Dysregulated myeloid differentiation in colitis is induced by inflammatory osteoclasts in a TNFα-dependent manner.

Inflammatory bowel disease (IBD) is characterized by very severe intestinal inflammation associated with extra-intestinal manifestations. One of the most critical ones is bone destruction, which remains a major cause of morbidity and a risk factor for osteopenia and osteoporosis in IBD patients. In various mouse models of IBD, we and other have demonstrated concomitant bone loss due to a significant increase in osteoclast activity. Besides bone resorption, osteoclasts are known to control hematopoietic niches in vivo and modulate inflammatory responses in vitro, suggesting they may participate in chronic inflammation in vivo. Here, using different models of colitis, we showed that osteoclast inhibition significantly reduced disease severity and that induction of osteoclast differentiation by RANKL contributed to disease worsening. Our results demonstrate a direct link between osteoclast activity and myeloid cell accumulation in the intestine during colitis. RNAseq analysis of osteoclasts from colitic mice revealed overexpression of genes involved in the remodeling of hematopoietic stem cell niches. We also demonstrated that osteoclasts induced hematopoietic progenitor proliferation accompanied by a myeloid skewing in the early phases of colitis, which was confirmed in a model of RANKL-induced osteoclastogenesis. Mechanistically, inhibition of TNF-α reduced the induction of myeloid skewing by OCL both in vitro and in vivo. Lastly, we observed that osteoclastic activity and the proportion of myeloid cells in the blood are positively correlated in patients with Crohn's disease. Collectively, our results shed light on a new role of osteoclasts in colitis in vivo, demonstrating they exert their colitogenic activity through an early action on hematopoiesis, leading to an increase in myelopoiesis sustaining gut inflammation.

Alterations in the mammary gland and tumor microenvironment of formerly obese mice.

BACKGROUND: Obesity is a risk factor for breast cancer, and women with obesity that develop breast cancer have a worsened prognosis. Within the mammary gland, obesity causes chronic, macrophage-driven inflammation and adipose tissue fibrosis. Weight loss is a recommended intervention to resolve obesity, but the impact of weight loss on the mammary gland microenvironment and in tumors has not been well identified. METHODS: To examine the effects of weight loss following obesity, mice were fed a high-fat diet for 16 weeks to induce obesity, then switched to a low-fat diet for 6 weeks. We examined changes in immune cells, including fibrocytes, which are myeloid lineage cells that have attributes of both macrophages and myofibroblasts, and collagen deposition within the mammary glands of non-tumor-bearing mice and within the tumors of mice that were transplanted with estrogen receptor alpha positive TC2 tumor cells. RESULTS: In formerly obese mice, we observed reduced numbers of crown-like structures and fibrocytes in mammary glands, while collagen deposition was not resolved with weight loss. Following transplant of TC2 tumor cells into the mammary glands of lean, obese, and formerly obese mice, diminished collagen deposition and cancer-associated fibroblasts were observed in tumors from formerly obese mice compared to obese mice. Within tumors of obese mice, increased myeloid-derived suppressor cells and diminished CD8+ T cells were identified, while the microenvironment of tumors of formerly obese mice were more similar to tumors from lean mice. When TC2 tumor cells were mixed with CD11b+CD34+ myeloid progenitor cells, which are the cells of origin for fibrocytes, and transplanted into mammary glands of lean and obese mice, collagen deposition within the tumors of both lean and obese was significantly greater than when tumor cells were mixed with CD11b+CD34- monocytes or total CD45+ immune cells. CONCLUSIONS: Overall, these studies demonstrate that weight loss resolved some of the microenvironmental conditions within the mammary gland that may contribute to tumor progression. Additionally, fibrocytes may contribute to early collagen deposition in mammary tumors of obese mice leading to the growth of desmoplastic tumors.

Alterations in the Mammary Gland and Tumor Microenvironment of Formerly Obese Mice.

Obesity is a risk factor for breast cancer, and women with obesity that develop breast cancer have a worsened prognosis. Within the mammary gland, obesity causes chronic, macrophage-driven inflammation and adipose tissue fibrosis. To examine the impact of weight loss on the mammary microenvironment, mice were fed high-fat diet to induce obesity, then switched to a low-fat diet. In formerly obese mice, we observed reduced numbers of crown-like structures and fibrocytes in mammary glands, while collagen deposition was not resolved with weight loss. Following transplant of TC2 tumor cells into the mammary glands of lean, obese, and formerly obese mice, diminished collagen deposition and cancer-associated fibroblasts were observed in tumors from formerly obese mice compared to obese mice. When TC2 tumor cells were mixed with CD11b+CD34+ myeloid progenitor cells, collagen deposition within the tumors was significantly greater compared to when tumor cells were mixed with CD11b+CD34- monocytes, suggesting that fibrocytes contribute to early collagen deposition in mammary tumors of obese mice. Overall, these studies show that weight loss resolved some of the microenvironmental conditions within the mammary gland that may contribute to tumor progression.

