Figure 7.

Adoptive transfer of Th9 cells restores lung inflammation in butyrate-treated OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The groups that received butyrate during sensitization were treated during challenge. Butyrate-treated mice also received an adoptive transfer (IP) of OT-II Th0, Th2, or Th9 cells 24 h before challenge initiation. Euthanasia was performed 24 h after the last challenge (A). Lungs were digested and cells stained with monoclonal antibodies to determine the frequency of eosinophils as shown by the bar graph (B). Alternatively, cells were stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO₂, and stained with monoclonal antibodies to determine the frequency of IL-9+CD4+ and IL-13+CD4+ T cells as shown by the bar graphs (C,D), respectively. Lung tissues were also stained with hematoxylin/eosin (H&E) and periodic acid–Schiff (PAS), scale bars: 100 μm. Thick and thin arrows indicate inflammatory infiltrates and mucus production, respectively (E). Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.