A Point Mutation in Cassette Relieves the Repression Regulation of CcpA Resulting in an Increase in the Degradation of 2,3-Butanediol in Lactococcus lactis.

In lactic acid bacteria, the global transcriptional regulator CcpA regulates carbon metabolism by repressing and activating the central carbon metabolism pathway, thus decreasing or increasing the yield of certain metabolites to maximize carbon flow. However, there are no reports on the deregulation of the inhibitory effects of CcpA on the metabolism of secondary metabolites. In this study, we identified a single-base mutant strain of Lactococcus lactis N8-2 that is capable of metabolizing 2,3-butanediol. It has been established that CcpA dissociates from the catabolite responsive element (cre) site due to a mutation, leading to the activation of derepression and expression of the 2,3-butanediol dehydrogenase gene cluster (butB and butA). Transcriptome analysis and quantitative polymerase chain reaction (Q-PCR) results showed significant upregulation of transcription of butB and butA compared to the unmutated strain. Furthermore, micro-scale thermophoresis experiments confirmed that CcpA did not bind to the mutated cre. Furthermore, in a bacterial two-plasmid fluorescent hybridization system, it was similarly confirmed that the dissociation of CcpA from cre eliminated the repressive effect of CcpA on downstream genes. Finally, we investigated the differing catalytic capacities of the 2,3-butanediol dehydrogenase gene cluster in L. lactis N8-1 and L. lactis N8-2 for 2,3-butanediol. This led to increased expression of butB and butA, which were deregulated by CcpA repression. This is the first report on the elimination of the deterrent effect of CcpA in lactic acid bacteria, which changes the direction of enzymatic catalysis and alters the direction of carbon metabolism. This provides new perspectives and strategies for metabolizing 2,3-butanediol using bacteria in synthetic biology.

Effective adsorption of nisin on the surface of polystyrene using hydrophobin HGFI.

Herein, a new method was demonstrated for effective immobilization of the antibacterial peptide nisin on Grifola frondosa hydrophobin (HGFI), without the need of any additional complex reaction. Hydrophobin can self-assemble as a monolayer to form continuous negative-charged surfaces with enhanced wettability and biocompatibility. Adding nisin solution to such hydrophobin surface created antibacterial surfaces. The quantification analysis revealed that more nisin could be adsorbed on the HGFI-coated than to control polystyrene surfaces at different pH values. This suggested that electronic attraction and wettability may play important roles in this process. The transmission electron microscopy, atomic force microscopy and fourier transform infrared (FTIR) analysis indicated the adsorption mode of nisin on the HGFI film, i.e., hydrophobins served as an adhesive layer for binding charged peptides to interfaces. The antibacterial activity of the treated surface was investigated via counting, a nucleic acid release test, scanning electron microscopy, and biofilm detection. These results indicated the excellent antibacterial activity of nisin adsorbed on the HGFI-coated surfaces. The activity retention of adsorbed nisin was demonstrated by immersing the modified substrates in a flowed liquid condition.

Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8.

Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS-PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology.

Engineering Lactococcus lactis as a multi-stress tolerant biosynthetic chassis by deleting the prophage-related fragment.

