Wnt-β-Catenin Signaling in Human Dendritic Cells Mediates Regulatory T-Cell Responses to Fungi via the PD-L1 Pathway

Abstract

The signaling pathways activated following interaction between dendritic cells (DCs) and a pathogen determine the polarization of effector T-cell and regulatory T-cell (Treg) responses to the infection. Several recent studies, mostly in the context of bacterial infections, have shown that the Wnt/β-catenin pathway plays a major role in imparting tolerogenic features in DCs and in promotion of Treg responses. However, the significance of the Wnt/β-catenin pathway's involvement in regulating the immune response to the fungal species is not known. Using Aspergillus fumigatus, a ubiquitous airborne opportunistic fungal species, we show here that fungi activate the Wnt/β-catenin pathway in human DCs and are critical for mediating the immunosuppressive Treg responses. Pharmacological inhibition of this pathway in DCs led to inhibition of maturation-associated molecules and interleukin 10 (IL-10) secretion without affecting the majority of the inflammatory cytokines. Furthermore, blockade of Wnt signaling in DCs suppressed DC-mediated Treg responses in CD4⁺ T cells and downregulated both tumor necrosis factor alpha (TNF-α) and IL-10 responses in CD8⁺ T cells. Mechanistically, induction of β-catenin pathway by A. fumigatus required C-type lectin receptors and promoted Treg polarization via the induction of programmed death-ligand 1 on DCs. Further investigation on the identity of fungal molecular patterns has revealed that the cell wall polysaccharides β-(1, 3)-glucan and α-(1, 3)-glucan, but not chitin, possess the capacity to activate the β-catenin pathway. Our data suggest that the Wnt/β-catenin pathway is a potential therapeutic target to selectively suppress the Treg response and to sustain the protective Th1 response in the context of invasive aspergillosis caused by A. fumigatus. IMPORTANCE The balance between effector CD4⁺ T-cell and immunosuppressive regulatory T-cell (Treg) responses determines the outcome of an infectious disease. The signaling pathways that regulate human CD4⁺ T-effector versus Treg responses to the fungi are not completely understood. By using Aspergillus fumigatus, a ubiquitous opportunistic fungal species, we show that fungi activate the Wnt/β-catenin pathway in human dendritic cells (DCs) that promotes Treg responses via induction of immune checkpoint molecule programmed death ligand 1 on DCs. Blockade of the Wnt/β-catenin pathway in DCs led to the selective inhibition of Treg without affecting the Th1 response. Dissection of the identity of A. fumigatus pathogen-associated molecular patterns (PAMPs) revealed that cell wall polysaccharides exhibit selectivity in their capacity to activate the β-catenin pathway in DCs. Our data thus provide a pointer that Wnt/β-catenin pathway represents potential therapeutic target to selectively suppress Treg responses and to sustain protective a Th1 response against invasive fungal diseases.

Keywords: Aspergillus fumigatus; DC-SIGN; PD-L1; Wnt signaling; Wnt-β-catenin; dectin-1; dectin-2; dendritic cells; human; regulatory T cells.

FIG 1

A. fumigatus swollen conidia and live conidia, as well as hyphae, activate the Wnt/β-catenin pathway in human (DCs). (A) Monocyte-derived DCs (0.5 × 10⁶ cells/mL) were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) and were either unstimulated (CA) or stimulated with swollen conidia (SC) (0.5 × 10⁶/mL) or dormant conidia (DOC) (0.5 × 10⁶/mL) for 24 h. The activation of Wnt/β-catenin pathway was assessed by immunoblotting of active β-catenin and p-GSK-3β proteins. β-Actin was used as a protein loading control. Representative blot and densitometric analyses of the blots for active β-catenin and p-GSK-3β proteins (mean ± standard error of the mean [SEM]; n = 6 independent donors) are presented. (B) Monocyte-derived DCs (0.5 × 10⁶ cells/mL) were cultured with GM-CSF and IL-4 and were either unstimulated (CA) or stimulated with germinating/hyphae morphotype (Hy; 0.5 × 10⁶/mL), SC (0.5 × 10⁶/mL), or live conidia (LC; 500/mL) for 24 h. Representative blot and densitometric analyses of the blots for active β-catenin and p-GSK-3β proteins (mean ± SEM; n = 3 independent donors) are presented. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test.

