A dominant role for the methyl-CpG-binding protein Mbd2 in controlling Th2 induction by dendritic cells

Peter C Cook, Heather Owen, Aimée M Deaton, Jessica G Borger, Sheila L Brown, Thomas Clouaire, Gareth-Rhys Jones, Lucy H Jones, Rachel J Lundie, Angela K Marley, Vicky L Morrison, Alexander T Phythian-Adams, Elisabeth Wachter, Lauren M Webb, Tara E Sutherland, Graham D Thomas, John R Grainger, Jim Selfridge, Andrew N J McKenzie, Judith E Allen, Susanna C Fagerholm, Rick M Maizels, Alasdair C Ivens, Adrian Bird, Andrew S MacDonald, Peter C Cook, Heather Owen, Aimée M Deaton, Jessica G Borger, Sheila L Brown, Thomas Clouaire, Gareth-Rhys Jones, Lucy H Jones, Rachel J Lundie, Angela K Marley, Vicky L Morrison, Alexander T Phythian-Adams, Elisabeth Wachter, Lauren M Webb, Tara E Sutherland, Graham D Thomas, John R Grainger, Jim Selfridge, Andrew N J McKenzie, Judith E Allen, Susanna C Fagerholm, Rick M Maizels, Alasdair C Ivens, Adrian Bird, Andrew S MacDonald

Abstract

Dendritic cells (DCs) direct CD4(+) T-cell differentiation into diverse helper (Th) subsets that are required for protection against varied infections. However, the mechanisms used by DCs to promote Th2 responses, which are important both for immunity to helminth infection and in allergic disease, are currently poorly understood. We demonstrate a key role for the protein methyl-CpG-binding domain-2 (Mbd2), which links DNA methylation to repressive chromatin structure, in regulating expression of a range of genes that are associated with optimal DC activation and function. In the absence of Mbd2, DCs display reduced phenotypic activation and a markedly impaired capacity to initiate Th2 immunity against helminths or allergens. These data identify an epigenetic mechanism that is central to the activation of CD4(+) T-cell responses by DCs, particularly in Th2 settings, and reveal methyl-CpG-binding proteins and the genes under their control as possible therapeutic targets for type-2 inflammation.

