IL-33 modulates inflammatory brain injury but exacerbates systemic immunosuppression following ischemic stroke

Abstract

Stroke triggers a complex inflammatory process in which the balance between pro- and antiinflammatory mediators is critical for the development of the brain infarct. However, systemic changes may also occur in parallel with brain inflammation. Here we demonstrate that administration of recombinant IL-33, a recently described member of the IL-1 superfamily of cytokines, promotes Th2-type effects following focal ischemic stroke, resulting in increased plasma levels of Th2-type cytokines and fewer proinflammatory (3-nitrotyrosine+F4/80+) microglia/macrophages in the brain. These effects of IL-33 were associated with reduced infarct size, fewer activated microglia and infiltrating cytotoxic (natural killer-like) T cells, and more IL-10-expressing regulatory T cells. Despite these neuroprotective effects, mice treated with IL-33 displayed exacerbated post-stroke lung bacterial infection in association with greater functional deficits and mortality at 24 hours. Supplementary antibiotics (gentamicin and ampicillin) mitigated these systemic effects of IL-33 after stroke. Our findings highlight the complex nature of the inflammatory mechanisms differentially activated in the brain and periphery during the acute phase after ischemic stroke. The data indicate that a Th2-promoting agent can provide neuroprotection without adverse systemic effects when given in combination with antibiotics.

Keywords: Cardiology; Immunotherapy; Inflammation; Mouse models; Stroke.

Figure 1. Infarct volume and functional outcome.

Representative coronal brain sections from mice treated with (A) vehicle (Veh), (B) recombinant IL-4, or (C) recombinant IL-33 after stroke (infarct areas are outlined in white). Total infarct volume (D), open field performance (E), hanging wire latency to fall (F), and clinical score at 24 hours after stroke (G). Data are presented as mean ± SEM in D–F and as median scores in G. *P < 0.05, 1-way ANOVA with Bonferroni’s post hoc test for D–F and Kruskal-Wallis test with Dunn’s post-hoc test for G. Infarct volume, n = 12–15; open field, n = 11–24; hanging wire, n = 12–29; and clinical score, n = 18–40.

Figure 2. Pro- and antiinflammatory cytokine expression in the brain and plasma after stroke.

Expression of (A) Il-1β, (B) Il-6, (C) Il-5, and (D) Il-13, in the ischemic hemisphere at 24 hours after stroke, quantified using qPCR. Circulating levels of (E) G-CSF, (F) TNF, (G) IL-6, and (H) IL-5 at 24 hours after stroke. Data are presented as mean ± SEM. *P < 0.05, 1-way ANOVA with Bonferroni post hoc test. Brain qPCR, n = 5–12; plasma cytokine: n = 2–12 (n = 2 for sham IL-33 only). Veh, Vehicle.

Figure 3. Leukocytes in the brain after stroke.

Flow cytometric quantification of (A) total leukocytes, (B) neutrophils, (C) monocytes, (D) CD4⁺ T cells, (E) CD8⁺ T cells, (F) CD4^–CD8^– double-negative T cells, (G) NK cells, (H) NKT cells, (I) CD4⁺ NKT cells, and (J) CD4^–CD8^– double-negative NKT cells in each hemisphere at 24 hours after stroke. (K) Immunohistochemical staining of CXCL16⁺ cells in the brain at 24 hours after stroke. Data are presented as mean ± SEM. *P < 0.05, 1-way ANOVA with Bonferroni’s post hoc test. Total leukocytes, n = 12–21; subsets, n = 8–13; CXCL16 immunohistochemistry, n = 8. S, sham; C, contralateral; I, ischemic; Veh, vehicle.

Figure 4. Microglial cells and macrophages in the brain after stroke.

Total number of microglial cells quantified using (A) flow cytometry and (B) immunohistochemical staining of CD68⁺ cells in the ischemic hemisphere at 24 hours after stroke. Total number of macrophages and/or microglia quantified using (C) flow cytometry and (D) immunohistochemical staining of F4/80⁺ cells in the ischemic hemisphere at 24 hours after stroke. (E) The number of F4/80⁺ cells that coexpress 3-nitrotyrosine (3-NT) in the ischemic hemisphere is also shown. Data are presented as mean ± SEM. *P < 0.05, 1-way ANOVA with Bonferroni’s post hoc test. Flow cytometry, n = 8–13; CD68, n = 8–10; F4/80 and 3-NT, n = 5–7. S, sham; C, contralateral; I, ischemic; M/M, microglia/macrophages.

Figure 5. Post-stroke infection and combined treatment with antibiotics.

(A) Mortality, (B) bacterial load in the lung at 24 hours after stroke, (C) clinical score, and (D) hanging wire latency to fall at 24 hours after stroke. Data are presented as mean ± SEM in B and D and as median score in C. *P < 0.05, 1-way ANOVA with Bonferroni’s post hoc test for B and D, and Kruskal-Wallis test with Dunn’s post hoc test for C. Bacterial load, n = 4–9; clinical score, n = 12–40; hanging wire, n = 11–25. Ab, antibiotics; ND, not detectable; Veh, vehicle; HW, hanging wire.

Figure 6. Infarct volumes in postischemic brains following combined treatment with antibiotics.

Representative coronal brain sections from mice treated with a combination of (A) vehicle and antibiotics, (D) vehicle alone, or (B and E) IL-33 and antibiotics after stroke; and total infarct volumes at (C) 24 hours or (F) 72 hours after stroke (infarct areas are outlined in white). *P < 0.05, Student’s unpaired t test. Infarct volume at 24 hours, n = 5–10; infarct volume at 72 hours, n = 13–14. Veh, vehicle; Ab, antibiotics.

Figure 7. Effects of low-dose IL-33 on functional outcomes and leukocyte subsets at 72 hours.

Survival (A) and clinical score (B) at 72 hours after stroke. Flow cytometric quantification of (C) FoxP3⁺CD4⁺ T cells, (D) IL-10⁺FoxP⁺CD4⁺ T cells, (E) percentage of ST2-expressing FoxP3⁺CD4⁺ T cells, (F) NK cells, and (G) IL-10⁺ NK cells in the ischemic hemisphere of IL-10^eGFPFoxp3^mRFP mice at 72 hours after stroke. Data are presented as mean ± SEM. *P < 0.05; Student’s unpaired t test. Flow cytometric analysis, n = 4–6. Veh, vehicle.