Acute invariant NKT cell activation triggers an immune response that drives prominent changes in iron homeostasis

Iron homeostasis is an essential biological process that ensures the tissue distribution of iron for various cellular processes. As the major producer of hepcidin, the liver is central to the regulation of iron metabolism. The liver is also home to many immune cells, which upon activation may greatly impact iron metabolism. Here, we focus on the role of invariant natural killer T (iNKT) cells, a subset of T lymphocytes that, in mice, is most abundant in the liver. Activation of iNKT cells with the prototypical glycosphingolipid antigen, α-galactosylceramide, resulted in immune cell proliferation and biphasic changes in iron metabolism. This involved an early phase characterized by hypoferremia, hepcidin induction and ferroportin suppression, and a second phase associated with strong suppression of hepcidin despite elevated levels of circulating and tissue iron. We further show that these changes in iron metabolism are fully dependent on iNKT cell activation. Finally, we demonstrate that the biphasic regulation of hepcidin is independent of NK and Kupfer cells, and is initially driven by the STAT3 infammatory pathway, whereas the second phase is regulated by repression of the BMP/SMAD signaling pathway. These fndings indicate that iNKT activation and the resulting cell proliferation infuence iron homeostasis

Acute invariant NKT cell activation triggers an immune response that drives prominent changes in iron homeostasis

© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen ses/by/4.0/.Iron homeostasis is an essential biological process that ensures the tissue distribution of iron for various cellular processes. As the major producer of hepcidin, the liver is central to the regulation of iron metabolism. The liver is also home to many immune cells, which upon activation may greatly impact iron metabolism. Here, we focus on the role of invariant natural killer T (iNKT) cells, a subset of T lymphocytes that, in mice, is most abundant in the liver. Activation of iNKT cells with the prototypical glycosphingolipid antigen, α-galactosylceramide, resulted in immune cell proliferation and biphasic changes in iron metabolism. This involved an early phase characterized by hypoferremia, hepcidin induction and ferroportin suppression, and a second phase associated with strong suppression of hepcidin despite elevated levels of circulating and tissue iron. We further show that these changes in iron metabolism are fully dependent on iNKT cell activation. Finally, we demonstrate that the biphasic regulation of hepcidin is independent of NK and Kupffer cells, and is initially driven by the STAT3 inflammatory pathway, whereas the second phase is regulated by repression of the BMP/SMAD signaling pathway. These findings indicate that iNKT activation and the resulting cell proliferation influence iron homeostasis.This work was supported by grants from the Canadian Institutes of Health Research (CIHR, Grant no. PJT-159775) and Natural Sciences and Engineering Research Council of Canada (NSERC, Grant RGPIN-2018-06442) to MMS. HH received a PhD scholarship from the NSERC. SL is a Research Scholars Emeritus awardee from the FRQS.info:eu-repo/semantics/publishedVersio

High-throughput refractive index-based microphotonic sensor for enhanced cellular discrimination

This paper presents a novel microphotonic sensor based on silicon technologies for high-throughput single cell measurements. It employs a highly sensitive Fabry-Pérot resonant cavity to extract cellular refractive index information. The integrated large cross-section rib waveguides provide a single-mode like behavior important for resonant cell sensing. Differentiated myeloid cells derived from a promyelocytic leukemia cell line were injected in a microchannel, sheathlessly focused using inertial forces and analyzed while flowing through the resonant cavity volume. Results were compared against a commercial flow cytometer and showed a substantial improvement on cellular discrimination. Thus, this sensor has the ability to discriminate cell populations, usually identified using fluorescent parameters, without any dyes and can reach measurement rate as high as 2000 cells per second. By harnessing the cell's effective volume refractive index, our device offers complementary measurements readily improving actual technologies and thus providing crucial information for research and clinical professionals

Three-dimensional assessment of oogenesis dynamic aspects and its regulation through miR-202 in the medaka Oryzias latipes

