Destruction of conditional insulinoma cell lines in NOD mice: a role for autoimmunity.

AIMS/HYPOTHESIS: betaTC-tet (H2(k)) is a conditional insulinoma cell line derived from transgenic mice expressing a tetracycline-regulated oncogene. Transgenic expression of several proteins implicated in the apoptotic pathways increase the resistance of betaTC-tet cells in vitro. We tested in vivo the sensitivity of the cells to rejection and the protective effect of genetic alterations in NOD mice. METHODS: betaTC-tet cells and genetically engineered lines expressing Bcl-2 (CDM3D), a dominant negative mutant of MyD88 or SOCS-1 were transplanted in diabetic female NOD mice or in male NOD mice with diabetes induced by high-dose streptozotocin. Survival of functional cell grafts in NOD-scid mice was also analyzed after transfer of splenocytes from diabetic NOD mice. Autoreactive T-cell hybridomas and splenocytes from diabetic NOD mice were stimulated by betaTC-tet cells. RESULTS: betaTC-tet cells and genetically engineered cell lines were all similarly rejected in diabetic NOD mice and in NOD-scid mice after splenocyte transfer. In 3- to 6-week-old male NOD mice treated with high-dose streptozotocin, the cells temporarily survived, in contrast with C57BL/6 mice treated with high-dose streptozotocin (indefinite survival) and untreated 3- to 6-week-old male NOD mice (rejection). The protective effect of high-dose streptozotocin was lost in older male NOD mice. betaTC-tet cells did not stimulate autoreactive T-cell hybridomas, but induced IL-2 secretion by splenocytes from diabetic NOD mice. CONCLUSION/INTERPRETATION: The autoimmune process seems to play an important role in the destruction of betaTC-tet cells in NOD mice. Genetic manipulations intended at increasing the resistance of beta cells were inefficient. Similar approaches should be tested in vivo as well as in vitro. High dose streptozotocin influences immune rejection and should be used with caution.

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.

Selective absence of CD8+ TCRalpha beta+ intestinal epithelial cells in transgenic mice expressing beta2-microglobulin-associated ligands exclusively on thymic cortical epithelium.

Whereas interactions between the TCRalpha beta and self MHC:peptide complexes are clearly required for positive selection of mature CD4(+) and CD8(+) T cells during intrathymic development, the role of self or foreign ligands in maintaining the peripheral T cell repertoire is still controversial. In this report we have utilized keratin 14-beta2-microglobulin (K14-beta2m)-transgenic mice expressing beta2m-associated ligands exclusively on thymic cortical epithelial cells to address the possible influence of TCR:ligand interactions in peripheral CD8(+) T cell homeostasis. Our data indicate that CD8(+) T cells in peripheral lymphoid tissues are present in normal numbers in the absence of self MHC class I:peptide ligands. Surprisingly, however, steady state homeostasis of CD8(+) T cells in the intestinal epithelium is severely affected by the absence of beta2m-associated ligands. Indeed TCRalpha beta(+) IEL subsets expressing CD8alpha beta or CD8alpha alpha are both dramatically reduced in K14-beta2m mice, suggesting that the development, survival or expansion of CD8(+) IEL depends upon interaction of the TCR with MHC class I:peptide or other beta2m-associated ligands elsewhere than on thymic cortical epithelium. Collectively, our data reveal an unexpected difference in the regulation of CD8(+) T cell homeostasis by beta2m-associated ligands in the intestine as compared to peripheral lymphoid organs.

Different epitopes of the LACK protein are recognized by V beta 4 V alpha 8 CD4+ T cells in H-2b and H-2d mice susceptible to Leishmania major.

After inoculation of Leishmania major, a rapid production of IL-4 by LACK-specific CD4+ T cells has been shown to drive Th2 cell development in susceptible mice i.e. BALB/c and C57BL/6 mice rendered susceptible by neutralization of IFN-gamma at the onset of infection. Here, we showed that peptide AA 156-173 induced an early IL-4 mRNA expression not only in BALB/c mice but also in resistant B10.D2 mice when IFN-gamma is neutralized. Epitope mapping of LACK protein demonstrated that peptide containing AA 293-305 induced early IL-4 mRNA transcripts in susceptible H-2b mice i.e. BALB/b and resistant C57BL/6 mice when IFN-gamma is neutralized. Stringently, the early IL-4 response to the H-2d (AA 156-173) or the H-2b (AA 293-305) epitopes occurred in V beta 4 V alpha 8 CD4+ T cells from either H-2d or H-2b susceptible mice, respectively.

