Publications by authors named "Nicolas Cagnard"

76 Publications

The association of Greig syndrome and mastocytosis reveals the involvement of hedgehog pathway in advanced mastocytosis.

Blood 2021 Aug 23. Epub 2021 Aug 23.

IMAGINE Institute- INSERM UMR 1163, Paris, France.

Mastocytosis is a heterogeneous disease characterized by an abnormal accumulation of mast cells (MCs) in one or several organs. Although a somatic KIT D816V mutation is detected in ~85% of patients, attempts to demonstrate its oncogenic effect alone have repeatedly failed, suggesting that additional pathways are involved in MC transformation. From three children presenting with both Greig cephalopolysyndactyly syndrome (GCPS, MIM#175700) and congenital mastocytosis, we demonstrated the involvement of the hedgehog (Hh) pathway in mastocytosis. GCPS is an extremely rare syndrome resulting from haploinsufficiency of GLI3, the major repressor of Hh family members. From these familial cases of mastocytosis, we demonstrate that the Hh pathway is barely active in normal primary MCs and overactive in neoplastic MCs. We show that GLI3 and KIT mutations have a synergistic, tumorigenic effect on the onset of mastocytosis in a GCPS mouse model. Finally, we show that Hh inhibitors suppress neoplastic MC proliferation in vitro and extend the survival time of aggressive systemic mastocytosis (ASM) mice. This work revealed, for the first time, the involvement of Hh signaling in the pathophysiology of mastocytosis and demonstrated the cooperative effects of the KIT and Hh oncogenic pathways in ASM, leading to the identification of new promising therapeutic targets.
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http://dx.doi.org/10.1182/blood.2020010207DOI Listing
August 2021

A monocyte/dendritic cell molecular signature of SARS-CoV-2-related multisystem inflammatory syndrome in children with severe myocarditis.

Med (N Y) 2021 Sep 14;2(9):1072-1092.e7. Epub 2021 Aug 14.

Sorbonne Université, UMS037, PASS, Plateforme de Cytométrie de la Pitié-Salpêtrière CyPS, 75013 Paris, France.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children is generally milder than in adults, but a proportion of cases result in hyperinflammatory conditions often including myocarditis.

Methods: To better understand these cases, we applied a multiparametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. Plasma cytokine and chemokine levels and blood cellular composition were measured, alongside gene expression at the bulk and single-cell levels.

Findings: The most severe forms of multisystem inflammatory syndrome in children (MIS-C) related to SARS-CoV-2 that resulted in myocarditis were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomics analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis characterized by sustained nuclear factor κB (NF-κB) activity and tumor necrosis factor alpha (TNF-α) signaling and associated with decreased gene expression of NF-κB inhibitors. We also found a weak response to type I and type II interferons, hyperinflammation, and response to oxidative stress related to increased HIF-1α and Vascular endothelial growth factor (VEGF) signaling.

Conclusions: These results provide potential for a better understanding of disease pathophysiology.

Funding: Agence National de la Recherche (Institut Hospitalo-Universitaire Imagine, grant ANR-10-IAHU-01; Recherche Hospitalo-Universitaire, grant ANR-18-RHUS-0010; Laboratoire d'Excellence ''Milieu Intérieur," grant ANR-10-LABX-69-01; ANR-flash Covid19 "AIROCovid" and "CoVarImm"), Institut National de la Santé et de la Recherche Médicale (INSERM), and the "URGENCE COVID-19" fundraising campaign of Institut Pasteur.
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http://dx.doi.org/10.1016/j.medj.2021.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363470PMC
September 2021

The alternative RelB NF-κB subunit is a novel critical player in diffuse large B-cell lymphoma.

Blood 2021 07 7. Epub 2021 Jul 7.

Université de Paris, NF-kappaB, Différenciation et Cancer, Paris, France

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid malignancy affecting adults. NF-kB transcription factor family is activated by two main pathways, the canonical and the alternative NF-kB activation pathways with different functions. The alternative NF-kB pathway leads to the activation of the transcriptionally active RelB NF-kB subunit. Alternative NF-kB activation status and its role in DLBCL pathogenesis remain undefined. Here, we reveal a frequent activation of RelB in a large cohort of DLBCL patients and cell lines, independently of their ABC or GCB subtypes. RelB activity defines a new subset of DLBCL patients with a peculiar gene expression profile and mutational pattern. Importantly, RelB activation does not correlate with the MCD genetic subtype, enriched for ABC tumors carrying MYD88L265P and CD79B mutations that cooperatively activate canonical NF-kB, thus indicating that current genetic tools to evaluate NF-kB activity in DLBCL do not provide information on the alternative NF-kB activation. Further, the newly defined RelB-positive subgroup of DLBCL patients exhibits a dismal outcome following immunochemotherapy. Functional studies revealed that RelB confers DLBCL cell resistance to DNA-damage induced apoptosis in response to doxorubicin, a genotoxic agent used in front-line treatment for DLBCL. We also show that RelB positivity is associated with high expression of cIAP2. Altogether, RelB activation can be used to refine the prognostic stratification of DLBCL and may contribute to subvert the therapeutic DNA damage response in a segment of DLBCL patients.
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http://dx.doi.org/10.1182/blood.2020010039DOI Listing
July 2021

A DL-4- and TNFα-based culture system to generate high numbers of nonmodified or genetically modified immunotherapeutic human T-lymphoid progenitors.

Cell Mol Immunol 2021 Jul 11;18(7):1662-1676. Epub 2021 Jun 11.

