An adult thymic stromal-cell suspension model for in vitro positive selection.

Presented here is a cell-suspension model for positive selection using thymocytes from alphabeta-TCR (H-2Db-restricted) transgenic mice specific to the lymphocytic choriomeningitis virus (LCMV) on a nonselecting MHC background (H-2d or TAP-1 -/-), cocultured with freshly isolated adult thymus stromal cells of the selecting MHC type. The thymic stromal cells alone induced positive selection of functional CD4- CD8+ cells whose kinetics and efficiency were enhanced by nominal peptide. Fibroblasts expressing the selecting MHC alone did not induce positive selection; however, together with nonselecting stroma and nominal peptide, there was inefficient positive. These results suggest multiple signaling in positive selection with selection events able to occur on multiple-cell types. The ease with which this model can be manipulated should greatly facilitate the resolution of the mechanisms of positive selection in normal and pathological states.

FGF-2 controls the differentiation of resident cardiac precursors into functional cardiomyocytes.

Recent evidence suggests that the heart possesses a greater regeneration capacity than previously thought. In the present study, we isolated undifferentiated precursors from the cardiac nonmyocyte cell population of neonatal hearts, expanded them in culture, and induced them to differentiate into functional cardiomyocytes. These cardiac precursors appear to express stem cell antigen-1 and demonstrate characteristics of multipotent precursors of mesodermal origin. Following infusion into normal recipients, these cells home to the heart and participate in physiological and pathophysiological cardiac remodeling. Cardiogenic differentiation in vitro and in vivo depends on FGF-2. Interestingly, this factor does not control the number of precursors but regulates the differentiation process. These findings suggest that, besides its angiogenic actions, FGF-2 could be used in vivo to facilitate the mobilization and differentiation of resident cardiac precursors in the treatment of cardiac diseases.

Processing of tumor-associated antigen by the proteasomes of dendritic cells controls in vivo T-cell responses.

Dendritic cells are unique in their capacity to process antigens and prime naive CD8(+) T cells. Contrary to most cells, which express the standard proteasomes, dendritic cells express immunoproteasomes constitutively. The melanoma-associated protein Melan-A(MART1) contains an HLA-A2-restricted peptide that is poorly processed by melanoma cells expressing immunoproteasomes in vitro. Here, we show that the expression of Melan-A in dendritic cells fails to elicit T-cell responses in vitro and in vivo because it is not processed by the proteasomes of dendritic cells. In contrast, dendritic cells lacking immunoproteasomes induce strong anti-Melan-A T-cell responses in vitro and in vivo. These results suggest that the inefficient processing of self-antigens, such as Melan-A, by the immunoproteasomes of professional antigen-presenting cells prevents the induction of antitumor T-cell responses in vivo.

Toward improved immunocompetence of adoptively transferred CD8+ T cells.

Adoptive transfer of autologous or allogenic T cells to patients is being used with increased frequency as a therapy for infectious diseases and cancer. However, many questions remain with regard to defining optimized procedures for preparation and selection of T cell populations for transfer. In a new study in this issue of the JCI, Gattinoni and colleagues used a TCR transgenic mouse model to examine in vitro-generated tumor antigen-specific CD8+ T cells at various stages of differentiation for their efficacy in adoptive immunotherapy against transplantable melanoma. The results confirm that CD8+ T cells progressively lose immunocompetence with prolonged in vitro cultivation and suggest that effector CD8+ T cells alone may be considerably less potent at protecting hosts with advanced tumors than are less differentiated T cells.

The cells and peripheral representation of sodium taste in mice.

Salt taste in mammals can trigger two divergent behavioural responses. In general, concentrated saline solutions elicit robust behavioural aversion, whereas low concentrations of NaCl are typically attractive, particularly after sodium depletion. Notably, the attractive salt pathway is selectively responsive to sodium and inhibited by amiloride, whereas the aversive one functions as a non-selective detector for a wide range of salts. Because amiloride is a potent inhibitor of the epithelial sodium channel (ENaC), ENaC has been proposed to function as a component of the salt-taste-receptor system. Previously, we showed that four of the five basic taste qualities-sweet, sour, bitter and umami-are mediated by separate taste-receptor cells (TRCs) each tuned to a single taste modality, and wired to elicit stereotypical behavioural responses. Here we show that sodium sensing is also mediated by a dedicated population of TRCs. These taste cells express the epithelial sodium channel ENaC, and mediate behavioural attraction to NaCl. We genetically engineered mice lacking ENaCalpha in TRCs, and produced animals exhibiting a complete loss of salt attraction and sodium taste responses. Together, these studies substantiate independent cellular substrates for all five basic taste qualities, and validate the essential role of ENaC for sodium taste in mice.