FIG 2

A. fumigatus swollen conidia induce secretion of Wnt1 and Wnt7a proteins in DCs. DCs (0.5 × 10⁶ cells/mL) were cultured with GM-CSF and IL-4 and were either stimulated with swollen conidia (SC) or dormant conidia (DOC), for 18 h, or kept unstimulated as a control (CA). (A) Expression of various canonical (WNT1, WNT3A, WNT3, and WNT8A) and noncanonical Wnt ligands (WNT5A, WNT7A, and WNT7B) (mean ± SEM; n = 6 donors) was analyzed by quantitative real-time reverse transcription-PCR (RT-PCR); fold change in the expression of these genes compared to that of the housekeeping gene GAPDH is plotted. (B) The amount (ng/mL) of Wnt1, Wnt3a, Wnt5a, Wnt7a, and Wnt7b in the cell-free supernatants (mean ± SEM; n = 6 donors) after 24 h of stimulation of DCs with swollen conidia or dormant conidia. *, P < 0.05; **, P < 0.01; ns, not significant; determined by one-way ANOVA followed by Tukey’s multiple-comparison test.

FIG 3

Wnt/β-catenin signaling is critical for A. fumigatus swollen conidia to induce maturation of DCs. DCs (0.5 × 10⁶ cells/mL) were cultured with GM-CSF and IL-4 and were either unstimulated (CA) or were treated with dimethyl sulfoxide (DMSO) or Wnt inhibitor (Wnt Inhi) for 2 h followed by stimulation with swollen conidia (SC) at a 1:1 ratio for 48 h. DC phenotype was analyzed by flow cytometry. (A) Representative histogram overlays showing the expression of CD83, CD80, CD86, CD40, and HLA-DR on DCs under various experimental conditions and median fluorescence intensities (MFI) (mean ± SEM; n = 8 donors) of those markers. (B) The amount (pg/mL) of secreted interleukin 6 (IL-6), IL-8, IL-10, IL-12, IL-1β, and tumor necrosis factor alpha (TNF-α) cytokines in the cell-free supernatant from the above-described experiments (mean ± SEM; n = 8 donors) are measured by an enzyme-linked immunosorbent assay (ELISA). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant as determined by one-way ANOVA test with Tukey’s multiple-comparison test.

FIG 4

Wnt/β-catenin signaling is critical for A. fumigatus swollen conidia-stimulated DCs to induce polarization of Treg response. DCs (0.5 × 10⁶ cells/mL) were cultured with GM-CSF and IL-4 and were either unstimulated (CA) or exposed to DMSO or Wnt inhibitor (Wnt Inhi) for 2 h, followed by stimulation with swollen conidia (SC). After 48 h, DCs were washed and cocultured with autologous naive CD4⁺ T cells for 5 days. The polarization of various T-cell subsets was analyzed by flow cytometry. Representative dot plots (% positive cells) and pooled data (mean ± SEM) from 4 to 10 donors showing the frequency of Th1 cells (IFN-γ⁺ CD4⁺), Th17 cells (IL-17A⁺ CD4⁺), Th2 (IL-4⁺ CD4⁺) cells, Tregs (CD25⁺ FoxP3⁺ CD127^low/neg CD4⁺), and IL-10-secreting CD4⁺ T cells (IL-10⁺ CD4⁺) were presented. *, P < 0.05; **, P < 0.01; ns, not significant as determined by one-way ANOVA with Tukey’s multiple-comparison test.

FIG 5

Wnt/β-catenin signaling in DCs regulate CD8⁺ T-cell response to A. fumigatus. DCs (0.5 × 10⁶ cells/mL) were cultured with GM-CSF and IL-4 and were either unstimulated (CA) or exposed to DMSO or Wnt inhibitor (Wnt Inhi) for 2 h followed by stimulation with swollen conidia (SC). After 48 h, DCs were washed and cocultured with autologous CD8⁺ T cells for 5 days. CD8⁺ T-cell response was analyzed based on the intracellular staining for the cytokines gamma interferon (IFN-γ), IL-2, IL-10, and TNF-α. Representative dot plots (% positive cells) and pooled data (mean ± SEM) from 4 donors were presented. *, P < 0.05; **, P < 0.01; ns, not significant as determined by one-way ANOVA with Tukey’s multiple-comparison test.

