Mutation screening of SEMA3A and SEMA7A in patients with congenital hypogonadotropic hypogonadism.

Background:Congenital hypogonadotropic hypogonadism (HH), a rare disorder characterized by absent, partial, or delayed puberty, can be caused by the lack or deficient number of hypothalamic gonadotropin-releasing hormone (GnRH) neurons. SEMA3A was recently implicated in the etiology of the disorder, and Sema7A-deficient mice have a reduced number of GnRH neurons in their brains.Methods:SEMA3A and SEMA7A were screened by Sanger sequencing in altogether 50 Finnish HH patients (34 with Kallmann syndrome (KS; HH with hyposmia/anosmia) and 16 with normosmic HH (nHH)). In 20 patients, mutation(s) had already been found in genes known to be implicated in congenital HH.Results:Three heterozygous variants (c.458A>G (p.Asn153Ser), c.1253A>G (p.Asn418Ser), and c.1303G>A (p.Val435Ile)) were found in SEMA3A in three KS patients, two of which also had a mutation in FGFR1. Two rare heterozygous variants (c.442C>T (p.Arg148Trp) and c.1421G>A (p.Arg474Gln)) in SEMA7A were found in one male nHH patient with a previously identified KISS1R nonsense variant and one male KS patient with a previously identified mutation in KAL1, respectively.Conclusion:Our results suggest that heterozygous missense variants in SEMA3A and SEMA7A may modify the phenotype of KS but most likely are not alone sufficient to cause the disorder.

Immune response mechanisms and protection against "Plasmodium falciparum" and "Plasmodium berghei"

Malaria is one of the most important tropical and infectious diseases causing many deaths and enormous social and economic consequences, particularly in the developing countries. Despite of widely use of anti-malaria drugs and insecticide, the development of successful vaccines constitutes one of the main strategies to control malaria transmission. Several proteins expressed from blood stage such as merozoite surface proteins (MSP] or liver stage as circumsporozoite protein (CSP) are shown to be the targets of immune responses in humans and in animals. Thus, several studies have illustrated that natural infection and laboratory immunizations of humans and animals with Plasmodium sporozoite (SPZ) and its derivate-proteins (peptides) can elicit protection and control of parasite infection. However, a clear understanding of immune response against defined Plasmodium proteins should be the prerequisite conditions before any development of appropriate vaccines. In this order, our study focused on the immune responses to MSP2 (dimorphic and C-terminal fragments) in human and mice; and the mechanisms by which mouse infected hepatocytes present Plasmodium antigens to CD8+ T-cells to induce protective immunity in mice.¦The first part of this work shows that infected hepatocytes can present Plasmodium antigens to PbCSP-specific CD8+ T-cells and induce a protective immunity in mice. Here, this was addressed in vivo and showed that the infected hepatocytes were able of stimulating of primed-and naive-CD8+ T-cell clones and induced fully protective immunity against SPZ challenge. The role of infected hepatocytes in antigen presentation was illustrated here by their graft into immuno-deficient mice and depletion of cosspresenting dentritic cells (DCs) that are known to have key role in the activation of CD8+ T-cells during the liver cycle stage of Plasmodium.¦The second part of this project concerned the fine specificity of Ab responses regarding D and C regions of the two allelic families of MSP2 (3D7 and FC27). Covering of the two regions by overlapping-20 mers led to delineate the epitopes in the different endemic areas and different age groups of donors. The major epitopes characterizing D or C regions were conserved in different endemic areas (P12/P13 and P15/P16 for the 3D7-D, P23/24 and P25/26 for the FC27-D; P29/P30 for the C region). This offers thus, the possibility of a multi-epitope vaccine design including the major epitopes from the two domains of the two allelic MSP2 families. On the other, the 20 mers, particularly some major epitopes of the 3D7-Dregion (P12, P13 and P16) belonged to the epitopes that presented a high probability to be associated with protection in the children group [1 to 5 year-old). In addition, D and C LSP purified Abs (pAbs) recognized merozoite derived polypeptides and native proteins. A crossreactivity activity of homologous pAbs against the heterologous was also illustrated between the two allelic MSP2 parasites. Finally, the functional analysis of D regions pAbs showed an inhibition of Plasmodium falciparum growth suggesting the functional biological activity of the D region pAbs in the control of malaria.¦The last part of this project aimed the evaluation of the immunogenicity of the D and C region LSPs of the two allelic MSP2 families in the presence of adjuvants for the possible use in clinical trial study in humans. The MSP2 LSP mixture showed that D and C were immunogenic and defined limited epitopes (whose intensity of immune responses) depending on the adjuvants and mouse strain for the D regions. The major epitopes characterizing the C region were usually conserved in different strains of mouse and adjuvants used. Furthermore, the single region (either with D or C) immunization of mice confirmed the immunogenicity and the presence of their limited epitopes. We concluded that the possibility to finely delineate in animals the immune responses to antigens might help to select optimal antigen/adjuvant combinations to be tested later in clinical trials. Thus, formulation of glucopyranosyl-lipid A stable emulsion, GLA-SE (toll like receptor (TLR) 4 agonist) and its different combination (CpG: TLR9 agonist and GDQ: LR7 agonist) with MSP2 LSP was better than with alum, montanide ISA 720 (Mt) and virosome. Immunization of mice with allelic LSP did not show a crossreactivity between the two allelic MSP2 parasites unlike as humans, suggesting that the crossreactivity could be acquired during natural infection of the population who are usually exposed to both allelic parasite forms (3D7 and FC27).¦Nevertheless, similar epitope of D (P12, P13 and P25) and C (P29) regions have been found both in mice and human. This offers an opportunity to compare their epitopes in naïve immunized donors with LSPs and naturally infected populations in the endemic areas.