Glucose sensing by the hepatoportal sensor is GLUT2-dependent: in vivo analysis in GLUT2-null mice.

In the preceding article, we demonstrated that activation of the hepatoportal glucose sensor led to a paradoxical development of hypoglycemia that was associated with increased glucose utilization by a subset of tissues. In this study, we tested whether GLUT2 plays a role in the portal glucose-sensing system that is similar to its involvement in pancreatic beta-cells. Awake RIPGLUT1 x GLUT2-/- and control mice were infused with glucose through the portal (Po-) or the femoral (Fe-) vein for 3 h at a rate equivalent to the endogenous glucose production rate. Blood glucose and plasma insulin concentrations were continuously monitored. Glucose turnover, glycolysis, and glycogen synthesis rates were determined by the 3H-glucose infusion technique. We showed that portal glucose infusion in RIPGLUT1 x GLUT24-/- mice did not induce the hypoglycemia observed in control mice but, in contrast, led to a transient hyperglycemic state followed by a return to normoglycemia; this glycemic pattern was similar to that observed in control Fe-mice and RIPGLUT1 x GLUT2-/- Fe-mice. Plasma insulin profiles during the infusion period were similar in control and RIPGLUT1 x GLUT2-/- Po- and Fe-mice. The lack of hypoglycemia development in RIPGLUT1 x GLUT2-/- mice was not due to the absence of GLUT2 in the liver. Indeed, reexpression by transgenesis of this transporter in hepatocytes did not restore the development of hypoglycemia after initiating portal vein glucose infusion. In the absence of GLUT2, glucose turnover increased in Po-mice to the same extent as that in RIPGLUT1 x GLUT2-/- or control Fe-mice. Finally, co-infusion of somatostatin with glucose prevented development of hypoglycemia in control Po-mice, but it did not affect the glycemia or insulinemia of RIPGLUT1 x GLUT2-/- Po-mice. Together, our data demonstrate that GLUT2 is required for the function of the hepatoportal glucose sensor and that somatostatin could inhibit the glucose signal by interfering with GLUT2-expressing sensing units.

Gliotransmission in the hippocampus, mechanisms and interaction with synaptic functions

