Because phosphoinositide 3-kinase (PI3K) plays a central role in cellular activation, proliferation, and survival, pharmacologic inhibitors targeting components of the PI3K pathway are actively being developed as therapeutics for the treatment of inflammatory disorders and cancer. These targeted drugs inhibit the activity of either PI3K itself or downstream protein kinases. However, a previously unexplored, alternate strategy is to activate the negative regulatory phosphatases in this pathway. The SH2-containing inositol-5-phosphatase SHIP1 is a normal physiologic counterregulator of PI3K in immune/hematopoietic cells that hydrolyzes the PI3K product phosphatidylinositiol-3,4,5-trisphosphate (PIP 3 ). We now describe the identification and characterization of potent and specific small-molecule activators of SHIP1. These compounds represent the first small-molecule activators of a phosphatase, and are able to activate recombinant SHIP1 enzyme in vitro and stimulate SHIP1 activity in intact macrophage and mast cells. Mechanism of activation studies with these compounds suggest that they bind a previously undescribed, allosteric activation domain within SHIP1. Furthermore, in vivo administration of these compounds was protective in mouse models of endotoxemia and acute cutaneous anaphylaxis, suggesting that SHIP1 agonists could be used therapeutically to inhibit the PI3K pathway. IntroductionIn response to extracellular signals, phosphoinositide 3-kinase (PI3K) becomes activated and phosphorylates phosphatidylinositol-4,5-bisphosphate (PI-4,5-P 2 ) within the plasma membrane to generate phosphatidylinositol-3,4,5-bisphosphate (PIP 3 ). PIP 3 then initiates a cascade of downstream signaling pathways by interacting with pleckstrin homology (PH) domain-containing proteins, such as protein kinase B (PKB, also known as Akt), that regulate cellular activation, proliferation and/or survival, depending on the cell type and stimulus. 1 Cellular levels of PIP 3 are normally tightly regulated by both PI3K and the 5Ј inositol phosphatases SHIP1 (SH2 domain-containing inositol phosphatase) and SHIP2, as well as the 3Ј inositol phosphatase PTEN, which dephosphorylates PIP 3 . 2,3 Of these, SHIP1 is unique in that its expression is restricted primarily to immune and hematopoietic cells. 2,4 SHIP's role in immune cell homeostasis is shown both by the myeloproliferative syndrome observed in SHIP1 Ϫ/Ϫ mice, as well as the hypersensitivity of SHIP1 Ϫ/Ϫ mice and cells to immune stimulation. 5,6 SHIP1 mediates signaling from the inhibitory Fc␥RIIB receptor, 7 and is important in terminating signal transduction from activating immune/hematopoietic cell receptor systems. 8 Diminished SHIP1 activity or expression has been observed in human inflammatory diseases 9 and hematopoietic malignancies. [10][11][12][13] Because dysregulated activation of the PI3K pathway contributes to inflammatory/immune disorders and cancer, intense efforts have been invested into the development of inhibitors of PI3K itself, as well as downstream protein kinas...

The hematopoietic system produces a large number of highly specialized cell types that are derived through a hierarchical differentiation process from a common stem cell population. miRNAs are critical players in orchestrating this differentiation. Here, we report the development and application of a high-throughput microfluidic real-time quantitative PCR (RT-qPCR) approach for generating global miRNA profiles for 27 phenotypically distinct cell populations isolated from normal adult mouse hematopoietic tissues. A total of 80,000 RT-qPCR assays were used to map the landscape of miRNA expression across the hematopoietic hierarchy, including rare progenitor and stem cell populations. We show that miRNA profiles allow for the direct inference of cell lineage relations and functional similarity. Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage. The analysis of miRNA expression in single hematopoietic cells further demonstrates that miRNA expression is very tightly regulated within highly purified populations, underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity.RT-qPCR | stem cell | hematopoiesis | microfluidic | single cell

There is a great deal of interest in determining what regulates the generation of classically activated (M1) vs alternatively activated (M2) macrophages (Mφs) because of the opposing effects that these two Mφ subsets have on tumor progression. We show herein that IL-3 and, to a lesser extent, GM-CSF skew murine Mφ progenitors toward an M2 phenotype, especially in the absence of SHIP. Specifically, the addition of these cytokines, with or without M-CSF, to adherence- or lineage-depleted (Lin−) SHIP−/− bone marrow (BM) cells induces high levels of the M2 markers, arginase I, and Ym1 in the resulting mature Mφs. These in vitro-derived mature Mφs also display other M2 characteristics, including an inability to enhance anti-CD3-stimulated splenic T cell secretion of IFN-γ and low IL-12 and high IL-10 production in response to LPS. Not surprisingly, given that IL-3 and GM-CSF utilize STAT5 to trigger many downstream signaling pathways, this M2 phenotype is suppressed when STAT5−/− BM cells are used. Unexpectedly, however, this M2 phenotype is also suppressed when STAT6−/− BM cells are used, suggesting that IL-4- or IL-13-induced signaling might be involved. Consistent with this, we found that IL-3 and GM-CSF stimulate the production of IL-4, especially from SHIP−/− Lin− BM cells, and that neutralizing anti-IL-4 Abs block IL-3-induced M2 skewing. Moreover, we found that basophil progenitors within the Lin− BM are responsible for this IL-3- and GM-CSF-induced IL-4 production, and that SHIP represses M2 skewing not by preventing skewing within Mφs themselves but by inhibiting IL-4 production from basophils.

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The SH2-containing inositol-5'-phosphatase, SHIP, represses the proliferation, survival, and activation of hematopoietic cells, in large part by translocating to membranes following extracellular stimulation and hydrolysing the phosphatidylinositol-3-kinase (PI3K)-generated second messenger PI-3,4,5-P3 (PIP3) to PI-3,4-P2. SHIP-/- mice have, as a result, an increased number of monocyte/macrophages because their progenitors display enhanced survival and proliferation, as well as more rapid differentiation. Interestingly, SHIP-/- mice do not display lipopolysaccharide (LPS)- or CpG oligonucleotide-induced tolerance because this blunting of inflammatory mediator production is contingent upon LPS- and CpG-induced upregulation of SHIP in their macrophages and mast cells. This upregulation is mediated via the production of autocrine-acting TGFbeta which is induced via the MyD88-dependent pathway. The increased levels of SHIP then inhibit both MyD88-dependent and independent signaling. Intriguingly, SHIP-/- peritoneal and alveolar macrophages produce less nitric oxide (NO) than wild-type macrophages because they have constitutively high arginase I levels and this enzyme competes with inducible nitric oxide synthase (iNOS) for the substrate L-arginine. It is likely that, in the face of chronically elevated PIP3 levels in their myeloid progenitors, SHIP-/- mice display a skewed development away from M1 (killer) macrophages towards M2 (healing) macrophages. This suggests that SHIP plays a critical role in programming macrophages.

Material Supplementary 0.DC1http://www.jimmunol.org/content/suppl/2010/02/12/jimmunol.090317

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