de los fármacos , Esclerosis Múltiple Recurrente-Remitente/inmunología , Receptor Cross-Talk/efectos de los fármacos , Receptor Toll-Like 7/inmunología , Adulto , Linfocitos B/metabolismo , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Factores Inmunológicos/uso terapéutico , Masculino , Monocitos/metabolismo , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Cross-Talk/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Plasmacytoid DCs (pDCs) are crucial mediators in the establishment of immunity against most viruses, given their extraordinary capacity to produce a massive quantity of type I IFN. In this study we investigate the response of pDCs to infection with EBV, a γ-herpes virus that persists with an asymptomatic infection in immunocompetent hosts, although in certain conditions it can promote development of cancers or autoimmune diseases. We show that high amounts of type I IFNs were released from isolated pDCs after exposure to EBV by a mechanism requiring TLRs and a functional autophagic machinery. We next demonstrate that EBV can infect pDCs via viral binding to MHC class II molecule HLA-DR and that pDCs express EBV-induced latency genes. Furthermore, we observe that EBV is able to induce activation but not maturation of pDCs, which correlates with an impaired TNF-α release. Accordingly, EBV-infected pDCs are unable to mount a full T-cell response, suggesting that impaired pDC maturation, combined with a concomitant EBV-mediated upregulation of the T-cell inhibitory molecules B7-H1 and ICOS-L, could represent an immune-evasion strategy promoted by the virus. These mechanisms might lead to persistence in immunocompetent hosts or to dysregulated immune responses linked to EBV-associated diseases.
Asunto(s)
Células Dendríticas/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/fisiología , Linfocitos T/inmunología , Receptor Toll-Like 9/inmunología , Autofagia/inmunología , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Células Cultivadas , Células Dendríticas/virología , Antígenos HLA-DR/metabolismo , Herpesvirus Humano 4/patogenicidad , Humanos , Evasión Inmune , Ligando Coestimulador de Linfocitos T Inducibles/genética , Ligando Coestimulador de Linfocitos T Inducibles/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Activación de Linfocitos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Internalización del Virus , Latencia del Virus/genética , Replicación ViralRESUMEN
Mycobacterium tuberculosis (Mtb) evades the immune response by impairing the functions of different antigen-presenting cells. We have recently shown that Mtb hijacks differentiation of monocytes into dendritic cells (DCs). To further characterize the mechanisms underlying this process, we investigated the consequences of inducing dendritic cell differentiation using interferon-α and granulocyte-macrophage colony-stimulating factor in the presence of supernatants (SNs) obtained from monocyte cultures treated with or without heat-inactivated Mtb. Although the SNs from control cultures do not interfere with the generation of fully differentiated DCs, monocytes stimulated with SNs from Mtb-stimulated cells (SN Mtb) remained CD14(+) and poorly differentiated into CD1a(+) cells. Among cytokines known to affect dendritic cell differentiation, we observed a robust production of interleukin-1ß, interleukin-6, interleukin-10 and tumor necrosis factor-α upon Mtb stimulation. However, only interleukin-10 neutralization through the addition of soluble interleukin-10 receptor reversed the inhibitory activity of SN Mtb. Accordingly, the addition of recombinant interleukin-10 was able to significantly reduce CD1a expression. The interaction of Mtb with differentiating monocytes rapidly activates p38 mitogen-activated protein kinase, signal transducer and activator of transcription pathways, which are likely involved in interleukin-10 gene expression. Taken together, our results suggest that Mtb may inhibit the differentiation of bystander non-infected monocytes into DCs through the release of interleukin-10. These results shed light on new aspects of the host-pathogen interaction, which might help to identify innovative immunological strategies to limit Mtb virulence.
