Examinando por Autor "Pacheco, Rodrigo"
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Ítem Cross-talk between T-cells and gut-microbiota in neurodegenerative disorders(Wolters Kluwer Medknow Publications, 2019-12) Pacheco, RodrigoThe emerging role of gut microbiota as a key player in the development of neurodegenerative disorders: Mammals have evolved together with commensal microbiota to establish a symbiotic relationship in which they regulate reciprocally by synthesizing and responding to several common chemical substances. In this regard, gut microbiota constitutes a consortium of bacteria that not only participates in the degradation of nutrients, but also produces metabolites, fatty acids and neurotransmitters that can act on the enzymes and receptors expressed in eukaryotic cells, which considerably affects the physiology of the host and contribute to maintaining homeostasis (Lyte, 2013). According to the important role that gut-microbiota plays in maintaining homeostasis, alterations in the composition of gut-microbiota (dysbiosis) have consistently been involved in the development of neuropsychiatric, metabolic, autoimmune and neurodegenerative disorders. With respect to this last point, human and animal model researches have shown that the presence of some precise bacteria or the absence of some beneficial components in the gut microbiota of genetically susceptible individuals could trigger the development of Parkinson’s disease (PD), Alzheimer’s disease (AD), multiple sclerosis (MS) or amyotrophic lateral sclerosis (ALS). For instance, an increase of Proteus mirabilis in the composition of gut microbiota and excess production of short-chain fatty acids in the intestinal mucosa have been shown to promote the development of PD in several animal models (Sampson et al., 2016; Choi et al., 2018). On the other hand, it has been demonstrated that the butyrate producing bacteria, Butyrivibrio Fibrisolvens, was selectively decreased in the intestinal microbiota of animals genetically susceptible to ALS, and the administration of butyrate significantly attenuated the disease development disease (Zhang et al., 2017). Notably, in a study conducted with 34 pairs of discordant twins for MS, the results show that the composition of the intestinal microbiota from healthy twins was different from that obtained from twins with MS (Berer et al., 2017). Furthermore, using a mouse model of MS that develop the disease spontaneously in a specific-pathogen-free environment, the experiment showed that the mice developed a severer disease when they received the transfer of gut-microbiota from twins with MS than from healthy twins (Berer et al., 2017). Significant dysbiosis has also been implicated in AD patients, including the alteration in the composition of several bacterial taxa, such as Bacteroides Actinobacteria, Ruminococcus, Lachnospiraceae and Selenomonadales (Zhuang et al., 2018). Supporting these data obtained from AD patients, studies conducted in mouse models and recently also in Drosophila have shown that dysbiosis of the intestinal microbiota produces cognitive deterioration and neurodegeneration. Thus, the composition of gut microbiota plays a fundamental role in controlling homeostasis in central nervous system (CNS), and avoiding neurodegenerative disorders.Ítem Cross-talk between T-cells and gut-microbiota in neurodegenerative disorders(2019-12) Pacheco, RodrigoÍtem Dopamine receptor D3 expression is altered in CD4+ T-cells from Parkinson's disease patients and its pharmacologic inhibition attenuates the motor impairment in a mouse model(Frontiers Media S.A., 2019-05) Elgueta, Daniela; Contreras, Francisco; Prado, Carolina; Montoya, Andro; Ugalde, Valentina; Chovar, Ornella; Villagra, Roque; Henríquez, Claudio; Abellanas, Miguel A.; Aymerich, María S.; Franco, Rarael; Pacheco, RodrigoNeuroinflammation constitutes a fundamental process involved in Parkinson's disease (PD). Microglial cells play a central role in the outcome of neuroinflammation and consequent neurodegeneration of dopaminergic neurons in the substantia nigra. Current evidence indicates that CD4+ T-cells infiltrate the brain in PD, where they play a critical role determining the functional phenotype of microglia, thus regulating the progression of the disease. We previously demonstrated that mice bearing dopamine receptor D3 (DRD3)-deficient CD4+ T-cells are completely refractory to neuroinflammation and consequent neurodegeneration induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study we aimed to determine whether DRD3-signalling is altered in peripheral blood CD4+ T-cells obtained from PD patients in comparison to healthy controls (HC). Furthermore, we evaluated the therapeutic potential of targeting DRD3 