L'ovaire est caractérisé par des processus de croissance, de stockage et de recrutement des ovocytes entraînant d'importants réarrangements cellulaires au cours du cycle de vie de la femelle. Chez le médaka Oryzias latipes, comme chez d'autres poissons téléostéens, ces mécanismes d'ovogenèse sont mal compris d'un point de vue quantitatif et restent souvent extrapolés à partir de fractions de l'organe entier. Par conséquent, la connaissance de la dynamique des populations d'ovocytes dans l'ovaire est limitée, alors qu'elle est essentielle pour comprendre la régulation de la fertilité, notamment chez les espèces à intervalles de ponte courts comme le médaka. Pour y remédier, nous avons développé une méthode d'analyse des ovaires entiers qui est exhaustive, accessible et également transférable à d'autres espèces. En combinant la transparisation des tissus, la microscopie confocale 3D et l'analyse d'images assistée par des algorithmes d'apprentissage profond, notre méthode a permis d'extraire des données quantitatives et morphologiques à différents stades de développement. L'analyse de l'évolution du contenu ovarien au cours du temps a révélé l'établissement de différentes réserves folliculaires au cours de la croissance/maturation de la femelle, indiquant la présence de deux recrutements majeurs qui alimentent progressivement une distribution folliculaire asynchrone chez l'adulte reproducteur. L'étude de la lignée miR-202-KO, dont la fertilité est réduite, a mis en évidence l'importance du maintien de la distribution asynchrone des follicules pour l'établissement d'un taux de fertilité normal. Les dérégulations des profils de distribution des follicules observées chez les femelles miR-202-KO sont cohérentes avec le rôle de miR- 202 dans la régulation du recrutement des follicules au milieu et à la fin de la croissance primaire. Grâce au développement de méthodes innovantes, cette étude a ainsi permis de mieux comprendre la dynamique ovarienne chez les poissons, ainsi que la manière dont miR-202 est impliqué dans sa régulation.The ovary is characterized by processes of oocyte growth, storage and recruitment, thus resulting in important cellular rearrangements during the female life cycle. In medaka Oryzias latipes, as in other teleost fish, these mechanisms of oogenesis are poorly understood from a quantitative point of view and often remain extrapolated from fractions of the whole organ. As a consequence, knowledge of oocyte population dynamics in the ovary is limited, although this is essential for understanding the regulation of fertility, most notably in species with short spawning intervals such as medaka. To address this, we have developed a method for analyzing whole ovaries that is comprehensive, accessible and also transferable to other species. By combining tissue clearing, 3D confocal microscopy and image analysis assisted by deep learning algorithms, our method allowed the extraction of quantitative and morphological data at different stages of development. The analysis of ovarian content changes over time revealed the establishment of different follicular reserves during female growth/maturation, indicating the presence of two major recruitments that progressively supply asynchronous follicular distribution in the reproducing adult. The study of the miR-202-KO line with reduced fertility has highlighted the importance of maintaining the asynchronous distribution of follicles for the establishment of a normal fertility rate. The deregulations of follicle distribution patterns observed in miR-202-KO females were consistent with a role for miR-202 in regulating follicle recruitment during mid and late primary growth. Through the development of innovative methods, this study provided a new understanding of ovarian dynamics in fish, as well as a better comprehension of how miR-202 is involved in its regulation

Exploration tridimensionnelle des aspects dynamiques de l’ovogenèse et de sa régulation par le miR-202 chez le médaka Oryzias latipes