SUMMARYAstrocytes represent the largest cell population in the human brain. In addition to a well established role as metabolic support for neuronal activity, in the last years these cells have been found to accomplish other important and, sometimes, unexpected functions. The tight enwrapping of synapses by astrocytic processes and the predominant expression of glutamate uptake carriers in the astrocytic rather than neuronal plasma membranes brought to the definition of a critical involvement of astrocytes in the clearance of glutamate from synaptic junctions. Moreover, several publications showed that astrocytes are able to release chemical transmitters (gliotransmitters) suggesting their active implication in the control of synaptic functions. Among gliotransmitters, the best characterized is glutamate, which has been proposed to be released from astrocytes in a Ca2+ dependent manner via exocytosis of synaptic-like microvesicles.In my thesis I present results leading to substantial advancement of the understanding of the mechanisms by which astrocytes modulate synaptic activity in the hippocampus, notably at excitatory synapses on dentate granule cells. I show that tumor necrosis factor- alpha (TNFa), a molecule that is generally involved in immune system functions, critically controls astrocyte-to-synapse communication (gliotransmission) in the brain. With constitutive levels of TNFa present, activation of purinergic G protein-coupled receptors in astrocytes, called P2Y1 receptors, induces localized intracellular calcium ([Ca2+]j) elevation in astrocytic processes (measured by two-photon microscopy) followed by glutamate release and activation of pre-synaptic NMDA receptors resulting in synaptic potentiation. In preparations lacking TNFa, astrocytes respond with identical [Ca2+]i elevations but fail to induce neuromodulation. I find that TNFa specifically controls the glutamate release step of gliotransmission. Addition of very low (picomolar) TNFa concentrations to preparations lacking the cytokine, promptly reconstitutes both normal exocytosis in cultured astrocytes and gliotransmission in hippocampal slices. These data provide the first demonstration that gliotransmission and its synaptic effects are controlled not only by astrocyte [Ca2+]i elevations but also by permissive/homeostatic factors like TNFa.In addition, I find that higher and presumably pathological TNFa concentrations do not act just permissively but instead become direct and potent triggers of glutamate release from astrocytes, leading to a strong enhancement of excitatory synaptic activity. The TNFa action, like the one observed upon P2Y1R activation, is mediated by pre-synaptic NMDA receptors, but in this case the effect is long-lasting, and not reversible. Moreover, I report that a necessary molecular target for this action of TNFa is TNFR1, one of the two specific receptors for the cytokine, as I found that TNFa was unable to induce synaptic potentiation when applied in slices from TNFR1 knock-out (Tnfrlv") mice. I then created a double transgenic mouse model where TNFR1 is knocked out in all cells but can be re-expressed selectively in astrocytes and I report that activation of the receptors in these cells is sufficient to reestablish TNFa-dependent long-lasting potentiation of synaptic activity in the TNFR1 knock-out mice.I therefore discovered that TNFa is a primary molecule displaying both permissive and instructive roles on gliotransmission controlling synaptic functions. These reports might have profound implications for the understanding of both physiological and pathological processes associated to TNFa production, including inflammatory processes in the brain.RÉSUMÉLes astrocytes sont les cellules les plus abondantes du cerveau humain. Outre leur rôle bien établi dans le support métabolique de l'activité neuronale, d'autres fonctions importantes, et parfois inattendues de ces cellules ont été mises en lumière au cours de ces dernières années. Les astrocytes entourent étroitement les synapses de leurs fins processus qui expriment fortement les transporteurs du glutamate et permettent ainsi aux astrocytes de jouer un rôle critique dans l'élimination du glutamate de la fente synaptique. Néanmoins, les astrocytes semblent être capables de jouer un rôle plus intégratif en modulant l'activité synaptique, notamment par la libération de transmetteurs (gliotransmetteurs). Le gliotransmetteur le plus étudié est le glutamate qui est libéré par l'exocytose régulée de petites vésicules ressemblant aux vésicules synaptiques (SLMVs) via un mécanisme dépendant du calcium.Les résultats présentés dans cette thèse permettent une avancée significative dans la compréhension du mode de communication de ces cellules et de leur implication dans la transmission de l'information synaptique dans l'hippocampe, notamment des synapses excitatrices des cellules granulaires du gyrus dentelé. J'ai pu montrer que le « facteur de nécrose tumorale alpha » (TNFa), une cytokine communément associée au système immunitaire, est aussi fondamentale pour la communication entre astrocyte et synapse. Lorsqu'un niveau constitutif très bas de TNFa est présent, l'activation des récepteurs purinergiques P2Y1 (des récepteurs couplés à protéine G) produit une augmentation locale de calcium (mesurée en microscopie bi-photonique) dans l'astrocyte. Cette dernière déclenche ensuite une libération de glutamate par les astrocytes conduisant à l'activation de récepteurs NMDA présynaptiques et à une augmentation de l'activité synaptique. En revanche, dans la souris TNFa knock-out cette modulation de l'activité synaptique par les astrocytes n'est pas bien qu'ils présentent toujours une excitabilité calcique normale. Nous avons démontré que le TNFa contrôle spécifiquement l'exocytose régulée des SLMVs astrocytaires en permettant la fusion synchrone de ces vésicules et la libération de glutamate à destination des récepteurs neuronaux. Ainsi, nous avons, pour la première fois, prouvé que la modulation de l'activité synaptique par l'astrocyte nécessite, pour fonctionner correctement, des facteurs « permissifs » comme le TNFa, agissant sur le mode de sécrétion du glutamate astrocytaire.J'ai pu, en outre, démontrer que le TNFa, à des concentrations plus élevées (celles que l'on peut observer lors de conditions pathologiques) provoque une très forte augmentation de l'activité synaptique, agissant non plus comme simple facteur permissif mais bien comme déclencheur de la gliotransmission. Le TNFa provoque 1'activation des récepteurs NMD A pré-synaptiques (comme dans le cas des P2Y1R) mais son effet est à long terme et irréversible. J'ai découvert que le TNFa active le récepteur TNFR1, un des deux récepteurs spécifiques pour le TNFa. Ainsi, l'application de cette cytokine sur une tranche de cerveau de souris TNFR1 knock-out ne produit aucune modification de l'activité synaptique. Pour vérifier l'implication des astrocytes dans ce processus, j'ai ensuite mis au point un modèle animal doublement transgénique qui exprime le TNFR1 uniquement dans les astrocytes. Ce dernier m'a permis de prouver que l'activation des récepteurs TNFR1 astrocytaires est suffisante pour induire une augmentation de l'activité synaptique de manière durable.Nous avons donc découvert que le TNFa possède un double rôle, à la fois un rôle permissif et actif, dans le contrôle de la gliotransmission et, par conséquent, dans la modulation de l'activité synaptique. Cette découverte peut potentiellement être d'une extrême importance pour la compréhension des mécanismes physiologiques et pathologiques associés à la production du TNFa, en particulier lors de conditions inflammatoires.RÉSUMÉ GRAND PUBLICLes astrocytes représentent la population la plus nombreuse de cellules dans le cerveau humain. On sait, néanmoins, très peu de choses sur leurs fonctions. Pendant très longtemps, les astrocytes ont uniquement été considérés comme la colle du cerveau, un substrat inerte permettant seulement de lier les cellules neuronales entre elles. Il n'y a que depuis peu que l'on a découvert de nouvelles implications de ces cellules dans le fonctionnement cérébral, comme, entre autres, une fonction de support métabolique de l'activité neuronale et un rôle dans la modulation de la neurotransmission. C'est ce dernier aspect qui fait l'objet de mon projet de thèse.Nous avons découvert que l'activité des synapses (régions qui permettent la communication d'un neurone à un autre) qui peut être potentialisée par la libération du glutamate par les astrocytes, ne peut l'être que dans des conditions astrocytaires très particulières. Nous avons, en particulier, identifié une molécule, le facteur de nécrose tumorale alpha (TNFa) qui joue un rôle critique dans cette libération de glutamate astrocytaire.Le TNFa est surtout connu pour son rôle dans le système immunitaire et le fait qu'il est massivement libéré lors de processus inflammatoires. Nous avons découvert qu'en concentration minime, correspondant à sa concentration basale, le TNFa peut néanmoins exercer un rôle indispensable en permettant la communication entre l'astrocyte et le neurone. Ce mode de fonctionnement est assez probablement représentatif d'un processus physiologique qui permet d'intégrer la communication astrocyte/neurone au fonctionnement général du cerveau. Par ailleurs, nous avons également démontré qu'en quantité plus importante, le TNFa change son mode de fonctionnement et agit comme un stimulateur direct de la libération de glutamate par l'astrocyte et induit une activation persistante de l'activité synaptique. Ce mode de fonctionnement est assez probablement représentatif d'un processus pathologique.Nous sommes également arrivés à ces conclusions grâce à la mise en place d'une nouvelle souche de souris doublement transgéniques dans lesquelles seuls les astrocytes (etnon les neurones ou les autres cellules cérébrales) sont capables d'être activés par le TNFa.

