IL-17A expression during infection with <i>Klebsiella pneumoniae.</i>

<p>Wild-type or Smart-17A mice were infected with 500–1000 <i>K. pneumoniae</i>. (A) At the indicated time points, cells were harvested from lungs and numbers of cells were enumerated. (B) Cells were isolated from the lungs of mice 2 days after infection and assayed for hNGFR expression. (C) The total number of hNGFR⁺ and YFP⁺ cells on day 2 post-infection were calculated and the percentage attributable to each cell population is shown in a pie graph. The percentage of background staining seen in a wild-type mouse under identical conditions was subtracted before performing all calculations to control for nonspecific staining. This experiment was repeated 3 times with n >3 mice at each time point. For bar and pie graphs, data from independent experiments were compiled. For flow cytometry, representative plots are shown.</p

IL-17A expression during experimental autoimmune encephalomyelitis.

<p>Wild-type or Smart-17A mice were immunized with MOG-CFA to induce EAE. (A) At the indicated time point, cells were harvested from the draining axial, brachial and inguinal lymph nodes (LN) at day 6 or spinal cords and cerebellums (CNS) at day 12 and numbers of cells were enumerated. (B) Cells were assayed for hNGFR expression. (C) The total numbers of hNGFR⁺ cells were calculated and the percentage attributable to each cell population is shown in a pie graph. The percentage of background staining seen in a wild-type mouse under identical conditions was subtracted before performing all calculations to control for nonspecific staining. (D) Cells were isolated from the LN or CNS and immediately stained for surface markers or restimulated for 5 hr with PMA/ionomycin and then stained for surface markers and/or intracellular expression of IL-17A. The experiments in A–C were repeated 3 times with n>2 mice at each time point. The experiment in D was repeated 2 times with n>5 mice at each time point. For bar and pie graphs, data from independent experiments were compiled. For flow cytometry, representative plots are shown. For the CNS data, mice were excluded that did not display symptoms of paralysis.</p

Expression of IL-17A from innate-like T lymphocytes cells can be induced by IL-1β and/or IL-23.

<p>(A) Smart-17A mice were inoculated intranasally with PBS or with 500 ng IL-1β, IL-23 or both cytokines. Lungs were harvested 8 hr later and cells were analyzed for hNGFR expression. Gates were set using a wild-type control. The experiment was repeated 2 times and representative data are shown. (B) Wild-type mice were infected with <i>K. pneumoniae</i> and levels of IL-1β and the IL-23 subunit p19 mRNA in whole lung homogenate were measured using quantitative PCR. Expression of GAPDH was used as a reference to define relative expression. The experiment was done twice and a representative experiment is shown, n = 3 for all groups.</p

Generation of Smart-17A mice.

<p>(A) Targeting strategy for the <i>il17a</i> locus. For detailed description, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039750#s4" target="_blank">Materials and Methods</a>. (B) CD4⁺ T cells were isolated from wild-type or Smart-17A mice and polarized under Th17 conditions for 4 days. hNGFR was detected using a surface antibody and IL-17A was assayed using intracellular cytokine staining after restimulation. A representative flow cytometry plot is shown from >5 comparable experiments.</p

Migratory DCs require MyD88 signaling to respond normally to i.n. exposure to flagellin or CpG ODN.

