Fig 2: Group 2 innate lymphoid cells (ILC2) elevate oxygen consumption (OCR) rate upon stimulation with activating cytokines. Bone marrow-derived group 2 innate lymphoid cells (ILC2) were seeded into Seahorse XFe96 microplates at a cell density of 100.000 cells/well and stimulated with either IL-7 only, IL-33 only, or a combination of IL-7 and IL-33 (A-C), or with IL-2 only, IL-33 only, or a combination of IL-2 and IL-33 (D-F). All cytokines were used at 10 ng/mL. After 24 hours of cytokine stimulation the Mito Stress Test assay was performed using the Seahorse Analyzer and oxygen consumption rates (OCR) (A, D), proton leak (B, E), basal and maximal respiration as well as the spare respiratory capacities (C, F) were determined. Data reporting the treatment with IL-33 are the same for (A-F). Data are shown as average ± standard deviation (SD) and are representative of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Fig 3: Determining cell seeding density for Seahorse metabolic assays. Bone marrow-derived group 2 innate lymphoid cells (ILC2) were seeded at three different cell densities (100.000 (100k), 75.000 (75k) or 50.000 (50k) per well) into Seahorse XFe96 microplates and stimulated with either IL-7 only (A), IL-2 only (B), IL-33 only (C), IL-7 and IL-33 (D), or with IL-2 and IL-33 (E). All cytokines were used at 10 ng/mL. After 24 hours of cytokine stimulation the Mito Stress Test assay was performed using the Seahorse Analyzer and oxygen consumption rates (OCR) were determined. Brightfield microscopy images of seeding densities of 100.000 cells/well are shown (A-E); size bars = 150mM. Data are shown as average ± standard deviation (SD) and are representative of three independent experiments.

Fig 4: Group 2 innate lymphoid cells (ILC2) augment ATP production upon stimulation with activating cytokines. (A-D) To determine ATP production bone marrow-derived group 2 innate lymphoid cells (ILC2) were seeded into Seahorse XFe96 microplates at a cell density of 100.000 cells/well and stimulated with either IL-7 only, IL-33 only, or a combination of IL-7 and IL-33 (A, B), or with IL-2 only, IL-33 only, or a combination of IL-2 and IL-33 (C, D). All cytokines were used at 10 ng/mL. After 24 hours of cytokine stimulation the Real-Time ATP Rate assay was performed using the Seahorse Analyzer. ATP production from oxidative phosphorylation as well as glycolysis were analyzed and depicted as a proportion of 100% and as absolute values. Data reporting the treatment with IL-33 are the same for (A-D). Data are shown as average ± standard deviation (SD) and are representative of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Fig 5: IL-33 inhibits tenocyte migration via MyD88 and TGFβ/Smad signaling in vitro and in vivo.(A) Flow cytometry quantification of ST2+ macrophages (CD11b+), T helper cells (CD4+), and tenocytes (ScxGFP+) isolated from injured neonatal tendons at 14 DPI. (B) Scratch assay quantification of tenocyte migration with cytokine treatment at 12 hours (n = 4 independent samples, one-way ANOVA with Tukey’s post hoc test). (C) Scratch assay quantification of tenocyte migration with MyD88 inhibition in the presence of IL-33. (D) Quantification of tenocytes treated with IL-33 for 2 hours and immunostained for phosphorylated SMAD2/3 (n = 160 to 200 cells from four independent samples, two-tailed Student’s t test) (n = 4 to 5 independent samples, one-way ANOVA with Tukey’s post hoc test). (E) ScxCreERT2; RosaT neonates were treated with recombinant IL-33. Immunostaining quantification of ScxLIN cells with IL-33 treatment at 14 DPI (n = 4 mice, two-tailed Student’s t test). (F) Nuclear immunostaining for pSMAD2/3 with IL-33 treatment at 14 DPI (n = 4 mice, two-tailed Student’s t test). For all quantifications, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.