Asunto(s)
Efecto Espectador , Diferenciación Celular , Células Dendríticas/citología , Células Dendríticas/inmunología , Interleucina-10/inmunología , Mycobacterium tuberculosis/fisiología , Tuberculosis/inmunología , Efecto Espectador/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Citocinas/biosíntesis , Humanos , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Mycobacterium tuberculosis/inmunología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Knowledge of the molecular events regulating the innate response to Mycobacterium tuberculosis (Mtb) is critical for understanding immunological pathogenesis and protection from tuberculosis. To this aim, the regulation and the expression of regulatory and proinflammatory cytokines were investigated in human primary monocytes upon Mtb infection. We found that Mtb-infected monocytes preferentially express a proinflammatory cytokine profile, including IL-6, TNF-α, and IL-1ß. Conversely, among the regulatory cytokines, Mtb elicited IL-10 and IL-23 release while no expression of IL-12p70, IL-27, and IFN-ß was observed. The analysis of the signalling pathways leading to this selective cytokine expression showed that in monocytes Mtb activates MAPK and NF-κB but is unable to stimulate IRF-3 phosphorylation, a transcription factor required for IL-12p35 and IFN-ß gene expression. Thus, by inducing a specific cytokine profile, Mtb can influence the immunoregulatory properties of monocytes, which represent important target of novel vaccinal strategies against Mtb infection.
Asunto(s)
Citocinas , Expresión Génica , Monocitos , Técnicas de Cultivo de Célula , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Expresión Génica/inmunología , Humanos , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Sistema de Señalización de MAP Quinasas/inmunología , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/microbiología , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosforilación , Transducción de Señal/genética , Transducción de Señal/inmunología , Balance Th1 - Th2RESUMEN
In human primary dendritic cells (DC) rapamycin-an autophagy inducer and protein synthesis inhibitor-overcomes the autophagy block induced by Mycobacterium tuberculosis (Mtb) and promotes a Th1 response via IL-12 secretion. Here, the immunostimulatory activity of rapamycin in Mtb-infected DC was further investigated by analyzing both transcriptome and translatome gene profiles. Hundreds of differentially expressed genes (DEGs) were identified by transcriptome and translatome analyses of Mtb-infected DC, and some of these genes were found further modulated by rapamycin. The majority of transcriptome-associated DEGs overlapped with those present in the translatome, suggesting that transcriptionally stimulated mRNAs are also actively translated. In silico analysis of DEGs revealed significant changes in intracellular cascades related to cytokine production, cytokine-induced signaling and immune response to pathogens. In particular, rapamycin treatment of Mtb-infected DC caused an enrichment of IFN-ß, IFN-λ and IFN-stimulated gene transcripts in the polysome-associated RNA fraction. In addition, rapamycin led to an increase of IL-12, IL-23, IL-1ß, IL-6, and TNF-α but to a reduction of IL-10. Interestingly, upon silencing or pharmacological inhibition of GSK-3ß, the rapamycin-driven modulation of the pro- and anti-inflammatory cytokine balance was lost, indicating that, in Mtb-infected DC, GSK-3ß acts as molecular switch for the regulation of the cytokine milieu. In conclusion, our study sheds light on the molecular mechanism by which autophagy induction contributes to DC activation during Mtb infection and points to rapamycin and GSK-3ß modulators as promising compounds for host-directed therapy in the control of Mtb infection.
Asunto(s)
Autofagia/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Mycobacterium tuberculosis/inmunología , Sirolimus/farmacología , Tuberculosis/tratamiento farmacológico , Autofagia/genética , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/inmunología , Perfilación de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Cultivo Primario de Células , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismo , Tuberculosis/inmunología , Tuberculosis/microbiologíaRESUMEN
Although data show the importance of type I interferons (IFNs) in the regulation of the innate and adaptive immunity elicited in response to viral, bacterial and parasitic infections, the functional activities of these cytokines during fungal infections are poorly understood. We examined here the impact of IFN-ß on the response of human monocyte-derived dendritic cells (DCs) infected in vitro with Aspergillus fumigatus. Having found that A. fumigatus-infected DCs do not express IFN-ß, we evaluated the effect of the exogenous addition of IFN-ß on the maturation of human DCs induced by the infection with A. fumigatus conidia. Although the phagocytosis of the fungus was not affected by IFN-ß treatment, the expression of CD86 and CD83 induced upon A. fumigatus challenge was enhanced in IFN-ß-conditioned DCs, which also showed an increased expression of IL-27 and IL-12p70, members of IL-12 family. Through these modifications, IFN-ß improved the capacity of DCs to promote an anti-Aspergillus T helper type 1 response, as evaluated by mixed leucocyte reaction, which plays a crucial role in the control of invasive aspergillosis. Our results identified a novel effect of IFN-ß on anti-Aspergillus immune responses which, in turn, might open new perspectives on the use of IFN-ß in immunotherapy for fungal infections aimed at enhancing the immunological functions of DCs.