<p>Expression of activation markers on migratory DCs in the lung-draining, mediastinal LNs of <i>Myd88</i>^<i>fl/fl</i> (<i>M</i>^<i>F</i>) and <i>Myd88</i>^<i>fl/fl</i> <i>CD11c-Cre</i> (<i>M</i>^<i>F</i> <i>CD11c-Cre</i>) mice one day after i.n. administration (d1) of OVA-AF647 or OVA-AF647 plus TLR ligand. (<b>A</b>) Migratory DCs were gated as CD11c⁺I-A^b(hi), then gated according to OVA-AF647 expression. (<b>B</b>) and (<b>C</b>) Comparison of different activation markers on migratory DCs between <i>M</i>^<i>F</i> and <i>M</i>^<i>F</i> <i>CD11c-Cre</i> mice treated i.n with OVA-AF647, OVA-AF647 plus flagellin (1 μg), or OVA-AF647 plus CpG (0.75 or 3 μg). (<b>B</b>) Representative histograms of different activation markers on migratory DCs that did take up OVA-AF647. (<b>C</b>) Level of expression (MFI) of activation markers on migratory DCs that did (OVA⁺) or did not (OVA^-) take up fluorescent OVA. (<b>D</b>) and (<b>E</b>) Comparison of different activation markers on migratory DCs between <i>M</i>^<i>F</i> and <i>M</i>^<i>F</i> <i>CD11c-Cre</i> treated i.n with OVA-AF647 or OVA-AF647 plus CpG ODN (0.75 μg). (<b>D</b>) Representative histograms of different activation markers on migratory DCs that did take up OVA-AF647. (<b>E</b>) Level of expression (MFI) of activation markers on migratory DCs that did (OVA⁺) or did not (OVA^-) take up fluorescent OVA. Data in (<b>B</b>) and (<b>C</b>) contain 3–4 mice per group and are representative of 3 independent experiments using OVA-AF647 and a fourth independent experiment using non-fluorescent OVA. Data in (<b>D</b>) and (<b>E</b>) contain 4–6 mice per group and are representative of two independent experiments. Negative control histograms (solid light gray) were from CD11c^-I-A^b- cells. Each circle represents an individual mouse. Error bars indicate mean +SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 using one-way anova with Bonferroni post-test.</p

Flagellin induces rapid production of inflammatory cytokines by AMs, LECs, Ly6C^hi monocytes, and CD103⁺ cDCs.

<p>Inflammatory gene mRNA inductions and IL-33 protein in whole lung tissue (<b>A-E</b>), enriched lung cell populations (<b>E</b>), or sorted cell populations from the lung (<b>F</b>) after one i.n. administration of OVA, OVA plus flagellin (1 μg), or OVA plus CpG (3 μg). mRNA inductions were normalized to <i>Hprt</i>. (<b>A</b>) Inflammatory gene mRNA inductions in whole lung tissue 2h after i.n. administration. (<b>B</b>) IL-33 protein in whole lung homogenates at the indicated time points. (<b>C-F</b>) Inflammatory gene mRNA inductions were measured in whole lung tissue (<b>C</b>, <b>D</b>), in fractionated lung epithelial or hematopoietic-derived cell populations (<b>E</b>), or in sorted cell populations (<b>F</b>) 2h after i.n. administration of <i>Myd88</i>^<i>fl/fl</i> mice (<i>M</i>^<i>F</i>), <i>Myd88</i>^<i>fl/fl</i> <i>CD11c-Cre</i> (<i>M</i>^<i>F</i> <i>CD11c-Cre</i>) mice, or <i>Myd88</i>^<i>fl/fl</i> <i>LysM-Cre</i> (<i>M</i>^<i>F</i> <i>LysM-Cre</i>) mice. Data in (<b>A</b>) contain 5 mice per group and are representative of two independent experiments, data in (<b>B</b>) contain 3 mice per group and are representative of two independent experiments at each time point, data in (<b>C</b>) contain 3–5 mice per group and are representative of three independent experiments, data in (<b>D</b>) contain 3 mice per group and are representative of two independent experiments, data in (<b>E</b>) contain 3–4 mice per group and are representative of two independent experiments, and data in (<b>F</b>) are pooled from two independent experiments with combined totals of 6–7 mice per group; each circle represents the data from sorted cells obtained from 3–4 mice. Error bars indicate mean +SD. In (<b>A</b>), statistical differences (P ≤ 0.05) are indicated with the following symbols: O vs. OFla (†), and OFla vs. OCpG (00B6).* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 using one-way anova with Bonferroni post-test (<b>A</b>-<b>D</b>) or Student’s <i>t</i>-test within the same tissue/cell population (<b>E</b>).</p

Primer pairs for quantitative PCR.

<p>Primer pairs for quantitative PCR.</p

Flagellin and CpG ODN induce robust innate inflammatory infiltrates in the lung.