Urokinase receptor (uPAR, CD87) is a platelet receptor important for kinetics and TNF-induced endothelial adhesion in mice.

BACKGROUND: Urokinase plasminogen activator receptor (uPAR, CD87) is a widely distributed 55-kD, glycoprotein I-anchored surface receptor. On binding of its ligand uPA, it is known to increase leukocyte adhesion and traffic. Using genetically deficient mice, we explored the role of uPAR in platelet kinetics and TNF-induced platelet consumption. METHODS AND RESULTS: Anti-uPAR antibody stained platelets from normal (+/+) but not from uPAR-/- mice, as seen by fluorescence-activated cell sorter analysis. 51Cr-labeled platelets from uPAR-/- donors survived longer than those from +/+ donors when injected into a +/+ recipient. Intratracheal TNF injection induced thrombocytopenia and a platelet pulmonary localization, pronounced in +/+ but absent in uPAR-/- mice. Aprotinin, a plasmin inhibitor, decreased TNF-induced thrombocytopenia. TNF injection markedly reduced the survival and increased the pulmonary localization of 51Cr-labeled platelets from +/+ but not from uPAR-/- donors, indicating that it is the platelet uPAR that is critical for their response to TNF. As seen by electron microscopy, TNF injection increased the number of platelets and polymorphonuclear neutrophils (PMNs) in the alveolar capillaries of +/+ mice, whereas in uPAR-/- mice, platelet trapping was insignificant and PMN trapping was slightly reduced. Platelets within alveolar capillaries of TNF-injected mice were activated, as judged from their shape, and this was evident in +/+ but not in uPAR-/- mice. CONCLUSIONS: These results demonstrate for the first time the critical role of platelet uPAR for kinetics as well as for activation and endothelium adhesion associated with inflammation.

TLR 2 modulates inflammation in zymosan-induced arthritis in mice

Résumé L'objectif de cette étude est la compréhension des mécanismes sous-jacents à l'inflammation articulaire dans un modèle murin d'arthrite induite par le zymosan (ZIA). En particulier, la participation du récepteur Toll 2 (TLR2) et du complément C3 a été recherchée. L'inflammation articulaire a été quantifiée par l'accumulation de Technetium (Tc) in vivo, et par histologie des articulations arthritiques. Les réponses humorales et cellulaires induites par le zymosan ont été quantifiées par la prolifération lymphocytaire in vitro et par la mesure de la production d'anticorps dirigés contre le zymosan in vivo. L'inflammation associée à l'arthrite induite au zymosan est, d'après le Tc-uptake, d'aspect biphasique, avec un pic après 1 jour, puis une deuxième phase plus tardive. La deuxième phase persiste jusqu'au 24 ème jour et est associée au développement d'une immunité spécifique contre le zymosan. Les souris déficientes pour TLR-2 présentent une réduction significative de l'inflammation articulaire précoce (jour 1) et tardive (jour 24), ainsi qu'une nette diminution de l'infiltrat inflammatoire dans la membrane synoviale. De plus, la prolifération de cellules du ganglion lymphatique ainsi que le taux d'IgG dirigés contre le zymosan sont diminués de façon significative après 25 jour d'arthrite chez les souris déficientes en TLR2 par rapport aux souris sauvages contrôles. Par contraste, chez les souris déficientes pour C3 on n'observe pas de différence dans l'uptake de Tc ou le scoring histologique par rapport à la lignée sauvage. Ces résultats montrent que l'arthrite induite au zymosan n'est pas seulement un modèle d'inflammation aigue, mais que l'inflammation synoviale persiste même après 25 jours. Ce modèle implique à la fois des mécanismes d'immunité innée et acquise. Le signalling via TLR 2 semble jouer in rôle dans l'immunité au zymosan et pourrait être responsable de la nature biphasique de ce modèle d'arthrite. Abstract The interplay between the innate and acquired immune systems in chronic inflammation is not well documented. We have investigated the mechanisms of inflammation in murine zymosan-induced arthritis (ZIA) in the light of recent data on the roles of Toll-like receptor 2 (TLR2) and Dentin-1 in the activation of monocyte/macrophages by zymosan. The severity of inflammation, joint histology, lymphocyte proliferation and antibody production in response to zymosan were analyzed in mice deficient in TLR2 and complement C3, and the effects of Dentin-1 inhibition by laminarin were studied. In comparison with wild-type animals, TLR2-deficient mice showed a significant decrease in the early (day 1) and late phases (day 24) of joint inflammation. C3-deficient mice showed no differences in technetium uptake or histological scoring. TLR2-deficient mice also showed a significant decrease in lymph node cell proliferation in response to zymosan and a lower IgG antibody response to zymosan at day 25 in comparison with wild-type controls, indicating that TLR2 signalling has a role in the development of acquired immune responses to zymosan. Although laminarin, a soluble β-glucan, was able to significantly inhibit zymosan uptake by macrophages in vitro, it had no effect on ZIA in vivo. These results show that ZIA is more prolonged than was originally described and involves both the innate and acquired immune pathways. C3 does not seem to have a major role in this model of joint inflammation.