confined to CD4+ T-cells by inducing the pharmacologic antagonism or the transcriptional inhibition of DRD3-signalling in a mouse model of PD induced by the chronic administration of MPTP and probenecid (MPTPp). In vitro analyses performed in human cells showed that the frequency of peripheral blood Th1 and Th17 cells, two phenotypes favoured by DRD3-signalling, were significantly increased in PD patients. Moreover, naïve CD4+ T-cells obtained from PD patients displayed a significant higher Th1-biased differentiation in comparison with those naïve CD4+ T-cells obtained from HC. Nevertheless, DRD3 expression was selectively reduced in CD4+ T-cells obtained from PD patients. The results obtained from in vivo experiments performed in mice show that the transference of CD4+ T-cells treated ex vivo with the DRD3-selective antagonist PG01037 into MPTPp-mice resulted in a significant reduction of motor impairment, although without significant effect in neurodegeneration. Conversely, the transference of CD4+ T-cells transduced ex vivo with retroviral particles codifying for an shRNA for DRD3 into MPTPp-mice had no effects neither in motor impairment nor in neurodegeneration. Notably, the systemic antagonism of DRD3 significantly reduced both motor impairment and neurodegeneration in MPTPp mice. Our findings show a selective alteration of DRD3-signalling in CD4+ T-cells from PD patients and indicate that the selective DRD3-antagonism in this subset of lymphocytes exerts a therapeutic effect in parkinsonian animals dampening motor impairment. Copyright © 2019 Elgueta, Contreras, Prado, Montoya, Ugalde, Chovar, Villagra, Henríquez, Abellanas, Aymerich, Franco and Pacheco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Ítem Dopamine receptor D3 signalling in astrocytes promotes neuroinflammation(BioMed Central Ltd., 2019-12) Montoya, Andro; Elgueta, Daniela; Campos, Javier; Chovar, Ornella; Falcón, Paulina; Matus, Soledad; Alfaro, Iván; Bono, María Rosa; Pacheco, RodrigoBackground: Neuroinflammation constitutes a pathogenic process leading to neurodegeneration in several disorders, including Alzheimer's disease, Parkinson's disease (PD) and sepsis. Despite microglial cells being the central players in neuroinflammation, astrocytes play a key regulatory role in this process. Our previous results indicated that pharmacologic-Antagonism or genetic deficiency of dopamine receptor D3 (DRD3) attenuated neuroinflammation and neurodegeneration in two mouse models of PD. Here, we studied how DRD3-signalling affects the dynamic of activation of microglia and astrocyte in the context of systemic inflammation. Methods: Neuroinflammation was induced by intraperitoneal administration of LPS. The effect of genetic DRD3-deficiency or pharmacologic DRD3-Antagonism in the functional phenotype of astrocytes and microglia was determined by immunohistochemistry and flow cytometry at different time-points. Results: Our results show that DRD3 was expressed in astrocytes, but not in microglial cells. DRD3 deficiency resulted in unresponsiveness of astrocytes and in attenuated microglial activation upon systemic inflammation. Furthermore, similar alterations in the functional phenotypes of glial cells were observed by DRD3 antagonism and genetic deficiency of DRD3 upon LPS challenge. Mechanistic analyses show that DRD3 deficiency resulted in exacerbated expression of the anti-inflammatory protein Fizz1 in glial cells both in vitro and in vivo. Conclusions: These results suggest that DRD3 signalling regulates the dynamic of the acquisition of pro-inflammatory and anti-inflammatory features by astrocytes and microglia, finally favouring microglial activation and promoting neuroinflammation. © 2019 The Author(s).Ítem Función del receptor de Dopamina D3 expresado en células T CD4+ en el desarrollo de la enfermedad de Parkinson(Universidad Andrés Bello, 2013) González Velozo, Hugo; Pacheco, Rodrigo; Rosembatt, MarioEvidencias recientes han demostrado que células T CD4+ infiltran en la substancia nigra en pacientes con enfermedad de Parkinson (EP), así como en modelos animales de la enfermedad. Estas células T CD4+ a través de sus fenotipos efectores inflamatorios promueven la activación microglial y contribuyen a la neurodegeneración de neuronas dopaminérgicas. La alteración de la expresión del receptor de dopamina D3 (D3R) en linfocitos de pacientes con EP ha sido correlacionada con la severidad de la enfermedad. Además, evidencias farmacológicas han asociado la estimulación de este receptor con la producción de IFN-y por células T CD4+humanas in vitro, citoquina involucrada en la estimulación y mantención de la respuesta glial proinflamatoria. En esta tesis se estudió el papel que juega el D3R expresado en células T CD4+ en la neurodegeneración dopaminérgica asociada a la EP utilizando un modelo murino de la enfermedad. Los resultados muestran que ratones deficientes en el D3R se encuentran significativamente protegidos de la pérdida neurona1 y la activación microglial inducidas por el tratamiento con 1 -methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). Notablemente, los ratones deficientes en el D3R recuperan la susceptibilidad a MPTP después de la transferencia de células T CD4+ purificadas de ratones normales. Además, ratones deficientes en la recombinasa RAGA, los cuales carecen de células T y que son resistentes a la neurodegeneración dopaminérgica inducida por MPTP, adquieren la susceptibilidad a MPTP cuando se les transfieren células T CD4+normales, pero no cuando se les transfieren células T CD4+ deficientes en D3R. Además, experimentos orientados a analizar el papel del D3R en la funcionalidad de las células T CD4+, revelaron que este receptor se acopla a señalización mediada por Gai y que favorece la activación celular y adquisición del fenotipo inflamatorio Thl. Así, los datos obtenidos en esta tesis indican que el D3R expresado en células T CD4+ juega un papel fundamental en la fisiopatología de la EP en un modelo animal.Ítem Identification of master regulator genes controlling pathogenic CD4+ T cell fate in inflammatory bowel disease through transcriptional network analysis(Scientific Reports, Volume 14, Issue 1 December 2024 Article number 10553, 2024-12) Jiménez, José M.; Contreras-Riquelme, J. Sebastián; Vidal, Pía M.; Prado, Carolina; Bastías, Macarena; Meneses, Claudio; Martín, Alberto J. M.; Perez-Acle, Tomás; Pacheco, RodrigoInflammatory bowel diseases (IBD) are a group of chronic inflammatory conditions of the gastrointestinal tract associated with multiple pathogenic factors, including dysregulation of the immune response. Effector CD4+ T cells and regulatory CD4+ T cells (Treg) are central players in maintaining the balance between tolerance and inflammation. Interestingly, genetic modifications in these cells have been implicated in regulating the commitment of specific phenotypes and immune functions. However, the transcriptional program controlling the pathogenic behavior of T helper cells in IBD progression is still unknown. In this study, we aimed to find master transcription regulators controlling the pathogenic behavior of effector CD4+ T cells upon gut inflammation. To achieve this goal, we used an animal model of IBD induced by the transfer of naïve CD4+ T cells into recombination-activating gene 1 (Rag1) deficient mice, which are devoid of lymphocytes. As a control, a group of Rag1−/− mice received the transfer of the whole CD4+ T cells population, which includes both effector T cells and Treg. When gut inflammation progressed, we isolated CD4+ T cells from the colonic lamina propria and spleen tissue, and performed bulk RNA-seq. We identified differentially up- and down-regulated genes by comparing samples from both experimental groups. We found 532 differentially expressed genes (DEGs) in the colon and 30 DEGs in the spleen, mostly related to Th1 response, leukocyte migration, and response to cytokines in lamina propria T-cells. We integrated these data into Gene Regulatory Networks to identify Master Regulators, identifying four up-regulated master gene regulators (Lef1, Dnmt1, Mybl2, and Jup) and only one down-regulated master regulator (Foxo3). The altered expression of master regulators observed in the transcriptomic analysis was confirmed by qRT-PCR analysis and found an up-regulation of Lef1 and Mybl2, but without differences on Dnmt1, Jup, and Foxo3. These two master regulators have been involved in T cells function and cell cycle progression, respectively. We identified two master regulator genes associated with the pathogenic behavior of effector CD4+ T cells in an animal model of IBD. These findings provide two new potential molecular targets for treating IBD. © The Author(s) 2024.Ítem IRE1α activation in bone marrow-derived dendritic cells modulates innate recognition of melanoma cells and favors CD8+ T cell priming(Frontiers Media S.A., 2019-01) Medel, Bernardita; Costoya, Cristobal; Fernandez, Dominique; Pereda, Cristian; Lladser, Alvaro; Sauma, Daniela; Pacheco, Rodrigo; Iwawaki, Takao; Salazar Onfray, Flavio; Osorio, FabiolaThe IRE1α/XBP1s signaling pathway is an arm of the unfolded protein response (UPR) that safeguards the fidelity of the cellular proteome during endoplasmic reticulum (ER) stress, and that has also emerged as a key regulator of dendritic cell (DC) homeostasis. However, in the context of DC activation, the regulation of the IRE1α/XBP1s axis is not fully understood. In this work, we report that cell lysates generated from melanoma cell lines markedly induce XBP1s and certain members of the UPR such as the chaperone BiP in bone marrow derived DCs (BMDCs). Activation of IRE1α endonuclease upon innate recognition of melanoma cell lysates was required for amplification of proinflammatory cytokine production and was necessary for efficient cross-presentation of melanoma-associated antigens without modulating the MHC-II antigen presentation machinery. Altogether, this work provides evidence indicating that ex-vivo activation of the IRE1α/XBP1 pathway in BMDCs enhances CD8+ T cell specific responses against tumor antigens. © 2007 - 2019 Frontiers Media S.A. All Rights Reserved.