Guidelines for translational research in heart failure

Heart failure (HF) remains a major cause of death and hospitalization worldwide. Despite medical advances, the prognosis of HF remains poor and new therapeutic approaches are urgently needed. The development of new therapies for HF is hindered by inappropriate or incomplete preclinical studies. In these guidelines, we present a number of recommendations to enhance similarity between HF animal models and the human condition in order to reduce the chances of failure in subsequent clinical trials. We propose different approaches to address safety as well as efficacy of new therapeutic products. We also propose that good practice rules are followed from the outset so that the chances of eventual approval by regulatory agencies increase. We hope that these guidelines will help improve the translation of results from animal models to humans and thereby contribute to more successful clinical trials and development of new therapies for HF.European Union [CardioNeT-ITN-289600, CardioNext-ITN-608027, FP7-IMI-JU-SAFET-115003]; Spanish Ministry of Economy [SAF2012-31451]; Regional Government of Madrid [2010-BMD-2321]; Spanish Ministry of Economy; Pro-CNIC Foundation; NIH [HL-120732, HL100401]; AHA [14SFRN20740000]; CPRIT [RP110486P3]; Leducq Foundation [11CVD04]; MINECO-SAF [2013-42962R]; Instituto Carlos III [TERCEL-RD-12/00190026, RIC12/00420024]S

Epigenetic priming of immune/inflammatory pathways activation and abnormal activity of cell cycle pathway in a perinatal model of white matter injury

Prenatal inflammatory insults accompany prematurity and provoke diffuse white matter injury (DWMI), which is associated with increased risk of neurodevelopmental pathologies, including autism spectrum disorders. DWMI results from maturation arrest of oligodendrocyte precursor cells (OPCs), a process that is poorly understood. Here, by using a validated mouse model of OPC maturation blockade, we provide the genome-wide ID card of the effects of neuroinflammation on OPCs that reveals the architecture of global cell fate issues underlining their maturation blockade. First, we find that, in OPCs, neuroinflammation takes advantage of a primed epigenomic landscape and induces abnormal overexpression of genes of the immune/inflammatory pathways: these genes strikingly exhibit accessible chromatin conformation in uninflamed OPCs, which correlates with their developmental, stage-dependent expression, along their normal maturation trajectory, as well as their abnormal upregulation upon neuroinflammation. Consistently, we observe the positioning on DNA of key transcription factors of the immune/inflammatory pathways (IRFs, NFkB), in both unstressed and inflamed OPCs. Second, we show that, in addition to the general perturbation of the myelination program, neuroinflammation counteracts the physiological downregulation of the cell cycle pathway in maturing OPCs. Neuroinflammation therefore perturbs cell identity in maturing OPCs, in a global manner. Moreover, based on our unraveling of the activity of genes of the immune/inflammatory pathways in prenatal uninflamed OPCs, the mere suppression of these proinflammatory mediators, as currently proposed in the field, may not be considered as a valid neurotherapeutic strategy

Overcoming Target Driven Fratricide for T Cell Therapy

Chimeric Antigen Receptor (CAR) T cells expressing the fusion of the NKG2D protein with CD3ζ (NKG2D-CAR T Cells) acquire a specificity for stress-induced ligands expressed on hematological and solid cancers. However, these stress ligands are also transiently expressed by activated T cells implying that NKG2D-based T cells may undergo self-killing (fratricide) during cell manufacturing or during the freeze thaw cycle prior to infusion in patients. To avoid target-driven fratricide and enable the production of NKG2D-CAR T cells for clinical application, two distinct approaches were investigated. The first focused upon the inclusion of a Phosphoinositol-3-Kinase inhibitor (LY294002) into the production process. A second strategy involved the inclusion of antibody blockade of NKG2D itself. Both processes impacted T cell fratricide, albeit at different levels with the antibody process being the most effective in terms of cell yield. While both approaches generated comparable NKG2D-CAR T cells, there were subtle differences, for example in differentiation status, that were fine-tuned through the phasing of the inhibitor and antibody during culture in order to generate a highly potent NKG2D-CAR T cell product. By means of targeted inhibition of NKG2D expression or generic inhibition of enzyme function, target-driven CAR T fratricide can be overcome. These strategies have been incorporated into on-going clinical trials to enable a highly efficient and reproducible manufacturing process for NKG2D-CAR T cells

