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.

Altered expression of the transcription factor Mef2c during retinal degeneration in Rpe65-/- mice.

Purpose. To investigate the role of the myocyte enhancer factor 2 (Mef2) transcription factor family in retinal diseases, Mef2c expression was assessed during retinal degeneration in the Rpe65(-/-) mouse model of Leber's congenital amaurosis (LCA). Mef2c-dependent expression of photoreceptor-specific genes was further addressed. Methods. Expression of Mef2 members was analyzed by oligonucleotide microarray, quantitative PCR (qPCR) and in situ hybridization. Mef2c-dependent transcriptional activity was assayed by luciferase assay in HEK293T cells. Results. Mef2c was the only Mef2 member markedly downregulated during retinal degeneration in Rpe65(-/-) mice. Mef2c mRNA level was decreased by more than 2 fold at 2 and 4 months and by 3.5 fold at 6 months in retinas of Rpe65(-/-) mice. Downregulation of Mef2c at the protein level was confirmed in Rpe65(-/-) retinas. The decrease in Mef2c mRNA levels in the developing Rpe65(-/-) retinas, from post-natal day (P)13 onward, was concomitant with the decreased expression of the rod-specific transcription factors Nrl and Nr2e3. Nrl was further shown to drive Mef2c transcriptional activity, supporting a physiological role for Mef2c in the retina. In addition, Mef2c appeared to act as a transcriptional repressor of its own expression, as well as those of the retina-specific retinal G-protein coupled receptor (Rgr), rhodopsin and M-opsin genes. Conclusions. These findings highlight the early altered regulation of the rod-specific transcriptional network in Rpe65-related disease. They further indicate that Mef2c may act as a novel transcription factor involved in the development and the maintenance of photoreceptor cells.

Establishment of a reliable laser-Induced Choroidal Neovascularization Animal Model

Purpose: Pathologic choroidal neovascularizations (CNV) are implicated in the wet form of age-related macular degeneration (ARMD). Abnormal vessel growth is also observed in disease when hypoxia and/or inflammation occur. Our goal is to establish a standard protocol of laser-induced CNV in mice that have different levels of pigmentation to identify the most reliable animal model.Methods: CNV was induced by 4 burns around the optic disk, using a green argon laser (100μm diameter spot size; 0,05 sec. duration) in C57/Bl6, DBA/1 and Balb/c to ascertain the efficacy of the method in function of retina pigmentation. Five different intensities were tested and Bruch's membrane disruption was identified by the appearance of a bubble at the site of photocoagulation. Fluorescein angiographies (FA) were undertaken 14 days post lesion and CNV area was quantified by immunohistochemistry on cryosections.Results: CNV retina area was related to spot intensity after laser injury. While 180mW and 200mW do not induce reliable CNV (respectively 27.85±0.35% and 29±1.67% of the retina surface), 260mW is required to induce 51,07±8.52% of CNV in C57/Bl6 mice. For the DBA/1 strain, less pigmented, 200mW was sufficient to induce 49.35±3.9% of CNV, indicating that lower intensity are required to induce CNV. Furthermore, an intensity of 180mW induced greater CNV (35.55±6.01%) than in C57/Bl6 mice. Nevertheless, laser did not induce reproducible 50% CNV in Balb/c albino mice for all intensities tested. Isolectin-B4 and GFAP stainings revealed neovessel formation and photoreceptor (PR) degeneration at the impact site. The presence of glia was observed throughout all the retinal layers and angiograms showed fluorescein leakage in pigmented mice.Conclusions: The establishment of a standard protocol to induce CNV and subsequent PR degeneration is of prime importance for the use of the laser-induced CNV model and will allow to evaluate the therapeutic potency of agents to prevent CNV and retinal degeneration.

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.

Modulation of the airway allergic response : characterization of the cellular and molecular mechanisms

