Myeloid Cell Development Research Articles

IgE receptor induces Stat5b serine phosphorylation in mast cells (HYP3P.346)

Abstract Signal Transducers and Activators of Transcription (STATs) are latent transcription factors that mediate a number of cellular responses including but not limited to cell survival, proliferation and differentiation. This protein family consists of seven members, STAT1-6 including two closely related molecules, STAT5a and STAT5b, that show 96% amino acid sequence homology and are critical for lymphoid, myeloid, and erythroid cell development and function. We recently identified Stat5b as the critical regulator of IgE-mediated cytokine production in mast cells. We found that Fyn-mediated Stat5 tyrosine phosphorylation is critical for Stat5-dependent effects. Our recent work demonstrates that Stat5b is also phosphorylated on serine residues during IgE signaling in mast cells. We find that STAT5b serine phosphorylation occurs through a Src-independent pathway requiring PI3-kinase function. Understanding the pathways leading to Stat5b serine phosphorylation and its role in the inflammatory response will yield new targets for mast cell-associated diseases.

Development of a novel method for isolation of four developmentally distinct myeloid derived dendritic cells pre-cursors: phenotypic and functional analyses (HEM3P.283)

Abstract Myeloid cell progenitors have the potential to give rise to dendritic cells which have a high capacity to stimulate adaptive immune responses as well as to cells with potent immunosuppressive activity. However, the stage in their development in which these functions are gained is poorly defined. Here, we developed a method for isolating progenitor populations at specific stages of development. Bone marrow cells were lineage depleted and cultured in GM-CSF to drive their differentiation toward a myeloid lineage for three days. Cells were then sorted based on Ly6C and CSF-1R (CD115) expression, two markers whose expression is known to change throughout myeloid ontogeny. We then monitored the developmental progression of each population based on the expression of these markers as well as other hallmarks of dendritic cell or myeloid suppressor cell phenotypes. We also measured the function of each population relative to antigen uptake and degradation as well as their response to stimulation by TLR agonists. Collectively, our results indicate that myeloid progenitors progress along this pathway: Ly6C-CD115- -> Ly6C+CD115- -> Ly6C+CD115+ -> Ly6C-CD115+ -> Ly6C-CD115+. Phenotypically, these populations represent CMP, MDSC, monocytes, immature DC, and more mature DC, respectively. These findings not only enhance our understanding of myeloid cell development and differentiation driven by GM-CSF, but also inform the design of GM-CSF-derived DC based vaccine strategies.

The dynamic lives of macrophage and dendritic cell subsets in atherosclerosis

Atherosclerosis, the major pathological process through which arterial plaques are formed, is a dynamic chronic inflammatory disease of large- and medium-sized arteries in which the vasculature, lipid metabolism, and the immune system all play integral roles. Both the innate and adaptive immune systems are involved in the development and progression of atherosclerosis but myeloid cells represent the major component of the burgeoning atherosclerotic plaque. Various myeloid cells, including monocytes, macrophages (MΦs), and dendritic cells (DCs) can be found within the healthy and atherosclerotic arterial wall, where they can contribute to or regulate inflammation. However, the precise behaviors and functions of these cells in situ are still active areas of investigation that continue to yield exciting and surprising new data. Here, we review recent progress in understanding of the complex biology of MΦs and DCs, focusing particularly on the dynamic regulation of these subsets in the arterial wall and novel, emerging functions of these cells during atherogenesis.

Spermidine/spermine N1-acetyltransferase activity associates with white blood cell count in myeloid leukemias

The metabolism of polyamines, the cationic small molecules essential for cell proliferation and differentiation, is altered in cancer cells and can be exploited in cancer diagnosis and therapy. Spermidine/spermine N(1)-acetyltransferase (SSAT), which regulates intracellular levels of polyamines by catabolizing spermidine and spermine, has a controversial role in the development of cancers. In this study, the polyamine metabolism and function of SSAT were characterized in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoid leukemia patient samples. Also, mice overexpressing SSAT and having a myeloproliferative phenotype were analyzed for their response to decitabine and histone deacetylase inhibitor trichostatin A. The presence of epigenetic factors in the bone marrow cells of SSAT mice was analyzed. Elevated levels of spermidine and spermine, as well as increased activity of SSAT, were detected in AML, CML, and acute lymphoid leukemia patients compared with the controls. However, we found SSAT activity to be associated with white blood cell count only in AML and CML patients. Decitabine treatment brought the peripheral blood and bone marrow cell counts of SSAT mice to the level of wild-type mice. Spermidine/spermine N(1)-acetyltransferase mice had increased histone methylation and an increased level of histone deacetylase 1 in their bone marrow cells. The study suggests that SSAT influences the development of myeloid malignancies, and epigenetic factors partly contribute to the SSAT overexpression-induced myeloproliferative disease in mice.

Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells

Human pluripotent stem cells offer a powerful system to study human biology and disease. Here, we report a system to both express transgenes specifically in ES cell derived hematopoietic cells and knockdown gene expression stably throughout the differentiation of ES cells. We characterize a CD43 promoter construct that when inserted into the AAVS1 “safe harbor” locus utilizing a zinc finger nuclease specifically drives GFP expression in hematopoietic cells derived from a transgenic ES cell line and faithfully recapitulates endogenous CD43 expression. In addition, using the same gene targeting strategy we demonstrate that constitutive expression of short hairpin RNAs within a microRNA backbone can suppress expression of PU.1, an important regulator of myeloid cell development. We show that PU.1 knockdown cell lines display an inhibition in myeloid cell formation and skewing towards erythroid development. Overall, we have generated a powerful system to track hematopoietic development from pluripotent stem cells and study gene function through hematopoietic specific gene expression and constitutive gene knockdown.

B- myb is an essential regulator of hematopoietic stem cell and myeloid progenitor cell development

The B-myb (MYBL2) gene is a member of the MYB family of transcription factors and is involved in cell cycle regulation, DNA replication, and maintenance of genomic integrity. However, its function during adult development and hematopoiesis is unknown. We show here that conditional inactivation of B-myb in vivo results in depletion of the hematopoietic stem cell (HSC) pool, leading to profound reductions in mature lymphoid, erythroid, and myeloid cells. This defect is autonomous to the bone marrow and is first evident in stem cells, which accumulate in the S and G2/M phases. B-myb inactivation also causes defects in the myeloid progenitor compartment, consisting of depletion of common myeloid progenitors but relative sparing of granulocyte-macrophage progenitors. Microarray studies indicate that B-myb-null LSK(+) cells differentially express genes that direct myeloid lineage development and commitment, suggesting that B-myb is a key player in controlling cell fate. Collectively, these studies demonstrate that B-myb is essential for HSC and progenitor maintenance and survival during hematopoiesis.

Pancreatic-carcinoma-cell-derived pro-angiogenic factors can induce endothelial-cell differentiation of a subset of circulating CD34+ progenitors

BackgroundCD34+ progenitor cells comprise both hematopoietic and endothelial progenitor cells. Recent studies suggest that circulating endothelial progenitor cells are recruited into the angiogenic vascular system of several cancers, including pancreatic carcinoma, and that they correlate with clinical progress. However, whether endothelial progenitor cell mobilization occurs in response to cytokine release by tumor cells is still unclear.MethodsThe chemotactic- and/or differentiating-activities of the poorly-differentiated pancreatic carcinoma cell line PT45, and of the immortal H6c7 cell line, a line of near-normal pancreatic duct epithelial cells, on endothelial progenitor cells were investigated in vitro using circulating CD34+ as model.ResultsThe study showed that Vascular Endothelial Growth Factor produced by PT45 cells and, at lesser extent, by H6c7 cells, predominantly chemoattract peripheral blood CD34+ expressing the type 2 relative receptor. Addition of PT45-conditioned medium to CD34+ cells, cultured under conditions supporting myeloid cell development, diverted the differentiation of a subset of these progenitor cells into cells expressing endothelial cell markers, such as CD146, CD105, VE-cadherin and von Willebrand Factor-related antigen. Moreover, these endothelial-like cells formed capillary networks in vitro, chiefly through the release of Angiopoietin-1 by PT45 cells.ConclusionsThe results demonstrate that pancreatic-carcinoma cells potentially attract circulating endothelial progenitor cells to the tumor site, by releasing high levels of pro-angiogenic factors such as Vascular Endothelial Growth Factor and Angiopoietin-1, and may direct the differentiation of these cell subsets of the CD34+ cell population into endothelial cells; the latter cells may become a component of the newly-formed vessels, contributing to angiogenesis-mediated tumor growth and metastasis.

