Chantal Charlet-Renard

Dialogue between Blastocyst hCG and Endometrial LH/hCG Receptor: Which Role in Implantation?

The specific interaction of blastocyst-derived human chorionic gonadotropin (hCG) and endometrial LH/hCG-R constitutes a fundamental component of the molecular dialogue at the materno-fetal interface. From our observations and studies from other groups, hCG was indeed shown to play a significant role in implantation and tolerance of the embryo, decidual differentiation and remodeling, as well as in placentation. The profile pattern of LH/hCG-R expression by endometrial epithelium correlates with the theoretical timing of the implantation window. Studies are currently being conducted in assisted medical procreation and in an animal model of implantation to establish the index of LH/hCG-R expression as a new biomarker of uterine receptivity for embryo implantation.

Persistent Infection of Human Thymic Epithelial Cells by Coxsackievirus B4

ABSTRACT Persistent replication of coxsackievirus B4 (CVB4) E2 (diabetogenic) and CVB4 JBV (nondiabetogenic) strains in thymic epithelial cell (TEC)-enriched cultures (≥95%) was proved by detection of positive- and negative-strand viral RNA by reverse transcription-PCR in extracted RNA from cell cultures, VP1 capsid protein detection by immunofluorescence (IF) staining, and release of infectious particles up to 30 days after infection without obvious cytolysis. By double-IF staining, cytokeratin-containing cells were shown to be susceptible to CVB4. The persistence of CVB4 was associated with a significantly increased rate of TEC proliferation (up to 70%) after 20 days of culture and a significantly increased chronic production of immunoreactive interleukin-6 (IL-6), leukemia inhibitory factor, and granulocyte-macrophage colony-stimulating factor in supernatant after 3 days of culture. The CVB4 replication and the release of cytokines were not restricted to the CVB4 E2 diabetogenic strain and did not depend on the genetic background of the host; however, TEC were more responsive to CVB4 E2 than CVB4 JBV as far as the production of cytokines.

Characterization of the Insulin-Like Growth Factor Axis in the Human Thymus

The components of the insulin‐like growth factor (IGF) axis have been investigated in the normal human thymus. Using ribonuclease protection assays (RPA), IGF‐II transcripts were detected in the normal human thymus. By reverse transcriptase polymerase chain reaction (RT‐PCR) analyses, promoters P3 and P4 were found to be active in the transcription of IGF2 gene within human thymic epithelial cells (TEC). No IGF‐II mRNA could be detected in human lymphoid Jurkat T cells with 30 cycles of RT‐PCR. By Northern blot analyses, IGFBP‐2 to ‐6 (but not IGFBP‐1) were found to be expressed in TEC with a predominance of IGFBP‐4. Interestingly, Jurkat T cells only express IGFBP‐2 but at high levels. The type 1 IGF receptor was detected in Jurkat T cells but not in human TEC. The identification of the components of the IGF axis within separate compartments of the human thymus adds further evidence for a role of this axis in the control of T‐cell development. The precise influence of thymic IGF axis upon T‐cell differentiation and immunological self‐tolerance however needs to be further investigated.

Neurohypophysial Peptides Stimulate the Phosphorylation of Pre-T Cell Focal Adhesion Kinases

Thymic oxytocin (OT) behaves as a cryptocrine signal targeted at the outer surface of thymic epithelial cell plasma membrane from where OT is able to interact with neurohypophysial peptide receptors expressed by pre-T cells. Immature T cells bear a receptor of the V1 subtype, while OT receptors are predominantly expressed by cytotoxic CD8+ lymphocytes. In both T cell types, neurohypophysial peptide receptors transduce OT via the phosphoinositide pathway. Protein tyrosine phosphorylation is an early event of T cell activation. Western blots of murine pre-T cells (RL12-NP line) proteins probed with anti-phosphotyrosine (PY-20) revealed a great number of proteins the phosphorylation of which increased either with OT or vasopressin treatment. Two were immunoprecipitated with anti-focal adhesion kinase (FAK) mAb 2A7 and were identified one as p125FAK and the other as a coprecipitating 130-kDa protein. The p125FAK is connected to the Ras/MAPK pathway and is also implicated in TCR/CD3 signalling in T cell. Another protein phosphorylated by OT in RL12-NP was identified as paxillin, a 68-kDa protein localised at focal adhesion sites and associated with p125FAK. These results indicate that phosphorylation of focal adhesion kinase may be induced in pre-T cell by thymic OT.

