Promoted Generation of T Helper 1-Like Regulatory T Cells After Transient Middle Cerebral Artery Occlusion in Type-2 Diabetic Mice

Abstract

ABSTRACT Background Regulatory T cells (Tregs) play a remarkable role in modulating post-ischemic neuroinflammation. However, the characteristics of Tregs in diabetic ischemic stroke remain unknown. Methods Transient middle cerebral artery occlusion (MCAO) was conducted on leptin receptor-mutated db/db mice and db/+ mice. The number, cytokine production, and signaling features of Tregs in peripheral blood and ipsilateral hemispheres were evaluated by flow cytometry. Treg plasticity was assessed by the adoptive transfer of splenic Tregs into mice. The effect of ipsilateral macrophages/microglia on Treg plasticity was determined by in vitro co-culture analysis. Results db/db mice had more infiltrating Tregs in their ipsilateral hemispheres than db/+ mice. Infiltrating Tregs in db/db mice expressed higher transforming growth factor-β (TGF-β), interleukin-10 (IL-10), forkhead box P3 (Foxp3), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and T-box expressed in T cells (T-bet) in comparison to infiltrating Tregs in db/+ mice, suggesting promoted generation of T helper 1 (Th1)-like Tregs in the brains of db/db mice after stroke. The post-ischemic brain microenvironment of db/db mice significantly up-regulated IFN-γ, TNF-α, T-bet, IL-10, and TGF-β in infiltrating Tregs. Moreover, ipsilateral macrophages/microglia remarkably enhanced the expression of IFN-γ, TNF-α, and T-bet but not IL-10 and TGF-β in Tregs. db/db macrophages/microglia were more potent in up-regulating IFN-γ, TNF-α, and T-bet than db/+ macrophages/microglia. Interleukin-12 (IL-12) blockage partially abolished the modulatory effect of macrophages/microglia on Tregs. Conclusion The generation of Th1-like Tregs was promoted in the brains of type 2 diabetic mice after stroke. Our study reveals significant Treg plasticity in diabetic stroke. Abbreviations: Foxp3: forkhead box P3; IFN-γ: interferon-γ; IL-10: interleukin-10; IL-12: interleukin-12; MCAO: middle cerebral artery occlusion; PBS: phosphate-buffered saline; STAT1: Signal transducer and activator of transcription 1; STAT5: Signal transducer and activator of transcription 1; T-bet: T-box expressed in T cells; TGF-β: transforming growth factor-β; Th1: T helper 1; TNF-α: tumor necrosis factor-α; Tregs: regulatory T cells. Foxp3: forkhead box P3; IFN-γ: interferon-γ; IL-10: interleukin-10; IL-12: interleukin-12; MCAO: middle cerebral artery occlusion; PBS: phosphate-buffered saline; STAT1: Signal transducer and activator of transcription 1; STAT5: Signal transducer and activator of transcription 1; T-bet: T-box expressed in T cells; TGF-β: transforming growth factor-β; Th1: T helper 1; TNF-α: tumor necrosis factor-α; Tregs: regulatory T cells.