Université de Paris, Imagine Institute, Laboratory of Human Lymphohematopoiesis, INSERM UMR 1163, Paris, France.

Several obstacles to the production, expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy. In the context of HSCT, delayed naïve T-cell recovery contributes to poor outcomes. A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human T-lymphoid progenitors (HTLPs), allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus. However, it is challenging to produce HTLPs in the high numbers required to meet clinical needs. Here, we found that adding tumor necrosis factor alpha (TNFα) to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival. This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy (including CAR T-cell therapy).
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http://dx.doi.org/10.1038/s41423-021-00706-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245454PMC
July 2021

Bi-allelic variants in IPO8 cause a connective tissue disorder associated with cardiovascular defects, skeletal abnormalities, and immune dysregulation.

Am J Hum Genet 2021 06 18;108(6):1126-1137. Epub 2021 May 18.

Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Parkville 3052, Melbourne, VIC, Australia; Department of Paediatrics, The University of Melbourne, 3010 Parkville, Melbourne, VIC, Australia.

Dysregulated transforming growth factor TGF-β signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-β-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-β signaling, ipo8 zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8 zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-β signaling during development and reinforces the existing link between TGF-β signaling and connective tissue defects.
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http://dx.doi.org/10.1016/j.ajhg.2021.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206386PMC
June 2021

Mobilized Multipotent Hematopoietic Progenitors Stabilize and Expand Regulatory T Cells to Protect Against Autoimmune Encephalomyelitis.

Front Immunol 2020 23;11:607175. Epub 2020 Dec 23.

Université de Paris, Inserm U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Paris, France.

Achieving immunoregulation expansion of Foxp3 regulatory CD4 T cells (Treg) remains challenging. We have shown that mobilization confers to multipotent hematopoietic progenitors (MPPs) the capacity to enhance Treg proliferation. Transcriptomic analysis of Tregs co-cultured with MPPs revealed enhanced expression of genes stabilizing the suppressive function of Tregs as well as the activation of IL-1β-driven pathways. Adoptive transfer of only 25,000 MPPs effectively reduced the development of experimental autoimmune encephalomyelitis (EAE), a pre-clinical model for multiple sclerosis (MS). Production of the pathogenic cytokines IL-17 and GM-CSF by spinal cord-derived CD4 T-cells in MPP-protected recipients was reduced while Treg expansion was enhanced. Treg depletion once protection by MPPs was established, triggered disease relapse to the same level as in EAE mice without MPP injection. The key role of IL-1β was further confirmed by the lack of protection against EAE in recipients of IL-1β-deficient MPPs. Mobilized MPPs may thus be worth considering for cell therapy of MS either per se or for enrichment of HSC grafts in autologous bone marrow transplantation already implemented in patients with severe refractory multiple sclerosis.
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http://dx.doi.org/10.3389/fimmu.2020.607175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786289PMC
June 2021

Unique inflammatory signature in haemophilic arthropathy: miRNA changes due to interaction between blood and fibroblast-like synoviocytes.

J Cell Mol Med 2020 12 7;24(24):14453-14466. Epub 2020 Nov 7.

Laboratory of Cellular and Molecular Mechanisms of Hematological Disorders and Therapeutical Implications, Paris Descartes - Sorbonne Paris Cité University, Labex GR-Ex, Imagine Institute, Inserm U1163, Paris, France.

In haemophilia, the recurrence of hemarthrosis leads to irreversible arthropathy termed haemophilic arthropathy (HA). However, HA is a unique form of arthropathy in which resident cells, such as fibroblast-like synoviocytes (FLS), come into direct contact with blood. Therefore, we hypothesized that FLS in HA could have a unique inflammatory signature as a consequence of their contact with blood. We demonstrated with ELISA and ELISPOT analyses that HA-FLS expressed a unique profile of cytokine secretion, which differed from that of non-HA-FLS, mainly consisting of cytokines involved in innate immunity. We showed that unstable cytokine mRNAs were involved in this process, especially through miRNA complexes as confirmed by DICER silencing. A miRNOME analysis revealed that 30 miRNAs were expressed differently between HA and non-HA-FLS, with most miRNAs involved in inflammatory control pathways or described in certain inflammatory diseases, such as rheumatoid arthritis or lupus. Analysis of transcriptomic networks, impacted by these miRNAs, revealed that protein processes and inflammatory pathways were particularly targeted in LPS-induced FLS, and in particular vascularization and osteoarticular modulation pathways in steady-state FLS. Our study demonstrates that the presence of blood in contact with FLS may induce durable miRNA changes that likely participate in HA pathophysiology.
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http://dx.doi.org/10.1111/jcmm.16068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753994PMC
December 2020

Tumor invasion in draining lymph nodes is associated with Treg accumulation in breast cancer patients.

Nat Commun 2020 06 29;11(1):3272. Epub 2020 Jun 29.

Institut Curie, PSL Research University, INSERM U932, F-75005, Paris, France.

