Fig 1.
SEA stimulates PGE2 secretion and primes Th2 responses independently of ω-1 in human moDCs.
(A) PGE2 concentration in supernatants from moDC cultures after stimulation with indicated reagents. Concentrations are determined based on an internal standard. Data represent mean ± SEM of 4 independent experiments. (B) moDCs stimulated with indicated lipids (concentration of 2.5 ng/mL for LXA4, PGE2, and PGD2; 12.5 μg/mL for 5-HETE, 8-HETE, and 11-HETE; 25 μg/mL 9-HODE and 13-HODE) were analyzed for Th2 polarizing potential as described in Materials and methods. The ratio of percentage of IL-4+ over percentage of IFN-γ+ T cells based on intracellular cytokine staining was calculated relative to the control condition. (C) moDCs were pulsed with indicated stimuli and subsequently cocultured with a CD40L-expressing cell line. Supernatants were collected after 24 h, and IL-12p70 concentration was determined by ELISA. (D) T-cell polarization was determined as in panel B. Top and bottom panels show representative flow cytometry plots of intracellular staining of CD4+ T cells for indicated cytokines, and the ratio of IL-4 over IFN-γ ratio of these plots based on 4 experiments. Numbers in plots represent frequencies of cells in indicated quadrants. (E) PGE2 levels as determined by LC-MS/MS in supernatants of moDCs stimulated with indicated stimuli. Data represent mean ± SEM of 3 independent experiments. (A) Statistical significance of different time points per condition compared to baseline (0 h) time point. “*” and “#”: P < 0.05; “**”: P < 0.01. (A) based on two-way ANOVA test or (B–D) for significantly different with the LPS control (*) or between-test conditions (#) based on paired analysis (paired Student t test). Underlying data can be found in S1 Data. CD4, cluster of differentiation 4; HETE, Hydroxyeicosatetraenoic acid; HODE, Hydroxyoctadecadienoic acid; IFN-γ, interferon γ; IL-4, interleukin 4; LC-MS/MS, liquid chromatography tandem mass spectrometry; LPS, lipopolysaccharide; LXA4, lipoxin A4; moDC, monocyte-derived DC; PGE2, prostaglandin E2; SEA, soluble egg antigen; Th2, T helper 2.
Fig 2.
ω-1–independent Th2 polarization by SEA is dependent on PGE2 synthesis by moDCs.
(A–C) T-cell polarization assay as described in Fig 1B. (A) Neutralizing anti-PGE2 antibody was added during stimulation of moDCs with indicated reagents or (B) during DC–T cell coculture. (C) EP2 and EP4 receptor inhibitors (EP2-I and EP4-I) were added during stimulation of moDCs with indicated stimuli. (A–C) Left: representative flow cytometry plots are shown of intracellular staining of CD4+ T cells for IL-4 and IFN-γ. Numbers in plots represent frequencies of cells in indicated quadrants. Right: these data were used to calculate the fold change in frequency of IL-4+ and IFN-γ+ T cells polarized by moDCs stimulated with indicated stimuli relative to the cytokine production by T cells polarized by LPS-stimulated moDCs, for which the values were set to 1. Bars represent mean ± SEM of at least 4 independent experiments. Significance was calculated based on the ratio of IL-4 over IFN-γ between conditions. *P < 0.05 and **P < 0.01 for significantly different from control conditions based on paired analysis (paired Student t test). Underlying data can be found in S1 Data. ω-1, omega-1; CD4, cluster of differentiation 4; EP2, prostaglandin E2 receptor 2; IL-4, interleukin 4; IFNγ, interferon γ; LPS, lipopolysaccharide; moDC, monocyte-derived DC; PGE2, prostaglandin E2; SEA, soluble egg antigen; Th2, T helper 2.
Fig 3.
OX40L is induced by SEA via PGE2 signaling and is required for Th2 induction.
(A, B) moDCs were stimulated as indicated for 48 h in the presence or absence of neutralizing anti-PGE2 antibody, after which expression of OX40L was analyzed by flow cytometry. The fold change based on geometric mean fluorescence is shown relative to LPS, which is set to 1 (dashed line). (A) PGE2 was taken along as positive control for OX40L expression and a representative histogram plot of OX40L expression is shown on the left. (C) T-cell polarization assay as described in Fig 2C. Neutralizing OX40L antibody was added during the DC–T cell coculture. Bar graphs represent means ± SEM of at least 6 independent experiments. “*”: P < 0.05: “**” and “##”: P < 0.01 for significant differences with the control conditions (*) or between-test condition (#) based on paired analysis (paired Student t test). Underlying data can be found in S1 Data. DC, dendritic cell; LPS, lipopolysaccharide; moDC, monocyte-derived DC; OX40L, OX40 ligand; PGE2, prostaglandin E2; SEA, soluble egg antigen; Th2, T helper 2.
Fig 4.
SEA promotes PGE2 synthesis and drives Th2 polarization via signaling through Dectin-1 and Dectin-2 in human moDCs.
