ST2⁺ Tregs suppress CD4⁺ T cell proliferation more effectively than ST2⁻ Tregs <i>in vitro</i>.

<p><i>(A)</i> Proliferation profiles of CellTrace-labelled WT CD25^- CD62L^hi CD4⁺ responder T cells (Tresp) co-cultured with WT ST2⁺ (black) and ST2⁻ (grey) CD25⁺ Tregs during an <i>in vitro</i> suppression assay at day 4 of culture. T cells were stimulated by APCs and anti-CD3 antibody with <i>(right column)</i> or without <i>(left column)</i> the addition of recombinant IL-33. Treg:Tresp ratios are indicated <i>(left)</i>. Percentage of divided cells and the division index (number in brackets) are shown in each histogram in the respective color. <i>(B)</i> Proliferation profile of Tresp cultured under the same conditions as in 3A but without Tregs, either with (grey) or without (black) the addition of anti-CD3 antibody. <i>(C)</i> MFI of the ST2 staining on all Tregs recovered from the cultures described in 3A. Data are representative of 2–3 independent experiments.</p

ST2⁺ Tregs preferentially home outside of secondary lymphoid organs and exhibit a highly activated phenotype.

<p>Flow cytometric analysis of the phenotype and frequency of WT ST2⁺ and ST2⁻ Foxp3⁺ Tregs in spleen, pLN, blood, lung, lamina propria of the small intestine (siLP) and colon (coLP): <i>(A)</i> Frequency of ST2⁺ Tregs <i>(left)</i> and MFI of the ST2 staining on the ST2⁺ Treg fraction <i>(right)</i>. <i>(B)</i> MFI of chemokine receptor and α4β7 staining on ST2⁺ and ST2⁻ Tregs. <i>(C)</i> KLRG1 and CD103 expression in ST2⁺ <i>(top)</i> and ST2⁻ <i>(bottom)</i> Tregs from spleen; quantified frequencies from indicated organs <i>(right)</i>. <i>(D)</i> Frequency of CD44^hi, CD62L^lo and CTLA-4⁺ T cells within ST2⁺ and ST2⁻ Treg populations. <i>(E)</i> MFI of the Foxp3 staining <i>(left)</i> and geometric mean index of GATA-3 <i>(right)</i> in ST2⁺ and ST2⁻ Tregs. <i>(F)</i> Quantification of mRNA expression of the indicated genes from FACS-sorted ST2⁺ and ST2⁻ CD25⁺ Tregs from spleen and pLN <i>ex vivo</i>. mRNA expression normalized to <i>Hprt</i> endogenous control. <i>(G)</i> Frequency of ST2⁺ and ST2⁻ Tregs with IL-10 production capability as detected by GFP expression from <i>B6</i>.<i>Foxp3</i>^<i>hCD2</i> <i>xIl10</i>^<i>gfp</i> reporter mice. Fig <i>2A</i>: Data are representative of at least 2 independent experiments. Bar graphs show the mean ± SD of at least 5 biological replicates. Fig <i>2B</i>: pooled data from 2 independent experiments with 3–5 biological replicates each. Bar graphs show the mean ± SD. Fig <i>2C–2E</i> and <i>2G</i>: Data are representative of at least 2 independent experiments. Scatter plots depict one mouse as individual dot with mean ± SD. Fig <i>2F</i>: pooled data from 2 independent experiments. Significance was tested using unpaired Student’s t test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; non-significant (ns) p > 0.05.</p

ST2⁺ Tregs express Th2 cytokines and suppress CD4⁺ T cell proliferation via IL-10 and TGFβ.

<p><i>(A-D)</i> ST2⁺ and ST2⁻ Tregs from spleen and lymph nodes of WT mice activated <i>in vitro</i> by plate-bound anti-CD3/anti-CD28 antibodies in the presence of IL-2 with or without recombinant IL-33 for 60–70 hours: <i>(A)</i> Fold change in the number of viable Tregs upon IL-33 treatment. <i>(B) Tgfb1</i> mRNA expression normalized to <i>Hprt</i> endogenous control. <i>(C)</i> Cytokine concentration in the supernatants as determined by cytometric bead array. <i>(D)</i> Geometric mean index of GATA-3 in stable ST2⁺ and ST2⁻ Tregs at the end of culture. <i>(E) In vitro</i> suppression assay with ST2⁺ and ST2⁻ Tregs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161507#pone.0161507.g003" target="_blank">Fig 3</a> (Treg:Tresp ratio 1:5) with addition of blocking anti-IL-10R antibody or TGFβRI inhibitor. The relative division index indicates the fold increase in division of Tresp upon treatment. Division index of untreated Tresp was set to 1 in each group. <i>(F)</i> Quantification of mRNA expression of the indicated genes from sorted ST2⁺ and ST2⁻ CD25⁺ Tregs <i>ex vivo</i>. mRNA expression normalized to <i>Hprt</i> endogenous control. Fig <i>4A</i>–<i>4C</i>, <i>4E</i> and <i>4F</i>, data pooled from 2–3 independent experiments each performed with 2 replicates per condition. Fig <i>4D</i> is representative of 2 independent experiments with at least 2 replicates per condition each. Bar graphs show the mean ± SD. Significance was tested using unpaired Student’s t test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; non-significant (ns) p > 0.05.</p

ST2⁺ Tregs arise independently of IL-33 signals.