Methylation status of CEBPA gene promoter in chronic myeloid leukemia

CCAAT/enhancer binding protein alpha is one of the crucial transcription factors for myeloid cell development that has been found to be involved in hematopoietic differentiation and leukemiogenesis. Recently, epigenetic regulation of CEBPA expression through DNA methylation has been demonstrated in leukemia. The aim of this study was to investigate the methylation status of CEBPA gene in chronic myeloid leukemia (CML) patients. The methylation status of CEBPA promoter was studied in 100 patients with CML and 98 normal healthy individuals from Hyderabad, India, using methylation-specific polymerase chain reaction. The aberrant methylation of CEBPA gene promoter was found in 32 of the 100 CML cases. A highly significant association was found between the frequency of CEBPA gene promoter hypermethylation and the CML stages (P = 0.017), but association with respect to age and gender of the patient was not found. The results suggest that aberrant methylation in the CpG island of the promoter region of this gene might be a common event in CML, and systemic expression studies will be needed to unfold the role of CEBPA promoter methylation in the development, progression, and prognosis of CML.

Activation Of EphrinB2/EphB4 Influences Myeloid Leukemia Cell Migration and Invasion

Eph receptors and ephrin ligands are cell-surface molecules capable of bidirectional signaling that control cell-cell interactions, migration and invasion. However, their role and regulation in myeloid leukemia cells remain to be defined. To address the hypothesis that Ephrin/EphB is an important regulator of myeloid leukemia cell migration and invasion, we first screened the mRNA levels of 23 eph and ligand ephrin RTK family members in myeloid leukemia cells (K562, HL-60, THP-1) and mononuclear cells from healthy donors, then found that EphB4, EphA5, EfnA1 highly expressed in most myeloid leukemia cells compared to healthy donors(P<0.05). Both the mRNA and protein levels of EphB4 and EphA5 were detected in 13 primary myeloid leukemia cells (5 from patients with extramedullary leukemia among 13 cases) and 10 mononuclear cells from healthy donors by real-time RT-PCR and Immunoblot analysis. The results showed that both the mRNA and protein levels of EphB4 and EphA5 were higher in 13 primary myeloid leukemia cells relative to the 10 healthy donors (P=0.046). Moreover, the EphB4 were highly expressed in 5 patients with extramedullary leukemia compared with 8 patients without extramedullary leukemia. These findings indicated that EphB4 and EphA5 expression were correlated with the development of myeloid leukemia cells, moreover, EphB4 may be closely related with myeloid leukemia cell migration or invasion. To further clarified the question, migration were determined in leukemia cell lines (K562 cells) which were treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins by transwell migration assay. Invasion were also determined by matrigel invasion assay. The results showed that, after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (1.8 to 2.5-fold, P<0.05), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.2 to 1.8-fold, P<0.05). However, after ephrinA1–Fc stimulation, the numbers of K562 cells migrating through transwell chamber didn't significantly increase compared to Fc proteins stimulation (P>0.05), and the numbers of K562 cells invading the matrigel also didn't enhanced (P>0.05). These findings indicated that ephrinB2–Fc could activate EphB4, leading to the change of myeloid leukemia cell migration and invasion. Further study may help to assess a promising potential of this protein to be used as a prognostic marker or as a target for a molecular therapy. Disclosures:No relevant conflicts of interest to declare.