The effects of high plasma levels of Aß1-42 on mononuclear macrophage in mouse models of Alzheimer's disease.

More and more evidences are proving that microglia play a crucial role in the pathogenesis of Alzheimer's disease (AD) and the plasma Aß1-42 levels significantly increased 15 years before the onset of dominantly inherited AD. However, the effects of high plasma levels of Aß1-42 on mononuclear macrophage, the peripheral counterparts of microglia, remain unclear. In the present study, we used APP/PS1 transgenic (Tg) mice and a parabiotic model of wild type (Wt) mice and Tg mice (Parabiotic Wt-Tg, Pa (Wt-Tg)) to investigate the effects of high plasma levels of Aß1-42 on peripheral mononuclear macrophage. Our results showed that in the early stage of Tg mice (7 months) and Pa (Wt-Tg) mice (4 months), the proportions of pro-inflammatory macrophages in peritoneal cavity, myeloid derived suppressor cells (MDSCs) in spleen, granulocyte-monocyte progenitors (GMPs) in bone marrow, and the plasma levels of interleukin-6 (IL-6) were significantly decreased. While the proportions of pro-inflammatory macrophages, MDSCs, GMPs, and the plasma levels of IL-6 and tumor necrosis factor (TNF)-α, as well as the numbers of bone marrow-derived macrophages (BMDMs) in mice brain were increased in the late stage of Tg mice (11 months) and Pa (Wt-Tg) mice (8 months). In addition, the proportions of monocytes in spleen and the proliferation of bone marrow cells (BMCs) were enhanced consistently, and the phagocytic function of macrophages kept stably after high plasma levels of Aß1-42 sustaining stimulation. These results demonstrated that high plasma levels of Aß1-42 play a biphasic regulating role at different stages of the disease, namely inhibiting effects on peripheral pro-inflammatory macrophages in the early stage of AD model, while promoting effects in the late stage of AD model. The mechanism behind this may be associated with their effects on MDSCs in spleen and myeloid progenitor cells in bone marrow. Therefore, intervening the effects of plasma Aß1-42 on pro-inflammatory macrophages might offer a new therapeutic approach to AD.

Fibrocytes enhance mammary gland fibrosis in obesity.

Obesity rates continue to rise, and obese individuals are at higher risk for multiple types of cancer, including breast cancer. Obese mammary fat is a site of chronic, macrophage-driven inflammation, which enhances fibrosis within adipose tissue. Elevated fibrosis within the mammary gland may contribute to risk for obesity-associated breast cancer. To understand how inflammation due to obesity enhanced fibrosis within mammary tissue, we utilized a high-fat diet model of obesity and elimination of CCR2 signaling in mice to identify changes in immune cell populations and their impact on fibrosis. We observed that obesity increased a population of CD11b+ cells with the ability to form myofibroblast-like colonies in vitro. This population of CD11b+ cells is consistent with fibrocytes, which have been identified in wound healing and chronic inflammatory diseases but have not been examined in obesity. In CCR2-null mice, which have limited ability to recruit myeloid lineage cells into obese adipose tissue, we observed reduced mammary fibrosis and diminished fibrocyte colony formation in vitro. Transplantation of myeloid progenitor cells, which are the cells of origin for fibrocytes, into the mammary glands of obese CCR2-null mice resulted in significantly increased myofibroblast formation. Gene expression analyses of the myeloid progenitor cell population from obese mice demonstrated enrichment for genes associated with collagen biosynthesis and extracellular matrix remodeling. Together these results show that obesity enhances recruitment of fibrocytes to promote obesity-induced fibrosis in the mammary gland.

Neonatal Hepatic Myeloid Progenitors Expand and Propagate Liver Injury in Mice.