Tumor-draining lymph node (TDLN) invasion by metastatic cells in breast cancer correlates with poor prognosis and is associated with local immunosuppression, which can be partly mediated by regulatory T cells (Tregs). Here, we study Tregs from matched tumor-invaded and non-invaded TDLNs, and breast tumors. We observe that Treg frequencies increase with nodal invasion, and that Tregs express higher levels of co-inhibitory/stimulatory receptors than effector cells. Also, while Tregs show conserved suppressive function in TDLN and tumor, conventional T cells (Tconvs) in TDLNs proliferate and produce Th1-inflammatory cytokines, but are dysfunctional in the tumor. We describe a common transcriptomic signature shared by Tregs from tumors and nodes, including CD80, which is significantly associated with poor patient survival. TCR RNA-sequencing analysis indicates trafficking between TDLNs and tumors and ongoing Tconv/Treg conversion. Overall, TDLN Tregs are functional and express a distinct pattern of druggable co-receptors, highlighting their potential as targets for cancer immunotherapy.
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http://dx.doi.org/10.1038/s41467-020-17046-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324591PMC
June 2020

IL-15 superagonist RLI has potent immunostimulatory properties on NK cells: implications for antimetastatic treatment.

J Immunother Cancer 2020 06;8(1)

Analyse Moléculaire, Modélisation et Imagerie de la Maladie Cancéreuse, Laboratoire d'Immunomonitoring en Oncologie, Gustave Roussy Institute, INSERM, CNRS, Paris-Saclay University, Villejuif, Île-de-France, France

Background: As the immune system is compromised in patients with cancer, therapeutic strategies to stimulate immunity appear promising, to avoid relapse and increase long-term overall survival. Interleukin-15 (IL-15) has similar properties to IL-2, but does not cause activation-induced cell death nor activation and proliferation of regulatory T cells (Treg), which makes it a serious candidate for anticancer immunotherapy. However, IL-15 has a short half-life and high doses are needed to achieve responses. Designed to enhance its activity, receptor-linker-IL-15 (RLI) (SO-C101) is a fusion molecule of human IL-15 covalently linked to the human IL-15Rα sushi+ domain currently assessed in a phase I/Ib clinical trial on patients with advanced/metastatic solid cancer.

Methods: We investigated the antimetastatic activity of RLI in a 4T1 mouse mammary carcinoma that spontaneously metastasizes and evaluated its immunomodulatory role in the metastatic lung microenvironment. We further characterized the proliferation, maturation and cytotoxic functions of natural killer (NK) cells in tumor-free mice treated with RLI. Finally, we explored the effect of RLI on human NK cells from healthy donors and patients with non-small cell lung cancer (NSCLC).

Results: RLI treatment displayed antimetastatic properties in the 4T1 mouse model. By characterizing the lung microenvironment, we observed that RLI restored the balance between NK cells and neutrophils (CD11b Ly6G Ly6C) that massively infiltrate lungs of 4T1-tumor bearing mice. In addition, the ratio between NK cells and Treg was strongly increased by RLI treatment. Further pharmacodynamic studies in tumor-free mice revealed superior proliferative and cytotoxic functions on NK cells after RLI treatment compared with IL-15 alone. Characterization of the maturation stage of NK cells demonstrated that RLI favored accumulation of CD11b CD27 KLRG1 mature NK cells. Finally, RLI demonstrated potent immunostimulatory properties on human NK cells by inducing proliferation and activation of NK cells from healthy donors and enhancing cytotoxic responses to NKp30 crosslinking in NK cells from patients with NSCLC.

Conclusions: Collectively, our work demonstrates superior activity of RLI compared with rhIL-15 in modulating and activating NK cells and provides additional evidences for a therapeutic strategy using RLI as antimetastatic molecule.
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http://dx.doi.org/10.1136/jitc-2020-000632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295443PMC
June 2020

MeCP2 is involved in random mono-allelic expression for a subset of human autosomal genes.

Biochim Biophys Acta Mol Basis Dis 2020 06 15;1866(6):165730. Epub 2020 Feb 15.

Assistance Publique - Hôpitaux de Paris, APHP, Centre Universitaire Paris, Hôpital Cochin, Laboratoire de Génétique et Biologie Moléculaires, Paris, France; Institut de Psychiatrie et de Neurosciences de Paris (IPNP), INSERM U1266, Team "Vulnérabilité aux troubles psychiatriques et addictifs", Université de Paris, Paris, France. Electronic address:

Widespread random monoallelic gene expression (RMAE) effects influence about 10% of human genes. However, the mechanisms by which RME of autosomal genes is established and those by which it is maintained both remain open questions. Because the choice of allelic expression is randomly performed cell-by-cell, the RMAE mechanism is not observable in non-clonal cell populations or in whole tissues. Several target genes of MeCP2, the gene involved in Rett syndrome (RTT), have been previously described as subject to RMAE, suggesting that MeCP2 may be involved in the establishment and/or maintenance of RME of autosomal genes. To improve our knowledge on this largely unknown phenomenon, and to study the role of MeCP2 in RMAE, we compared RMA gene expression profiles in clonal cell cultures expressing wild-type MeCP2 versus mutant MeCP2 from a RTT patient carrying a pathogenic non-sense variant. Our data clearly demonstrated that MeCP2 deficiency does not affect significantly allelic gene expression of X-linked genes, imprinted genes as well as the RMAE profile in the majority of genes. However, the functional deficiency in MeCP2 appeared to disrupt the mono-allelic or the bi-allelic expression of at least 49 genes allowing us to define a specific signature of MECP2 mutated clones.
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http://dx.doi.org/10.1016/j.bbadis.2020.165730DOI Listing
June 2020

Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs.

Blood 2020 04;135(15):1219-1231

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA.

In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or β hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.
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http://dx.doi.org/10.1182/blood.2019002350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146019PMC
April 2020

Congenital cystic adenomatoid malformations of the lung: an epithelial transcriptomic approach.

