Scientific Resources

Although microbial infections can alter steady-state hematopoiesis,the mechanisms that drive such changes are not well understood. We addressed a role for IFN-γ signaling in infection-induced bone marrow suppression and anemia in a murine model of human monocytic ehrlichiosis,an emerging tick-borne disease. Within the bone marrow of Ehrlichia muris-infected C57BL/6 mice,we observed a reduction in myeloid progenitor cells,as defined both phenotypically and functionally. Infected mice exhibited a concomitant increase in developing myeloid cells within the bone marrow,an increase in the frequency of circulating monocytes,and an increase in splenic myeloid cells. The infection-induced changes in progenitor cell phenotype were critically dependent on IFN-γ,but not IFN-α,signaling. In mice deficient in the IFN-γ signaling pathway,we observed an increase in myeloid progenitor cells and CDllb(lo)Gr1(lo) promyelocytic cells within the bone marrow,as well as reduced frequencies of mature granulocytes and monocytes. Furthermore,E. muris-infected IFN-γR-deficient mice did not exhibit anemia or an increase in circulating monocytes,and they succumbed to infection. Gene transcription studies revealed that IFN-γR-deficient CDllb(lo)Gr1(lo) promyelocytes from E. muris-infected mice exhibited significantly reduced expression of irf-1 and irf-8,both key transcription factors that regulate the differentiation of granulocytes and monocytes. Finally,using mixed bone marrow chimeric mice,we show that IFN-γ-dependent infection-induced myelopoiesis occurs via the direct effect of the cytokine on developing myeloid cells. We propose that,in addition to its many other known roles,IFN-γ acts to control infection by directly promoting the differentiation of myeloid cells that contribute to host defense. View Publication

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome,a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction,but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14,the ribosomal protein gene deleted in the 5q-syndrome,or RPS19,the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect,whereas nutlin-3,a compound that activates p53 through inhibition of HDM2,selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome,we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53,providing a basis for the failure of erythropoiesis in the 5q-syndrome,DBA,and perhaps other bone marrow failure syndromes. View Publication

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia in the United States and globally. Despite the availability of pneumococcal capsular polysaccharide (PPS) and protein conjugate-based vaccines,the prevalence of antibiotic-resistant pneumococcal strains,serotype (ST) replacement in nonconjugate vaccine strains,and uncertainty as to whether the PPS vaccine that is used in adults protects against pneumonia emphasize the need for continued efforts to understand the nature of protective PPS antibody responses. In this study,we generated mouse monoclonal antibodies (MAbs) to a conjugate consisting of the PPS of serotype 8 (PPS8) S. pneumoniae and tetanus toxoid. Thirteen MAbs,including four IgMs that bound to PPS8 and phosphorylcholine (PC) and five IgMs and four IgG1s that bound to PPS8 but not PC,were produced,and their nucleotide sequences,epitope and fine specificity,and efficacy against lethal challenge with ST8 S. pneumoniae were determined. MAbs that bound to PPS8 exhibited gene use that was distinct from that exhibited by MAbs that bound to PC. Only PPS8-binding MAbs that did not bind PC were protective in mice. All 13 MAbs used germ line variable-region heavy (V(H)) and light (V(L)) chain genes,with no evidence of somatic hypermutation. Our data reveal a relationship between PPS specificity and V(H) gene use and MAb efficacy in mice. These findings provide insight into the relationship between antibody molecular structure and function and hold promise for the development of novel surrogates for pneumococcal vaccine efficacy. View Publication

Bruton tyrosine kinase (Btk) is essential for B cell development and function and also appears to be important for myeloid cells. The bone marrow of Btk-deficient mice shows enhanced granulopoiesis compared with that of wild-type mice. In purified granulocyte-monocyte-progenitors (GMP) from Btk-deficient mice,the development of granulocytes is favored at the expense of monocytes. However,Btk-deficient neutrophils are impaired in maturation and function. Using bone marrow chimeras,we show that this defect is cell-intrinsic to neutrophils. In GMP and neutrophils,Btk plays a role in GM-CSF- and Toll-like receptor-induced differentiation. Molecular analyses revealed that expression of the lineage-determining transcription factors C/EBPα,C/EBPβ,and PU.1,depends on Btk. In addition,expression of several granule proteins,including myeloperoxidase,neutrophilic granule protein,gelatinase and neutrophil elastase,is Btk-dependent. In the Arthus reaction,an acute inflammatory response,neutrophil migration into tissues,edema formation,and hemorrhage are significantly reduced in Btk-deficient animals. Together,our findings implicate Btk as an important regulator of neutrophilic granulocyte maturation and function in vivo. View Publication

