Research Article: PU.1 Is Essential for CD11c Expression in CD8+/CD8− Lymphoid and Monocyte-Derived Dendritic Cells during GM-CSF or FLT3L-Induced Differentiation

Date Published: December 20, 2012

Publisher: Public Library of Science

Author(s): Xue-Jun Zhu, Zhong-Fa Yang, Yaoyu Chen, Junling Wang, Alan G. Rosmarin, Silke Appel. http://doi.org/10.1371/journal.pone.0052141

Abstract

Dendritic cells (DCs) regulate innate and acquired immunity through their roles as antigen-presenting cells. Specific subsets of mature DCs, including monocyte-derived and lymphoid-derived DCs, can be distinguished based on distinct immunophenotypes and functional properties. The leukocyte integrin, CD11c, is considered a specific marker for DCs and it is expressed by all DC subsets. We created a strain of mice in which DCs and their progenitors could be lineage traced based on activity of the CD11c proximal promoter. Surprisingly, we observed levels of CD11c promoter activity that were similar in DCs and in other mature leukocytes, including monocytes, granulocytes, and lymphocytes. We sought to identify DNA elements and transcription factors that regulate DC-associated expression of CD11c. The ets transcription factor, PU.1, is a key regulator of DC development, and expression of PU.1 varies in different DC subsets. GM-CSF increased monocyte-derived DCs in mice and from mouse bone marrow cultured in vitro, but it did not increase CD8+ lymphoid-derived DCs or B220+ plasmacytoid DCs. FLT3L increased both monocyte-derived DCs and lymphoid-derived DCs from mouse bone marrow cultured in vitro. GM-CSF increased the 5.3 Kb CD11c proximal promoter activity in monocyte-derived DCs and CD8+ lymphoid-derived DCs, but not in B220+ plasmacytoid DCs. In contrast, FLT3L increased the CD11c proximal promoter activity in both monocyte-derived DCs and B220+ plasmacytoid DCs. We used shRNA gene knockdown and chromatin immunoprecipitation to demonstrate that PU.1 is required for the effects of GM-CSF or FLT3L on monocyte-derived DCs. We conclude that both GM-CSF and FLT3L act through PU.1 to activate the 5.3 Kb CD11c proximal promoter in DCs and to induce differentiation of monocyte-derived DCs. We also confirm that the CD11c proximal promoter is not sufficient to direct lineage specificity of CD11c expression, and that additional DNA elements are required for lineage-specific CD11c expression.

Partial Text

Dendritic cells (DCs) are bone marrow-derived cells that play crucial roles in regulating and integrating innate and adaptive immune responses. DCs sample the environment, recognize invading pathogens through pattern recognition Toll-like receptors (TLRs), and initiate protective T cell responses by presenting antigens to lymphocytes. Because of their highly developed antigen presenting capacity, DCs have drawn attention for use in cell therapy [1], [2]. DCs are a heterogeneous group of cells that have been classified into distinct subsets, primarily based on patterns of cell surface antigen expression. The integrin αL chain, CD11c, is considered relatively specific for DCs. CD11c is expressed on all mature DC subsets in mouse, and its expression increases as DCs mature from DC progenitors. Subsets of mature DCs in blood and the lymphatic system include conventional DCs (cDCs; CD11c+CD4−CD8+CD11b−MHCII+, or CD11c+CD4+CD8−CD11b−MHCII+), plasmacytoid DCs (pDCs; CD11c+B220+Ly6C+), and monocyte-derived DCs (CD11c+CD11b+CD8−CD4−MHCII+) [3], [4], [5].

The leukocyte integrin, CD11c, is expressed by DCs and is considered to be a relatively specific marker of this lineage. In this study we demonstrated that expression of GFP driven by the 5.3 kb proximal promoter of mouse CD11c did not accurately reflect expression of native CD11c. We generated mice that are transgenic for both CD11c-Cre and Rosa26-loxP-STOP-loxP YFP, which can trace activation of the CD11c proximal promoter during DC differentiation. We found that the CD11c promoter was also active in HSCs, hematopoietic progenitor cells, and in all categories of mature leukocytes. Thus, the CD11c proximal promoter is neither sufficient nor specific for driving CD11c expression in DCs, and we conclude that additional promoter or enhancer regions of CD11c must be required to control its specific expression pattern in DCs. Treatment of bone marrow cells in vitro with GM-CSF or FLT3L increased CD11c expression in monocyte-derived (CD11b+) DCs. In vivo, GM-CSF activated the CD11c promoter in CD11b+ DCs and in CD8+ DCs, but not in B220+ pDCs. We showed that the transcription factors, Irf-4 and Pu.1, bound to the CD11c proximal promoter, and that Pu.1 knock down reduced GM-CSF or FLT3L-induced CD11c promoter activation. Together with the observation that expression of Pu.1 is high in CD8+ and CD11b+ cDCs, but low in B220+ pDCs, we conclude that high level expression of Pu.1 is essential for the control of CD11c expression in CD11b+ and in CD8+ cDCs.

Source:

http://doi.org/10.1371/journal.pone.0052141