BACKGROUND: In bioengineering, growth of microorganisms is limited because of environmental and industrial stresses during fermentation. This study aimed to construct a nisin-producing chassis Lactococcus lactis strain with genome-streamlined, low metabolic burden, and multi-stress tolerance characteristics. RESULTS: The Cre-loxP recombination system was applied to reduce the genome and obtain the target chassis strain. A prophage-related fragment (PRF; 19,739 bp) in the L. lactis N8 genome was deleted, and the mutant strain L. lactis N8-1 was chosen for multi-stress tolerance studies. Nisin immunity of L. lactis N8-1 was increased to 6500 IU/mL, which was 44.44% higher than that of the wild-type L. lactis N8 (4500 IU/mL). The survival rates of L. lactis N8-1 treated with lysozyme for 2 h and lactic acid for 1 h were 1000- and 10,000-fold higher than that of the wild-type strain, respectively. At 39 â„ƒ, the L. lactis N8-1 could still maintain its growth, whereas the growth of the wild-type strain dramatically dropped. Scanning electron microscopy showed that the cell wall integrity of L. lactis N8-1 was well maintained after lysozyme treatment. Tandem mass tags labeled quantitative proteomics revealed that 33 and 9 proteins were significantly upregulated and downregulated, respectively, in L. lactis N8-1. These differential proteins were involved in carbohydrate and energy transport/metabolism, biosynthesis of cell wall and cell surface proteins. CONCLUSIONS: PRF deletion was proven to be an efficient strategy to achieve multi-stress tolerance and nisin immunity in L. lactis, thereby providing a new perspective for industrially obtaining engineered strains with multi-stress tolerance and expanding the application of lactic acid bacteria in biotechnology and synthetic biology. Besides, the importance of PRF, which can confer vital phenotypes to bacteria, was established.

Restructured Lactococcus lactis strains with emergent properties constructed by a novel highly efficient screening system.

BACKGROUND: After 2.83% genome reduction in Lactococcus lactis NZ9000, a good candidate host for proteins production was obtained in our previous work. However, the gene deletion process was time consuming and laborious. Here, we proposed a convenient gene deletion method suitable for large-scale genome reduction in L. lactis NZ9000. RESULTS: Plasmid pNZ5417 containing a visually selectable marker PnisZ-lacZ was constructed, which allowed more efficient and convenient screening of gene deletion mutants. Using this plasmid, two large nonessential DNA regions, L-4A and L-5A, accounting for 1.25% of the chromosome were deleted stepwise in L. lactis 9k-3. When compared with the parent strain, the mutant L. lactis 9k-5A showed better growth characteristics, transformability, carbon metabolic capacity, and amino acids biosynthesis. CONCLUSIONS: Thus, this study provides a convenient and efficient system for large-scale genome deletion in L. lactis through application of visually selectable marker, which could be helpful for rapid genome streamlining and generation of restructured L. lactis strains that can be used as cell factories.

Co-expression of Nisin Z and Leucocin C as a Basis for Effective Protection Against Listeria monocytogenes in Pasteurized Milk.

Nisin, an important bacteriocin from Lactococcus lactis subsp., is primarily active against various Gram-positive bacteria. Leucocin C, produced by Leuconostoc carnosum 4010, is a class IIa bacteriocin used to inhibit the growth of Listeria monocytogenes. Because two bacteriocins have different modes of action, the combined use of them could be a potential strategy for effective inhibition of foodborne pathogens. In this study, L. lactis N8-r-lecCI (N8 harboring lecCI gene) coexpressing nisin-leucocin C was constructed based on the food-grade carrier L. lactis N8. Production of both bacteriocins was stably maintained. Antimicrobial measurements showed that the recombinant strain is effectively against Listeria monocytogenes and Staphylococcus aureus and moderately against Salmonella enterica serovar Enteritidis and Escherichia coli because of its stronger antibacterial activity than the parental strain, this result first demonstrated that the co-expression of nisin and leucocin C results in highly efficient antimicrobial activity. The checkerboard assay showed that the antibacterial activity of L. lactis N8-r-lecCI supernatant was enhanced in the presence of low concentration of EDTA. Analysis of the scanning electron microscope image showed the biggest cellular morphology change in L. monocytogenes treated with a mixture of EDTA and L. lactis N8-r-lecCI supernatant. The practical effect was verified in pasteurized milk through time-kill assay. The L. lactis N8-r-lecCI strain expressing both nisin and leucocin C has a promising application prospect in pasteurized milk processing and preservation because of its strong antibacterial activity.

Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000.