Ítem Myeloid CD11c+ antigen-presenting cells ablation prevents hypertension in response to angiotensin II plus high-salt diet(Lippincott Williams and Wilkins, 2018) Hevia, Daniel; Araos, Patricio; Prado, Carolina; Luppichini, Eugenia Fuentes; Rojas, MacArena; Alzamora, Rodrigo; Cifuentes-Araneda, Flavia; Gonzalez, Alexis A.; Amador, Cristian A.; Pacheco, Rodrigo; Michea, LuisIncreasing evidence shows that antigen-presenting cells (APCs) are involved in the development of inflammation associated to hypertension. However, the potential role of APCs in the modulation of renal sodium transport has not been addressed. We hypothesized that APCs participate in renal sodium transport and, thus, development of high blood pressure in response to angiotensin II plus a high-salt diet. Using transgenic mice that allow the ablation of CD11chigh APCs, we studied renal sodium transport, the intrarenal renin-angiotensin system components, blood pressure, and cardiac/renal tissue damage in response to angiotensin II plus a high-salt diet. Strikingly, we found that APCs are required for the development of hypertension and that the ablation/restitution of APCs produces rapid changes in the blood pressure in mice with angiotensin II plus a high-salt diet. Moreover, APCs were necessary for the induction of intrarenal renin-angiotensin system components and affected the modulation of natriuresis and tubular sodium transporters. Consistent with the prevention of hypertension, the ablation of APCs also prevented cardiac hypertrophy and the induction of several indicators of renal and cardiac damage. Thus, our fndings indicate a prominent role of APCs as modulators of blood pressure by mechanisms including renal sodium handling, with kinetics that suggest the involvement of tubular cell functions in addition to the modulation of inflammation and adaptive immune response. © 2017 The Authors.Ítem Papel del receptor de dopamina D5 expresado en células del sistema inmune en el desarrollo de esclerosis múltiple.(Universidad Andrés Bello, 2013) Prado Terrazas, Carolina Elizabeth; Pacheco, Rodrigo; Facultad de Ciencias BiológicasRESUMEN: Los linfocitos T (LT) CD4+ son fundamentales en dirigir la respuesta inmune antígeno (Ag)-específica y la tolerancia. Las células dendríticas (DCs) son las responsables de activar LT vírgenes y promover su diferenciación hacia el fenotipo funcional más apropiado. Debido a su importante rol en el control de la inmunidad, la activación y diferenciación de DCs y LT CD4+ requieren de mecanismos altamente regulados. Numerosos estudios han demostrado que la dopamina (DA), no sólo media interacciones en el sistema nervioso, sino que también puede contribuir a la modulación de la inmunidad. En esta tesis, se estudió el papel que este neurotransmisor juega como un regulador de la función de DCs y LT CD4+ y su consecuente participación en la regulación de la respuesta inmune in vivo. Específicamente, este trabajo se enfoca en el análisis del rol del receptor de DA D5 (D5R) expresado en DCs y LT CD4+ en la modulación de la función de estas células. Para estudiar in vivo la relevancia de la expresión del D5R en ambas poblaciones se utilizó un modelo animal de autoinmunidad, análogo a la esclerosis múltiple (EM) en humanos. Respecto a las DCs, los resultados obtenidos indican que expresan la maquinaria necesaria para sintetizar y almacenar DA. También expresan los receptores de DA (DARs) D1R, D2R, D3R y D5R, pero sólo este último modula su expresión dependiendo del estado de maduración de las DCs. La ausencia del D5R en DCs resulta en una menor producción de IL-12 e IL-23, y por ende, una menor capacidad de inducir la expansión de LT CD4+ Th17. In vivo la transferencia de DCs deficientes en el D5R (D5RKO) en ratones recipientes normales (WT) reduce la severidad de la Encefalomielitis Autoinmune Experimental (EAE), debido a una menor frecuencia de LT CD4+ Th17 que infiltran el sistema nervioso central (SNC). Los LT CD4+ vírgenes (Tn) y reguladores naturales (nTregs) también poseen la maquinaria necesaria para sintetizar y almacenar DA; sin embargo, a nivel de proteína expresan preferencialmente el D1R y el D5R. En Tn la expresión del D5R no sería importante para la activación y diferenciación a fenotipos efectores/reguladores. En nTregs en cambio, la ausencia de este receptor resulta en una disminución parcial en la actividad supresora de estas células in vitro e in vivo en el desarrollo de EAE. En conjunto estos resultados indican que el D5R expresado en DCs es capaz de modular el desarrollo de una respuesta autoinmune in vivo, promoviendo la producción de IL-23 necesaria para la diferenciación terminal y expansión de la población Th17. En cambio, el D5R expresado en nTregs favorece parcialmente la capacidad supresora de éstas células.