Anti-inflammatory effect of cholera toxin B subunit in experimental stroke

Background: Cholera toxin B subunit (CTB) has multifaceted immunoregulatory functions. Immunity plays an important role in the mechanism of stroke. However, little is known about whether CTB is beneficial for stroke. Methods: CTB was administered intraperitoneally after ischemia to rats subjected to transient focal ischemia. Infarct volumes, body weight loss, and neurologic deficits were measured. Cytokines, microglia/macrophage activation, and transcriptional factors in the ischemic brain were tested. The mRNA expressions of IL-1 beta and TNF-alpha were tested in the microglia/macrophage isolated from the ischemic hemisphere gamma delta T cells, IL-17-producing gamma delta T cells, Th17 cells, and regulatory T (Treg) cells in the ischemic brain were tested gamma delta T cells and Treg cells in the peripheral blood were also evaluated. Results: CTB reduced infarct volumes, neurologic deficits, and body weight loss after ischemia. At 24 h after ischemia, CTB downregulated the levels of IL-1 beta, TNF-alpha, NF-kB p65, phosphorylated-ERK1/2, and microglia/macrophage activation and suppressed NF-kB binding activity, but did not affect the level of ERK1/2. The mRNA expressions of IL-1 beta and TNF-alpha in the microglia/macrophage isolated from the ischemic hemisphere were suppressed after CTB therapy. In the ischemic hemisphere, CTB treatment reduced the levels of gamma delta T cells, IL-17-producing gamma delta T cells, and IL-17 at both 24 and 72 h after ischemia, while Th17 cells were not affected. After CTB treatment, the levels of Treg cells, TGF-beta, and IL-10 remained unchanged at 24 h and upregulated at 72 h after ischemia. Inactivation of Treg cells using anti-CD25 attenuated the increase of TGF-beta and IL-10 induced by CTB at 72 h after ischemia. In the peripheral blood, CTB increased Treg cells and suppressed gamma delta T cells at 24 h after ischemia. And then at 72 h after ischemia, it increased Treg cells but did not impact gamma delta T cells. CTB had no effect on cytokines, transcription factors, infiltrating gamma delta T cells, and Treg cells in the brain of shams. In the peripheral blood of shams, CTB increased Treg cells at both 24 and 72 h, while it did not affect gamma delta T cells. Conclusions: CTB decreased neurologic impairment and tissue injury after cerebral ischemia via its immunomodulatory functions, including inhibiting microglia/macrophage activation, suppressing gamma delta T cells, and inducing production of Treg cells, thus regulating the secretion of related cytokines. Suppression of NF-kB and ERK1/2 pathways is involved in the neuroprotective mechanism of CTB.