Fig. 4

Induction of Dusp10 suppresses IL-5 production in ILC2. a Schematic representation of wild-type Dusp10 (WT) and C409S mutant. b–g Freshly prepared ILC2s purified form the spleen of IL-33-injected BALB/c mice were infected with a Mock (empty vector), Dusp10, or C409S mutant-IRES-hNGFR-containing retrovirus and then cultured with IL-7, IL-25 and IL-33. 5 days after infection, hNGFR-positive ILC2 cells were enriched by cell sorting. b Western blot analysis of DUSP10 in freshly prepared ILC2s and Dusp10-overexpressing ILC2s. The arbitrary ratio of the intensity of DUSP10 to that of tubulin is shown. c Freshly prepared ILC2s were infected with a Dusp10, or C409S mutant-IRES-hNGFR-containing retrovirus and then cultured with IL-7, IL-25, and IL-33. 5 days after infection, intracellular-staining profiles of IL-5 and IL-13 in hNGFR⁺ ILC2s are shown. Cells were purified and then stimulated with IL-7 plus IL-33 for 6 h with or without 30 min treatment of SB203850 before stimulation. The percentages of IL-5 positive cells in hNGFR⁺ ILC2s were shown (mean ± SD; n = 3 for each groups). d Quantitative RT-PCR analysis of Il5 and Il13 in hNGFR⁺ ILC2s. Cells were stimulated with IL-7 plus IL-33 for 4 h. e ELISA analysis of the culture supernatant from 3 × 10⁴ cells of hNGFR⁺ ILC2s. Those cells were sorted as in d, and then stimulated with IL-7/33 for 12 h. f Intracellular-staining of phospho-p38 of hNGFR⁺ ILC2s after stimulation with IL-7 + IL-33 for 30 min. Mock represents empty vector introduction. Graph shows mean MFI ± SD values of phospho-p38 in hNGFR⁺ ILC2s (n = 4), separately. g Phosphorylated p38 (p-p38) in freshly prepared ILC2s with Dusp10-overexpression after stimulation with IL-7 + IL-33 for 30 min. The arbitrary ratio of the intensity of p-p38 to that of tubulin is shown. More than four independent experiments were performed and showed similar results (b–g) (**p < 0.01; *p < 0.05; Mann–Whitney U test; b–g). d, f, g Three technical replicates were performed with quantitative RT-PCR and ELISA analysis