After <i>Mtb</i> infection, IL-22 expression is IL-23 dependent.

<p>Experimental mice were infected with <i>Mtb</i> via the aerosol route. (<b>A</b>) Before and at different time points of infection with approx. 100 CFU <i>Mtb</i>, gene expression of <i>Il22</i>, <i>Il12b</i>, <i>Il17a</i>, <i>Il17f</i>, and <i>Il23p19</i> was quantified by real time RT-PCR in lung homogenates of C57BL/6 mice based on the expression of <i>Hprt</i>. Data represent mean ± SD of 5 mice per group. (<b>B</b>) Twenty days after infection with approx. 1000 CFU <i>Mtb</i> of C57BL/6 (black symbols) and IL-23p19^−/− (white symbols) mice, gene expression of <i>Il22</i> was quantified by real time RT-PCR in lung homogenates based on the expression of <i>Hprt</i>. Data represent mean ± SD for of 5 mice per group. Statistical analysis was performed using the Student’s <i>t</i> test defining differences between C57BL/6 and IL-23p19^−/− mice as significant (***, p<0.001).</p

TH1 and TH17 immune responses in IL-22^−/− mice after low dose infection with <i>Mtb</i>.

<p>C57BL/6 mice (black bars) and IL-22^−/− (white bars) mice were infected with approx. 100 CFU <i>Mtb</i> via the aerosol route. At different time points, single cell suspensions of lungs were prepared, restimulated with anti-CD3/CD28 and stained for flow cytometric analysis. (<b>A</b>) Representative dot plots of restimulated cells from C57BL/6 mice stained with isotype control antibodies. (<b>B</b>) Representative dot plots of unstimulated (left panel) or restimulated (right panel) IFNγ- and IL-17A-producing CD44⁺CD4⁺ and CD8⁺ cells isolated at day 22 after infection. (<b>C</b>) Frequencies of IFNγ^negIL-17A⁺, IFNγ⁺IL-17A^neg, and IFNγ⁺IL-17A⁺CD4/CD8⁺ cells. Data represent mean ± SD of 4–5 mice per group. Statistical analysis was performed using a two-way ANOVA defining differences between C57BL/6 and IL-22^−/− mice as significant (*, p<0.05; ***, p<0.001).</p

The course of low dose <i>Mtb</i> infection in IL-22^−/− mice.

<p>C57BL/6 mice (black symbols) and IL-22^−/− (white symbols) mice were infected with approx. 100 CFU <i>Mtb</i> via the aerosol route. At (<b>A</b>) earlier and (<b>B</b>) later time points, mycobacterial colony enumeration assays were performed in lungs, spleen, and liver. Data represent mean ± SD of 4–5 mice per group. Statistical analysis was performed using two-way ANOVA defining differences between C57BL/6 and IL-22^−/− mice as significant (*, p<0.05; **, p<0.01; ***, p<0.001). (<b>C</b>) Survival of 10–13 infected mice per group. Statistical analysis was performed using the log-rank test.</p

The course of high dose <i>Mtb</i> infection in IL-22^−/− mice.

<p>C57BL/6 mice (black symbols) and IL-22^−/− (white symbols) mice were infected with approx. 1000 CFU <i>Mtb</i> via the aerosol route. At (<b>A</b>) earlier and (<b>B</b>) later time points, mycobacterial colony enumeration assays were performed in lungs, spleen, and liver. Data represent mean ± SD of 5 mice per group. Statistical analysis was performed using two-way ANOVA defining differences between C57BL/6 and IL-22^−/− mice as significant (**, p<0.01). (<b>C</b>) Survival of 10 infected mice per group. Statistical analysis was performed using the log-rank test.</p

Antigen-specific production of IFNγ and IL-17A by CD4⁺ T cells of low dose-infected IL-22^−/− mice.

<p>C57BL/6 mice (black bars) and IL-22^−/− (white bars) mice were infected with approx. 100 CFU <i>Mtb</i> via the aerosol route. At earlier (left panel) and later (right panel) time points, the frequency of Esat-6_1–20 specific (<b>A</b>, <b>B</b>) IFNγ- and (<b>C</b>, <b>D</b>) IL-17A-producing cells in (<b>A</b>, <b>C</b>) cell suspensions enriched for CD4⁺ T cells or in (<b>B</b>, <b>D</b>) whole cell suspensions from lungs was determined by ELISPOT assay. Data represent mean ± SD of 4–5 mice per group. Statistical analysis was performed using a two-way ANOVA defining differences between C57BL/6 and IL-22^−/− mice as significant (*, p<0.05).</p

Macrophage effector response in IL-22^−/− mice after low dose <i>Mtb</i> infection.

