Plasticité fonctionnelle des cellules « Natural Killer » déciduales dans les conditions physiologiques et infectieuses

Au début de la grossesse, des cellules Natural Killer (NK) sont massivement recrutées au niveau de l'utérus et constituent la population immunitaire maternelle majoritaire au niveau du site d'implantation embryonnaire, la decidua basalis. Les cellules NK déciduales (dNK) présentent des propriétés uniques par rapport aux autres populations de cellules NK. Leur fonction principale est l'aide à l'implantation embryonnaire et au bon déroulement de la grossesse. Les fonctions des cellules NK sont contrôlées par les nombreux récepteurs activateurs et inhibiteurs qu'elles expriment. Les récepteurs de cytotoxicité naturels (NCR) NKp30, NKp44 et NKp46 sont exprimés aussi bien par les cellules dNK que les cellules NK du sang périphérique (pNK). Toutefois, leur fonction diffère entre les deux populations. Ainsi, bien que les cellules NK soient des cellules cytotoxiques, ce potentiel est parfaitement contrôlé dans la decidua, afin d'éviter des dommages aux cellules fœtales semi-allogéniques. Pourtant le fœtus est susceptible d'être infecté in utero par des pathogènes de la mère. Durant la première partie de ma thèse, nous avons analysé l'implication des cellules dNK dans la protection du fœtus contre les infections congénitales par le cytomégalovirus humain (HCMV). Nous avons mis au point un nouveau modèle d'infection de cellules stromales de decidua in vitro, afin d'étudier les mécanismes mis en jeu lors d'une réponse immune protectrice dans l'utérus gestant. Nous avons montré que les cellules dNK forment des synapses immunologiques activatrices avec des cellules cibles autologues infectées par le HCMV. Elles acquièrent un potentiel cytotoxique et sont capables de lyser les cibles infectées après engagement de récepteurs activateurs, notamment NKG2D et NKG2C. En réponse aux cellules infectées par le HCMV, les cellules dNK modifient leur profil de sécrétion de cytokines et de chimiokines. Cette première partie nous a permis de suggérer que les cellules dNK sont capables d'apporter une protection au fœtus contre les transmissions verticales de certains pathogènes. Durant la second partie de ma thèse, je me suis intéressé aux mécanismes moléculaires responsables des différences fonctionnelles entre cellules dNK et cellules pNK, notamment en ce qui concerne les NCR. Les gènes qui codent les trois NCR subissent un épissage alternatif qui donne naissance à plusieurs variants. Les protéines issues de cet épissage sont très proches en termes de séquence mais elles présentent des différences fonctionnelles. Nous avons ainsi pu montrer que les cellules dNK expriment différemment les transcrits alternatifs de NKp30 et NKp44 par rapport aux cellules pNK. Nous avons également pu démontrer que l'épissage " sélectif " des transcrits des NCR est sous l'influence de l'environnement cytokinique particulier de la decidua basalis. En effet, des cellules pNK cultivées dans des cocktails de cytokines définis modifient le profil d'épissage des ARNm de NKp30 et NKp44. L'environnement de la grossesse pourrait donc influer sur l'expression et la fonction des récepteurs des cellules NK, pour les adapter à leur rôle dans le bon déroulement de la grossesse.During the first trimester of pregnancy, Natural Killer cells massively infiltrate the pregnant uterus and constitute the principal immune population at the fetal implantation site, called decidua basalis. Decidual NK cells (dNK) are a unique NK cell population. They play an important role in the establishment and maintenance of pregnancy mainly through vascular remodeling of the uterine arteries. dNK cells establish privileged contacts with fetal-derived trophoblasts via NK cell receptor engagement. NK cell effector functions are tightly controlled by a large panel of activating and inhibitory receptors. Natural cytotoxicity receptors (NCRs) NKp30, NKp44 and NKp46 are expressed by dNK cells and pNK cells, but their functions are quite different between these two NK cell populations. NK cells are robust cytotoxic effectors of the immune system. However, the cytotoxic potential of dNK cells is perfectly controlled to ensure protection of semi-allogenic fetal cells. Pathogens such as human Cytomegalovirus (HCMV) can be transmitted to the fetus across materno-fetal interfaces. In the first part of my PhD thesis, we investigated the role of dNK cells against the development of HCMV congenital infections. We developed a new in vitro autologous culture system of decidual stromal cells and dNK cells, to dissect the protective immune response in pregnant uterus. We showed that dNK cells can form activating immunological synapse with autologous HCMV-infected target cells. Engagement of NKG2D and NKG2C activating receptors trigger dNK cell cytotoxic functions against HCMV-infected cells. The dNK cell cytokine/chemokine secretion profile was modified after co-culture with HCMV-infected targets. All together, our data suggest that dNK cells can protect the fetus from vertically transmitted pathogens during pregnancy. During the second part of my PhD thesis, we dissected molecular mechanisms that orchestrate the functional differences between dNK cells and pNK cells, notably NCRs functions. NCRs genes are transcribed in multiples mRNA splice variants that can be translated to different functional proteins. Although there is a high sequence homology, the resulting proteins are endowed with huge functional differences. We demonstrated that dNK cells differentially express NKp30 and NKp44 transcripts compared to pNK cells. To clarify the role of the microenvironment in the "selective" splicing of NCR transcripts, we used a defined cytokine cocktail that characterize the decidual milieu. We showed that pNK cells are able to adjust their alternative splicing for NKp30 and NKp44 mRNAs. Our data suggest that the pregnant uterus micro-environnement can influence NK cell receptor expression and function, to render them able to support pregnancy

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

The up side of decidual natural killer cells: new developments in immunology of pregnancy

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Gene silencing by EZH2 suppresses TGF-β activity within the decidua to avert pregnancy-adverse wound healing at the maternal-fetal interface.