BACKGROUND: The implementation of novel chassis organisms to be used as microbial cell factories in industrial applications is an intensive research field. Lactococcus lactis, which is one of the most extensively studied model organisms, exhibits superior ability to be used as engineered host for fermentation of desirable products. However, few studies have reported about genome reduction of L. lactis as a clean background for functional genomic studies and a model chassis for desirable product fermentation. RESULTS: Four large nonessential DNA regions accounting for 2.83% in L. lactis NZ9000 (L. lactis 9 k) genome (2,530,294 bp) were deleted using the Cre-loxP deletion system as the first steps toward a minimized genome in this study. The mutants were compared with the parental strain in several physiological traits and evaluated as microbial cell factories for heterologous protein production (intracellular and secretory expression) with the red fluorescent protein (RFP) and the bacteriocin leucocin C (LecC) as reporters. The four mutants grew faster, yielded enhanced biomass, achieved increased adenosine triphosphate content, and diminished maintenance demands compared with the wild strain in the two media tested. In particular, L. lactis 9 k-4 with the largest deletion was identified as the optimum candidate host for recombinant protein production. With nisin induction, not only the transcriptional efficiency but also the production levels of the expressed reporters were approximately three- to fourfold improved compared with the wild strain. The expression of lecC gene controlled with strong constitutive promoters P5 and P8 in L. lactis 9 k-4 was also improved significantly. CONCLUSIONS: The genome-streamlined L. lactis 9 k-4 outcompeted the parental strain in several physiological traits assessed. Moreover, L. lactis 9 k-4 exhibited good properties as platform organism for protein production. In future works, the genome of L. lactis will be maximally reduced by using our specific design to provide an even more clean background for functional genomics studies than L. lactis 9 k-4 constructed in this study. Furthermore, an improved background will be potentially available for use in biotechology.

Isolation of strong constitutive promoters from Lactococcus lactis subsp. lactis N8.

The synthesis of heterologous proteins in Lactococcus lactis is strongly influenced by the promoter selected for the expression. The nisin A promoter is commonly used for induced expression of proteins in L. lactis, whereas few constitutive promoters (P45 and the weaker P32) have been used for protein expression studies. In this study, eight different putative strong constitutive promoters were identified through transcriptional analysis of L. lactis N8 and were investigated for their capability to drive nisZ gene expression with promoters P45 and P32 as control. Four strong promoters (P8, P5, P3 and P2) were identified as having a transcriptional activity that was higher than that of P45 through RT-qPCR and agar-diffusion experiments. In addition, these four promoters were fused to the erythromycin resistant gene (ermC) with promoter P45 as control and inserted into the backbone of the pNZ8048 vector. The transcriptional efficiencies of promoters P8, P5, P2 and P3 were all higher than promoter P45 based on the obtained MIC50 values and they all showed different activity levels. In conclusion, four strong constitutive promoters with a wide range of promoter activities were identified and are suitable for protein production in L. lactis.

The origin and evolution of mutations in acute myeloid leukemia.

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.

Expression of the G-CSF receptor in monocytic cells is sufficient to mediate hematopoietic progenitor mobilization by G-CSF in mice.

Granulocyte colony-stimulating factor (G-CSF), the prototypical mobilizing cytokine, induces hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow in a cell-nonautonomous fashion. This process is mediated, in part, through suppression of osteoblasts and disruption of CXCR4/CXCL12 signaling. The cellular targets of G-CSF that initiate the mobilization cascade have not been identified. We use mixed G-CSF receptor (G-CSFR)-deficient bone marrow chimeras to show that G-CSF-induced mobilization of HSPCs correlates poorly with the number of wild-type neutrophils. We generated transgenic mice in which expression of the G-CSFR is restricted to cells of the monocytic lineage. G-CSF-induced HSPC mobilization, osteoblast suppression, and inhibition of CXCL12 expression in the bone marrow of these transgenic mice are intact, demonstrating that G-CSFR signals in monocytic cells are sufficient to induce HSPC mobilization. Moreover, G-CSF treatment of wild-type mice is associated with marked loss of monocytic cells in the bone marrow. Finally, we show that bone marrow macrophages produce factors that support the growth and/or survival of osteoblasts in vitro. Together, these data suggest a model in which G-CSFR signals in bone marrow monocytic cells inhibit the production of trophic factors required for osteoblast lineage cell maintenance, ultimately leading to HSPC mobilization.