Neutrophil-derived CCL3 is essential for the rapid recruitment of dendritic cells to the site of Leishmania major inoculation in resistant mice.

Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3(-/-) mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.

Leishmania major-specific B cells are necessary for Th2 cell development and susceptibility to L. major LV39 in BALB/c mice.

B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (microMT). Whereas BALB/c microMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c microMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c microMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c microMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.

The C76R transmembrane activator and calcium modulator cyclophilin ligand interactor mutation disrupts antibody production and B-cell homeostasis in heterozygous and homozygous mice.

BackgroundMutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B-cell defects observed in patients with common variable immunodeficiency therefore remains unclear.ObjectiveWe sought to define the functional consequences of the C104R mutation on B-cell function.MethodsWe performed in vitro studies of TACI C104R expression and signaling. A knock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge.ResultsC104R expression and ligand binding are significantly diminished when the mutant protein is expressed in 293T cells or in patients' cell lines. This leads to defective nuclear factor κB activation, which is proportionally restored by reintroduction of wild-type TACI. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis.ConclusionsThese data show that the C104R mutation and its murine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturb B-cell function with lymphoproliferation and immunoglobulin production defects.

Decreased expression of peroxisome proliferator-activated receptor alpha and liver fatty acid binding protein after partial hepatectomy of rats and mice.

BACKGROUND AIMS: Marked changes in metabolism, including liver steatosis and hypoglycemia, occur after partial hepatectomy. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear hormone receptor that is activated by fatty acids and involved in hepatic fatty acid metabolism and regeneration. Liver fatty acid binding protein (LFABP) is an abundant protein in liver cytosol whose expression is regulated by PPAR alpha. It is involved in fatty acid uptake and diffusion and in PPAR alpha signaling. The aim of this study was to investigate the expression of PPAR alpha and LFABP during liver regeneration. METHODS: Male Sprague-Dawley rats and male C57 Bl/6 mice were subjected to 2/3 hepatectomy and LFABP and PPAR alpha mRNA and protein levels were measured at different time points after surgery. The effect of partial hepatectomy was followed during 48 h in rats and 72 h in mice. RESULTS: PPAR alpha mRNA and protein levels were decreased 26 h after hepatectomy of rats. The LFABP mRNA and protein levels paralleled those of PPAR alpha and were also decreased 26 h after hepatectomy. In mice, the mRNA level was decreased after 36 and 72 h after hepatectomy. In this case, LFABP mRNA levels decreased more slowly after partial hepatectomy than in rats. CONCLUSIONS: A marked decrease in PPAR alpha expression may be important for changed gene expression, e.g. LFABP, and metabolic changes, such as hypoglycemia, during liver regeneration.

Anti-leishmania effector functions of CD4+ Th1 cells and early events instructing Th2 cell development and susceptibility to Leishmania major in BALB/c mice.

Resistance and susceptibility to infection with the intracellular parasite, Leishmania major, are mediated by parasite-specific CD4+ Th1 and Th2 cells, respectively. It is well established that the protective effect of parasite-specific CD4+ Th1 cells is largely dependent upon the IFN-gamma produced. However, recent results indicate that the effect of Th1 cells on resolution of lesions induced by L. major in genetically resistant mice also requires a functional Fas-FasL pathway of cytotoxicity. In contrast to resistant mice, susceptible BALB/c mice develop aberrant Th2 responses following infection with L. major and consequently suffer progressive disease. These outcomes clearly depends upon the production of interleukin 4 (IL-4) early after infection. We have shown that a burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hrs after infection, occurs within CD4+ T cells that express V beta 4-V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and Th1 responses occurred following infection. The LACK antigen of L. major induced comparable IL-4 production in V beta 4-V alpha 8 CD4+ T cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4-V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex parasite. The IL-4 produced rapidly by these CD4+ T cells induces within 48 hours a state of unresponsiveness to IL-12 among parasite-specific CD4+ T cell precursors by downregulating the IL-12 receptor beta 2 chain expression.

Less mortality but more relapses in experimental allergic encephalomyelitis in CD8-/- mice.

