SLAMF8 Downregulates Mouse Macrophage Microbicidal Mechanisms via PI3K Pathways

SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.910112/ full#supplementary-materialACKNOWLEDGMENTS SR-P is a PhD student belonging to the Official Doctoral Program in Biomedicine of the University of Granada. The authors thank Dr. Ana Santos Carro and Dr. David Porcell from the Center of Technical Instrumentation, University of Granada, for their excellent technical assistance with confocal microscopy, and Dr. M.C. Ruiz-Ruiz and Dr. Silvia Calpe-Flores for revising the manuscript.Signaling lymphocytic activation molecule family 8 (SLAMF8) is involved in the negative modulation of NADPH oxidase activation. However, the impact of SLAMF8 downregulation on macrophage functionality and the microbicide mechanism remains elusive. To study this in depth, we first analyzed NADPH oxidase activation pathways in wild-type and SLAMF8-deficient macrophages upon different stimulus. Herein, we describe increased phosphorylation of the Erk1/2 and p38 MAP kinases, as well as increased phosphorylation of NADPH oxidase subunits in SLAMF8-deficient macrophages. Furthermore, using specific inhibitors, we observed that specific PI3K inhibition decreased the differences observed between wild-type and SLAMF8-deficient macrophages, stimulated with either PMA, LPS, or Salmonella typhimurium infection. Consequently, SLAMF8-deficient macrophages also showed increased recruitment of small GTPases such as Rab5 and Rab7, and the p47phox subunit to cytoplasmic Salmonella, suggesting an impairment of Salmonella-containing vacuole (SCV) progression in SLAMF8-deficient macrophages. Enhanced iNOS activation, NO production, and IL-6 expression were also observed in the absence of SLAMF8 upon Salmonella infection, either in vivo or in vitro, while overexpression of SLAMF8 in RAW264.7 macrophages showed the opposite phenotype. In addition, SLAMF8-deficient macrophages showed increased activation of Src kinases and reduced SHP-1 phosphate levels upon IFNγ and Salmonella stimuli in comparison to wild-type macrophages. In agreement with in vitro results, Salmonella clearance was augmented in SLAMF8-deficient mice compared to that in wild-type mice. Therefore, in conclusion, SLAMF8 intervention upon bacterial infection downregulates mouse macrophage activation, and confirmed that SLAMF8 receptor could be a potential therapeutic target for the treatment of severe or unresolved inflammatory conditions.Plan Estatal de Investigación Científica y Ténica y de Innovación, ISCIII Subdirección General de Evaluación y Fomento de la Investigación, Ministerio de Economía y Competitividad, Spain (Grants PI16/01642 and PI10/01096

Modulación de los mecanismos de activación del macrófago durante la infección por "Trypanosoma cruzi": invasión celular e inhibición de la maduración del macrófago

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 19-02-2006The mechanism by which T. cruzi modulates macrophage function during the acute phase of Chagas` disease is of great importance to understand the immunopathogenesis of this disease. In this work we have analyzed several aspects of this, such as the regulation of different signal transduction pathways involved in the control of immunomodulatory molecules and the process of entry of T. cruzi into macrophages, both in vivo and in vitro infected cells. Primarily we analyzed the activation status of macrophages in BALB/c mice in response to T. cruzi acute infection with circulating blood tripomastigotes. We analyzed in peritoneal macrophages from acutely infected the expression of different molecules required relevant for the infection such as TNF-α, COX-2 and iNOS. The infection induced the expression of all them with different kinetics being the peak of TNF-α production coincident with the highest parasitemia, while COX-2 and iNOS are expressed in a later times. As this response is normally regulated in macrophages by kinases such as p38-MAPK and PI3K, we studied their activation/phosphorylation during the course of infection. PI3K activity was inhibited upon infection, while p38- MAPK was induced coinciding with COX-2 and iNOS induction during the course of the infection in vivo. A second aim was to determine the effect of T. cruzi infection on antigen presentation in vivo as well as in vitro. Expression of co-stimulatory molecules CD80 and CD86 in peritoneal macrophages from infected BALB/c mice was strongly inhibited at the end of the acute phase. However, the expression of MHCII expression increased transiently during the peak of parasitemia but subsequently returned to basal levels. This effect on the expression co-stimulatory molecules was also observed in our in vitro model of infection, where we found that T. cruzi infection inhibited CD80 and CD86 cell surface expression independently of p38-MAPK activation. Infection experiments in vitro also suggest that the intracellular replicative form of Τ. cruzi is inhibiting the expression of MHCII on the cell surface in response to IFN-γ, interfering this way with the antigen presentation to T CD4+ cells by macrophages. Finally, we have analyzed the molecules that are involved in the endocytic pathway that T. cruzi uses to enter into the macrophage. We chose Rab5 as a candidate marker since it is one of the first molecules implicated in phagosome maturation. We have observed that T. cruzi infection induces the binding of Rab5 to early phagosomes containing live parasite through PI3K activation, confirming that the activation of Rab5 in the PI3K pathway is essential for the entrance of T. cruzi into the cell. Using various knockout mice, we have found that certain cell surface receptors, such as TLR2, TLR4 and SLAM (CD150), are involved in the entry and activation of macrophages by T. cruzi. Activation of Rab5 and fusion of early endosomes by T. cruzi infection involved TLR2 and SLAM receptors but was independent of TLR4. In summary, we have established two phases during T. cruzi infection of the macrophage that occur in vivo and can be further reproduced more detailed in vitro. A first stage involves macrophage interaction with the parasite, followed by infection, and a second stage that involves intracellular replication. Both of these processes are altered by T. cruzi, which interferes with the mechanisms of macrophage activation and likely uses it for its intracellular replication and development