ABSTRACT Allergic asthma is a major complication of atopy. Its severity correlates with the presence of activated T lymphocytes and eosinophils in the bronchoalveolar lavage fluid (BALF). Mechanisms that protect against asthma are poorly understood. Based on oral models of mucosal tolerance induction, models using the nasal route showed that uptake of important amounts of antigen can induce tolerance and reverse the allergic phenotype. 1L-10 producing regulatory T cells were proposed as key players in tolerance induction, but other players, e.g. dendritic cells (DC), B cells and epithelial cells may have to be taken into consideration. The objective of the present study is to characterize the effects of a therapeutic intranasal treatment (INT) in a murine model of asthma and to determine, in this model, the cellular and molecular mechanisms leading to protection against asthma. First, we established an asthma model by sensitizing the BALB/c mouse to ovalbumin (OVA) by two intraperitoneal injections of alum-adsorbed OVA and three inhalations of aerosolized OVA. Then OVA was applied to the nasal mucosa of OVA- sensitized mice. Mice were later re-exposed to OVA aerosols to assess the protection induced by OVA INT. OVA sensitization induced strong eosinophil recruitment, OVA-specific T cell proliferation and IgE production. Three intranasal treatments at 24-hour intervals with 1.5 mg OVA drastically reduced inflammatory cell recruitment into the BALF and inhibited OVA-specific IgE production upon allergen re-exposure. T cell proliferation in ex vivo bronchial lymph node (BLN) cells was inhibited, as well as TH2 cytokine production. Protection against OVA-induced bronchial inflammation was effective for an extended period of time and treated mice resisted a second re-exposure. Transfer of CD4+ cells from BLN and lungs of OVA-treated mice protected asthmatic recipient mice from subsequent aerosol challenge indicating an involvement of CD4+ T regulatory cells in this protection. RESUME L'asthme allergique est une manifestation clinique majeure de l'atopie. La sévérité de l'asthme est liée à la présence de lymphocytes T activés ainsi que d'éosinophiles dans le lavage broncho-alvéolaire (LBA). Les mécanismes permettant de se prémunir contre l'asthme sont mal connus. Basés sur des modèles muqueux d'induction de tolérance par la voie orale, des modèles utilisant la voie nasale ont montré que d'importantes quantités d'antigène peuvent induire une tolérance et ainsi reverser le phénotype allergique. Des cellules régulatrices produisant de l'IL-10 pourraient jouer un rôle clé dans l'induction de la tolérance mais d'autres acteurs tels que les cellules dendritiques, les cellules B et les cellules épithéliales doivent aussi être prises en compte. L'objectif de la présente étude est de caractériser les effets d'un traitement intranasal thérapeutique dans un modèle murin d'asthme et de déterminer dans ce modèle les mécanismes cellulaires et moléculaires conférant une protection contre l'asthme. En premier lieu, un modèle d'asthme allergique a été établi en sensibilisant des souris BALB/c à l'ovalbumine (OVA) par deux injections intraperitonéales d'OVA adsorbé sur de l'alum et trois séances d'OVA en aérosol. Dans un second temps, de l'OVA a été administrée sur la muqueuse nasale des souris sensibilisées à l'OVA. Les souris furent ensuite challengées par des aérosols d'OVA afin d'évaluer la protection conférée par le traitement intranasal à l'OVA. La sensibilisation à l'OVA a induit un fort recrutement d'éosinophiles, une réponse proliférative des cellules T à l'OVA ainsi qu'une production d'lgE spécifiques. Trois traitements intranasaux à 24 heures d'intervalle avec 1.5 mg d'OVA ont permis de réduire drastiquement le recrutement des cellules inflammatoires dans le LBA ainsi que d'inhiber la production d'lgE spécifiques à l'OVA produits lors d'une ré-exposition à l'OVA. La prolifération en réponse à l'OVA de cellules extraites ex vivo de ganglions bronchiques a, elle aussi, été inhibée de même que la production de cytokines TH2. La protection contre l'inflammation provoquée par l'aérosol est efficace pour une longue période et les souris traitées résistent à une seconde ré- exposition. Le transfert de cellules CD4+ issues de ganglions bronchiques et de poumons de souris traitées à l'OVA protège les souris asthmatiques receveuses contre les effets inflammatoires d'un aérosol, indiquant que des cellules T CD4+ régulatrices pourraient être impliquées dans cette protection. RESUME DESTINE A UN LARGE PUBLIC L'asthme est une affection des voies respiratoires qui se caractérise par une contraction de la musculature des voies aériennes, une production de mucus et d'anticorps de l'allergie (IgE). On parle d'asthme allergique lorsque les facteurs déclenchant l'asthme sont des allergènes inhalés tels que acariens, pollens ou poils d'animaux. Le système immunitaire des patients asthmatiques a un défaut de programmation qui le rend réactif à des substances qui sont normalement inoffensives. Le traitement actuel de l'asthme repose sur le soulagement des symptômes grâce à des produits à base de stéroïdes. Les techniques permettant de reprogrammer le système immunitaire (immunothérapie) ne sont pas efficaces pour tous les antigènes et prennent beaucoup de temps. En conséquence, il est nécessaire de mieux comprendre les mécanismes sous-tendant une telle reprogrammation afin d'en améliorer le rendement et l'efficacité. Dans ce but, des modèles d'immunothérapie ont été mis au point chez la souris. Ils permettent une plus grande liberté d'investigation. Dans cette étude, un modèle d'asthme allergique dans la souris a été établi par une sensibilisation à un antigène particulier : l'ovalbumine (OVA). Ce modèle présente les caractéristiques principales de l'asthme humain : recrutement de cellules inflammatoires dans les poumons, augmentation de la production d'anticorps et de la résistance des bronches aux flux respiratoires. Cette souris asthmatique a ensuite été traitée par application nasale d'OVA. Comparées aux souris non traitées, les souris traitées à l'OVA ont moins de cellules inflammatoires dans leurs poumons et produisent moins d'anticorps IgE. D'autres marqueurs inflammatoires sont aussi fortement diminués. Des cellules de poumons ou de ganglions bronchiques prélevées sur des souris traitées injectées dans des souris asthmatiques améliorent les symptômes de l'asthme. Ces cellules pourraient donc avoir un rôle régulateur dans l'asthme. Les caractériser et les étudier afin d'être capable de les générer est crucial pour les futures thérapies de l'asthme.

