Identification of small RNAs in Staufen2 complexes from rat brain

MikroRNAs (miRNAs) sind ~22 nt lange, nicht kodierende RNAs, welche Gene post-transkriptionell regulieren können, indem sie die Translation hemmen oder den Abbau der mRNA auslösen. Im Nervensystem spielen miRNAs eine zentrale Rolle in der Entwicklung und wahrscheinlich auch bei der synaptischen Plastizität. Hierfür sind RNA-bindende Proteine (RBPs), welche mRNAs an die Synapsen lokalisieren, ebenso wichtig wie eine Stimulus-abhängige lokale Proteinsynthese. Während des Transports ist die Translation der mRNAs in neuronalen Ribonukleoprotein-Partikeln (RNPs) unterbunden, was entweder durch Regulator-Proteine oder durch miRNAs bewerkstelligt werden kann. Das im Kiebler Labor untersuchte Protein Staufen2 (Stau2) gehört zur Familie der dsRBPs und wird hauptsächlich im Nervensystem exprimiert. In der Taufliege Drosophila melanogaster spielt es eine Rolle bei der Gedächtnisbildung. In hippokampalen Neuronen stellen Staufen-Proteine wichtige Komponenten in RNA-enthaltenden Partikeln dar, welche sich bis in die distalen Dendriten erstrecken. Kürzlich wurde ein Zusammenhang zwischen Stau2 und miRNAs vorgeschlagen, da der Export von Stau2 aus dem Kern von Exportin-5 abhängig ist, dem primären Exportfaktor für Vorläufer-miRNAs. Deshalb war die Hauptaufgabe dieses Projekts zu untersuchen, ob kleine nicht-kodierende RNAs, im Besonderen miRNAs, in Stau2 Partikeln enthalten sind. Über Immunoprezipitationen mit affinitätsgereinigten Antikörpern gegen Stau2 gelang es, Stau2 RNPs mit allen enthaltenen Proteinen und RNAs zu isolieren. Auf Proteinebene wurden verschiedene RISC-assoziierte Komponenten wie Ago, PACT, FMRP und eIF6 in Stau2 Komplexen gefunden, was auf eine Beteiligung von miRNAs hindeutet. Auf RNA-Ebene gelang mittels radioaktiver Markierung von RNA der Nachweis kleiner RNAs mit einer Größe von 20-22 nt in den Stau2 Komplexen. Dies ist ein weiterer Hinweis auf einen Zusammenhang zwischen miRNAs und Stau2 Partikeln. Zusätzlich wurde das Vorhandensein von miR-18, 26a, 128a, 134 und let-7c in Stau2 Komplexen mittels RT-PCR nachgewiesen. In Folge soll die in den Stau2 Immunoprezipitationen vorkommende RNA kloniert und sequenziert werden, um eine vollständige Liste aller Stau2-assoziierten miRNAs zu erhalten. Eine Hypothese um den Zusammenhang zwischen miRNAs und Stau2 zu erklären, wäre dass miRNAs eventuell eine Funktion in der Inhibierung der Translation von mRNAs in den Stau2 Komplexen erfüllen. Während des Transports sind mRNAs in RNPs durch miRNAs gebunden, was die Translation verhindert. Dies wird später durch einen spezifischen Stimulus aufgehoben, welcher eine lokale Translation an der Synapse ermöglicht.microRNAs (miRNAs) are ~22 nt long non-coding RNAs which are involved in post-transcriptional gene regulation via mRNA silencing or degradation. miRNAs are abundant in the nervous system, where they have key roles in development and possibly in synaptic plasticity. Both RNA localization as well as stimulus-dependent local protein synthesis critically contribute to synaptic plasticity. During transport, the silencing of mRNAs within neuronal ribonucleoprotein particles (RNPs) can be achieved either by translational regulator-proteins or by miRNAs. Staufen2 (Stau2), an RNA-binding protein (RBP) investigated in the Kiebler lab, belongs to the family of dsRBPs and is mainly expressed in the nervous system. Staufen was shown to be involved in memory formation in Drosophila melanogaster. In hippocampal neurons, Staufen proteins are core components of distinct RNA-containing particles that reach out to distal dendrites. Recent evidence suggested a link between Stau2 and miRNAs. The export of Stau2 from the nucleus depends on Exportin-5, the main export factor for precursor-miRNAs. Therefore, this project aimed at investigating whether small non-coding RNAs, especially miRNAs, are part of Stau2-containing complexes in the rat brain. Immunoprecipitation, using affinity-purified anti-Stau2 antibodies, was performed to isolate Stau2 RNPs including all associated proteins and RNAs. On the protein level, the presence of different RISC-associated components like Ago, PACT, FMRP and eIF6 in Stau2 complexes suggested a participation of the miRNA pathway. On the RNA level, the specific association of small RNAs at the size of 20-22 nt with Stau2 RNPs in E17 rat brain provided further evidence for a connection between miRNAs and Stau2 complexes, as shown by radioactive labeling of RNA isolated after Stau2 immunoprecipitation. In addition, RT-PCR proved the presence of 5 candidate miRNAs, miR 18, 26a, 128a, 134 and let-7c, in Stau2 complexes. In the future, RNA isolated from Stau2 immunoprecipitations will be cloned and sequenced to obtain a complete list of miRNAs associated with Stau2 complexes. A hypothesis to explain this association between miRNAs and Stau2 would be that the miRNA translationally silences its target mRNAs during transport in Stau2 complexes. Upon a specific stimulus, the suppression would be relieved allowing translation to occur at the synapse