The Receptor Slamf1 on the Surface of Myeloid Lineage Cells Controls Susceptibility to Infection by Trypanosoma cruzi

Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, causes severe myocarditis often resulting in death. Here, we report that Slamf1-/- mice, which lack the hematopoietic cell surface receptor Slamf1, are completely protected from an acute lethal parasite challenge. Cardiac damage was reduced in Slamf1-/- mice compared to wild type mice, infected with the same doses of parasites, as a result of a decrease of the number of parasites in the heart even the parasitemia was only marginally less. Both in vivo and in vitro experiments reveal that Slamf1-defIcient myeloid cells are impaired in their ability to replicate the parasite and show altered production of cytokines. Importantly, IFN-γ production in the heart of Slamf1 deficient mice was much lower than in the heart of wt mice even though the number of infiltrating dendritic cells, macrophages, CD4 and CD8 T lymphocytes were comparable. Administration of an anti-Slamf1 monoclonal antibody also reduced the number of parasites and IFN-γ in the heart. These observations not only explain the reduced susceptibility to in vivo infection by the parasite, but they also suggest human Slamf1 as a potential target for therapeutic target against T. cruzi infection.Fondo de Investigación Sanitaria (PI040993); Ministerio de Ciencia e Innovación (SAF2005-02220, SAF2007-61716 and SAF2010-18733); European Union (Eicosanox and ChagasEpiNet); CSIC-CONICET; BSCH/UAM; Comunidad de Madrid S2010/BMD-2332; RED RECAVA RD06/0014/1013; RED RICET RD06/0021/0016; Fundacion Ramon Areces; Government of PanamaPeer reviewe

Cutting edge: Slamf8 is a negative regulator of Nox2 activity in macrophages

Slamf8 (CD353) is a cell surface receptor that is expressed upon activation of macrophages (MΦs) by IFN-γ or bacteria. In this article, we report that a very high NADPH oxidase (Nox2) enzyme activity was found in Slamf8 -/- MΦs in response to Escherichia coli or Staphylococcus aureus, as well as to PMA. The elevated Nox2 activity in Slamf8 -/- MΦs was also demonstrated in E. coli or S. aureus phagosomes by using a pH indicator system and was further confirmed by a reduction in the enzyme activity after transfection of the receptor into Slamf8-deficient primary MΦs or RAW 264.7 cells. Upon exposure to bacteria or PMA, protein kinase C activity in Slamf8 -/- MΦs is increased. This results in an enhanced phosphorylation of p40phox, one key component of the Nox2 enzyme complex, which, in turn, leads to greater Nox2 activity. Taken together, the data show that, in response to inflammation-associated stimuli, the inducible receptor Slamf8 negatively regulates inflammatory responses. Copyright © 2012 by The American Association of Immunologists, Inc.NIH (AI-15066); Ministry of Education and Science of Spain (SAF2006-08529; SAF2007-62562); Crohn’s and Colitis Foundation of AmericaPeer Reviewe

I think, therefore I really learn. An educational methodology to boost learning and thinking skills