Asunto(s)
Aspergillus fumigatus/inmunología , Células Dendríticas/inmunología , Interferón beta/farmacología , Células TH1/inmunología , Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proliferación Celular , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Células Dendríticas/virología , Expresión Génica/genética , Expresión Génica/inmunología , Humanos , Interferón beta/genética , Interferón beta/metabolismo , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Lipopolisacáridos/farmacología , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Células TH1/citologíaRESUMEN
Pyrogen content is a key quality feature that must be checked in all injectable products, including vaccines. Four tests are currently available in the European Pharmacopoeia to monitor pyrogen/endotoxin presence: the rabbit pyrogen test (RPT), the bacterial endotoxin test, the recombinant factor C test, and the monocyte activation test (MAT). Here, we explored the possibility to replace the RPT with the MAT in the quality control of a vaccine against tick-borne encephalitis virus (TBEV). The testing was carried out using cryopreserved peripheral blood mononuclear cells as cell source. IL-6 release was selected as readout for the detection of both endotoxin and non-endotoxin contaminants. MAT applicability for pyrogen testing of the TBEV vaccine was assessed through preparatory tests and resulted in the establishment of a very sensitive assay (limit of detection (LOD) = 0.04 EU/mL; sensitivity = 0.1 EU/mL). Both quantitative Method A and semiquantitative Method B were used for data analysis. Our studies revealed that for a vaccine without intrinsic pyrogenicity, such as that against TBEV, sensitivity (the lowest endotoxin value of the standard curve) should be used instead of LOD to define a stable maximum valid dilution of the product. In conclusion, we describe the challenges of MAT implementation for anti-TBEV vaccine following the current Ph. Eur. chapter 2.6.30 and propose a re-evaluation of the validity criteria of Methods A and B in order to set a semi-quantitative or limit test suitable for those products for which a reference lot comparison analysis is not applicable or favorable.
Asunto(s)
Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Endotoxinas/toxicidad , Monocitos/efectos de los fármacos , Pirógenos/toxicidad , Vacunas Virales/efectos adversos , Alternativas a las Pruebas en Animales , Animales , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Control de Calidad , Conejos , Vacunas Virales/inmunología , Vacunas Virales/normasRESUMEN
BACKGROUND: Multiple sclerosis (MS) afflicts more than 2.5 million individuals worldwide and this number is increasing over time. Within the past years, a great number of disease-modifying treatments have emerged; however, efficacious treatments and a cure for MS await discovery. Thymosins, soluble hormone-like peptides produced by the thymus gland, can mediate immune and non-immune physiological processes and have gained interest in recent years as therapeutics in inflammatory and autoimmune diseases. METHODS: Pubmed was searched with no time constraints for articles using a combination of the keywords "thymosin/s" or "thymus factor/s" AND "multiple sclerosis", mesh terms with no language restriction. RESULTS: Here, we review the state-of-the-art on the effects of thymosins on MS and its experimental models. In particular, we describe what is known in this field on the roles of thymosin-α1 (Tα1) and -ß4 (Tß4) as potential anti-inflammatory as well as neuroprotective and remyelinating molecules and their mechanisms of action. CONCLUSION: Based on the data that Tα1 and Tß4 act as anti-inflammatory molecules and as inducers of myelin repair and neuronal protection, respectively, a possible therapeutic application in MS for Tα1 and Tß4 alone or combined with other approved drugs may be envisaged. This approach is reasonable in light of the current clinical usage of Tα1 and data demonstrating the safety, tolerability and efficacy of Tß4 in clinical practice.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Modelos Animales de Enfermedad , Esclerosis Múltiple/tratamiento farmacológico , Timosina/uso terapéutico , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Humanos , Fármacos Neuroprotectores/uso terapéutico , Células Precursoras de Oligodendrocitos/efectos de los fármacos , Investigación Biomédica Traslacional , Resultado del TratamientoRESUMEN
