mTOR Inhibitors Prevent CMV Infection through the Restoration of Functional αß and γδ T cells in Kidney Transplantation.

BACKGROUND: The reported association of mTOR-inhibitor (mTORi) treatment with a lower incidence of cytomegalovirus (CMV) infection in kidney transplant recipients (KTR) who are CMV seropositive (R+) remains unexplained. METHODS: The incidence of CMV infection and T-cell profile was compared between KTRs treated with mTORis and mycophenolic acid (MPA), and in vitro mTORi effects on T-cell phenotype and functions were analyzed. RESULTS: In KTRs who were R+ and treated with MPA, both αß and γδ T cells displayed a more dysfunctional phenotype (PD-1+, CD85j+) at day 0 of transplantation in the 16 KTRs with severe CMV infection, as compared with the 17 KTRs without or with spontaneously resolving CMV infection. In patients treated with mTORis (n=27), the proportion of PD-1+ and CD85j+ αß and γδ T cells decreased, when compared with patients treated with MPA (n=44), as did the frequency and severity of CMV infections. mTORi treatment also led to higher proportions of late-differentiated and cytotoxic γδ T cells and IFNγ-producing and cytotoxic αß T cells. In vitro, mTORis increased proliferation, viability, and CMV-induced IFNγ production of T cells and decreased PD-1 and CD85j expression in T cells, which shifted the T cells to a more efficient EOMESlow Hobithigh profile. In γδ T cells, the mTORi effect was related to increased TCR signaling. CONCLUSION: Severe CMV replication is associated with a dysfunctional T-cell profile and mTORis improve T-cell fitness along with better control of CMV. A dysfunctional T-cell phenotype could serve as a new biomarker to predict post-transplantation infection and to stratify patients who should benefit from mTORi treatment. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Proportion of CMV Seropositive Kidney Transplant Recipients Who Will Develop a CMV Infection When Treated With an Immunosuppressive Regimen Including Everolimus and Reduced Dose of Cyclosporine Versus an Immunosuppressive Regimen With Mycophenolic Acid and Standard Dose of Cyclosporine A (EVERCMV), NCT02328963.

CD154 Induces Interleukin-6 Secretion by Kidney Tubular Epithelial Cells under Hypoxic Conditions: Inhibition by Chloroquine.

Inflammation is a major contributor to tubular epithelium injury in kidney disorders, and the involvement of blood platelets in driving inflammation is increasingly stressed. CD154, the ligand of CD40, is one of the mediators supporting platelet proinflammatory properties. Although hypoxia is an essential constituent of the inflammatory reaction, if and how platelets and CD154 regulate inflammation in hypoxic conditions remain unclear. Here, we studied the control by CD154 of the proinflammatory cytokine interleukin- (IL-) 6 secretion in short-term oxygen (O2) deprivation conditions, using the HK-2 cell line as a kidney tubular epithelial cell (TEC) model. IL-6 secretion was markedly stimulated by CD154 after 1 to 3 hours of hypoxic stress. Both intracellular IL-6 expression and secretion were stimulated by CD154 and associated with a strong upregulation of IL-6 mRNA and increased transcription. Searching for inhibitors of CD154-mediated IL-6 production by HK-2 cells in hypoxic conditions, we observed that chloroquine, a drug that has been repurposed as an anti-inflammatory agent, alleviated this induction. Therefore, CD154 is a potent early stimulus for IL-6 secretion by TECs in O2 deprivation conditions, a mechanism likely to take part in the deleterious inflammatory consequences of platelet activation in kidney tubular injury. The inhibition of CD154-induced IL-6 production by chloroquine suggests the potential usefulness of this drug as a therapeutic adjunct in conditions associated with acute kidney injury.

MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate.

Pluripotent mouse embryonic stem cells (mESCs), maintained in the presence of the leukemia inhibitory factor (LIF) cytokine, provide a powerful model with which to study pluripotency and differentiation programs. Extensive microarray studies on cultured cells have led to the identification of three LIF signatures. Here we focus on muscle ras oncogene homolog (MRAS), which is a small GTPase of the Ras family encoded within the Pluri gene cluster. To characterise the effects of Mras on cell pluripotency and differentiation, we used gain- and loss-of-function strategies in mESCs and in the Xenopus laevis embryo, in which Mras gene structure and protein sequence are conserved. We show that persistent knockdown of Mras in mESCs reduces expression of specific master genes and that MRAS plays a crucial role in the downregulation of OCT4 and NANOG protein levels upon differentiation. In Xenopus, we demonstrate the potential of Mras to modulate cell fate at early steps of development and during neurogenesis. Overexpression of Mras allows gastrula cells to retain responsiveness to fibroblast growth factor (FGF) and activin. Collectively, these results highlight novel conserved and pleiotropic effects of MRAS in stem cells and early steps of development.

The "StemDif Sensor Test": A Straightforward, Non-Invasive Assay to Characterize the Secreted Stemness and/or Differentiation Activities of Tumor-Derived Cancer Cell Lines.

