Supplementary Figure S1. Flow cytometric analysis of the expression of Thy1 in NH cells. Flow cytometric analysis of the expression of T1/ST2 and Thy1 in NH cells derived from the lungs of naïve mice. Data are representatives of three independent experiments.
Supplementary Figure S2. Analysis of NH cells and Type-2 myeloid cells in IL-25- or IL-33-administered mice. Flow cytometric analysis of NH cells and Type-2 myeloid (T2M) cells in the lungs. IL-25 (0.5 g/animal) or IL-33 (0.5 g/animal) was administered intranasally for 3 consecutive days in C57BL/6 mice. Anti-IL-17RB antibody was kindly provided by Hiroshi Watarai. Data are representatives of two independent experiments.
Supplementary Figure S3. Corticosteroids attenuate the accumulation of NH cells and reduce airway inflammation in a mouse model of ovalbumin- (OVA) induced asthma. (a) OVA (10 ng) was administered intranasally for 3 consecutive days to mice sensitized with OVA and alum. Some mice were treated with intraperitoneal injections of dexamethasone (Dex, 0.5-5 mg/kg) at 24 and 1 hr prior to OVA administration. (b,c) Numbers of eosinophils in the BALF (b) and NH cells in the lung (c) 4 days after the final administration of OVA or vehicle (PBS). Mean ± SEM, n = 3 for each group. (d,e) Protein levels of IL-5 (d) and IL-13 (e) in the lung. Mean ± SEM, n = 3 for each group. (f) Intracellular staining of IL-5 and IL-13 in CD4 + T cells and NH cells. (g) Histology of the airways stained with hematoxylin & eosin (left panels) or periodic acid Schiff (PAS)-alcian blue (right panels). Scale bar indicates 100 m. NS: not significant. *P < 0.05, **P < 0.01 (Student s t-test). Data are representatives of three independent experiments.
Supplementary Figure S4. Analysis of the main source of type-2 cytokine in several asthma model mice. Intracellular staining of IL-5 and IL-13 in lineage marker-positive and negative lymphoid cells in the lung treated with IL-33 (0.5 g/animal i.n. for 3 days), OVA (10 ng i.n. in sensitized animals), dexamethasone (Dex, 5 mg/kg i.p.), or their combination (OVA+IL-33; OVA 10 ng and IL-33 0.1 g i.n. in OVA-sensitized animals). Data are representatives of two independent experiments.
Supplementary Figure S5. TSLP and IL-33 expression in the lungs of OVA and IL-33-induced model mice. (a) Tslp mrna expression (normalized to Gapdh mrna) and (b) TSLP protein levels in the lungs of mice treated with OVA + IL-33. (c) Tslp mrna expression (normalized to Gapdh mrna) in the lungs of mice treated OVA alone or IL-33 alone and (d) IL-33 mrna expression (normalized to Gapdh mrna) in the lungs of mice treated with OVA. Mean ± SEM, n = 3 for each group. *P < 0.05, ***P < 0.001, NS: not significant (Student s t-test). Data are representatives of two independent experiments. Primer sequences of Tslp; 5 - GCCATTTCCTGAGTACCGTC-3, 5 - TTCACTCCCCGACAAAACAT-3, Il33; 5 -ATGAGACCTAGAATGAAGTATTC-3 and 5 -GCACAGGCGTTTTACTGCATT-3.
Supplementary Figure S6. TSLPR, IL-2R, and IL-7R expression in NH cells and Th2 cells. (a) Tslpr mrna expression (normalized to Gapdh mrna), (b) Il2ra mrna expression (normalized to Gapdh mrna), and (c) Il7ra mrna expression (normalized to Gapdh mrna) in NH cells and Th2 cells. Mean ± SEM, n = 3 for each group. *P < 0.05, ***P < 0.001 (Student s t-test). Data are representatives of two independent experiments.primer sequences of Tslpr; 5 -ACTGTGACATCACCCCGTG-3, 5 -GACATGGCATGGGCACTC-3, Il2ra; 5 -AGGAGAGGGCTTTGAATGTG-3 and 5 -TTGCTGATGTTGGGGTTTCT-3, and Il7ra; 5 -CATTTCACTCGTAAAAGAGCCC-3 and 5 -TGGAAGTGGATGGAAGTCAA-3.
Supplementary Figure S7. Anti-TSLP antibody did not suppress accumulation of eosinophils and NH cells without dexamethasone in OVA+IL-33 model mice. OVA and IL-33 were administered to OVA-sensitized mice in combination with an anti-tslp antibody (25 g) or an isotype antibody. (a) Number of eosinophils in the BALF and (b) NH cells in the lung. Mean + SEM, n = 3 for each group. NS: not significant. Data are representatives of two independent experiments.
