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. 2011 Jan 7;6(1):e15723.
doi: 10.1371/journal.pone.0015723.

Macrophage Activation and Differentiation Signals Regulate schlafen-4 Gene Expression: Evidence for Schlafen-4 as a Modulator of Myelopoiesis

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Free PMC article

Macrophage Activation and Differentiation Signals Regulate schlafen-4 Gene Expression: Evidence for Schlafen-4 as a Modulator of Myelopoiesis

Wendy J van Zuylen et al. PLoS One. .
Free PMC article

Abstract

Background: The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity.

Methodology/principal findings: Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM) by the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS), the TLR3 agonist Poly(I∶C), and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN)-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1⁻/⁻ BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1)-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens.

Conclusions: Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the myeloid lineage in vivo perturbs myelopoiesis. We hypothesise that the down-regulation of Slfn4 gene expression during macrophage differentiation is a necessary step in development of this lineage.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Induction of Slfn expression by LPS in BMM, and in the CIA mouse model.
(A) BMM were stimulated with LPS over a time course (0h no treatment control, 2h, 4h, 8h, 24h), RNA was extracted and reverse transcribed, and the expression of Slfn1, Slfn2, Slfn4, Slfn5, Slfn8, and Slfn9 was determined using quantitative real-time PCR. Data (mean + SEM) are combined from 3 independent experiments. * P<0.05 compared to control; ** P<0.01 compared to control; *** P<0.001 compared to control. (B) RNA from whole joints (disease affected, score 3; or unaffected, score 0) from mice with CIA was used to synthesise cDNA. mRNA expression of Slfn1, Slfn2, Slfn4, Slfn5, Slfn8, Slfn9, csf1r, and Emr1 was determined using real-time PCR. The data, displayed as fold induction relative to control (score 0), are combined from two independent experiments (mean + range).
Figure 2
Figure 2. Putative regulatory elements within the Slfn4 promoter.
The Slfn4 proximal promoter is a TATA-containing promoter with putative binding sites for STAT1, IRF family members, PU.1 and AP1. The TSS, designated +1, is marked with an arrow.
Figure 3
Figure 3. Slfn4 mRNA is induced via autocrine type I IFN and is down-regulated during macrophage differentiation.
(A) BMM were stimulated with LPS (10 ng/ml), dsRNA Poly(I∶C) (30 µg/ml), Imiquimod (15 µg/ml), Zymosan (150 µg/ml), synthetic phosphorothioate CpG oligonucleotide 1668S (0.3 µM) and non-stimulatory control oligonucleotide 1668S-GC (0.3 µM), Pam3Cys (15 ng/ml), or IFN-γ (500 pg/ml) over a 24h time course. The data, displayed as fold induction relative to control (0h), are combined from two independent experiments (mean + range). (B) BMM from IFNAR-1−/− and wild type mice were stimulated with LPS over a time course. The data are combined from two independent experiments (mean + range). C) HMDM were stimulated with LPS over a time course (0h no treatment control, 2h, 6h, 24h). The data are combined from eight independent donors (mean + SEM). (D, E) Bone marrow progenitors were differentiated into BMM in the presence of CSF-1 over a 6-day time course. Data are combined from three independent experiments (mean + SEM). Slfn4 (A–D) and csf1r (E) mRNA expression was determined using quantitative real-time PCR. * P<0.05 compared to 0h or day 0 control; ** P<0.01 compared to 0h or day 0 control; *** P<0.001 compared to 0h or day 0 control.
Figure 4
Figure 4. Macrophage-specific expression of the UAS-Slfn4-V5 transgene.
(A) Elements of the UAS-Slfn4-V5 transgene include six UAS, a kozak sequence, and the open reading frame of Slfn4 followed by a V5-tag. Upon crossing of the UAS-Slfn4-V5 mouse with the MacBlue mouse, the offspring (MacBlue/UAS-Slfn4-V5 mice) contain the GAL4-expressing module, the GAL4-reporting module, and the UAS-Slfn4-V5 transgene. GAL4/VP16 protein binding to both the UAS induces the expression of ECFP and Slfn4-V5 specifically in cells of the myeloid lineage. (B and C) RNA from bone marrow (BM) or BMM from the offspring of four UAS-Slfn4-V5 founder lines (F1–F4) was extracted and cDNA was prepared. Slfn4 mRNA levels relative to hprt were determined by quantitative real-time PCR. Data are combined from at least two independent experiments (mean + range) and are presented as expression relative to BM controls to enable normalization across different the transgenic lines (B), or as expression relative to hprt (no normalization across the transgenic lines) (C).
Figure 5
Figure 5. Slfn4 over-expression did not alter bone marrow proliferation or cell viability.
Bone marrow cells from MacBlue/UAS-Slfn4-V5 mice and littermate controls were cultured in semisolid methylcellulose medium in the presence (+) or absence (−) of CSF-1. The size (A) and frequency (B) of colonies were examined on day 14. Data are combined from six independent experiments and are displayed as mean + SEM. (C) Bone marrow cells from MacBlue/UAS-Slfn4-V5 mice and littermate controls were cultured in the presence of CSF-1 over a 7-day time course and cell survival was measured on each day by MTT assay. Data are combined from three independent experiments and are displayed as mean ± SEM.
Figure 6
Figure 6. Slfn4 over-expression caused splenomegaly.
(A) Macroscopic appearance of spleens from Slfn4 over-expressing mice (right) and from MacBlue littermate controls (left). (B) Organ weights are expressed as percentage of body weight. Data are combined from four independent experiments and are displayed as mean + SEM.
Figure 7
Figure 7. Increased neutrophils and macrophages in the livers and spleens of MacBlue/UAS-Slfn4-V5 mice.
(A) Hematoxylin and eosin staining was performed on liver paraffin sections. Arrowheads indicate aggregates of neutrophils in the liver. Liver sections from MacBlue/UAS-Slfn4-V5 and MacBlue littermate controls were also stained for F4/80 (brown). Arrows indicate F4/80 expressing cells surrounding aggregates of neutrophils. (B) Hematoxylin and eosin staining was also performed on spleen paraffin sections. Arrows indicate clusters of megakaryocytes. Spleen sections from MacBlue/UAS-Slfn4-V5 and MacBlue littermate controls were also stained for F4/80 (brown). Sections were examined using an Olympus BX-51 microscope with a DP-70 digital camera and DP controller imaging software (Olympus). RP, red pulp; WP, white pulp.
Figure 8
Figure 8. Increased macrophages and granulocytes in spleen from MacBlue/UAS-Slfn4-V5 mice.
Splenocytes from MacBlue littermate controls (A) and MacBlue/UAS-Slfn4-V5 mice (B) were stained for the cell surface markers F4/80 (macrophage), Ly-6G (granulocyte), B220 (CD45R, B cell), and CD3ε (T cell). The samples were analysed by FACS and quadrants were set based upon isotype control profiles. Data are representative of two independent experiments.

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