Th2 polarization induced by SEA is mediated via Dectin-2 and Syk signaling in vivo.

<p>(A–D) WT or CD11c^ΔSyk 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.g004" target="_blank">Fig 4F</a>. (G) ROS production by indicated BMDCs was determined as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.g005" target="_blank">Fig 5E</a>, 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. “*”: <i>P</i> < 0.05; “**” and “##”: <i>P</i> < 0.01; “***” and “###”: <i>P</i> < 0.001 for significant differences with the control conditions (*) or between-test condition (#) based on unpaired analysis (unpaired Student <i>t</i> test). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.s009" target="_blank">S1 Data</a>. 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.