<p>C57BL/6 mice (black bars) and IL-22^−/− (white bars) mice were infected with approx. 100 CFU <i>Mtb</i> via the aerosol route. At (<b>A</b>) earlier and (<b>B</b>) later time points, gene expression of <i>Ifng</i>, <i>Nos2</i>, and <i>Lrg47</i> was quantified by real time RT-PCR in lung homogenates of C57BL/6 mice based on the expression of <i>Hprt</i>. Data represent mean ± SD of 5 mice per group. Statistical analysis was performed using a two-way ANOVA defining differences between C57BL/6 and IL-22^−/− mice as significant (**, p<0.01).</p

The inflammatory cytokine responses in IL-22^−/− mice after low dose <i>Mtb</i> infection.

<p>C57BL/6 mice (black bars) and IL-22^−/− (white bars) mice were infected with approx. 100 CFU <i>Mtb</i> via the aerosol route. At (<b>A</b>) earlier and (<b>B</b>) later time points, gene expression of <i>Il12b</i>, <i>Tnf</i>, <i>Il6</i>, and <i>Il10</i> was quantified by real time RT-PCR in lung homogenates of C57BL/6 mice based on the expression of <i>Hprt</i>. Data represent mean ± SD of 5 mice per group. Statistical analysis was performed using a two-way ANOVA defining differences between C57BL/6 and IL-22^−/− mice as significant (*, p<0.05).</p

In experimental TB, IL-22 is mainly expressed by IFNγ-producing cells.

<p>Experimental mice were infected with approx. 100 CFU <i>Mtb</i> via the aerosol route. (<b>A</b>, <b>B</b>). After 22 days of infection, lung cells of C57BL/6 mice were restimulated with anti-CD3/CD28 or left unstimulated. Cells were subsequently stained for CD90.2, CD4, IL-17A, IFNγ and IL-22, analyzed by flow cytometry and the frequencies of cytokine-producing cells were compared. (<b>A</b>) Representative dot plots and frequencies of cytokine-producing CD4^neg (left panel, left graph) and CD4⁺ (right panel, right graph) cells gated for CD90.2⁺. The frequencies of gated cytokine-producing cells are shown and represent means ± SD of 5 mice. (<b>B</b>) Representative dot plots of control stainings (<i>i</i>, isotype controls; <i>ii</i>, unstimulated cells; <i>iii</i>, staining in IL-22^−/− mice) and of cytokine-producing cells indicating G1 (IFNγ⁺IL-22^neg), G2 (IFNγ⁺IL-22⁺) and G3 (IFNγ^negIL-22⁺) for further analysis. The absolute numbers and frequencies of gated cytokine-producing cells are shown and represent means ± SD of 5 mice. Statistical analysis was performed using two-way ANOVA defining differences between different cytokine-producing cell populations as significant (***, p<0.001).</p

The recruitment of macrophages, granulocytes, and activation of T cells in IL-22^−/− mice after low dose <i>Mtb</i> infection.

<p>C57BL/6 mice (black bars) and IL-22^−/− (white bars) mice were infected with approx. 100 CFU <i>Mtb</i> via the aerosol route. At different time points, single cell suspensions of lungs were prepared, counted and stained for flow cytometric analysis. (<b>A</b>) Numbers of total infiltrating cells, macrophages (CD11b⁺CD11c⁺MHCII⁺Gr-1^neg), granulocytes (CD11b⁺CD11c^negMHCII^negGr-1⁺) and T cells (CD4⁺ or CD8⁺). (<b>B</b>) Representative dot plots of naive (CD4/8⁺CD62L^neg/+CD44^neg), effector memory (CD4/8⁺CD62L^negCD44⁺), and central memory (CD4/8⁺CD62L⁺CD44⁺) T cells at two representative time points (22 and 254 days after infection). (<b>C</b>) Analysis of T cell populations described in (<b>B</b>) during the course of <i>Mtb</i> infection. Data in (<b>A</b>) and (<b>C</b>) represent mean ± SD of 4–5 mice per group. Statistical analysis was performed using a two-way ANOVA defining differences between C57BL/6 and IL-22^−/− mice as significant (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Inverse correlation of Mx1 protein levels and viral load in lungs of mice lacking functional receptors for IFN-α/β, IFN-λ or both.

<p>Groups of mice were infected with 10⁵ pfu of SC35M-ΔNS1 and either killed at (A) 48 hours post infection to determine viral titers in the lung or at (B) 20 hours post infection to determine Mx1 protein levels by western blotting. Two animals of each group are shown. Actin-normalized Mx1 signal intensities are indicated. The calculated value of the wild-type mice was set to 100%. (*: p<0.05), ***: p<0.001).</p