Long-term, multilineage hematopoiesis occurs in the combined absence of beta-catenin and gamma-catenin

The canonical Wnt signaling pathway plays key roles in stem-cell maintenance, progenitor cell expansion, and lineage decisions. Transcriptional responses induced by Wnt depend on the association of either beta-catenin or gamma-catenin with lymphoid enhancer factor/T cell factor transcription factors. Here we show that hematopoiesis, including thymopoiesis, is normal in the combined absence of beta- and gamma-catenin. Double-deficient hematopoietic stem cells maintain long-term repopulation capacity and multilineage differentiation potential. Unexpectedly, 2 independent ex vivo reporter gene assays show that Wnt signal transmission is maintained in double-deficient hematopoietic stem cells, thymocytes, or peripheral T cells. In contrast, Wnt signaling is strongly reduced in thymocytes lacking TCF-1 or in nonhematopoietic cells devoid of beta-catenin. These data provide the first evidence that hematopoietic cells can transduce canonical Wnt signals in the combined absence of beta- and gamma-catenin

Selective changes in the neurofilament and microtubule cytoskeleton of NF-H/LacZ mice.

This study focused mainly on changes in the microtubule cytoskeleton in a transgenic mouse where beta-galactosidase fused to a truncated neurofilament subunit led to a decrease in neurofilament triplet protein expression and a loss in neurofilament assembly and abolished transport into neuronal processes in spinal cord and brain. Although all neurofilament subunits accumulated in neuronal cell bodies, our data suggest an increased solubility of all three subunits, rather than increased precipitation, and point to a perturbed filament assembly. In addition, reduced neurofilament phosphorylation may favor an increased filament degradation. The function of microtubules seemed largely unaffected, in that tubulin and microtubule-associated proteins (MAP) expression and their distribution were largely unchanged in transgenic animals. MAP1A was the only MAP with a reduced signal in spinal cord tissue, and differences in immunostaining in various brain regions corroborate a relationship between MAP1A and neurofilaments.

Phosphorylation regulates FOXC2-mediated transcription in lymphatic endothelial cells.

One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development.

Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere.