BACKGROUND: Biliary atresia (BA) is a progressive pediatric inflammatory disease of the liver that leads to cirrhosis and necessitates liver transplantation. The rapid progression from liver injury to liver failure in children with BA suggests that factors specific to the perinatal hepatic environment are important for disease propagation. Hematopoietic stem and progenitor cells (HSPCs) reside in the fetal liver and are known to serve as central hubs of inflammation. We hypothesized that HSPCs are critical for the propagation of perinatal liver injury (PLI). METHODS: Newborn BALB/c mice were injected with rhesus rotavirus (RRV) to induce PLI or with PBS as control. Livers were compared using histology and flow cytometry. To determine the effects of HSPCs on PLI, RRV-infected neonatal mice were administered anti-CD47 and anti-CD117 to deplete HSPCs. RESULTS: PLI significantly increased the number of common myeloid progenitors and the number of CD34+ hematopoietic progenitors. Elimination of HSPCs through antibody-mediated myeloablation rescued animals from PLI and significantly increased survival (RRV+isotype control 36.4% vs. RRV+myeloablation 77.8%, Chi-test = 0.003). CONCLUSIONS: HSPCs expand as a result of RRV infection and propagate PLI. Targeting of HSPCs may be useful in preventing and treating neonatal inflammatory diseases of the liver such as BA.

Platelet-derived immuno-inflammatory indices show best performance in early prediction of COVID-19 progression.

BACKGROUND: Coronavirus disease 2019 (COVID-19) profoundly affects the immune and hematopoietic systems with various degrees of reactive changes in the blood cell counts. Immuno-inflammatory indices are considered a simple and effective tool in the prediction of COVID-19 outcomes. We aimed to evaluate and compare the usefulness of leukocyte and platelet counts-based immuno-inflammatory indices on admission to hospital in predicting COVID-19 progression and mortality. METHODS: A total of 945 patients were enrolled. In addition to blood cell counts, we assessed hemogram-derived immuno-inflammatory indices in relation to COVID-19 progression and death. The indices were tested by analysis of variance, receiver operating characteristic curve analysis, and binomial logistic regressions. RESULTS: Patients with severe COVID-19 had significantly higher counts of neutrophils, eosinophils, and large immature cells (LIC), while decreased counts of platelets and monocytes. Lymphopenia was found in all of the patients, but without significant association with the outcomes. Patients with a LIC count ≥0.265 x 09 /L had 54.7% more odds of having COVID-19 progression. In multivariable analyses, platelets/neutrophil-to-lymphocyte ratio (P/NLR) and platelets-to-neutrophil radio (P/N) were significant independent predictors of COVID-19 progression and mortality. The odds of a poor outcome were two times higher in cases with P/NLR < 43 x 109 /L and P/N < 29 x 109 /L. CONCLUSION: Indices that include platelet count in combination with neutrophil and/or lymphocyte counts displayed the best discriminatory ability and prognostic value of COVID-19 outcomes. Additionally, LIC showed promising results in the early identification of severe COVID-19.

Molecular mechanisms of aberrant neutrophil differentiation in glycogen storage disease type Ib.

Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is caused by a deficiency in glucose-6-phosphate transporter (G6PT). Neutropenia in GSD-Ib has been known to result from enhanced apoptosis of neutrophils. However, it has also been raised that neutrophil maturation arrest in the bone marrow would contribute to neutropenia. We now show that G6pt-/- mice exhibit severe neutropenia and impaired neutrophil differentiation in the bone marrow. To investigate the role of G6PT in myeloid progenitor cells, the G6PT gene was mutated using CRISPR/Cas9 system, and single cell-derived G6PT-/- human promyelocyte HL-60 cell lines were established. The G6PT-/- HL-60s exhibited impaired neutrophil differentiation, which is associated with two mechanisms: (i) abnormal lipid metabolism causing a delayed metabolic reprogramming and (ii) reduced nuclear transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ) in G6PT-/- HL-60s. In this study, we demonstrated that G6PT is essential for neutrophil differentiation of myeloid progenitor cells and regulates PPARγ activity.

Notch1 activation enhances proliferation via activation of cdc2 and delays differentiation of myeloid progenitors.

