Essential Roles of C/EBPβ in Survival of Ly6C^– monocytes

Accumulating evidences have shown that mouse monocytes can be divided into two subsets, based on the expression of a surface marker Ly6C. Although distinct functions of Ly6C⁺ monocytes (also called classical or inflammatory monocytes) and Ly6C^– monocytes (also known as patrolling monocytes) have been gradually uncovered, molecular mechanisms which govern development of these monocytes remain largely unknown.

We have previously reported the requirement of CCAAT Enhancer Binding Protein β (C/EBPβ), a leucine zipper transcription factor, for ‘emergency’ granulopoiesis (Nat Immunol, 2006, J Immunol, 2012, Leukemia 2013). C/EBPβ is also known to play roles in the differentiation and function of macrophages. However, involvement of C/EBPβ in monocyte development has not been fully investigated. The aim of this study is to elucidate the roles of C/EBPβ in monopoiesis.

First, we measured C/EBPβ mRNA expression in purified hematopoietic stem cells, myeloid progenitors and monocyte subsets, and found that monocytes, especially Ly6C^– monocytes, expressed C/EBPβ mRNA at extremely higher level than any of other cell types examined.

When we analyzed peripheral blood, the frequencies of total monocytes (CD11b⁺ CD115⁺ cells) in C/EBPβ^–/– mice was significantly lower than those in wild type (WT) mice (4.24±2.71% in WT mice vs. 0.72±0.50% in C/EBPβ^–/– mice, p<0.001). Of note, Ly6C^– monocytes were almost absent in peripheral blood of C/EBPβ^–/– mice (0.67±0.57% in WT mice vs. 0.017±0.021% in C/EBPβ^–/– mice).

In order to clarify whether the defects in C/EBPβ^–/– monopoiesis were cell-intrinsic or cell-extrinsic, we generated mixed bone marrow (BM) chimeras by reconstituting lethally irradiated mice (CD45.1⁺) with BM cells from WT (CD45.1⁺) mice together with the equal number of BM cells from either WT or C/EBPβ^–/– (CD45.2⁺) mice. Six weeks after reconstitution, we confirmed that C/EBPβ^–/– BM-derived Ly6C^– monocytes were absent in peripheral blood of the recipient mice, suggesting that monopoiesis in C/EBPβ^–/– mice is impaired in a cell-intrinsic manner.

A recent report revealed that MX1-Cre transgenic system can be used for monocyte specific deletion of genes of interest, as MX1 is highly expressed by monocytes (Hashimoto D et al. Immunity 2013). In MX1-Cre⁺ C/EBPβ^{floxed/floxed} mice, the number of monocytes were decreased to the level similar to C/EBPβ^–/– mice, suggesting that C/EBPβ is specifically required in monocytes rather than other progenitors during monopoiesis.

As cell cycle status of myeloid progenitors and monocytes did not differ between WT mice and C/EBPβ^–/– mice, we evaluated apoptosis by flow cytometry. The frequencies of late apoptotic/dead cells within Ly6C^– monocytes in peripheral blood of C/EBPβ^–/– mice were significantly higher than those in peripheral blood of WT mice (5.84±2.90% in WT mice vs. 50.4±22.4% in C/EBPβ^–/– mice, p<0.001). These enhanced apoptosis of C/EBPβ^–/– Ly6C^– monocyte was partially reversed by retroviral transduction of Bcl2 gene.

Previous reports have shown that Nr4a1, CX3CR1 and S1PR5 are required for survival or BM egress of Ly6C^– monocytes. We found that mRNA expressions of these factors are severely reduced in C/EBPβ^–/– Ly6C^– monocytes. These results suggested that C/EBPβ maintains survival of Ly6C^– monocytes through direct or indirect association of these molecules.

Collectively, our data strongly indicate that C/EBPβ is essential for survival of Ly6C^– monocytes. We are currently investigating the molecular mechanisms involved in the enhanced apoptosis of of Ly6C^– monocytes in C/EBPβ^–/– mice.