Figure 4: IL-33 is produced by FALC stromal cells.

(a) Pieces of lung, mediastinum, omentum or gonadal adipose tissue (GAT) were cultured for 1 h at 37 °C and spontaneous release of IL-33 in the supernatant was measured by ELISA. Data are representative of four independent experiments, symbols represent individual mice, n=5. (b) Representative whole-mount immunofluorescence staining of mediastinal FALCs from BALB/c mice showing IL-33 (red), 4,6-diamidino-2-phenylindole (DAPI; blue) and Gp38 (green). Higher magnification shows expression of IL-33 in nucleus (n) and cytoplasmic/membranous area (*). Scale bar, 50 μm. (c,d) Flow cytometric analysis of digested pericardial FALCs from naive and day 11 Ls-infected C57BL/6 mice showing intracellular IL-33 expression by CD45⁻Gp38⁺ stromal and CD45⁺ haematopoietic cells (c), and % of stromal cells and haematopoietic cells expressing IL-33 (d). (e) Naive and day 11 Ls-infected C57BL/6 mediastinum were cultured for 1 h at 37 °C and spontaneous release of IL-33 in the supernatant was measured by ELISA. (f) Representative whole-mount immunofluorescence staining of mediastinal FALCs from naive and day 11 Ls-infected C57BL/6 mice showing IL-33 (red), DAPI (blue) and Gp38 (green) and quantification of the % area of IL-33 staining. Scale bar, 50 μm. Data in (c–f) are pooled from two experiments, symbols in d,e represent individual mice, n=8 or 10 per group, symbols in f represent individual clusters. ns not significant, ***P<0.001, ****P<0.0001 (normally distributed data analysed by one-way ANOVA with Sidak multiple comparisons post test, non-normally distributed data analysed using the Kruksal–Wallis test with Dunn’s multiple comparisons post test). Error bars represent mean with s.e.m. in all graphs.