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

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 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

Primer pairs for quantitative PCR.

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

Different polarizations of CD4 T cells in the lung after i.n. sensitization with antigen and either flagellin or CpG ODN.

<p>(<b>A</b>-<b>C</b>) Presence of activated T_H2 cells and IL-4-producing basophils in the lung of OVA- rechallenged mice. IL-4 reporter (4get/KN2) mice were administered i.n. with OVA, OVA plus 1 μg flagellin, or OVA plus 3 μg CpG ODN, and challenged i.n. with OVA. On d21, expression of IL-4 reporters by lung CD4 T cells and basophils was determined. (<b>A</b>) Numbers of lung CD4 T cells and basophils. (<b>B</b>) Representative flow cytometry plots of IL-4 production (hCD2⁺) by IL-4 competent (GFP⁺) CD4 T cells (CD1d-tet^-CD3⁺CD4⁺GFP⁺) and basophils (CD1d-tet^-CD3^-CD49b⁺SSC^loGFP⁺). (<b>C</b>) Percentages IL-4⁺ (hCD2⁺) of IL-4 competent (GFP⁺) CD4 T cells and basophils, percentages GFP⁺IL-4⁺(hCD2⁺) of total CD4 T cells, and numbers of GFP⁺IL-4⁺(hCD2⁺) CD4 T cells and basophils. (<b>D</b>-<b>F</b>) Presence of activated T_H1 cells and IFN-γ-expressing CD8 T cells in the lungs of rechallenged mice. IFN-γ reporter (GREAT) mice were administered i.n. with OVA, OVA plus 5 μg flagellin, or OVA plus 3 μg CpG ODN, and challenged with i.n. OVA. On d16, expression of IFN-γ reporter by lung CD4 and CD8 T cells was determined. (<b>D</b>) Numbers of lung CD4 and CD8 T cells. (<b>E</b>) Representative flow cytometry plots of IFN-γ (YFP⁺) by CD4 and CD8 T cells. (<b>F</b>) Percentages and numbers of IFN-γ⁺ CD4 and CD8 T cells. (<b>G</b>-<b>I</b>) Presence of activated T_H17 cells and IL-17A-expressing γδ T cells in the lungs of rechallenged mice. IL-17 reporter (SMART-17A) mice were administered i.n. with OVA, OVA plus 1 μg flagellin, or OVA plus 3 μg CpG ODN, and challenged with i.n. OVA. On d17, expression of the IL-17 reporter by lung CD4 and γδ T cells was determined. (<b>G</b>) Total numbers of lung CD4 and γδ T cells. (<b>H</b>) Representative flow cytometry plots of IL-17 (hNGFR⁺) expression by CD4 and γδ T cells. (<b>I</b>) Percentages and numbers of hNGFR⁺ CD4 and γδ T cells. Data in (<b>A</b>-<b>C</b>) contain four mice per group and are representative of one of three independent experiments, data in (<b>D</b>-<b>F</b>) contain 4–5 mice per group and are representative one of two independent experiments, data in (<b>G</b>-<b>I</b>) are pooled from three independent experiments with combined totals of 11–13 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

The CpG ODN-induced type 1 immune response is dependent on IL-12 and MyD88 signaling in cDCs and/or AMs.

