Contributions of IL-25 and IL-33 to eosinophil recruitment in lungs.

<p>The enzymatic activity and mRNA expression levels of EPO and MPO in BAL fluids and lungs, respectively, <u>from</u> wild-type (WT), IL-25^-/- mice and IL-33^-/- mice shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134226#pone.0134226.g003" target="_blank">Fig 3</a>. Data are pooled from three independent experiments, each of which gave similar results. Data show the mean + SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001 vs. the corresponding values for PBS-treated mice. †<i>P</i> < 0.05 and NS (not significant) vs. the indicated group (WT vs. IL-25^-/-, or WT vs. IL-33^-/-).</p

Expression of IL-25 and IL-33 mRNA in the skin after tape-stripping and in the lungs after antigen challenge.

<p>Dorsal skin was collected from wild-type (WT) mice, IL-25^-/- mice and IL-33^-/- mice 6 hours after tape-stripping followed by with or without a patch containing PBS or OVA, while lungs were harvested from WT mice sensitized EC with OVA 24 hours after the last intranasal challenge with OVA. mRNA was isolated from the skin and lungs, and the expression levels of IL-25 and IL-33 mRNA were determined by quantitative PCR. (A) The IL-25 and IL-33 mRNA expression levels in the skin (n = 3–5). *<i>P</i> < 0.05 vs. the corresponding values for a group without stripping (a group without stripping vs. a group with tape-stripping). ‡ P<0.05 vs. the indicated group (a group without stripping and patch vs. a group without stripping and with patch containing OVA). †<i>P</i> < 0.05 and ††<i>P</i> < 0.01 vs. the indicated group (WT vs. IL-25^-/-, or WT vs. IL-33^-/-). NS means “not significant” between the indicated groups. (B) The IL-25 and IL-33 mRNA expression levels in the lungs (PBS, n = 8; OVA, n = 24). Data show the mean + SEM. *<i>P</i> < 0.05 and **<i>P</i> < 0.01 (PBS group vs. OVA group).</p

IL-25 and IL-33 are important for expression of Th2-type/associated cytokines and chemokines, but not Th17-type cytokines, after the last intranasal OVA challenge, in lungs from mice sensitized epicutaneously (EC) with OVA.

<p>Total mRNA was isolated from the lungs of wild-type (WT), IL-25^-/- mice and IL-33^-/- mice as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134226#pone.0134226.g002" target="_blank">Fig 2E</a>. The expression of mRNA for cytokines and chemokines was determined by quantitative PCR. (A) WT (PBS, n = 8; OVA, n = 24) and IL-25^-/- mice (PBS, n = 8; OVA, n = 28). (B) WT (PBS, n = 10; OVA, n = 20) and IL-33^-/- mice (PBS, n = 11; OVA, n = 20). Data are pooled from three independent experiments, each of which gave similar results. Data show the mean + SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001 vs. the corresponding values for PBS-treated mice. †<i>P</i> < 0.05, ††<i>P</i> < 0.01 and NS (not significant) vs. the indicated group (WT vs. IL-25^-/-, or WT vs. IL-33^-/-).</p

IL-25 and IL-33 are not required for airway hyperresponsiveness after the last intranasal OVA challenge in mice sensitized epicutaneously (EC) with OVA.

<p>Twenty-four hours after the last inhalation of OVA or PBS, mice sensitized EC with OVA were monitored for pulmonary resistance in response to methacholine. (A) Wild-type (WT) (PBS, n = 7; OVA, n = 7) and IL-25^-/- mice (PBS, n = 7; OVA, n = 7). (B) WT (PBS, n = 5; OVA, n = 8) and IL-33^-/- mice (PBS, n = 5; OVA, n = 12). (C, D) The levels of OVA-specific IgE in sera from mice after intranasal challenge were determined by ELISA. Data show the mean + SEM. *<i>P</i> < 0.05 and **<i>P</i> < 0.01 vs. the corresponding values for PBS-treated mice (A, B). NS (not significant) vs. the indicated group (WT vs. IL-25^-/-, or WT vs. IL-33^-/-)(C, D).</p

IL-25 and IL-33 are crucial for development of airway eosinophilia, but not neutrophilia, after the last intranasal OVA challenge in mice sensitized epicutaneously (EC) with OVA.