FIG 6

Wnt/β-catenin signaling regulates the A. fumigatus-induced expression of PD-L1 on DCs. DCs (0.5 × 10⁶ cells/mL) were cultured with GM-CSF and IL-4 and were either left unstimulated (CA) or treated with DMSO or Wnt inhibitor (Wnt Inhi) for 2 h followed by stimulation with swollen conidia (SC). After 48 h, the expressions of CD273 (PD-L2), CD275 (ICOSL), CD252 (OX-40L), and CD274 (PD-L1) were examined by flow cytometry. (A) Expression of CD273, CD275 (% positive cells), and CD252 (% positive cells and median fluorescence intensity [MFI]) on A. fumigatus swollen conidia-stimulated DCs (mean ± SEM; n = 2 in duplicates) with or without inhibition of Wnt signaling. (B) Expression of CD274 (PD-L1) on swollen conidia-stimulated DCs with or without inhibition of Wnt signaling. Representative histogram overlays, percent positive cells and MFI of CD274 (mean ± SEM; n = 8 independent donors) were indicated. (C) PD-L1 blockade abrogates the ability of swollen conidia-stimulated DCs to induce Treg expansion. DCs (0.5 × 10⁶ cells/mL) were cultured with GM-CSF and IL-4 and were either unstimulated (CA) or stimulated with swollen conidia (SC). After 48 h, cells were washed and stimulated DCs were incubated with either blocking monoclonal antibodies to PD-L1 or isotype control antibodies and then cocultured with autologous naive CD4⁺ T cells for 5 days. The frequency of Tregs was analyzed by flow cytometry. The data (mean ± SEM) from 4 donors were presented. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant as determined by one-way ANOVA followed by Tukey’s multiple-comparison test.

FIG 7

A. fumigatus cell wall glucans but not chitin activate Wnt/β-catenin signaling in DCs. DCs were cultured with GM-CSF and IL-4 and were either unstimulated (CA) or stimulated with swollen conidia (SC) (0.5 × 10⁶/mL), or with α-(1, 3)-glucan (1 μg/mL/0.5 × 10⁶ cells), or with β-(1, 3)-glucan (1 μg/mL/0.5 × 10⁶ cells), or with chitin (1 μg/mL/0.5 × 10⁶ cells) for 24 h. Activation of Wnt/β-catenin pathway was analyzed by immunoblotting of active β-catenin and p-GSK-3β. Actin was used as a loading control. Representative blots and densitometric analyses (mean ± SEM; n = 4) are displayed. *, P < 0.05; **, P < 0.01; ns, not significant; as determined by one-way ANOVA followed by Dunnett’s multiple-comparison test.

FIG 8

Induction of β-catenin pathway by A. fumigatus in DCs requires C-type lectin receptors. DCs were cultured with GM-CSF and IL-4 and were either left unstimulated (CA) or stimulated with swollen conidia (SC) or were treated with EDTA (A), or anti-Dectin-1 monoclonal antibody (B), or anti-DC-SIGN monoclonal antibody or anti-Dectin-2 monoclonal antibody (C), or Syk inhibitor (D) for 1 h before stimulating with swollen conidia. GAPDH (A) or β-actin (B to D) were used as loading controls. As EDTA chelates divalent cations such as Ca⁺² and Mg⁺², it could affect β-actin polymerization and stability. Hence, GAPDH was used as loading control for the experiment shown in panel A. Representative blots and densitometric analysis (mean ± SEM) of active β-catenin and p-GSK-3β from n = 6 (A and B), n = 3 (C) and n = 4 (D) experiments are presented. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant as determined by one-way ANOVA and Dunnett’s multiple-comparison test. The “#” symbol denotes irrelevant bands.