Melatonin Promotes Oligodendroglial Maturation of Injured White Matter in Neonatal Rats

OBJECTIVE:To investigate the effects of melatonin treatment in a rat model of white matter damage (WMD) in the developing brain. Additionally, we aim to delineate the cellular mechanisms of melatonin effect on the oligodendroglial cell lineage. METHODS:A unilateral ligation of the uterine artery in pregnant rat at the embryonic day 17 induces fetal hypoxia and subsequent growth restriction (GR) in neonatal pups. GR and control pups received a daily intra-peritoneal injection of melatonin from birth to post-natal day (P) 3. RESULTS:Melatonin administration was associated with a dramatic decrease in microglial activation and astroglial reaction compared to untreated GR pups. At P14, melatonin prevented white matter myelination defects with an increased number of mature oligodendrocytes (APC-immunoreactive) in treated GR pups. Conversely, melatonin was not found to be associated with an increased density of total oligodendrocytes (Olig2-immunoreactive), suggesting that melatonin is able to promote oligodendrocyte maturation but not proliferation. These effects appear to be melatonin-receptor dependent and were reproduced in vitro. INTERPRETATION:These data suggest that melatonin has a strong protective effect on developing damaged white matter through decreased microglial activation and oligodendroglial maturation leading to a normalization of the myelination process. Consequently, melatonin should be a considered as an effective neuroprotective candidate not only in perinatal brain damage but also in inflammatory and demyelinating diseases observed in adults

IL-9 promotes mucosal inflammation and anti-microbial responses directly or via IL-13 induction