A little-appreciated feature of early pregnancy is that embryo implantation and placental outgrowth do not evoke wound-healing responses in the decidua, the specialized endometrial tissue that surrounds the conceptus. Here, we provide evidence that this phenomenon is partly due to an active program of gene silencing mediated by EZH2, a histone methyltransferase that generates repressive histone 3 lysine 27 trimethyl (H3K27me3) histone marks. We find that pregnancies in mice with EZH2-deficient decidual stromal cells frequently fail by mid-gestation, with the decidua showing ectopic myofibroblast formation, peri-embryonic collagen deposition, and gene expression profiles associated with transforming growth factor β (TGF-β)-driven fibroblast activation and fibrogenic extracellular matrix (ECM) remodeling. Analogous responses are observed when the mutant decidua is surgically wounded, while blockade of TGF-β receptor signaling inhibits the defects and improves reproductive outcomes. Together, these results highlight a critical feature of reproductive success and have implications for the context-specific control of TGF-β-mediated wound-healing responses elsewhere in the body

Circumvention of luteolysis reveals parturition pathways in mice dependent upon innate type 2 immunity.

Although mice normally enter labor when their ovaries stop producing progesterone (luteolysis), parturition can also be triggered in this species through uterus-intrinsic pathways potentially analogous to the ones that trigger parturition in humans. Such pathways, however, remain largely undefined in both species. Here, we report that mice deficient in innate type 2 immunity experienced profound parturition delays when manipulated endocrinologically to circumvent luteolysis, thus obliging them to enter labor through uterus-intrinsic pathways. We found that these pathways were in part driven by the alarmin IL-33 produced by uterine interstitial fibroblasts. We also implicated important roles for uterine group 2 innate lymphoid cells, which demonstrated IL-33-dependent activation prior to labor onset, and eosinophils, which displayed evidence of elevated turnover in the prepartum uterus. These findings reveal a role for innate type 2 immunity in controlling the timing of labor onset through a cascade potentially relevant to human parturition

Human Cytomegalovirus Infection Elicits New Decidual Natural Killer Cell Effector Functions

International audienceDuring the first trimester of pregnancy the uterus is massively infiltrated by decidual natural killer cells (dNK). These cells are not killers, but they rather provide a microenvironment that is propitious to healthy placentation. Human cytomegalovirus (HCMV) is the most common cause of intrauterine viral infections and a known cause of severe birth defects or fetal death. The rate of HCMV congenital infection is often low in the first trimester of pregnancy. The mechanisms controlling HCMV spreading during pregnancy are not yet fully revealed, but evidence indicating that the innate immune system plays a role in controlling HCMV infection in healthy adults exists. In this study, we investigated whether dNK cells could be involved in controlling viral spreading and in protecting the fetus against congenital HCMV infection. We found that freshly isolated dNK cells acquire major functional and phenotypic changes when they are exposed to HCMV-infected decidual autologous fibroblasts. Functional studies revealed that dNK cells, which are mainly cytokines and chemokines producers during normal pregnancy, become cytotoxic effectors upon their exposure to HCMV-infected autologous decidual fibroblasts. Both the NKG2D and the CD94/NKG2C or 2E activating receptors are involved in the acquired cytotoxic function. Moreover, we demonstrate that CD56 pos dNK cells are able to infiltrate HCMV-infected trophoblast organ culture ex-vivo and to co-localize with infected cells in situ in HCMV-infected placenta. Taken together, our results present the first evidence suggesting the involvement of dNK cells in controlling HCMV intrauterine infection and provide insights into the mechanisms through which these cells may operate to limit the spreading of viral infection to fetal tissues

Natural cytotoxicity receptor splice variants orchestrate the distinct functions of human natural killer cell subtypes

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H3K27me3 dynamics dictate evolving uterine states in pregnancy and parturition.

Uncovering the causes of pregnancy complications such as preterm labor requires greater insight into how the uterus remains in a noncontractile state until term and then surmounts this state to enter labor. Here, we show that dynamic generation and erasure of the repressive histone modification tri-methyl histone H3 lysine 27 (H3K27me3) in decidual stromal cells dictate both elements of pregnancy success in mice. In early gestation, H3K27me3-induced transcriptional silencing of select gene targets ensured uterine quiescence by preventing the decidua from expressing parturition-inducing hormone receptors, manifesting type 1 immunity, and most unexpectedly, generating myofibroblasts and associated wound-healing responses. In late gestation, genome-wide H3K27 demethylation allowed for target gene upregulation, decidual activation, and labor entry. Pharmacological inhibition of H3K27 demethylation in late gestation not only prevented term parturition, but also inhibited delivery while maintaining pup viability in a noninflammatory model of preterm parturition. Immunofluorescence analysis of human specimens suggested that similar regulatory events might occur in the human decidua. Together, these results reveal the centrality of regulated gene silencing in the uterine adaptation to pregnancy and suggest new areas in the study and treatment of pregnancy disorders

Genotype specific pathogenicity of hepatitis E virus at the human maternal-fetal interface

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