Injury induces the recruitment of bone marrow-derived cells (BMDCs) that contribute to the repair and regeneration process. The behavior of BMDCs in injured tissue has a profound effect on repair,but the regulation of BMDC behavior is poorly understood. Aberrant recruitment/retention of these cells in wounds of diabetic patients and animal models is associated with chronic inflammation and impaired healing. BMD Gr-1(+)CD11b(+) cells function as immune suppressor cells and contribute significantly to tumor-induced neovascularization. Here we report that Gr-1(+)CD11b(+) cells also contribute to injury-induced neovascularization,but show altered recruitment/retention kinetics in the diabetic environment. Moreover,diabetic-derived Gr-1(+)CD11b(+) cells fail to stimulate neovascularization in vivo and have aberrant proliferative,chemotaxis,adhesion,and differentiation potential. Previously we demonstrated that gene transfer of HOXA3 to wounds of diabetic mice is taken up by and expressed by recruited BMDCs. This is associated with a suppressed inflammatory response,enhanced neovascularization,and accelerated wound healing. Here we show that sustained expression of Hoxa3 in diabetic-derived BMD Gr-1(+)CD11b(+) cells reverses their diabetic phenotype. These findings demonstrate that manipulation of adult stem/progenitor cells ex vivo could be used as a potential therapy in patients with impaired wound healing. View Publication

Recent data indicate that a variety of regulatory molecules active in embryonic development may also play a role in the regulation of early hematopoiesis. Here we report that the human Vent-like homeobox gene VENTX,a putative homolog of the Xenopus xvent2 gene,is a unique regulatory hematopoietic gene that is aberrantly expressed in CD34(+) leukemic stem-cell candidates in human acute myeloid leukemia (AML). Quantitative RT-PCR documented expression of the gene in lineage positive hematopoietic subpopulations,with the highest expression in CD33(+) myeloid cells. Notably,expression levels of VENTX were negligible in normal CD34(+)/CD38(-) or CD34(+) human progenitor cells. In contrast to this,leukemic CD34(+)/CD38(-) cells from AML patients with translocation t(8,21) and normal karyotype displayed aberrantly high expression of VENTX. Gene expression and pathway analysis demonstrated that in normal CD34(+) cells enforced expression of VENTX initiates genes associated with myeloid development and down-regulates genes involved in early lymphoid development. Functional analyses confirmed that aberrant expression of VENTX in normal CD34(+) human progenitor cells perturbs normal hematopoietic development,promoting generation of myeloid cells and impairing generation of lymphoid cells in vitro and in vivo. Stable knockdown of VENTX expression inhibited the proliferation of human AML cell lines. Taken together,these data extend our insights into the function of embryonic mesodermal factors in human postnatal hematopoiesis and indicate a role for VENTX in normal and malignant myelopoiesis. View Publication

CRKL (CRK-like) is an adapter protein predominantly phosphorylated in cells that express the tyrosine kinase p210(BCR-ABL),the fusion product of a (9;22) chromosomal translocation causative for chronic myeloid leukemia. It has been unclear,however,whether CRKL plays a functional role in p210(BCR-ABL) transformation. Here,we show that CRKL is required for p210(BCR-ABL) to support interleukin-3-independent growth of myeloid progenitor cells and long-term outgrowth of B-lymphoid cells from fetal liver-derived hematopoietic progenitor cells. Furthermore,a synthetic phosphotyrosyl peptide that binds to the CRKL SH2 domain with high affinity blocks association of endogenous CRKL with the p210(BCR-ABL) complex and reduces c-MYC levels in K562 human leukemic cells as well as in mouse hematopoietic cells transformed by p210(BCR-ABL) or the imatinib-resistant mutant T315I. These results indicate that the function of CRKL as an adapter protein is essential for p210(BCR-ABL)-induced transformation. View Publication

Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that IFN-α plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). In this study,we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs,characterized by transcriptional repression of IL-1α and β,IL-1R1,and vascular endothelial growth factor A,and upregulation of IL-1R antagonist and the decoy receptor IL-1R2. IL-1β promotes significant improvement in the functional capacity of lupus EPCs/CACs,therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated,at least in part,by increases in EPC/CAC proliferation,by decreases in EPC/CAC apoptosis,and by preventing the skewing of CACs toward nonangiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs,and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis,but not anti-neutrophil cytoplasmic Ab-positive vasculitis,showed this pathway to be operational in vivo,with increased IL-1R antagonist,downregulation of vascular endothelial growth factor A,and glomerular and blood vessel decreased capillary density,compared with controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1-dependent pathways. This could promote atherosclerosis and loss of renal function in this disease. View Publication

Adhesion properties of hematopoietic stem cells (HSCs) in the bone marrow (BM) niches control their migration and affect their cell-cycle dynamics. The serum response factor (Srf) regulates growth factor-inducible genes and genes controlling cytoskeleton structures involved in cell spreading,adhesion,and migration. We identified a role for Srf in HSC adhesion and steady-state hematopoiesis. Conditional deletion of Srf in BM cells resulted in a 3-fold expansion of the long- and short-term HSCs and multipotent progenitors (MPPs),which occurs without long-term modification of cell-cycle dynamics. Early differentiation steps to myeloid and lymphoid lineages were normal,but Srf loss results in alterations in mature-cell production and severe thrombocytopenia. Srf-null BM cells also displayed compromised engraftment properties in transplantation assays. Gene expression analysis identified Srf target genes expressed in HSCs,including a network of genes associated with cell migration and adhesion. Srf-null stem cells and MPPs displayed impair expression of the integrin network and decreased adherence in vitro. In addition,Srf-null mice showed increase numbers of circulating stem and progenitor cells,which likely reflect their reduced retention in the BM. Altogether,our results demonstrate that Srf is an essential regulator of stem cells and MPP adhesion,and suggest that Srf acts mainly through cell-matrix interactions and integrin signaling. View Publication

In up to one-third of patients with acute myeloid leukemia,a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM),and this is thought to function in cancer pathogenesis. Here,we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases,caspase-6 and -8,through direct interaction with their cleaved,active forms,but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8-mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia. View Publication

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here,we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML),an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models,Cre-lox technology,and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML,while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells,such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently,myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells,resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation,and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity. View Publication

The 8p11 myeloproliferative syndrome (EMS),also referred to as stem cell leukemia/lymphoma,is a chronic myeloproliferative disorder that rapidly progresses into acute leukemia. Molecularly,EMS is characterized by fusion of various partner genes to the FGFR1 gene,resulting in constitutive activation of the tyrosine kinases in FGFR1. To date,no previous study has addressed the functional consequences of ectopic FGFR1 expression in the potentially most relevant cellular context,that of normal primary human hematopoietic cells. Herein,we report that expression of ZMYM2/FGFR1 (previously known as ZNF198/FGFR1) or BCR/FGFR1 in normal human CD34(+) cells from umbilical-cord blood leads to increased cellular proliferation and differentiation toward the erythroid lineage in vitro. In immunodeficient mice,expression of ZMYM2/FGFR1 or BCR/FGFR1 in human cells induces several features of human EMS,including expansion of several myeloid cell lineages and accumulation of blasts in bone marrow. Moreover,bone marrow fibrosis together with increased extramedullary hematopoiesis is observed. This study suggests that FGFR1 fusion oncogenes,by themselves,are capable of initiating an EMS-like disorder,and provides the first humanized model of a myeloproliferative disorder transforming into acute leukemia in mice. The established in vivo EMS model should provide a valuable tool for future studies of this disorder. View Publication