The generation and analysis of combined legless 1 and 2 knock-out mice

Summary The Wnt signaling pathway plays an important role during development and also for maintaining tissue homeostasis due to its function in proliferation, differentiation and cell fate decisions. Wnt ligands bind to Frizzled receptors and activate a signaling cascade that results in the stabilization of β-Catenin, a key component of the pathway. β-Catenin translocates to the nucleus, where, together with a transcription factor of the Tcf/Lef family, it activates the expression of target genes. Legless and Pygopus are two recently discovered essential components of the Wnt pathway in Drosophila, which may mediate the nuclear import and retention of beta-Catenin and/or contribute directly to the activation of Wnt target genes. To address the function of Legless in the mouse, we have generated compound constitutive and conditional knockout alleles of the two homologues legless 'I (bc1-9) and 2. We have induced the deletion of legless in self-renewing tissues such as the gastrointestinal tract, the mammary gland and the skin during adulthood and constitutively in the embryo. The present thesis focused on the consequences of the inactivation of legless in epithelial homeostasis as well as in a regeneration model and its comparison to pygopus. Deletion of neither legless nor pygopus in the adult small intestine resulted in any apparent anomaly, contrasting expectations from the phenotype caused by over-expression of Dickkopf, a Wnt inhibitor (Pinto et al., 2003). These observations indicate that canonical Wnt signaling might not be indispensable for normal gastrointestinal epithelium homeostasis, or that, in this context, Legless and Pygopus are not essential components of the Wnt pathway. However, the regeneration of the colonic epithelium after DSS induced damage was markedly impaired in legless, but not in pygopus deficient mice. Thus, unlike in Drosophila, deletion of mammalian legless and pygopus resulted in different phenotypes, suggesting that Legless might interact with as yet unidentified partners in addition to Pygopus. Resumé La voie de signalisation Wnt joue un rôle important au cours du développement ainsi que pour le maintien de l' homéostase tissulaire due à sa fonction durant la prolifération, la différentiation et les décisions sur l'avenir des cellules. Les ligands de Wnt se lient aux récepteurs Frizzled et activent une cascade de signalisation résultant en la stabilisation de β-Catenin, un composant central de cette voie. β-Catenin est transloquée dans le noyau ou, avec l'aide des facteurs de transcription de la famille Tcf/lef, elle active la transcription des gènes cibles. Legless et Pygopus sont deux composants récemment découverts et essentiels de la voie de signalisation Wnt chez la Drosophile qui pourraient être des médiateurs de l'import et de la rétention nucléaire de bêta-catenin et/ou contribuer directement a l'activation des gènes cibles. Afin de comprendre la fonction de Legless chez la souris, nous avons généré simultanément les allèles « knock-out » constitutifs et conditionnels des deux homologues legless 1 (bc1-9) et 2. Nous avons induit la délétion de legless dans des tissus capables de s'auto renouveler comme le tract gastro-intestinal, la glande mammaire et la peau chez l'adulte et nous avons supprimé constitutivement legless chez l'embryon. La présente thèse est concentrée sur les conséquences de l'inactivation de legless au cours de l' homéostase épithéliale ainsi que dans un modèle de régénération et sur sa comparaison avec pygopus. Ni la délétion de legless ni celle de pygopus dans l'intestin adulte n'ont résulté en quelque anomalie, contrastant nos attentes provenant des phénotypes causes par la surexpression de Dickkpof, un inhibiteur de Wnt (Pinto et al., 2003). Ces observations indiquent que la voie de signalisation Wnt/β-Catenin pourrait ne pas être indispensable à l' homéostase normale du tract gastro-intestinal, ou que, dans ce contexte, Legless et Pygopus ne sont pas des composants essentiels de la vole Wnt. Cependant, la régénération de l'épithélium du colon après induction de son endommagement au DSS fut dramatiquement diminuée chez legless mais pas chez les souris mutantes pour pygopus. Ainsi, a la différence de chez la Drosophile, la délétion de legless et pygopus chez les mammifères a résulté en des phénotypes différents, suggérant que Legless pourrait interagir avec d'autres partenaires, encore non identifies, que Pygopus.

