Fig 2: IL-33 protects from mortality and epithelial barrier disruption during C. difficile infection. C57BL6 mice (a–f) or ST2-/- mice (f–i) were treated with an antibiotic cocktail prior to infection with R20291 and treated by IP injection with recombinant IL-33 (rIL-33) or vehicle control (PBS) for 5 days prior to infection. a Survival curves (b), weight loss (c), and clinical scores after infection and treatment. (d, e) Infected cecal tissue was examined with IL-33 treatment and without (day 3). d Representative epithelial damage (H&E) of treatment groups assessed by (e) blinded scoring of infected tissue (H&E). f Peak C. difficile bacterial burden in cecal contents after infection (day 2) of IL-33-treated mice or ST2-/- mice compared with the vehicle or wild-type controls. g–i ST2-/- on C57BL6 background and C57BL6 controls were cohoused for 3 weeks prior to R20291 infection. g Survival curves (h) weight loss and (i) clinical scores were assessed. a, g Comparison made by log rank test (a, n = 35, 37; g, n = 21, 20). b–e, h–i Comparison made by Student's t test (b, n = 39; c, n = 29, 30; e, n = 10; h, n = 27, 26; i, n = 20). f Comparisons for C. difficile burden were made by Mann–Whitney U tests (f, n = 11, 12, 8, 7). a–c, g–i The data combined from three independent experiments. d–f The data representative of three independent experiments. Statistical significance is demarked as *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bar is 50 µm. Error bar indicates SEM

Fig 3: IL-33-ILC2 crosstalk is protective during C. difficile infection. a–c Rag2-/- and Rag2-/-?c-/- were infected with R20291 and treated with or without IL-33. a Survival curves, (b) Rag2-/- weight loss, and (c) Rag2-/-?c-/- weight loss after IL-33 treatment and infection. d, e Immune cell profiling of ILCs within the colon of IL-33-treated or untreated mice during R20291 infection. ILCs were subsetted into ILC2s (Lin- CD45+ CD90+ CD127+ GATA+ ST2+); ILC1s (Lin- CD45+ CD90+ CD127+Tbet+), and ILC3s (Lin- CD45+ CD90+ CD127+RORyT+) by flow cytometry. d Representative dot plots of ILC subsets within the colon of PBS controls or IL-33-treated mice during infection (day 2). e Enumeration of the number of ILC2s in the colon of IL-33-treated C57BL6 or Rag2-/- mice during R20291 infection. f, g Adoptive transfer of ex vivo expanded and sorted purified ILC2s from uninfected IL-33 treated mice into naive Rag2-/-?c-/- mice. f Survival curves and (g) weight loss after ILC2 transfer and R20291 infection. h Pearson correlation between the frequency of ILC2s and the percent weight loss during R20291 infection. a, f Comparison made by log rank test (a, n = 6, 7, 10, 10; f, n = 11, 10). (b, c, e, g) Comparison made by two-tailed Student's t test (b, n = 8; c, n = 17,15; e, n = 10, 10, 4, 4; g, n = 10). h Comparison made by Spearman correlation (h, n = 39). a–c The data representative of two independent experiments. d–h The data representative of three independent experiments. Statistical significance is demarked as *P < 0.05, **P < 0.01, and ***P < 0.001. Error bar indicates SEM

Fig 4: IL-33 protects RAG2–/– mice and upregulates Gata3+ and ST2+ ILC2s.a–g RAG2–/– mice (C57BL/6 background) were treated with IL-33 and PBS followed by an amebic challenge. On day 3 post amebic challenge, mice were sacrificed to collect cecal content and cecal tissue. a Infection rate determined by amebic culture of cecal content. b E. histolytica DNA measured by qPCR from cecal content. c Weight loss. d Flow cytometry gating to determine the total number of ILC1, ILC2, and ILC3. e Total number of Gata3+ and ST2+ ILC2s in cecal tissue. f Number of ILC1. g Number of ILC3. a–c Data pooled from two independent experiments (n = 5–6). d–g Data representative of two independent experiments (n = 5–9). Statistical significance was determined by Fisher’s exact test and unpaired t test. Error bars indicate SEM.

Fig 5: The differential function of CCR2 and CCR4 in lung and gut ILC2s.a, b Deficiency of CCR2 abrogated the ILC2 counts in the lung (a) and gut (b). WT or Ccr2-/- mice were subjected to the i.p. injection of IL-25 or IL-33 followed by flow cytometry analysis. The cell frequency of indicated ILC2 subsets was calculated and shown as mean ± SD (right panel). (n = 3 for each group) (P = 0.0072, 0.0059, 0.0056 for a, P = 0.0040, 0.0066, 0.0012, 0.0196 for b). c CCR2 is required for the ILC2 relocalization from BM to the lung. ILC2Ps (Lin-CD127+Sca1+ST2+KLRG1-) and ILC2s (Lin-CD127+Sca1+ST2+KLRG1+) from BM of WT or Ccr2-/- mice were analyzed by flow cytometry. The indicated ILC2 subsets was calculated and shown as mean ± SD (right panel). (n = 3 for each group) (P = 0.0376, 0.0064, 0.0021). d Adoptive transfer of WT and Ccr2-/- ILC2Ps. ILC2Ps (Lin-CD127+Sca1+ST2+KLRG1-) were isolated from BM of WT and Ccr2-/- mice. WT and Ccr2-/- ILC2Ps were 1:1 mixed (5 × 104 for each) and i.v. injected into B-NDG mice. One week after transfer, the recipient mice were i.p. injected with 400 ng/mouse/day IL-33 for three days. The cell frequency of ILC2s in the lung and gut were calculated and shown as mean ± SD. (n = 3 for each group). (P = 0.0039, 0.0086, 0.0050 by Two-tailed unpaired Student’s t-test). The schematic diagram was created with BioRender.com. e Scatter plot comparing the gene expression pattern between WT ILC2s versus Ccr2-/- ILC2s from the lung and gut. ILC2s (Lin-CD45+CD127+KLRG1+) were isolated from WT or Ccr2-/- mice after treatment with IL-33 and subjected to bulk mRNA sequencing. Blue dots, upregulated genes in Ccr2-/- ILC2s; red dots, upregulated genes in WT ILC2s. Representative differentially expressed genes (DEGs) are depicted. f WT or Ccr4-/- mice were i.p. injected with IL-25 or IL-33 for three days. ILC2s from the lung and gut were analyzed by flow cytometry and cell frequency was shown as mean ± SD. (n = 3 for each group) (P = 0.0031 by Two-tailed unpaired Student’s t-test). g Comparison of gene expression in lung iILC2s from WT versus Ccr4-/- mice. iILC2s (Lin-CD45+CD127+ST2-KLRG1hi) were isolated from the lung of WT or Ccr4-/- mice after treatment with IL-25 followed by bulk mRNA sequencing. Blue dots, upregulated genes in Ccr4-/- ILC2s; red dots, upregulated genes in WT ILC2s. Representative DEGs are depicted. Data are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01 by Two-tailed unpaired Student’s t-test. Source data are provided as a Source Data file.