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. 2023 Aug 7;220(10):e20230133. doi: 10.1084/jem.20230133

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© 2023 Shao et al.

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Figure 1. — d-cAMP promotes ILC3 activation and suppresses the expression and activity of FOXO1. (A) Proliferation of ILC3s was not affected by d-cAMP treatment. Viable ILC3s (Lin⁻NK1.1⁻KLRG1⁻CD45^lowCD127⁺CD90.2^hi, Lin = CD3, CD19, CD11b, CD11c, Gr-1, TER-119, TCRβ, TCRγδ) were isolated from the lamina propria of the small intestine of WT mice and treated with vehicle or the indicated concentrations of d-cAMP in RPMI1640 complete medium supplemented with 25 ng ml⁻¹ IL-7 and 25 ng ml⁻¹ IL-23 for 16 h. The cell counts of ILC3s were analyzed by flow cytometry and shown as means ± SD. ns, not significant, by one-way ANOVA. n = 5 for each group. (B) d-cAMP promoted the activation of ILC3s. The isolated ILC3s were treated with the indicated concentrations of d-cAMP. The levels of IL-17A and IL-22 in supernatant of cell culture were analyzed by ELISA and shown as means ± SD. ns, not significant, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001, by one-way ANOVA. n = 5 for each group. (C) d-cAMP elevated the levels of inflammatory cytokines of ILC3s. The isolated ILC3s were treated with the indicated concentrations of d-cAMP. The proportion and numbers of IL-17A–, IL-17F–, and IL-22–positive ILC3s were analyzed by flow cytometry and shown as means ± SD. ns, not significant, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001, by one-way ANOVA. n = 5 for each group. (ILC3 = Lin⁻NK1.1⁻KLRG1⁻CD45⁺CD127⁺RORγt⁺, Lin = CD3, CD19, CD11b, CD11c, Gr-1, TER-119, TCRβ, TCRγδ). (D and E) d-cAMP decreased the level of FOXO1 and promoted its phosphorylation. The isolated ILC3s were treated with the indicated concentrations of d-cAMP. Medians of fluorescence intensity (MFI) of FOXO1 (D) and phosphorylated FOXO1 (p-FOXO1) in ILC3s (E) were determined by flow cytometry and shown as means ± SD. The representative flow cytometry plots are shown on the left. ns, not significant, **, P < 0.01, ****, P < 0.0001, by one-way ANOVA. n = 5 for each group. (F) ILCs in small intestine from WT mice were isolated by Magnetic Cell Sorting system (BioLegend) and treated with vehicle or d-cAMP (50 μM) for 16 h. ILCs were stained with propidium iodide (PI) for cell nucleus and antibodies against FOXO1, Lin (CD3, CD19, CD11b, CD11c, Gr-1, TER-119, TCRβ, TCRγδ, NK1.1, and KLRG1), CD45, and RORγt. The FOXO1 nuclear translocation was examined by imaging flow cytometry. Scale bar, 5 μm. n = 5 for each group. Data represent at least three independent experiments.