The Thymic Repertoire of Neuroendocrine-Related Self Antigens: Biological Role in T-Cell Selection and Pharmacological Implications

Thymic epithelium, including nurse cells (TEC/TNC), as well as other thymic stromal cells (macrophages and dentritic cells), express a repertoire of polypeptide belonging to various neuroendocrine protein families (such as the neurophypophysial, tachykinin, neurotensin and insulin families). A hierarchy of dominance exists in the organization of the thymic repertoire of neuroendocrine precursors. Oxytocin (OT) is more expressed in the TEC/TNC than vasopressin (VP); insulin-like growth factor 2 (IGF-2) thymic expression predominates over IGF-1, and much more over (pro)insulin. Thus, OT was proposed to be the self antigen of the neurohypophysial family, and IGF-2 the self antigen precursor of the insulin family. The dual role of the thymus in T-cell life and death is recapitulated at the level of the thymic neuroendocrine protein repertoire. Indeed, thymic polypeptides behave as accessory signals involved in T-cell development and positive selection according to the cryptocrine model of signaling. Moreover, thymic neuroendocrine polypeptides are the source of self antigens presented by thymic MHC molecules to developing pre-T cells. This presentation might induce the negative selection of T cells bearing a randomly rearranged antigen receptor (TCR) oriented against neuroendocrine families. Using an animal model of autoimmune type 1 diabetes (BB rat), we have shown a defect in intrathymic expression of the self antigen of the insulin family (IGF-2) and in IGF-2-mediated T-cell education to recognize and tolerate the insulin family. Altogether these studies have enlighted the crucial role played by the thymus in the induction of the central self tolerance of neuroendocrine families. The tolerogenic properties of thymic self peptides could be used in a novel type of vaccination for the prevention of autoimmune diseases.

Inhibin and related peptides: Mechanisms of action and regulation of secretion☆

The structure of inhibin is known; it consists of a heterodimer composed of one alpha and one beta subunit. The homodimer of beta A (beta A-beta A) and the heterodimer beta A-beta B, called activin A and B, respectively, stimulate the release and synthesis of FSH by gonadotrophs. Inhibin exerts effects at the hypophyseal, hypothalamic, and gonadal levels. Produced by granulosa cells in the female and by Sertoli cells in the male, inhibin synthesis is stimulated by FSH and reduced by hypophysectomy and progesterone. At present, there is no evidence for a signal from germinal cells to modify inhibin production. Inhibin secretion evolves in parallel with follicular maturation and aromatase activity, whereas luteinization arrests its production. Nevertheless, important differences in the regulation of inhibin secretion seem to exist from one species to another. Sperm inhibin levels can be correlated with spermatozoa number. Administration of inhibin to sheep induces either anovulation or an increase in the rate of ovulation depending on the scheme of treatment.

Aire and Foxp3 Expression in a Particular Microenvironment for T Cell Differentiation

Objective: The thymus is the primary lymphoid organ responsible for T cell development and the establishment of central self-tolerance. Among thymic epithelial cells, thymic nurse cells (TNC) interact closely with immature thymocytes and constitute a special microenvironment for T cell differentiation and selection. In addition, TNC express neuroendocrine self-antigens such as oxytocin and insulin-like growth factor-2, whose intrathymic transcription is regulated by the autoimmune regulator gene/protein (Aire). Both effector and natural regulatory T cell (nTreg) lineages develop in the thymus, but the mechanisms leading to nTreg selection in the thymus are still unclear. Foxp3 is the most specific nTreg marker that is required for nTreg functional activity, but not for engagement into the Treg lineage. Aire has been suggested to be a potential factor implicated in this role. The objective of this study was to characterize Aire and Foxp3 expression in TNC/thymocyte complexes. Methods:Aire and Foxp3 expression was investigated by RT-qPCR in TNC/thymocyte complexes isolated by enzymatic digestion and sedimentation. Aire and Foxp3 proteins were located by confocal microscopy and specific immunocytochemistry. Results: Both Aire and Foxp3 transcripts were detected in TNC/thymocyte complexes. Foxp3 was detected in the nucleus of thymocytes internalized into TNC. Aire was located mainly in TNC cytoplasm and, although to a lower degree, in the nucleus of some TNC-associated thymocytes. Conclusions: Aire and Foxp3 are present in the particular TNC microenvironment which has previously been shown to support thymic selection. The differential localization of these two markers suggests a role for TNC in nTreg development.

Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

Abstract Neurohypophysial oxytocin (OT) and vasopressin (VP) genes are transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

Programming of neuroendocrine self in the thymus and its defect in the development of neuroendocrine autoimmunity.

For centuries after its first description by Galen, the thymus was considered as only a vestigial endocrine organ until the discovery in 1961 by Jacques FAP Miller of its essential role in the development of T (thymo-dependent) lymphocytes. A unique thymus first appeared in cartilaginous fishes some 500 million years ago, at the same time or shortly after the emergence of the adaptive (acquired) immune system. The thymus may be compared to a small brain or a computer highly specialized in the orchestration of central immunological self-tolerance. This was a necessity for the survival of species, given the potent evolutionary pressure imposed by the high risk of autotoxicity inherent in the stochastic generation of the diversity of immune cell receptors that characterize the adaptive immune response. A new paradigm of “neuroendocrine self-peptides” has been proposed, together with the definition of “neuroendocrine self.” Neuroendocrine self-peptides are secreted by thymic epithelial cells (TECs) not according to the classic model of neuroendocrine signaling, but are processed for presentation by, or in association with, the thymic major histocompatibility complex (MHC) proteins. The autoimmune regulator (AIRE) gene/protein controls the transcription of neuroendocrine genes in TECs. The presentation of self-peptides in the thymus is responsible for the clonal deletion of self-reactive T cells, which emerge during the random recombination of gene segments that encode variable parts of the T cell receptor for the antigen (TCR). At the same time, self-antigen presentation in the thymus generates regulatory T (Treg) cells that can inhibit, in the periphery, those self-reactive T cells that escaped negative selection in the thymus. Several arguments indicate that the origin of autoimmunity directed against neuroendocrine glands results primarily from a defect in the intrathymic programming of self-tolerance to neuroendocrine functions. This defect may be genetic or acquired, for example during an enteroviral infection. This novel knowledge of normal and pathologic functions of the thymus constitutes a solid basis for the development of a novel type of tolerogenic/negative self-vaccination against type 1 diabetes (T1D).

The Somatotrope Growth Hormone-Releasing Hormone/Growth Hormone/Insulin-Like Growth Factor-1 Axis in Immunoregulation and Immunosenescence

Most scientific reports debate the thymotropic and immuno-stimulating properties of the somatotrope growth hormone-releasing hormone (GHRH)/growth hormone (GH)/insulin-like growth factor (IGF)-1 axis, but there is still some disagreement about the physiological role of this axis in basal conditions. Moreover, some authors have hypothesized that the physiological role of the somatotrope axis only appears in stressful conditions (such as sepsis or infective and inflammatory diseases). This chapter will provide an extended overview of the expression of the components (signals and receptors) of the somatotrope axis and their properties on cells of the innate and adaptive immune system. It will also summarize some clinical studies suggesting a benefit for a short-term GH treatment in acute immunodeficiencies, and the importance of GH supplementation in adult GH deficiency. A new transgenic mouse model, the hypothalamic GHRH-deficient (Ghrh-/-) mouse, which exhibits a severe deficiency of the somatotrope axis, will be presented since it will be of great help in further deciphering the regulation by the GHRH/GH/IGF-1 axis on both immune development and function. Finally, we will discuss the implication of aging-related somatopause in relation to the general context of Immunosenescence.