Deletion of Sirt3 does not affect atherosclerosis but accelerates weight gain and impairs rapid metabolic adaptation in LDL receptor knockout mice: implications for cardiovascular risk factor development.

Sirt3 is a mitochondrial NAD(+)-dependent deacetylase that governs mitochondrial metabolism and reactive oxygen species homeostasis. Sirt3 deficiency has been reported to accelerate the development of the metabolic syndrome. However, the role of Sirt3 in atherosclerosis remains enigmatic. We aimed to investigate whether Sirt3 deficiency affects atherosclerosis, plaque vulnerability, and metabolic homeostasis. Low-density lipoprotein receptor knockout (LDLR(-/-)) and LDLR/Sirt3 double-knockout (Sirt3(-/-)LDLR(-/-)) mice were fed a high-cholesterol diet (1.25 % w/w) for 12 weeks. Atherosclerosis was assessed en face in thoraco-abdominal aortae and in cross sections of aortic roots. Sirt3 deletion led to hepatic mitochondrial protein hyperacetylation. Unexpectedly, though plasma malondialdehyde levels were elevated in Sirt3-deficient mice, Sirt3 deletion affected neither plaque burden nor features of plaque vulnerability (i.e., fibrous cap thickness and necrotic core diameter). Likewise, plaque macrophage and T cell infiltration as well as endothelial activation remained unaltered. Electron microscopy of aortic walls revealed no difference in mitochondrial microarchitecture between both groups. Interestingly, loss of Sirt3 was associated with accelerated weight gain and an impaired capacity to cope with rapid changes in nutrient supply as assessed by indirect calorimetry. Serum lipid levels and glucose tolerance were unaffected by Sirt3 deletion in LDLR(-/-) mice. Sirt3 deficiency does not affect atherosclerosis in LDLR(-/-) mice. However, Sirt3 controls systemic levels of oxidative stress, limits expedited weight gain, and allows rapid metabolic adaptation. Thus, Sirt3 may contribute to postponing cardiovascular risk factor development.

The role of Toll-like Receptor (TLR) 9 in the development of a T-helper (Th)1/Th2 response in the murine model of infection with " Leishmania major "