<p>Phenotype of Tregs in naive WT, <i>Il1rl1</i>^<i>-/-</i> and <i>Il33</i>^<i>-/-</i> mice: <i>(A)</i> ST2 and Foxp3 expression by splenic CD4⁺ T cells of one representative naive WT mouse; quadrant numbers indicate the average frequency ± SD in 4 mice. <i>(B)</i> Frequencies and total numbers of FoxP3⁺ Tregs in spleen (Spl), peripheral lymph nodes (pLN) and lung. <i>(C)</i> Frequency of ST2 expression in Tregs of spleen and lung. <i>(D)</i> Total number of ST2⁺ Tregs in spleen, pLN and lung. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161507#pone.0161507.g002" target="_blank">Fig <i>2A</i>, <i>2C</i> and <i>2D</i></a>: Data are representative of at least 2 independent experiments. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161507#pone.0161507.g002" target="_blank">Fig <i>2B</i></a>: Pooled data from 2 independent experiments, each with 4 mice per genotype. Bar graphs show the mean ± SD of at least 4 individual mice. Significance was tested using unpaired Student’s t test. Asterisks indicate significance; all others non-significant. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.</p

Galectin-9 inhibits PMA- and ionomycin-dependent degranulation of HMC-1 cells.

<p>(a, b) HMC-1 cells were treated with 0, 0.25, 0.5 or 1 µM (a) and 0 or 0.5 µM (b) recombinant human galectin-9 (rhGal-9) for 30 min. The cells were then stimulated with 0.1 µg/ml PMA +1 µg/ml ionomycin for 30 min. The level of degranulation was assessed from the activity of β-hexosaminidase in the culture supernatant and plotted as the percent release. (c) The number of viable cells in (b) was determined by trypan blue staining. (d) The proportion of propidium iodide-negative and annexin V-positive apoptotic cells in (b) was assessed by flow cytometry. (e) The relative level of degranulation per live HMC-1 cells was determined as (b)/(c). Data show the mean ± SD of triplicate samples and are a representative result of three (a) or two (b–e) independent experiments. *p<0.05, **p<0.01 versus PMA+ionomycin alone.</p

Histological analysis for infiltration of leukocytes and hyperplasia of goblet cells.

<p>Histological analysis of nasal mucosa (maxillary turbinate) from wild-type, IL-25^−/− and IL-33^−/− mice 48 hours after the last inhalation of HDM or PBS. (A) H&E staining. After inhalation of HDM, but not PBS, eosinophils were observed in the nasal mucosa of wild-type and IL-25^−/− mice but hardly detectable in mucosa from IL-33^−/− mice. Arrowheads indicate eosinophils. (B) PAS staining. After inhalation of HDM, but not PBS, goblet cell hyperplasia was similarly observed in the nasal mucosa of both wild-type and IL-25^−/− mice, but it was markedly reduced in IL-33^−/− mice compared with wild-type mice. Arrowheads indicate PAS-positive goblet cells. Data show a representative result from 3–7 mice in each group (bar  = 20 µm).</p

Gal-9 induces cytokine and chemokine production by HMC-1 cells.

<p>ELISA was performed to determine the levels of IL-6, IL-8 and MCP-1 in the culture supernatants of HMC-1 cells (<b>a</b>), HMC-1 cells pre-treated with 20 mM lactose or sucrose (<b>b</b>), HMC-1 cells pre-treated with recombinant human TIM-3/Fc (rhTIM-3/Fc) or control human IgG (human IgG) (<b>c</b>) and HMC-1 cells pre-treated with ERK inhibitor (PD98059) or its control (SB202474) (<b>d</b>) after 18 hours’ stimulation with 0, 0.25, 0.5 or 1 µM recombinant human Galectin-9 (rhGal-9). Data show the mean ± SD of triplicate samples and are a representative result of three independent experiments. *p<0.05 and/or **p<0.01 versus 0 µM rhGal-9 (<b>a–d</b>), and †p<0.05 and/or ††p<0.01 versus sucrose (<b>b</b>), control human IgG (<b>c</b>) or ERK inhibitor control (<b>d</b>).</p

IL-27 plays an important role in expansion, differentiation, and mobilization of LSK cells to control malaria infection.