The ovary is characterized by processes of oocyte growth, storage and recruitment, thus resulting in important cellular rearrangements during the female life cycle. In medaka Oryzias latipes, as in other teleost fish, these mechanisms of oogenesis are poorly understood from a quantitative point of view and often remain extrapolated from fractions of the whole organ. As a consequence, knowledge of oocyte population dynamics in the ovary is limited, although this is essential for understanding the regulation of fertility, most notably in species with short spawning intervals such as medaka. To address this, we have developed a method for analyzing whole ovaries that is comprehensive, accessible and also transferable to other species. By combining tissue clearing, 3D confocal microscopy and image analysis assisted by deep learning algorithms, our method allowed the extraction of quantitative and morphological data at different stages of development. The analysis of ovarian content changes over time revealed the establishment of different follicular reserves during female growth/maturation, indicating the presence of two major recruitments that progressively supply asynchronous follicular distribution in the reproducing adult. The study of the miR-202-KO line with reduced fertility has highlighted the importance of maintaining the asynchronous distribution of follicles for the establishment of a normal fertility rate. The deregulations of follicle distribution patterns observed in miR-202-KO females were consistent with a role for miR-202 in regulating follicle recruitment during mid and late primary growth. Through the development of innovative methods, this study provided a new understanding of ovarian dynamics in fish, as well as a better comprehension of how miR-202 is involved in its regulation.L'ovaire est caractérisé par des processus de croissance, de stockage et de recrutement des ovocytes entraînant d'importants réarrangements cellulaires au cours du cycle de vie de la femelle. Chez le médaka Oryzias latipes, comme chez d'autres poissons téléostéens, ces mécanismes d'ovogenèse sont mal compris d'un point de vue quantitatif et restent souvent extrapolés à partir de fractions de l'organe entier. Par conséquent, la connaissance de la dynamique des populations d'ovocytes dans l'ovaire est limitée, alors qu'elle est essentielle pour comprendre la régulation de la fertilité, notamment chez les espèces à intervalles de ponte courts comme le médaka. Pour y remédier, nous avons développé une méthode d'analyse des ovaires entiers qui est exhaustive, accessible et également transférable à d'autres espèces. En combinant la transparisation des tissus, la microscopie confocale 3D et l'analyse d'images assistée par des algorithmes d'apprentissage profond, notre méthode a permis d'extraire des données quantitatives et morphologiques à différents stades de développement. L'analyse de l'évolution du contenu ovarien au cours du temps a révélé l'établissement de différentes réserves folliculaires au cours de la croissance/maturation de la femelle, indiquant la présence de deux recrutements majeurs qui alimentent progressivement une distribution folliculaire asynchrone chez l'adulte reproducteur. L'étude de la lignée miR-202-KO, dont la fertilité est réduite, a mis en évidence l'importance du maintien de la distribution asynchrone des follicules pour l'établissement d'un taux de fertilité normal. Les dérégulations des profils de distribution des follicules observées chez les femelles miR-202-KO sont cohérentes avec le rôle de miR- 202 dans la régulation du recrutement des follicules au milieu et à la fin de la croissance primaire. Grâce au développement de méthodes innovantes, cette étude a ainsi permis de mieux comprendre la dynamique ovarienne chez les poissons, ainsi que la manière dont miR-202 est impliqué dans sa régulation

An end-to-end pipeline based on open source deep learning tools for reliable analysis of complex 3D images of Medaka ovaries

Computational analysis of bio-images by deep learning (DL) algorithms has made exceptional progress in recent years and has become much more accessible to non specialists with the development of ready-to-use tools. The study of oogenesis mechanisms and female reproductive success in fish has also recently benefited from the development of efficient three-dimensional (3D) imaging protocols on entire ovaries. Such large datasets have a great potential for the generation of new quantitative data on oogenesis but are, however, complex to analyze due to imperfect fluorescent signals and the lack of efficient image analysis workflows. Here, we applied two open-source DL tools, Noise2Void and Cellpose, to analyze the oocyte content of medaka ovaries at larvae and adult stages. These tools were integrated into end-to-end analysis pipelines that include image pre-processing, cell segmentation, and image post-processing to filter and combine labels. Our pipelines thus provide effective solutions to accurately segment complex 3D images of entire ovaries with either irregular fluorescent staining or low autofluorescence signal. In the future, these pipelines will be applicable to extensive cellular phenotyping in fish for developmental or toxicology studies

C-Eci: A Cubic-Eci combined clearing method for 3D follicular content analysis In the ovary

Deciphering mechanisms of oocyte development in female fishes still remains challenging and a comprehensive overview of this process at the level of the organ is still needed. The recent development optical tissue clearing methods have tremendously boosted the 3D imaging of large size biological samples that are naturally opaque. However, no attempt of clearing on fish ovary that accumulates extremely high concentration of lipids within oocytes has been reported to date. To face with this ovarian-specific challenge, we combined two existing clearing methods, the non-toxic solvent-based Eci method for efficient clearing and the CUBIC method to enhance lipid removal and reduce non-specific staining. The methyl green fluorescent dye was used to stain nuclei and delineate follicles. Using this procedure (named C-Eci), ovaries of both medaka and trout could be imaged in 3D and all follicles analyzed. To our knowledge this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-Eci methods thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further morphological studies

A comprehensive 3D approach to unravel follicular growth dynamics that governs fecundity in medaka fish (Oryzias latipes)

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