<p>Mice were sensitized EC with OVA three times and then treated intranasally with OVA or PBS for 3 consecutive days. Twenty-four hours after the last inhalation of OVA or PBS, BALFs and lungs were collected. (A, B) Lung sections from wild-type (WT) and IL-25^-/- mice were stained with hematoxylin-eosin (H-E) (A) and PAS (B). (C, D) Lung sections from WT and IL-33^-/- mice were stained with H-E (C) and PAS (D). Scale bars = 100 mm. The data show representative results from 10–14 mice in each experimental group, as indicated. (E) The numbers of BAL cells from WT (PBS, n = 8; OVA, n = 24) and IL-25^-/- mice (PBS, n = 8; OVA, n = 28), and from WT (PBS, n = 10; OVA, n = 20) and IL-33^-/- mice (PBS, n = 11; OVA, n = 20). Data are pooled from three independent experiments, each of which gave similar results. Data show the mean + SEM (E, F). *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001 vs. the corresponding values for PBS-treated mice. †<i>P</i> < 0.05 and NS (not significant) vs. the indicated group (WT vs. IL-25^-/-, or WT vs. IL-33^-/-).</p

IL-25 and IL-33 are important for expression of Th2-type/associated cytokines and chemokines, but not Th17-type cytokines, after the last intranasal OVA challenge, in lungs from mice sensitized epicutaneously (EC) with OVA.

<p>Total mRNA was isolated from the lungs of wild-type (WT), IL-25^-/- mice and IL-33^-/- mice as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134226#pone.0134226.g002" target="_blank">Fig 2E</a>. The expression of mRNA for cytokines and chemokines was determined by quantitative PCR. (A) WT (PBS, n = 8; OVA, n = 24) and IL-25^-/- mice (PBS, n = 8; OVA, n = 28). (B) WT (PBS, n = 10; OVA, n = 20) and IL-33^-/- mice (PBS, n = 11; OVA, n = 20). Data are pooled from three independent experiments, each of which gave similar results. Data show the mean + SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001 vs. the corresponding values for PBS-treated mice. †<i>P</i> < 0.05, ††<i>P</i> < 0.01 and NS (not significant) vs. the indicated group (WT vs. IL-25^-/-, or WT vs. IL-33^-/-).</p

Normal Th2- and Th17-type cytokine production by DLN and spleen cells of IL-25^-/- and IL-33^-/- mice sensitized epicutaneously (EC) with OVA.

<p>DLN and spleens were collected from mice 24 hours after the last EC sensitization with OVA. DLN and spleen cells were cultured with OVA in PBS or PBS alone. ELISA was performed to determine the levels of IL-4, IL-5, IL-13 and IL-17 in the culture supernatants of the DLN cells from wild-type (WT) mice and IL-25^-/- mice (A) and from WT mice and IL-33^-/- mice (B), and the spleen cells from WT mice and IL-25^-/- mice (C) and from WT mice and IL-33^-/- mice (D). Data show the mean + SEM (n = 8–23). *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001 vs. the corresponding values for the culture with PBS alone. NS means “not significant” between the indicated group (WT vs. IL-25^-/-, or WT vs. IL-33^-/-).</p

Effects of Y-27632 on pCTB viability.

<p>pCTBs were cultured in the absence (A) and presence (B) of 10 μM Y-27632 for 96 h. Cytoplasmic F-actin was stained with Alexa Fluor 488-conjugated phalloidin, and nuclei were stained with DAPI. Scale bar = 50 μm. (C) The cytoplasmic area was measured using Hybrid Cell Count software (Keyence). Data are expressed as μm². Each point shows the mean of 10 fields from one condition (from six separate placentas, n = 6). Statistical differences were determined with Wilcoxon’s matched-pairs signed rank test. (D) Mitochondrial activity was measured by WST-8 assay, and data are expressed as O.D. 450 nm. The data are from 3 separate experiments using pCTBs from 3 different placentas. All data are shown as the mean ± SD. *, <i>P</i> < 0.05.</p

Effects of a Rac 1 inhibitor, NSC-23766, and a PKA inhibitor, H-89, on fusion and expression of fusogenic genes and hCG-β in pCTBs.

