C/EBPβ Is Required for Survival of Ly6C^- Monocytes after Committment to Monocyte Lineage through Upregulation of Csf1r

Monopoiesis is the process in which hematopoietic stem cells (HSCs) continuously give rise to monocytes. Accumulating evidence has identified cellular constituents of monopoiesis. Common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), macrophage-dendritic cell precursors (MDPs) and common monocyte progenitors (cMoPs) are the intermediates during the differentiation of HSCs into mature monocytes. In mice, CD11b⁺ CD115⁺ monocytes are further divided into two subsets based on the expression of Ly6C. Classical monocytes express Ly6C on their surface. By contrast, Ly6C⁻ patrolling monocytes have been recently identified, and the molecular mechanisms which regulate the development and homeostasis of Ly6C⁻monocytes still remain elusive.

C/EBPβ is a leucine zipper transcription factor which regulates stress-induced granulopoiesis (Hirai et al. Nat Immunol, 2006, Hayashi et al. Leukemia 2013). We have recently found that peripheral blood (PB) monocytes are significantly reduced in steady-state Cebpb^−/− mice (Tamura et al. Biochem Biophys Res Commun, 2015). In addition, last year at this meeting, we have reported that cell death of Ly6C⁻ monocytes was accelerated through reduced expression of Csf1r (encoding a receptor for M-CSF) in Cebpb^−/− mice. Here in this study, we determined the precise developmental stage where C/EBPβ is mandatory for survival of Ly6C⁻ monocytes, and investigated the mechanism of Csf1r regulation by C/EBPβ.

A recent publication demonstrated that Mx1 is preferentially expressed by monocytes and a Mx1 promoter-mediated conditional system targets monocytes without inoculation of polyI:C (Hashimoto et al. Immunity, 2013), suggesting that Mx1-Cre Cebpb^f/f mouse is ideal to evaluate the monocyte-specific requirement for C/EBPβ. We confirmed that upregulation of Cebpb mRNA during monopoiesis was significantly impaired after cMoP stage in Mx1-Cre⁺Cebpb^f/f mice. In order to exclude the possible involvement of Cebpβ deficient microenvironment, bone marrow (BM) cells of Mx1-Cre⁺Cebpβ^f/f mice (CD45.2⁺) were transplanted into lethally irradiated CD45.1⁺ wild type mice. The frequencies of Ly6C⁻ monocytes in the recipients of Mx1-Cre⁺Cebpb^f/f BM cells were significantly reduced when compared to mice that received Mx1-Cre⁻Cebpb^f/f BM cells (Figure). These results strongly suggest that C/EBPβ is specifically required after commitment to monocytes.

In order to investigate the molecular mechanisms involved in the regulation of Csf1r by C/EBPβ, we utilized a combination of a promoter and an enhancer region located in the first intron of Csf1r gene (Fms intronic regulatory element: FIRE) for reporter assay (Pridans et al. Mol Ther Methods Clin Dev, 2014). These regulatory elements contain at least 2 consensus binding sites for C/EBPβ (one in the promoter and the other in the enhancer). C/EBPβ significantly enhanced the reporter activity of the regulatory elements in a dose-dependent manner, and introduction of mutations into either of the consensus binding sites abrogated the reporter activity. Next, we engineered EML cells, a mouse HSC line, to express C/EBPβ-estrogen receptor (ER) fusion protein or ER alone. Nuclear translocation of C/EBPβ-ER in the presence of tamoxifen resulted in significant increase of Csf1r mRNA and protein. Using these cells, we performed chromatin immunoprecipitation PCR. Upon treatment with tamoxifen, significant enrichment of C/EBPβ at the promoter region and the FIRE region was observed. These data indicated that C/EBPβ regulates Csf1r through direct binding to these regulatory elements.

Collectively, these results demonstrate that C/EBPβ supports survival of Ly6C⁻ monocytes after commitment to monocyte lineage through direct regulation of Csf1r, which is critical for survival and differentiation of monocytes.

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