Androgens show sex-dependent differences in myelination in immune and non-immune murine models of CNS demyelination

Abstract Neuroprotective, anti-inflammatory, and remyelinating properties of androgens are well-characterized in demyelinated male mice and men suffering from multiple sclerosis. However, androgen effects mediated by the androgen receptor (AR), have been only poorly studied in females who make low androgen levels. Here, we show a predominant microglial AR expression in demyelinated lesions from female mice and women with multiple sclerosis, but virtually undetectable AR expression in lesions from male animals and men with multiple sclerosis. In female mice, androgens and estrogens act in a synergistic way while androgens drive microglia response towards regeneration. Transcriptomic comparisons of demyelinated mouse spinal cords indicate that, regardless of the sex, androgens up-regulate genes related to neuronal function integrity and myelin production. Depending on the sex, androgens down-regulate genes related to the immune system in females and lipid catabolism in males. Thus, androgens are required for proper myelin regeneration in females and therapeutic approaches of demyelinating diseases need to consider male-female differences

Hypercholesterolemia Negatively Regulates P2X7-Induced Cellular Function in CD4+ and CD8+ T-Cell Subsets from B6 Mice Fed a High-Fat Diet

We have previously showed that plasma membrane cholesterol and GM1 ganglioside content are responsible for the opposite sensitivity of mouse leukemic T cells to ATP. We also reported that the sensitivity of CD4+ and CD8+ T cells to ATP depends on their stage of differentiation. Here, we show that CD4+ and CD8+ T cells from B6 mice express different levels of membrane GM1 and P2X7 but similar levels of cholesterol. Thus, in CD4+ T cells, membrane cholesterol content negatively correlated with ATP/P2X7-induced CD62L shedding but positively correlated with pore formation, phosphatidylserine externalization, and cell death. By contrast, in CD8+ T cells, cholesterol, GM1, and P2X7 levels negatively correlated with all these ATP/P2X7-induced cellular responses. The relationship between cholesterol and P2X7-induced cellular responses was confirmed by modulating cholesterol levels either ex vivo or through a high-fat diet. Membrane cholesterol enrichment ex vivo led to a significant reduction in all P2X7-induced cellular responses in T cells. Importantly, diet-induced hypercholesterolemia in B6 mice was also associated with decreased sensitivity to ATP in CD4+ and CD8+ T cells, highlighting the relationship between cholesterol intake and the amplitudes of P2X7-induced cellular responses in T cells

Impact du cholestérol circulant et membranaire sur les fonctions cellulaires induites par P2X7 et la sévérité de pathologies auto-immunes de type lupique

During my thesis, I focused on the influence of membrane dynamics on the functionality of P2X7 in T lymphocytes in relation to the differences in SLE development in MRL/lpr and B6/lpr lupus mice. Thus, I hypothesized that the circulating cholesterol level could influence the organization of membrane signalling microdomains of normal and pathological T cell and be responsible for the differences in sensitivity of the P2X7 receptor to ATP4-. I therefore researched, in vitro and in vivo, the existence of links between the regulation of cholesterol metabolism and the functionality of the P2X7 receptor according to activation and differentiation state of T lymphocytes. I developed techniques for modulating and quantifying membrane cholesterol levels as well as protocols for determining the location of the P2X7 receptor within membrane domains. I was thus able to demonstrate, in vitro, on tumor T lymphocyte lines, the influence of the membrane cholesterol level, basal or modulated, on the cellular functions induced by the P2X7 receptor. I was also able to demonstrate, ex vivo, a variation in membrane cholesterol levels depending on activation and differentiation state of normal or pathological T lymphocytes and that these differences impact the functionality of P2X7. In addition, I have shown in normal B6 mice subjected to a cholesterol-enriched diet that the membrane cholesterol level of T lymphocytes and especially their sensitivity to ATP4- is dependent on the circulating cholesterol level. Finally, cholesterol lowering treatments as well as cholesterol enriched diet are currently being performed to assess the impact of circulating cholesterol levels on the severity of autoimmune pathologies in MRL/lpr and B6/lpr mice.Au cours de ma thèse, je me suis intéressé à l'influence de la dynamique membranaire sur la fonctionnalité de P2X7 dans les lymphocytes T en lien avec les différences de développement du LED chez les souris lupiques MRL/lpr et B6/lpr. Ainsi, j'ai émis l'hypothèse que le taux de cholestérol circulant pourrait influencer l'organisation des microdomaines membranaires de signalisation des lymphocytes T normaux et pathologiques et être responsable des différences de sensibilité du récepteur P2X7 à l'ATP4-. J'ai donc recherché, in vitro et in vivo, l'existence de liens entre la régulation du métabolisme du cholestérol et la fonctionnalité du récepteur P2X7 selon l'état d'activation et de différenciation des lymphocytes T. Pour ce faire, j'ai mis au point les techniques de modulation et de quantification des taux de cholestérol membranaire ainsi que les protocoles permettant de déterminer la localisation du récepteur P2X7 au sein des domaines membranaires. J'ai ainsi pu démontrer, in vitro, sur des lignées lymphocytaires T tumorales, l'influence du taux de cholestérol membranaire, basal ou modulé, sur les fonctions cellulaires induites par le récepteur P2X7. J'ai également pu mettre en évidence, ex vivo, une variation des taux de cholestérol membranaire selon l'état d'activation et de différenciation des lymphocytes T normaux ou pathologiques et que ces différences impactent la fonctionnalité de P2X7. De plus, j'ai montré chez des souris normales B6 soumises à un régime enrichi en cholestérol que le taux de cholestérol membranaire des lymphocytes T et surtout leur sensibilité à l'ATP4- est dépendante du taux de cholestérol circulant. Enfin, des traitements hypo et hyper cholestérolémiants sont actuellement réalisés pour évaluer l'impact du taux de cholestérol circulant sur la sévérité des pathologies auto-immunes des souris MRL/lpr et B6/lpr