Understanding Staphylococcus aureus (S. aureus)-host immune system interaction is crucial to meet the tremendous medical need associated with this life-threatening bacterial infection. Given the crucial role of dendritic cells (DC) in dictating immune responses upon microbial challenge, we investigated how the bacterial viability and the conservation of structural integrity influence the response of human DC to S. aureus. To this end, human primary DC were stimulated with the methicillin-resistant S. aureus USA300 live strain, USA300 inactivated by heat (HI), ultraviolet irradiation (UVI), or paraformaldehyde treatment (PFAI) and subsequently analyzed for cell phenotype and immune-modulatory properties. Although no differences in terms of DC viability and maturation were observed when DC were stimulated with live or inactivated bacteria, the production of IL-12, IL-23, and other cytokines differed significantly. The Th1 and Th17 expansion was also more pronounced in response to live vs. inactivated S. aureus. Interestingly, cytokine production in DC treated with live and inactivated USA300 required phagocytosis, whereas blocking endosomal Toll-like receptor signaling mainly reduced the cytokine release by live and HI USA300. A further analysis of IFN-ß signaling revealed the induction of a cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING)-independent and IRF3-dependent signaling pathway(s) in UVI-stimulated DC. This study underscores the capacity of human DC to discriminate between live and inactivated S. aureus and, further, indicates that DC may represent a valuable experimental setting to test different inactivation methods with regard to the retention of S. aureus immunoregulatory properties. These and further insights may be useful for the development of novel therapeutic and prophylactic anti-S. aureus vaccine strategies.
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Supervivencia Celular/inmunología , Citocinas/inmunología , Células Dendríticas/inmunología , Staphylococcus aureus/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Células Cultivadas , Citocinas/biosíntesis , Humanos , Activación de Linfocitos/inmunologíaRESUMEN
The roles of plasmacytoid dendritic cells (pDCs) and their response to interferon (IFN)-beta therapy in multiple sclerosis (MS) patients are poorly understood. We identified pDC accumulation in white matter lesions and leptomeninges of MS brains and abundant expression of the Type I IFN-induced protein MxA, mainly in perivascular CD3+ lymphocytes in lesions, indicating Type I IFN production by activated pDCs. The pDC chemoattractant chemerin was detected in intralesional cerebrovascular endothelial cells, and the chemerin receptor was expressed on infiltrating leukocytes, including pDCs. The effect of IFN-beta on pDC phenotype and function was evaluated in MS patients before and during IFN-beta treatment. Although IFN-beta did not modify the frequency and immature phenotype of circulating pDC, they showed lower expression of major histocompatibility complex Class II and blood-dendritic cell antigen 2 molecules and upregulation of CD38 and B7H1 costimulatory molecules. On exposure to CpG (a site where cytosine [C] lies next to guanine [G] in the DNA sequence [the p indicates that C and G are connected by a phosphodiester bond]) oligodeoxynucleotides in vitro, pDCs from IFN-beta-treated MS patients showed reduced expression of the pDC maturation markers CD83 and CD86 molecules; in vitro IFN-beta treatment of pDCs from healthy donors resulted in lower secretion of proinflammatory cytokines, including IFN-alpha, and a decreased ability to stimulate allogeneic T cells in response to maturative stimuli. These data indicate that IFN-beta modulates the immunologic functions of pDC, thus identifying pDCs as a novel target of IFN-beta therapy in MS patients.
