Shared and Distinct Functions of the Transcription Factors IRF4 and IRF8 in Myeloid Cell Development

Interferon regulatory factor (IRF) 8 and IRF4 are structurally-related, hematopoietic cell-specific transcription factors that cooperatively regulate the differentiation of dendritic cells and B cells. Whilst in myeloid cells IRF8 is known to modulate growth and differentiation, the role of IRF4 is poorly understood. In this study, we show that IRF4 has activities similar to IRF8 in regulating myeloid cell development. The ectopic expression of IRF4 in myeloid progenitor cells in vitro inhibits cell growth, promotes macrophages, but hinders granulocytic cell differentiation. We also show that IRF4 binds to and activates transcription through the IRF-Ets composite sequence (IECS). Furthermore, we demonstrate that Irf8-/-Irf4-/- mice exhibit a more severe chronic myeloid leukemia (CML)-like disease than Irf8-/- mice, involving a disproportionate expansion of granulocytes at the expense of monocytes/macrophages. Irf4-/- mice, however, display no obvious abnormality in myeloid cell development, presumably because IRF4 is expressed at a much lower level than IRF8 in granulocyte-macrophage progenitors. Our results also suggest that IRF8 and IRF4 have not only common but also specific activities in myeloid cells. Since the expression of both the IRF8 and IRF4 genes is downregulated in CML patients, these results may add to our understanding of CML pathogenesis

<i>Irf4</i> and <i>Irf8</i> transcript levels in myeloid and other hematopoietic cells.

<p>Expression levels of endogenous <i>Irf4</i> and <i>Irf8</i> mRNAs in common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), macrophages (MΦ), granulocytes (Gr), T cells, B cells, pDCs, and cDCs. Two to 10 mice were used to obtain RNA from each cell type. The pooled RNAs were analyzed in triplicate by qRT-PCR (mean ± standard deviation) using the standard curve method. Cells from WT mice (A) or mutant mice (B) were analyzed. *<i>P</i><0.01 (Student's <i>t</i>-test).</p

Specific activities of IRF4 and IRF8 in macrophages.

<p>(A) Genes specifically induced by IRF4 or IRF8. Tot2 cells transduced with empty MSCV-puro, MSCV-IRF4FLAG-puro or MSCV-IRF8FLAG-puro were analyzed by qRT-PCR on day 3 (mean ± standard deviation). Data are representative of two independent experiments with similar results. (B) Distinct patterns of cytokine gene induction in IRF4- and IRF8-transduced macrophages. Cells transduced with MSCVs were stimulated on day 5 with 1 µg/ml LPS or 1 µg/ml CpG-B for 5 h, and analyzed by qRT-PCR using the ΔΔCT method (mean ± standard deviation). Two repeat experiments gave similar results. *<i>P</i><0.01 (Student's <i>t</i>-test).</p

IRF4 induces macrophage-related genes and growth arrest during macrophage differentiation.

<p>(A) Induction of macrophage-related genes. Transcript levels in MSCV-transduced cells on day 5 were analyzed by qRT-PCR in triplicate. Data were analyzed using the ΔΔCT method and normalized by the <i>Gapdh</i> levels and shown as values relative to those in empty vector-transduced cells (mean ± standard deviation; representative of three independent experiments with similar results). *<i>P</i><0.01 (Student's <i>t</i>-test). (B) Total viable cell yields (left panel) and cell cycle profiles (on day 4, right panel) after the transduction of MSCVs. Data are expressed as mean ± standard deviation of three independent experiments. *<i>P</i><0.01 (Student's <i>t</i>-test).</p

A CML-like disease in mice deficient for <i>Irf8</i> and <i>Irf4</i>.

<p>(A) Spleens (left panel) and spleen weight of WT, <i>Irf8</i>^-/-, <i>Irf4</i>^-/-, and DKO mice. Values are mean ± standard deviation from measurements of 5 to 6 spleens of each genotype. *<i>P</i><0.01 (Student's <i>t</i>-test). (B) Flow cytometric analysis of granulocytes (CD11b⁺ Gr1⁺) and macrophages (F4/80⁺) in bone marrow cells (upper panels) and splenocytes (lower panels). Numbers indicate the percentages of granulocytes and macrophages. Data are representative of three independent experiments with similar results. (C) The absolute numbers of granulocytes and macrophages per femur (upper part) or spleen (lower part). The ratios of granulocytes to macrophages are shown in the right panels. Values are mean ± standard deviation from 3 to 5 mice of each genotype. *<i>P</i><0.01 and **<i>P</i><0.05 (Student's <i>t</i>-test).</p

IRF4 targets the IECS.

<p>(A) Reporter assays of transcription through the IECS. Tot2 cells were transduced with SIRV-IECS-Ld40-GFP or SIRV-mIECS-Ld40-GFP and then with empty MSCV-CD8t, MSCV-IRF4-CD8t, or MSCV-IRF8-CD8t. The promoter activities in CD8⁺ cells were analyzed on day 2 after the transduction of MSCVs. The activity is shown as mean fluorescent intensity (MFI) of GFP signals (mean ± standard deviation of three independent experiments). *<i>P</i><0.01 (Student's <i>t</i>-test). (B, C) ChIP assays for binding to the IECS (B) or a gene promoter containing an IECS (C). Cells transduced with SIRVs and MSCVs were analyzed by ChIP assays on day 3 after the transduction of MSCVs. Chromatin was precipitated by anti-IRF4 antibody, anti-IRF8 antibody, or normal goat IgG. Precipitated DNA was analyzed by qPCR in triplicate using primers that amplified the IECS sequence in SIRVs or those that amplified the IECS region of the <i>Cst3</i> gene promoter (mean ± standard deviation). Data are representative of three independent experiments. *<i>P</i><0.01 (Student's <i>t</i>-test).</p

IRF4 drives the differentiation of myeloid progenitors towards macrophages.

<p>(A) Wright-Giemsa stain of Tot2 cells transduced with empty MSCV-puro, MSCV-IRF4FLAG-puro or MSCV-IRF8FLAG-puro (original magnification, x 600). (B) Surface marker analysis. Cells on day 6 were stained with the indicated antibodies or isotype control antibodies and analyzed by flow cytometry. Note that differentiated macrophages have higher autofluorescence than undifferentiated cells. (C) Phagocytic activity. Cells on day 6 were incubated with fluorescein-labeled <i>E. coli</i> bioparticles at 37°C or 4°C for 2 h and analyzed by flow cytometry. (D) Immunoblotting analysis of FLAG-tagged IRFs. β-tubulin expression is shown as a loading control.</p