Differential Expression Of TREM-1 In Myelogenous Leukemia Cells

ObjectivesTriggering receptor expressed on myeloid cells (TREM) -1 is a receptor as a member of the immunoglobulin superfamily expressed on the cell-surface of neutrophils, monocytes and macrophages. This receptor amplifies the inflammatory response, activating the signaling pathway. TREM-1 expression is associated with mature myeloid cell development. TREM-1 is shed from the membrane of activated macrophages without the transmembrane and intracellular domains, and can be found as soluble TREM (sTREM)-1. Soluble TREM-1 is thought to negatively regulate TREM receptor signaling. Some studies currently reported that TREM-1 regulates the malignant behavior of cancer cells in lung cancer and HCC. However, no related studies about the role of TREM-1 in leukemia have been carried out. The aims of this study was investigated the TREM-1 expression in myelogenous leukemia cells. MethodsThirty-five patients with AML, twenty-five patients with CML and a control group of eleven healthy people were subjected to the study. TREM-1 expressions on the surfaces of leukemia cells were measured by flow cytometry. Plasma sTREM-1 levels were measured by ELISA. ResultsIn this study, our results provide the first evidence that TREM-1 was differentially expressed in myelogenous leukemia cells. The TREM-1 mean ratio of median fluorescence intensity (mean ratio of MFI) was 3.13±0.88 and 2.52±0.40 in CML and AML patients, respectively. The TREM-1 mean ratio of MFI was 3.03±1.40 in myelogenous leukemia cell lines (K562, HL60, THP-1). The TREM-1 mean ratio of MFI was 5.37±0.88 in healthy controls. Compared to healthy controls, myelogenous leukemia cells had decreased TREM-1 expressions (P<0.001). The TREM-1 mean ratio of MFI was 4.89±0.60 in patients who are in complete remission after Novartis's Gleevec therapy. Compared with CML patient groups, patients who are in complete remission after Gleevec therapy had rising TREM-1 expressions (P<0.01). TREM-1 expressions of patients who are in complete remission after Gleevec therapy were slightly lower than the healthy controls, but this did not reach significance. No significant difference in TREM-1 expressions was seen between AML and CML patient groups, male and female patient groups, and cells derived from peripheral blood and bone marrow of the same leukemia patients (p>0.1).In addition, the plasma sTREM-1 levels were measured by ELISA. sTREM-1 levels was 48.54±57.63pg/mL for AML group and 43.72±23.93pg/mL for CML group. Results indicated that plasma sTREM-1 levels significantly higher in AML and CML patients than that in healthy controls (P<0.01). However, there was no significant difference in plasma sTREM-1 levels observed in AML patient group compared with CML patient group, male patients group compared with female patients group, and plasma from peripheral blood compared with plasma from bone marrow of the same leukemia patients (p>0.1).An ongoing project focuses on the relationship between the function of TREM-1 and occurrence, progression and prognosis of myelogenous leukemia, advances will be reported in time. ConclusionTREM-1 expression on leukemia cells was significantly lower in patients with AML and CML than those in healthy controls and patients in complete remission had increased TREM-1 expression. Patients with AML and CML had increased plasma soluble TREM-1. The TREM-1 expression on leukemia cells had an inverse correlation with plasma sTREM-1 level in AML and CML patients. Disclosures:No relevant conflicts of interest to declare.

Transcriptome Profiling Reveals Insight into Distinct Immune Responses to Aeromonas salmonicida in Gill of Two Rainbow Trout Strains

The fish gills represent a crucial organ for the communication with the aquatic environment. Transcriptional changes in gills of two hatchery rainbow trout strains in response to injection with the potent pathogen Aeromonas salmonicida were detected by global gene expression profiling using a 4×44K oligonucleotide microarray. Emphasis was placed on "day 3 postinfection" representing a decisive time point for the resolution of inflammation. The comparison of features and pathways differentially regulated in branchial tissues revealed that the local breeding strain BORN and imported American rainbow trout apply common and specific immune strategies. In gills of infected BORN trout, we observed a dynamic regulation of genes controlling NF-κB pathways and the induction of factors promoting the development of myeloid cells, whereas an increased expression of lysozyme and immunoglobulin genes was obvious in gills of infected import trout. In order to prove the relevance of the array-predicted candidates as well as well-known immune genes for gill immunity, a subsequent in vitro experiment was conducted. Altogether, we uncovered dynamic but moderate changes in the expression of a broad range of immune-relevant features implying the gill's involvement in pathogen defense strategies.

Establishment of a Human Allergy Model Using Human IL-3/GM-CSF–Transgenic NOG Mice

