Analysis of cytokine production and surface markers of infiltrating mononuclear cells in the lungs.

<p>Panel A shows the expression of CD25 and CD44 among IL-22 producing cells. Panel B shows the IFN-γ and IL-17A production from sensitized and challenged mouse lungs. Panel C shows the expression of Rorgt. Rorc-eYFP mice were sensitized and challenged and the lung infiltrating mononuclear cells were analyzed for the expression of YFP.</p

Intracellular cytokine staining of mononuclear cells in the lung.

<p>Cells were isolated from either challenged only (c) or sensitized and challenged (s/c) <i>Il22</i> deficient and congenic wild-type controls (IL-22^+/+). Panel A shows a representative plot of intracellular IL-5 and IFN-γ staining. Panel B shows cell counts for IL-4 positive, IFN-γ negative (IL-4⁺IFN⁻), IL-5 positive, IFN-γ negative (IL-5⁺IFN⁻), IFN-γ positive, IL-5 negative (IFN⁺IL-5⁻) and IL-17A positive, IFN-γ negative (IL-17A⁺IFN⁻) cells. Each dot represent a single mouse. Data from 2 independent experiments are given. * p<0.05 compared to all other groups.</p

Histology score and PAS-positive cells in airway epithelium.

<p>Peribronchial inflammation was graded by a semi-quantitative score (no inflammation = 0 to severe inflammation = 4). For each slide 5 randomly chosen areas were scored. Goblet cell metaplasia is expressed as number of PAS-positive cells per mm of basement membrane (BM). Mean values±SEM are given, n = 12 per group. N.D.: not detectable.</p><p>*p<0.05 compared to sens/chall PBS.</p

IL-22 expression is increased during allergic airway inflammation.

<p>Panel A: Expression of IL-22 and IL-22 R1 (IL-22 Rc) was assessed in lung tissue of challenged only (chall, n = 6) and sensitized and challenged (sens/chall, n = 6) animals. Total RNA was isolated 24 hours after the last challenge, reverse transcribed, and gene expression analyzed by PCR with specific primers for IL-22R1. Data are shown as fold induction relative to expression in naïve animals after normalization to GAPDH. Mean±SEM from 2 independent experiments are given. * p<0.05. Panel B: Levels of IL-22 in BAL fluid 48 hrs following the last challenge in challenged only (chall, n = 6) and sensitized and challenged (sens/chall, n = 6) animal. Mean±SEM from 2 independent experiments are given. * p<0.05. Panel C and D: IL-22 intracellular staining in lung cells 24 hrs following the last exposure in sensitized and challenged (top row) and challenged only (bottom row) animals and frequency of CD45⁺IL-22⁺ cells in lung tissue each dot represents a single mouse from 2 independent experiments. * p<0.05.</p

Administration of rIL-22 reduces AHR and airway inflammation.

<p>Airway responsiveness (panel A), cell counts in BAL fluid (panel B) and lung tissue inflammation and goblet cell metaplasia (panel C) were assessed in mice 48 h after the last airway challenge. Mice which were sensitized and challenged (sens/chall, n = 12) showed increased airway reactivity and numbers of eosinophils in BAL fluid compared to challenged only mice (chall, n = 5), where no eosinophils were detectable. Intranasal treatment of sensitized and challenge animals with 0.1 µg recombinant IL-22 (IL-22 0.1 µg, n = 12) showed little effects on AHR and inflammation. In contrast, mice treated with either 1 µg (IL-22 1 µg, n = 12) or 10 µg (IL-22 1 µg, n = 12) of recombinant IL-22 showed decreased AHR and number of eosinophils in BAL fluid. Means±SEM are given, *p<0.05 compared to sens/chall. <i>Panel C:</i> Tissue inflammation was evaluated 48 hrs following the last challenge using hematoxylin and eosin staining (HE) and PAS staining for goblet cells in challenged only mice (chall), non-treated sensitized and challenged mice (sens/chall) and sensitized and challenged animals treated with 10 µg of recombinant IL-22 (rIL-22). Final magnifications 100× and 400× for inserts. <i>Panels D and E:</i> Levels of IL-13 (panel D) and CCL17 (panel E) were measured in BAL fluid by ELISA 48 h after the last challenge. Means±SEM of challenged only mice (chall, <i>n</i> = 5), non-treated sensitized and challenged mice (sens/chall, n = 12), and sensitized and challenged mice treated with 0.1 µg (0.1 µg IL-22, n = 12), 1 µg (1 µg IL-22, n = 12) and 10 µg (10 µg IL-22, n = 12) of rIl-22, respectively. Mean±SEM are given. * p<0.05 compared to sens/chall and 0.1 µg IL-22.</p

Serum immunoglobulin titers.