Ítem Purinergic Signaling as a Regulator of Th17 Cell Plasticity(PUBLIC LIBRARY SCIENCE, 2016-06) Fernández, Dominique; Flores-Santibáñez, Felipe; Neira, Jocelyn; Osorio-Barrios, Francisco; Tejón, Gabriela; Nuñez, Sarah; Hidalgo, Yessia; Fuenzalida, Maria Jose; Meza, Daniel; Ureta, Gonzalo; Lladser, Alvaro; Pacheco, Rodrigo; Acuña-Castillo, Claudio; Guixé, Victoria; Quintana, Francisco J.; Bono, Maria Rosa; Rosemblatt, Mario; Sauma, DanielaT helper type 17 (Th17) lymphocytes, characterized by the production of interleukin-17 and other pro-inflammatory cytokines, are present in intestinal lamina propria and have been described as important players driving intestinal inflammation. Recent evidence, supporting the notion of a functional and phenotypic instability of Th17 cells, has shown that Th17 differentiate into type 1 regulatory (Tr1) T cells during the resolution of intestinal inflammation. Moreover, it has been suggested that the expression of CD39 ectonucleotidase endows Th17 cells with immunosuppressive properties. However, the exact role of CD39 ectonucleotidase in Th17 cells has not been studied in the context of intestinal inflammation. Here we show that Th17 cells expressing CD39 ectonucleotidase can hydrolyze ATP and survive to ATP-induced cell death. Moreover, in vitro-generated Th17 cells expressing the CD39 ectonucleotidase produce IL-10 and are less pathogenic than CD39 negative Th17 cells in a model of experimental colitis in Rag-/- mice. Remarkably, we show that CD39 activity regulates the conversion of Th17 cells to IL-10-producing cells in vitro, which is abrogated in the presence of ATP and the CD39-specific inhibitor ARL67156. All these data suggest that CD39 expression by Th17 cells allows the depletion of ATP and is crucial for IL-10 production and survival during the resolution of intestinal inflammation.Ítem Rol del heterómero CCR9:DRDS en la migración de linfocitos T CD4+(Universidad Andrés Bello, 2021) Campos Acuña, Javier; Pacheco, RodrigoLas enfermedades inflamatorias intestinales son un conjunto de patologías que están caracterizadas por una desregulación de la respuesta inmune y una reducción de los niveles homeostáticos de dopamina en el tracto digestivo. Este desbalance se debe principalmente a un cambio en las subpoblaciones de linfocitos T CD4+ efectores y reguladores que mantienen una respuesta controlada en contra de la microbiota intestinal. La disminución de los niveles de dopamina favorece la diferenciación y expansión linfocitaria de los fenotipos inflamatorios Th1 y Th17. Además, durante la progresión de estas patologías, se produce un aumento de la migración linfocitaria hacia las zonas de inflamación, promovida por un aumento de la producción de MAdCAM-1 y CCL25 en el colon, ligandos de la integrina a4b7 y el receptor acoplado a proteína G, CCL25, respectivamente. Con respecto a esto, hemos observado que ratones linfopénicos reconstituidos con linfocitos T CD4 + carentes del receptor D5 de dopamina (DRD5) son resistentes a la colitis crónica inducida por la transferencia adoptiva de células T vírgenes. Los linfocitos transferidos migran de forma poco eficiente al colon a pesar de mantener una expresión elevada de la molécula CCR9. En este contexto, nuestro laboratorio ha sugerido la existencia de heterómeros formados por CCR9 y DRD5 en las células T CD4 + que infiltran la mucosa intestinal en condiciones de colitis inflamatoria en ratón. A través de ensayos de BRET y de ligación por proximidad, hemos observado que tanto CCR9 como el DRD5 se expresan en estrecha proximidad en la membrana de células Jurkat transducidas con dichas proteínas o en células T CD4+ efectoras en el colon de ratones en un modelo de colitis crónica. Finalmente, observamos que, si interrumpíamos la formación del heterómero con péptidos análogos a los segmentos transmembrana involucrados en la interface de interacción entre ambos receptores, los linfocitos T CD4 + con tropismo intestinal pierden su capacidad migratoria hacia el colon. De esta forma, creemos que el heterómero conformado por CCR9: DRDS actuaría de manera distinta a los protómeros que lo conforman, promoviendo la migración linfocitaria hacia el colon en condiciones de inflamación intestinal.