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. 2015 Aug 15;195(4):1723-31.
doi: 10.4049/jimmunol.1402276. Epub 2015 Jun 29.

Opposing Roles of Dectin-1 Expressed on Human Plasmacytoid Dendritic Cells and Myeloid Dendritic Cells in Th2 Polarization

HyeMee Joo  1 , Katherine Upchurch  2 , Wei Zhang  1 , Ling Ni  1 , Dapeng Li  1 , Yaming Xue  1 , Xiao-Hua Li  1 , Toshiyuki Hori  3 , Sandra Zurawski  1 , Yong-Jun Liu  1 , Gerard Zurawski  2 , SangKon Oh  4
Affiliations

Opposing Roles of Dectin-1 Expressed on Human Plasmacytoid Dendritic Cells and Myeloid Dendritic Cells in Th2 Polarization

HyeMee Joo et al. J Immunol. .

Abstract

Dendritic cells (DCs) can induce and control host immune responses. DC subset-dependent functional specialties and their ability to display functional plasticity, which is mainly driven by signals via pattern recognition receptors, identify DCs as immune orchestrators. A pattern recognition receptor, Dectin-1, is expressed on myeloid DCs and known to play important roles in Th17 induction and activation during fungal and certain bacterial infections. In this study, we first demonstrate that human plasmacytoid DCs express Dectin-1 in both mRNA and protein levels. More interestingly, Dectin-1-activated plasmacytoid DCs promote Th2-type T cell responses, whereas Dectin-1-activated myeloid DCs decrease Th2-type T cell responses. Such contrasting outcomes of Th2-type T cell responses by the two DC subsets are mainly due to their distinct abilities to control surface OX40L expression in response to β-glucan. This study provides new insights for the regulation of host immune responses by Dectin-1 expressed on DCs.