The development of animal models that mimic human allergic responses is crucial to study the pathophysiology of disease and to generate new therapeutic methodologies. Humanized mice reconstituted with human immune systems are essential to study human immune reactions in vivo and are expected to be useful for studying human allergies. However, application of this technology to the study of human allergies has been limited, largely because of the poor development of human myeloid cells, especially granulocytes and mast cells, which are responsible for mediating allergic diseases, in conventional humanized mice. In this study, we developed a novel transgenic (Tg) strain, NOD/Shi-scid-IL2rγ(null) (NOG), bearing human IL-3 and GM-CSF genes (NOG IL-3/GM-Tg). In this strain, a large number of human myeloid cells of various lineages developed after transplantation of human CD34⁺ hematopoietic stem cells. Notably, mature basophils and mast cells expressing FcεRI were markedly increased. These humanized NOG IL-3/GM-Tg mice developed passive cutaneous anaphylaxis reactions when administered anti-4-hydroxy-3-nitrophenylacetyl IgE Abs and 4-hydroxy-3-nitrophenylacetyl. More importantly, a combination of serum from Japanese cedar pollinosis patients and cedar pollen extract also elicited strong passive cutaneous anaphylaxis responses in mice. Thus, to our knowledge, our NOG IL-3/GM-Tg mice are the first humanized mouse model to enable the study of human allergic responses in vivo and are excellent tools for preclinical studies of allergic diseases.

Induction of Functional Human Macrophages from Bone Marrow Promonocytes by M-CSF in Humanized Mice

Engraftment of human CD34⁺ hematopoietic stem/progenitor cells into immunodeficient mice leads to robust reconstitution of human T and B cells but not monocytes and macrophages. To identify the cause underlying the poor monocyte and macrophage reconstitution, we analyzed human myeloid cell development in humanized mice and found that it was blocked at the promonocyte stage in the bone marrow. Expression of human M-CSF or GM-CSF by hydrodynamic injection of cytokine-encoding plasmid completely abolished the accumulation of promonocytes in the bone marrow. M-CSF promoted the development of mature monocytes and tissue-resident macrophages whereas GM-CSF did not. Moreover, correlating with an increased human macrophages at the sites of infection, M-CSF-treated humanized mice exhibited an enhanced protection against influenza virus and Mycobacterium infection. Our study identifies the precise stage at which human monocyte/macrophage development is blocked in humanized mice and reveals overlapping and distinct functions of M-CSF and GM-CSF in human monocyte and macrophage development. The improved reconstitution and functionality of monocytes/macrophages in the humanized mice following M-CSF expression provide a superior in vivo system to investigate the role of macrophages in physiological and pathological processes.

Down-Regulation of the Oncogene PTTG1 via the KLF6 Tumor Suppressor during Induction of Myeloid Differentiation

The aberrant expression of proto-oncogenes is involved in processes that are responsible for cellular proliferation and the inhibition of myeloid differentiation in acute myeloid leukemia (AML). Pituitary Tumor-Transforming gene 1 (PTTG1), an oncogenic transcription factor, is abundantly expressed in various human cancers and hematopoietic malignancies. However, its expression in normal leukocytes and most normal tissues is very low or undetectable. The mechanism by which PTTG1 overexpression modifies myeloid cell development and promotes leukemogenesis remain unclear. To investigate the mechanistic links between PTTG1 overexpression and leukemia cell differentiation, we utilized phorbol 12-myristate 13-acetate (PMA), a well-known agent that triggers monocyte/macrophage differentiation, to analyze the expression patterns of PTTG1 in PMA-induced myeloid differentiation. We found that PTTG1 is down-regulated at the transcriptional level in PMA-treated HL-60 and THP1 cells. In addition, we identified a binding site for a tumor suppressor protein, Kruppel-like factor 6 (KLF6), in the PTTG1 promoter. We found that KLF6 could directly bind and repress PTTG1 expression. In HL-60 and THP1 cells, KLF6 mRNA and protein levels are up-regulated with a concordant reduction of PTTG1 expression upon treatment with PMA. Furthermore, KLF6 knockdown by shRNA abolished the suppression of PTTG1 and reduced the activation of the differentiation marker CD11b in PMA-primed cells. The protein kinase C (PKC) inhibitor and the MAPK/ERK kinase (MEK) inhibitor significantly blocked the potentiation of PMA-mediated KLF6 induction and the down-regulation of PTTG1, indicating that PTTG1 is suppressed via the activation of PKC/ERK/KLF6 pathway. Our findings suggest that drugs that increase the KLF6 inhibition of PTTG1 may have a therapeutic application in AML treatment strategies.

