Person:

Silberstein, Leslie

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 (3,4,5)-trisphosphate in transfused neutrophils with PTEN inhibitor SF1670, providing a therapeutic strategy for improving the efficacy of granulocyte transfusion.

The phosphatidylinositol-3,4,5-triphosphate (PIP3) binding function of pleckstrin homology (PH) domain is essential for the activation of oncogenic Akt/PKB kinase. Following the PIP3-mediated activation at the membrane, the activated Akt is subjected to other regulatory events, including ubiquitination-mediated deactivation. Here, by identifying and characterizing an allosteric inhibitor, SC66, we show that the facilitated ubiquitination effectively terminates Akt signaling. Mechanistically, SC66 manifests a dual inhibitory activity that directly interferes with the PH domain binding to PIP3 and facilitates Akt ubiquitination. A known PH domain-dependent allosteric inhibitor, which stabilizes Akt, prevents the SC66-induced Akt ubiquitination. A cancer-relevant Akt1 (e17k) mutant is unstable, making it intrinsically sensitive to functional inhibition by SC66 in cellular contexts in which the PI3K inhibition has little inhibitory effect. As a result of its dual inhibitory activity, SC66 manifests a more effective growth suppression of transformed cells that contain a high level of Akt signaling, compared with other inhibitors of PIP3/Akt pathway. Finally, we show the anticancer activity of SC66 by using a soft agar assay as well as a mouse xenograft tumor model. In conclusion, in this study, we not only identify a dual-function Akt inhibitor, but also demonstrate that Akt ubiquitination could be chemically exploited to effectively facilitate its deactivation, thus identifying an avenue for pharmacological intervention in Akt signaling.

Hematopoietic stem cells (HSCs) reside in complex bone marrow microenvironments, where niche-induced signals regulate hematopoiesis. Focal adhesion kinase (Fak) is a nonreceptor protein tyrosine kinase that plays an essential role in many cell types, where its activation controls adhesion, motility, and survival. Fak expression is relatively increased in HSCs compared to progenitors and mature blood cells. Therefore, we explored its role in HSC homeostasis. We have used the Mx1-CreLinducible conditional knockout mouse model to investigate the effects of Fak deletion in bone marrow compartments. The total number as well as the fraction of cycling (Lin^-Sca-1^+c-kit^+) (LSK) cells is increased in (Fak^{-/-}) mice compared to controls, while hematopoietic progenitors and mature blood cells are unaffected. Bone marrow cells from (Fak^{-/-}) mice exhibit enhanced, long-term (i.e., 20-week duration) engraftment in competitive transplantation assays. Intrinsic Fak function was assessed in serial transplantation assays, which showed that HSCs ((Lin^-Sca-1^+c-kit^+CD34^-Flk-2^-) cells) sorted from (Fak^{-/-}) mice have similar self-renewal and engraftment ability on a per-cell basis as wild-type HSCs. When Fak deletion is induced after engraftment of (Fak^{fl/fl}Mx1-Cre^+) bone marrow cells into wild-type recipient mice, the number of LSKs is unchanged. In conclusion, Fak inactivation does not intrinsically regulate HSC behavior and is not essential for steady- state hematopoiesis. However, widespread Fak inactivation in the hematopoietic system induces an increased and activated HSC pool size, potentially as a result of altered reciprocal interactions between HSCs and their microenvironment.

A dysfunctional bone marrow (BM) microenvironment is thought to contribute to the development of hematologic diseases. However, functional replacement of pathologic BM microenvironment through BM transplantation has not been possible. Furthermore, the study of hematopoietic inductive BM microenvironment is hampered by the lack of a functional nonhematopoietic reconstitution system. Here, we show that a deficiency of SH2-containing inositol-5'-phosphatase-1 (SHIP) in a nonhematopoietic host microenvironment enables its functional reconstitution by wild-type donor cells. This microenvironment reconstitution normalizes hematopoiesis in peripheral blood and BM and alleviates pathology of spleen and lung in the SHIP-deficient recipients. SHIP-deficient BM contains a significantly smaller population of multipotent stromal cells with distinct properties, which may contribute to the reconstitution by wild-type cells. We further demonstrate that it is the nonhematopoietic donor cells that are responsible for the reconstitution. Thus, we have established a nonhematopoietic BM microenvironment reconstitution system to functionally study specific cell types in hematopoietic niches.

Hematopoietic stem cells (HSCs) are the best-characterized tissue-specific stem cells, yet experimental study of HSCs remains challenging, as they are exceedingly rare and methods to purify them are cumbersome. Moreover, genetic tools for specifically investigating HSC biology are lacking. To address this we sought to identify genes uniquely expressed in HSCs within the hematopoietic system and to develop a reporter strain that specifically labels them. Using microarray profiling we identified several genes with HSC-restricted expression. Generation of mice with targeted reporter knock-in/knock-out alleles of one such gene, Fgd5, revealed that though Fgd5 was required for embryonic development, it was not required for definitive hematopoiesis or HSC function. Fgd5 reporter expression near exclusively labeled cells that expressed markers consistent with HSCs. Bone marrow cells isolated based solely on Fgd5 reporter signal showed potent HSC activity that was comparable to stringently purified HSCs. The labeled fraction of the Fgd5 reporter mice contained all HSC activity, and HSC-specific labeling was retained after transplantation. Derivation of next generation mice bearing an Fgd5-CreERT2 allele allowed tamoxifen-inducible deletion of a conditional allele specifically in HSCs. In summary, reporter expression from the Fgd5 locus permits identification and purification of HSCs based on single-color fluorescence.