Mice lacking in CD8 were generated from homologous recombination in embryonal stem cells at the CD8 locus and bred with the experimental allergic encephalomyelitis (EAE)-susceptible PL/JH-2u through four backcross generations to investigate the role of CD8+ T cells in this model of multiple sclerosis. The disease onset and susceptibility were similar to those of wild-type mice. However, the mutant mice had a milder acute EAE, reflected by fewer deaths, but more chronic EAE, reflected by a higher frequency of relapse. This suggests that CD8+ T lymphocytes may participate as both effectors and regulators in this animal model.

IL-4 rapidly produced by V beta 4 V alpha 8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice.

BALB/c mice develop aberrant T helper 2 (Th2) responses and suffer progressive disease after infection with Leishmania major. These outcomes depend on the production of interleukin-4 (IL-4) early after infection. Here we demonstrate that the burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hr after infection, occurs within CD4+ T cells that express V beta 4 V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient BALB/c mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and T helper 1 responses occurred following infection. Recombinant LACK antigen from L. major induced comparable IL-4 production in V beta 4 V alpha 8 CD4+ cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4 V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex organism.

A fetal sheep liver extract containing immunostimulatory substances including LPS acts as leukocyte activator in cells of LPS responder and non responder mice.

Purified fractions from a fetal sheep liver extract (FSLE) were investigated, in a murine model, for induction of leukocyte stimulating activities. The fractions FSLE-1 and FSLE-2 induced splenocyte proliferation in vitro in C57Bl/10ScSn (LPS responder) mice comparable to LPS, and in C57Bl/10ScCr (LPS non responder) mice. They also stimulated the release of nitrogen radicals in bone marrow-derived macrophages (BMDM) from several mouse inbred strains including both C57Bl/10ScSn and C57Bl/10ScCr mice. Stimulation of NO production could be blocked by L-NMMA, an inhibitor of iNOS, and enhanced by the simultaneous addition of IFN-gamma. Moreover, stimulation of macrophages by FSLE-1 and FSLE-2 induced a cytostatic effect of the activated macrophages for Abelson 8-1 tumor cells. The stimulatory activity of the purified fractions is partially due to trace amounts of LPS derived from the fetal liver extract which was enriched during purification. Our results may help to explain the beneficial effect of the extract in patients which has been observed clinically.

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.

A multilevel approach to understanding circadian rhythmicity : social and sleep-dependent effects in ants and mice

