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

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

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

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 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

IL-33 induces TRAF6-dependent cytokine production by mast cells.

<p>BMCMCs obtained from B6J-WT mice (A) and B6J-WT and -TLR4^−/− mice (B; left panels) and FLCMCs obtained from B6J-WT and -TRAF6^−/− mice (B; right panels) were cultured in the presence of various concentration of rmIL-33 (A) or in the presence and absence of 100 ng/ml rmIL-33 for 6 h (for TNF measurement) and 24 h (for IL-6 and IL-13 measurement). The levels of IL-6, IL-13 and/or TNF in the culture supernatants were determined by ELISA. Data show the mean + SD (n = 3). *p<0.05 vs. WT.</p

IL-33 enhances LPS-mediated cytokine production by macrophages.

<p>TGC-induced peritoneal macrophages derived from B6J-WT mice (A–D) and B6N-WT and -IL-33^−/− mice (E) were cultured in the presence and absence of 100 ng/ml LPS, with and without 100 ng/ml IL-33, for 9, 24 and/or 48 h. (A, E) The levels of IL-6 in the culture supernatants by ELISA. (B) The percentage of PI-positive cells by flow cytometry. (C) LDH levels in the culture supernatants. (D) The number of IL-33-secreting cells by ELISPOT. Data show the mean +/± SEM (n = 3 [A] or 4 [B–E]). *p<0.05 vs. the indicated group (A) or Medium (B–E), and ^†p<0.05 vs. 24 h (C, D) or WT (E). P+I = PMA+ionomycin.</p

Effects of anti-ST2 mAbs on cytokine production by IL-33-stimulated BMCMCs.

<p>B6J-WT BMCMCs were stimulated with 0–1,000 ng/ml (A) or 100 ng/ml (B) rmIL-33 in the presence of 40 µg/ml of several anti-ST2 mAbs or isotype control rat IgG for 24 h. The levels of IL-6 and IL-13 in the culture supernatants were determined by ELISA. Data show the mean + SEM (n = 3). *p<0.05 vs. rat IgG+IL-33. The expression of ST2 on the cell surface of BALB-WT and ST2^−/− BMCMCs was determined using several distinct anti-ST2 mAb clones. Representative data by flow cytometry are shown (C). Shaded area indicates isotype-matched control IgG staining, and bold line indicates anti-ST2 mAb staining.</p