Phosphatidylinositol 3,4,5-trisphosphate ((PIP_3)) is a second messenger that is involved in a number of cell activities including cell growth, proliferation, and motility. (PIP_3) is produced by PI3K and regulated by PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP lipid phosphatases. Evidence from our experiments shows that enhanced (PIP_3) production results in elevated neutrophil recruitment under inflammatory conditions. However, the mechanism of this elevation is not well understood. We used intravital video microscopy to investigate neutrophil recruitment in the cremaster venules of wild-type and PTEN knockout (KO) mice. Neutrophil transmigration was augmented in PTEN KO mice 4 h after TNF-α intrascrotal injection. PTEN KO neutrophils also showed significantly enhanced transmigration 2 h after MIP-2 intrascrotal injection, an effect that dramatically decreased when PI3K or Src kinase inhibitor treatments preceded MIP-2 stimulation. Similarly, fMLP superfusion of the cremaster muscle lead to enhanced emigration in PTEN KO mice. The observed elevation in neutrophil emigration was likely caused by increased speed of crawling, crossing the venular wall, and migrating through the muscular tissue in PTEN KO mice because the effect of PTEN depletion on neutrophil rolling or adhesion was minimal. Interestingly, chemoattractant-induced release of gelatinase and elastase was also elevated in PTEN null neutrophils, providing a potential mechanism for the enhanced neutrophil migration in the PTEN KO mice. Collectively, these results demonstrate that PTEN deletion in neutrophils enhances their invasivity and recruitment to inflamed sites more likely by raising the cell physical capability to cross the vascular and tissue barriers.

Cytokine-induced neutrophil mobilization from the bone marrow to circulation is a critical event in acute inflammation, but how it is accurately controlled remains poorly understood. In this study, we report that CXCR2 ligands are responsible for rapid neutrophil mobilization during early-stage acute inflammation. Nevertheless, although serum CXCR2 ligand concentrations increased during inflammation, neutrophil mobilization slowed after an initial acute fast phase, suggesting a suppression of neutrophil response to CXCR2 ligands after the acute phase. We demonstrate that granulocyte colony-stimulating factor (G-CSF), usually considered a prototypical neutrophil-mobilizing cytokine, was expressed later in the acute inflammatory response and unexpectedly impeded CXCR2-induced neutrophil mobilization by negatively regulating CXCR2-mediated intracellular signaling. Blocking G-CSF in vivo paradoxically elevated peripheral blood neutrophil counts in mice injected intraperitoneally with Escherichia coli and sequestered large numbers of neutrophils in the lungs, leading to sterile pulmonary inflammation. In a lipopolysaccharide-induced acute lung injury model, the homeostatic imbalance caused by G-CSF blockade enhanced neutrophil accumulation, edema, and inflammation in the lungs and ultimately led to significant lung damage. Thus, physiologically produced G-CSF not only acts as a neutrophil mobilizer at the relatively late stage of acute inflammation, but also prevents exaggerated neutrophil mobilization and the associated inflammation-induced tissue damage during early-phase infection and inflammation.

Background: Microtubule drugs are effective anti-cancer agents, primarily due to their ability to induce mitotic arrest and subsequent cell death. However, some cancer cells are intrinsically resistant or acquire a resistance. Lack of apoptosis following mitotic arrest is thought to contribute to drug resistance that limits the efficacy of the microtubule-targeting anti-cancer drugs. Genetic or pharmacological agents that selectively facilitate the apoptosis of mitotic arrested cells present opportunities to strengthen the therapeutic efficacy. Methodology and Principal Findings: We report a natural product Celastrol targets tubulin and facilitates mitotic cell death caused by microtubule drugs. First, in a small molecule screening effort, we identify Celastrol as an inhibitor of neutrophil chemotaxis. Subsequent time-lapse imaging analyses reveal that inhibition of microtubule-mediated cellular processes, including cell migration and mitotic chromosome alignment, is the earliest events affected by Celastrol. Disorganization, not depolymerization, of mitotic spindles appears responsible for mitotic defects. Celastrol directly affects the biochemical properties of tubulin heterodimer in vitro and reduces its protein level in vivo. At the cellular level, Celastrol induces a synergistic apoptosis when combined with conventional microtubule-targeting drugs and manifests an efficacy toward Taxol-resistant cancer cells. Finally, by time-lapse imaging and tracking of microtubule drug-treated cells, we show that Celastrol preferentially induces apoptosis of mitotic arrested cells in a caspase-dependent manner. This selective effect is not due to inhibition of general cell survival pathways or mitotic kinases that have been shown to enhance microtubule drug-induced cell death. Conclusions and Significance: We provide evidence for new cellular pathways that, when perturbed, selectively induce the apoptosis of mitotic arrested cancer cells, identifying a potential new strategy to enhance the therapeutic efficacy of conventional microtubule-targeting anti-cancer drugs.

SUMMARY Gasdermin D (GSDMD) is considered a proinflammatory factor that mediates pyroptosis in macrophages to protect hosts from intracellular bacteria. Here, we reveal that GSDMD deficiency paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, which established GSDMD as a negative regulator of innate immunity. In contrast to its activation in macrophages, in which activated inflammatory caspases cleave GSDMD to produce an N-terminal fragment (GSDMD-cNT) to trigger pyroptosis, GSDMD cleavage and activation in neutrophils was caspase independent. It was mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE), released from cytoplasmic granules into the cytosol in aging neutrophils. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived NT fragment (GSDMD-eNT) that induced lytic cell death as efficiently as GSDMD-cNT. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a potential target for antibacterial and anti-inflammatory therapies.