Respir Res 2020 Feb 4;21(1):43. Epub 2020 Feb 4.

Service de Pneumologie et d'Allergologie Pédiatriques, AP-HP, Hôpital Universitaire Necker-Enfants Malades, 75743 Cedex 15, Paris, France.

Background: The pathophysiology of congenital cystic adenomatoid malformations (CCAM) of the lung remains poorly understood.

Aim: This study aimed to identify more precisely the molecular mechanisms limited to a compartment of lung tissue, through a transcriptomic analysis of the epithelium of macrocystic forms.

Methods: Tissue fragments displaying CCAM were obtained during planned surgical resections. Epithelial mRNA was obtained from cystic and normal areas after laser capture microdissection (LCM). Transcriptomic analyses were performed and the results were confirmed by RT-PCR and immunohistochemistry in independent samples.

Results: After controlling for RNA quality, we analysed the transcriptomes of six cystic areas and five control areas. In total, 393 transcripts were differentially expressed in the epithelium, between CCAM and control areas. The most highly redundant genes involved in biological functions and signalling pathways differentially expressed between CCAM and control epithelium included TGFB2, TGFBR1, and MAP 2 K1. These genes were considered particularly relevant as they have been implicated in branching morphogenesis. RT-qPCR analysis confirmed in independent samples that TGFBR1 was more strongly expressed in CCAM than in control tissues (p < 0.03). Immunohistochemistry analysis showed TGFBR1 (p = 0.0007) and TGFB2 (p < 0.02) levels to be significantly higher in the epithelium of CCAM than in that of control tissues.

Conclusions: This compartmentalised transcriptomic analysis of the epithelium of macrocystic lung malformations identified a dysregulation of TGFB signalling at the mRNA and protein levels, suggesting a possible role of this pathway in CCAM pathogenesis.

Trial Registration: ClinicalTrials.gov Identifier: NCT01732185.
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http://dx.doi.org/10.1186/s12931-020-1306-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001206PMC
February 2020

CBFβ-SMMHC Affects Genome-wide Polycomb Repressive Complex 1 Activity in Acute Myeloid Leukemia.

Cell Rep 2020 01;30(2):299-307.e3

Université Paris Descartes Sorbonne Cité, Institut Necker Enfants Malades (INEM), Institut National de Recherche Médicale (INSERM) U1151, Paris, France; Laboratory of Onco-Hematology, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Necker-Enfants Malades, Paris, France; Systems Biology Ireland, School of Medicine, University College Dublin, Dublin, Ireland; National Children's Research Centre, Children's Health Ireland at Crumlin, Dublin, Ireland. Electronic address:

Mutations and deletions of polycomb repressive complex (PRC) components are increasingly recognized to affect tumor biology in a range of cancers. However, little is known about how genetic alterations of PRC-interacting molecules such as the core binding factor (CBF) complex influence polycomb activity. We report that the acute myeloid leukemia (AML)-associated CBFβ-SMMHC fusion oncoprotein physically interacts with the PRC1 complex and that these factors co-localize across the AML genome in an apparently PRC2-independent manner. Depletion of CBFβ-SMMHC caused substantial increases in genome-wide PRC1 binding and marked changes in the association between PRC1 and the CBF DNA-binding subunit RUNX1. PRC1 was more likely to be associated with actively transcribed genes in CBFβ-SMMHC-expressing cells. CBFβ-SMMHC depletion had heterogeneous effects on gene expression, including significant reductions in transcription of ribosomal loci occupied by PRC1. Our results provide evidence that CBFβ-SMMHC markedly and diversely affects polycomb recruitment and transcriptional regulation across the AML genome.
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http://dx.doi.org/10.1016/j.celrep.2019.12.026DOI Listing
January 2020

A variant erythroferrone disrupts iron homeostasis in -mutated myelodysplastic syndrome.

Sci Transl Med 2019 07;11(500)

Université de Paris, Paris 75006, France.

Myelodysplastic syndromes (MDS) with ring sideroblasts are hematopoietic stem cell disorders with erythroid dysplasia and mutations in the splicing factor gene. Patients with MDS with mutations often accumulate excessive tissue iron, even in the absence of transfusions, but the mechanisms that are responsible for their parenchymal iron overload are unknown. Body iron content, tissue distribution, and the supply of iron for erythropoiesis are controlled by the hormone hepcidin, which is regulated by erythroblasts through secretion of the erythroid hormone erythroferrone (ERFE). Here, we identified an alternative transcript in patients with MDS with the mutation. Induction of this transcript in primary -mutated bone marrow erythroblasts generated a variant protein that maintained the capacity to suppress hepcidin transcription. Plasma concentrations of ERFE were higher in patients with MDS with an gene mutation than in patients with wild-type MDS. Thus, hepcidin suppression by a variant ERFE is likely responsible for the increased iron loading in patients with -mutated MDS, suggesting that ERFE could be targeted to prevent iron-mediated toxicity. The expression of the variant transcript that was restricted to -mutated erythroblasts decreased in lenalidomide-responsive anemic patients, identifying variant ERFE as a specific biomarker of clonal erythropoiesis.
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http://dx.doi.org/10.1126/scitranslmed.aav5467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005358PMC
July 2019

Ttc7a regulates hematopoietic stem cell functions while controlling the stress-induced response.

Haematologica 2020 01 19;105(1):59-70. Epub 2019 Apr 19.

