A Novel CD135+ Subset of Mouse Monocytes with a Distinct Differentiation Pathway and Antigen-Presenting Properties.

The mononuclear phagocyte system (MPS), composed of monocytes/macrophages and dendritic cells (DCs), plays a critical role at the interface of the innate and adaptive immune systems. However, the simplicity of MPS has been challenged recently by discoveries of novel cellular components. In the current study, we identified the CD135+ subset of monocytes as a novel class of APCs in mice. CD135+ monocytes were readily found in the bone marrow, spleen, and peripheral blood at steady state, and they expressed markers specific to DCs, including MHC class II and CD209a, along with markers for monocytes/macrophages. In addition, this subset phagocytosed bacteria and activated naive T lymphocytes, fulfilling the criteria for APCs. CD135+ monocytes were derived directly from macrophage DC progenitors, not from common monocyte progenitors or other monocytes, suggesting that these are distinct from conventional monocytes. These findings facilitate our understanding of the MPS network that regulates immune responses for host defense.

C/EBPß isoforms sequentially regulate regenerating mouse hematopoietic stem/progenitor cells.

The transcription factor CCAAT enhancer-binding protein ß (C/EBPß) is required for stress-induced granulopoiesis at the level of hematopoietic stem/progenitor cells (HSPCs); however, its role and mechanisms of action in HSPCs are unknown. In this study, we assessed the regulation and functions of C/EBPß in HSPCs, especially under stress conditions. After 5-fluorouracil treatment or bone marrow transplantation, Cebpb-/- HSPCs exhibited impaired cell-cycle activation and myeloid differentiation at the early and late phases of regeneration, respectively, whereas at steady state, Cebpb deficiency did not affect HSPCs. C/EBPß was upregulated in response to hematopoietic stress, especially in CD150high long term-hematopoietic stem cells (LT-HSCs). Intracellular flow cytometric analysis that detected distinct domains of C/EBPß revealed that, among the 3 isoforms of C/EBPß, liver-enriched inhibitory protein (LIP) was upregulated in LT-HSCs prior to liver-enriched activating protein (LAP)/LAP* during regeneration. Early upregulation of LIP promoted cell-cycle entry of LT-HSCs by positively regulating Myc and expanded the HSPCs pool. Subsequent myeloid differentiation of amplified HSPCs was mediated by LAP/LAP*, which were upregulated at a later phase of regeneration. Collectively, our findings show that stress-induced sequential upregulation of C/EBPß isoforms is critical for fine-tuning the proliferation and differentiation of regenerating HSPCs.

C/EBPß is a critical mediator of IFN-α-induced exhaustion of chronic myeloid leukemia stem cells.

Even in the era of ABL tyrosine kinase inhibitors, eradication of chronic myeloid leukemia (CML) stem cells is necessary for complete cure of the disease. Interferon-α (IFN-α) has long been used for the treatment of chronic-phase CML, but its mechanisms of action against CML stem cells remain unclear. We found that IFN-α upregulated CCAAT/enhancer binding protein ß (C/EBPß) in BCR-ABL-expressing mouse cells by activating STAT1 and STAT5, which were recruited to a newly identified 3' distal enhancer of Cebpb that contains tandemly aligned IFN-γ-activated site elements. Suppression or deletion of the IFN-γ-activated site elements abrogated IFN-α-dependent upregulation of C/EBPß. IFN-α induced differentiation and exhaustion of CML stem cells, both in vitro and in vivo, in a C/EBPß-dependent manner. In addition, IFN-α upregulated C/EBPß and induced exhaustion of lineage- CD34+ cells from CML patients. Collectively, these results clearly indicate that C/EBPß is a critical mediator of IFN-α-induced differentiation and exhaustion of CML stem cells.

C/EBPß is required for survival of Ly6C- monocytes.

The transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) is highly expressed in monocytes/macrophages. However, its roles in monopoiesis are largely unknown. Here, we investigated the roles of C/EBPß in monopoiesis. Further subdivision of monocytes revealed that Cebpb messenger RNA was highly upregulated in Ly6C- monocytes in bone marrow. Accordingly, the number of Ly6C- monocytes was significantly reduced in Cebpb-/- mice. Bone marrow chimera experiments and Mx1-Cre-mediated deletion of Cebpb revealed a cell-intrinsic and monocyte-specific requirement for C/EBPß in monopoiesis. In Cebpb-/- mice, turnover of Ly6C- monocytes was highly accelerated and apoptosis of Ly6C- monocytes was increased. Expression of Csf1r, which encodes a receptor for macrophage colony-stimulating factor, was significantly reduced in Ly6C- monocytes of Cebpb-/- mice. C/EBPß bound to positive regulatory elements of Csf1r and promoted its transcription. Collectively, these results indicate that C/EBPß is a critical factor for Ly6C- monocyte survival, at least in part through upregulation of Csf1r.

Cyclic AMP responsive element binding proteins are involved in 'emergency' granulopoiesis through the upregulation of CCAAT/enhancer binding protein ß.

In contrast to the definitive role of the transcription factor, CCAAT/Enhancer binding protein α (C/EBPα), in steady-state granulopoiesis, previous findings have suggested that granulopoiesis during emergency situations, such as infection, is dependent on C/EBPß. In this study, a novel lentivirus-based reporter system was developed to elucidate the molecular switch required for C/EBPß-dependency. The results demonstrated that two cyclic AMP responsive elements (CREs) in the proximal promoter region of C/EBPß were involved in the positive regulation of C/EBPß transcription during granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced differentiation of bone marrow cells. In addition, the transcripts of CRE binding (CREB) family proteins were readily detected in hematopoietic stem/progenitor cells. CREB was upregulated, phosphorylated and bound to the CREs in response to GM-CSF stimulation. Retroviral transduction of a dominant negative CREB mutant reduced C/EBPß mRNA levels and significantly impaired the proliferation/differentiation of granulocyte precursors, while a constitutively active form of CREB facilitated C/EBPß transcription. These data suggest that CREB proteins are involved in the regulation of granulopoiesis via C/EBPß upregulation.

Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences.

The efficacy and specificity of small interfering RNAs (siRNAs) are largely dependent on the siRNA sequence. Since only empirical strategies are currently available for predicting these parameters, simple and accurate methods for evaluating siRNAs are needed. To simplify such experiments, target genes are often tagged with reporters for easier readout. Here, we used a bicistronic vector expressing a target gene and green fluorescent protein (GFP) to create a system in which the effect of an siRNA sequence was reflected in the GFP expression level. Cells were transduced with the bicistronic vector, expression vectors for siRNA and red fluorescent protein (RFP). Flow cytometric analysis of the transduced cells revealed that siRNAs for the target gene silenced GFP from the bicistronic vector, but did not silence GFP transcribed without the target gene sequence. In addition, the mean fluorescence intensities of GFP on RFP-expressing cells correlated well with the target gene mRNA and protein levels. These results suggest that this flow cytometry-based method enables us to quantitatively evaluate the efficacy and specificity of siRNAs. Because of its simplicity and effectiveness, this method will facilitate the screening of effective siRNA target sequences, even in high-throughput applications.