Neutralization of IL-22 compromises early A. fumigatus lung clearance. (A) C57BL/6 WT mice were challenged intratracheally with 5 × 10⁷ to 7 × 10⁷ A. fumigatus conidia. Six hours after challenge, the mice were administered 50 μg of goat anti-mouse IL-22 or goat IgG antibodies intratracheally. IL-22 levels were quantified in lung homogenates 24 h after challenge by ELISA. Shown are cumulative data from two independent studies (n = 5 mice per group per time point). The data are expressed as mean pg/ml plus SEM. ** indicates a P value of <0.01 (unpaired two-tailed Student's t test). (B) Real-time PCR analysis of A. fumigatus 18S rRNA levels in the lungs of WT mice administered anti-IL-22 or isotype control antibodies. Shown are cumulative data from two independent studies (n = 5 mice per group per time point). The data are expressed as mean A. fumigatus 18S rRNA plus SEM. ** indicates a P value of <0.01 (unpaired two-tailed Student's t test).

Impaired antifungal activity in lung lavage fluid from A. fumigatus-challenged Dectin-1-deficient and IL-22-deficient mice. (A) C57BL/6 WT, Dectin-1-deficient (Dectin-1 KO), and IL-22-deficient (IL-22 KO) mice were challenged intratracheally with 5 × 10⁷ to 7 × 10⁷ A. fumigatus conidia, and 24 h after exposure, bronchoalveolar lavage was performed. The lung lavage fluid was processed to remove cells and A. fumigatus, and then, 50 μl of clarified lavage fluid from each strain was incubated with 1 × 10⁵ A. fumigatus conidia (in 150 μl of RPMI supplemented with 10% FBS and 1% penicillin-streptomycin) for 4 h at 37°C. Thereafter, the contents of the well was subjected to total RNA extraction using the MasterPure yeast RNA purification kit and analyzed for A. fumigatus viability. For each experiment, the percent above WT was calculated by dividing the A. fumigatus 18S rRNA units in Dectin-1-deficient and IL-22-deficient cultures by the A. fumigatus 18S rRNA units in WT cultures. WT values were set at 100. Shown are cumulative data from eight independent studies. * and *** indicate P values of <0.05 and <0.001, respectively (paired two-tailed Student's t test). (B) C57BL/6 WT, Dectin-1-deficient, and IL-22-deficient mice were challenged intratracheally with 5 × 10⁷ to 7 × 10⁷ A. fumigatus conidia, and 24 h after exposure, lungs were collected and homogenized and lipocalin 2 levels were quantified in the clarified homogenates by ELISA. Shown are cumulative data from two independent studies (n = 4 to 5 per group). ** and *** indicate P values of <0.01 and <0.001, respectively (unpaired two-tailed Student's t test). (C) C57BL/6 WT and lipocalin 2-deficient (Lcn2 KO) mice were challenged intratracheally with 5 × 10⁷ to 7 × 10⁷ A. fumigatus conidia, and 24 h after exposure, the lung fungal burden was assessed by real-time PCR analysis of A. fumigatus 18S rRNA levels. Shown are cumulative data from two independent studies (n = 5 mice per group). The data are expressed as mean A. fumigatus 18S rRNA plus SEM.

IL-22 production after A. fumigatus challenge is dependent on Dectin-1. (A) C57BL/6 WT and Dectin-1-deficient (KO) mice were challenged intratracheally with 5 × 10⁷ to 7 × 10⁷ A. fumigatus conidia, and 48 h after exposure, IL-22 levels were quantified in lung homogenates by ELISA. The data are expressed as mean pg/ml plus standard error of the mean (SEM). Shown are cumulative data from three independent studies (n = 5 mice/group for each study). *** indicates a P value of <0.001 (unpaired two-tailed Student's t test). (B) C57BL/6 WT and Dectin-1-deficient (KO) mice were challenged intratracheally with 5 × 10⁷ to 7 × 10⁷ A. fumigatus conidia, and 18 h after exposure, lungs were collected and enzymatically digested. Single-cell suspensions were isolated, and 1 × 10⁶ cells were cultured for 24 h in a volume of 0.2 ml. IL-22 levels were quantified in clarified coculture supernatants by ELISA. Shown are cumulative data from four independent studies. The data are expressed as mean pg/ml plus SEM. *** indicates a P value of <0.001 (unpaired two-tailed Student's t test).