Accumulating evidence indicates that the Notch signaling pathway has crucial roles in the control of fate decision and differentiation in numerous cell types. However, the role of Notch signaling in regulating proliferation and differentiation of myeloid progenitor cells remains controversial. To elucidate this issue, we modulated Notch activity through transducing a constitutively activated form of Notch1 and/or a dominant-negative form of MAML1 (DNMAML1) into myeloid progenitor 32D cells and assessed their effects on cell proliferation and differentiation. We found that Notch1 activation enhances proliferation and delays granulocytic differentiation of 32D cells. The enhanced proliferation due to activated Notch1 signaling was associated with upregulation of c-Myc, followed by decreased expression of p21 and p27, and increased cdc2 kinase activity, through a mechanism that was not blocked by DNMAML1. Conversely, Notch1 activation significantly delayed granulocytic differentiation and maintained a part of myeloid progenitor cells in an immature stage, and this Notch1-mediated effect was dependent on MAML. The Notch1-induced effects on mye myeloid cell proliferation and differentiation were likely mediated by induction of c-Myc and repression of PU.1, respectively. Thus, Notch1 signaling plays an important part in modulating proliferation and differentiation in MAML-independent and -dependent manners and promoting expansion of myeloid progenitors.

Mapping the CLEC12A expression on myeloid progenitors in normal bone marrow; implications for understanding CLEC12A-related cancer stem cell biology.

The C-type lectin domain family 12, member A (CLEC12A) receptor has emerged as a leukaemia-associated and cancer stem cell marker in myeloid malignancies. However, a detailed delineation of its expression in normal haematopoiesis is lacking. Here, we have characterized the expression pattern of CLEC12A on the earliest stem- and myeloid progenitor subsets in normal bone marrow. We demonstrate distinct CLEC12A expression in the classically defined myeloid progenitors, where on average 39.1% (95% CI [32.5;45.7]) of the common myeloid progenitors (CMPs) expressed CLEC12A, while for granulocyte-macrophage progenitors and megakaryocyte-erythroid progenitors (MEPs), the average percentages were 81.0% (95% CI [76.0;85.9]) and 11.9% (95% CI [9.3;14.6]), respectively. In line with the reduced CLEC12A expression on MEPs, functional assessment of purified CLEC12A+/- CMPs and MEPs in the colony-forming unit assay demonstrated CLEC12A+ subsets to favour non-erythroid colony growth. In conclusion, we provide evidence that the earliest CLEC12A+ cell in the haematopoietic tree is the classically defined CMP. Furthermore, we show that CLEC12A-expressing CMPs and MEPs are functionally different than their negative counterparts. Importantly, these data can help determine which cells will be spared during CLEC12A-targeted therapy, and we propose CLEC12A to be included in future studies of myeloid cancer stem cell biology.

[Effect of nerve growth factor on osteogenic potential of type 2 diabetic mice bone marrow stromal cell in vitro].

Objective: To study the effects of nerve growth factor (NGF) on the proliferation, osteogenic differentiation and mineralization of type 2 diabetic mice bone marrow stromal cell (BMSC), providing basis for clinical application of NGF. Methods: Three 8-week-old male db/db mice and two 8-week-old male C57BL/6J mice were used in the study. BMSC derived from femur were cultured though adherence method. BMSC of C57BL/6J mice and db/db mice was divided into normal group and diabetic group to conduct the osteogenic potential experiment, named experiment one. In experiment two, diabetic BMSC was divided into 3 groups: diabetic control group, NGF group, and K252a+NGF group [K252a was the inhibitor of tyrosine kinase A (TrkA), which was the high affinity receptor of NGF], to investigate effect of NGF on osteogenic potential of diabetic mice BMSC. After seeding BMSC, K252a was added into K252a+NGF group, then NGF was added 30 min later. NGF was added into NGF group and K252a+NGF group, but not diabetic control group. The proliferation of BMSC at 1, 3, 5 and 7 d in experiment one and the proliferation of BMSC at 1, 2 and 3 d in experiment two were evaluated through methyl thiazolyl tetrazolium, and the level of alkaline phosphatase (ALP) at 3, 5 and 7 d in both experiments were measured. After being osteogenic induced for 14 d, mineralized nodules in both experiments were quantitated by alizarin red calcium stain. Five holes were set in every group, and all experiments were repeated 3 times. Results: The BMSC proliferation of diabetic group was significantly higher than that of the normal group at 3, 5 and 7 d (P<0.05). After being osteogenic inducted for 3, 5 and 7 d, ALP level of diabetic group were significantly lower than that of normal group (P<0.05). After being osteogenic inducted for 14 d, calcium nodule count of diabetic group [(23.1±6.4) nodule/field] were significantly lower than that of normal group [(36.9±7.9) nodule/field](P<0.05). At 1, 2 and 3 d, BMSC proliferations of diabetic control group, NGF group and K252a+NGF group were not statistically different (P>0.05). After being osteogenic inducted for 3 and 5 d, ALP level of NGF group was significantly higher than that of diabetic control group (P<0.05). After being osteogenic inducted for 3, 5, and 7 d, ALP level of K252a+NGF group was significantly lower than that of NGF group (P<0.05) and diabetic control group (P<0.05). After being osteogenic induced for 14 d, calcium nodule count of NGF group [(45.2±6.8) nodule/field] was significantly more than that of diabetic control group [(23.1±6.4) nodule/field](P<0.05); while calcium nodule count of K252a+NGF group [(18.0±4.5) nodule/field] was significantly less than that of NGF group (P<0.05) and diabetic control group (P<0.05). Conclusions: The differentiation and mineralization of type 2 diabetic mice BMSC was significantly reduced. NGF promoted the osteoblastic differentiation and mineralization of diabetic mice BMSC in viro though combining with TrkA.

Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis.

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34+CD38+CD33+ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3+ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.

Murine Retrovirally-Transduced Bone Marrow Engraftment Models of MLL-Fusion-Driven Acute Myelogenous Leukemias (AML).

MLL-rearranged leukemia represents approximately 5% to 10% of adult acute myelogenous leukemia (AML) and nearly half of all infant/pediatric acute leukemia cases. These leukemias have a poor prognosis, and there are no approved therapeutic options. The rearrangement in the MLL gene leads to aberrant expression of MLL-fusion proteins. These are transforming in murine bone marrow and, in particular, on stem cells and myeloid progenitors derived from bone marrow or fetal liver. The commonality of the MLL fusions is the in-frame fusion of 8 to 11 N-terminal exons of MLL1 (KMT2a) with the C-terminus of a partner fusion gene. Currently, over 80 different fusion partners are known. The protocols detailed in this unit focus on bone marrow-derived models only, using one particular MLL fusion, MLL-AF9. These models have proven effective for drug screening to predict clinical response. © 2017 by John Wiley & Sons, Inc.

Mesenchymal Stem Cells Modulate Differentiation of Myeloid Progenitor Cells During Inflammation.

Mesenchymal stem cells (MSCs) possess distinct immunomodulatory properties and have tremendous potential for use in therapeutic applications in various inflammatory diseases. MSCs have been shown to regulate pathogenic functions of mature myeloid inflammatory cells, such as macrophages and neutrophils. Intriguingly, the capacity of MSCs to modulate differentiation of myeloid progenitors (MPs) to mature inflammatory cells remains unknown to date. Here, we report the novel finding that MSCs inhibit the expression of differentiation markers on MPs under inflammatory conditions. We demonstrate that the inhibitory effect of MSCs is dependent on direct cell-cell contact and that this intercellular contact is mediated through interaction of CD200 expressed by MSCs and CD200R1 expressed by MPs. Furthermore, using an injury model of sterile inflammation, we show that MSCs promote MP frequencies and suppress infiltration of inflammatory cells in the inflamed tissue. We also find that downregulation of CD200 in MSCs correlates with abrogation of their immunoregulatory function. Collectively, our study provides unequivocal evidence that MSCs inhibit differentiation of MPs in the inflammatory environment via CD200-CD200R1 interaction. Stem Cells 2017;35:1532-1541.

Assessment of Drug Sensitivity in Hematopoietic Stem and Progenitor Cells from Acute Myelogenous Leukemia and Myelodysplastic Syndrome Ex Vivo.

Current understanding suggests that malignant stem and progenitor cells must be reduced or eliminated for prolonged remissions in myeloid neoplasms such as acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS). Multicolor flow cytometry has been widely used to distinguish stem and myeloid progenitor cells from other populations in normal and malignant bone marrow. In this study, we present a method for assessing drug sensitivity in MDS and AML patient hematopoietic stem and myeloid progenitor cell populations ex vivo using the investigational Nedd8-activating enzyme inhibitor MLN4924 and standard-of-care agent cytarabine as examples. Utilizing a multicolor flow cytometry antibody panel for identification of hematopoietic stem cells, multipotent progenitors, common myeloid progenitors, granulocyte-monocyte progenitors, and megakaryocyte-erythroid progenitors present in mononuclear cell fractions isolated from bone marrow aspirates, we compare stem and progenitor cell counts after treatment for 24 hours with drug versus diluent. We demonstrate that MLN4924 exerts a cytotoxic effect on MDS and AML stem and progenitor cell populations, whereas cytarabine has more limited effects. Further application of this method for evaluating drug effects on these populations ex vivo and in vivo may inform rational design and selection of therapies in the clinical setting. Stem Cells Translational Medicine 2017;6:840-850.

Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells.

The concentration and composition of cardiolipin (CL) in mitochondria are altered in age-related heart disease, Barth Syndrome, and other rare genetic disorders, resulting in mitochondrial dysfunction. To explore whether exogenous CL can be delivered to cells, CL was combined with apolipoprotein A-I to generate water-soluble, nanoscale complexes termed nanodisks (ND). Mass spectrometry of HL60 myeloid progenitor cell extracts revealed a 30-fold increase in cellular CL content following incubation with CL-ND. When CL-ND containing a fluorescent CL analogue was employed, confocal microscopy revealed CL localization to mitochondria. The ability of CL-ND to elicit a physiological response was examined in an HL60 cell culture model of Barth Syndrome neutropenia. siRNA knockdown of the phospholipid transacylase, tafazzin (TAZ), induced apoptosis in these cells. When TAZ knockdown cells were incubated with CL-ND, the apoptotic response was attenuated. Thus, CL-ND represent a potential intervention strategy for replenishment of CL in Barth Syndrome, age-related heart disease, and other disorders characterized by depletion of this key mitochondrial phospholipid.

Twist-1, a novel regulator of hematopoietic stem cell self-renewal and myeloid lineage development.

Transcription factor Twist-1 plays essential roles in specification and differentiation of mesoderm-derived tissues. Growing evidences now link Twist-1 to the acquisition of stem-cell-like properties. However, the role of Twist-1 in hematopoietic stem cell (HSC) remains largely uncharacterized. We report that Twist-1 is more highly expressed in murine HSC and its expression declines with differentiation. To investigate Twist-1 gene function, retroviral-mediated overexpression or removal experiments are performed. Competitive repopulation studies demonstrate that enforced expression of Twist-1 in HSC-enriched Lin(-) c-Kit(+) Sca-1(+) (LKS) cells results in an increase in the size of the G(0) population, and in their reconstitution ability after the first and a second transplantation. Conversely, removal of Twist-1 in LKS cells impairs their ability to repopulate. In addition, increased Twist-1 expression causes a shift toward production of myeloid cells. Twist-1 transduction in LKS cells activates myeloid lineage-determining factors PU.1 and GATA-1 and downregulates lymphoid factor GATA-3 in vitro, suggesting that Twist-1-mediated myeloid skewing occurs in hematopoietic stem and progenitor cells (HSPCs). These findings indicate that Twist-1 is not only involved in the maintenance of HSC dormancy and self-renewal capacity but also implicated in the myeloid lineage fate choice of HSPCs. Exploration of the underlying mechanisms reveals that Runx1/c-Mpl/Tie2 regulatory pathway could possibly account for the observed effects caused by Twist-1 overexpression. Our study provides the first evidence supporting a role for Twist-1 in hematopoiesis.

Expressions of transcription factors in goldfish (Carassius auratus L.) macrophages and their progenitors.

The development of macrophages is a highly regulated process requiring coordination amongst transcription factors. The presence/absence, relative levels, antagonism, or synergy of all transcription factors involved is critical to directing lineage cell fate and differentiation. While relative levels of many key myeloid transcription factors have been determined in mammalian macrophage differentiation, a similar set of studies have yet to be conducted in a teleost system. In this study, we report on the mRNA levels of transcription factors (cebpa, cjun, cmyb, egr1, gata1, gata2, gata3, lmo2, mafb, pax5, pu.1 and runx1) in sorted goldfish progenitor cells, monocytes, and macrophages from primary kidney macrophage cultures. The mRNA levels of runx1 and pu.1 were significantly higher, gata3 and pax5 mRNA levels were lower, in monocytes compared to progenitors, and the mRNA levels of cjun, egr1, gata2, gata3, mafb and pax5 were significantly decreased in macrophages compared to progenitor cells. The relative mRNA levels of the interferon regulatory factor family of transcription factors, irf1, irf2, irf5, irf7, irf8 and irf9 in sorted progenitors, monocytes and macrophages were also measured. In contrast to other irf family transcription factors examined, irf8 mRNA levels were increased in monocytes compared to progenitors by greater than three-fold, suggesting that irf8 is important for monopoiesis. Lastly, we show the differential regulation of myeloid transcription factor mRNA levels in sorted progenitor cells from 1, 2, or 3-day old cultures in response to the recombinant goldfish growth factors, rgCSF-1 and rgKITLA.