Fig 1: IL-25 administration restores BBB integrity following TBI. A-D Western blot analysis of TJs (ZO-1, Occludin, Claudin-5) in the ipsilateral cortex 3 days after TBI: Representative blots (A), ZO-1 quantification (B), Occludin quantification (C), Claudin-5 quantification (D) (n = 6 per group). E Immunofluorescence of ZO-1 (red) co-stained with CD31 (endothelial cells) (green) in the ipsilateral cortex 3 days after TBI. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. F Immunofluorescence of TUNEL (green) co-stained with CD31 (endothelial cells) (red) in the ipsilateral cortex 3 days after TBI. Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm. G Quantification of endothelial expression of ZO-1 in the ipsilateral cortex 3 days after TBI (n = 6 per group). H Quantification of endothelial expression of TUNEL in the ipsilateral cortex 3 days after TBI (n = 6 per group). I Representative macroscopic images of Evans Blue extravasation (blue areas) in brain tissue 3 days after TBI. J Quantitative analysis of Evans Blue leakage in ipsilateral hemispheres (n = 6 per group). K The brain water content of mice 3 days after TBI (n = 6 per group). L Representative T2-weighted MRI of whole brains 3 days after TBI. Data presented as mean ± SD; ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig 2: IL-25 expression peaked at day 3 after TBI and predominantly localized to neuron and BMECs. A Temporal profile of IL-25 expression in a murine TBI model. B-C IL-25 cytokine levels in ipsilateral cerebral cortex tissue (B) and serum (C) of mice (n = 6 per group). D Schematic workflow of TBI modeling and therapeutic administration in mice. E-F IL-25 cytokine levels in ipsilateral cerebral cortex tissue (E) and serum (F) of mice at 3 days after TBI (n = 6 per group). G Representative immunofluorescence images showing IL-25 (green) co-stained with NeuN (neuron), CD31 (endothelial cells), GFAP (astrocytes), or Iba-1 (microglia) (red) in the ipsilateral cortex 3 days after TBI and co-localization ratio (Pearsons) (n = 6 per group). Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. Data presentation: Mean ± SD; ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig 3: IL-13 improves BBB function by reducing CXCL-10 expression. A-B Cytokine array analysis identified CXCL-10 as one of the significantly downregulated cytokines in the IL-13-treated groups (n = 3 per group). C-D CXCL-10 expression in bEnd.3 cells subjected to OGD/R ± IL-25, IL-5, or IL-13: Representative Western blot (C) and quantification (D) (n = 6 per group). E-F CXCL-10 levels in ipsilateral cortex 3 days after TBI: Representative blot (E) and quantification (F) (n = 6 per group). G Experimental workflow of TBI modeling and therapeutic administration. H-K Western blot analysis of TJs (ZO-1, Occludin, Claudin-5) in the ipsilateral cortex 3 days after TBI: Representative blots (H), ZO-1 quantification (I), Occludin quantification (J), Claudin-5 quantification (K) (n = 6 per group). Data presented as mean ± SD; ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig 4: IL-25 improves neurological recovery after TBI. A Schematic workflow of TBI modeling, therapeutic administration, and behavioral assessment timeline. B-C Longitudinal assessment of sensorimotor function: mNSS (B), Latency to fall on accelerating rotarod (C) (n = 6 per group). D-G Morris Water Maze cognitive evaluation after TBI: Representative swim paths (D), Escape latency during acquisition trials (days 1–5) (E), Platform crossings during probe trial (days 6) (F), Time spent in target quadrant during probe trial (days 6) (G) (n = 6 per group). Data presented as mean ± SD; ns: not significant; when the TBI + PBS group was compared to the Sham group, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, when the TBI + IL-25 group was compared to the TBI + PBS group, *p < 0.05, **p < 0.01, **p < 0.01, ***p < 0.001
Fig 5: IL-25 activates ILC2s and stimulates the production of downstream factors IL-5/IL-13. A Single-Cell transcriptomic atlas of after TBI mice brain UMAP projection of 29,046 single cells isolated from cerebral cortex tissues of Sham (7,963), TBI + PBS (9,332 ), and TBI + IL-25 (11,751) groups. Colors represent distinct cell populations identified through unsupervised clustering. B Proportions of BBB-associated cells (endothelial cells, astrocytes, pericytes) across Sham, TBI + PBS, and TBI + IL-25 groups. C Heatmap visualization of differentially expressed genes in endothelial cells isolated from Sham, TBI + PBS, and TBI + IL-25 treatment groups. D KEGG network pathway enrichment analysis of differentially expressed genes in endothelial cells from the SHAM, TBI + PBS, and TBI + IL-25 groups. E Integrated KEGG pathway analysis of the three groups. F Cell-cell communication analysis depicting differential gene expression between endothelial cells and ILC2s. G Flow cytometry gating strategy for ILC2 identification: lymphocytes were selected by FSC/SSC, followed by detection of Lin-CD45+CD127+ST2+ cells. H-I Representative flow plots (H) and quantitative analysis (I) of cerebral cortex ILC2s at 3 days after TBI (n = 3 per group). J-M Cytokine levels in ipsilateral cerebral cortex tissue and serum at 3 days after TBI: IL-5 (cerebral cortex) (J), IL-5 (serum) (K), IL-13 (cerebral cortex) (L), IL-13 (serum) (M) (n = 6 per group). Data presented as mean ± SD; ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Supplier Page from Abcam for Recombinant Mouse IL-25 protein