Suppression of CXCL12 production by bone marrow osteoblasts is a common and critical pathway for cytokine-induced mobilization.

Current evidence suggests that hematopoietic stem/progenitor cell (HSPC) mobilization by granulocyte colony-stimulating factor (G-CSF) is mediated by induction of bone marrow proteases, attenuation of adhesion molecule function, and disruption of CXCL12/CXCR4 signaling in the bone marrow. The relative importance and extent to which these pathways overlap or function independently are uncertain. Despite evidence of protease activation in the bone marrow, HSPC mobilization by G-CSF or the chemokine Grobeta was abrogated in CXCR4(-/-) bone marrow chimeras. In contrast, HSPC mobilization by a VLA-4 antagonist was intact. To determine whether other mobilizing cytokines disrupt CXCR4 signaling, we characterized CXCR4 and CXCL12 expression after HSPC mobilization with Flt3 ligand (Flt3L) and stem cell factor (SCF). Indeed, treatment with Flt3L or SCF resulted in a marked decrease in CXCL12 expression in the bone marrow and a loss of surface expression of CXCR4 on HSPCs. RNA in situ and sorting experiments suggested that the decreased CXCL12 expression is secondary to a loss of osteoblast lineage cells. Collectively, these data suggest that disruption of CXCR4 signaling and attenuation of VLA-4 function are independent mechanisms of mobilization by G-CSF. Loss of CXCL12 expression by osteoblast appears to be a common and key step in cytokine-induced mobilization.

Csf3r mutations in mice confer a strong clonal HSC advantage via activation of Stat5.

A fundamental property of leukemic stem cells is clonal dominance of the bone marrow microenvironment. Truncation mutations of CSF3R, which encodes the G-CSF receptor (G-CSFR), are implicated in leukemic progression in patients with severe congenital neutropenia. Here we show that expression of a truncated mutant Csf3r in mice confers a strong clonal advantage at the HSC level that is dependent upon exogenous G-CSF. G-CSF-induced proliferation, phosphorylation of Stat5, and transcription of Stat5 target genes were increased in HSCs isolated from mice expressing the mutant Csf3r. Conversely, the proliferative advantage conferred by the mutant Csf3r was abrogated in myeloid progenitors lacking both Stat5A and Stat5B, and HSC function was reduced in mice expressing a truncated mutant Csf3r engineered to have impaired Stat5 activation. These data indicate that in mice, inappropriate Stat5 activation plays a key role in establishing clonal dominance by stem cells expressing mutant Csf3r.

Lentiviral-mediated RNAi inhibition of Sbds in murine hematopoietic progenitors impairs their hematopoietic potential.

Shwachman-Diamond syndrome (SDS) is a rare multisystem disorder characterized by exocrine pancreatic insufficiency, multilineage hematopoietic dysfunction, and metaphyseal chondrodysplasia. Bone marrow dysfunction is present in nearly all patients with SDS, with neutropenia being the most common abnormality. The majority of patients with SDS have mutations in the Shwachman Bodian Diamond syndrome (SBDS) gene. We have developed a strategy to examine the consequences of lentiviral-mediated RNA interference (RNAi) of Sbds on hematopoiesis. Here, we report that both Sbds RNA and protein expression can be efficiently inhibited in primary murine hematopoietic cells using lentiviral-mediated RNAi. Inhibition of Sbds results in a defect in granulocytic differentiation in vitro and impairs myeloid progenitor generation in vivo. In addition, short-term hematopoietic engraftment was impaired, which is due in part to reduced homing of hematopoietic progenitors to the bone marrow. Finally, we show that inhibition of Sbds is associated with a decrease in circulating B lymphocytes, despite evidence of normal B lymphopoiesis. These data provide the first evidence that loss of Sbds is sufficient to induce abnormalities in hematopoiesis.

STAT3 governs distinct pathways in emergency granulopoiesis and mature neutrophils.

