IL-10 production by WKO CD1d^highCD5⁺ Breg.

<p>a: Representative flow-cytometry histograms showing IL-10-producing cells identified within CD1d^highCD5⁺ Breg cells of younger (<6 months old) and older (>6 months old) mice before and 5 hrs after stimulation with LPS, PMA, and ionomycin. b: Percentage of IL-10-positive cells within CD1d^highCD5⁺ Breg. White bars indicate the percentage (±SD) of IL-10-positive cells before stimulation (Before). Black bars indicate the percentage (±SD) IL-10 positive cells 5 hrs after stimulation with LPS, PMA and ionomycin (After). Each histogram represents data observed in 10 to 27 mice. c: IL-10 production after stimulation of CD1d^highCD5⁺ Breg cells with LPS, PMA and ionomycin. Bar graphs indicate mean (±SD) of 3–5 experiments in which CD1d^highCD5⁺ Breg cells were pooled from 6–10 mice were analyzed. *<i>p</i><0.05, **<i>p</i><0.002 (Student’s <i>t</i>-test).</p

Characteristics of CD1d^highCD5⁺ Breg cells in WKO mice.

<p>a: Representative results of the percentages of CD1d^highCD5⁺ Breg cells detected within total splenic CD19⁺ B cells of WT and WKO mice. b: Total number of splenic CD1d^highCD5⁺ Breg cells in younger (<6 months old) and older (>6 months old) mice. Average numbers (±SD) of CD1d^highCD5⁺ Breg cells from 23–49 mice are shown. *<i>p</i><0.002 (Student’s <i>t</i>-test).</p

CD1d^highCD5⁺ Breg suppression activity.

<p>a: Representative results showing the fluorescent intensity of CD69 expression on WT CD4⁺ T cells and CD1d^highCD5⁺ Breg cells 72 hrs after co-culture with anti-CD3/CD28. b: Fluorescence intensity of CD69 expression on the surface of CD4⁺ T cells and CD1d^highCD5⁺ Breg cells. Fluorescence intensity was expressed as delta mean fluorescence intensity (ΔMFI), which was calculated by subtracting the intensity of expression in unstimulated cells from that of cultured cell populations. Bar graphs indicate mean ± SD. *<i>p</i><0.05 (Student <i>t</i>-test). c: Concentrations of cytokine (IFN- and TNF-) measured in the medium of CD1d^highCD5⁺ Breg cells and CD4⁺ T cells co-cultured with LPS, PMA and ionomycin. Bar graphs indicate mean ±SD of 3–5 experiments in which CD1d^highCD5⁺ Breg cells pooled from 6–10 mice were analyzed. *<i>p</i><0.05, **<i>p</i><0.02 (Student’s <i>t</i>-test).</p

Activation status of CD1d^highCD5⁺ Breg cells before/after LPI stimulation.

<p>a: Representative flow-cytometry histograms showing the expression of CD69 on CD1d^highCD5⁺ Breg cells. Gray curves indicate isotype staining of the CD1d^highCD5⁺ Breg cells. Thin and thick lines indicate CD69 expression before and after LPI stimulation, respectively. b: CD69 expression intensity on CD1d^highCD5⁺ Breg cells before LPI stimulation expressed as geometric mean. c: Change in intensity of CD69 expression on CD1d^highCD5⁺ Breg cells after LPI stimulation. Data are expressed as ΔMFI, calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139729#pone.0139729.g003" target="_blank">Fig 3b</a>. Data are mean and SD of samples obtained from 6–10 mice in each assays and repeated 3–5 times. *<i>p</i><0.001 (Student’s <i>t</i>-test).</p

Deregulated JNK signaling enhances apoptosis during hyperthermia

c-Jun N-terminal kinases (JNKs) comprise a subfamily of mitogen-activated protein kinases (MAPKs). The JNK group is known to be activated by a variety of stimuli. However, the molecular mechanism underlying heat-induced JNK activation is largely unknown. The aim of this study was to clarify how JNK activity is stimulated by heat. The expression levels of various MAPK members in HeLa cells, with or without hyperthermia treatment, were evaluated via western blotting. The kinase activity of MAPK members was assessed through in vitro kinase assays. Cell death was assessed in the absence or presence of siRNAs targeting MAPK-related members. Hyperthermia decreased the levels of MAP3Ks, such as ASK1 and MLK3 which are JNK kinase kinase members, but not those of the downstream MAP2K/SEK1 and MAPK/JNK. Despite the reduced or transient phosphorylation of ASK1, MLK3, or SEK1, downstream JNK was phosphorylated in a temperature-dependent manner. In vitro kinase assays demonstrated that heat did not directly stimulate SEK1 or JNK. However, the expression levels of DUSP16, a JNK phosphatase, were decreased upon hyperthermia treatment. DUSP16 knockdown enhanced the heat-induced activation of ASK1–SEK1–JNK pathway and apoptosis. JNK was activated in a temperature-dependent manner despite reduced or transient phosphorylation of the upstream MAP3K and MAP2K. Hyperthermia-induced degradation of DUSP16 may induce activation of the ASK1–SEK1–JNK pathway and subsequent apoptosis.</p

Additional file 2: Figure S2. of Fli1-haploinsufficient dermal fibroblasts promote skin-localized transdifferentiation of Th2-like regulatory T cells

Signal intensity of IL-33. Signal intensity of IL-33 was analyzed and summarized. To quantify signal intensity of IL-33, color images were converted to grayscale, and then the brightness was measured in five different randomly selected fibroblasts and epidermal areas per specimen. WT Wild-type mice; Basal Under physiological condition; BLM Bleomycin-treated. (PDF 57 kb

Additional file 1: Figure S1. of Fli1-haploinsufficient dermal fibroblasts promote skin-localized transdifferentiation of Th2-like regulatory T cells

Scatterplots of Fig. 1b. Scatterplots of the proportions of IL-4-, IL-13-, IL17A-, and IFN-γ-producing splenic Tregs from bleomycin (BLM)-treated wild-type and Fli1 +/− mice (n = 6). (PDF 592 kb