<p><b>(A-G)</b> pCTBs were cultured in the presence and absence of 10 μM Y-27632 and/or 50 μM NSC-23766 for 96 h. (A, B) Cytoplasmic F-actin was stained with Alexa Fluor 488-conjugated phalloidin, and nuclei were stained with DAPI. (A) pCTBs cultured with Y-27632 (10 μM), (B) pCTBs cultured with Y-27632 (10 μM) and NSC-23766 (50 μM). Scale bar = 50 μm. (C) The cytoplasmic area was measured using Hybrid Cell Count software (Keyence). Each bar shows the mean of 10 fields in each well, and the data are representative of the experiment using 3 pCTBs from 3 different donors. (D, E) Plasma membrane protein desmoplakin I was stained with Alexa Fluor 555, and nuclei were stained with DAPI. (D) pCTBs cultured with Y-27632 (10 μM), (E) pCTBs cultured with Y-27632 (10 μM) and NSC-23766 (50 μM). Scale bar = 50 μm. (F) The fusion index was calculated. Each column shows the mean of 10 fields in one well, and the data are representative of the experiment using 3 pCTBs from 3 different donors. (G) Total RNA was extracted, and the mRNA expression levels for GCM1, syncytin-1, syncytin-2 and CGB are shown. Each column shows the mean of the results for 3 pCTBs from 3 different donors, and the fold change was calculated against each control sample. (H-N) pCTBs were cultured in the presence and absence of 10 μM Y-27632 and/or 10 μM H-89 for 96 h. (H, I) Cytoplasmic F-actin was stained with Alexa Fluor 488-conjugated phalloidin, and nuclei were stained with DAPI. (H) pCTBs cultured with Y-27632 (10 μM), (I) pCTBs cultured with Y-27632 (10 μM) and H-89 (10 μM). Scale bar = 50 μm. (J) The cytoplasmic area was measured using Hybrid Cell Count software (Keyence). Each column shows the mean of 10 fields in one well, and the data are representative of the experiment using 3 pCTBs from 3 different donors. (K, L) Plasma membrane protein desmoplakin I was stained with Alexa Fluor 555, and nuclei were stained with DAPI. (K) pCTBs cultured with Y-27632 (10 μM), (L) pCTBs cultured with Y-27632 (10 μM) and H-89 (10 μM). Scale bar = 50 μm. (M) The fusion index was calculated. Each column shows the mean of 10 fields in one well, and the data are representative of the experiment using 3 pCTBs from 3 different donors. (N) Total RNA was extracted, and mRNA expression levels for GCM1, syncytin-1, syncytin-2 and CGB are shown. Each column shows the mean of the results for 5 pCTBs from 5 different donors, and the fold change was calculated against each control sample. Error bar shows SD. *, <i>P</i> < 0.05. **, <i>P</i> < 0.005. ***, <i>P</i> < 0.0005. ****, <i>P</i> < 0.0001. ns, not significant.</p

The effect of Y-27632 on pCTB proliferation.

<p>pCTBs were cultured with and without Y-27632 (10 μM) for 24, 48, 72 and 96 h. HeLa cells were also cultured and tested as a positive control. (A) Ki-67 protein in pCTBs and HeLa cells was detected by immunofluorescence staining. The Ki-67-positive cell number was divided by the total nuclei number and shown as Ki-67+ cells. Each point was the average of pCTBs from 3 different donors, and one condition was the mean of 10 randomly selected fields in one well. The HeLa cell data were the mean of 10 randomly selected fields. (B) [³H] thymidine was added to the culture medium of pCTBs with and without Y-27632 and HeLa cells for 6 hours. Each dot in the figure represents the mean of pCTBs from 4 different donors, and the data of each donor were the mean of triplicate determinations. The HeLa cell data are the mean of triplicate determinations. Data are expressed as the mean ± SD. Statistical analyses were performed only between pCTBs with and without Y-27632 by using Student’s t-test with correction for multiple comparisons using Holm-Sidak method. ns, not significant.</p