Kim Theilgaard-Mönch

Upregulation of Flt3 Expression within the Bone Marrow Lin−Sca1+c-kit+ Stem Cell Compartment Is Accompanied by Loss of Self-Renewal Capacity

Flt3 has emerged as a potential regulator of hematopoietic stem cells (HSC). Sixty percent of cells in the mouse marrow Lin(-)Sca1(+)c-kit(+) HSC pool expressed flt3. Although single cell cloning showed comparable high proliferative, myeloid, B, and T cell potentials of Lin(-)Sca1(+)c-kit(+)flt3(+) and Lin(-)Sca1(+)c-kit(+)flt3(-) cells, only Lin(-)Sca1(+)c-kit(+)flt3(-) cells supported sustained multilineage reconstitution. In striking contrast, Lin(-)Sca1(+)c-kit(+)flt3(+) cells rapidly and efficiently reconstituted B and T lymphopoiesis, whereas myeloid reconstitution was exclusively short term. Unlike c-kit, activation of flt3 failed to support survival of HSC, whereas only flt3 mediated survival of Lin(-)Sca1(+)c-kit(+)flt3(+) reconstituting cells. Phenotypic and functional analysis support that Lin(-)Sca1(+)c-kit(+)flt3(+) cells are progenitors for the common lymphoid progenitor. Thus, upregulation of flt3 expression on Lin(-)Sca1(+)c-kit(+) HSC cells is accompanied by loss of self-renewal capacity but sustained lymphoid-restricted reconstitution potential.

Key Role of flt3 Ligand in Regulation of the Common Lymphoid Progenitor but Not in Maintenance of the Hematopoietic Stem Cell Pool

The first lineage commitment step of hematopoietic stem cells (HSC) results in separation into distinct lymphoid and myeloid differentiation pathways, reflected in the generation of common lymphoid and myeloid progenitors (CLP and CMP, respectively). In this report we present the first evidence for a nonredundant regulator of this process, in that adult mice deficient in expression of the flt3 ligand (FL) have severely (10-fold) reduced levels of the CLP, accompanied by reductions in the earliest identifiable B and T cell progenitors. In contrast, CMP and HSC are unaffected in FL-deficient mice. Noteworthy, CLP express high levels of both the flt3 receptor and ligand, indicating a potential autocrine role of FL in regulation of the earliest lymphoid commitment step from HSC.

NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements

Nonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance process that eliminates mRNAs containing premature termination codons (PTCs). NMD has been hypothesized to impact on several aspects of cellular function; however, its importance in the context of a mammalian organism has not been addressed in detail. Here we use mouse genetics to demonstrate that hematopoietic-specific deletion of Upf2, a core NMD factor, led to the rapid, complete, and lasting cell-autonomous extinction of all hematopoietic stem and progenitor populations. In contrast, more differentiated cells were only mildly affected in Upf2-null mice, suggesting that NMD is mainly essential for proliferating cells. Furthermore, we show that UPF2 loss resulted in the accumulation of nonproductive rearrangement by-products from the Tcrb locus and that this, as opposed to the general loss of NMD, was particularly detrimental to developing T-cells. At the molecular level, gene expression analysis showed that Upf2 deletion led to a profound skewing toward up-regulated mRNAs, highly enriched in transcripts derived from processed pseudogenes, and that NMD impacts on regulated alternative splicing events. Collectively, our data demonstrate a unique requirement of NMD for organismal survival.

The Transcriptional Activation Program of Human Neutrophils in Skin Lesions Supports Their Important Role in Wound Healing

