Fig 2: The changes of IL-33 and ST2 expression after RNS. (A,B) RNS induced a reduction of IL-33 and ST2 expression in both cortex and hippocampus tissues. (C) The optical densities of the two types of protein bands mentioned above were quantitatively analyzed. (D) Real-time PCR analysis of IL-33 mRNA in both cortex and hippocampus tissues. (E) Immunofluorescence staining was performed to demonstrate the changes of IL-33 and ST2 expression in cortex tissue. Bar 50 μm. (F) Semi-quantitative analysis of IL-33 or ST2 positive cells relative to the total number of cells. The data were expressed as means ± SEM (n = 6). ##P < 0.01 vs. Sham + PBS group, #P < 0.05 vs. Sham + PBS group. **P < 0.01 vs. RNS + PBS group, *P < 0.05 vs. RNS + PBS group. No significance (NS) was observed between RNS + IL-33 group and RNS + PBS group. Experiments are representative of three independent experiments.

Fig 3: IL-33 dampens inflammation and skews gut type-2 immunity during CDI. a–c Immune profiling of myeloid cells within the colon of IL-33-treated or untreated mice. a, b Quantification of the frequency of N = neutrophils (CD45+ CD11b+ Ly6g+ Ly6c+), M = monocytes (CD45+ CD11b+ Ly6g- Ly6c+), and E = eosinophils (CD45+ CD11b+ SiglecF+ Ly6g−) in the colon of (a) antibiotic-treated mice or (b) R20291-infected mice. c Representative dot plots of myeloid cells within the colon of IL-33-treated (right plots) vs. untreated (left plots) mice. d, e Cytokine protein expression in cecal tissue during C. difficile infection. d Proinflammatory IL-1β, IL-6, and IL-23 expression and (e) anti-inflammatory IL-10, IL-4, and IL-5 expression with and without IL-33 treatment. f–h Enumeration of mucin+ goblet cells with and without IL-33 treatment by microscopy. f Blinded quantification of number of PAS+ goblet cells per colon crypt during R20291 infection (day 2). g, h Representative PAS+ goblet cell staining of the (g) vehicle or (h) IL-33 treated mice. a–e Comparison made by two-tailed student t test as described in methods (a, n = 7; b, n = 17,18, d, n = 17,16,8; e, n = 15,16; f, n = 9,10). The data representative of three independent experiments. b–e Two combined 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 negatively regulated RNS-induced ERS responses. (A,B) Administration of IL-33 alone reversed RNS-induced up-regulation of the expression level of ERS related-protein GRP-78, while Anti-IL-33 alone treatment further up-regulated the expression level of GRP-78. However, the combined treatment of IL-33 and Anti-IL-33 reversed the effect of IL-33 alone or Anti-IL-33 on the expression level of GRP-78 in both cortex and hippocampus at 72 h post-RNS. (C,D) Optical densities of the protein bands were quantitatively analyzed, and normalized with loading control β-actin. The data were expressed as means ± SEM (n = 4–6). ##P < 0.01 vs. Sham + PBS group, #P < 0.05 vs. Sham + PBS group. **P < 0.01 vs. PBS group, *P < 0.05 vs. PBS group. &P < 0.05 vs. RNS + IL-33 group. $$P < 0.01 vs. RNS + Anti-IL-33 group. Experiments are representative of three independent experiments.

Fig 5: 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).