(A) cPLA2 activity 8 h after stimulation. Zymosan was taken along as a positive control for cPLA2 activation. (B) Protein expression of COX-1 and COX-2 were assessed by western blot. β-actin was used as housekeeping protein. One of 3 experiments is shown. (C) Following 1 h pre-incubation with specific inhibitors for cPLA2 (Pyr.) or COX-1 and COX-2 (SC and ind., respectively), moDCs were stimulated for 12 h with LPS plus SEAΔα-1/ω-1, and supernatants were collected for PGE2 determination by LC-MS/MS. (D) At the indicated time points after stimulation with depicted stimuli, phosphorylation of ERK was determined by flow cytometry. A representative flow cytometry plot of intracellular staining for phospho-ERK is shown on the left. (E) PGE2 levels were determined as in panel C. U0216 was used as inhibitor of ERK. (F) moDCs were treated 45 min with indicated blocking antibodies or isotype controls after which the cells were incubated with PF-647–labeled SEA. Antigen binding/uptake was analyzed by flow cytometry and plotted as relative differences. A representative flow cytometry plot of SEA uptake is depicted on the left. (G) PGE2 levels were assessed as in panel C, following pre-incubation with blocking antibodies as described in panel F. (H) Syk and (I, J) ERK phosphorylation were determined as described in panel D following pre-incubation with blocking antibodies as described in panel F or panel J with Syk inhibitor R406. Representative flow cytometry plots of Syk (panel H) and ERK (panel I, J) phosphorylation is shown on the left. (K) PGE2 levels were assessed as in panel C. (L, M) moDCs were pre-incubated with indicated blocking antibodies, followed by 48 h stimulation with LPS plus SEAΔα-1/ω-1, after which OX40L expression was determined by flow cytometry. Data are based on geometric mean florescence. (M) Cells described in panel L were used for T-cell polarization assay as described in Fig 2A. Data represent mean ± SEM of 2 (panel D, H, I) or at least 3 independent experiments (panel A, C–G, J–M) and are shown relative to control conditions, which are set to 1 (panel A, C–E, G–M) or 100% (F). “*” and “#”: P < 0.05; “**” and “##”: P < 0.01; “***” and “###”: P < 0.001 for significant differences with the control (*) or between-test condition (#) based on paired analysis (paired Student t test). Underlying data can be found in S1 Data. COX, cyclooxygenase; cPLA2, cytosolic phospholipase A2; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; ERK, extracellular signal-regulated kinase; ind., Indometacin; LC-MS/MS, liquid chromatography tandem mass spectrometry; LPS, lipopolysaccharide; moDC, monocyte-derived DC; MR, mannose receptor; OX40L, OX40 ligand; PGE2, prostaglandin E2; Pyr., Pyrrophenone; SC, SC236; SEA, soluble egg antigen; Syk, spleen tyrosine kinase; Th2, T helper 2.
Fig 5.
SEA-induced ROS production by moDCs results in PGE2 isomer synthesis that contributes to Th2 polarization.
(A) T-cell polarization assay as described in Fig 2A in the presence of COX inhibitors S and I. Bars represent mean ± SEM of at least 3 independent experiments. (B) LC-MS/MS trace showing the transition m/z 351 → 271; the detected IsoPs are indicated by numbers. (C) Tandem MS spectrum of isomer 2, showing the fragment ion m/z 189, characteristic for 15-series IsoPs. (D) Showing the MS/MS spectrum of isomer 3, possibly identifying this isomer as a 5-series IsoP, based on the fragment ions m/z 115, 217, and 191. (E) ROS generation was determined by flow cytometry (H2-DCFDA) of moDCs pulsed for 6 h with indicated reagents. On the left, representative histograms for ROS induction are shown. On the right, the geometric mean fluorescence of these signals is enumerated and shown as fold change relative to unstimulated moDCs (dashed line set to 1). (F) ROS production was quantified as in panel E following pretreatment with general ROS scavenger NAC or R406 for 1 h. (G) moDCs were stimulated with indicated PGs with or without anti-PGE2 after which a T-cell polarization assay was performed as described in Fig 2A. (H) moDCs were stimulated with indicated reagents in the presence following 1 h pre-incubation with COX inhibitors (S and I) and NAC after which a T-cell polarization assay was performed as described in Fig 2A. Bars represent mean ± SEM of at least 3 independent experiments. “*” and “#”: P < 0.05: “**” and “#”: P < 0.01 for significant differences with the LPS control (*) or between-test conditions (#) based on paired analysis (paired Student t test). Underlying data can be found in S1 Data. COX, cyclooxygenase; H2-DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; I, indomethacin; IsoP, isoprostane; LC-MS/MS, liquid chromatography tandem mass spectrometry; LPS, lipopolysaccharide; moDC, monocyte-derived DC; NAC, N-acetyl-L-cysteine; PGE2, prostaglandin E2; ROS, reactive oxygen species; S, SC236; SEA, soluble egg antigen; Th2, T helper 2.