ABSTRACT Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA'-'lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize expressing the Bacillus thuringiensis insecticidal gene cry1Ab and the near-isogenic parent line. Plant age had a significant impact on gene expression. Using maize as a model, expression of the phlA'-'lacZ reporter gene peaked at 24 h after planting of pregerminated seedlings, and dropped to a fourth of that value within 48 h, remaining at that level throughout 22 days of plant growth. Root infection by Pythium ultimum stimulated bacterial gene expression on both cucumber and maize, and this was independent of differences in rhizosphere colonization on these host plants. To our knowledge, this is the first comprehensive evaluation of how biotic factors that commonly confront bacterial inoculants in agricultural systems (host genotype, host age, and pathogen infection) modulate the expression of key biocontrol genes for disease suppression.

The inositol Inpp5k 5-phosphatase affects osmoregulation through the vasopressin-aquaporin 2 pathway in the collecting system.

Inositol Inpp5k (or Pps, SKIP) is a member of the inositol polyphosphate 5-phosphatases family with a poorly characterized function in vivo. In this study, we explored the function of this inositol 5-phosphatase in mice and cells overexpressing the 42-kDa mouse Inpp5k protein. Inpp5k transgenic mice present defects in water metabolism characterized by a reduced plasma osmolality at baseline, a delayed urinary water excretion following a water load, and an increased acute response to vasopressin. These defects are associated with the expression of the Inpp5k transgene in renal collecting ducts and with alterations in the arginine vasopressin/aquaporin-2 signalling pathway in this tubular segment. Analysis in a mouse collecting duct mCCD cell line revealed that Inpp5k overexpression leads to increased expression of the arginine vasopressin receptor type 2 and increased cAMP response to arginine vasopressin, providing a basis for increased aquaporin-2 expression and plasma membrane localization with increased osmotically induced water transport. Altogether, our results indicate that Inpp5k 5-phosphatase is important for the control of the arginine vasopressin/aquaporin-2 signalling pathway and water transport in kidney collecting ducts.

The alpha(1A/C)- and alpha(1B)-adrenergic receptors are required for physiological cardiac hypertrophy in the double-knockout mouse.

Catecholamines and alpha(1)-adrenergic receptors (alpha(1)-ARs) cause cardiac hypertrophy in cultured myocytes and transgenic mice, but heart size is normal in single KOs of the main alpha(1)-AR subtypes, alpha(1A/C) and alpha(1B). Here we tested whether alpha(1)-ARs are required for developmental cardiac hypertrophy by generating alpha(1A/C) and alpha(1B) double KO (ABKO) mice, which had no cardiac alpha(1)-AR binding. In male ABKO mice, heart growth after weaning was 40% less than in WT, and the smaller heart was due to smaller myocytes. Body and other organ weights were unchanged, indicating a specific effect on the heart. Blood pressure in ABKO mice was the same as in WT, showing that the smaller heart was not due to decreased load. Contractile function was normal by echocardiography in awake mice, but the smaller heart and a slower heart rate reduced cardiac output. alpha(1)-AR stimulation did not activate extracellular signal-regulated kinase (Erk) and downstream kinases in ABKO myocytes, and basal Erk activity was lower in the intact ABKO heart. In female ABKO mice, heart size was normal, even after ovariectomy. Male ABKO mice had reduced exercise capacity and increased mortality with pressure overload. Thus, alpha(1)-ARs in male mice are required for the physiological hypertrophy of normal postnatal cardiac development and for an adaptive response to cardiac stress.

A novel Th cell epitope of Candida albicans mediates protection from fungal infection.

Fungal pathogens are a frequent cause of opportunistic infections. They live as commensals in healthy individuals but can cause disease when the immune status of the host is altered. T lymphocytes play a critical role in pathogen control. However, specific Ags determining the activation and function of antifungal T cells remain largely unknown. By using an immunoproteomic approach, we have identified for the first time, to our knowledge, a natural T cell epitope from Candida albicans. Isolation and sequencing of MHC class II-bound ligands from infected dendritic cells revealed a peptide that was recognized by a major population of all Candida-specific Th cells isolated from infected mice. Importantly, human Th cells also responded to stimulation with the peptide in an HLA-dependent manner but without restriction to any particular HLA class II allele. Immunization of mice with the peptide resulted in a population of epitope-specific Th cells that reacted not only with C. albicans but also with other clinically highly relevant species of Candida including the distantly related Candida glabrata. The extent of the reaction to different Candida species correlated with their degree of phylogenetic relationship to C. albicans. Finally, we show that the newly identified peptide acts as an efficient vaccine when used in combination with an adjuvant inducing IL-17A secretion from peptide-specific T cells. Immunized mice were protected from fatal candidiasis. Together, these results uncover a new immune determinant of the host response against Candida ssp. that could be exploited for the development of antifungal vaccines and immunotherapies.