Laboratory of Normal and Pathological Homeostasis of the Immune System, INSERM UMR 1163, Imagine Institute, Paris

The molecular machinery that regulates the balance between self-renewal and differentiation properties of hematopoietic stem cells (HSC) has yet to be characterized in detail. Here we found that the tetratricopeptide repeat domain 7 A (Ttc7a) protein, a putative scaffold protein expressed by HSC, acts as an intrinsic regulator of the proliferative response and the self-renewal potential of murine HSC Loss of Ttc7a consistently enhanced the competitive repopulating ability of HSC and their intrinsic capacity to replenish the hematopoietic system after serial cell transplantations, relative to wildtype cells. Ttc7a-deficient HSC exhibit a different transcriptomic profile for a set of genes controlling the cellular response to stress, which was associated with increased proliferation in response to chemically induced stress and myeloablative stress Our results therefore revealed a previously unrecognized role of Ttc7a as a critical regulator of HSC stemness. This role is related, at least in part, to regulation of the endoplasmic reticulum stress response.
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http://dx.doi.org/10.3324/haematol.2018.207100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939534PMC
January 2020

The class 3 PI3K coordinates autophagy and mitochondrial lipid catabolism by controlling nuclear receptor PPARα.

Nat Commun 2019 04 5;10(1):1566. Epub 2019 Apr 5.

Institut Necker-Enfants Malades (INEM), 75014, Paris, France.

The class 3 phosphoinositide 3-kinase (PI3K) is required for lysosomal degradation by autophagy and vesicular trafficking, assuring nutrient availability. Mitochondrial lipid catabolism is another energy source. Autophagy and mitochondrial metabolism are transcriptionally controlled by nutrient sensing nuclear receptors. However, the class 3 PI3K contribution to this regulation is unknown. We show that liver-specific inactivation of Vps15, the essential regulatory subunit of the class 3 PI3K, elicits mitochondrial depletion and failure to oxidize fatty acids. Mechanistically, transcriptional activity of Peroxisome Proliferator Activated Receptor alpha (PPARα), a nuclear receptor orchestrating lipid catabolism, is blunted in Vps15-deficient livers. We find PPARα repressors Histone Deacetylase 3 (Hdac3) and Nuclear receptor co-repressor 1 (NCoR1) accumulated in Vps15-deficient livers due to defective autophagy. Activation of PPARα or inhibition of Hdac3 restored mitochondrial biogenesis and lipid oxidation in Vps15-deficient hepatocytes. These findings reveal roles for the class 3 PI3K and autophagy in transcriptional coordination of mitochondrial metabolism.
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http://dx.doi.org/10.1038/s41467-019-09598-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451001PMC
April 2019

Early Acute Microvascular Kidney Transplant Rejection in the Absence of Anti-HLA Antibodies Is Associated with Preformed IgG Antibodies against Diverse Glomerular Endothelial Cell Antigens.

J Am Soc Nephrol 2019 04 8;30(4):692-709. Epub 2019 Mar 8.

Bristol Renal, Bristol Heart Institute, Translational Health Sciences, Bristol Medical School, University of Bristol, Great Britain.

Background: Although anti-HLA antibodies (Abs) cause most antibody-mediated rejections of renal allografts, non-anti-HLA Abs have also been postulated to contribute. A better understanding of such Abs in rejection is needed.

Methods: We conducted a nationwide study to identify kidney transplant recipients without anti-HLA donor-specific Abs who experienced acute graft dysfunction within 3 months after transplantation and showed evidence of microvascular injury, called acute microvascular rejection (AMVR). We developed a crossmatch assay to assess serum reactivity to human microvascular endothelial cells, and used a combination of transcriptomic and proteomic approaches to identify non-HLA Abs.

Results: We identified a highly selected cohort of 38 patients with early acute AMVR. Biopsy specimens revealed intense microvascular inflammation and the presence of vasculitis (in 60.5%), interstitial hemorrhages (31.6%), or thrombotic microangiopathy (15.8%). Serum samples collected at the time of transplant showed that previously proposed anti-endothelial cell Abs-angiotensin type 1 receptor (AT1R), endothelin-1 type A and natural polyreactive Abs-did not increase significantly among patients with AMVR compared with a control group of stable kidney transplant recipients. However, 26% of the tested AMVR samples were positive for AT1R Abs when a threshold of 10 IU/ml was used. The crossmatch assay identified a common IgG response that was specifically directed against constitutively expressed antigens of microvascular glomerular cells in patients with AMVR. Transcriptomic and proteomic analyses identified new targets of non-HLA Abs, with little redundancy among individuals.

Conclusions: Our findings indicate that preformed IgG Abs targeting non-HLA antigens expressed on glomerular endothelial cells are associated with early AMVR, and that cell-based assays are needed to improve risk assessments before transplant.
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http://dx.doi.org/10.1681/ASN.2018080868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442343PMC
April 2019

Tetratricopeptide repeat domain 7A is a nuclear factor that modulates transcription and chromatin structure.

Cell Discov 2018 13;4:61. Epub 2018 Nov 13.

1Laboratory of Normal and Pathological Homeostasis of the Immune System, INSERM UMR 1163, Imagine Institute, Paris, France.