To investigate the cellular fate and function of polymorphonuclear neutrophilic granulocytes (PMNs) attracted to skin wounds, we used a human skin-wounding model and microarray technology to define differentially expressed genes in PMNs from peripheral blood, and PMNs that had transmigrated to skin lesions. After migration to skin lesions, PMNs demonstrated a significant transcriptional response including genes from several different functional categories. The up-regulation of anti-apoptotic genes concomitant with the down-regulation of proapoptotic genes suggested a transient anti-apoptotic priming of PMNs. Among the up-regulated genes were cytokines and chemokines critical for chemotaxis of macrophages, T cells, and PMNs, and for the modulation of their inflammatory responses. PMNs in skin lesions down-regulated receptors mediating chemotaxis and anti-microbial activity, but up-regulated other receptors involved in inflammatory responses. These findings indicate a change of responsiveness to chemotactic and immunoregulatory mediators once PMNs have migrated to skin lesions and have been activated. Other effects of the up-regulated cytokines/chemokines/enzymes were critical for wound healing. These included the breakdown of fibrin clots and degradation of extracellular matrix, the promotion of angiogenesis, the migration and proliferation of keratinocytes and fibroblasts, the adhesion of keratinocytes to the dermal layer, and finally, the induction of anti-microbial gene expression in keratinocytes. Notably, the up-regulation of genes, which activate lysosomal proteases, indicate a priming of skin lesion-PMNs for degradation of phagocytosed material. These findings demonstrate that migration of PMNs to skin lesions induces a transcriptional activation program, which regulates cellular fate and function, and promotes wound healing.

Regulation of human neutrophil granule protein expression

The function of the mature polymorphonuclear neutrophil is dependent on its granules, each with its characteristic content of proteins. The granule proteins are formed at different stages during maturation of neutrophils from myeloblasts to segmented cells. The regulation of granule protein expression is controlled by a number of transcription factors, many of which are also essential for commitment of multipotent hematopoietic stem cells to lineage-committed myeloid progenitor cells and for differentiation of these progenitor cells; among these, PU.1 and C/EBPalpha stand out as critical for all granule proteins whereas AML-1 is critical for primary granule protein expression and C/EBPepsilon for secondary and tertiary granule protein expression.

Loss of C/EBP alpha cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage

CCAAT/enhancer binding protein (C/EBP)α is a myeloid-specific transcription factor that couples lineage commitment to terminal differentiation and cell cycle arrest, and is found mutated in 9% of patients who have acute myeloid leukemia (AML). We previously showed that mutations which dissociate the ability of C/EBPα to block cell cycle progression through E2F inhibition from its function as a transcriptional activator impair the in vivo development of the neutrophil granulocyte and adipose lineages. We now show that such mutations increase the capacity of bone marrow (BM) myeloid progenitors to proliferate, and predispose mice to a granulocytic myeloproliferative disorder and transformation of the myeloid compartment of the BM. Both of these phenotypes were transplantable into lethally irradiated recipients. BM transformation was characterized by a block in granulocyte differentiation, accumulation of myeloblasts and promyelocytes, and expansion of myeloid progenitor populations—all characteristics of AML. Circulating myeloblasts and hepatic leukocyte infiltration were observed, but thrombocytopenia, anemia, and elevated leukocyte count—normally associated with AML—were absent. These results show that disrupting the cell cycle regulatory function of C/EBPα is sufficient to initiate AML-like transformation of the granulocytic lineage, but only partially the peripheral pathology of AML.

Neutrophils and keratinocytes in innate immunity—cooperative actions to provide antimicrobial defense at the right time and place

The human neutrophil is a professional phagocyte of fundamental importance for defense against microorganisms, as witnessed by the life‐threatening infections occurring in patients with neutropenia or with defects that result in decreased microbicidal activity of the neutrophil [ 1 , 2 ]. Likewise, the skin and mucosal surfaces provide important barriers against infections. Traditionally, these major defense systems, the epithelial cells and the neutrophils, have been viewed as limited in their armory: The epithelial cells provide defense by constituting a physical barrier, and the neutrophils provide instant delivery of preformed antimicrobial substances or on‐the‐spot assembly of the multicomponent reduced nicotinamide adenine dinucleotide phosphate oxidase from stored components for the generation of reactive oxygen metabolites. Recent research has shown that epithelial cells are highly dynamic and able to generate antimicrobial peptides in response not only to microbial infection itself [ 3 4 5 6 ] but more importantly, to the growth factors that are called into play when the physical barrier is broken, and the risk of microbial infection is imminent [ 7 ]. Likewise, the neutrophil changes its profile of actively transcribed genes when it diapedeses into wounded skin [ 8 ]. This results in generation of signaling molecules, some of which support the growth and antimicrobial potential of keratinocytes and epithelial cells. This paper will highlight some recent advances in this field.