Ítem Rol del sistema de dos componentes ArcAB en la supervivencia de Salmonella Typhimurium a las condiciones ambientales encontradas dentro de macrófagos, neutrófilos y en la evasión de la respuesta inmune adaptativa(Universidad Andrés Bello, 2019) Pardo Esté, Coral; Saavedra, Claudia; Pacheco, Rodrigo; Facultad de Ciencias de la VidaDurante su ciclo infectivo los patógenos utilizan varias estrategias que le permiten adaptarse a las diferentes condiciones a las que se enfrentan durante su ciclo infectivo dentro del hospedero. En este contexto, el sistema de dos componentes ArcAB es uno de los mecanismos que posee el patógeno bacteriano Salmone!la enterica serovar Typhimurium para responder de forma eficiente a condiciones cambiantes de oxígeno, lo que le permite a la bacteria adaptarse de manera eficaz. Particularmente, en esta investigación nos propusimos evaluar la participación que tiene ArcAB en la capacidad de supervivencia de la bacteria al ambiente intracelular, específicamente frente a Especies Reactivas de Oxígeno (ROS), así como también en la evasión de la respuesta inmune adaptativa. Para esto, se determinaron las condiciones asociadas a la presencia de ROS en las que la bacteria se encuentra dentro de los fagocitos. Posteriormente, evaluamos la intensidad del daño oxidativo intracelular, el cual está relacionado con la presencia del gen que codifica para el factor transcripcional ArcA en el genoma de la bacteria, así como también en la activación de la enzima mieloperoxidasa, la cual es la principal responsable de la producción de ROS dentro de los neutrófilos. Del mismo modo, determinamos que el factor transcripcional ArcA participa activamente en la modulación de la respuesta a nivel transcripcional de Salmone!la en la vacuola fagocítica. También encontramos que ArcA está involucrado en la defensa contra el daño oxidativo dado que regula genes claves que están relacionados con la virulencia y el metabolismo bacteriano, que son indispensables para la supervivencia de la bacteria. Por lo tanto, en conjunto estos resultados nos permiten afirmar que el sistema de dos componentes ArcAB es necesario para que Salmone!la Typhimurium sobreviva dentro de fagocitos como macrófagos y neutrófilos, a través de la modulación de su expresión génica eficiente frente a ROS. Así, estos resultados muestran el potencial que tiene ArcA como candidata para su utilización biotecnológica en la industria de producción de alimentos y la medicina aplicada. La utilización de cepas atenuadas de Salmone!la puede usarse como modelo para la generación de vacunas contra salmonelosis, una enfermedad con alta prevalencia en países en vías de desarrollo. Además, estas cepas también tienen el potencial de ser utilizadas como carriers en terapia sitio dirigida, en enfermedades crónicas o cáncer donde es necesario llevar moléculas a lugares específicos dentro del organismo.Ítem Smoking promotes exacerbated inflammatory features in dendritic cells of chilean rheumatoid arthritis patients(Sociedad Medica de Santiago, 2018-02) Prado, Carolina; Iruretagoyena, Mirentxu; Burgos, Paula I.; Pacheco, RodrigoBackground: The dual potential to promote tolerance or inflammation when facing self-antigens makes dendritic cells (DCs) fundamental players in autoimmunity. There is an association between smoking and DCs maturation in patients with rheumatoid arthritis (RA). However, ethnicity is a key factor in autoimmune disorders. Aim: To evaluate phenotypic and functional alterations of DCs obtained from Chilean patients with RA as compared to healthy controls (HC). In second term, to compare the inflammatory behaviour of DCs between smoker and non-smoker patients. Material and Methods: Monocyte-derived DCs and T-cells were obtained from blood samples isolated from 30 HC and 32 RA-patients, 14 of which were currently smokers and 18 non-smokers. Several maturation surface markers were evaluated in DCs, including HLA-DR, CD40, CD80, CD83 and CD86. Furthermore, autologous co-cultures of DCs and T-cells were carried out and then T-cell proliferation, and expansion of Th1, Th17 and Tregs were analysed. Results: Compared with HC, RA-patients displayed increased HLA-DR expression in DCs, which was manifested mainly in patients with moderate-to- high disease activity scores (DAS28). Furthermore, RA-patients presented a stronger Th17-expansion and a correlation between DAS28 and Th1-expansion. Both effects were mainly observed in patients in remission or with a low DAS28. Moreover, smoker RA-patients displayed enhanced HLA-DR and CD83 expression in DCs as well as an exacerbated Th17-expansion and a correlation between DAS28 and Th1-expansion. Conclusions: These findings suggest that smoking enhances the inflammatory behaviour of DCs and the consequent Th1 and Th17-mediated response in patients with RA. © 2018, Sociedad Medica de Santiago. All Rights reserved.