Supplementary Figure S8. Anti-TSLP antibody partially suppresses corticosteroid-resistant airway hyperresponsiveness. Airway hyperresponsiveness was measured by FlexiVent system using methacholine (Mch, 25-100 mg/ml). n = 4 for each group. Data are representatives of two independent experiments.
Supplementary Figure S9. Neither anti-il-2 nor anti-il-7 antibody ameliorates corticosteroid resistance in OVA + IL-33 model mice. OVA, IL-33 and dexamethasone (Dex, 5 mg/kg) were administered to OVA-sensitized mice with either with an anti-il-2 antibody (25 µg), an anti-il-7 antibody (25 µg), an anti-tslp antibody (25 µg), an isotype control antibody (a,b), or their combination. Graphs represent the number of eosinophils in the BALF (a, c) and NH cells in the lung (b, d) 4 days after the final administration of OVA/IL-33/Dex/antibodies. Mean ± SEM, n = 3-5 for each group. NS: not significant. Data are representatives of two independent experiments.
Supplementary Figure S10. Comparison of the corticosteroid sensitivity between wild-type and Tslpr -/- NH cells. Bone marrow cells from wild-type (WT, CD45.1) and Tslpr -/- (CD45.2) mice were co-transferred into WT (CD45.1 x CD45.2) mice after 6 Gy irradiation (TSLPR -/- ), while control mice (BL/6) received bone marrow cells from WT (CD45.1) and WT (CD45.2) mice. Lung NH cells (Lin - Thy-1 + lymphoid cells) in dexamethasone-treated OVA + IL-33 model were stained intracellularly for IL-5 and IL-13 (a), and the ratio of CD45.2 + to CD45.1 + NH cells positive for IL-5 (b) and IL-13 (c) was determined. n = 3 for each group. Data are representatives of two independent experiments.
Supplementary Figure S11. TSLP-induced GATA3 expression was decreased by dexamethasone. Western blot analysis of GATA3 in NH cells cultured with IL-33 (10 ng/ml), TSLP (10 ng/ml), dexamethasone (Dex, 10-8 M), or their combination for 12 hrs. TATA-binding protein (TBP) is shown as a loading control. Data are representatives of two independent experiments.
Supplementary Figure S12. Full blots for figure panels. (a,b) Full blots for Fig 6a. (c,d,e) Full blots for Fig 6b. (g,h) Full blots for Supplementary Fig S9.
Supplementary Table S1. Antibody sources and dilutions. Antigen Clone Label Source Dilution Bcl-2 C-2 purified Santa Cruz Biotechnology 1:100 Bcl-xL 54H6 purified Cell Signaling Technology 1:100 p-stat5 (Tyr694) D47E7 purified Cell Signaling Technology 1:1000 TBP polyclonal purified Cell Signaling Technology 1:1000 CD4 GK1.5 biotin BD Pharmingen 1:300 CD8α 53-6.7 biotin BD Pharmingen 1:200 CD11c HL3 biotin BD Pharmingen 1:100 CD11b M1/70 biotin BD Pharmingen 1:500 CD19 1D3 biotin BD Pharmingen 1:300 NK1.1 PK136 biotin BD Pharmingen 1:200 Gr-1 RB6-8C5 biotin BD Pharmingen 1:500 TER119 TER119 biotin BD Pharmingen 1:200 B220 RA3-6B2 biotin BD Pharmingen 1:300 Sca-1 E13-161 Pacific Blue BD Pharmingen 1:300 CD25 PC61 PerCP Cy5.5 BD Pharmingen 1:200 Thy-1.2 53-2.1 PE BD Pharmingen 1:1000 c-kit 2B8 PE Cy7 ebioscience 1:200 FcεRI Mar-1 biotin ebioscience 1:200 IL-5 TRFK5 PE ebioscience see * IL-13 ebio13a APC ebioscience see * IL-7R A7R34 APC ebioscience 1:200 T1/ST2 DJ8 FITC MD Bioscience 1:200 IL-17RB B5F6 biotin Dr. Hiroshi Watarai see ** CD16/CD32 2.4G2 purified purified in house 1:200 For FACS analysis, 50μl of each diluted antibodies were added to 150μl of cell suspension. * 20μl of diluted antibodies were added to 80μl cell suspension for intracellular cytokine staining. ** 1μl of antibodies were directly added on the pellet.