La surexpression d'interleukine (IL) -9 induit un phénotype de type asthmatique, comprenant une augmentation de sécretion de mucus, une mastocytose, une éosinophilie pulmonaire, une hyperéactivité bronchique, ainsi que l'induction de cytokines Th2, telles que l'IL-4, l'IL-5 et l'IL-13. Cependant, les souris surexprimant l'IL-13 ont le même type de profil asthmatique. Dès lors, afin de déterminer le rôle exact de l'IL-13 dans les effets de l'IL-9, des souris surexprimant l'IL-9 ont été croisées avec des souris déficientes en IL-13. Dans ces animaux, l'IL-9 peut encore induire l'infiltration pulmonaire de mastocytes et de lymphocytes B. Cependant, si l'éosinophilie systémique induite par l'IL-9 au niveau de la cavité péritonéale n'est pas diminuée en l'absence d'IL-13, ce n'est pas le cas de l'éosinophilie pulmonaire qui recquiert la présence d'IL-13, notamment pour la production de chémokines. De même, la production de mucus et l'induction de gènes épithéliaux par l'IL-9 dépendent complètement de l'expression d'IL-13. Des expériences de transfert de cellules hématopoiétiques dans des souris déficientes en récepteur IL-9 ont démontré que l'IL-13 n'était pas un cofacteur agisant en synergie avec l'IL-9, mais bien un médiateur des activités de l'IL-9 sur les cellules épithéliales pulmonaires. Dans l'intestin, la production de mucus par les cellules caliciformes ainsi que l'expression de gènes épitheliaux sont également médiées par l'IL-13. Mais de plus, l'IL-9 induit l'hyperplasie des cellules de Paneth, des cellules epitheliales spécialisées dans les défenses innées de l'intestin. Ces cellules, normalement uniquement présentes dans le fond des glandes de Lieberkühn, sont également observables dans le colon des souris surexprimant l'IL-9. Comme les autres activités de l'IL-9 sur des cellules épithéliales, cet effet de l'IL-9 est dépendant de l'IL-13. Le fait qu'une mastocytose soit également observée au niveau de la muqueuse intestinale suggérait que l'IL-9 pourrait aussi jouer un rôle dans l'inflammation intestinale. Afin d'en savoir plus sur les activités de l'IL-9 durant l'inflammation, nous avons utilisé un modèle de colite expérimentale induite par un transfert de cellules CD4+CD45RBhigh dans des souris SCID immunodéprimées. La surexpression d'IL-9, aussi bien par les souris receveuses que par les cellules injectées, excacerbe la colite, comme en témoignent la dégradation de l'architecture du colon et l'augmentation d'expression de gènes inflammatoires. Ces données indiquent donc que l'IL-9 et l'IL-13 sont impliquées dans les réponses immunitaires innées des muqueuses.Increased Interleukin (IL) -9 expression induces an asthma-like phenotype, including mucus overproduction, mastocytosis, lung eosinophilia, airway hyperresponsiveness, and increased production of other Th2 cytokines such as IL-4, IL-5 and IL-13. In order to determine the exact role of IL-13 in this phenotype, as IL-13 overexpressing mice develop the same asthmatic profil, mice overexpressing IL-9 were crossed with IL-13-deficient mice. In these animals, IL-9 could still induce mastocytosis and B lymphocyte infiltration of the lungs. While IL-9-induced eosinophilia in the peritoneal cavity was not diminished in absence of IL-13, IL-13 was required for IL-9 to increase eosinophil chemoattractant expression, and therefore, lung eosinophilia. However, mucus production and upregulation of lung epithelial genes upon IL-9 overexpression were completely abolished in absence of IL-13. Additional experiments of hematopoietic cell transfer into IL-9 receptor deficient mice indicated that IL-13 was a direct mediator, and not a cofactor, of IL-9 effects on lung epithelial cells. In the same line of observations, in colon from IL-9 transgenic mice, intestinal mucin genes together with goblet cells-associated genes were upregulated in an IL-13-dependent way. Additionnally, typical Paneth cell markers were found to have an enhanced expression in response to IL-9. Histochemical staining of Paneth cells by phloxine/tartrazine confirmed that IL-9 induced Paneth cells hyperplasia in Lieberkühn glands of the small intestine, and in the colonic mucosa, where this cell type is normally absent. The upregulation of these Paneth cell-specific genes by IL-9 required IL-13, like for the other epithelial effects of IL-9. The observation that IL-9-overexpressing mice showed increased mast cell numbers in the intestinal mucosa suggested that this cytokine might also play a role in intestinal inflammation. We addressed the potential role of IL-9 during the onset of intestinal inflammation, by using the experimental colitis model induced by transfer of naive CD4+ CD45RBhigh T cells into immunodeficient SCID mice. Based on histological observations and inflammatory genes expression analysis, IL-9 overexpression either by the transferred CD4+ T cells, or by recipient SCID mice, was found to exacerbate colitis. Taken together, these data indicates that IL-9 and IL-13 are involved in the regulation of innate immune response in lung as well in intestines.Thèse de doctorat en sciences biomédicales et pharmaceutiques (SBIM 3) -- UCL, 200

Neuroinflammation in preterm babies and autism spectrum disorders

International audienc

IL-9 exerts biological function on antigen-experienced murine T cells and exacerbates colitis induced by adoptive transfer.

IL-9 is involved in various T cell-dependent inflammatory models including colitis, encepahlitis, and asthma. However, the regulation and specificity of IL-9 responsiveness by T cells during immune responses remains poorly understood. Here, we addressed this question using two different models: experimental colitis induced by transfer of naive CD4 CD45RB T cells into immunodeficient mice, and OVA-specific T cell activation. In the colitis model, constitutive IL-9 expression exacerbated inflammation upon transfer of CD4 CD45RB T cells from WT but not from Il9r mice, indicating that IL-9 acts directly on T cells. Suprisingly, such naïve CD4 CD45RB T cells failed to express the Il9r or respond to IL-9 in vitro, in contrast with CD4 CD45RB T cells. By using OVA-specific T cells, we observed that T cells acquired the capacity to respond to IL-9 along with CD44 upregulation, after long-lasting (5 to 12 days) in vivo antigenic stimulation. Il9r expression was associated with Th2 and Th17 phenotypes. Interestingly, in contrast to the IL-2 response, antigen restimulation downregulated IL-9 responsiveness. Taken together, our results demonstrate that IL-9 does not act on naïve T cells but that IL-9 responsiveness is acquired by CD4 T cells after in vivo activation and acquisition of memory markers such as CD44