Fig 6.
Th2 polarization induced by SEA is mediated via Dectin-2 and Syk signaling in vivo.
(A–D) WT or CD11cΔSyk mice were injected with SEA (panel A, B), SEAΔα-1/ω-1 (panel C), or ω-1 (panel D) in the hind footpad, and draining pLNs were analyzed 7 d later. (A, C, D) pLN cells were restimulated with PMA/Ionomycin in the presence of brefeldin A, and CD4+ T cells were stained for indicated intracellular cytokines and percentage cytokine-positive CD4+ T cells enumerated. Based on these data, the ratio between the percentage IL-4+ over IFN-γ+ CD4+ T cells was determined as a measure for overall skewing towards Th2. (B) pLN cells were restimulated with SEA for 72 h, and cytokine levels in culture supernatants were determined. (A–D) Bar graphs represent mean ± SEM of 5 to 6 mice per group and are representative of 2 (panel A–C) or 1 (panel D) experiment. (E) BMDCs cultured from BM from WT, Dectin-1−/−, or Dectin-2−/− mice were pulsed overnight with SEAΔα-1/ω-1 and injected into hind footpads after which CD4+ T-cell responses were analyzed as in panel A. Representative flow cytometry plots of intracellular staining of CD4+ T cells are depicted, of which the data are enumerated in bar graphs representing mean ± SEM of 2 independent experiments with 3 to 6 mice per group. (F) SEA binding and uptake was determined as in Fig 4F. (G) ROS production by indicated BMDCs was determined as described in Fig 5E, 1 h after stimulation with SEAΔα-1/ω-1. Based on MFI, bar graphs represent fold change relative to control condition, which is set to 1. (H) BMDCs were stimulated as indicated for 18 h after which expression of OX40L was analyzed by flow cytometry. Representative plots are depicted, of which the data are enumerated in bar graphs and shown as fold change relative to control condition, which is set to 1, based on percentage positive cells. (F–H) Bar graphs represent mean of duplicates or triplicates ± SEM of 2 independent experiments. “*”: P < 0.05; “**” and “##”: P < 0.01; “***” and “###”: P < 0.001 for significant differences with the control conditions (*) or between-test condition (#) based on unpaired analysis (unpaired Student t test). Underlying data can be found in S1 Data. ω-1, omega-1; BMDC, bone marrow–derived DC; CD4, cluster of differentiation 4; H2-DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; IFN-γ, interferon γ; IL-4, interleukin 4; MFI, mean fluorescence intensity; nd/pLN, nondraining/popliteal lymph node; OX40L, OX40 ligand; PBS, phosphate buffered saline; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species; SEA, soluble egg antigen; Syk, spleen tyrosine kinase; Th2, T helper 2; WT, wild-type.
Fig 7.
Dectin-2 signaling is required for induction of a Th2 response during S. mansoni infection.
WT and Dectin-2−/− mice were infected with S. mansoni. After 8 wk of infection, cells from spleens (A) or mLNs (B) were restimulated with SEA or anti-CD3/CD28 for 72 h, and cytokine levels were analyzed in supernatants by Luminex or ELISA. Bars represent mean ± SEM of combined data of at least 2 or 3 independent experiments with 5 to 10 mice per group. (C) Granuloma sizes around eggs trapped in the liver of 8-wk–infected mice were assessed in Masson blue–stained liver sections. Data are based on 10 mice per group. Number of worms (D) and liver and intestinal eggs (E) in mice infected with S. mansoni for 8 wk. (F) IL-1β protein levels in livers of mice infected with S. mansoni for 8 wk. **P < 0.01 and ***P < 0.001 for significant differences relative to the control mice based on unpaired analysis (unpaired Student t test). Underlying data can be found in S1 Data. CD3, cluster of differentiation 3; IL-1β, interleukin 1β; mLN, mesenteric lymph node; SEA, soluble egg antigen; Th2, T helper 2; WT, wild-type.
Fig 8.
Proposed model of S. mansoni–driven Th2 polarization.
S. mansoni egg antigens, but not ω-1, interact with Dectin-1 and Dectin-2 expressed by DCs to promote 2 intracellular pathways in moDCs in an Syk-dependent manner: ERK-cPLA2-COX and ROS activity that culminate in PGE2 and PGE2 isomer synthesis, respectively. Both PGE2 and its isomers can then bind to EP2 and EP4 in an autocrine loop to trigger OX40L expression, which endows the DCs with the capacity to prime a Th2 response. ω-1, omega-1; COX, cyclooxygenase; cPLA2, cytosolic phospholipase A2; DC, dendritic cell; EP2, prostaglandin E2 receptor 2; ERK, extracellular signal-regulated kinase; moDC, monocyte-derived DC; OX40L, OX40 ligand; PGE2, prostaglandin E2; ROS, reactive oxygen species; Syk, spleen tyrosine kinase; Th2, T helper 2.