Loss of Mef2D function enhances TLR induced IL-10 production in macrophages

Abstract

Mef2 transcription factors comprise a family of four different isoforms that regulate a number of processes including neuronal and muscle development. While roles for Mef2C and Mef2D have been described in B-cell development their role in immunity has not been extensively studied. In innate immune cells such as macrophages, TLRs drive the production of both pro- and anti-inflammatory cytokines. IL-10 is an important anti-inflammatory cytokine produced by macrophages and it establishes an autocrine feedback loop to inhibit pro-inflammatory cytokine production. We show here that macrophages from Mef2D knockout mice have elevated levels of IL-10 mRNA induction compared with wild-type cells following LPS stimulation. The secretion of IL-10 was also higher from Mef2D knockout macrophages and this correlated to a reduction in the secretion of TNF, IL-6 and IL-12p40. The use of an IL-10 neutralising antibody showed that this reduction in pro-inflammatory cytokine production in the Mef2D knockouts was IL-10 dependent. As the IL-10 promoter has previously been reported to contain a potential binding site for Mef2D, it is possible that the binding of other Mef2 isoforms in the absence of Mef2D may result in a higher activation of the IL-10 gene. Further studies with compound Mef2 isoforms would be required to address this. We also show that Mef2D is highly expressed in the thymus, but that loss of Mef2D does not affect thymic T-cell development or the production of IFNγ from CD8 T cells.

Keywords: intracellular signaling; macrophages; mef2D; toll-like receptors.

Figure 1. Generation of Mef2D knockout mice

The Mef2d knockout was generated using the strategy shown in panel (A). A targeting vector was used to insert loxP sites upstream of Mef2D and in intron 2 via homologous recombination in ES cells. Targeted ES cells were used to generate mice carrying the targeted locus, which were then crossed to mice expressing Flpe to remove the neomycin resistance cassette and then Cre to delete exons 1 and 2 in the Mef2D gene, as described in the ´Materials and Methods’. To confirm the knockout was successful, protein extracts from the thymus, spleen, liver, lymph node, hippocampus, cortex and cerebellum of wild type and Mef2D knockout mice were immunoblotted for Mef2D (B). Levels of p38α MAPK were also determined to confirm equal loading of sample between the wild-type and Mef2D knockout lysates. ns indicates a non-specific band.

Figure 2. Mef2D is not required for T- or B-cell development

Thymi were isolated from wild-type and Mef2D knockout mice. Thymocytes were stained for Thy1, CD4 and CD8 and analysed by flow cytometry. The numbers of thymocytes is shown in panel (A). Cells were gated on Thy1 expression and the percentage of CD4 and CD8 positive cells is shown in panel (B), with quantification from four wild-type and 5 Mef2D knockout mice shown on the left and representative plots on the right. In panels (C–E), spleens were isolated from nine wild-type and nine Mef2D knockout mice and red blood cells lysed as described in the ‘Materials and Methods’ section and then cells stained for CD19, TCRβ, CD4 and CD8. Number of splenocytes is shown in panel (C). The percentage of T cells and B cells in the spleen is shown in panel (D). The percentage of CD4 and CD8 cells in the T cell (TCRβ+ve) cells is shown in panel (E). In panels (F and G), CD8 T cells were expanded from splenocytes ex vivo as described in the ‘Materials and Methods’ section. The number of cells present over time in cultures from wild-type and Mef2D knockout mice is shown in (F). On day 7, cells were either left unstimulated or stimulated with either anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) or PdBu (20 ng/ml) and ionomycin (0.5 µg/ml) for 4 h. Levels of interferon γ secreted into the media were measured by ELISA panel (G). For panels (F and G), data show mean and standard deviation of cultures from 4 mice per genotype. A P value of less than 0.05 (two-tailed Student’s t-test) is indicated by *.

