Expression physiopathologique du gène WNK1 responsable de l'hypertension hyperkaliémique familiale (élucidation du mécanisme mutationnel)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

WNK1 et WNK4, nouveaux acteurs de l’homéostasie hydrosodée

L’étude d’une forme rare d’hypertension artérielle de transmission mendélienne, l’hypertension hyperkaliémique familiale (HHF), a récemment permis d’identifier des mutations dans les gènes WNK1 et WNK4, qui codent pour des protéines appartenant à une nouvelle famille de sérine-thréonine kinases (with no lysine [K] kinase). Plusieurs éléments du tableau clinique de l’HHF, caractérisé par une hyperkaliémie, une hyperchlorémie et une grande sensibilité aux diurétiques thiazidiques, sont en faveur d’une anomalie du transport ionique dans le tubule distal rénal. En accord avec cette hypothèse, WNK1 et WNK4 sont fortement exprimés dans cette partie du néphron. On les retrouve également dans de nombreux épithéliums impliqués dans le transport du chlore, tels que celui du côlon. In vitro, WNK4 règle à la fois le transport de Na+, K+ et Cl-, et pourrait donc constituer une voie de régulation importante des transports ioniques rénal et extra-rénal.Arterial hypertension is a complex trait influenced by a variety of environmental and genetic factors. Several approaches can be used to identify its susceptibility genes : one is to study rare monogenic forms of hypertension, like familial hyperkalemic hypertension (FHH). Also known as pseudohypoaldosteronism type 2 or Gordon syndrome, FHH is characterized by hypertension, hyperkalemia despite normal renal glomerular filtration rate, abnormalities which are particularly sensitive to thiazide diuretics. Mild hyperchloremia, metabolic acidosis, and suppressed plasma renin activity are associated findings. Despite its phenotypic and genetic heterogeneity, mutations in two related genes, WNK1 and WNK4, were recently identified. These genes belong to a newly identified family of serine-threonine (with no lysine [K]) kinases. Both are highly expressed in the kidney and in a variety of epithelia involved in chloride transport. It has thus been postulated that these two kinases could be implicated in a new pathway of ionic transport regulation. Several studies have very recently confirmed this hypothesis in vitro, in Xenopus oocytes or kidney cell lines. They have shown that, in the renal distal tubule, WNK4 inhibits sodium reabsorption and potassium secretion, via inhibition of NCC (thiazide-sensitive Na+-Cl- cotransporter) and K+ channel ROMK activity, respectively. Interestingly, FHH mutations have opposite effects : while they lead to loss of NCC inhibition, they increase ROMK inhibition. Moreover, they also increase paracellular permeability to chloride of MDCK cells. WNK4 also inhibits apical and basal chloride transporters present in extra-renal epithelia, such as CFEX and Na+-K+-2 Cl-, respectively. It is also interesting to note that the WNK4-mediated negative regulation of NCC activity is in turn inhibited by WNK1. By its role on several transporters, WNK4 appears as a putative key regulator of ionic transport and blood pressure

Single cell resolution of Plasma Cell fate programming in health and disease

International audienceLong considered a homogeneous population dedicated to antibody secretion, plasma cell phenotypic and functional heterogeneity is increasingly recognised. Plasma cells were first segregated based on their maturation level, but the complexity of this subset might well be underestimated by this simple dichotomy. Indeed, in the last decade new functions have been attributed to plasma cells including but not limited to cytokine secretion. However, a proper characterization of plasma cell heterogeneity has remained elusive partly due to technical issues and cellular features that are specific to this cell type. Cell intrinsic and cell extrinsic signals could be at the origin of this heterogeneity. Recent advances in technologies like single cell RNA-seq, ATAC-seq or ChIP-seq on low cell numbers helped to elucidate the fate decision in other cell lineages and similar approaches could be implemented to evaluate the heterogeneous fate of activated B cells in health and disease. Here, we summarized published work shedding some lights on the stimuli and genetic program shaping B cell terminal differentiation at the single cell level in mice and men. We also discuss the fate and heterogeneity of plasma cells during immune responses, vaccination and in the frame of human plasma cell disorders

IL-2 requirement for human plasma cell generation: coupling differentiation and proliferation by enhancing MAPK-ERK signaling.

International audienceMature B cell differentiation involves a well-established transcription factor cascade. However, the temporal dynamics of cell signaling pathways regulating transcription factor network and coordinating cell proliferation and differentiation remain poorly defined. To gain insight into the molecular processes and extrinsic cues required for B cell differentiation, we set up a controlled primary culture system to differentiate human naive B cells into plasma cells (PCs). We identified T cell-produced IL-2 to be critically involved in ERK1/2-triggered PC differentiation. IL-2 drove activated B cell differentiation toward PC independently of its proliferation and survival functions. Indeed, IL-2 potentiated ERK activation and subsequent BACH2 and IRF8 downregulation, sustaining BLIMP1 expression, the master regulator for PC differentiation. Inhibition of the MAPK-ERK pathway, unlike STAT5 signaling, impaired IL-2-induced PC differentiation and rescued the expression profile of BACH2 and IRF8. These results identify IL-2 as a crucial early input in mature B cell fate commitment

Committed Human CD23-Negative Light-Zone Germinal Center B Cells Delineate Transcriptional Program Supporting Plasma Cell Differentiation