Cancer stem cells are a subpopulation of tumor cells characterized by their ability to self-renew, induce tumors upon engraftment in animals and exhibit strong resistance to chemotherapy and radiotherapy. These cells exhibit numerous characteristics in common with embryonic stem cells, expressing some of their markers, typically absent in non-pathological adult differentiated cells. The aim of this study was to investigate the potential of conditioned media from cancer stem cells to modulate the fate of Leukemia Inhibitory Factor (LIF)-dependent murine embryonic stem cells (mESCs) as a way to obtain a direct readout of the secretome of cancer cells. A functional assay, "the StemDif sensor test", was developed with two types of cancer stem cells derived from grade IV glioblastoma (adult and pediatric) or from gastric adenocarcinoma. We show that conditioned media from the selection of adult but not pediatric Glioma-Inducing Cells (GICs) maintain mESCs' pluripotency in correlation with LIF secretion and activation of STAT3 protein. In contrast, conditioned media from gastric adenocarcinoma cells display LIF-independent stemness and differentiation activities on mESC. Our test stands out for its user-friendly procedures, affordability and straightforward output, positioning it as a pioneering tool for in-depth exploration of cancer stem cell secretome characteristics.

Uncovering the Anticancer Potential of Murine Cytomegalovirus against Human Colon Cancer Cells.

Human cytomegalovirus (HCMV) components are often found in tumors, but the precise relationship between HCMV and cancer remains a matter of debate. Pro-tumor functions of HCMV were described in several studies, but an association between HCMV seropositivity and reduced cancer risk was also evidenced, presumably relying on recognition and killing of cancer cells by HCMV-induced lymphocytes. This study aimed at deciphering whether CMV influences cancer development in an immune-independent manner. Using immunodeficient mice, we showed that systemic infection with murine CMV (MCMV) inhibited the growth of murine carcinomas. Surprisingly, MCMV, but not HCMV, also reduced human colon carcinoma development in vivo. In vitro, both viruses infected human cancer cells. Expression of human interferon-ß (IFN-ß) and nuclear domain (ND10) were induced in MCMV-infected, but not in HCMV-infected human colon cancer cells. These results suggest a decreased capacity of MCMV to counteract intrinsic defenses in the human cellular host. Finally, immunodeficient mice receiving peri-tumoral MCMV therapy showed a reduction of human colon cancer cell growth, albeit no clinical sign of systemic virus dissemination was evidenced. Our study, which describes a selective advantage of MCMV over HCMV to control human colon cancer, could pave the way for the development of CMV-based therapies against cancer.

Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives.

BACKGROUND: Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency. RESULTS: By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii) LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of Pluri and Lifind genes, and revealed their differential modulation by Oct4 or Nanog master genes. Individual knock down of a selection of Pluri and Lifind genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed. CONCLUSION: We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment), which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG) is still expressed and which might be down-regulated together for driving cells towards differentiation.

Overexpression of Leap2 impairs Xenopus embryonic development and modulates FGF and activin signals.

Besides its widely described function in the innate immune response, no other clear physiological function has been attributed so far to the Liver-Expressed-Antimicrobial-Peptide 2 (LEAP2). We used the Xenopus embryo model to investigate potentially new functions for this peptide. We identified the amphibian leap2 gene which is highly related to its mammalian orthologues at both structural and sequence levels. The gene is expressed in the embryo mostly in the endoderm-derived tissues. Accordingly it is induced in pluripotent animal cap cells by FGF, activin or a combination of vegT/ß-catenin. Modulating leap2 expression level by gain-of-function strategy impaired normal embryonic development. When overexpressed in pluripotent embryonic cells derived from blastula animal cap explant, leap2 stimulated FGF while it reduced the activin response. Finally, we demonstrate that LEAP2 blocks FGF-induced migration of HUman Vascular Endothelial Cells (HUVEC). Altogether these findings suggest a model in which LEAP2 could act at the extracellular level as a modulator of FGF and activin signals, thus opening new avenues to explore it in relation with cellular processes such as cell differentiation and migration.

LIF-dependent signaling: new pieces in the Lego.

LIF, a member of the IL6 family of cytokine, displays pleiotropic effects on various cell types and organs. Its critical role in stem cell models (e.g.: murine ES, human mesenchymal cells) and its essential non redundant function during the implantation process of embryos, in eutherian mammals, put this cytokine at the core of many studies aiming to understand its mechanisms of action, which could benefit to medical applications. In addition, its conservation upon evolution raised the challenging question concerning the function of LIF in species in which there is no implantation. We present the recent knowledge about the established and potential functions of LIF in different stem cell models, (embryonic, hematopoietic, mesenchymal, muscle, neural stem cells and iPSC). We will also discuss EVO-DEVO aspects of this multifaceted cytokine.

The LIF cytokine: towards adulthood.

The aim of this article is to recapitulate the key features of leukaemia inhibitory factor cytokine (LIF), to review its numerous physiological effects and to comment on the most recent data. We will also present results of transcriptome analyses, which have highlighted different categories of LIF targets, identified in murine embryonic stem (ES) cells and early derivatives. We hope to stimulate new research fields on this puzzling cytokine, which, forty years after its discovery, has still not disclosed all its secrets.