Figure 3. Direct effects of SCFAs on ILCs.

(a) Activation of signaling molecules (Arf2, Stat3, Stat5 and rS6) in ILCs. Rag1^−/− colonic LP cells were cultured for 4h with IL-7 and SCFAs, and phosphorylation of indicated molecules in Lin⁻ CD127⁺ CD90⁺ ILCs was assessed. (b) Effects of small molecule inhibitors of Gαi, PI3K, mTOR and MEK1/2 on SCFA-mediated ILC proliferation. Rag1^−/− spleen cells were cultured in an ILC3 condition (IL-7, IL-23 and IL-1β) for 4 days with the inhibitors, and numbers of Lin⁻ CD90⁺ CD127⁺ ILCs were determined by flow cytometry. (c) Detection of membrane PIP₃ in ILCs by expressing GFP-PH^Akt. CD90⁺ ILCs were enriched with magnetic sorting and were transfected with a plasmid expressing GFP-PH^Akt and activated for 16h with cytokines (IL-7, IL-1β and IL-23) and TSA (20 nM) followed by paraformaldehyde fixation and staining with anti-CD90 and DRAQ5 for confocal microscopy. (d) Ffar2-dependent phosphorylation of Atf2, Stat3, and Stat5 in ILCs. LI LP ILC3, isolated from WT and Ffar2^−/− mice, were stained for phosphorylation of the molecules. Pooled data obtained from at least 3 different experiments (n=4-6) are shown. *Significant differences (p<0.05) between groups.