Fig. 8: FFAR3 signaling partially reprograms human ILC2s towards an anti-inflammatory effector state in vitro. | Nature Communications

Fig. 8: FFAR3 signaling partially reprograms human ILC2s towards an anti-inflammatory effector state in vitro.

From: Genetic diversity of Collaborative Cross mice implicates FFAR3 as a target for ILC2 anti-inflammatory reprogramming

Fig. 8

A Isolation of ILC2s from human PBMCs with representative gating for flow sorting (pre-gated on live, singlet lymphocytes). ILC2s from each donor evenly split between conditions (DMSO vehicle vs AR420626) and cultured with IL-33, TSLP, and IL-2 for 6 days. B Total ILC2s (n = 5 for 1 µM AR420626, n = 7 for 5 µM AR420626, n = 6 for 10 µM AR420626; same n for each endpoint in B–F). Values for AR:Veh = ratio of means between AR420626 and vehicle groups (5 µM: * p = 0.031, 10 µM: **** p < 0.0001). C Percent viability of ILC2s measured by propidium iodide (PI) negativity percentage. D Concentration of IL-5 (5 µM: ** p = 0.0018, 10 µM: * p = 0.031), E IL-13 (5 µM: ** p = 0.0033, 10 µM: ** p = 0.0080), and F IL-10 in the supernatant. Lowest values for IL-10 at each concentration = below the detection, multiple overlapping samples. G Representative flow plot of EGFR expression on human ILC2s with 5 µM AR420626 or vehicle. H Flow plot of EGFR expression on human ILC2s with IL-33, TSLP, and IL-2 at Days 4 and 6. I Total cell number and J viability (PI–) of human ILC2s cultured with IL-33, TSLP, IL-2 ± AR420626 and gefitinib at different doses (n = 4; gefitinib treatment showed no significant effect by binomial test). K IL-5 and L IL-13 in supernatant of human ILC2s (n = 4 IL-5; n = 3 IL-13). All IL-13 values for one donor were below detection. Statistical testing for K and L assessed whether gefitinib reversed AR420626-mediated IL-5 and IL-13 suppression. IL-5 (4/4) and IL-13 (3/3) levels were higher with at least one gefitinib dose compared to AR alone. Binomial testing yielded p = 0.0625 (IL-5) and p = 0.125 (IL-13). Shapiro–Wilk tests assessed normality of paired differences in (B–F, H). Normality not rejected: ratio paired Student’s t-test was used; rejected: Wilcoxon signed-rank test. Ratio two-tailed paired Student’s t-test for all analyses in (B–F, H) except 8.B–5 µM, 8.C–1 µM, 8.D–10 µM, and 8.F–5 µM, which used Wilcoxon signed-rank test (two-tailed). ns not significant.

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