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Figures

FIGURE 1
FIGURE 1
Human pDCs from the blood, tonsil, and spleen express Dectin-1. (A) pDCs and mDCs from the blood of healthy donors were stained with αDectin-1 mAb. (B-C) pDCs and mDCs from tonsil (B) and spleen (C) were stained with αDectin-1 mAb. (D) Summary for hDectin-1 expression on pDCs (left panel) and mDCs (right panel) from blood, tonsil, and spleen. Each dot represents data from a single donor. Mean fluorescence intensity (MFI) of isotype control antibody was subtracted.
FIGURE 2
FIGURE 2
hDectin-1 ligation by β-glucan activates pDCs via Syk. pDCs from blood were cultured overnight in the presence of none, curdlan, R848, or CpG-B. (A) FSC and SSC scatter gram of pDCs. (B) Annexin V staining of cells gated in (A). (C) Summary of three independent experiments of A and B. Error bars represent SD. (D) Expression levels of CD80, CD83, CD86, HLA-DR, and CD40 were measured. (E) Cytokine and chemokine levels. (F) pDCs were treated with a Syk inhibitor, R406, for 1h and then cultured overnight with 100 μg/ml curdlan. The amount of IL-6 in the culture supernatants was assessed. (G) pDCs were treated with 20 μg/ml anti-hDectin-1 antibody and then incubated overnight with 100 μg/ml curdlan. The amount of IL-6 in the culture supernatants was measured. In (E-G), error bars represent SD of triplicate assays. Two independent experiments showed similar results. (H) The amount of cysteinyl leukotriene (Cys-LT) secreted from pDCs stimulated with curdlan or house dust mite Dermatophagoides farina (Df) extract. FACS-sorted pDCs from two healthy donors were tested in duplicate assays (Mean ± SD).
FIGURE 3
FIGURE 3
hDectin-1-activated pDCs promote Th2 induction and Th2-type cytokine secretion by CD4+ T cells. (A) Allogeneic naïve CD4+ T cells were co-cultured for 7 days with untreated or curdlan-treated blood pDCs. Intracellular cytokine expression, IL-13 and IL-5 in upper panel and IFNγ and IL-17 in lower panel, during stimulation with PMA/ionomycin were measured. (B) Summary of data in experiment (A) performed with cells from different donors. (C) Surface OX40L expression on pDCs cultured overnight in medium alone or in the presence of either curdlan and control IgG or curdlan and anti-IFNα antibody. Cells were also stained with anti-CD86 antibody. (D) Summary of data in experiment (C) performed with cells from different donors. (E) The amount of IFNα secreted from blood pDCs cultured overnight with the indicated amounts of curdlan. Error bars indicate SD of triplicate assays. Two separate experiments showed similar results. (F) Allogeneic naïve CD4+ T cells were co-cultured for 7 days with untreated pDCs or curdlan-treated pDCs in the presence of control IgG or anti-OX40L antibody for 7 days. Intracellular IL-13 and IL-5 (upper panel), as well as IFNγ and IL-17 (lower panel), during stimulation with PMA/ionomycin were measured. (G) Summary of data in (F) from five independent experiments. (H) Cytokines secreted from T cells (E) during 48 h restimulation with PMA/ionomycin. Error bars indicate SD of triplicate assays. Two independent experiments showed similar results. ANOVA for (B), (D) and (G) and t-test for (H) were used for testing statistical significance of the data.
FIGURE 4
FIGURE 4
hDectin-1-activated mDCs decrease Th2 induction and Th2-type cytokine secretion by CD4+ T cells. (A) Blood mDCs were cultured overnight in medium alone or either in the presence of curdlan and control IgG or curdlan and anti-IL-10/IL-10R. Cells were stained with anti-CD86 and anti-OX40L antibodies. (B) Summarized data of (A) from four independent experiments using cells from different donors. (C) Levels of IL-10 secreted from mDCs cultured overnight with the indicated amounts of curdlan. Error bars indicate SD of triplicate assays. Two independent experiments showed similar results. (D) Allogeneic naïve CD4+ T cells were co-cultured for 7 days with untreated pDCs and curdlan-treated pDCs (left panels) in the presence of control IgG or anti-OX40L antibody. T cells were stained for intracellular IL-13/IL-5 (upper panels) as well as IFNγ/IL-17 (lower panels) expression during stimulation with PMA/ionomycin. (E) Summarized data of experiment (D) performed with cells from different donors. (F) Cytokines secreted from T cells (E) during 48 h restimulation with PMA/ionomycin. Error bars indicate SD of triplicate assays. Two independent experiments showed similar results. ANOVA for (B) and (E) and t-test for (F) were used for testing statistical significance of the data.
FIGURE 5
FIGURE 5
hDectin-1-activated pDCs and mDCs increase and decrease influenza virus-specific memory Th2 responses, respectively. (A) Binding of anti-hDectin-1-HA1 fusion protein to pDCs. Purified pDCs were incubated for 15 min with the indicated amounts of anti-hDectin-1-HA1 or control IgG4-HA1. Cells were further stained with anti-human IgG-FITC and sorted by FACS. (B) Autologous CD4+ T cells were co-cultured for 7 days with 2 μg/ml anti-hDectin-1-HA1-loaded pDCs in the presence or absence of curdlan plus anti-OX40L or curdlan plus control IgG. T cells were then restimulated with 1 μM HA1-derived peptides for 48 h. Cytokine levels in the culture supernatants were measured. Error bars indicate mean±SD of triplicate assay. (C) Summarized data of experiment (B) performed with cells from four different donors. Statistical significance was tested with ANOVA. (D) Autologous CD4+ T cells were co-cultured for 7 days with 2 μg/ml anti-hDectin-1-HA1-loaded mDCs in the presence or absence of curdlan. Cytokines secreted from T cells stimulated with PMA/ionomycin were assessed. (E) Summarized data of experiment (D) performed with cells from different donors. Statistical significance was tested with ANOVA. (F) Intracellular cytokine expression of total CD4+ T cells restimulated for 5 h with PMA/ionomycin in (D); IL-13 and IL-5 (upper panel) and IFNγ and IL-17 (lower panel). (G) CD4+ T cells were co-cultured for 7 days with 2 μg/ml anti-hDectin-1-HA1-loaded and curdlan-treated mDCs in the presence of γ-irradiated L cells or OX40L-L cells. Cytokines secreted from T cells stimulated for 48 h with 1 μM of the indicated peptides were assessed. Error bars indicate SD of triplicate assays. Two repeat experiments showed similar results. (H) Intracellular cytokine expression (IL-13/IL-5 in the upper panel and IFNγ/IL-17 in the lower panel) of T cells in (G) during 5 h restimulation with PMA/ionomycin.
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