A loss-of-function mutation in tetratricopeptide repeat domain 7A (TTC7A) is a recently identified cause of human intestinal and immune disorders. However, clues to related underlying molecular dysfunctions remain elusive. It is now shown based on the study of TTC7A-deficient and wild-type cells that TTC7A is an essential nuclear protein. It binds to chromatin, preferentially at actively transcribed regions. Its depletion results in broad range of epigenomic changes at proximal and distal transcriptional regulatory elements and in altered control of the transcriptional program. Loss of WT_TTC7A induces general decrease in chromatin compaction, unbalanced cellular distribution of histones, higher nucleosome accessibility to nuclease digestion along with genome instability, and reduced cell viability. Our observations characterize for the first time unreported functions for TTC7A in the nucleus that exert a critical role in chromatin organization and gene regulation to safeguard healthy immune and intestinal status.
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http://dx.doi.org/10.1038/s41421-018-0061-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232119PMC
November 2018

Role of in neural stem cell differentiation and neural lineage determination: relevance for neurodevelopmental disorders.

Mol Autism 2018 19;9:38. Epub 2018 Jun 19.

1INSERM UMR 1163, Laboratory of Molecular and pathophysiological bases of cognitive disorders, Imagine Institute, Necker-Enfants Malades Hospital, 24 Boulevard du Montparnasse, 75015 Paris, France.

Background: MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. miRNAs have emerged as important modulators of brain development and neuronal function and are implicated in several neurological diseases. Previous studies found upregulation is the most common miRNA deregulation event in neurodevelopmental disorders such as autism spectrum disorder (ASD), epilepsy, and intellectual disability (ID). Yet, how upregulation affects the developing fetal brain remains unclear.

Methods: We analyzed the expression of in the temporal lobe of ASD children using Taqman assay. To assess the role of in early brain development, we generated and characterized stably induced H9 human neural stem cell (H9 hNSC) overexpressing using various cell and molecular biology techniques.

Results: We first showed that upregulation occurs early during childhood in the ASD brain. In H9 hNSC, overexpression enhances neurite outgrowth and branching and favors differentiation into neuronal like cells. Expression analyses revealed that 10% of the transcriptome was deregulated and organized into two modules critical for cell cycle control and neuronal differentiation. Twenty known or predicted targets of were significantly deregulated in the modules, acting as potential drivers. The two modules also display distinct transcription profiles during human brain development, affecting regions relevant for ASD including the neocortex, amygdala, and hippocampus. Cell type analyses indicate markers for pyramidal, and interneurons are highly enriched in the deregulated gene list. Up to 40% of known markers of newly defined neuronal lineages were deregulated, suggesting that could participate also in the acquisition of neuronal identities.

Conclusion: Our results demonstrate the dynamic roles of in early neuronal development and provide new insight into the molecular events that link overexpression to impaired neurodevelopment. This, in turn, may yield new therapeutic targets and strategies.
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http://dx.doi.org/10.1186/s13229-018-0219-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6011198PMC
October 2018

Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin.

Nat Commun 2018 02 22;9(1):773. Epub 2018 Feb 22.

INSERM UMR1163, Imagine Institute, Paris Descartes University, 75015, Paris, France.

Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, postnatally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.
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http://dx.doi.org/10.1038/s41467-018-03124-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823889PMC
February 2018

Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors.

Eur Heart J 2018 05;39(20):1835-1847

Institut National de la Santé et de la Recherche Médicale (INSERM), UMRS-970, Paris Centre de Recherche Cardiovasculaire, 56, rue Leblanc, 75015 Paris, France.

Aims: We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act.

Methods And Results: The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P < 0.001; left ventricular end diastolic volume: -4%, P = 0.002), and increased LVEF (+14%, P < 0.0001) relative to baseline values. Gene profiling revealed that EV-treated hearts were enriched for tissue reparative pathways.

Conclusion: Extracellular vesicles secreted by iPSC-Pg are effective in the treatment of CHF, possibly, in part, through their specific miRNA signature and the associated stimulation of distinct cardioprotective pathways. The processing and regulatory advantages of EV could make them effective substitutes for cell transplantation.
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http://dx.doi.org/10.1093/eurheartj/ehy012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251654PMC
May 2018

BAFF and CD4 T cells are major survival factors for long-lived splenic plasma cells in a B-cell-depletion context.

Blood 2018 04 29;131(14):1545-1555. Epub 2018 Jan 29.

Institut Necker-Enfants Malades-INSERM U1151/Centre National de la Recherche (CNRS) UMR8633, Université Paris Descartes, Faculté de Médecine, Paris, France.

Previous data have suggested that B-cell-depletion therapy may induce the settlement of autoreactive long-lived plasma cells (LLPCs) in the spleen of patients with autoimmune cytopenia. To investigate this process, we used the AID-CreERT2-EYFP mouse model to follow plasma cells (PCs) engaged in an immune response. Multiplex polymerase chain reaction at the single-cell level revealed that only a small fraction of splenic PCs had a long-lived signature, whereas PCs present after anti-CD20 antibody treatment appeared more mature, similar to bone marrow PCs. This observation suggested that, in addition to a process of selection, a maturation induced on B-cell depletion drove PCs toward a long-lived program. We showed that B-cell activating factor (BAFF) and CD4 T cells play a major role in the PC survival niche, because combining anti-CD20 with anti-BAFF or anti-CD4 antibody greatly reduce the number of splenic PCs. Similar results were obtained in the lupus-prone NZB/W model. These different contributions of soluble and cellular components of the PC niche in the spleen demonstrate that the LLPC expression profile is not cell intrinsic but largely depends on signals provided by the splenic microenvironment, implying that interfering with these components at the time of B-cell depletion might improve the response rate in autoimmune cytopenia.
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http://dx.doi.org/10.1182/blood-2017-06-789578DOI Listing
April 2018

Type I interferon-mediated autoinflammation due to DNase II deficiency.