Transformation kinetics and microstructures of Ti17 titanium alloy during continuous cooling

International audienceWe have investigated the microstructure evolutions in the Ti17 near Click to view the MathML source titanium alloy during heat treatments. The phase transformation has first been studied experimentally by combining X-ray diffraction analysis, electrical resistivity and microscopy observations. From a series of isothermal treatments, a IT diagram has been determined, which takes into account the different morphologies. Then, a Johnson–Mehl–Avrami–Kolmogorov (JMAK) model has been successfully used to describe the phase transformation kinetics during either isothermal or cooling treatments. Finally, the coupling of the JMAK model to the finite element software ZeBuLoN allowed us to investigate the evolution of the spatial distribution of the different morphologies during the cooling of an aircraft engine shaft disk after forging

Graft-versus-host disease, but not graft-versus-leukemia immunity, is mediated by GM-CSF–licensed myeloid cells

Allogeneic hematopoietic cell transplantation (allo-HCT) not only is an effective treatment for several hematologic malignancies but can also result in potentially life-threatening graft-versus-host disease (GvHD). GvHD is caused by T cells within the allograft attacking nonmalignant host tissues; however, these same T cells mediate the therapeutic graft-versus-leukemia (GvL) response. Thus, there is an urgent need to understand how to mechanistically uncouple GvL from GvHD. Using preclinical models of full and partial MHC-mismatched HCT, we here show that the granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by allogeneic T cells distinguishes between the two processes. GM-CSF drives GvHD pathology by licensing donor-derived phagocytes to produce inflammatory mediators such as interleukin-1β and reactive oxygen species. In contrast, GM-CSF did not affect allogeneic T cells or their capacity to eliminate leukemic cells, retaining undiminished GvL responses. Last, tissue biopsies and peripheral blood mononuclear cells from patients with grade IV GvHD showed an elevation of GM-CSF–producing T cells, suggesting that GM-CSF neutralization has translational potential in allo-HCT

The IFN-γ-Inducible GTPase, Irga6, Protects Mice against Toxoplasma gondii but Not against Plasmodium berghei and Some Other Intracellular Pathogens

Clearance of infection with intracellular pathogens in mice involves interferon-regulated GTPases of the IRG protein family. Experiments with mice genetically deficient in members of this family such as Irgm1(LRG-47), Irgm3(IGTP), and Irgd(IRG-47) has revealed a critical role in microbial clearance, especially for Toxoplasma gondii. The in vivo role of another member of this family, Irga6 (IIGP, IIGP1) has been studied in less detail. We investigated the susceptibility of two independently generated mouse strains deficient in Irga6 to in vivo infection with T. gondii, Mycobacterium tuberculosis, Leishmania mexicana, L. major, Listeria monocytogenes, Anaplasma phagocytophilum and Plasmodium berghei. Compared with wild-type mice, mice deficient in Irga6 showed increased susceptibility to oral and intraperitoneal infection with T. gondii but not to infection with the other organisms. Surprisingly, infection of Irga6-deficient mice with the related apicomplexan parasite, P. berghei, did not result in increased replication in the liver stage and no Irga6 (or any other IRG protein) was detected at the parasitophorous vacuole membrane in IFN-γ-induced wild-type cells infected with P. berghei in vitro. Susceptibility to infection with T. gondii was associated with increased mortality and reduced time to death, increased numbers of inflammatory foci in the brains and elevated parasite loads in brains of infected Irga6-deficient mice. In vitro, Irga6-deficient macrophages and fibroblasts stimulated with IFN-γ were defective in controlling parasite replication. Taken together, our results implicate Irga6 in the control of infection with T. gondii and further highlight the importance of the IRG system for resistance to this pathogen

T-bet or not T-bet: Taking the last bow on the autoimmunity stage

The search for the encephalitogenic factor driving pathogenic T cells in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis (MS), and psoriasis has proven to be a long and difficult mission, which is not yet completed. In this issue of the European Journal of Immunology, the importance of the transcription factor T-bet, previously shown to be essential for the induction of autoimmune disease in mice, is challenged. Two independent groups, O'Connor et al. [Eur. J. Immunol. 2013. 43:2818-2823] report] and Grifka-Walk et al. [Eur. J. Immunol. 2013. 43:2824-2831], report that T-bet is not mandatory for T cells to cause experimental autoimmune encephalomyelitis (EAE), which serves as a paradigmatic T-cell-mediated autoimmune disease. Both groups found that T-bet KO mice were fully susceptible to develop EAE, both after immunization with self-antigen and after adoptive transfer of IL-23-polarized autoaggressive T cells. T-bet deficiency mediated the loss of IFN-γ expression but retained or even enhanced GM-CSF and IL-17 production by central nervous system (CNS)-infiltrating T cells. These findings indicate that we have lost the last transcriptional regulator previously held to be required for the generation of autoimmune pathogenic T cells

