Minocycline Alleviates White Matter Injury following Intracerebral Hemorrhage by Regulating CD4+ T Cell Differentiation via Notch1 Signaling Pathway

Abstract

Neuroinflammation is a major reason for white matter injury (WMI) after intracerebral hemorrhage (ICH). Apart from microglia/macrophage activation, T cells also play an important role in regulating immune responses after ICH. In a previous study, we have revealed the role of minocycline in modulating microglia/macrophage activation after ICH. However, the exact mechanisms of minocycline in regulating T cells differentiation after ICH are still not well understood. Hence, this study explored the relationship between minocycline and CD4+ T cell differentiation after ICH. Piglet ICH model was used to investigate naive CD4+ T cell differentiation and T cells signal gene activation after ICH with immunofluorescence and whole transcriptome sequencing. Naive CD4+ T cells and primary oligodendrocyte coculture model were established to explore the effect and mechanism of minocycline in modulating CD4+ T cell differentiation after ICH. Flow cytometry was used to indicate CD4+ T cell differentiation after ICH. The mechanism of minocycline in modulating CD4+ T cell differentiation was demonstrated with immunofluorescence and western blot. Double immunostaining of representative CD4+ T cell marker CD3 and different subtype CD4+ T cell assisted proteins (IL17, IL4, Foxp3, and IFNγ) demonstrated naive CD4+ T cell differentiation in piglet after ICH. Whole transcriptome sequencing for perihematomal white matter sorted from piglet brains indicated T cell signal gene activation after ICH. The results of luxol fast blue staining, immunofluorescent staining, and electron microscopy showed that minocycline alleviated white matter injury after ICH in piglets. For our in vitro model, minocycline reduced oligodendrocyte injury and neuroinflammation by regulating CD4+ T cell differentiation after ICH. Moreover, minocycline increased the expression of NOTCH1, ACT1, RBP-J, and NICD1 in cultured CD4+ T cell when stimulated with hemoglobin. Hence, minocycline treatment could modulate naive CD4+ T cell differentiation and attenuate white matter injury via regulating Notch1 signaling pathway after ICH.