<p>(A-D) Reduced induction of LSK cell population in <i>WSX-1</i>-deficient mice after malaria infection, accompanied by increased parasitemia and comparable production of IFN-γ. WT or <i>WSX-1</i>-deficient mice were infected with the blood stage of <i>P</i>. <i>berghei</i> XAT. Seven days later, parasitemia (A) and serum IFN-γ level (B) were determined, and LSK populations in the BM and spleen were analyzed by flow cytometry, and representative dot plots of c-Kit and Sca-1 in the Lin⁻ population are shown (C). Cell number of the LSK cell population was counted (D). (E) Augmented potential of LSK cells to differentiate into myeloid cells by malaria infection. LSK cells (1 × 10³) in the BM and spleen of the malaria-infected or non-infected WT mice were purified and differentiated into myeloid cells <i>in vitro</i> by IL-3 and SCF, and cell number of differentiated cells was counted. (F-G) Reduced cell number of neutrophils in <i>WSX-1</i>-deficient mice after malaria infection. The BM and spleen cells were analyzed for expression of Gr-1 and CD11b at 7 days after the infection (F), and cell number of neutrophils (Gr-1⁺CD11b⁺) was counted (G). (H-I) Decreased parasitemia in the <i>WSX-1</i>-deficient mice transferred with LSK cells purified from BM cells of the malaria-infected WT mice. LSK cells purified from BM cells of the infected WT CD45.1 mice were transferred into non-lethally irradiated <i>WSX-1</i>-deficient CD45.2 mice 7 days before infection. Neutrophil population in the BM and spleen was analyzed by flow cytometry, and representative dot plots of CD11b and Gr-1 in the CD45.1⁺ population are shown (H). Parasitemia was measured 4 and 7 days after the infection (I). Data are shown as mean ± SEM (n = 3–9) and are representative of at least two independent experiments. *<i>P</i> < 0.05, ***<i>P</i> < 0.005.</p

IL-27 and SCF expand CD34⁻CD150⁺ LSK cells into multipotent myeloid progenitor cells.

<p>(A-B) Enhanced expansion of CD34⁻ LSK cells by IL-27 and SCF. LSK cells from WT mice were divided into two populations according to the expression of CD34, CD34⁻ LSK, and CD34⁺ LSK cells, and each population (1 × 10²) was stimulated with IL-27 and SCF. One to 4 weeks later, these stimulated cells were analyzed by flow cytometry; representative dot plots of c-Kit and Sca-1 in the Lin⁻ population at 2 weeks are shown (A). Cell numbers of these stimulated cells were counted with time course (B). (C-E) Augmented expansion of CD34⁻CD150⁺ LSK cells by IL-27 and SCF. LSK cells were further divided into eight populations (F1-F8) according to the expression of CD34, CD150, and CD41 (C), and each population (50 cells) purified by sorting was stimulated with IL-27 and SCF. One to 4 weeks later, these stimulated cells were analyzed by flow cytometry. Representative dot plots of c-Kit and Sca-1 in the Lin⁻ population at 4 weeks are shown, and the cell number of the LSK cell population in these stimulated cells was counted with time course (D). LSK populations (1 × 10³) purified from primary or IL-27/SCF-expanded F1, F4, and F5 LSK cells were differentiated into myeloid cells by IL-3 and SCF, and cell number was counted (E). Data are shown as mean ± SEM (n = 3–4) and are representative of two to three independent experiments. *<i>P</i> < 0.05, ***<i>P</i> < 0.005.</p

Malaria infection enhances IL-27 expression through IFN-γ production to promote the expansion, differentiation, and mobilization of LSK cells.

<p>(A-B) Indispensable role for IFN-γ in the expansion of LSK cells after malaria infection. WT and <i>IFN-γ</i>-deficient mice were infected with the blood stage of <i>P</i>. <i>berghei</i> XAT. Seven days later, parasitemia was determined (A) and LSK populations were analyzed by flow cytometry (B). (C-D) IFN-γ-dependent induction of IL-27 p28 subunit expression by malaria infection. RNA was prepared 7 days after the infection and analyzed for expression of <i>p28</i> by real-time RT-PCR (C), and serum p28 levels were determined by ELISA (D). (E-G) Decreased parasitemia and augmented expansion of LSK cell population in <i>IFN-γ</i>-deficient mice by IL-27. <i>IFN-γ</i>-deficient mice were hydrodynamically injected with IL-27-expression vector or control vector at days 0 and 4 after infection; at day 7, parasitemia was measured (E), LSK population was analyzed by flow cytometry (F), and cell numbers of LSK cells and neutrophils were counted (G). Data are shown as mean ± SEM (n = 3–5) and are representative of at least two independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.005.</p