International audienceB cell affinity maturation occurs in the germinal center (GC). Light-zone (LZ) GC B cells (B-GC-cells) interact with follicular dendritic cells (FDCs) and compete for the limited, sequential help from T follicular helper cells needed to escape from apoptosis and complete their differentiation. The highest-affinity LZ B-GC-cells enter the cell cycle and differentiate into PCs, following a dramatic epigenetic reorganization that induces transcriptome changes in general and the expression of the PRDM1 gene in particular. Human PC precursors are characterized by the loss of IL-4/STAT6 signaling and the absence of CD23 expression. Here, we studied the fate of human LZ B-GC-cells as a function of their CD23 expression. We first showed that CD23 expression was restricted to the GC LZ, where it was primarily expressed by FDCs; less than 10% of tonsil LZ B-GC-cells were positive. Sorted LZ B-GC-cells left in culture and stimulated upregulated CD23 expression but were unable to differentiate into PCs - in contrast to cells that did not upregulate CD23 expression. An in-depth analysis (including single-cell gene expression) showed that stimulated CD23-negative LZ B-GC-cells differentiated into plasmablasts and time course of gene expression changes delineates the transcriptional program that sustains PC differentiation. In particular, we identified a B cell proliferation signature supported by a transient MYC gene expression. Overall, the CD23 marker might be of value in answering questions about the differentiation of normal B-GC-cells and allowed us to propose an instructive LZ B-GC-cells maturation and fate model

Citrulline enteral administration markedly reduces immunosuppressive extrafollicular plasma cell differentiation in a preclinical model of sepsis

International audienceThe sustained immunosuppression associated with severe sepsis favours an increased susceptibility to secondary infections and remains incompletely understood. Plasmablast and plasma cell subsets, whose primary function is to secrete antibodies, have emerged as important suppressive populations expanded during sepsis. In particular, sepsis supports CD39(hi) plasmablast metabolic reprogramming associated with adenosine-mediated suppressive activity. Arginine deficiency has been linked to increased risk of secondary infections in sepsis. Overcoming arginine shortage by citrulline administration efficiently improves sepsis-induced immunosuppression and secondary infections in the cecal ligation and puncture murine model. Here we aimed to determine the impact of citrulline administration on B cell suppressive responses in sepsis. We demonstrate that restoring arginine bioavailability through citrulline administration, markedly reduces the dominant extrafollicular B cell response, decreasing the immunosuppressive LAG3(+) and CD39(+) plasma cell populations, and restoring splenic follicles. At the molecular level, the IRF4/MYC-mediated B cell reprogramming required for extrafollicular plasma cell differentiation is shunted in splenic B cells of mice fed with citrulline. Our study reveals a prominent impact of nutrition on B cell responses, and plasma cell differentiation and further supports the development of citrulline-based clinical studies to prevent sepsis associated immune dysfunction. This article is protected by copyright. All rights reserved

The diversity of the plasmablast signature across species and experimental conditions: A meta-analysis

International audienceAntibody-secreting cells (ASC) are divided into two principal subsets, including the long-lived plasma cell (PC) subset residing in the bone marrow and the short-lived subset, also called plasmablast (PB). PB are described as a proliferating subset circulating through the blood and ending its differentiation in tissues. Due to their inherent heterogeneity, the molecular signature of PB is not fully established. The purpose of this study was to decipher a specific PB signature in humans and mice through a comprehensive meta-analysis of different data sets exploring the PB differentiation in both species and across different experimental conditions. The present study used recent analyses using whole RNA sequencing in prdm1-GFP transgenic mice to define a reliable and accurate PB signature. Next, we performed similar analysis using current data sets obtained from human PB and PC. The PB-specific signature is composed of 155 and 113 genes in mouse and human being, respectively. Although only nine genes are shared between the human and mice PB signature, the loss of B-cell identity such as the down-regulation of PAX5, MS4A1, (CD20) CD22 and IL-4R is a conserved feature across species and across the different experimental conditions. Additionally, we observed that the IRF8 and IRF4 transcription factors have a specific dynamic range of expression in human PB. We thus demonstrated that IRF4/IRF8 intranuclear staining was useful to define PB in vivo and in vitro and able to discriminate between atypical PB populations and transient states

Cardiovascular Expression of the Mouse WNK1 Gene during Development and Adulthood Revealed by a BAC Reporter Assay

Large deletions in WNK1 are associated with inherited arterial hypertension. WNK1 encodes two types of protein: a kidney-specific isoform (KS-WNK1) lacking kinase activity and a ubiquitously expressed full-length isoform (L-WNK1) with serine threonine kinase activity. Disease is thought to result from hypermorphic mutations increasing the production of one or both isoforms. However, the pattern of L-WNK1 expression remains poorly characterized. We generated transgenic mice bearing a murine WNK1 BAC containing the nlacZ reporter gene for monitoring L-WNK1 expression during development and adulthood. We observed previously unsuspected early expression in the vessels and primitive heart during embryogenesis, consistent with the early death of WNK1(−/−) mice. The generalized cardiovascular expression observed in adulthood may also suggest a possible kidney-independent role in blood pressure regulation. The second unsuspected site of L-WNK1 expression was the granular layer and Purkinje cells of the cerebellum, suggesting a role in local ion balance or cell trafficking. In the kidney, discordance between endogenous L-WNK1 and transgene expression suggests that either cis-regulatory elements important for physiological renal expression lie outside the BAC sequence or that illegitimate interactions occur between promoters. Despite this limitation, this transgenic model is a potentially valuable tool for the analysis of spatial and temporal aspects of WNK1 expression and regulation