Résumé françaisLa majorité des organismes vivants sont soumis à l'alternance du jour et de la nuit, conséquence de la rotation de la terre autour de son axe. Ils ont développé un système interne de mesure du temps, appelé horloge circadienne, leur permettant de s'adapter et de synchroniser leur comportement et leur physiologie aux cycles de lumière. Cette dernière est considérée comme étant le signal majeur entraînant l'horloge interne et. par conséquent, les rythmes journaliers d'éveil et de sommeil. Outre sa régulation circadienne, le sommeil est contrôlé par un processus homéostatique qui détermine son besoin. La contribution de ces deux processus dans le fonctionnement cellulaire du cerveau n'a pas encore été investiguée. La mesure de l'amplitude ainsi que de la prévalence des ondes delta de l'EEG (activité delta) constitue un index très fiable du besoin de sommeil. Il a été démontré que cette activité est génétiquement déterminée et associée à un locus de trait quantitatif situé sur le chromosome 13 de la souris.Grâce à des expériences de privation de sommeil et d'analyses de transcriptome du cerveau dans trois souches de souris présentant diverses réponses à la privation de sommeil, nous avons trouvé que Homerla, localisé dans la région d'intérêt du chromosome 13, est le meilleur marqueur du besoin de sommeil. Homerla est impliqué dans la récupération de l'hyperactivité neuronale induite par le glutamate, grâce à son effet tampon sur le calcium intracellulaire. Une fonction fondamentale du sommeil pourrait donc être de protéger le cerveau et de lui permettre de récupérer après une hyperactivité neuronale imposée par une veille prolongée.De plus, nous avons montré que 2032 transcrits sont exprimés rythmiqueraent dans le cerveau de la souris, parmi lesquels seulement 391 le restent après que les animaux aient été privés de sommeil à différents moments au cours des 24 heures. Cette observation montre clairement que la plupart des changements rythmiques au niveau du transcriptome dépendent du sommeil et non de l'horloge circadienne et souligne ainsi l'importance du sommeil dans la physiologie des mammifères.La plupart des expériences concernant les rythmes circadiens ont été réalisées sur des individus isolés en négligeant l'effet du contexte social sur les comportements circadiens. Les espèces sociales, telles que les fourmis, se caractérisent par une division du travail où une répartition des tâches s'effectue entre ses membres. De plus, certaines d'entre elles doivent être pratiquées en continu comme les soins au couvain tandis que d'autres requièrent une activité rythmique comme le fourragement. Ainsi la fourmi est un excellent modèle pour l'étude de 1 influence du contexte social sur les rythmes circadiens.A ces fins, nous avons décidé d'étudier les rythmes circadiens chez une espèce de fourmi Camponotus fellah et de caractériser au niveau moléculaire son horloge circadienne. Nous avons ainsi développé un système vidéo permettant de suivre l'activité locomotrice de tous les individus d'une colonie. Nos résultats montrent que, bien que la plupart des fourmis soient arythmiques à l'intérieur de la colonie, elles développent d'amples rythmes d'activité en isolation. De plus, ces rythmes disparaissent presque aussitôt que la fourmi est réintroduite dans la colonie. Cette rythmicité observée en isolation semble être générée par l'horloge circadienne car elle persiste en condition constante (obscurité totale). Nous avons ensuite regardé si cette apparente arythmie observée dans la colonie résultait d'un effet masquant des interactions sociales sur les rythmes circadiens d'activité. Nos résultats suggèrent que l'horloge interne est fonctionnelle dans la colonie mais que l'expression de ses rythmes au niveau comportemental est inhibée par les interactions sociales. Les analyses moléculaires du statut de l'horloge dans différents contextes sociaux sont actuellement en cours. Le contexte social semble donc un déterminant majeur du comportement circadien chez la fourmi.AbstractAlmost all living organisms on earth are subjected to the alternance of day and night re-sulting from the rotation of the earth around its axis. They have evolved with an internal timing system, termed the circadian clock, enabling them to adapt and synchronize their behavior and physiology to the daily changes in light and related environmental parame¬ters. Light is thought to be the major cue entraining the circadian clock and consequently the rhythms of rest/activity. In addition to its circadian dependent timing, sleep is reg¬ulated by a homeostatic process that determines its need. The contribution of these two processes in the cellular functioning of the brain has not yet been considered. A highly reliable index of the homeostatic process of sleep is the measure of the amplitude and prevalence of the EEG delta waves (delta activity). It has been shown that sleep need, measured by delta activity, is genetically determined and associated with a Quantitative Trait Locus (QTL) located on the mouse chromosome 13. By using sleep deprivation and brain transcriptome profiling in three inbred mouse strains showing different responses to sleep loss, we found that Homerla, localized within this QTL region is the best transcrip¬tional marker of sleep need. Interestingly Homerla is primarily involved in the recovery from glutamate-induced neuronal hyperactivity by its buffering effect on intracellular cal¬cium. A fundamental function of sleep may therefore reside in the protection and recovery of the brain from a neuronal hyperactivity imposed by prolonged wakefulness.Moreover, time course gene expression experiments showed that 2032 brain tran¬scripts present a rhythmic variation, but only 391 of those remain rhythmic when mice are sleep deprived at four time points around the clock. This finding clearly suggests that most changes in gene transcription over the day are sleep-wake dependent rather than clock dependent and underlines the importance of sleep in mammalian physiology.In the second part of this PhD, I was interested in the social influence on circadian behavior. Most experiments done in the circadian field have been performed on isolated individuals and have therefore ignored the effect of the social context on circadian behav-ior. Eusocial insect species such as ants are characterized by a division of labor: colony tasks are distributed among individuals, some of them requiring continuous activity such as nursing or rhythmic ones such as foraging. Thus ants represent a suitable model to study the influence of the social context on the circadian clock and its output rhythms.The aim of this part was to address the effect of social context on circadian rhythms in the ant species Camponotus fellah and to characterize its circadian clock at the molecu¬lar level. We therefore developed a video tracking system to follow the locomotor activity of all individuals in a colony. Our results show that most ants are arrhythmic within the colony, but develop, when subjected to social isolation, strong rhythms of activity that intriguingly disappear when individuals are reintroduced into the colony. The rhythmicity observed in isolated ants seems to be driven by the circadian clock as it persists under constant conditions (complete darkness). We then tested whether the apparent arrhyth- micity in the colony stemmed from a masking effect of social interactions on circadian rhythms. Indeed, we found that circadian clocks of ants in the colony are functional but their expression at the behavioral level is inhibited by social interactions. The molecular assessment of the circadian clock functional state in the different social context is still under investigation. Our results suggest that social context is a major determinant of circadian behavior in ants.