IL-33/ST2 Axis Protects Against Traumatic Brain Injury Through Enhancing the Function of Regulatory T Cells

Abstract

Traumatic brain injury (TBI) is a devastating condition due to its long-term sequelae on neurological functions. Inflammatory responses after TBI are critical for injury expansion and repair. Recent research in central nervous system (CNS) disorders reveals the importance of IL-33 and its receptor (ST2) as an alarmin system to initiate immune responses. This study explored the role of IL-33/ST2 signaling in TBI. TBI was induced in adult male C57BL/6J mice using a controlled cortical impact (CCI) model. We found that the expression of IL-33 increased in the injured brain and blood, and ST2 was elevated in the circulating and infiltrating regulatory T cells (Tregs) early after TBI. ST2 deficient mice exhibited reduced Treg numbers in the blood and brain 5 days after TBI. The brain lesion size was enlarged in ST2 knockout mice, which was accompanied by deteriorated sensorimotor function 5 days after TBI. In contrast, post-TBI treatment with IL-33 (2 μg/30 g body weight, intranasal) for 3 days significantly reduced brain lesion size and improved neurological functions 5 days after TBI. Meanwhile, IL-33 treatment increased ST2 expression in circulating and brain infiltrating Tregs. To further explore the involvement of Tregs in IL-33/ST2-mediated neuroprotection, Tregs were depleted by CD25 antibody injection. The absence of Tregs significantly reduced the protective effect of IL-33 after TBI. In vitro study confirmed that IL-33 (50 ng/ml) increased the production of IL-10 and TGFβ from activated Tregs and boosted the inhibitory effect of Tregs on T effector cell proliferation. Taken together, this study suggests that the activation of IL-33/ST2 signaling reduces brain lesion size and alleviates functional deficits after TBI at least partially through regulating the Treg response. IL-33 may represent a new immune therapeutic strategy to improve TBI outcomes.

Keywords: IL-33; ST2; neuroprotection; regulatory T cells; traumatic brain injury.

Figure 1

TBI results in increased expression of IL-33 and ST2. (A) Representative images of IL-33 (green) staining in a brain slice collected 5d after CCI. More IL-33⁺ cells were observed in the lesioned side (ipsilateral) of the brain. The region enclosed by the red box in the left image was enlarged in the right image. Scale bar, 150 μm. (B) Quantification of IL-33⁺ cells in the ipsilateral lesioned area and corresponding contralateral brain area 5d after CCI. (C, D) IL-33 protein levels were measured in the brain lysates (C) and blood (D) collected 1 and 3 days after CCI. (E) Double staining of IL-33 (green) in APC⁺ oligodendrocytes (red), GFAP⁺ astrocytes (red), Iba1⁺ microglia/macrophages (red) and NeuN⁺ neurons (red) in ipsilateral brains 5 days after CCI. Scale bar, 40 μm. Nuclei were stained blue with 4′,6-diamidino-2-phenylindole (DAPI). All images are representative of four animals. (F) Representative gating strategy for T lymphocytes, monocytes, neutrophils, and dendritic cells in the blood 5d after CCI. (G) Quantification of total CD3⁺ T cells, CD4⁺ T cells, CD4⁺CD25⁺Foxp3⁺ Tregs, and ST2⁺ Tregs in blood 5d after CCI or sham operation. (H) Quantification of total CD11b⁺ cells, CD11c⁺CD11b⁺ dendritic cells, CD11b⁺Ly6G⁺ neutrophils, and CD11b⁺F4/80⁺ macrophages in blood 5d after CCI or sham operation. *p < 0.05, **p < 0.001, ***p < 0.001, Student’s t test (B, C, G, H) or one-way ANOVA and post hoc Bonferroni (D).

Figure 2

ST2 knockout (KO) reduces Treg frequencies, enlarges brain lesion size and exacerbates functional deficits after TBI. (A) Representative gating strategy for CD4⁺ T cells, CD4⁺CD25⁺Foxp3⁺ Tregs, and ST2⁺ Tregs in the WT brain after CCI. (B) Quantification of CD4⁺CD25⁺Foxp3⁺ Tregs, ST2⁺ Tregs, and median fluorescence intensity (MFI) of ST2 in Tregs in the sham WT brain and WT brains collected 3d and 7d after CCI. n = 4/group. (C-M) Male C57/BL6 WT and ST2 KO mice were subjected to CCI. n = 6-8/group. (C) Representative images of crystal violet-stained brain sections collected 5d post-injury in WT and ST2 KO mice. Brain tissue loss was quantified. (D–H) Sensorimotor functions after CCI were assessed in WT and ST2 KO mice. (D) Adhesive removal test. The latency to remove the tape from the impaired forepaw was recorded. (E) Foot fault test was quantified as foot fault rate, which is the ratio of the total number of foot faults for the left forelimb to the total movement number of the left forelimb. (F) Hanging wire test. (G) Rotarod test. The latency to fall off the rotating rod was recorded. (H) Cylinder test. The asymmetric rate was calculated as described in Methods. (I) Quantification of total CD3⁺ T cells, CD4⁺ T cells, CD4⁺CD25⁺Foxp3⁺ Tregs, and MFI of ST2 in Tregs in the blood 5d after CCI in WT and ST2 KO mice. (J) Quantification of total CD11b⁺ cells, CD11c⁺CD11b⁺ dendritic cells, CD11b⁺Ly6G⁺ neutrophils, and CD11b⁺F4/80⁺ macrophages in the blood 5d after CCI in WT and ST2 KO mice. (K, L) Plasma IL-10 (K) and TGFβ (L) levels were measured by ELISA 5d after CCI in WT and ST2 KO mice. (M) Quantification of CD4⁺CD25⁺Foxp3⁺ Tregs and ST2⁺ Tregs in the brain 5d after CCI in WT and ST2 KO mice. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test (C, I, J, K, L, M), one-way ANOVA (B) or two-way repeated measures ANOVA (D–H) and post hoc Bonferroni.