Nat Commun 2017 12 19;8(1):2176. Epub 2017 Dec 19.

Pathology Department, Hôpital Necker-Enfants Malades, Assistance Publique-Hôpitaux de Paris, Paris, 75015, France.

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans.
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http://dx.doi.org/10.1038/s41467-017-01932-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736616PMC
December 2017

Generation of adult human T-cell progenitors for immunotherapeutic applications.

J Allergy Clin Immunol 2018 04 5;141(4):1491-1494.e4. Epub 2017 Dec 5.

Human Lymphohaematopoiesis Laboratory, INSERM U1163, Paris, France; University of Paris Descartes-Sorbonne Paris Cité, IMAGINE Institute, Paris, France; Biotherapy Clinical Investigation Centre, Necker Children's Hospital, Paris, France. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2017.10.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887055PMC
April 2018

UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells.

Nat Commun 2017 11 21;8(1):1640. Epub 2017 Nov 21.

Faculté de médecine Paris Descartes, Université Paris Descartes, 75015, Paris, France.

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8 T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs.
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http://dx.doi.org/10.1038/s41467-017-01601-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696382PMC
November 2017

Mutations in ACTRT1 and its enhancer RNA elements lead to aberrant activation of Hedgehog signaling in inherited and sporadic basal cell carcinomas.

Nat Med 2017 Oct 4;23(10):1226-1233. Epub 2017 Sep 4.

Genoscope (CEA), CNRS UMR 8030, University of Evry, Evry, France.

Basal cell carcinoma (BCC), the most common human cancer, results from aberrant activation of the Hedgehog signaling pathway. Although most cases of BCC are sporadic, some forms are inherited, such as Bazex-Dupré-Christol syndrome (BDCS)-a cancer-prone genodermatosis with an X-linked, dominant inheritance pattern. We have identified mutations in the ACTRT1 gene, which encodes actin-related protein T1 (ARP-T1), in two of the six families with BDCS that were examined in this study. High-throughput sequencing in the four remaining families identified germline mutations in noncoding sequences surrounding ACTRT1. These mutations were located in transcribed sequences encoding enhancer RNAs (eRNAs) and were shown to impair enhancer activity and ACTRT1 expression. ARP-T1 was found to directly bind to the GLI1 promoter, thus inhibiting GLI1 expression, and loss of ARP-T1 led to activation of the Hedgehog pathway in individuals with BDCS. Moreover, exogenous expression of ACTRT1 reduced the in vitro and in vivo proliferation rates of cell lines with aberrant activation of the Hedgehog signaling pathway. In summary, our study identifies a disease mechanism in BCC involving mutations in regulatory noncoding elements and uncovers the tumor-suppressor properties of ACTRT1.
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http://dx.doi.org/10.1038/nm.4368DOI Listing
October 2017

Klhl6 Deficiency Impairs Transitional B Cell Survival and Differentiation.

J Immunol 2017 10 14;199(7):2408-2420. Epub 2017 Aug 14.

Équipe Développement du Systéme Immunitaire, Institut Necker-Enfant Malades, INSERM U1151-CNRS UMR8253, Faculté de Médecine Paris Decartes, Université Paris Descartes, Sorbone Paris Cité, 75993 Paris Cedex 14, France;

Klhl6 belongs to the KLHL gene family, which is composed of an N-terminal BTB-POZ domain and four to six Kelch motifs in tandem. Several of these proteins function as adaptors of the Cullin3 E3 ubiquitin ligase complex. In this article, we report that Klhl6 deficiency induces, as previously described, a 2-fold reduction in mature B cells. However, we find that this deficit is centered on the inability of transitional type 1 B cells to survive and to progress toward the transitional type 2 B cell stage, whereas cells that have passed this step generate normal germinal centers (GCs) upon a T-dependent immune challenge. Klhl6-deficient type 1 B cells showed a 2-fold overexpression of genes linked with cell proliferation, including most targets of the anaphase-promoting complex/cyclosome complex, a set of genes whose expression is precisely downmodulated upon culture of splenic transitional B cells in the presence of BAFF. These results thus suggest a delay in the differentiation process of Klhl6-deficient B cells between the immature and transitional stage. We further show, in the BL2 Burkitt's lymphoma cell line, that KLHL6 interacts with Cullin3, but also that it binds to HBXIP/Lamtor5, a protein involved in cell-cycle regulation and cytokinesis. Finally, we report that KLHL6, which is recurrently mutated in B cell lymphomas, is an off-target of the normal somatic hypermutation process taking place in GC B cells in both mice and humans, thus leaving open whether, despite the lack of impact of Klhl6 deficiency on GC B cell expansion, mutants could contribute to the oncogenic process.
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http://dx.doi.org/10.4049/jimmunol.1700708DOI Listing
October 2017

Atorvastatin reduces β-Adrenergic dysfunction in rats with diabetic cardiomyopathy.

PLoS One 2017 20;12(7):e0180103. Epub 2017 Jul 20.

Sorbonne Universités, UPMC Univ Paris 06, UMR INSERM 1166, IHU ICAN, and Department of Anesthesiology and Critical Care Medicine, Hôpital Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris (APHP), Paris, France.

