Figure 6.
Impact of IL-33–mediated challenge on ILC2-associated epigenetic marker in lung and liver. (A) NK cells and ILC2 were isolated from the liver or lung of mice under homeostatic conditions or after i.p. treatment with 300 ng IL-33 for three consecutive days (liver) or i.n. treatment for three consecutive days with 250 ng IL-33 (lung). Graphs show frequencies and total numbers of Gata3+ ILC2 within Lin−CD127+ cells. Data shown were generated from n = 4–5 independent cell sorts with n = 7–8 mice per sort. Statistical significance was analyzed using unpaired two-tailed Student’s t test with *, P ≤ 0.05 and ****, P ≤ 0.0001. (B) Pyrosequencing results show the methylation value of selected ILC2-associated marker regions in liver NK cells and ILC2 of both naive and IL-33–treated mice. The mean methylation value of all CpG motifs within each region was calculated and transformed into a color-coded box, ranging from blue (100% methylation) and white (50% methylation) to yellow (0% methylation). Data for untreated mice were generated by pooling ILC2 from several cell sorts with n = 20–30 mice per sort. n = 2 independent experiments were conducted for IL-33 challenge with n = 16 mice. (C) ILC2 were isolated from the lung of naive or IL-33–challenged mice and restimulated with PMA/ionomycin in vitro for intracellular cytokine staining. Representative flow cytometry plots of Gata3+ ILC2 producing IL-5/IL-13 from control (PBS) and IL-33–challenged mice. Graphs show frequencies and total cell numbers of Gata3+IL-5+IL-13+ ILC2. Data shown from n = 2 independent experiments with n = 3 mice per group. Statistical significance was analyzed using unpaired two-tailed Student’s t test with *, P ≤ 0.05 and **, P ≤ 0.01. (D) Pyrosequencing results show the methylation value of ILC2-associated marker regions in lung NK cells and ILC2 of both naive and IL-33–treated mice. The mean methylation value of all CpG motifs within each region was calculated and transformed into a color-coded box, ranging from blue (100% methylation) and white (50% methylation) to yellow (0% methylation). Data for untreated mice were generated by pooling ILC2 from several cell sorts with n = 10–20 mice per sort. n = 2 independent experiments were conducted for IL-33 challenge with n = 5 mice.

Impact of IL-33–mediated challenge on ILC2-associated epigenetic marker in lung and liver. (A) NK cells and ILC2 were isolated from the liver or lung of mice under homeostatic conditions or after i.p. treatment with 300 ng IL-33 for three consecutive days (liver) or i.n. treatment for three consecutive days with 250 ng IL-33 (lung). Graphs show frequencies and total numbers of Gata3+ ILC2 within Lin−CD127+ cells. Data shown were generated from n = 4–5 independent cell sorts with n = 7–8 mice per sort. Statistical significance was analyzed using unpaired two-tailed Student’s t test with *, P ≤ 0.05 and ****, P ≤ 0.0001. (B) Pyrosequencing results show the methylation value of selected ILC2-associated marker regions in liver NK cells and ILC2 of both naive and IL-33–treated mice. The mean methylation value of all CpG motifs within each region was calculated and transformed into a color-coded box, ranging from blue (100% methylation) and white (50% methylation) to yellow (0% methylation). Data for untreated mice were generated by pooling ILC2 from several cell sorts with n = 20–30 mice per sort. n = 2 independent experiments were conducted for IL-33 challenge with n = 16 mice. (C) ILC2 were isolated from the lung of naive or IL-33–challenged mice and restimulated with PMA/ionomycin in vitro for intracellular cytokine staining. Representative flow cytometry plots of Gata3+ ILC2 producing IL-5/IL-13 from control (PBS) and IL-33–challenged mice. Graphs show frequencies and total cell numbers of Gata3+IL-5+IL-13+ ILC2. Data shown from n = 2 independent experiments with n = 3 mice per group. Statistical significance was analyzed using unpaired two-tailed Student’s t test with *, P ≤ 0.05 and **, P ≤ 0.01. (D) Pyrosequencing results show the methylation value of ILC2-associated marker regions in lung NK cells and ILC2 of both naive and IL-33–treated mice. The mean methylation value of all CpG motifs within each region was calculated and transformed into a color-coded box, ranging from blue (100% methylation) and white (50% methylation) to yellow (0% methylation). Data for untreated mice were generated by pooling ILC2 from several cell sorts with n = 10–20 mice per sort. n = 2 independent experiments were conducted for IL-33 challenge with n = 5 mice.

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