1.1 SUMMARY The role of the non-specific innate immune system is as important as the elaboration of the adaptive immune system in the initiation of an immune response to pathogens. The role of the Toll-like receptors (TLRs) in the innate immune response to virus and bacterial pathogens is widely recognised, however, little is known about the role of TLRs in host defence against eukaryotic pathogens. Immunologic investigations on the marine model of infection with Leishmania major (L. major) have correlated the outcome of the disease with expansion of different subsets of CD4+ cells, designated Th1 and Th2. The resistance of C57BL/6, CBA and C3H/He mice is linked with an IL-12 driven Th1 response. In BALB/c mice the susceptibility correlates with an IL-4 driven Th2 response. The initial event promoting the development of a Th1 or Th2 response still remains elusive. Recently, the contribution of the TLR signalling pathway in the innate and acquired immune response to infection with the intracellular protozoan parasite L. major has been demonstrated. Thus, the purpose of this study is to determine whether TLRs may play a role in influencing the outcome of the infection by directing the development of a Th1 or a Th2 response during infection with L, major parasites, in resistant C57BL/6 and susceptible BALB/c mice, respectively. We demonstrated that MyD88, the major TLR adaptor molecule is necessary for C57BL/6 to develop a resistant Th1 response following L. major infection. Our data show the essential role of MyD88 in the establishment of a protective Th1 response. We subsequently aimed to determine which TLRs may be involved in the protective response. Since TLR2 and TLR4 have shown to have a potential role for Leishmania recognition, we analysed the course of infection in TLR2 and TLR4 deficient mice on a C57BL/6 resistant background following L. major infection. Our results clearly demonstrate that TLR2 or TLR4 aze dispensable to control the outcome of the disease as the TLR2 and TLR4 knockout mice developed a protective Th1 response. With the aim of determining a potential TLR candidate important in the initiation of the Thl response, we assessed the mRNA expression of different TLRs (TLR1 to TLR9) using quantitative real-time RT-PCR at different time points during the first week of infection. The results clearly showed an upregulation of TLR7 and TLR9 mRNA expression during the early phase of infection in resistant C57BL/6 mice but not in susceptible BALB/c mice. To provide in vivo evidence for the role for, these TLRs in the outcome of cutaneous leishmaniasis, studies using TLR7 and TLR9 deficient mice on a resistant C57BL/6 background were performed. The TLR7 deficient mice developed a resistance phenotype that was comparable with C57BL/6 wild type mice. Thus, the presence of TLR7 is not indispensable for the development of a Th1 response and resistance to infection. On the contrary, TLR9 deficient mice on the C57BL/6 resistant background showed high variability in the outcome of the disease. Although some mice behave as resistant C57BL/6 mice, half of them developed high lesion following infection and showed a decrease in IFN-γ production and an increase in IL-4 as compared to wild type mice. These results suggest that TLR9 may be involved in the control of infection. To test the hypothesis that regulatory T cells (Treg) are playing a role in the high variability in the disease outcome in TLR9 deficient mice, depletion of CD4+CD25+ T cells with a specific antibody three days before infection with L. major were performed Interestingly, these treated mice developed large lesions, low IL-4 and decreased IFN-γ producion when compared to untreated mice. A better understanding of the mechanism by which Treg cells influence the outcome of the disease in TLR9 deficient mice following L. major infection is currently under investigation. Altogether, this study demonstrates the importance of TLR9 in the induction of a protective T'h1 response, a process that is involved in the resolution of the lesion induced by L. major infection. 1.2 RÉSUMÉ Le rôle de la réponse immunitaire innée a longtemps été négligé quant à l'impact qu'elle pourrait avoir dans l'initiation d'une réponse immune adaptative efficace dirigée contre un pathogène. Si l'importance des récepteurs Toll-like (TLR) du système inné dans la reconnaissance des virus et bactéries a été démontrée, son rôle dans la défense contre les pathogènes eucaryotes reste encore très élusif. Récemment, il a été montré que les voies de signalisation provenant de l'activation des TLRs pouvaient initier la réponse immunitaire innée et adaptative après une infection avec le parasite protozoaire Leishmania major (L. major). Dans un modèle marin d'infection avec L. major alors que la plupart des souches de souris telles que C57BL/6 sont résistantes à l'infection et développent une réponse immunitaire de type T helper 1 (Th1) induite par IL-12, peu de souches dont les BALB/c sont sensibles et développent une réponse Th2 induite par IL-4. La différentiation Th1/Th2 est un événement qui prend place de manière définitive lors de la première semaine après infection. Les événements précoces promouvant le développement d'une réponse Th1 ou Th2 n'étant pas connus, l'objectif de ce travail a été de démontrer un rôle des TLRs dans l'initiation d'une réponse immune innée et adaptative suite à l'infection par L. major. Nous avons démontré que MyD88, une molécule importante dans le processus de signalisation des TLRs, est nécessaire pour que les souris résistantes C57BL/6 développent une réponse Th1 protectrice. L'importance du rôle de TLR2 et TLR4 dans la reconnaissance du parasite Leishmania ayant été démontrée, nous avons privilégié l'analyse de la réponse immunitaire suite à une infection in vivo de souris déficiente en TLR2 ou TLR4 sur un fond génétique résistant. Les résultats obtenus montrent que la présence de ces récepteurs n'est pas indispensable pour le contrôle de l'infection et la polarisation d'une réponse Th1 caractéristique de la résistance à L. major. Cependant d'autres TLRs peuvent aussi activer la