The role of SHIP in the development and activation of mouse mucosal and connective tissue mast cells

Although SHIP is a well-established suppressor of IgE plus Ag-induced degranulation and cytokine production in bone marrow-derived mast cells (BMMCs), little is known about its role in connective tissue (CTMCs) or mucosal (MMCs) mast cells. In this study, we compared SHIP's role in the development as well as the IgE plus Ag and TLR-induced activation of CTMCs, MMCs, and BMMCs and found that SHIP delays the maturation of all three mast cell subsets and, surprisingly, that it is a positive regulator of IgE-induced BMMC survival. We also found that SHIP represses IgE plus Ag-induced degranulation of all three mast cell subsets and that TLR agonists do not trigger their degranulation, whether SHIP is present or not, nor do they enhance IgE plus Ag-induced degranulation. In terms of cytokine production, we found that in MMCs and BMMCs, which are poor producers of TLR-induced cytokines, SHIP is a potent negative regulator of IgE plus Ag-induced IL-6 and TNF-α production. Surprisingly, however, in splenic or peritoneal derived CTMCs, which are poor producers of IgE plus Ag-induced cytokines, SHIP is a potent positive regulator of TLR-induced cytokine production. Lastly, cell signaling and cytokine production studies with and without LY294002, wortmannin, and PI3Kα inhibitor-2, as well as with PI3K p85α(-/-) BMMCs and CTMCs, are consistent with SHIP positively regulating TLR-induced cytokine production via an adaptor-mediated pathway while negatively regulating IgE plus Ag-induced cytokine production by repressing the PI3K pathway

From nanoliter to large-scale bioreactors: How integrated technologies bring antibody treatments to patients faster

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Studien über die Funktion und Regulation von SHIP

The 145 kDa protein, SHIP, is a hemopoietic-restricted negative regulator of the PI3-Kinase pathway and plays a central role both in hemopoiesis and the activation of innate immune cells. Our goal in this thesis was to extend the well established role that SHIP plays in BMMC development and activation to other MC subsets. We found that SHIP affected MC properties in many different ways. For example, in the absence of SHIP, MCs generally matured more quickly and were more prone to degranulation. Interestingly, survival in the absence of growth factors was not affected by SHIP in most MC subsets but, in the presence of IgE, WT BMMCs survived far better than SHIP-/- BMMCs. However, there was no difference between WT and SHIP-/- MMCs. In addition, SHIP appeared to be a positive regulator of TLR-mediated cytokine production and a negative regulator of FcεR1-mediated activation. However, in WT BMMCs there was synergy between TLR and FcεR1 activation, which was not seen in WT MMCs, suggesting that even MCs from the same subset can acquire distinct properties during their differentiation. Also, the synergistic effects were the strongest in SHIP-/- cells, indicating an important role for SHIP in the integration and processing of different signals. We also found that SHIP was a potent negative regulator of IL-4 production. However, this likely was of more importance in basophils than MCs. Since SHIP activity is regulated in large part by its protein levels and since several cancers down-regulate SHIP to increase their survival we also explored how SHIP protein levels are down-regulated. Our studies with inducible BCR-ABL expressing cells and with IL-4-induced BMmacs, revealed that SHIP was degraded through the proteasome since treatment with the proteasome inhibitor MG-132 prevented SHIP protein levels from declining. We also found that the ubiquitin ligases Cbl and Cbl-b interacted with SHIP, suggesting they are involved in ubiquitinating SHIP. Studies in which we lysed BMmacs with non-ionic detergents revealed that SHIP was “set up” for rapid proteasomal degradation since MG-132 prevented lysis-induced SHIP degradation. Tyrosine phosphorylation of SHIP seemed to be required for its breakdown in both cell systems since its degradation was prevented by inhibiting Src kinases with PP2. Also, at least in BCR-ABL expressing cells, there was a strong inverse relationship between total and phosphorylated SHIP protein levels. We also found that induction of SHIP degradation required Stat6 signalling, at least in IL-4 treated BMmacs, since this effect was abrogated in Stat6-/- BMmacs. Thus, in this study we confirmed SHIP’s central role in MC development and FcεR1-mediated activation and extended these studies to TLR- mediated activation. We also identified key factors involved in inducing and mediating SHIP breakdown. This might be important in designing new therapeutic strategies that exploit SHIP’s central role in regulating immune responses.Das hämatopoetisch exprimierte Protein SHIP ist ein negativer Regulator des PI3-Kinase Signalweges und ein wichtiger Regulator der Blutzellenbildung sowie der Aktivierung von Zellen des angeborenen Immunsystems. Ein Ziel dieser Arbeit war es, die gut erforschte Rolle von SHIP in primären, vom Knochenmark (bone marrow, BM) differenzierten Mastzellen (MCs) auch in anderen MCmodellen zu analysieren. Es konnte gezeigt werden, dass SHIP die Eigenschaften von MCs auf verschiedene Art und Weise prägt. SHIP erwies sich als ein negativer Regulator der Differenzierung und Degranulierung von MCs, hatte aber keinen signifikanten Einfluss auf das Überleben von MCs in der Abwesenheit von Wachstumsfaktoren. Für die TLR Aktivierung war SHIP ein positiver und für die FcεR1 Aktivierung ein negativer Regulator. In BMMCs and MMCs, zwei verschiedenen Modellsystemen für den gleichen MCsubtyp, wurden zuweilen auch unterschiedliche Einflüsse von SHIP beobachtet. Dies könnte darauf Hinweisen, dass MCs vom gleichen Subtyp während ihrer Differenzierung unterschiedliche Eigenschaften erlangen können. SHIP hatte auch einen negativen Effekt auf die IL-4 Synthese, was aber wichtiger in Basophilen zu sein schien. SHIPs Phosphataseaktivität wird überwiegend durch dessen Expressionslevel reguliert, und einige Krebsarten regulieren SHIP herunter, um sich dadurch einen Überlebensvorteil zu verschaffen. Daher war es eine weitere Zielsetzung dieser Arbeit, mechanistische Einblicke, die zur Runterregulierung von SHIP führen, zu erlangen. Es konnte sowohl in BCR-ABL exprimierenden Ba/F3 Zellen, als auch in IL-4 behandelten, vom BM differenzierten, Makrophagen (BMmacs) gezeigt werden, dass SHIP durch das Proteasom abgebaut wird. Dieser Abbau wurde durch den Proteasominhibitor MG-132 unterdrueckt. Die Ubiquitinligasen Cbl und Cbl-b wurden als Kandidaten für die Ubiquitinierung von SHIP identifiziert. Lysierungsexperimente mit nichtionischen Detergenzien in BMmacs zeigten, dass SHIP sehr rapide durch das Proteasom abgebaut werden kann, da dies ebenfalls durch MG-132 inhibiert wurde. SHIPs Tyrosinphosphorylierung wurde in beiden untersuchten Zellsystemen als essentiell für dessen Abbau identifiziert, da der Src Kinaseinhibitor PP2 SHIPs Phosphorylierung und Abbau inhibierte. Zudem wurde in BCR-ABL exprimierenden Ba/F3 Zellen eine disproportionale Beziehung zwischen absoluten und phosphorylierten SHIP beobachtet. In BMmacs wurde der Stat6 Signalweg als essentiell für den IL-4 induzierten SHIP Abbau identifiziert. Im Rahmen dieser Arbeit haben wir die wichtige Funktion von SHIP in der Entwicklung und FcεR1 induzierten Aktivierung von MCs bestätigt und auf die TLR induzierte Aktivierung ausgeweitet. Es wurden auch erste Einblicke in die molekularen Mechanismen, die zum Abbau von SHIP führen, erlangt. Die Ergebnisse dieser Studie leisten somit einen wichtigen Beitrag in der Entwicklung von neuen Tumortherapien, die die zentrale Funktion von SHIP als Ansatzpunkt haben