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Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/inmunología , Células Dendríticas/efectos de los fármacos , Interferón beta/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Adulto , Antígenos CD/análisis , Antígenos CD/metabolismo , Antígeno B7-H1 , Biomarcadores/análisis , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Corteza Cerebral/fisiopatología , Quimiocinas/efectos de los fármacos , Quimiocinas/inmunología , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/inmunología , Células Dendríticas/inmunología , Femenino , Proteínas de Unión al GTP/efectos de los fármacos , Proteínas de Unión al GTP/inmunología , Humanos , Factores Inmunológicos/farmacología , Factores Inmunológicos/uso terapéutico , Péptidos y Proteínas de Señalización Intercelular , Interferón beta/uso terapéutico , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/fisiopatología , Proteínas de Resistencia a Mixovirus , Fibras Nerviosas Mielínicas/efectos de los fármacos , Fibras Nerviosas Mielínicas/inmunología , Fibras Nerviosas Mielínicas/ultraestructura , Fenotipo , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunologíaRESUMEN
The transcription factor STAT-4 plays a pivotal role in the IL-12-mediated development of naive CD4+ T cells into the Th1 phenotype. Initially thought to be restricted to the lymphoid lineage, STAT-4 was subsequently shown to be expressed in the myeloid compartment, mainly in activated monocytes, macrophages, and dendritic cells (DC). Here, we have studied STAT-4 in human monocyte-derived DC, and we demonstrated that its expression can be induced by multiple stimuli, such as the ligands for TLR-4, TLR-2, and TLR-3, different pathogens, CD40 ligand, and the proinflammatory cytokines TNF-alpha and IL-1beta. It is interesting that we found that STAT-4 is tyrosine-phosphorylated in response to type I IFN but not IL-12 in human mature DC. Cloning and functional analysis of the STAT-4 promoter showed that a NF-kappaB binding site, localized at -969/-959 bp upstream of the transcriptional start site, is involved in the regulation of this gene in primary human DC. EMSAs using a probe containing this NF-kappaB binding sequence and chromatin immunoprecipitation indicated that p65/p50 and p50/p50 dimers were the main NF-kappaB/Rel proteins involved in STAT-4 gene expression in maturing DC. The mutation of this kappaB site or the overexpression of the repressor IkappaBalpha exerted an inhibitory effect on a STAT-4 promoter-driven reporter as well as on STAT-4 expression. Altogether, these results indicate that STAT-4 can be finely tuned along with DC maturation through NF-kappaB activation and that its induction may be involved in preparing the DC to be receptive to the cytokine environment present in lymphoid organs.
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Células Dendríticas/metabolismo , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción STAT4/metabolismo , Secuencia de Bases , Sitios de Unión , Diferenciación Celular , Células Cultivadas , Células Dendríticas/fisiología , Humanos , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , Factor de Transcripción STAT4/genética , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Given the role played by chemokines in the selective homing of immune cells, we sought to characterize the profile of chemokines produced by human dendritic cells (DC) following in vitro Aspergillus fumigatus infection and their ability to recruit cells involved in the antifungal defense. At the onset of A. fumigatus infection, DC released elevated amounts of CXCL8 that promote the migration of polymorphonuclear cells (PMN). Moreover, soluble factors released from A. fumigatus-infected DC increased also the surface expression of two activation markers, CD11b and CD18, on PMN. A. fumigatus infection resulted also in CCL3, CCL4, CXCL10 and CCL20 productions that induce the migration of effector memory Th1 cells. Moreover, the late expression of CCL19 suggests that A. fumigatus-infected DC could be implicated in the migration of CCR7+ naïve T cells and mature DC in lymph nodes. Together these results suggested the involvement of human DC in the regulation of innate and adaptive immunity against A. fumigatus through the recruitment of cells active in the fungal destruction.
