Fig. 4: Paneth cells respond to IL-33 by promoting regeneration with EGF.

a Egf expression in WT and IL-33 KO mouse small intestine (SI) at baseline and five days after TBI (10 Gy), measured by qPCR (n = 6 mice/group, combined from two independent experiments). b Villous and crypt expression of Egf at baseline and five days after TBI (n = 6 mice/group, combined from two experiments). c RiboTag hemagglutinin (HA) lineage labeling. Left: immunofluorescence of HA⁺ cells 20 h (Olfm4-Ribo) or 5 days (Lyz-Ribo) after tamoxifen injection in vivo; anti-HA staining in pink for Olfm4-Ribo (with anti-lysozyme co-stain in green) and anti-HA staining in red for Lyz-Ribo; representative of staining from 5 mice. Right: qPCR profiling of the ISC compartment using anti-HA immunoprecipitates from RiboTag mice. d qPCR of RiboTag-isolated mRNA from ISCs and Paneth cells at baseline and five days after TBI (10 Gy); Egf, n = 4 mice/group from two experiments; Il1rl1 (sST2), n = 4 mice/group from two experiments; Il33, n = 7 mice/group from three experiments. e qPCR for Egf in SI organoids and in sort-purified Paneth cells +/- exposure to IL-33 (n = 8 wells/group). f, g Quantification and imaging of Lgr5-LacZ⁺ ISCs in ileum five days after TBI (10 Gy). Representative images show anti-β-gal immunofluorescent staining (green) and DAPI nuclear stain (blue) five days after TBI (10 Gy). f WT reporter mice +/− gefitinib treatment (1 mg/mouse) or vehicle (daily for three days starting the day of irradiation). Data combined from two experiments (n = 6 mice/group). g IL-33 KO reporter mice +/− treatment with EGF (10 µg/mouse) or PBS (daily for three days starting the day of irradiation). Data combined from two experiments (n = 6 mice/group). h Proposed model of IL-33-mediated regulation of regeneration within the ISC compartment after radiation injury: IL-33 was produced by epithelial stem cells after irradiation, and Paneth cells responded to IL-33 by increasing production of EGF, driving epithelial proliferation, crypt maintenance, and ISC recovery after damage. IL-33 also induced a negative feedback response, promoting expression of its own negative regulator sST2. Comparisons performed using a two-tailed Mann-Whitney U test (b, d), Kruskal-Wallis multiple comparison testing (a, e), or ANOVA multiple comparison testing (f, g); *p < 0.05, **p < 0.01, ***p < 0.001; scale bars: 100 µm. Source data for graphs are provided in the Source Data file. The exact p values are as follows: (a) WT vs KO, 10 Gy, p = 0.0003; (b) Villi, CTRL vs 10 Gy, p = 0.98; crypts, CTRL vs 10 Gy, p = 0.026; (d) Egf, Olfm4 Unirradiated vs 10 Gy, p = 0.17; Egf, Lyz Unirradiated vs 10 Gy, p = 0.026; sST2, Olfm4 Unirradiated vs 10 Gy, p = 0.23; sST2, Lyz Unirradiated vs 10 Gy, p = 0.029; Il33, Olfm4 Unirradiated vs 10 Gy, p = 0.0012; Il33, Lyz Unirradiated vs 10 Gy, p > 0.99; (e) CTRL vs Organoids, p = 0.047; CTRL vs IL-33, p = 0.047; (f) Unirradiated vs Vehicle, p = 0.0092; Unirradiated vs Gefinitib, p = < 0.0001; Vehicle vs Gefinitib, p = 0.03; (g) Unirradiated vs Vehicle, p = < 0.0001; Unirradiated vs EGF, p = 0.0022; Vehicle vs EGF, p = 0.011.