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.

Hepatic deficiency in transcriptional cofactor TBL1 promotes liver steatosis and hypertriglyceridemia.

The aberrant accumulation of lipids in the liver ("fatty liver") is tightly associated with several components of the metabolic syndrome, including type 2 diabetes, coronary heart disease, and atherosclerosis. Here we show that the impaired hepatic expression of transcriptional cofactor transducin beta-like (TBL) 1 represents a common feature of mono- and multigenic fatty liver mouse models. Indeed, the liver-specific ablation of TBL1 gene expression in healthy mice promoted hypertriglyceridemia and hepatic steatosis under both normal and high-fat dietary conditions. TBL1 deficiency resulted in inhibition of fatty acid oxidation due to impaired functional cooperation with its heterodimerization partner TBL-related (TBLR) 1 and the nuclear receptor peroxisome proliferator-activated receptor (PPAR) α. As TBL1 expression levels were found to also inversely correlate with liver fat content in human patients, the lack of hepatic TBL1/TBLR1 cofactor activity may represent a molecular rationale for hepatic steatosis in subjects with obesity and the metabolic syndrome.

Segmental copy number variation shapes tissue transcriptomes.

Copy number variation (CNV) is a key source of genetic diversity, but a comprehensive understanding of its phenotypic effect is only beginning to emerge. We have generated a CNV map in wild mice and classical inbred strains. Genome-wide expression data from six major organs show not only that expression of genes within CNVs tend to correlate with copy number changes, but also that CNVs influence the expression of genes in their vicinity, an effect that extends up to half a megabase. Genes within CNVs show lower expression and more specific spatial expression patterns than genes mapping elsewhere. Our analyses reveal differential constraint on copy number changes of genes expressed in different tissues. Dosage alterations of brain-expressed genes are less frequent than those of other genes and are buffered by tighter transcriptional regulation. Our study provides initial evidence that CNVs shape tissue transcriptomes on a global scale.

Angiopoietin 2 regulates the transformation and integrity of lymphatic endothelial cell junctions.

Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell-cell junctions that form during lymphatic development.

Atherosclerosis in ApoE-deficient mice progresses independently of the NLRP3 inflammasome.

The interleukin-1 (IL-1) family of cytokines has been implicated in the pathogenesis of atherosclerosis in previous studies. The NLRP3 inflammasome has recently emerged as a pivotal regulator of IL-1β maturation and secretion by macrophages. Little is currently known about a possible role for the NLRP3 inflammasome in atherosclerosis progression in vivo. We generated ApoE-/- Nlrp3-/-, ApoE-/- Asc-/- and ApoE-/- caspase-1-/- double-deficient mice, fed them a high-fat diet for 11 weeks and subsequently assessed atherosclerosis progression and plaque phenotype. No differences in atherosclerosis progression, infiltration of plaques by macrophages, nor plaque stability and phenotype across the genotypes studied were found. Our results demonstrate that the NLRP3 inflammasome is not critically implicated in atherosclerosis progression in the ApoE mouse model.

Interferon-gamma impacts at multiple points during the progression of autoimmune diabetes.

The role of interferon-gamma in autoimmune diabetes was assessed by breeding a null mutation of the interferon-gamma receptor alpha chain into the nonobese diabetic mouse strain, as well as into a simplified T cell receptor transgenic model of diabetes. In contrast to a previous report on abrogation of the interferon-gamma gene, mutation of the gene encoding its receptor led to drastic effects on disease in both mouse lines. Nonobese diabetic mice showed a marked inhibition of insulitis-both the kinetics and penetrance-and no signs of diabetes; the transgenic model exhibited near-normal insulitis, but this never evolved into diabetes, either spontaneously or after experimental provocation. This failure could not be explained by perturbations in the ratio of T helper cell phenotypes; rather, it reflected a defect in antigen-presenting cells or in the islet beta cell targets.