HemaExplorer: a database of mRNA expression profiles in normal and malignant haematopoiesis

The HemaExplorer (http://servers.binf.ku.dk/hemaexplorer) is a curated database of processed mRNA Gene expression profiles (GEPs) that provides an easy display of gene expression in haematopoietic cells. HemaExplorer contains GEPs derived from mouse/human haematopoietic stem and progenitor cells as well as from more differentiated cell types. Moreover, data from distinct subtypes of human acute myeloid leukemia is included in the database allowing researchers to directly compare gene expression of leukemic cells with those of their closest normal counterpart. Normalization and batch correction lead to full integrity of the data in the database. The HemaExplorer has comprehensive visualization interface that can make it useful as a daily tool for biologists and cancer researchers to assess the expression patterns of genes encountered in research or literature. HemaExplorer is relevant for all research within the fields of leukemia, immunology, cell differentiation and the biology of the haematopoietic system.

Highly glycosylated α1‐acid glycoprotein is synthesized in myelocytes, stored in secondary granules, and released by activated neutrophils

α‐1‐Acid glycoprotein (AGP) is an acute‐phase protein produced by hepatocytes and secreted into plasma in response to infection/injury. We recently assessed the transcriptional program of terminal granulocytic differentiation by microarray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/metamyelocytes (MYs), and BM neutrophils. These analyses demonstrated a transient, high mRNA expression of genuine secondary/tertiary granule proteins and AGP in MYs. In agreement with this, immunocytochemistry revealed the presence of AGP protein and the secondary granule protein lactoferrin in cells from the MY stage and throughout granulocytic differentiation. Immunoelectron microscopy demonstrated the colocalization of AGP and lactoferrin in secondary granules of neutrophils. This finding was substantiated by the failure to detect AGP and lactoferrin in blood cells from a patient with secondary/tertiary (specific) granule deficiency. In addition, Western blot analysis of subcellular fractions isolated from neutrophils revealed that neutrophil‐derived AGP, localized in secondary granules, was abundant and highly glycosylated compared with endocytosed, plasma‐derived AGP localized in secretory vesicles. Exocytosis studies further demonstrated a marked release of AGP and lactoferrin by activated neutrophils. Finally, induction of CCAAT/enhancer‐binding protein (C/EBP)‐ɛ in a myeloid cell line was shown to increase AGP transcript levels, indicating that AGP expression in myeloid cells, like in hepatocytes, is partially regulated by members of the C/EBP family. Overall, these findings define AGP as a genuine secondary granule protein of neutrophils. Hence, neutrophils, which constitute the first line of defense, are likely to serve as the primary local source of AGP at sites of infection or injury.

Comparing cancer vs normal gene expression profiles identifies new disease entities and common transcriptional programs in AML patients

Gene expression profiling has been used extensively to characterize cancer, identify novel subtypes, and improve patient stratification. However, it has largely failed to identify transcriptional programs that differ between cancer and corresponding normal cells and has not been efficient in identifying expression changes fundamental to disease etiology. Here we present a method that facilitates the comparison of any cancer sample to its nearest normal cellular counterpart, using acute myeloid leukemia (AML) as a model. We first generated a gene expression-based landscape of the normal hematopoietic hierarchy, using expression profiles from normal stem/progenitor cells, and next mapped the AML patient samples to this landscape. This allowed us to identify the closest normal counterpart of individual AML samples and determine gene expression changes between cancer and normal. We find the cancer vs normal method (CvN method) to be superior to conventional methods in stratifying AML patients with aberrant karyotype and in identifying common aberrant transcriptional programs with potential importance for AML etiology. Moreover, the CvN method uncovered a novel poor-outcome subtype of normal-karyotype AML, which allowed for the generation of a highly prognostic survival signature. Collectively, our CvN method holds great potential as a tool for the analysis of gene expression profiles of cancer patients.