voie de signalisation MyD88 dépendante. L'expression de l'ARNm des différents TLRs dans les ganglions drainant de souris sensibles et résistantes pendant la première semaine d'infection a été déterminée par PCR quantitative en temps réel. Les résultats obtenus montrent que l'ARNm de TLR7 et TLR9 était régulé positivement suite à l'infection par L. major chez les souris résistantes C57BL/6 alors qu'aucune modulation n'était détectable chez les souris sensibles BALB/c. Le rôle des récepteurs TLR7 et TLR9 a donc été évalué par l'infection par L. major des souris déficientes en TLR7 et TLR9 sur fond génétique C57BL/6. Nos résultats ont clairement démontré que les souris déficientes en TLR7 montrent une réponse immunitaire identique à celle des souris résistantes C57BL/6, signifiant que TLR7 n'est pas indispensable au développement d'une Th1 ainsi qu'au contrôle de la parasitémie. Paz contre, les souris déficientes en TLR9 sur un fond génétique résistant ont montré une grande variabilité dans la réponse à l'infection. En effet, la moitié des souris deviennent sensibles à l'infection, ceci étant associé à une diminution dans la production d'IFN-γ et à une augmentation de la production d'IL-4. Ces résultats suggèrent que TLR9 est impliqué dans le contrôle de la lésion et de la réponse immunitaire suite à l'infection avec L. major. Cependant les résultats avec les souris déficientes en TLR9 montrant une grande hétérogénéité et une balance Th1/Th2 instable, nous avons émis l'hypothèse que les cellules T régulatrices pouvaient être impliquées dans ce phénomène. Nous avons effectivement constaté qu'après déplétion des cellules CD4+CD25+, les souris déficientes en TLR9 développent des lésions aussi grandes que les souris BALB/c après infection par L. major. Cependant le nombre de parasites reste le même que chez les souris C57BL/6. De plus la production d'IL-4 ainsi que celle d'IFN-γ reste extrêment bas. Les mécanismes régulateurs impliqués dans ce processus sont en cours d'analyse. Ce travail met en évidence l'importance du TLR9 dans le développement d'une réponse Th1 lors d'une infection avec L. major, un processus nécessaire pour la résistance à l'infection. 1.3 RESUME POUR UN LARGE PUBLIC La leishmaniose est une maladie parasitaire répandue dans le monde entier et touchant plus de 88 pays. L'incidence mondiale de la leishmaniose cutanée et de 1 à 1,5 million de nouveaux cas par année. Plus de 12 millions de personnes sont affectées par la maladie et 350 millions de personnes sont une population à risque. Un modèle marin d'infection avec Leishmania major (L. major) a été établi qui reproduit plusieurs tableaux cliniques observés dans le cas de la leishmaniose cutanée chez l'homme. L'analyse de la réponse immunitaire dans les souris infectées par L. major a permis de distinguer deux groupes : les souris de la plupart des souches telles que C57BL/6 sont résistantes à l'infection et développent une réponse immunitaire de type T helper 1 (Th1), alors que quelques souches dont les BALB/c sont sensibles et développent une réponse de type Th2. La réponse immune adaptative dans le modèle d'infection avec L. major à été largement étudiée. Cependant, les événements précoces déterminants pour le développement d'une réponse Th1 ou Th2 restent encore très flous. Récemment, plusieurs publications ont montré que les récepteurs Toll-like (TLR) peuvent contribuer à l'initiation de la réponse immunitaire lors d'une infection avec le parasite intracellulaire L. major. Dans ce travail de thèse, nous avons étudié le rôle de MyD88, une molécule importante dans le processus de signalisation des TLRs, dans la réponse immune suite à une infection avec L. major. En l'absence de MyD88, les souris normalement résistantes à l'infection avec L. major deviennent sensibles et développent des lésions importantes. Ces souris ne sont plus capables de développer une réponse Thl, normalement caractéristique de leur phénotype résistant. Nous avons ensuite tenté de comprendre quels TLRs, plus précisément, pouvait être impliqué dans ce processus. Malgré quelques évidences démontrant que TLR2 et TLR4 pouvaient avoir un rôle important dans l'initiation d'une réponse immunitaire adaptative à Leishmania, nous avons montré que, in vivo après infection avec L. major, la déficience d'un de ces récepteurs n'était pas suffisante à faire basculer la réponse immunitaire. Les souris C57BL/6 déficient en TLR2 ou TLR4 peuvent parfaitement contrôler l'évolution de la maladie. De plus, ces souris, malgré l'absence de TLR2 ou TLR4, sont capables de monter une parfaite réponse Thl. Etant donné que TLR2 et TLR4 n'étaient pas essentiels pour la résistance à la maladie, nous avons analysé les TLRs, parmi les 12 décrits qui pouvaient être indispensables au développement d'une réponse de type Th1 associée à la résistance à l'infection par Leishmania. Nos expériences ont montré que l'expression de l'ARN messager (ARNm) de TLR7 et TLR9 était modulée suite à l'infection par L. major chez la souris résistante C57BL/6 alors qu'aucune modulation n'était visible chez les souris sensible BALB/c. Pensant que ces TLRs pourraient jouer un rôle dans la réponse immunitaire au parasite, nous avons étudié l'évolution de l'infection dans les souris déficientes en TLR7 et TLR9. Nos résultats ont clairement démontré que TLR7 n'était pas indispensable à la résistance au parasite alors que l'absence de TLR9 avait des conséquences radicales sur le contrôle de la lésion et de la réponse immunitaire suite à l'infection avec L. major. Ce travail révèle ainsi l'importance du TLR9 dans le développement d'une réponse Th1 lors d'une infection avec L. major, un processus nécessaire pour la résistance à l'infection. Il est a noté que nos résultats sont en accord avec le fait que les motifs CpG, qui sont des immunostimulateurs interagissant avec le TLR9, ont une activité adjuvante importante dans la préparation de vaccins contre la leishmaniose. Une meilleure compréhension des mécanismes immunologiques impliquant le TLR9 dans la reconnaissance du parasite est alors indispensable pour le développement de vaccins thérapeutiques efficaces.