Ítem Stat3 activation in combination with nf-kappab inhibition induces tolerogenic dendritic cells with high therapeutic potential to attenuate collagen-induced arthritis(Journal of Immunology Research, 2019) Prado, Carolina; Ugalde, Valentina; González, Hugo; Figueroa, Alicia; López, Ernesto; Lladser, Alvaro; Pacheco, RodrigoDendritic cells (DCs) have the ability to induce tolerance or inflammation in response to self-Antigens, which makes them fundamental players in autoimmunity. In this regard, immunogenic DCs produce IL-12 and IL-23 favouring the acquisition of Th1 and Th17 inflammatory phenotypes, respectively, by autoreactive CD4+ T-cells, thus promoting autoimmunity. Conversely, tolerogenic DCs produce IL-10 and TGF-β, inducing the generation of CD4+ T-cells with suppressive activity (Treg), which promote tolerance to self-constituents. Previous studies have shown that STAT3 signalling in DCs attenuates the production of proinflammatory cytokines, whilst NF-kB activation promotes it. In this study, we aimed to generate DCs displaying strong and constitutive tolerogenic profile to be used as immunotherapy in autoimmunity. To this end, we transduced bone marrowderived DCs with lentiviral particles codifying for a constitutively active version of STAT3 (constitutively active STAT3 (STAT3ca)) or with a constitutive repressor of NF-kB (IkBα superrepressor (IkBαSR)), and their therapeutic potential was evaluated in a mouse model of arthritis induced by collagen (CIA). Our results show that STAT3ca transduction favoured the production of the anti-inflammatory mediator IL-10, whereas IkBαSR transduction attenuated the expression of the proinflammatory cytokine IL-23 in DCs. Moreover, both STAT3ca-Transduced and IkBαSR-Transduced DCs separately exerted a mild but significant therapeutic effect reducing the severity of CIA development. Furthermore, when DCs were transduced with both STAT3ca and IkBSR together, they reduced CIA manifestation significantly stronger than when transduced with only STAT3ca or IkBαSR separately. These results show STAT3 and NF-kB as two important and complementary regulators of the tolerogenic behaviour of DCs, which should be considered as molecular targets in the design of DC-based suppressive immunotherapies for the treatment of autoimmune disorders.Ítem T-cell-driven inflammation as a mediator of the gut-brain axis involved in Parkinson's disease(Frontiers Media S.A., 2019) Campos-Acuña, Javier; Elgueta, Daniela; Pacheco, RodrigoParkinson's disease (PD) is a neurodegenerative disorder affecting mainly the dopaminergic neurons of the nigrostriatal pathway, a neuronal circuit involved in the control of movements, thereby the main manifestations correspond to motor impairments. The major molecular hallmark of this disease corresponds to the presence of pathological protein inclusions called Lewy bodies in the midbrain of patients, which have been extensively associated with neurotoxic effects. Importantly, different research groups have demonstrated that CD4+ T-cells infiltrate into the substantia nigra of PD patients and animal models. Moreover, several studies have consistently demonstrated that T-cell deficiency results in a strong attenuation of dopaminergic neurodegeneration in animal models of PD, thus indicating a key role of adaptive immunity in the neurodegenerative process. Recent evidence has shown that CD4+ T-cell response involved in PD patients is directed to oxidised forms of α-synuclein, one of the main constituents of Lewy bodies. On the other hand, most PD patients present a number of non-motor manifestations. Among non-motor manifestations, gastrointestinal dysfunctions result especially important as potential early biomarkers of PD, since they are ubiquitously found among confirmed patients and occur much earlier than motor symptoms. These gastrointestinal dysfunctions include constipation and inflammation of the gut mucosa and the most distinctive pathologic features associated are the loss of neurons of the enteric nervous system and the generation of Lewy bodies in the gut. Moreover, emerging evidence has recently shown a pivotal role of gut microbiota in triggering the development of PD in genetically predisposed individuals. Of note, PD has been positively correlated with inflammatory bowel diseases, a group of disorders involving a T-cell driven inflammation of gut mucosa, which is strongly dependent in the composition of gut microbiota. Here we raised the