Elevating Akt activation is an obvious clinical strategy to prevent progressive neuronal death in neurological diseases. However, this endeavor has been hindered because of the lack of specific Akt activators. Here, from a cell-based high-throughput chemical genetic screening, we identified a small molecule SC79 that inhibits Akt membrane translocation, but paradoxically activates Akt in the cytosol. SC79 specifically binds to the PH domain of Akt. SC79-bound Akt adopts a conformation favorable for phosphorylation by upstream protein kinases. In a hippocampal neuronal culture system and a mouse model for ischemic stroke, the cytosolic activation of Akt by SC79 is sufficient to recapitulate the primary cellular function of Akt signaling, resulting in augmented neuronal survival. Thus, SC79 is a unique specific Akt activator that may be used to enhance Akt activity in various physiological and pathological conditions.

Summary The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis, and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, however the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.

Summary Scratching triggers skin flares in atopic dermatitis (AD). We demonstrate that scratching of human skin, and tape stripping of mouse skin, causes neutrophil influx. This influx in mice was largely dependent on the generation of leukotriene B4 (LTB4) by neutrophils and their expression of the LTB4 receptor BLT1. Allergic skin inflammation in response to epicutaneous (EC) application of ovalbumin to tape-stripped skin was severely impaired in Ltb4r1−/− mice, and required expression of BLT1 on both T cells and non-T cells. Co-transfer of WT neutrophils, but not neutrophils deficient in BLT1 or the LTB4 synthesizing enzyme LTA4H, restored the ability of WT CD4+ effector T cells to transfer allergic skin inflammation to Ltb4r1−/− recipients. Pharmacologic blockade of LTB4 synthesis inhibited allergic skin inflammation elicited by cutaneous antigen challenge in previously EC-sensitized mice. Our results demonstrate that a neutrophil-T cell axis reliant on LTB4-BLT1 interaction is required for allergic skin inflammation.

The centrosome-nucleus attachment is a prerequisite for faithful chromosome segregation during mitosis. We addressed the function of the nuclear envelope (NE) protein Sun-1 in centrosome-nucleus connection and the maintenance of genome stability in Dictyostelium discoideum. We provide evidence that Sun-1 requires direct chromatin binding for its inner nuclear membrane targeting. Truncation of the cryptic N-terminal chromatinbinding domain of Sun-1 induces dramatic separation of the inner from the outer nuclear membrane and deformations in nuclear morphology, which are also observed using a Sun-1 RNAi construct. Thus, chromatin binding of Sun-1 defines the integrity of the nuclear architecture. In addition to its role as a NE scaffold, we find that abrogation of the chromatin binding of Sun-1 dissociates the centrosome-nucleus connection, demonstrating that Sun-1 provides an essential link between the chromatin and the centrosome. Moreover, loss of the centrosomenucleus connection causes severe centrosome hyperamplification and defective spindle formation, which enhances aneuploidy and cell death significantly. We highlight an important new aspect for Sun-1 in coupling the centrosome and nuclear division during mitosis to ensure faithful chromosome segregation.Key words: aneuploidy, centrosome hyperamplification, nuclear envelope architecture, spindle formation defects, Unc-84 The nuclear envelope (NE) separates the nuclear compartment from the cytoplasm. It is composed of two membranes: the outer nuclear membrane (ONM) and the inner nuclear membrane (INM). The lumen between the two membranes is the perinuclear space (PNS). The ONM is continuous with the endoplasmic reticulum (ER), whereas the INM harbors a unique set of proteins. INM and ONM proteins can interact within the PNS. Underneath the INM, the nuclear lamina is located, which is formed by intermediate filament (IF) proteins and associated proteins. The lamina forms the nucleoskeleton and associates with the INM, chromatin and nuclear pore complexes. Proteins of the NE have important roles. They are involved in nuclear migration and positioning and are essential for many processes such as mitosis, meiosis, differentiation and cell migration. Furthermore, several of the NE proteins have been associated with inherited diseases (1,2). Research in mammalian cells and in Caenorhabditis eleganshas identified conserved components of the NE that link the nucleoskeleton to the cytoskeleton. In C. elegans, two putative INM proteins, matefin/SUN-1 and UNC-84, bind to the nuclear lamina and extend their C-terminus into the PNS where they interact with the C-termini of KASH domain proteins (Klarsicht/Anc-1/Syne homology, designated KASH domain). Matefin/SUN-1 and UNC-84 belong to the SUN family of proteins based on the presence of the conserved SUN (Sad1/UNC-84 homology) domain at their C-terminus. KASH domain proteins are type II transmembrane proteins of the NE and have been identified as molecular linkers connecting the nucleus to actin filaments [filamentous acti...

