Panel A shows Predicting Associated Transcription factors from Annotated Affinities (PASTAA) of differentially regulated genes from ILC2s of Il33 KO versus WT mice. Panel B presents a heatmap of transcription factors expressed in small intestinal ILC2s from Il33 KO and Il1rl1 KO compared with WT mice. Red indicates higher expression and blue indicates lower expression. Panel C shows normalized RNA-seq counts of the gene Prdm1 across Il33 KO, Il1rl1 KO, and WT groups, with reduced expression in KO mice. Panel D displays relative expression of Prdm1 in stimulated small intestinal ILC2s under indicated conditions, showing increased expression after IL-33 stimulation. Panels E and F show flow cytometry plots and quantification of Blimp1-YFP and Blimp1-eGFP expression in lung ILC2 cultures treated with IL-7 alone or IL-7 plus IL-33. IL-33 increases Blimp-1 expression. Panels G and H show Blimp-1 reporter expression in small intestine and mesenteric lymph node (mLN) ILC2s at steady state in reporter mice. Panel I presents histograms of Blimp1-YFP expression in lung ILC2s after PBS or rIL-33 treatment, showing higher expression with IL-33. Panel J shows relative Prdm1 expression in ILC2s from small intestine, lung, and mLN tissues. Panels K and L include western blot images and quantification of Blimp-1 protein in ILC2s from Il33 KO and Il1rl1 KO compared with WT mice, confirming reduced protein levels in KO mice. Panel M shows Prdm1 expression in small intestinal ILC2s from Nmur1Cre Prdm1fl/fl mice compared with control mice. Panels N and O show frequencies of ILC2s and eosinophils in small intestine, lung, and mLN from Nmur1Cre Prdm1fl/fl and control mice.
IL-33 regulates Blimp-1 in ILC2s. (A) Predicting associated transcription factors from annotated affinities (PASTAA) of differentially regulated genes from small intestinal ILC2s of Il33 KO vs. WT mice, showing potential transcription factor regulation. (B) Heatmap of transcription factors differentially regulated in small intestinal ILC2s of Il33 KO and Il1rl1 KO vs. WT mice. (C) Normalized counts of the gene Prdm1 across all KOs. (A–C)n = 4 mice per group. (D) Relative expression of Prdm1 in stimulated small intestinal ILC2s. Cells were stimulated for 3 days with IL-7, IL-7 + IL-33, or IL-7 + IL-25. Data are representative of two independent experiments; n = 3–5 mice per group. (E and F) Flow cytometric plots and quantification of Blimp-1–YFP (E) and Blimp-1–eGFP (F) expression in lung ILC2s following in vitro IL-7 and IL-33 or IL-7 only stimulation, assessed in WT and Blimp-1–YFP or Blimp-1–eGFP mice. (G and H) Flow cytometric plots and quantification of Blimp-1 expression in ILC2s from small intestine (Si) and lungs of Blimp-1–YFP (G) or Blimp-1–eGFP (H) reporter mice in steady state. (E–H)n = 3–4 mice per group. (I) Histograms of Blimp-1–YFP–expressing ILC2s from the lungs and mLN after stimulation with PBS (Ctrl) or rIL-33, n = 2–3 mice per group. (J) Relative expression of the gene Prdm1 in ILC2s from indicated tissues. Data are representative of two independent experiments; n = 3–4 mice per group. (K and L) Representative western blot from Il33−/− and WT mice (K) and from Il1rl1−/− and WT mice (L) and quantification of K and L. Data are representative of two independent experiments; n = 3–5 mice per group. (M) Relative expression of Prdm1 in small intestinal ILC2s from Prdm1fl/fl and Nmur1CrePrdm1fl/fl mice; n = 4 mice per group. (N and O) Quantification of flow cytometric analysis of ILC2s (N) and eosinophils (Eos) (O) from Nmur1CrePrdm1fl/fl and littermate control (Prdm1fl/fl) mice from the indicated organs. Data are representative of two independent experiments; n = 3–5 mice per group. Mean ± SD; Student’s t test (G, H, J, K, L, M, N, and O) or one-way ANOVA (C–F); ns, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData F2.