MicroRNA-223 dose levels fine tune proliferation and differentiation in human cord blood progenitors and acute myeloid leukemia

A precise understanding of the role of miR-223 in human hematopoiesis and in the pathogenesis of acute myeloid leukemia (AML) is still lacking. By measuring miR-223 expression in blasts from 115 AML patients, we found significantly higher miR-223 levels in patients with favorable prognosis, whereas patients with low miR-223 expression levels were associated with worse outcome. Furthermore, miR-223 was hierarchically expressed in AML subpopulations, with lower expression in leukemic stem cell-containing fractions. Genetic depletion of miR-223 decreased the leukemia initiating cell (LIC) frequency in a myelomonocytic AML mouse model, but it was not mandatory for rapid-onset AML. To relate these observations to physiologic myeloid differentiation, we knocked down or ectopically expressed miR-223 in cord-blood CD34+ cells using lentiviral vectors. Although miR-223 knockdown delayed myeloerythroid precursor differentiation in vitro, it increased myeloid progenitors in vivo following serial xenotransplantation. Ectopic miR-223 expression increased erythropoiesis, T lymphopoiesis, and early B lymphopoiesis in vivo. These findings broaden the role of miR-223 as a regulator of the expansion/differentiation equilibrium in hematopoietic stem and progenitor cells where its impact is dose- and differentiation-stage-dependent. This also explains the complex yet minor role of miR-223 in AML, a heterogeneous disease with variable degree of myeloid differentiation

MicroRNA-223 dose levels fine tune proliferation and differentiation in human cord blood progenitors and acute myeloid leukemia

A precise understanding of the role of miR-223 in human hematopoiesis and in the pathogenesis of acute myeloid leukemia (AML) is still lacking. By measuring miR-223 expression in blasts from 115 AML patients, we found significantly higher miR-223 levels in patients with favorable prognosis, whereas patients with low miR-223 expression levels were associated with worse outcome. Furthermore, miR-223 was hierarchically expressed in AML subpopulations, with lower expression in leukemic stem cell–containing fractions. Genetic depletion of miR-223 decreased the leukemia initiating cell (LIC) frequency in a myelomonocytic AML mouse model, but it was not mandatory for rapid-onset AML. To relate these observations to physiologic myeloid differentiation, we knocked down or ectopically expressed miR-223 in cord-blood CD34(+) cells using lentiviral vectors. Although miR-223 knockdown delayed myeloerythroid precursor differentiation in vitro, it increased myeloid progenitors in vivo following serial xenotransplantation. Ectopic miR-223 expression increased erythropoiesis, T lymphopoiesis, and early B lymphopoiesis in vivo. These findings broaden the role of miR-223 as a regulator of the expansion/differentiation equilibrium in hematopoietic stem and progenitor cells where its impact is dose- and differentiation-stage-dependent. This also explains the complex yet minor role of miR-223 in AML, a heterogeneous disease with variable degree of myeloid differentiation