<p>Mice were sensitized and challenged as described in Methods. Serum levels of immunoglobulins were assessed 48 h after the last challenge. Mean values±SEM are given. <i>Il22^−/−</i>: C57Bl/6 IL-22 deficient mice, <i>Il22^+/+</i>: congenic wild-type control mice. N.D.: not detectable.</p><p>#p<0.05 compared to <i>Il22^+/+</i>chall and <i>Il22^−/−</i> chall.</p

Analysis of cytokines and chemokines in IL-22^−/− and congenic controls.

<p>Levels of IL-5. IL-13, IL-10, IFN-γ (IFN) and CCL-17 in BAL fluid, TSLP and IL-33 in lung homogenate and expression of CCL26 in whole lung were analyzed in challenged only (c) and sensitized and challenged (s/c) <i>Il22</i> deficient (IL-22^−/− c, n = 12; IL-22^−/− s/c, n = 13) and congenic wild-type controls (IL-22^+/+ c, n = 12; IL-22^+/+ s/c, n = 13). Each dot represents a single mouse, bar represents mean. Data are from 2 independent experiments. * p<0.05 compared to challenged groups, # p<0.05 compared to all other groups.</p

IL-22 deficient animals display increased AHR and airway inflammation.

<p>Airway resistance (panel A) and dynamic compliance (panel B) in challenged only wild-type (IL-22^+/+ chall, n = 12), challenged only IL-22 deficient (IL-22^−/− chall, n = 12), sensitized and challenged wild-type (IL-22^+/+ sens/chall, n = 13) and sensitized and challenged IL-22 deficient (IL-22^−/− sens/chall, n = 13) mice. Results are expressed as mean±SEM from 2 independent experiments. # p<0.01, *p<0.05. Panel C shows differential cell counts for eosinophils in BAL fluid. Each dot represents a single mouse, bar represents mean. # p<0.05 compared to IL-22^+/+ chall and IL-22^−/− chall; * p<0.05 compared to all other groups. N.D.: not detectable. Panel D: Tissue inflammation was evaluated 48 hrs following the last challenge using hematoxylin and eosin staining (HE) and PAS staining for goblet cells in challenged only wild-type mice (IL-22^+/+ chall), sensitized and challenge wild-type mice (IL-22^+/+ sens/chall), challenged only IL-22 deficient mice (IL-22^−/− chall) and sensitized and challenged IL-22 deficient (IL-22^−/− sens/chall). Final magnifications 100× and 400× for inserts. Panel E shows histology score and panel F number of goblet cells per mm of basement membrane for challenged only (c) and sensitized and challenged (s/c) wild-type (IL-22^+/+) and IL-22 deficient (IL-22^−/−) animals. Each groups contains 12 animals. Means±SEM are given. *p<0.01 compared to IL-22^+/+ c and IL-22^−/− c. # p<0.05 compared to all other groups.</p

IL-22 expression is increased during specific T cell responses in the lung.

<p>Panel A: IL-22 intracellular staining in lung cells 24 hrs after inhaled exposure of OT II mice with either PBS (PBS) or OVA for 3 consecutive days. Panel A shows IL-22 a representative intracellular staining of CD45⁺ cells. Panel B: Numbers of CD45⁺ IL22⁺ cells from 2 independent experiments, each dot represents a single mouse, bar represents Mean, * p<0.05; Panel C: levels of IL-22 in BAL fluid, mean±SEM are shown, n = 4 from 2 independent experiments, Panel D: representative IL-22 intracellular staining in lung cells 24 hrs after inhaled exposure of OT II mice OVA for 3 consecutive days. Panel E: ScaI and CD90 expression in lung cells 24 hrs after inhaled exposure of OT II mice. Red dots represent IL-22 positive cells, black dots represent IL-22 negative cells.</p