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Aspergilosis/inmunología , Aspergillus fumigatus/patogenicidad , Quimiocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Infiltración Neutrófila , Células TH1/inmunología , Aspergilosis/microbiología , Aspergillus fumigatus/inmunología , Quimiocina CCL19 , Quimiocina CCL20 , Quimiocinas/inmunología , Quimiocinas CC/metabolismo , Quimiotaxis de Leucocito/inmunología , Células Dendríticas/metabolismo , Humanos , Interleucina-8/metabolismo , Activación de Linfocitos , Proteínas Inflamatorias de Macrófagos/metabolismoRESUMEN
In human monocyte-derived dendritic cells (DC), infection with Mycobacterium tuberculosis and viruses or stimulation with Toll-like receptor type 3 and 4 agonists causes the release of type I interferon (IFN). Here, we describe that the IFN-beta released upon stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C) is responsible for a rapid and sustained signal transducer and activator of transcription 1 and 2 activation and expression of IFN-stimulated genes, such as the transcription factor IFN regulatory factor 7 and the chemokine CXC chemokine ligand 10. The autocrine production of IFN-beta from LPS and poly I:C-matured DC (mDC) induced a temporary saturation of the response to type I IFN and a marked decline in the level of the two IFN receptor (IFNAR) subunits. It is interesting that we found that upon clearing of the released cytokines, LPS-stimulated DC reacquired full responsiveness to IFN-beta but only partial responsiveness to IFN-alpha, and their maturation process was unaffected. Monitoring of surface and total levels of the receptor subunits showed that maximal expression of IFNAR2 resumed within 24 h of clearing, and IFNAR1 expression remained low. Thus, mDC can modulate their sensitivity to two IFN subtypes through a differential regulation of the IFNAR subunits.
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Células Dendríticas/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Inductores de Interferón/farmacología , Interferón-alfa/farmacología , Interferón beta/farmacología , Proteínas de la Membrana/biosíntesis , Receptores de Interferón/biosíntesis , Anticuerpos Monoclonales/farmacología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Quimiocina CXCL10 , Quimiocinas CXC/fisiología , Regulación hacia Abajo/efectos de los fármacos , Endotoxinas/farmacología , Humanos , Interferón alfa-2 , Proteínas de la Membrana/genética , Monocitos/citología , Poli I-C/farmacología , Receptor de Interferón alfa y beta , Receptores de Interferón/genética , Proteínas Recombinantes/farmacología , Factor de Transcripción STAT1/fisiología , Factor de Transcripción STAT2/fisiología , Transcripción GenéticaRESUMEN
The opportunistic pathogen Staphylococcus aureus (S. aureus) is a major cause of nosocomial- and community-acquired infections. In addition, many antibiotic-resistant strains are emerging worldwide, thus, there is an urgent unmet need to pinpoint novel therapeutic and prophylactic strategies. In the present study, we characterized the impact of infection with the pandemic methicillin-resistant USA300 S. aureus strain on human primary dendritic cells (DC), key initiators and regulators of immune responses. In particular, among staphylococcal virulence factors, the function of EsxA and EsxB, two small acidic dimeric proteins secreted by the type VII-like secretion system Ess (ESAT-6-like secretion system), was investigated in human DC setting. A comparative analysis of bacterial entry, replication rate as well as DC maturation, apoptosis, signaling pathway activation and cytokine production was performed by using wild type (wt) USA300 and three isogenic mutants carrying the deletion of esxA (ΔesxA), esxB (ΔesxB), or both genes (ΔesxAB). The S. aureus mutant lacking only the EsxA protein (ΔesxA) stimulated a stronger pro-apoptotic phenotype in infected DC as compared to wt USA300, ΔesxAB, and ΔesxB strains. When the mutant carrying the esxB deletion (ΔesxB) was analyzed, a higher production of both regulatory and pro-inflammatory mediators was found in the infected DC with respect to those challenged with the wt counterpart and the other esx mutants. In accordance with these data, supernatant derived from ΔesxB-infected DC promoted a stronger release of both IFN-γ and IL-17 from CD4+ T cells as compared with those conditioned with supernatants derived from wild type USA300-, ΔesxAB-, and ΔesxA-infected cultures. Although, the interaction of S. aureus with human DC is not yet fully understood, our data suggest that both cytokine production and apoptotic process are modulated by Esx factors, thus indicating a possible role of these proteins in the modulation of DC-mediated immunity to S. aureus.