hypothesis that T-cell driven inflammation, which mediates dopaminergic neurodegeneration in PD, is triggered in the gut mucosa. Accordingly, we discuss how structural components of commensal bacteria or how different mediators produced by gut-microbiota, including short-chain fatty acids and dopamine, may affect the behaviour of T-cells, triggering the development of T-cell responses against Lewy bodies, initially confined to the gut mucosa but later extended to the brain. Copyright © 2019 Campos-Acuña, Elgueta and Pacheco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Ítem T-cell-mediated regulation of neuroinflammation involved in neurodegenerative diseases(BioMed Central Ltd., 2014) González, Hugo; Pacheco, RodrigoNeuroinflammation is involved in several neurodegenerative disorders and emerging evidence indicates that it constitutes a critical process that is required for the progression of neurodegeneration. Microglial activation constitutes a central event in neuroinflammation. Furthermore, microglia can not only be activated with an inflammatory and neurotoxic phenotype (M1-like phenotype), but they also can acquire a neurosupportive functional phenotype (M2-like phenotype) characterised by the production of anti-inflammatory mediators and neurotrophic factors. Importantly, during the past decade, several studies have shown that CD4+ T-cells infiltrate the central nervous system (CNS) in many neurodegenerative disorders, in which their participation has a critical influence on the outcome of microglial activation and consequent neurodegeneration. In this review, we focus on the analysis of the interplay of the different sub-populations of CD4+ T-cells infiltrating the CNS and how they participate in regulating the outcome of neuroinflammation and neurodegeneration in the context of Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and multiple sclerosis. In this regard, encephalitogenic inflammatory CD4+ T-cells, such as Th1, Th17, GM-CSF-producer CD4+ T-cells and γδT-cells, strongly contribute to chronic neuroinflammation, thus perpetuating neurodegenerative processes. In contrast, encephalitogenic or meningeal Tregs and Th2 cells decrease inflammatory functions in microglial cells and promote a neurosupportive microenvironment. Moreover, whereas some neurodegenerative disorders such as multiple sclerosis, Parkinson's disease and Alzheimer's disease involve the participation of inflammatory CD4+ T-cells 'naturally', the physiopathology of other neurodegenerative diseases, such as amyotrophic lateral sclerosis, is associated with the participation of anti-inflammatory CD4+ T-cells that delay the neurodegenerative process. Thus, current evidence supports the hypothesis that the involvement of CD4+ T-cells against CNS antigens constitutes a key component in regulating the progression of the neurodegenerative process. © González and Pacheco; licensee BioMed Central Ltd.Ítem The histone demethylase inhibitor GSK-J4 limits inflammation through the induction of a tolerogenic phenotype on DCs(Academic Press, 2016-12) Doñas, Cristian; Carrasco, Macarena; Fritz, Macarena; Prado, Carolina; Tejón, Gabriela; Osorio-Barrios, Francisco; Manríquez, Valeria; Reyes, Paz; Pacheco, Rodrigo; Bono, María Rosa; Loyola, Alejandra; Rosemblatt, MarioAs it has been established that demethylation of lysine 27 of histone H3 by the lysine-specific deme thylase JMJD3 increases immune responses and thus elicits inflammation, we hypothesize that inhibition of JMJD3 may attenuate autoimmune disorders. We found that in vivo administration of GSK-J4, a selective inhibitor of JMJD3 and UTX, ameliorates the severity of experimental autoimmune encephalomyelitis (EAE). In vitro experiments revealed that the anti-inflammatory effect of GSK-J4 was exerted through an effect on dendritic cells (DCs), promoting a tolerogenic profile characterized by reduced expression of costimulatory molecules CD80/CD86, an increased expression of tolerogenic molecules CD103 and TGF-b1, and reduced secretion of proin flammatory cytokines IL-6, IFN-g, and TNF. Adoptive transfer of GSK-J4-treated DCs into EAE mice reduced the clinical manifestation of the disease and decreased the extent of inflammatory CD4þ T cells infiltrating the central nervous system. Notably, Treg generation, stability, and suppressive activity were all exacerbated by GSK-J4-treated DCs without affecting Th1 and Th17 cell production. Our data show that GSK-J4-mediated modulation of inflammation is achieved by a direct effect on DCs and that systemic treatment with GSK-J4 or adoptive transfer of GSK-J4-treated DCs ex vivo may be promising approaches for the treatment of inflammatory and autoimmune disorders. © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).