Zymosan induced arthritis in mice is biphasic and acts through the TLR2 pathway

Zymosan induced arthritis is thought to be dependent on activation of the alternative pathway of complement and is short lived. Recently it has been demonstrated that zymosan is capable of activating the innate immune system via toll-like receptor 2 (TLR2) and TLR6. These receptors play a role in linking the innate to the adaptive immune response. We have therefore reinvestigated the mechanisms by which zymosan induces arthritis by analyzing the kinetic of inflammation, the joint histology, lymphocyte proliferation in wild type and TLR2 deficient mice.

Species-Specific Recognition of Aspergillus fumigatus by Toll-like Receptor 1 and Toll-like Receptor 6.

Background. Aspergillus fumigatus causes invasive aspergillosis, a potentially fatal infection in oncohematological patients. Innate immune detection of A. fumigatus involves Toll-like receptor (TLR) 4 and TLR2, which forms a heterodimer with either TLR1 or TLR6. The role of those coreceptors in Aspergillus sensing is unknown. Methods. Cytokine production was measured in bone marrow-derived macrophages (BMDMs) from wild-type (WT) and TLR-deficient mice after incubation with a WT and an immunogenic RodA-deficient (ΔrodA-47) strain of A. fumigatus and in lungs from these mice after intranasal mold inoculation. Aspergillus fumigatus-mediated NF-κB activation was measured in HEK293T cells transfected with plasmids expressing mouse or human TLRs. Results. Bone marrow-derived macrophages from TLR1- and TLR6-deficient mice produced lower amounts of interleukin 12p40, CXCL2, interleukin 6, and tumor necrosis factor α than BMDMs from WT mice after stimulation with A. fumigatus. Lungs from TLR1- and TLR6-deficient mice had diminished CXCL1 and CXCL2 production and increased fungal burden after intranasal inoculation of ΔrodA A. fumigatus compared with lungs from WT mice. ΔrodA strain-mediated NF-κB activation was observed in HEK293T cells expressing mouse TLR2/1, mouse TLR2/6, and human TLR2/1 but not human TLR2/6. Conclusions. Innate immune detection of A. fumigatus is mediated by TLR4 and TLR2 together with TLR1 or TLR6 in mice and TLR1 but not TLR6 in humans.

Cannabinoid withdrawal syndrome is reduced in pre-proenkephalin knock-out mice

The functional interactions between the endogenous cannabinoid and opioid systems were evaluated in pre-proenkephalin-deficient mice. Antinociception induced in the tail-immersion test by acute Delta9-tetrahydrocannabinol was reduced in mutant mice, whereas no difference between genotypes was observed in the effects induced on body temperature, locomotion, or ring catalepsy. During a chronic treatment with Delta9-tetrahydrocannabinol, the development of tolerance to the analgesic responses induced by this compound was slower in mice lacking enkephalin. In addition, cannabinoid withdrawal syndrome, precipitated in Delta9-tetrahydrocannabinol-dependent mice by the injection of SR141716A, was significantly attenuated in mutant mice. These results indicate that the endogenous enkephalinergic system is involved in the antinociceptive responses of Delta9-tetrahydrocannabinol and participates in the expression of cannabinoid abstinence.

Activity of the δ-Opioid Receptor Is Partially Reduced, Whereas Activity of the κ-Receptor Is Maintained in Mice Lacking the μ-Receptor

Previous pharmacological studies have indicated the possible existence of functional interactions between μ-, δ- and κ-opioid receptors in the CNS. We have investigated this issue using a genetic approach. Here we describe in vitro and in vivo functional activity of δ- and κ-opioid receptors in mice lacking the μ-opioid receptor (MOR). Measurements of agonist-induced [35S]GTPγS binding and adenylyl cyclase inhibition showed that functional coupling of δ- and κ-receptors to G-proteins is preserved in the brain of mutant mice. In the mouse vas deferens bioassay, deltorphin II and cyclic[d-penicillamine2,d-penicillamine5] enkephalin exhibited similar potency to inhibit smooth muscle contraction in both wild-type and MOR −/− mice. δ-Analgesia induced by deltorphin II was slightly diminished in mutant mice, when the tail flick test was used. Deltorphin II strongly reduced the respiratory frequency in wild-type mice but not in MOR −/− mice. Analgesic and respiratory responses produced by the selective κ-agonist U-50,488H were unchanged in MOR-deficient mice. In conclusion, the preservation of δ- and κ-receptor signaling properties in mice lacking μ-receptors provides no evidence for opioid receptor cross-talk at the cellular level. Intact antinociceptive and respiratory responses to the κ-agonist further suggest that the κ-receptor mainly acts independently from the μ-receptor in vivo. Reduced δ-analgesia and the absence of δ-respiratory depression in MOR-deficient mice together indicate that functional interactions may take place between μ-receptors and central δ-receptors in specific neuronal pathways.

APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells.

