Fig. 6: DR3 signaling induces non-canonical NF-κB pathway in activated VAT-derived ILC2s.

In vivo activated VAT-derived murine ILC2s (aILC2s) were cultured in presence of recombinant mouse (rm) IL-2, rmIL-7 and rmIL-33 and stimulated with DR3 agonist (5 µg/mL) or isotype control for 24 h. Total RNA was isolated and sequenced. a Volcano plot comparison representing whole transcriptome gene expression of sorted WT ILC2s treated with either isotype control or DR3 agonist (5 µg/mL) for 24 h ex vivo. Differentially expressed genes (described as statistically significant adjusted p-value < 0.05) with changes of at least 1.5-fold change (FC) are shown in yellow (upregulated) and blue (downregulated). Relevant differentially expressed genes are identified. b Heat plot of all differentially expressed genes. c Selected cytokine and cytokine receptor genes and transcription factors (d) plotted as the normalized counts in isotype-treated cohort compared to DR3 stimulated cohort. Notable ILC2 related genes are labeled and highlighted in red. Gray area represents region of 1.5-fold change in gene expression. e Upregulated (red) and downregulated (green) genes in the canonical and non-canonical NF-κB pathways. f Representative expression of NF-κB p52 (f) and NF-κB p65 (g) in isolated ILC2s from mice challenged with IL-33 and cultured ex vivo for 24 hrs with DR3 agonist (red) or isotype control (blue), n = 6 mice. The staining FMO control is shown as grey. The corresponding quantification are presented as Mean Fluorescence Intensity (MFI), and the error bars denote the mean ± SD. Statistical analysis, two-tailed student’s t-test (f, g); n.s.: p-value of <0.05 was considered as non-significant.