<p>(<b>A</b>), (<b>B</b>) Neutrophil accumulation in the airways one day after a single i.n. administration (d1) of OVA (O) or OVA plus flagellin (1 μg) (OFla) in wildtype, <i>Tlr5</i>^<i>-/-</i><i>Tlr11</i>^<i>-/-</i>, and <i>Nlrc4</i>^<i>-/-</i> mice (<b>A</b>) or in wildtype, <i>Tlr5</i>^<i>-/-</i><i>Tlr11</i>^<i>-/-</i> and <i>Tlr4</i>^<i>-/-</i> mice (<b>B</b>), as assessed by flow cytometry of the cells in the BAL fluid. (<b>C</b>) Cellular composition of the innate inflammatory infiltrate in the lung one day after the third i.n. sensitization (d3) with OVA, OVA plus flagellin (1 μg), or OVA plus CpG ODN (3 μg) (OCpG). Data in (<b>A</b>) contain 4 mice per group and are representative of two independent experiments, data in (<b>B</b>) contain 4 mice per group and are representative of two independent experiments, and data in (<b>C</b>) contain 3–4 mice per group and are representative of three independent experiments. Error bars indicate mean +SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 using one-way anova with Bonferroni post-test. In (<b>A</b>) and (<b>B</b>), all groups of OVA-treated mice have statistically significant different values compared to OVA plus flagellin-treated wild-type mice (<b>A</b> and <b>B</b>), <i>Nlrc4</i>^<i>-/-</i> (<b>A</b>), and <i>Tlr4</i>^<i>-/-</i> mice (<b>B</b>) (*** P ≤ 0.001 for all comparisons; not indicated on the panel).</p

Multiple cytokines are likely involved in flagellin-induced T cell responses.

<p><i>Myd88</i>^<i>fl/fl</i> (<i>M</i>^<i>F</i>) and <i>Myd88</i>^<i>fl/fl</i> <i>CD11c-Cre</i> expressing the 4get/KN2 reporter were treated with anti-TSLP (IgG2a), anti-IL-33R (IgG1), and/or appropriate control antibodies (rat IgG2a, rat IgG1), one day before initial sensitization (i.p. 250 μg anti-TSLP, 160 μg anti-IL-33R, or corresponding amounts of appropriate isotype controls), and anti-IL-33R or rat IgG1 again on d2 (i.p. 160 μg). These mice were then administered i.n. OVA or OVA plus flagellin (1 μg) on d0, 1, and 2. On d6, expression of IL-4 (GFP⁺hCD2⁺) by CD4 T cells in the mediastinal LN was examined using the same gating strategy as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167693#pone.0167693.g005" target="_blank">Fig 5</a>. (<b>A</b>) Numbers of CD4 T cells, (<b>B</b>) percentages and (<b>C</b>) numbers of GFP⁺IL-4⁺(hCD2⁺) CD4 T cells. Data are pooled from three independent experiments, one of which did not have the anti-TSLP and IL-33R treatment group, with combined totals of 7–11 mice per group. The comparisons between <i>M</i>^<i>F</i> mice treated i.n. with OVA or OVA plus flagellin, and <i>M</i>^<i>F</i> <i>CD11c-Cre</i> mice treated i.n. with OVA plus flagellin are representative of three additional independent experiments. Each circle represents one individual mouse. Error bars indicate mean +SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 using one-way anova with Bonferroni post-test.</p

Flagellin, but not CpG ODN, promotes development of IL-4-producing CD4 T cells in the draining LN.

<p>4get/KN2 reporter mice were administered OVA, OVA plus flagellin (1 μg), or OVA plus CpG (0.75 μg) i.n. on d0, 1 and 2. In addition, to block IL-12 action in some mice, mice were given anti-IL-12 p40 or control antibody (rat IgG2a) twice, one day before initial sensitization (700 μg i.p.) and again on d2 (300 μg i.p.). On d6, expression of IL-4 reporters (GFP⁺hCD2⁺) by CD4 T cells was examined in the mediastinal LN. (<b>A</b>) Gating strategy of CD4 T cells (CD4⁺), activated CD4 T cells (CD4⁺CD44^hiB220^-CD62L^-), and T_FH cells (CD4⁺CD44^hiB220^-CD62L^-PD-1⁺CXCR5⁺). (<b>B</b>) Numbers of CD4 T cells, percentages and numbers of GFP⁺IL-4⁺(hCD2⁺) CD4 T cells. (<b>C</b>) Percentages and numbers of activated CD4 T cells, percentages and numbers of T_FH cells, and percentages and numbers of GFP⁺IL-4⁺(hCD2⁺) T_FH. Data are pooled from two independent experiments with combined totals of 8 mice per group. Each circle represents one individual mouse. Error bars indicate mean +SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 using one-way anova with Bonferroni post-test.</p