Figure 3

IL-33 treatment reduces brain lesion and functional deficits after TBI and adjusts peripheral Treg responses. (A) Experimental design. C57/BL6 mice were treated with IL-33 (2 μg/30 g body weight) or the same volume of PBS vehicle intranasally 2h after CCI and repeated daily for 2 more days. n = 6-8/group. (B) Tissue loss was evaluated in coronal brain sections stained by crystal violet 5d after TBI. Representative images show six crystal violet-stained brain sections, spanning from 1.10 mm anterior to bregma to 1.94 mm posterior to bregma with the same interval. (C–H) Sensorimotor function was evaluated by the adhesive removal test (C, D), foot fault test (E), hanging wire test (F), rotarod test (G) and the cylinder test (H). (I) Flow cytometry quantification of total CD3⁺ T cells, CD4⁺ T cells, CD4⁺CD25⁺Foxp3⁺ Tregs, ST2⁺ Tregs, and MFI of ST2 in Tregs in blood 5d after CCI. (J) Quantification of total CD11b⁺ cells, CD11c⁺CD11b⁺ dendritic cells, CD11b⁺Ly6G⁺ neutrophils, and CD11b⁺F4/80⁺ macrophages in the blood 5d after TBI. (K–L) Plasma IL-10 (K) and TGFβ (L) levels were measured by ELISA 5d after CCI. (M–N) Quantification of CD4⁺CD25⁺Foxp3⁺ Tregs (M) and ST2⁺ Tregs in the brain 5d after CCI. (O) Expression levels of CD25, CTLA4 and GITR in Tregs in the brain 5d after CCI. *p < 0.05, **p < 0.01, ***p < 0.001. Student’s t test (I–N), one-way (B) or two-way repeated measures ANOVA (C–H) and post hoc Bonferroni.

Figure 4

Treg is essential for the neuroprotective effect of the IL-33/ST2 axis. (A) Experimental design. C57/BL6 mice were treated with isotype IgG Veh (300 µg) or anti-CD25 mAb (300 µg) 2d prior to CCI. Some anti-CD25 mAb-treated mice received IL-33 (2 μg/30 g body weight) intranasally 2h after CCI and repeated daily for two more days. n = 6-7/group. (B) Flow cytometry confirms the reduction of Tregs in blood of CD25 Ab treated mice 5d after CCI. (C) Left: representative brain sections of crystal violet staining show the tissue loss 5d post TBI. Right: Quantification of total volume of tissue loss. (D–I) Sensorimotor function was evaluated by the adhesive removal test (D, E), foot fault test (F), hanging wire test (G), rotarod test (H), and the cylinder test (I). (J, K) Plasma IL-10 (J) and TGFβ (K) levels were measured by ELISA 5d after CCI. *p < 0.05, **p < 0.01, ***p < 0.001. One-way (B, C, J, K) or two-way repeated measures ANOVA (D–I) and post hoc Bonferroni.

Figure 5

IL-33 enhances the production of anti-inflammatory cytokines from Tregs and boosts their suppressive effects on T effector cells. Tregs and Teff were prepared from the spleens collected from the WT or ST2 KO mice 5d after CCI. Tregs were cultured with anti-CD3 and anti-CD28 for 2 days followed by IL-33 (50 ng/ml) or PBS treatment for 24h. (A, B) IL-10 (A) and TGFβ (B) levels in conditioned media were measured by ELISA. One-way ANOVA and post hoc Sidak. (C) The effect of Tregs on Teff proliferation was measured by BrdU incorporation. Vehicle or IL-33 treated Tregs were added at a ratio of 1:1, 1:2, 1:4, 1:8, and 1:16, to the number of Teff. Cells were incubated for 2d. Suppression of Teff proliferation was determined using a BrdU cell proliferation kit. n = 8 per condition. (D) Flow cytometry analysis of CTLA4, GITR and CD25. n = 4. Two-way ANOVA and post hoc Bonferroni (A–C) and Student’s t test (D). *p < 0.05, **p < 0.01, ***p < 0.001.