Figure 3: IL-9 activates mast cells to produce IL-2.

C57BL/6 or Cftr^−/− mice (six per group) infected intranasally with live A. fumigatus conidia were evaluated at different days after infection (dpi) for (a) deposition of DNA on lung epithelial cells by TUNEL, resulting in bright DNA staining; (b) detection of c-Kit⁺FcɛR⁺ and c-Kit⁺IL-9⁺ lung mast cells (MC) by flow cytometry (numbers refer to percentages of positive cells); (c) toluidine blue, relative MC number mm⁻² and, in the inset, immunohistochemical staining for chymase- and tryptase-positive MC in lung section. Photographs were taken with a high-resolution microscope (Olympus DP71) equipped with a × 40 objective and (in the inset) a × 100 objective and with EVOS FL Color Imaging System with a × 60 objective (immunohistochemical staining). (d) Toluidine blue stain and transcription factors expression (PCR with reverse transcription) of c-Kit⁺ cells magnetically isolated from lung of uninfected C57BL/6 mice and pulsed with live A. fumigatus conidia. (e) Cytokine production (mean values±s.d., ELISA on culture supernatants) by purified lung c-Kit⁺ cells, pulsed with A. fumigatus and stimulated with IgE, IL-9 and IL-33; (f) detection of c-Kit⁺IL-2⁺, CD90.2⁺IL-2⁺ and CD4⁺IL-2⁺ lung cells by flow cytometry (numbers refer to percentages of positive cells). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, conidia-pulsed versus unpulsed c-Kit⁺ cells, stimulated versus unstimulated c-Kit⁺ cells and Cftr^−/− versus C57BL/6 c-Kit⁺ cells (data represent pooled results or representative images from three experiments, Two-way ANOVA, Bonferroni post test). Mcpt1, mast cell protease 1; Mcpt6, tryptase beta 2; Rorc, retinoic acid receptor–related orphan receptor C; Rora, RAR-related orphan receptor alpha; Tbet, T box expressed in T cells; Tph1, tryptophan hydroxylase 1.