Para propiciar un mejor aprendizaje es necesario facilitar un procesamiento profundo. Este requiere que el material a aprender se procese, se comprenda, se conecte con conocimiento previo, se aplique a situaciones nuevas o se transforme. Por otra parte, vivimos en un mundo en evolución constante, con aparición de nuevas profesiones (ciencia de datos, por ejemplo) y cambios frecuentes en la forma de ejercer las profesiones existentes. Tenemos delante grandes retos (pandemia, sostenibilidad ambiental), complejidad creciente (el sistema económico mundial es enormemente complejo), etc. Ante esta situación, las habilidades de pensamiento y solución de problemas resultan más necesarias que nunca. Sin embargo, pensar es una actividad compleja y abstracta, que necesitamos aprender, contando con métodos de enseñanza que faciliten su aprendizaje. Por ello se propone una metodología que emplea el pensamiento como base de un procesamiento profundo, mejorando el aprendizaje, a la par que se aprenden las destrezas del pensamiento. El pensamiento es, por tanto, parte de una metodología docente, ya que contribuye a un mejor aprendizaje y, de forma colateral, es objeto de aprendizaje en sí mismo, como herramienta general para mejorar el desempeño en ámbitos muy diversos de la vida personal, académica y profesional. La metodología propuesta tiene como fin la facilitación del aprendizaje y el desarrollo del pensamiento, mediante materiales visuales y manipulativos, apoyado por un aprendizaje mediado de personas expertas. Los materiales visuales desarrollados fueron del estilo de los empleados, entre otros, por Robert Swartz. Se trata de plantillas que ayudan a explicitar distintos procesos de pensamiento. Los materiales manipulativos utilizaron simulaciones computacionales (al estilo de la propuesta metodológica SimGuide) adecuadas para procesos educativos, dadas sus facilidades de visualización y experimentación intuitivas. Estos procesos se potencian y facilitan no sólo mejorando o desarrollando habilidades del pensamiento como bien señala la divulgadora Julia Galef, sino que también lo hacen mediante estilos cognitivos (perspectiva exploradora más que defensiva), emociones (como la curiosidad ante lo sorprendente o el disfrute de aprender o resolver problemas), actitudes y valores (como la apertura y la flexibilidad cognitivas) y aspectos de autoimagen y autoestima (como la estima y la seguridad personal, más allá de estar acertados o equivocados respecto a temas concretos). El objetivo de esta nueva metodología es doble: por un lado, emplear actividades de pensamiento en tanto que facilitadores de un mejor aprendizaje, gracias al procesamiento profundo que facilita y, por otro, enseñar a pensar, como herramienta profesional fundamental. Las habilidades de pensamiento y solución de problemas tienen, además, gran aplicación en la vida cotidiana de las personas. Se obtuvieron resultados bastante satisfactorios del buen funcionamiento de la metodología propuesta.In order to improve learning, it is necessary to facilitate deep processing. For that to happen, the material to be learned needs to be processed, understood, connected with previous knowledge, applied to new situations, or transformed. Additionally, we live in a world in constant evolution, with the emergence of new professions (data science, for example) and frequent changes in the way existing professions are practiced. We face major challenges (pandemics, environmental sustainability), increasing complexity (the global economic system is highly complex), etc. In such a case, thinking and problem-solving skills are key. However, thinking is a complex and abstract activity, which we need to learn, relying on teaching methods facilitating that learning. Therefore, we propose a methodology that uses thinking as the basis for deep processing, enhancing learning while learning thinking skills. Thinking is, therefore, part of a teaching methodology, since it contributes to better learning and, collaterally, is itself an object of learning. That way, thinking sills become a general tool to improve performance in very diverse areas of personal, academic and professional life. The proposed methodology aims at facilitating learning and the development of thinking, through visual and manipulative materials. This is supported by mediated learning from experts. The visual materials were arranged in the Robert Swartz’s style. These templates help to make different thinking processes explicit. The manipulative materials make use of computational simulations (like in SimGuide methodology) suitable for educational processes, given their ease of visualization and intuitive and manipulative experimentation. These processes are enhanced and facilitated not only by improving or developing thinking skills, as pointed out by Julia Galef, but also through cognitive styles (exploratory rather than defensive perspective), emotions (such as curiosity about the surprising or the enjoyment of learning or solving problems), attitudes and values (such as cognitive openness and flexibility) and aspects of self-image and self-esteem (such as personal esteem and security, beyond being right or wrong about specific topics). There were two main objectives: 1) To use thinking activities as facilitators of better learning, due to the deep processing it facilitates, and 2) to teach thinking as a fundamental professional tool. Thinking and problem-solving skills also have great application in people's daily lives. Finally, assessment results came in broad support of the proposed methodology good performance.Sección Deptal. de Investigación y Psicología en Educación (Psicología)Fac. de PsicologíaFALSEsubmitte

forestsschool: classroom in nature

Las aulas en la naturaleza se han transformado en un modelo educativo en el que se aprende de manera vivencial en contacto con la naturaleza, en un entorno en el que los niños puedan manifestar todos sus intereses y desarrollar su parte cognitiva, emocional y social. Este proyecto tiene un potencial impacto en un curriculo universitario interdisciplinar y de aplicación multicompetencial para docentes y alumnos, y puede favorecer no sólo la formación complementaria de nuestros alumnos universitarios de infantil, sino que los docentes de asignaturas diferentes pero complementarias podamos establecer estrategias de cooperación a modo transversal que enriquezcan nuestras propuestas curriculares, haciendo coincidentes en momentos puntuales de nuestra docencia la impartición de contenidos que se desarrollan sobre un mismo denominador común, en el mismo espacio y al mismo tiempo, con el resultado final de poder aprender y adquirir competencias a través de una educación de calidad, en ciudades y comunidades sostenibles, ecológicas y acordes con los preceptos de los ODS que se llevan ejecutando con eficacia desde hace mucho tiempo en la comunidades educativas de países centroeuropeos, donde esta metodología ya está implantada en la educación superior para formar profesionales, y con una red extensa de centros de infantil homologados que la llevan a cabo.Depto. de Didáctica de las Lenguas, Artes y Educación FísicaFac. de EducaciónFALSEsubmitte