Impact of circulating and plasma membrane cholesterol on P2X7 induced cellular responses and lupus severity

Au cours de ma thèse, je me suis intéressé à l'influence de la dynamique membranaire sur la fonctionnalité de P2X7 dans les lymphocytes T en lien avec les différences de développement du LED chez les souris lupiques MRL/lpr et B6/lpr. Ainsi, j'ai émis l'hypothèse que le taux de cholestérol circulant pourrait influencer l'organisation des microdomaines membranaires de signalisation des lymphocytes T normaux et pathologiques et être responsable des différences de sensibilité du récepteur P2X7 à l'ATP4-. J'ai donc recherché, in vitro et in vivo, l'existence de liens entre la régulation du métabolisme du cholestérol et la fonctionnalité du récepteur P2X7 selon l'état d'activation et de différenciation des lymphocytes T. Pour ce faire, j'ai mis au point les techniques de modulation et de quantification des taux de cholestérol membranaire ainsi que les protocoles permettant de déterminer la localisation du récepteur P2X7 au sein des domaines membranaires. J'ai ainsi pu démontrer, in vitro, sur des lignées lymphocytaires T tumorales, l'influence du taux de cholestérol membranaire, basal ou modulé, sur les fonctions cellulaires induites par le récepteur P2X7. J'ai également pu mettre en évidence, ex vivo, une variation des taux de cholestérol membranaire selon l'état d'activation et de différenciation des lymphocytes T normaux ou pathologiques et que ces différences impactent la fonctionnalité de P2X7. De plus, j'ai montré chez des souris normales B6 soumises à un régime enrichi en cholestérol que le taux de cholestérol membranaire des lymphocytes T et surtout leur sensibilité à l'ATP4- est dépendante du taux de cholestérol circulant. Enfin, des traitements hypo et hyper cholestérolémiants sont actuellement réalisés pour évaluer l'impact du taux de cholestérol circulant sur la sévérité des pathologies auto-immunes des souris MRL/lpr et B6/lpr.During my thesis, I focused on the influence of membrane dynamics on the functionality of P2X7 in T lymphocytes in relation to the differences in SLE development in MRL/lpr and B6/lpr lupus mice. Thus, I hypothesized that the circulating cholesterol level could influence the organization of membrane signalling microdomains of normal and pathological T cell and be responsible for the differences in sensitivity of the P2X7 receptor to ATP4-. I therefore researched, in vitro and in vivo, the existence of links between the regulation of cholesterol metabolism and the functionality of the P2X7 receptor according to activation and differentiation state of T lymphocytes. I developed techniques for modulating and quantifying membrane cholesterol levels as well as protocols for determining the location of the P2X7 receptor within membrane domains. I was thus able to demonstrate, in vitro, on tumor T lymphocyte lines, the influence of the membrane cholesterol level, basal or modulated, on the cellular functions induced by the P2X7 receptor. I was also able to demonstrate, ex vivo, a variation in membrane cholesterol levels depending on activation and differentiation state of normal or pathological T lymphocytes and that these differences impact the functionality of P2X7. In addition, I have shown in normal B6 mice subjected to a cholesterol-enriched diet that the membrane cholesterol level of T lymphocytes and especially their sensitivity to ATP4- is dependent on the circulating cholesterol level. Finally, cholesterol lowering treatments as well as cholesterol enriched diet are currently being performed to assess the impact of circulating cholesterol levels on the severity of autoimmune pathologies in MRL/lpr and B6/lpr mice

Hypercholesterolemia Negatively Regulates P2X7-Induced Cellular Function in CD4⁺ and CD8⁺ T-Cell Subsets from B6 Mice Fed a High-Fat Diet