<p>(<b>A</b>) Percentages of CD4 T cells, CD8 T cells, γδ T cells, NK T cells, and NK cells producing IFN-γ (YFP⁺) in GREAT reporter mice one day after third i.n. administration (d3) of OVA, OVA plus flagellin (5 xg), or OVA plus CpG ODN (3 μg). (<b>B</b>) <i>Il12b</i> and (<b>C</b>) <i>Il12a</i> mRNA induction in whole lung tissue 2 h after one i.n. administration of OVA, OVA plus flagellin (1 μg), or OVA plus CpG ODN (3 μg) in wild-type mice as measured by qPCR. Samples were normalized to <i>Hprt</i> mRNA. (<b>D-F</b>) GREAT reporter mice were treated with anti-IL-12 p40 or with control antibody (rat IgG2a), one day before initial sensitization (700 μg intraperitoneally (i.p.)) and again on d2 (300 μg i.p.) (<b>E, F</b>). These mice were sensitized i.n. with OVA or OVA plus CpG ODN (0.75 μg) on d0, 1 and 2 and rechallenged with OVA alone on d15. (<b>D, E</b>) Percentages of IFN-γ (YFP⁺) lymphocytes in the lung on d3 (<b>D</b>) and d16 (<b>E</b>). (<b>F</b>) Serum levels of OVA-specific IgG2c on d16. (<b>G</b>) <i>Il12b</i> mRNA induction in whole lung tissue 2 h after one i.n. administration of OVA, or OVA plus CpG ODN (3 μg) in <i>Myd88</i>^<i>fl/fl</i> mice (<i>M</i>^<i>F</i>) or <i>Myd88</i>^<i>fl/fl</i><i>CD11c-Cre</i> (<i>M</i>^<i>F</i> <i>CD11c-Cre</i>) as measured by qPCR. Samples were normalized to <i>Hprt</i> mRNA. <b>(H-I)</b> Inductions of <i>Il12b</i> (<b>H</b>) and <i>Il12a</i> (<b>I</b>) mRNAs in sorted cell populations from lung tissue 2 h after i.n. treatment as in (<b>B</b>). (<b>J-L</b>) <i>M</i>^<i>F</i> or <i>M</i>^<i>F</i> <i>CD11c-Cre</i> mice expressing the GREAT reporter were sensitized i.n. with OVA or OVA plus 0.75 μg CpG ODN on d0, 1, and 2, and rechallenged on d15 with OVA alone. (<b>J, K</b>) Percentages of IFN-γ reporter⁺ lymphocytes in the lung on d3 (<b>J</b>) and on d16 (<b>K</b>). (<b>L</b>) Serum levels of OVA-specific IgG2c on d16. Data in (<b>A</b>) are pooled from two independent experiments with combined totals of 7–8 mice per group, data in (<b>B</b>) contain 5 mice per group and are representative of two independent experiments, data in (<b>C</b>) contain 4 mice per group and are representative of two independent experiments, data in (<b>D</b>) are pooled from two independent experiments with 6–7 mice per group, and similar results were obtained in a third independent experiment, data in (<b>E, F</b>) contain 3–4 mice per group and are representative of one of three independent experiments, data in (<b>G</b>) contain 4 mice per group and are representative of two independent experiments, data in (<b>H, I</b>) are pooled from two independent experiments with combined totals of 6–7 mice per group, data in (<b>J</b>) are pooled from two independent experiments with combined totals of 5 or 8 mice per group, and data in (<b>K, L</b>) are pooled from three independent experiments with combined totals of 10 or 12 mice per group. Each circle represents one individual mouse except in (<b>H, I</b>), in which each circle represents 3–4 mice pooled before cell sorting. 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

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

MyD88 Shapes Vaccine Immunity by Extrinsically Regulating Survival of CD4⁺ T Cells during the Contraction Phase

<div><p>Soaring rates of systemic fungal infections worldwide underscore the need for vaccine prevention. An understanding of the elements that promote vaccine immunity is essential. We previously reported that Th17 cells are required for vaccine immunity to the systemic dimorphic fungi of North America, and that Card9 and MyD88 signaling are required for the development of protective Th17 cells. Herein, we investigated where, when and how MyD88 regulates T cell development. We uncovered a novel mechanism in which MyD88 extrinsically regulates the survival of activated T cells during the contraction phase and in the absence of inflammation, but is dispensable for the expansion and differentiation of the cells. The poor survival of activated T cells in <i>Myd88</i>^-/- mice is linked to increased caspase3-mediated apoptosis, but not to Fas- or Bim-dependent apoptotic pathways, nor to reduced expression of the anti-apoptotic molecules Bcl-2 or Bcl-xL. Moreover, TLR3, 7, and/or 9, but not TLR2 or 4, also were required extrinsically for MyD88-dependent Th17 cell responses and vaccine immunity. Similar MyD88 requirements governed the survival of virus primed T cells. Our data identify unappreciated new requirements for eliciting adaptive immunity and have implications for designing vaccines.</p></div

Binding of <i>Aspergillus</i> conidia to airway mucins is FleA dependent.

<p>(A) Z-stack confocal images showing binding of WT <i>A</i>. <i>fumigatus</i> conidia (TJMP 131.5) to mucin and that <i>ΔfleA</i> conidia have very limited binding. (B) Quantitative binding of WT <i>A</i>. <i>fumigatus</i> conidia to mucin or <i>ΔfleA</i> deletion mutants. (C) Quantitative binding of WT <i>A</i>. <i>flavus</i> conidia to mucin or <i>ΔfleA</i> deletion mutants. Note that <i>A</i>. <i>fumigatus</i>-mucin interactions were investigated using GFP labeled conidia whereas the <i>A</i>. <i>flavus</i>-mucin interactions were investigated using Calcofluor white-stained conidia (since the <i>A</i>. <i>flavus</i> conidia lack GFP). The data shown in panels B and C (27 replicates) reflects the mean ± SD of three independent experiments.</p