Background: In the diabetic heart the β-adrenergic response is altered partly by down-regulation of the β1-adrenoceptor, reducing its positive inotropic effect and up-regulation of the β3-adrenoceptor, increasing its negative inotropic effect. Statins have clinical benefits on morbidity and mortality in diabetic patients which are attributed to their "pleiotropic" effects. The objective of our study was to investigate the role of statin treatment on β-adrenergic dysfunction in diabetic rat cardiomyocytes.

Methods: β-adrenergic responses were investigated in vivo (echocardiography) and ex vivo (left ventricular papillary muscles) in healthy and streptozotocin-induced diabetic rats, who were pre-treated or not by oral atorvastatin over 15 days (50 mg.kg-1.day-1). Micro-array analysis and immunoblotting were performed in left ventricular homogenates. Data are presented as mean percentage of baseline ± SD.

Results: Atorvastatin restored the impaired positive inotropic effect of β-adrenergic stimulation in diabetic hearts compared with healthy hearts both in vivo and ex vivo but did not suppress the diastolic dysfunction of diabetes. Atorvastatin changed the RNA expression of 9 genes in the β-adrenergic pathway and corrected the protein expression of β1-adrenoceptor and β1/β3-adrenoceptor ratio, and multidrug resistance protein 4 (MRP4). Nitric oxide synthase (NOS) inhibition abolished the beneficial effects of atorvastatin on the β-adrenoceptor response.

Conclusions: Atorvastatin restored the positive inotropic effect of the β-adrenoceptor stimulation in diabetic cardiomyopathy. This effect is mediated by multiple modifications in expression of proteins in the β-adrenergic signaling pathway, particularly through the NOS pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180103PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519044PMC
September 2017

A rare castration-resistant progenitor cell population is highly enriched in Pten-null prostate tumours.

J Pathol 2017 09 28;243(1):51-64. Epub 2017 Jul 28.

Institut Necker Enfants Malades (INEM), Inserm U1151-CNRS UMR 8253, University Paris Descartes, Sorbonne Paris Cité, Faculty of Medicine, Paris, France.

Castration-resistant prostate cancer is a lethal disease. The cell type(s) that survive androgen deprivation remain poorly described, despite global efforts to understand the various mechanisms of therapy resistance. We recently identified in wild-type (WT) mouse prostates a rare population of luminal progenitor cells that we called LSC according to their FACS profile (Lin /Sca-1 /CD49f ). Here, we investigated the prevalence and castration resistance of LSC in various mouse models of prostate tumourigenesis (Pb-PRL, Pten , and Hi-Myc mice). LSC prevalence is low (∼8%, similar to WT) in Hi-Myc mice, where prostatic androgen receptor signalling is unaltered, but is significantly higher in the two other models, where androgen receptor signalling is decreased, rising up to more than 80% in Pten prostates. LSC tolerate androgen deprivation and persist or are enriched 2-3 weeks after castration. The tumour-initiating properties of LSC from Pten mice were demonstrated by regeneration of tumours in vivo. Transcriptomic analysis revealed that LSC represent a unique cell entity as their gene expression profile is different from luminal and basal/stem cells, but shares markers of each. Their intrinsic androgen signalling is markedly decreased, explaining why LSC tolerate androgen deprivation. This also illuminates why Pten tumours are castration-resistant since LSC represent the most prevalent cell type in this model. We validated CK4 as a specific marker for LSC on sorted cells and prostate tissues by immunostaining, allowing for the detection of LSC in various mouse prostate specimens. In castrated Pten prostates, there was significant proliferation of CK4 cells, further demonstrating their key role in castration-resistant prostate cancer progression. Taken together, this study identifies LSC as a probable source of prostate cancer relapse after androgen deprivation and as a new therapeutic target for the prevention of castrate-resistant prostate cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.4924DOI Listing
September 2017

Early born neurons are abnormally positioned in the doublecortin knockout hippocampus.

Hum Mol Genet 2017 01;26(1):90-108

INSERM UMR-S 839, Paris.

Human doublecortin (DCX) mutations are associated with severe brain malformations leading to aberrant neuron positioning (heterotopia), intellectual disability and epilepsy. The Dcx protein plays a key role in neuronal migration, and hippocampal pyramidal neurons in Dcx knockout (KO) mice are disorganized. The single CA3 pyramidal cell layer observed in wild type (WT) is present as two abnormal layers in the KO, and CA3 KO pyramidal neurons are more excitable than WT. Dcx KO mice also exhibit spontaneous epileptic activity originating in the hippocampus. It is unknown, however, how hyperexcitability arises and why two CA3 layers are observed.Transcriptome analyses were performed to search for perturbed postnatal gene expression, comparing Dcx KO CA3 pyramidal cell layers with WT. Gene expression changes common to both KO layers indicated mitochondria and Golgi apparatus anomalies, as well as increased cell stress. Intriguingly, gene expression analyses also suggested that the KO layers differ significantly from each other, particularly in terms of maturity. Layer-specific molecular markers and BrdU birthdating to mark the final positions of neurons born at distinct timepoints revealed inverted layering of the CA3 region in Dcx KO animals. Notably, many early-born 'outer boundary' neurons are located in an inner position in the Dcx KO CA3, superficial to other pyramidal neurons. This abnormal positioning likely affects cell morphology and connectivity, influencing network function. Dissecting this Dcx KO phenotype sheds light on coordinated developmental mechanisms of neuronal subpopulations, as well as gene expression patterns contributing to a bi-layered malformation associated with epilepsy.
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http://dx.doi.org/10.1093/hmg/ddw370DOI Listing
January 2017
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