Granulocyte colony-stimulating factor (G-CSF) is essential for the host response to bacterial infection by controlling neutrophil production in the bone marrow. The G-CSF receptor (G-CSFR) activates the Jak/STAT pathway, although little is understood about how these signals regulate basal and stress-induced granulopoiesis. We examined STAT3 function in granulocytes using a bone marrow conditional knockout mouse model. Our results show that STAT3 has a crucial role in emergency granulopoiesis and mature neutrophil function. STAT3-deficient mice have an aberrant response to G-CSF in vivo, characterized by failure to accumulate immature granulocytes and an increased ratio of mature to immature neutrophils in the bone marrow, peripheral blood, and spleen. Acute neutrophil mobilization is impaired in STAT3-deficient mice as judged by their failure to up-regulate circulating neutrophils following short-term G-CSF exposure. STAT3 also controls neutrophil chemotactic responses to natural ligands for CXCR2 and regulates the magnitude of chemoattractant-induced actin polymerization. These functions of STAT3 are independent of its principal target gene Socs3, which encodes a crucial feedback inhibitor of cytokine signaling. Our results demonstrate the existence of distinct STAT3 target pathways in neutrophils required for granulopoiesis and innate immunity.

Src family kinases are important negative regulators of G-CSF-dependent granulopoiesis.

Granulocyte colony-stimulating factor (G-CSF) is the principal cytokine regulating granulopoiesis. Truncation mutations of the G-CSF receptor (G-CSFR) are associated with the development of acute myeloid leukemia in patients with severe congenital neutropenia. Although increased proliferative signaling by a representative G-CSFR truncation mutation (termed d715) has been documented, the molecular basis for this hyperproliferative phenotype has not been fully characterized. Given the accumulating evidence implicating Src family kinases in the transduction of cytokine receptor signals, the role of these kinases in the regulation of G-CSF signaling was examined. We show that Hck and Lyn, Src family kinases expressed in myeloid cells, are negative regulators of granulopoiesis that act at distinct stages of granulocytic differentiation. Whereas Hck regulates the G-CSF-induced proliferation of granulocytic precursors, Lyn regulates the production of myeloid progenitors. Interestingly, d715 G-CSFR myeloid progenitors were resistant to the growth-stimulating effect of treatment with a Src kinase inhibitor. Together, these data establish Lyn and Hck as key negative regulators of granulopoiesis and raise the possibility that loss of Src family kinase activation by the d715 G-CSFR may contribute to its hyperproliferative phenotype.

G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow.

Accumulating evidence indicates that interaction of stromal cell-derived factor 1 (SDF-1/CXCL12 [CXC motif, ligand 12]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During granulocyte colony-stimulating factor (G-CSF)-induced HPC mobilization, CXCL12 protein expression in the bone marrow decreases. Herein, we show that in a series of transgenic mice carrying targeted mutations of their G-CSF receptor and displaying markedly different G-CSF-induced HPC mobilization responses, the decrease in bone marrow CXCL12 protein expression closely correlates with the degree of HPC mobilization. G-CSF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. Cell sorting experiments showed that osteoblasts and to a lesser degree endothelial cells are the major sources of CXCL12 production in the bone marrow. Interestingly, osteoblast activity, as measured by histomorphometry and osteocalcin expression, is strongly down-regulated during G-CSF treatment. However, the G-CSF receptor is not expressed on osteoblasts; accordingly, G-CSF had no direct effect on osteoblast function. Collectively, these data suggest a model in which G-CSF, through an indirect mechanism, potently inhibits osteoblast activity resulting in decreased CXCL12 expression in the bone marrow. The consequent attenuation of CXCR4 signaling ultimately leads to HPC mobilization.

G-CSF induced progenitor mobilization in mice with PIGA- blood cells.