Summary The cellular mechanisms controlling infection-induced emergency granulopoiesis are poorly defined. Here we found that reactive oxygen species (ROS) concentrations in the bone marrow (BM) were elevated during acute infection in a phagocytic NADPH oxidase-dependent manner in myeloid cells. Gr1+ myeloid cells were uniformly distributed in the BM, and all c-Kit+ progenitor cells were adjacent to Gr1+ myeloid cells. Inflammation-induced ROS production in the BM played a critical role in myeloid progenitor expansion during emergency granulopoiesis. ROS elicited oxidation and deactivation of phosphatase and tensin homolog (PTEN), resulting in up-regulation of PtdIns(3,4,5)P3 signaling in BM myeloid progenitors. We further revealed that BM myeloid cell-produced ROS stimulated proliferation of myeloid progenitors via a paracrine mechanism. Taken together, our results establish that phagocytic NADPH oxidase-mediated ROS production by BM myeloid cells plays a critical role in mediating emergency granulopoiesis during acute infection.

Inositol phosphates (InsP) are widely produced throughout animal and plant tissues. Diphosphoinositol pentakisphosphate (InsP7) contains an energetic pyrophosphate bond. Here, we demonstrate that disruption of InsP6K1, one of the three mammalian InsP6Ks that convert InsP6 to InsP7, confers enhanced PtdIns(3,4,5)P3-mediated membrane translocation of Akt pleckstrin homology (PH) domain and thus augments downstream PtdIns(3,4,5)P3 signaling in murine neutrophils. Consequently, these neutrophils exhibited elevated phagocytic and bactericidal capabilities and amplified NADPH oxidase-mediated superoxide production. These phenotypes were replicated in human primary neutrophils with pharmacologically inhibited InsP6Ks. By contrast, increasing intracellular InsP7 amounts blocked chemoattractant-elicited PH domain membrane translocation and dramatically suppressed PtdIns(3,4,5)P3-mediated cellular events in neutrophils. These findings establish a role for InsP7 in signal transduction and provide a mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.

The clinical outcome of granulocyte transfusion therapy is often hampered by short ex vivo shelf life, inefficiency of recruitment to sites of inflammation, and poor pathogen-killing capability of transplanted neutrophils. Here, using a recently developed mouse granulocyte transfusion model, we revealed that the efficacy of granulocyte transfusion can be significantly increased by elevating intracellular phosphatidylinositol (3,4,5)-trisphosphate signaling with a specific phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor SF1670. Neutrophils treated with SF1670 were much sensitive to chemoattractant stimulation. Neutrophil functions, such as phagocytosis, oxidative burst, polarization, and chemotaxis, were augmented after SF1670 treatment. The recruitment of SF1670-pretreated transfused neutrophils to the inflamed peritoneal cavity and lungs was significantly elevated. In addition, transfusion with SF1670-treated neutrophils led to augmented bacteria-killing capability (decreased bacterial burden) in neutropenic recipient mice in both peritonitis and bacterial pneumonia. Consequently, this alleviated the severity of and decreased the mortality of neutropenia-related pneumonia. Together, these observations demonstrate that the innate immune responses can be enhanced and the severity of neutropenia-related infection can be alleviated by augmenting phosphatidylinositol ( IntroductionSevere infection frequently occurs when the number of neutrophils in the blood is too low (neutropenia). 1,2 The blood of healthy adults contains approximately 1500 to 7000 neutrophils/mm 3 . Neutropenia is defined as an absolute neutrophil count (ANC) of Ͻ 1500/mm 3 for non-African Americans, or Ͻ 1200/mm 3 for African Americans. The risk of infection begins to increase at an ANC Ͻ1000/mm 3 . It is considered as severe neutropenia when the ANC falls below 500/mm 3 . One common cause of severe neutropenia is chemotherapy, which is extensively used to treat various hematologic malignancies and solid tumors. Neutropenia-associated infection is the most important dose-limiting toxicity of this therapeutic treatment, impacting on the quality of life and clinical outcomes, with the potential to cause death. [1][2][3] Neutropenia-related infections have been treated with broadspectrum antibiotic therapy and granulocyte colony-stimulating factor (G-CSF) therapy. However, not all patients respond to antibiotic treatment. G-CSF therapy often does not work before the bone marrow is recovered and is associated with side effects such as bone pain, headache, fatigue, and nausea. [1][2][3][4] Granulocyte transfusion also has been considered a therapeutic modality for lifethreatening bacterial and fungal infections in severe neutropenic patients. [5][6][7][8] The possibility of enhancing host immune defenses by infusion of neutrophil has been explored for Ͼ 70 years. Numerous studies demonstrated that transfusion of granulocyte concentrates obtained without growth factor stimulation or G-CSF-mobilized neutrophils is of benefit f...

Luo et al. report that CXCR2 ligands are responsible for rapid neutrophil mobilization during early-stage acute inflammation and that G-CSF suppresses this mobilization by negatively regulating CXCR2-mediated intracellular signaling.