GM-CSF in Neuroinflammation: Licensing Myeloid Cells for Tissue Damage

Multiple sclerosis (MS) is the prototypical inflammatory disease of the central nervous system (CNS). MS lesions harbor different immune cells, but the contribution of individual cell types to disease etiology and progression is not well understood. In experimental autoimmune encephalomyelitis (EAE), auto-reactive helper T (Th) cells instigate CNS inflammation by acting on myeloid cells via the production of granulocyte-macrophage colony-stimulating factor (GM-CSF). Recent reports have implicated myeloid cells in both the inflammatory process and as executers of tissue damage in the CNS. We review these findings here, and integrate them into a model wherein GM-CSF produced by Th cells coordinates monocyte recruitment to the CNS, and differentiation into pathogenic effectors. We discuss the implications of this model to current therapies for MS, and outline important areas of further inquiry

Dysregulation of the Cytokine GM-CSF Induces Spontaneous Phagocyte Invasion and Immunopathology in the Central Nervous System

Chronic inflammatory diseases are influenced by dysregulation of cytokines. Among them, granulocyte macrophage colony stimulating factor (GM-CSF) is crucial for the pathogenic function of T cells in preclinical models of autoimmunity. To study the impact of dysregulated GM-CSF expression in vivo, we generated a transgenic mouse line allowing the induction of GM-CSF expression in mature, peripheral helper T (Th) cells. Antigen-independent GM-CSF release led to the invasion of inflammatory myeloid cells into the central nervous system (CNS), which was accompanied by the spontaneous development of severe neurological deficits. CNS-invading phagocytes produced reactive oxygen species and exhibited a distinct genetic signature compared to myeloid cells invading other organs. We propose that the CNS is particularly vulnerable to the attack of monocyte-derived phagocytes and that the effector functions of GM-CSF-expanded myeloid cells are in turn guided by the tissue microenvironment

Sorafenib promotes graft-versus-leukemia activity in mice and humans through IL-15 production in FLT3-ITD-mutant leukemia cells

Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD leukemia cells. This synergized with the allogeneic CD8 T cell response, leading to long-term survival in six mouse models of FLT3-ITD AML. Sorafenib-related IL-15 production caused an increase in CD8CD107aIFN-γ T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8 T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT

Prediction of the kinetics of the phase transformations and the associated microstructure during continuous coolingin the Ti17

2nd international conference on thermal process modelling and computer simulation, Nancy, 31 mars-2 avril 2003The aim of this paper is to present recent experimental results and related simulation about the $\beta$ -> $\alpha$GB + $\alpha$WGB and -> $\alpha$WI transformations which occur in the Ti17 alloy during the thermal treatments following the heating in the $\beta$ phase field. These phase transformations were experimentally studied under isothermal conditions in samples with negligible thermal gradients. The IT diagram was obtained, on the basis of electrical resistivity measurements and microstructural SEM observations. The kinetics of the phase transformation was further numerically simulated for continuous cooling on the basis of a formerly developed model giving the amount of each morphology ($\alpha$WGB, $\alpha$WI). Experimental and calculated results are compared

Helicobacter pylori-specific Protection Against Inflammatory Bowel Disease Requires the NLRP3 Inflammasome and IL-18

BACKGROUND The Gram-negative bacterium Helicobacter pylori is a constituent of the human gastric microbiota. Chronic infection with H. pylori causes gastritis and predisposes to gastric carcinoma but has also been inversely linked to various allergic and chronic inflammatory conditions. In particular, large meta-analyses have documented an inverse association between H. pylori infection and the risk of developing ulcerative colitis and Crohn's disease. METHODS We investigated possible protective effects of experimental H. pylori infection and of regular treatment with H. pylori extract in 2 mouse models of colitis and in mouse models of type I diabetes and multiple sclerosis. The mechanism of protection was examined in mouse strains lacking specific innate immune recognition pathways and cytokines. RESULTS We show here that experimental infection with H. pylori and administration of regular doses of H. pylori extract both alleviate the clinical and histopathological features of dextran sodium sulfate-induced chronic colitis and of T-cell transfer-induced colitis. High resolution endoscopy of the protected animals revealed the accumulation of large amounts of colonic mucus upon H. pylori exposure, which could be attributed to transcriptional activation of the mucin 2 gene. The protection against dextran sodium sulfate-induced colitis was dependent on the NLRP3 inflammasome and interleukin-18 signaling. Other autoimmune diseases, i.e., experimental autoimmune encephalomyelitis and type I diabetes, were not controlled by H. pylori. CONCLUSIONS In summary, we propose here that the immunomodulatory activity of an ancient constituent of the gut microbiota, H. pylori, may be exploited for the prevention and/or treatment of inflammatory bowel diseases