The persistence of serum IgG antibodies elicited in human infants is much shorter than when such responses are elicited later in life. The reasons for this rapid waning of antigen-specific antibodies elicited in infancy are yet unknown. We have recently shown that adoptively transferred tetanus toxoid (TT)-specific plasmablasts (PBs) efficiently reach the bone marrow (BM) of infant mice. However, TT-specific PBs fail to persist in the early-life BM, suggesting that they fail to receive the molecular signals that support their survival/differentiation. Using a proliferation-inducing ligand (APRIL)- and B-cell activating factor (BAFF) B-lymphocyte stimulator (BLyS)-deficient mice, we demonstrate here that APRIL is a critical factor for the establishment of the adult BM reservoir of anti-TT IgG-secreting cells. Through in vitro analyses of PB/plasma cell (PC) survival/differentiation, we show that APRIL induces the expression of Bcl-X(L) by a preferential binding to heparan sulfate proteoglycans at the surface of CD138(+) cells. Last, we identify BM-resident macrophages as the main cells that provide survival signals to PBs and show that this function is slowly acquired in early life, in parallel to a progressive acquisition of APRIL expression. Altogether, this identifies APRIL as a critical signal for PB survival that is poorly expressed in the early-life BM compartment.

Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence.

Fungal infections represent a serious threat, particularly in immunocompromised patients. Interleukin-1beta (IL-1beta) is a key pro-inflammatory factor in innate antifungal immunity. The mechanism by which the mammalian immune system regulates IL-1beta production after fungal recognition is unclear. Two signals are generally required for IL-1beta production: an NF-kappaB-dependent signal that induces the synthesis of pro-IL-1beta (p35), and a second signal that triggers proteolytic pro-IL-1beta processing to produce bioactive IL-1beta (p17) via Caspase-1-containing multiprotein complexes called inflammasomes. Here we demonstrate that the tyrosine kinase Syk, operating downstream of several immunoreceptor tyrosine-based activation motif (ITAM)-coupled fungal pattern recognition receptors, controls both pro-IL-1beta synthesis and inflammasome activation after cell stimulation with Candida albicans. Whereas Syk signalling for pro-IL-1beta synthesis selectively uses the Card9 pathway, inflammasome activation by the fungus involves reactive oxygen species production and potassium efflux. Genetic deletion or pharmalogical inhibition of Syk selectively abrogated inflammasome activation by C. albicans but not by inflammasome activators such as Salmonella typhimurium or the bacterial toxin nigericin. Nlrp3 (also known as NALP3) was identified as the critical NOD-like receptor family member that transduces the fungal recognition signal to the inflammasome adaptor Asc (Pycard) for Caspase-1 (Casp1) activation and pro-IL-1beta processing. Consistent with an essential role for Nlrp3 inflammasomes in antifungal immunity, we show that Nlrp3-deficient mice are hypersusceptible to Candida albicans infection. Thus, our results demonstrate the molecular basis for IL-1beta production after fungal infection and identify a crucial function for the Nlrp3 inflammasome in mammalian host defence in vivo.

TCR-MHC class II interaction is required for peripheral expansion of CD4 cells in a T cell-deficient host.

It is well established that T cell-deficient nude and SCID mice can be reconstituted by i.v. injection of small numbers of purified peripheral CD4+ T cells; however, the requirements for expansion of the transferred T cells in such systems are not clear. We show here that blood and lymphoid organs of MHC class II-deficient mice (which selectively lack mature CD4+ T cells) cannot be reconstituted by transfer of purified splenic CD4+ T cells, whereas TCRalpha-deficient mice (which lack both CD4+ and CD8+ mature T cells) are readily reconstituted. The failure of CD4+ T cell reconstitution in MHC class II-deficient mice was not due to the presence of CD8+ T cells, since similar results were obtained in TCRalpha-MHC class II double-deficient mice. Consistent with most previous studies CD4+ T cells in reconstituted TCRalpha-deficient mice had a diverse TCR Vbeta repertoire and were predominantly of an activated/memory (CD44high) phenotype. Collectively our data demonstrate that the expansion of peripheral CD4+ T cells in a T cell-deficient host is dependent upon interactions of the TCR with MHC class II.

Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice.

This study demonstrates that endogenously produced interferon gamma (IFN-gamma) forms the basis of a tumor surveillance system that controls development of both chemically induced and spontaneously arising tumors in mice. Compared with wild-type mice, mice lacking sensitivity to either IFN-gamma (i.e., IFN-gamma receptor-deficient mice) or all IFN family members (i.e., Stat1-deficient mice) developed tumors more rapidly and with greater frequency when challenged with different doses of the chemical carcinogen methylcholanthrene. In addition, IFN-gamma-insensitive mice developed tumors more rapidly than wild-type mice when bred onto a background deficient in the p53 tumor-suppressor gene. IFN-gamma-insensitive p53(-/-) mice also developed a broader spectrum of tumors compared with mice lacking p53 alone. Using tumor cells derived from methylcholanthrene-treated IFN-gamma-insensitive mice, we found IFN-gamma's actions to be mediated at least partly through its direct effects on the tumor cell leading to enhanced tumor cell immunogenicity. The importance and generality of this system is evidenced by the finding that certain types of human tumors become selectively unresponsive to IFN-gamma. Thus, IFN-gamma forms the basis of an extrinsic tumor-suppressor mechanism in immunocompetent hosts.