Figures

Figure 1. Mbd2 regulates expression of several…
Figure 1. Mbd2 regulates expression of several key DC pathways associated with APC function.
(a) WT or Mbd2−/− BMDCs cultured on multichamber glass slides were stained with phalloidin (green) and 4,6-diamidino-2-phenylindole (blue) and surface area analysed by confocal microscopy. Photomicrographs are representative images from five fields in one experiment of three (scale bars, 38 μM (top panel) and 7 μM (bottom panel)). CD11c staining on WT (blue) and Mbd2−/− (red) BMDCs (one of six experiments) was assessed by flow cytometry. (b) Heat map showing the mRNA signature of WT versus Mbd2−/− BMDCs (119 genes, log2 normalized intensity, twofold change-filtered, P<0.05 (moderated t-test), three biological replicates per genotype). (c) Heat map showing 18 downregulated Mbd2−/− versus WT BMDCs genes selected based on putative function following network analysis. (d) Heat map showing nine upregulated Mbd2−/− versus WT BMDCs genes selected based on putative function following network analysis. (e) To validate microarray data, mRNA expression of genes of interest were assessed by qPCR (normalized against Hprt, a.u.), surface protein expression measured by flow cytometry and secreted protein by ELISA, comparing WT and Mbd2−/− BMDCs. Results are mean+s.e.m. (three replicate wells, one of at least six experiments). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). a.u., arbitrary units; gMFI, geometric mean fluorescence intensity.
Figure 2. H3K9/K14 acetylation of genes controlled…
Figure 2. H3K9/K14 acetylation of genes controlled by Mbd2.
(a) ChIP sequencing composite profile showing H3K9/K14ac or input DNA signal (as read coverage) for WT (blue) and Mbd2−/− (red) BMDCs for a selection of genes with significantly altered mRNA profiles. Signal is displayed from −3 kb to +3 kb surrounding each annotated transcription start site (TSS). (b) Genome screenshots representing H3K9/K14ac signal in WT (blue) and Mbd2−/− (red) BMDCs at selected gene loci (Ccl17, Cd40, Irf4 and Mrc1). RefSeq genes are in black.
Figure 3. Mbd2 regulates DC antigen uptake…
Figure 3. Mbd2 regulates DC antigen uptake and interaction with CD4+ T cells.
(a,b) WT (blue) or Mbd2−/− (red) BMDCs were cultured for 4 days with CFSE-labelled OT-II T cells in the presence of OVA peptide (a) or protein (b). T-cell proliferation was assessed by flow cytometric analysis of CFSE dilution (six wells per group, one of five experiments). (c) Adhesion of WT CD4+ T cells to WT or Mbd2−/− BMDCs under shear flow conditions (n=mean of T-cell adhesion combined from three separate experiments). (d) WT (blue) or Mbd2−/− (red) BMDCs were incubated with DQ-OVA at 37 or 4 °C, and uptake assessed by flow cytometry (five wells per group, one of more than five experiments). **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test (a,b) or ANOVA (c,d). gMFI, geometric mean fluorescence intensity.
Figure 4. Mbd2 is vital for optimal…
Figure 4. Mbd2 is vital for optimal Th2 but not for Th1 induction in vivo.
(a,b) WT (grey) or Mbd2−/− (black) mice were injected s.c. with PBS, S. mansoni eggs (a) or heat-killed S. typhimurium (St) (b). Seven days later the draining pLNs were harvested, cells restimulated for 72 h with SEA or St and cytokine secretion assessed by ELISA (five mice per group, one of two to four experiments). (c,d) Mbd2ΔDC (black) mice or littermate controls (grey) were injected s.c. with PBS, S. mansoni eggs (c) or heat-killed S. typhimurium (St) (d). Seven days later the draining pLNs were harvested, cells restimulated for 72 h with SEA or St and cytokine secretion assessed by ELISA (five to six mice per group, one of two to three experiments). Bar graphs show mean+s.e.m. *P<0.05, **P<0.01 (Student's t-test). pLNs, popliteal lymph nodes; s.c., subcutaneously.
Figure 5. DC expression of Mbd2 is…
Figure 5. DC expression of Mbd2 is vital for optimal Th2 induction and development.
(a,b) CD4+GFP− T cells from KN2xIL-10eGFP or KN2xIL-13eGFP mice were cultured for 3 days with WT (blue) or Mbd2−/− (red) BMDCs, anti-CD3 mAb±IL-4 (Th2 conditions) and assessed for IL-4 (huCD2) protein production, and IL-10 and IL-13 mRNA expression by flow cytometry (five to six replicate culture wells, one of six experiments). (c) CD4+GFP− T cells from KN2xIL-13eGFP mice were cultured for 3 days with WT (grey) or Mbd2−/− (black) BMDCs, anti-CD3 mAb±IL-4 (Th2 conditions) and supernatants assessed for IL-10 and IL-13 protein secretion by ELISA. (d,e) CD4+GFP− T cells from IL-10eGFP mice were cultured for 3 days with WT (grey) or Mbd2−/− (black) DCs, anti-CD3 mAb±IL-12 (Th1 conditions) or IL-6/TGF-β/IL-23 (Th17 conditions) and supernatants assessed for IFNγ and IL-17 protein secretion by ELISA or IL-10 mRNA expression was determined by flow cytometry (four replicate wells per group, one of three experiments). (f,g) WT (grey) or Mbd2−/− (black) BMDCs were cultured overnight in medium alone (M) or with SEA and injected s.c. into WT mice. Seven days later the pLNs were harvested, cells restimulated for 72 h with SEA and cytokine secretion assessed by ELISA (four to five mice per group, one of three experiments). Bar graphs show mean+s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (ANOVA). mAb, monoclonal antibody; s.c., subcutaneously; TGF-β, transforming growth factor-β.
Figure 6. DC expression of Mbd2 is…
Figure 6. DC expression of Mbd2 is required for induction of pulmonary allergic inflammation.
(a) WT mice were sensitized by intranasal administration of WT (blue or grey in b–f) or Mbd2−/− (red or black in b–f) BMDCs that had been cultured overnight in medium alone (M) or with HDM, then intranasally challenged with 5 μg HDM on days 14 and 15 post BMDC transfer and tissues harvested on day 17. (b) BAL fluid cells were counted, cytospin preparations stained and 200 cells per slide counted for specific cell types. (c) Lung tissue cells were isolated and eosinophilia, neutrophilia and monocyte infiltration assessed by flow cytometry. (d) Cytokines in BAL fluid were measured by ELISA. (e) Lung tissue mRNA expression was assessed by qPCR (normalized against Hprt, a.u.). (f) Proportions of TCRβ+CD4+Foxp3+ Treg cells (Treg) and activated effector TCRβ+CD4+Foxp3−CD44+CD69+ T cells (Teff) in lung tissues were assessed by flow cytometry. (g) Representative lung sections from recipients of WT or Mbd2−/− HDM-BMDCs, stained with hematoxylin and eosin (scale bar, 50 μM). Bar graphs show mean+s.e.m. (three to six mice per group, one of six experiments). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (ANOVA). a.u., arbitrary units.

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