Pathogenic Vibrio activate NLRP3 inflammasome via cytotoxins and TLR/nucleotide-binding oligomerization domain-mediated NF-kappa B signaling.

Vibrio vulnificus and Vibrio cholerae are Gram-negative pathogens that cause serious infectious disease in humans. The beta form of pro-IL-1 is thought to be involved in inflammatory responses and disease development during infection with these pathogens, but the mechanism of beta form of pro-IL-1 production remains poorly defined. In this study, we demonstrate that infection of mouse macrophages with two pathogenic Vibrio triggers the activation of caspase-1 via the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was mediated by hemolysins and multifunctional repeat-in-toxins produced by the pathogenic bacteria. NLRP3 activation in response to V. vulnificus infection required NF-kappaB activation, which was mediated via TLR signaling. V. cholerae-induced NLRP3 activation also required NF-kappaB activation but was independent of TLR stimulation. Studies with purified V. cholerae hemolysin revealed that toxin-stimulated NLRP3 activation was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-kappaB activation. Our results identify the NLRP3 inflammasome as a sensor of Vibrio infections through the action of bacterial cytotoxins and differential activation of innate signaling pathways acting upstream of NF-kappaB.

Expression of T cell receptor genes in the thymus: localization of transcripts in situ and comparison of mature and immature subsets.

We have analyzed the expression of T cell receptor (TcR) genes in the thymus using in situ RNA hybridizations with probes to the constant regions of the TcR alpha, beta, gamma and delta chains. Localization of transcripts revealed low TcR alpha mRNA levels in the thymus cortex and very low levels in the subcapsular region. In contrast, TcR beta message was very abundant in the cortex. TcR gamma or delta mRNA+ thymocytes showed a scattered, predominantly cortical localization. In contrast to gamma, TcR delta transcripts were abundant in the subcapsular region. Control experiments with sorted TcR alpha/beta or gamma/delta cells revealed a detection efficiency of 75%-85% for the respective TcR mRNA and data on TcR gene expression in mature, CD3+ thymocytes were consistent with previous reports. The analysis of immature, CD3- thymocyte subsets, however, revealed a virtual absence of TcR alpha transcripts and an unexpectedly high proportion of cells (14%-29%) expressing the gene for the TcR delta chain. The data are discussed in view of current models of lineage relationships in the thymus.

IL-2/anti-IL-2 antibody complexes show strong biological activity by avoiding interaction with IL-2 receptor alpha subunit CD25.

IL-2 is crucial to T cell homeostasis, especially of CD4(+) T regulatory cells and memory CD8(+) cells, as evidenced by vigorous proliferation of these cells in vivo following injections of superagonist IL-2/anti-IL-2 antibody complexes. The mechanism of IL-2/anti-IL-2 antibody complexes is unknown owing to a lack of understanding of IL-2 homeostasis. We show that IL-2 receptor alpha (CD25) plays a crucial role in IL-2 homeostasis. Thus, prolongation of IL-2 half-life and blocking of CD25 using antibodies or CD25-deficient mice led in combination, but not alone, to vigorous IL-2-mediated T cell proliferation, similar to IL-2/anti-IL-2 antibody complexes. These data suggest an unpredicted role for CD25 in IL-2 homeostasis.

Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas-mediated cell death in mice.

Tissue transglutaminase (TG2) is a protein cross-linking enzyme known to be expressed by hepatocytes and to be induced during the in vivo hepatic apoptosis program. TG2 is also a G protein that mediates intracellular signaling by the alpha-1b-adrenergic receptor (AR) in liver cells. Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibodies to mice causes both disseminated endothelial cell apoptosis and fulminant hepatic failure. Here we report that an intraperitoneal dose of anti-Fas antibodies, which is sublethal for wild-type mice, kills all the TG2 knock-out mice within 20 hours. Although TG2-/- thymocytes exposed to anti-Fas antibodies die at the same rate as wild-type mice, TG2-/- hepatocytes show increased sensitivity toward anti-Fas treatment both in vivo and in vitro, with no change in their cell surface expression of Fas, levels of FLIP(L) (FLICE-inhibitory protein), or the rate of I-kappaBalpha degradation, but a decrease in the Bcl-xL expression. We provide evidence that this is the consequence of the impaired AR signaling that normally regulates the levels of Bcl-xL in the liver. In conclusion, our data suggest the involvement of adrenergic signaling pathways in the hepatic regeneration program, in which Fas ligand-induced hepatocyte proliferation with a simultaneous inhibition of the Fas-death pathway plays a determinant role.