Myeloid cell-specific TRAF3-deficient mice exhibit normal myeloid cell development and homeostasis (P1196)

Abstract Myeloid cells, including monocytes, macrophages and granulocytes, are crucial players of innate immunity and inflammation. These cells constitutively or inducibly express a number of receptors of the TNF receptor superfamily and Toll-like receptors (TLRs), whose signals are transduced by TRAF molecules. In light of the in vitro evidence that TRAF3 is required for TLRs-induced type I interferon production and for CD40-induced IL-12 production in macrophages, we generated myeloid cell-specific TRAF3-/- (M-TRAF3-/-; TRAF3flox/floxLysM+/Cre) mice to investigate the in vivo functions of TRAF3 in myeloid cells. We examined immune cell populations in the bone marrow, spleen, and peritoneal cavity in M-TRAF3-/- mice at the age of 8-12 weeks. No significant differences in the frequencies or numbers of immune cell populations such as B cells, T cells, granulocytes and macrophages were found between M-TRAF3-/- and littermate control (LMC) mice. TRAF3-/- macrophages also expressed similar levels of MHC class II, CD80 and CD86 as compared to LMC macrophages, suggesting that no endogenous activation of macrophages occurs. Our findings indicate that specific ablation of TRAF3 in myeloid cells neither affected myeloid cell maturation nor changed the homeostasis of macrophages and neutrophils. In contrast, activation of macrophages induced by TLRs was altered in the absence of TRAF3. Our ongoing study will further elucidate the in vivo functions of TRAF3 in innate immunity and inflammation.

The pur protein family: genetic and structural features in development and disease.

The Pur proteins are an ancient family of sequence-specific single-stranded nucleic acid-binding proteins. They bind a G-rich element in either single- or double-stranded nucleic acids and are capable of displacing the complementary C-rich strand. Recently several reports have described Pur family member knockouts, mutations, and disease aberrations. Together with a recent crystal structure of Purα, these data reveal conserved structural features of these proteins that have been adapted to serve functions unique to higher eukaryotes. In humans Pur proteins are critical for myeloid cell development, muscle development, and brain development, including trafficking of mRNA to neuronal dendrites. Pur family members have been implicated in diseases as diverse as cancer, premature aging, and fragile-X mental retardation syndrome.

Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer

Two major populations of myeloid-derived suppressor cells (MDSC), monocytic MDSC (M-MDSC) and polymorphonuclear MDSC (PMN-MDSC) regulate immune responses in cancer and other pathologic conditions. Under physiologic conditions, Ly6ChiLy6G− inflammatory monocytes, which are the normal counterpart of M-MDSC, differentiate into macrophages and dendritic cells (DCs). PMN-MDSC is the predominant group of MDSC that accumulates in cancer. Here we show that a large proportion of M-MDSC in tumor-bearing mice acquired phenotypic, morphological and functional features of PMN-MDSC. Acquisition of this phenotype, but not the functional attributes of PMN-MDSC, was mediated by transcriptional silencing of the retinoblastoma (Rb) gene through epigenetic modifications mediated by histone deacetylase 2 (HDAC-2). These data demonstrate novel mechanism regulation of myeloid cells in cancer.

Scratching the surface: signaling and routing dynamics of the CSF3 receptor

Following activation by their cognate ligands, cytokine receptors undergo intracellular routing towards lysosomes where they are degraded. Cytokine receptor signaling does not terminate at the plasma membrane, but continues throughout the endocytotic pathway. The modes of internalization and intracellular trafficking of specific receptors, the level of recycling towards the plasma membrane, the type of protein modifications (phosphorylation, ubiquitination) and the enzymes involved in these processes are remarkably diverse. This heterogeneity may contribute to the fine-tuning of signal amplitudes and duration from different receptors. The colony-stimulating factor 3 receptor (CSF3R) is unique for its balanced signaling output, first leading to proliferation of myeloid progenitors, followed by a cell cycle arrest and granulocytic differentiation. The mechanisms associated with CSF3R signal modulation, involving receptor lysine ubiquitination and redox-controlled phosphatase activities, are to a large extent confined to the signaling endosome. Interactions between signaling endosomes and the endoplasmic reticulum play a key role in this process. Here, we review the mechanisms of intracellular routing of CSF3R, their consequences for myeloid blood cell development and their implications for myeloid diseases.