OBJECTIVE: In patients with paroxysmal nocturnal hemoglobinuria (PNH) a proportion of blood cells are deficient in glycosyl phosphatidylinositol (GPI) anchored proteins due to a mutation in the PIGA gene. Previous studies showed that in PNH the majority of circulating early progenitor cells were normal but after G-CSF were mainly, of the PNH phenotype. This suggested that GPI-linked proteins contribute to the regulation of progenitor trafficking from bone marrow to peripheral blood. METHODS: To test this hypothesis we studied progenitor cells in bone marrow, spleen, and peripheral blood in response to G-CSF in mice genetically engineered to have a proportion of blood cells deficient in GPI-linked proteins (LF mice). RESULTS: In contrast to humans, LF and wild-type mice have comparable numbers of progenitor cells in bone marrow, spleen, and peripheral blood. Similarly, in LF mice the proportion of PIGA- progenitor cells in peripheral blood corresponds the proportion of PIGA- progenitor cells measured in bone marrow and spleen. After G-CSF the number of circulating progenitors significantly increased but the proportion of PIGA- cells remained the same in peripheral blood,bone marrow, and spleen. CONCLUSIONS: Our data indicate that under basal laboratory conditions the lack of GPI-linked protein does not cause a retention of progenitor cells in the bone marrow. This implies that the preferential circulation of normal progenitor cells in patients with PNH requires an additional component that most likely is provided by the altered microenvironment of the underlying bone marrow failure.

Characterization of hematopoietic progenitor mobilization in protease-deficient mice.

Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment, where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis, HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly, HPC mobilization by G-CSF was normal in MMP-9-deficient mice, NE x CG-deficient mice, or mice lacking dipeptidyl peptidase I, an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover, combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE x CG-deficient mice, indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12 alpha protein expression in the bone marrow of Ne x CG-deficient mice, indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively, these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization.

Pivotal role of granulocyte colony-stimulating factor in the development of progenitors in the common myeloid pathway.

Granulocyte colony-stimulating factor (G-CSF) is the principal cytokine regulating granulopoiesis. G-CSF receptor-deficient mice (G-CSFR-/-) are neutropenic but have only a modest reduction of committed myeloid progenitors. Since it is likely that compensatory mechanisms are induced by the severe neutropenia present in G-CSFR-/- mice, a competitive repopulation assay was performed. These data show that under basal conditions, G-CSF drives nearly all of granulopoiesis through multiple mechanisms. Most importantly, G-CSFR signals regulate the production and/or maintenance of committed-myeloid progenitors. Surprisingly, G-CSFR signals also play a significant role in the regulation of primitive multipotential progenitors in vivo. The contribution of G-CSFR-/- cells to the hematopoietic stem cell compartment is modestly reduced. Moreover, a marked decrease in the contribution of G-CSFR-/- cells to other progenitors in the myeloid pathway, including erythroid and megakaryocytic progenitors, is observed. In contrast, relative to the hematopoietic stem cell compartment, the contribution of G-CSFR-/- cells to the lymphoid lineages is increased. These data suggest that G-CSFR signals may play a role in directing the commitment of primitive hematopoietic progenitors to the common myeloid lineage. Thus, regulation of G-CSF levels may provide a mechanism for directing primitive hematopoietic progenitors into the common myeloid lineage in response to environmental stresses.

G-CSF is an essential regulator of neutrophil trafficking from the bone marrow to the blood.

Neutrophils are released from the bone marrow in a regulated fashion to maintain homeostatic levels in the blood and to respond to physiological stresses, including infection. We show that under basal conditions granulocyte colony-stimulating factor (G-CSF) is an essential regulator of neutrophil release from the bone marrow. Nonredundant signals generated by the membrane-proximal 87 amino acids of the G-CSF receptor (G-CSFR) are sufficient to mediate this response. Surprisingly, G-CSFR expression on neutrophils is neither necessary nor sufficient for their mobilization from the bone marrow, suggesting that G-CSF induces neutrophil mobilization indirectly through the generation of trans-acting signals. Evidence is provided suggesting that downregulation of stromal cell-derived factor 1 expression in the bone marrow may represent such a signal.