IL-22-deficient mice have impaired A. fumigatus lung clearance. (A) C57BL/6 WT and IL-22-deficient (IL-22 KO) mice were challenged intratracheally with 5 × 10⁷ to 7 × 10⁷ A. fumigatus conidia, and 24 h after exposure, the lung fungal burden was assessed by real-time PCR analysis of A. fumigatus 18S rRNA levels. Shown are cumulative data from three independent studies (n = 5 mice per group). The data are expressed as mean A. fumigatus 18S rRNA plus SEM. ** indicates a P value of <0.01 (unpaired two-tailed Student's t test). (B) Levels of IL-1α, TNF-α, CCL3, and CCL4 were quantified in lung homogenates collected 24 h postinfection by Bio-Plex. Shown are cumulative data from three independent studies (n = 5 mice per group per time point). The data are expressed as mean pg/ml plus SEM. * and *** indicate P values of <0.05 and <0.001, respectively (unpaired two-tailed Student's t test). (C) Lung cells were isolated via bronchoalveolar lavage, Fc blocked, stained with a LIVE/DEAD staining kit, and then stained with fluorochrome-conjugated CD11b and Ly-6G. Shown are representative data from one of two independent studies. The data are expressed as the absolute number of live cells in lung lavage fluid. * indicates a P value of <0.05 (unpaired two-tailed Student's t test). (D) IL-12p40 and IL-12p70 were quantified in lung homogenates collected 24 h postinfection by Bio-Plex. Shown are cumulative data from three independent studies (n = 5 mice per group per time point). The data are expressed as mean pg/ml plus SEM. * and ** indicate P values of <0.05 and 0.01, respectively (unpaired two-tailed Student's t test). (E and F) Levels of CXCL9 and CXCL10 (E) and IL-17A (F) were quantified in lung homogenates collected 24 h postinfection by ELISA. Shown are cumulative data from three independent studies (n = 5 mice per group per time point). The data are expressed as mean pg/ml plus SEM. ** indicates a P value of <0.01 (unpaired two-tailed Student's t test).

IL-22 production by lung cells in response to A. fumigatus is independent of IL-1β, IL-6, and IL-18 but requires IL-23. (A) Lung cells were isolated as described in Materials and Methods, and 1 × 10⁶ cells were cultured for 24 h in a volume of 0.2 ml. Neutralizing antibodies against IL-1β, IL-6, IL-18, and IL-23 were added at a final concentration of 2 to 5 μg/ml at the beginning of the culture. Rat (IL-1β, IL-6, and IL-18) or goat (IL-23) isotype antibodies were included as a control. IL-22 levels were quantified in clarified coculture supernatants by ELISA. Shown are cumulative data from two independent studies for each condition (isotype and neutralizing antibody) run in triplicate. The data are expressed as mean pg/ml plus SEM. *** indicates a P value of <0.001 (unpaired two-tailed Student's t test); ns, not significant. (B) Lung cells were isolated from WT and KO mice as described, and 1 × 10⁶ cells were cultured for 24 h in a volume of 0.2 ml. Recombinant murine IL-23 was added at 1 and 10 ng/ml at the beginning of the culture. The controls included lung cells cultured in the absence of IL-23. IL-22 levels were quantified in clarified coculture supernatants by ELISA. Shown are cumulative data from three independent studies. The data are expressed as mean pg/ml plus SEM. *, **, and *** indicate P values of 0.05, 0.01, and 0.001, respectively (unpaired two-tailed Student's t test).