Figure 3.

Reduced numbers of type 2 innate lymphoid cells (ILC2s) isolated from SPC^Cre/CARMA3^F/F mice. (A) SPC^Cre/CARMA3^+/+ and SPC^Cre/CARMA3^F/F mice received a single dose of either PBS or 100 μg A. alternata intranasally and were harvested 24 hours later. Protein levels of IL-33 in the BAL quantified by ELISA. Data are means ± SEM of eight mice per group from two experiments. *P < 0.05 (A. alternata treatment compared with the same genotype that was treated with PBS or SPC^Cre/CARMA3^+/+–A. alternata compared with SPC^Cre/CARMA3^F/F–A. alternata). (B–C) Normal human bronchial epithelial (NHBE) cells were grown on an air–liquid interface postinfection with lentivirus containing a nontargeting shRNA (scRNA) or a CARMA3-targeting shRNA. These cells were then stimulated with either media or 100 μg A. alternata for 6 hours for RNA analysis (B) or 0.5 hour for protein (C). Values are the means ± SEM of three to six samples. This experiment was repeated twice. *P < 0.05 (A. alternata treatment compared with the same targeting shRNA that was treated with media or scRNA–A. alternata compared with CARMA3 shRNA–A. alternata). SPC^Cre/CARMA3^+/+ and SPC^Cre/CARMA3^F/F mice received either PBS or 100 μg A. alternata intranasally on Days 1–4. (D–F) On Day 5, the lungs were isolated and the number of lineage⁻ CD45⁺ Sca-1⁺ KLRG1⁺ ILC2s, lineage⁻ CD45⁺ Sca-1⁺ KLRG1⁺ Thy1.2⁺ T1ST2⁺ CD25⁺ ILC2s, and lineage⁻ CD45⁺ Sca-1⁺ KLRG1⁺ Thy1.2⁺ T1ST2⁺ CD25⁻ ILC2s were determined by flow cytometry. (G–I) The percentage of total lung cells of lineage⁻ CD45⁺ Sca-1⁺ KLRG1⁺ ILC2s, lineage⁻ CD45⁺ Sca-1⁺ KLRG1⁺ Thy1.2⁺ T1ST2⁺ CD25⁺ ILC2s, and lineage⁻ CD45⁺ Sca-1⁺ KLRG1⁺ Thy1.2⁺ T1ST2⁺ CD25⁻ ILC2s were determined by flow cytometry. Data are means ± SEM of eight mice per group from two experiments. *P < 0.05 (A. alternata treatment compared with the same genotype that was treated with PBS or SPC^Cre/CARMA3^+/+–A. alternata compared with SPC^Cre/CARMA3^F/F–A. alternata).