Fig 2: Immunometabolic regulation of ILCs after TBI.(A) Phosphorylated AMPKα1 (p-AMPKα1), a measure of AMPK activation, was assessed in meningeal cells at 24 hours after sham/TBI in WT mice. Isolated meninges were assessed by forward scatter (FSC)/side scatter (SSC), and selected populations were further analyzed for p-AMPKα. Scatterplots depicting the % total p-AMPKα+ cells are indicative of suppressed AMPK activation within the meninges after TBI. (B) AMPKα1–global KO (AMPKα1–/–) mice showed higher frequencies of all ILC subtypes after TBI, as compared with WT mice, with most pronounced increases noted for ILC1 and ILC3. (C) Intracisternal administration of IL-33 (1 μg) increased meningeal expression of p-AMPKα after TBI, as compared with placebo treatment in both WT mice and in Rag1–/– mice, which lack mature B and T lymphocytes, but possess functional ILC. Conversely, p-AMPKα was unaffected by IL-33 treatment in Rag1–/– IL2rg–/– mice, which lack both mature lymphocytes and ILC2. (D) Intracisternal administration of IL-33 (1 μg) increased meningeal expression of ILC2 and suppressed both ILC1 and ILC3 expansion at day 5 after TBI in WT and Rag1–/– mice, as compared with placebo (PBS). In contrast, IL-33 did not affect ILC number in Rag1–/– IL2rg–/– mice, which lack ILC2. Meningeal tissue was analyzed by flow cytometry. (E) The stimulatory effects of intracisternal IL-33 on ILC2 frequency were lost in AMPKα1–/– mice, as compared with WT mice. For all panels, quantified data are presented as the mean ± SD from n = 6 mice/group. For each panel, data were compared within each ILC subtype using a 2-tailed Student’s t test (*P < 0.05, **P < 0.01, ****P < 0.0001).

Fig 3: 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 4: IL-33 is upregulated by host in response to C. difficile infection. Mice were infected with R20291 (CDT+) or attenuated R20291_cdtb (CDT-) and whole-cecal tissue transcriptomic analyses was performed on day 3 post infection. a Heatmap of genes upregulated (red) or downregulated (blue) in response to CDT toxin. b Volcano plot highlighting IL-33 (blue) among genes altered (>0.5 logFC = red). c ELISA of IL-33 protein within the cecal tissue (day 3) of R20291 infected, R20291_cdtb infected, or uninfected antibiotic-treated (ABX) mice. d Enriched pathways of the top upregulated transcripts (log FC > 0.5; p < 0.05) listed by significance (−log10(p)) and created using the ConsensusPathDP database. (a, b, n = 5 biologically independent animals). c Comparison made by ANOVA (n = 13, 13, 20 combined from two independent experiments). Individual n numbers in each panel are shown in same order as their corresponding groups from left to right, and this arrangement is applied to other relevant figures in the text. Statistical significance is demarked as *P < 0.05, **P < 0.01, and ***P < 0.001, throughout the text. Error bar indicates SEM

Fig 5: IL-33 signaling is dysregulated during human CDI and targetable with FMT or HSP therapy. a–c Immunohistochemistry staining of IL-33 from colon tissue biopsies of six CDI− patients and six CDI+ patients. a Representative image from a CDI− patient biopsy. b Representative image from a CDI+ patient biopsy. c Quantification of IL-33-positive cells per field view taken as an average of five blinded images from each patient biopsy and quantified on Image-J. d–f Analysis of systemic sST2 (IL-33 decoy receptor) in the serum of CDI patients by ELISA. d Patients were stratified based on their WBC into severe vs. non-severe CDI, and sST2 was assessed between the two groups. e Spearman correlation between WBC and sST2 concentration. f Survival curves of CDI+ patients stratified into high (sST2 > 55,000 pg/ml) or low sST2 (sST2 < 55,000 pg/ml). g–i Mice treated with a broad-spectrum antibiotic cocktail (ABX) were orally gavaged with a (g) murine FMT 2x or a (i) purified human spore preparation (HSP) isolated from a normal human donor and IL-33 protein within the cecal tissue was measured by ELISA. h Microbiota diversity in cecal contents of untreated donors and antibiotic-treated FMT recipients (Simpson index y axis). c, d Comparison made by Mann–Whitney test (c, n = 6, d, n = 109, 53). e Comparison made by Spearman correlation. f Comparison made by log rank test (e, f, n = 160). g–i Comparison made by ANOVA for multiple comparisons (g, n = 17,16, 8, 10; h, n = 12,10,10; i, n = 5). g–h Data representative of two 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