We have previously showed that plasma membrane cholesterol and GM1 ganglioside content are responsible for the opposite sensitivity of mouse leukemic T cells to ATP. We also reported that the sensitivity of CD4+ and CD8+ T cells to ATP depends on their stage of differentiation. Here, we show that CD4+ and CD8+ T cells from B6 mice express different levels of membrane GM1 and P2X7 but similar levels of cholesterol. Thus, in CD4+ T cells, membrane cholesterol content negatively correlated with ATP/P2X7-induced CD62L shedding but positively correlated with pore formation, phosphatidylserine externalization, and cell death. By contrast, in CD8+ T cells, cholesterol, GM1, and P2X7 levels negatively correlated with all these ATP/P2X7-induced cellular responses. The relationship between cholesterol and P2X7-induced cellular responses was confirmed by modulating cholesterol levels either ex vivo or through a high-fat diet. Membrane cholesterol enrichment ex vivo led to a significant reduction in all P2X7-induced cellular responses in T cells. Importantly, diet-induced hypercholesterolemia in B6 mice was also associated with decreased sensitivity to ATP in CD4+ and CD8+ T cells, highlighting the relationship between cholesterol intake and the amplitudes of P2X7-induced cellular responses in T cells

The Smoothened agonist SAG Modulates the Male and Female Peripheral Immune Systems Differently in an Immune Model of Central Nervous System Demyelination

Both Hedgehog and androgen signaling pathways are known to promote myelin regeneration in the central nervous system. Remarkably, the combined administration of agonists of each pathway revealed their functional cooperation towards higher regeneration in demyelination models in males. Since multiple sclerosis, the most common demyelinating disease, predominates in women, and androgen effects were reported to diverge according to sex, it seemed essential to assess the existence of such cooperation in females. Here, we developed an intranasal formulation containing the Hedgehog signaling agonist SAG, either alone or in combination with testosterone. We show that SAG promotes myelin regeneration and presumably a pro-regenerative phenotype of microglia, thus mimicking the effects previously observed in males. However, unlike in males, the combined molecules failed to cooperate in the demyelinated females, as shown by the level of functional improvement observed. Consistent with this observation, SAG administered in the absence of testosterone amplified peripheral inflammation by presumably activating NK cells and thus counteracting a testosterone-induced reduction in Th17 cells when the molecules were combined. Altogether, the data uncover a sex-dependent effect of the Hedgehog signaling agonist SAG on the peripheral innate immune system that conditions its ability to cooperate or not with androgens in the context of demyelination

Coopération fonctionnelle des voies de signalisation Hedgehog et des androgènes au cours de la myélinisation développementale et réparatrice

International audienceAbstract Hedgehog morphogens control fundamental cellular processes during tissue development and regeneration. In the central nervous system (CNS), Hedgehog signaling has been implicated in oligodendrocyte and myelin production, where it functions in a concerted manner with other pathways. Since androgen receptor (AR) plays a key role in establishing the sexual phenotype of myelin during development and is required for spontaneous myelin regeneration in the adult CNS, we hypothesized the existence of a possible coordination between Hedgehog and androgen signals in oligodendrocyte and myelin production. Here, we report complementary activities of both pathways during early postnatal oligodendrogenesis further revealing that persistent Hedgehog signaling activation impedes myelin production. The data also uncover prominent pro‐myelinating activity of testosterone and involvement of AR in the control of neural stem cell commitment toward the oligodendroglial lineage. In the context of CNS demyelination, we provide evidence for the functional cooperation of the pathways leading to acceleration of myelin regeneration that might be related to their respective role on microglial and astroglial responses, higher preservation of axonal integrity, lower neuroinflammation, and functional improvement of animals in an immune model of CNS demyelination. Strong decreases of deleterious cytokines in the CNS (GM‐CSF, TNF‐α, IL‐17A) and spleen (IL‐2, IFN‐γ) stand as unique features of the combined drugs while the potent therapeutic activity of testosterone on peripheral immune cells contributes to increase tolerogenic CD11c + dendritic cells, reduce the clonal expansion of conventional CD4 + T cells and increase CD4 + Foxp3 + regulatory T cells. Altogether, these data might open promising perspectives for demyelinating diseases

DataSheet_1_P2X7 purinergic receptor plays a critical role in maintaining T-cell homeostasis and preventing lupus pathogenesis.docx

The severe lymphoproliferative and lupus diseases developed by MRL/lpr mice depend on interactions between the Faslpr mutation and MRL genetic background. Thus, the Faslpr mutation causes limited disease in C57BL/6 mice. We previously found that accumulating B220+ CD4–CD8– double negative (DN) T cells in MRL/lpr mice show defective P2X7 receptor ( P2X7)-induced cellular functions, suggesting that P2X7 contributes to T-cell homeostasis, along with Fas. Therefore, we generated a B6/lpr mouse strain (called B6/lpr-p2x7KO) carrying homozygous P2X7 knockout alleles. B6/lpr-p2x7KO mice accumulated high numbers of FasL-expressing B220+ DN T cells of CD45RBhighCD44high effector/memory CD8+ T-cell origin and developed severe lupus, characterized by leukocyte infiltration into the tissues, high levels of IgG anti-dsDNA and rheumatoid factor autoantibodies, and marked cytokine network dysregulation. B6/lpr-p2x7KO mice also exhibited a considerably reduced lifespan. P2X7 is therefore a novel regulator of T-cell homeostasis, of which cooperation with Fas is critical to prevent lymphoaccumulation and autoimmunity.</p