IRF-8 Controls Melanoma Progression by Regulating the Cross Talk between Cancer and Immune Cells within the Tumor Microenvironment

The transcription factor interferon regulatory factor-8 (IRF-8) is crucial for myeloid cell development and immune response and also acts as a tumor suppressor gene. Here, we analyzed the role of IRF-8 in the cross talk between melanoma cells and tumor-infiltrating leukocytes. B16-F10 melanoma cells transplanted into IRF-8-deficient (IRF-8(-/-)) mice grow more rapidly, leading to higher numbers of lung metastasis, with respect to control animals. These events correlated with reduced dendritic cell and T cell infiltration, accumulation of myeloid-derived suppressor cells and a chemokine/chemokine receptor expression profile within the tumor microenvironment supporting tumor growth, angiogenesis, and metastasis. Noticeably, primary tumors developing in IRF-8(-/-) mice displayed a clear-cut inhibition of IRF-8 expression in melanoma cells. Injection of the demethylating agent 5-aza-2'-deoxycytidine into melanoma-bearing IRF-8(-/-) animals induced intratumoral IRF-8 expression and resulted in the re-establishment of a chemokine/ chemokine receptor pattern favoring leukocyte infiltration and melanoma growth arrest. Importantly, intrinsic IRF-8 expression was progressively down-modulated during melanoma growth in mice and in human metastatic melanoma cells with respect to primary tumors. Lastly, IRF-8 expression in melanoma cells was directly modulated by soluble factors, among which interleukin-27 (IL-27), released by immune cells from tumor-bearing mice. Collectively, these results underscore a key role of IRF-8 in the cross talk between melanoma and immune cells, thus revealing its critical function within the tumor microenvironment in regulating melanoma progression and invasiveness.

Impairment of Myeloid Dendritic Differentiation and Role in the Support of Splenic Dysmegakaryopoiesis in Patients with Primary Myelofibrosis

AbstractAbstract 1762 IntroductionThe primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by myeloproliferation, splenomegaly with hematopoietic metaplasia and dysmegakaryopoieisis. We have previously described an increase in Flt3 ligand (FL) in plasma and spleen of patients with PMF and its role in the dysmegakaryopoiesis (Desterke and al, Cancer Res, 2011). Account the importance of FL in development of splenic myeloid dendritic cells (mDC), we studied the differentiation of mDC in patients and its potential impact on dysmegakaryopoiesis. Patients and MethodsThe mDCs were obtained from cell culture of blood and spleen mononuclear cells in presence of fetal calf serum (FCS) and bacterial lipolysaccharides (LPS). The megakaryocytes were obtained by culturing CD34+ cells from blood or spleen in specific medium serum free in presence of IL-3, IL-6, IL-11 and Tpo. Gene expression was quantified by microarray and RT-QPCR, protein analysis by immunofluorescence and flow cytometry, and migration experiments were performed in Boyden chamber. ResultsTranscriptome of circulating CD34+ cells from PMF patients showed an increase in expression of genes encoding for integrin CD11c and also TLR4 and a decrease in the expression of gene encoding TLR9: suggesting the presence of progenitor mDCs in the blood. These results have been confirmed 1/ in cytometry by an increase in the number of CD34+ CD11c+ HLA-Dr+ cells in the blood; 2/ in cell culture by the presence of adherent cell colonies positives for TLR4+ CD11c+ HLA-Dr+ in the blood. The in vitro differentiation of mDC cells and the proportion of mature mDCs HLA-Dr+ CD11c+ cells are decreased in blood of PMF patients. Myeloid nature of circulating DCs was confirmed by the absence expression of the plasmacytoid membrane marker CD123 and by an increased of TLR4 and myeloid PU-1 (myeloid transcription factor) expression in opposite to a decreased of plasmacytoid markers: IL-23, HMGB1 and TLR9. Moreover in PMF patients, circulating adherent mDCs overexpressed CXCL12 chemokine and also FL which have abnormal chemottractant ability with respect to PMF megakaryocytic precursors still expressing flt3 receptor. Finally, in PMF patients, coculture of MK with mDC promotes their survival, differentiation and maturation (MK ploidy and transcriptional program). Primary results confirmed the presence of these mDCs precursors (CD34+ CD11c+ HLA-Dr+) in the spleen of PMF patients which harbored also an extramedullary megakaryopoiesis. These mDCs precursors are absent of the spleen from healthy subjects. ConclusionOur results show an increased presence of mDC progenitor population CD34+ HLA-Dr+ CD11c+ in the blood and the spleen of PMF patients. They also suggest that these cells are involved in migration, survival and differentiation/maturation of megakaryocytes, particulary in the spleen of patients. Disclosures:No relevant conflicts of interest to declare.