IL-13 mediates in vivo IL-9 activities on lung epithelial cells but not on hematopoietic cells.

Increased IL-9 expression, either systemically or under the control of lung-specific promoter, induces an asthma-like phenotype, including mucus overproduction, mastocytosis, lung eosinophilia, and airway hyperresponsiveness. These activities correlate with increased production of other Th2 cytokines such as IL-4, IL-5, and IL-13 in IL-9 Tg mice. To determine the exact role of IL-13 in this phenotype, mice overexpressing IL-9 were crossed with IL-13-deficient mice. In these animals, IL-9 could still induce mastocytosis and B lymphocyte infiltration of the lungs. Although IL-9-induced eosinophilia in the peritoneal cavity was not diminished in the absence of IL-13, IL-13 was required for IL-9 to increase eotaxin expression and lung eosinophilia. Mucus production and up-regulation of lung epithelial genes upon IL-9 overexpression were completely abolished in the absence of IL-13. Using hemopoietic cell transfer experiments with recipients that overexpressed IL-9 but were deficient in the IL-9 receptor (IL-9R), we could demonstrate that the effect of IL-9 on lung epithelial cells is indirect and could be fully restored by transfer of hemopoietic cells expressing IL-9R. Mucus production by lung epithelial cells was only up-regulated when hemopoietic cells simultaneously expressed functional IL-9R and IL-13 genes, indicating that IL-13 is not a cofactor but a direct mediator of the effect of IL-9 on lung epithelial cells. Taken together, these data indicate that IL-9 can promote asthma through IL-13-independent pathways via expansion of mast cells, eosinophils, and B cells, and through induction of IL-13 production by hemopoietic cells for mucus production and recruitment of eosinophils by lung epithelial cells

The Impact of Mouse Preterm Birth Induction by RU-486 on Microglial Activation and Subsequent Hypomyelination

Preterm birth (PTB) represents 15 million births every year worldwide and is frequently associated with maternal/fetal infections and inflammation, inducing neuroinflammation. This neuroinflammation is mediated by microglial cells, which are brain-resident macrophages that release cytotoxic molecules that block oligodendrocyte differentiation, leading to hypomyelination. Some preterm survivors can face lifetime motor and/or cognitive disabilities linked to periventricular white matter injuries (PWMIs). There is currently no recommendation concerning the mode of delivery in the case of PTB and its impact on brain development. Many animal models of induced-PTB based on LPS injections exist, but with a low survival rate. There is a lack of information regarding clinically used pharmacological substances to induce PTB and their consequences on brain development. Mifepristone (RU-486) is a drug used clinically to induce preterm labor. This study aims to elaborate and characterize a new model of induced-PTB and PWMIs by the gestational injection of RU-486 and the perinatal injection of pups with IL-1beta. A RU-486 single subcutaneous (s.c.) injection at embryonic day (E)18.5 induced PTB at E19.5 in pregnant OF1 mice. All pups were born alive and were adopted directly after birth. IL-1beta was injected intraperitoneally from postnatal day (P)1 to P5. Animals exposed to both RU-486 and IL-1beta demonstrated microglial reactivity and subsequent PWMIs. In conclusion, the s.c. administration of RU-486 induced labor within 24 h with a high survival rate for pups. In the context of perinatal inflammation, RU-486 labor induction significantly decreases microglial reactivity in vivo but did not prevent subsequent PWMIs