Neutrophils are a major component of the innate immune response. Their homeostasis is maintained, in part, by the regulated release of neutrophils from the bone marrow. Constitutive expression of the chemokine CXCL12 by bone marrow stromal cells provides a key retention signal for neutrophils in the bone marrow through activation of its receptor, CXCR4. Attenuation of CXCR4 signaling leads to entry of neutrophils into the circulation through unknown mechanisms. We investigated the role of CXCR2-binding ELR + chemokines in neutrophil trafficking using mouse mixed bone marrow chimeras reconstituted with Cxcr2 -/-and WT cells. In this context, neutrophils lacking CXCR2 were preferentially retained in the bone marrow, a phenotype resembling the congenital disorder myelokathexis, which is characterized by chronic neutropenia. Additionally, transient disruption of CXCR4 failed to mobilize Cxcr2 -/-neutrophils. However, neutrophils lacking both CXCR2 and CXCR4 displayed constitutive mobilization, showing that CXCR4 plays a dominant role in neutrophil trafficking. With regard to CXCR2 ligands, bone marrow endothelial cells and osteoblasts constitutively expressed the ELR + chemokines CXCL1 and CXCL2, and CXCL2 expression was induced in endothelial cells during G-CSF-induced neutrophil mobilization. Collectively, these data suggest that CXCR2 signaling is a second chemokine axis that interacts antagonistically with CXCR4 to regulate neutrophil release from the bone marrow.

Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are less clear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect associated with induction of toll-like receptor (TLR) expression and signaling. The G-CSF-mediated expansion of HSCs is reduced in mice lacking TLR2, TLR4 or the TLR signaling adaptor MyD88. Induction of HSC quiescence is abrogated in mice lacking MyD88 or in mice treated with antibiotics to suppress intestinal flora. Finally, loss of TLR4 or germ free conditions mitigates the G-CSF-mediated HSC repopulating defect. These data suggest that low level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling.

E-selectin is an inducible cell adhesion molecule which mediates rolling of neutrophils on the endothelium, an early event in the development of an inflammatory response. Inhibition of selectin-mediated rolling is a possible means for controlling inflammation-induced diseases, and several classes of compounds have been tested for this use. We describe here the use of recombinant peptide library screening for identification and optimization of novel ligands which bind to E-selectin. Several of these peptides bind with K d values in the low nanomolar range and block E-selectin-mediated adhesion of neutrophils in static and flow-cell assays. Administration of the peptide to mice undergoing an acute inflammatory response reduced the extent of neutrophil transmigration to the site of inflammation, demonstrating the utility of this compound as a potential therapeutic. The identification of a peptide ligand for E-selectin suggests that the complete natural ligand for this adhesion molecule may include protein as well as carbohydrate moieties.E-selectin is a cell adhesion molecule which is induced on the surface of endothelial cells in response to inflammatory cytokines (1, 2). Binding of E-selectin to its ligand expressed on the surface of circulating neutrophils initiates rolling, an early step in the recruitment of these cells to a site of injury or inflammation (3, 4). The sequence of E-selectin (2,5,6) shows that it is a member of the mammalian C-type lectin family (7), and its three-dimensional structure shows strong similarity to mannose-binding protein (8). The carbohydrate structure sialyl Lewis x (sLe x ; 1 Neu5Ac␣2-3Gal␤1-4[Fuc␣1-3]GlcNAc) has been identified as a ligand which binds to the lectin domain at the N terminus of E-selectin (9 -12). However, natural ligands may also contain protein (13,14) or other carbohydrate structures. Inhibition of neutrophil adhesion to endothelium is an attractive approach to controlling inflammation-mediated diseases such as rheumatoid arthritis or psoriasis (15). Several potential therapeutics have been tested for their ability to inhibit the E-selectin-neutrophil adhesion event, including carbohydrate-based molecules (16, 17), antibodies (18), soluble E-selectin (19), and selectin-Ig chimeras (20). While these molecules have been useful to show the utility of selectin blockers for treating inflammation, each has significant drawbacks as a therapeutic, including short in vivo half-life, potential immunogenicity, high cost, and other possible side effects. A further limitation of these approaches is the lack of an efficient means to improve the pharmaceutical properties of these molecules.In the past few years, several methods for creating and screening vast libraries of recombinant peptides have been developed (21-24). These libraries have been used to discover novel peptide ligands for several proteins, including antibodies (21, 24, 25), receptors (26, 27), and lectins (28, 29), as well as novel enzyme substrates (30 -33). We report here the use of recombinant peptide dis...

Chaperonin-containing TCP-1 (CCT or TRiC) is a multi-subunit complex that folds many of the proteins essential for cancer development. CCT is expressed in diverse cancers and could be an ideal therapeutic target if not for the fact that the complex is encoded by eight distinct genes, complicating the development of inhibitors. Few definitive studies addressed the role of specific subunits in promoting the chaperonin's function in cancer. To this end, we investigated the activity of CCT2 (CCTβ) by overexpressing or depleting the subunit in breast epithelial and breast cancer cells. We found that increasing total CCT2 in cells by 1.3-1.8-fold using a lentiviral system, also caused CCT3, CCT4, and CCT5 levels to increase. Likewise, silencing cct2 gene expression by ~50% caused other CCT subunits to decrease. Cells expressing CCT2 were more invasive and had a higher proliferative index. CCT2 depletion in a syngeneic murine model of triple negative breast cancer (TNBC) prevented tumor growth. These results indicate that the CCT2 subunit is integral to the activity of the chaperonin and is needed for tumorigenesis. Hence CCT2 could be a viable target for therapeutic development in breast and other cancers. The hallmarks of cancer (uncontrolled proliferation, genomic instability, metastasis, etc.) reveal the complex nature of this disease and the challenges faced developing effective therapeutics 1,2. Cancer does, however, have an "Achilles heel" and that is its dependency or addiction on major cellular events or processes like transcription, translation, splicing, protein degradation and protein-folding 3. In healthy cells, such conserved and essential processes are rigorously regulated by the proteostasis network (PN) to ensure proteome balance. In order to maintain proteome integrity, the cellular proteome must be synthesized, folded into its native structure, and, when no longer needed, degraded and the amino acids recycled 4,5. Chaperones and chaperonins are key players in the PN 6. Unlike healthy, non-transformed cells, the PN of cancer cells is taxed to produce proteins involved in survival, angiogenesis, migration, proliferation which are essential for tumor formation, progression and metastasis. Cancer cells have a higher dependency on molecular chaperones and are uniquely challenged due to imbalances caused by chromosomal abnormalities and overexpression of oncogenes, ultimately leading to cellular stress 7. As example, inhibitors of Heat Shock Protein 90 (HSP90) showed promising outcomes in the treatment of metastatic breast cancer 8. However, despite being in clinical trials since 1998, the success of HSP90 inhibitors in clinical trials remains mixed 9-11. Reasons such as dose-limiting toxicity, incomplete inhibition of HSP90, and insufficient downregulation of client proteins impeded the clinical use of current HSP90 inhibitors 12,13. In recent years,