Figure 3. Mef2D is not essential for myeloid cell development

Spleens were isolated from five wild-type and four Mef2D knockout mice and red blood cells lysed as described in the ‘Materials and Methods’ section and then cells stained for CD19, TCRβ and the indicated markers. Representative plots to identify CD11b / Gr1 positive neutrophils are shown in panel (A), CD11b / F4/80 positive macrophages in panel (B) and Cd11b / CD11c positive conventional dendritic cells in panel (C). Average cell numbers and standard deviation for the three cell populations are shown in panel (D). A P value of less than 0.05 (two-tailed Student’s t-test) is indicated by *.

Figure 4. Mef2D knockout does not inhibit TLR4 induced signalling in macrophages

(A) Bone marrow derived macrophages were isolated from wild-type and Mef2D knockout mice. The levels of Mef2A, Mef2C and Mef2D mRNA relative to GAPDH mRNA were then determined by qPCR. Error bars represent the standard deviation of independent cultures from four mice per genotype. (B–E) BMDMs were stimulated with 100 ng/ml LPS for the indicated times. Total RNA was isolated and the levels of nur77 (B), egr1 (C) and nurr1 (D) mRNA determined by qPCR. Fold change is expressed relative to the level in unstimulated wild-type macrophages. Error bars represent standard deviation from independent cultures from three mice per genotype. A P value (two-tailed Student’s t-test) between wild-type and Mef2D knockout cells of less than 0.001 is indicated by ** and less than 0.05 by *. (E) Cells were lysed and samples run on SDS poly-acrylamide gels and blotted for the indicated phospho or total proteins (E). ns indicates a non-specific band detected by the phospho MSK1 antibody.

Figure 5. Mef2D knockout results in decreased pro-inflammatory production in LPS stimulated macrophages

Bone marrow derived macrophages were isolated from wild-type and Mef2D knockout mice. Cells were stimulated with 100 ng/ml LPS for the indicated times. The levels of TNF (A), IL-6 (B) and IL-12p40 (C) secreted into the media are shown in panel (B). Total RNA was isolated and the levels of TNF (D), IL-6 (E) and IL-12p40 (F) mRNA determined by qPCR. Values are expressed relative to the level in unstimulated wild type macrophages (D–F). Error bars represent standard deviation from independent cultures from three mice per genotype. A P value (two tailed Student’s t-test) between wild-type and Mef2D knockout cells of less than 0.001 is indicated by ***, less than 0.01 by ** and less than 0.05 by *.

Figure 6. Mef2D acts as a negative regulator of LPS induced IL-10 production

Bone marrow derived macrophages were isolated from wild type and Mef2D knockout mice. Cells were stimulated with 100 ng/ml LPS for the indicated times. Total RNA was isolated and the levels of IL-10 mRNA determined by qPCR. Values are expressed relative to the level in unstimulated wild type macrophages (A). The levels of IL-10 secreted into the media are shown in (B). Error bars represent standard deviation from independent cultures from 3 mice per genotype. A p value (2 tailed Students ttest) between wild type and knockout cells of less than 0.001 is indicated by *** and less than 0.05 by *.

Figure 7. Decreased LPS induced pro-inflammatory cytokine production in Mef2D knockout macrophages is dependent on IL-10

Bone marrow derived macrophages were isolated from wild-type and Mef2D knockout mice. Cells were stimulated with 100 ng/ml LPS for 8 h in the presence or absence of a neutralising antibody against IL-10. The levels of TNF (A), IL-6 (B) and IL-12p40 (C) secreted into the media are shown in panel (B). Total RNA was isolated and the levels of TNF (D), IL-6 (E) and IL-12p40 (F) mRNA determined by qPCR. Values are expressed relative to the level in unstimulated wild-type macrophages (D–F). Error bars represent standard deviation from independent cultures from three mice per genotype. A P value (Student’s t-test) between wild-type and knockout cells of less than 0.001 is indicated by ***, less than 0.01 by ** and less than 0.05 by *.