Fig. 4: Strain-specific ILC2 phenotypes between CC030 and C57BL/6J implicate Ffar3 as a candidate regulator. | Nature Communications

Fig. 4: Strain-specific ILC2 phenotypes between CC030 and C57BL/6J implicate Ffar3 as a candidate regulator.

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

Fig. 4

A ILC2s from lungs of C57BL/6J and CC030 after 4 days of culture with IL-33, TSLP, and IL-2 (n = 3 per strain) ** p = 0.0016. B CFSE dilution between ILC2s after 48 h of culture with IL-33, TSLP, and IL-2. C Proliferation index and D division index after 48 h culture as in (B) (n = 4 per strain) **** p < 0.0001. E IL-13 in supernatant of ILC2s cultured per (A) (n = 3 per strain) ** p = 0.0018. F IL-13 expression by intracellular cytokine staining. MFI = mean fluorescence intensity (n = 3 per strain) * p = 0.011. G Representative flow plots of active caspase+ ILC2s, pre-gated on live cells (numbers above gates = %). H Quantification of active caspase+ ILC2s as in (G) (n = 4 per strain) *** p = 0.0004. I Representative flow plot of early/late apoptosis by Annexin V and propidium iodide (PI) staining on ILC2s 6 days post culture. J Early and late apoptotic K ILC2s (n = 5 per strain) *** p = 0.0004, ** p = 0.0019. L Apoptotic pathways enriched in naïve C57BL/6J vs CC030 ILC2s. M ILC2s as % of live lung cells and N as % of innate lymphocytes (Lineage⁻, CD90⁺) (n = 3 per strain) ** p = 0.0027, **** p < 0.0001. O IL-5 and P IL-13 in lung homogenate after 4 days of Alt Ex challenge (n = 4 C57BL/6J, 5 CC030) *** p = 0.0006, **** p < 0.0001. Q Total eosinophils in bronchoalveolar lavage (BAL) fluid after 4 days of Alt Ex challenge (n = 5 per strain, also in Supplementary Fig. 4 C) ** p = 0.0027. R Diagram implicating candidate genes in Ch.7 ILC2 locus. S Ffar3 expression by normalized counts from bulk RNA-seq (DESeq2 DGE analysis, Wald test (two-tailed) with Benjamini–Hochberg correction; **** p = 1.76 × 10−12). T qPCR for Ffar3 from RNA of flow-sorted naïve CC030 ILC2s from various tissues (n = 5 mice; one sample each from bone marrow, adipose, and colon had insufficient RNA). A, C–F, H, J, K, M–Q, S, T: bar height = mean, error bars = SD. A, C–F, M–P, S–T: each representative of two independent experiments, H, J, K, Q, S, T: each representative of one independent experiment. Significance for (A), (C–F), (H), (J, K), (L–Q), assessed by two-tailed unpaired Student’s t-test; for (T) by one-way ANOVA with Tukey multiple comparisons (two-tailed).

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