ST2, amemberoftheinterleukin(IL)1receptorfamily,anditsligandIL-33playcriticalrolesinimmuneregulationandinflammatoryresponses. This study explores the roles of endogenous IL-33/ST2 signaling in ischemic brain injury and elucidates the underlying mechanisms of action. The expression of IL-33 rapidly increased in oligodendrocytes and astrocytes after 60 min transient middle cerebral artery occlusion (tMCAO). ST2 receptor deficiency exacerbated brain infarction 3 d after tMCAO as well as distal permanent MCAO. ST2 deficiency also aggravated neurologicaldeficitsupto7daftertMCAO.Conversely,intracerebroventricularinfusionsofIL-33aftertMCAOattenuatedbraininfarction.Flow cytometry analyses demonstrated high levels of ST2 expression on microglia, and this expression was dramatically enhanced after tMCAO. The absence of ST2 enhanced the expression of M1 polarization markers on microglia/macrophages, and impaired the expression of M2 polarization markers after tMCAO. In vitro studies on various types of cultures and coculture systems confirmed that IL-33/ST2 signaling potentiated expression of IL-10 and other M2 genes in primary microglia. The activation of ST2 on microglia led to a protective phenotype that enhanced neuronal survival against oxygen glucose deprivation. Further in vitro studies revealed that IL-33-activated microglia released IL-10, and that this was critical for their neuroprotective effects. Similarly, intracerebroventricular infusions of IL-33 into IL-10 knockout mice failed to provide neuroprotection against tMCAO in vivo. These results shed new light on the IL-33/ST2 axis as an immune regulatory mechanism that serves as a natural brake on the progression of ischemic brain injury.

Inflammation mediated by the peripheral infiltration of inflammatory cells plays an important role in intracerebral hemorrhage (ICH) induced secondary injury. Previous studies have indicated that regulatory T lymphocytes (Tregs) might reduce ICH-induced inflammation, but the precise mechanisms that contribute to ICH-induced inflammatory injury remain unclear. Our results show that the number of Tregs in the brain increases after ICH. Inducing Tregs deletion using a CD25 antibody or Foxp3 DTR -mice increased neurological deficient scores (NDS), the level of inflammatory factors, hematoma volumes, and neuronal degeneration. Meanwhile, boosting Tregs using a CD28 super-agonist antibody reduced the inflammatory injury. Furthermore, Tregs depletion shifted microglia/macrophage polarization toward the M1 phenotype while boosting Tregs shifted this transition toward the M2 phenotype. In vitro, a transwell co-culture model of microglia and Tregs indicated that Tregs changed the polarization of microglia, decreased the expression of MHC-II, IL-6, and TNF-a and increased CD206 expression. IL-10 originating from Tregs mediated the microglia polarization by increasing the expression of Glycogen Synthase Kinase 3 beta (GSK3b), which phosphorylates and inactivates Phosphatase and Tensin homologue (PTEN) in microglia, TGF-b did not participate in this conversion. Thus, Tregs ameliorated ICH-induced inflammatory injury by modulating microglia/macrophage polarization toward the M2 phenotype through the IL-10/GSK3b/PTEN axis.

White matter (WM) occupies a large volume of the human cerebrum and is mainly composed of myelinated axons and myelin-producing glial cells. The myelinated axons within WM are the structural foundation for efficient neurotransmission between cortical and subcortical areas. Similar to neuron-enriched gray matter areas, WM undergoes a series of changes during the process of aging. WM malfunction can induce serious neurobehavioral and cognitive impairments. Thus, age-related changes in WM may contribute to the functional decline observed in the elderly. In addition, aged WM becomes more susceptible to neurological disorders, such as stroke, traumatic brain injury (TBI), and neurodegeneration. In this review, we summarize the structural and functional alterations of WM in natural aging and speculate on the underlying mechanisms. We also discuss how age-related WM changes influence the progression of various brain disorders, including ischemic and hemorrhagic stroke, TBI, Alzheimer’s disease, and Parkinson’s disease. Although the physiology of WM is still poorly understood relative to gray matter, WM is a rational therapeutic target for a number of neurological and psychiatric conditions.

Delayed thrombolytic treatment with recombinant tissue plasminogen activator (tPA) may exacerbate blood-brain barrier breakdown after ischaemic stroke and lead to lethal haemorrhagic transformation. The immune system is a dynamic modulator of stroke response, and excessive immune cell accumulation in the cerebral vasculature is associated with compromised integrity of the blood-brain barrier. We previously reported that regulatory T cells, which function to suppress excessive immune responses, ameliorated blood-brain barrier damage after cerebral ischaemia. This study assessed the impact of regulatory T cells in the context of tPA-induced brain haemorrhage and investigated the underlying mechanisms of action. The number of circulating regulatory T cells in stroke patients was dramatically reduced soon after stroke onset (84 acute ischaemic stroke patients with or without intravenous tPA treatment, compared to 115 age and gender-matched healthy controls). Although stroke patients without tPA treatment gradually repopulated the numbers of circulating regulatory T cells within the first 7 days after stroke, post-ischaemic tPA treatment led to sustained suppression of regulatory T cells in the blood. We then used the murine suture and embolic middle cerebral artery occlusion models of stroke to investigate the therapeutic potential of adoptive regulatory T cell transfer against tPA-induced haemorrhagic transformation. Delayed administration of tPA (10 mg/kg) resulted in haemorrhagic transformation in the ischaemic territory 1 day after ischaemia. When regulatory T cells (2 × 106/mouse) were intravenously administered immediately after delayed tPA treatment in ischaemic mice, haemorrhagic transformation was significantly decreased, and this was associated with improved sensorimotor functions. Blood-brain barrier disruption and tight junction damages were observed in the presence of delayed tPA after stroke, but were mitigated by regulatory T cell transfer. Mechanistic studies demonstrated that regulatory T cells completely abolished the tPA-induced elevation of MMP9 and CCL2 after stroke. Using MMP9 and CCL2 knockout mice, we discovered that both molecules partially contributed to the protective actions of regulatory T cells. In an in vitro endothelial cell-based model of the blood-brain barrier, we confirmed that regulatory T cells inhibited tPA-induced endothelial expression of CCL2 and preserved blood-brain barrier integrity after an ischaemic challenge. Lentivirus-mediated CCL2 knockdown in endothelial cells completely abolished the blood-brain barrier protective effect of regulatory T cells in vitro. Altogether, our studies suggest that regulatory T cell adoptive transfer may alleviate thrombolytic treatment-induced haemorrhage in stroke victims. Furthermore, regulatory T cell-afforded protection in the tPA-treated stroke model is mediated by two inhibitory mechanisms involving CCL2 and MMP9. Thus, regulatory T cell adoptive transfer may be useful as a cell-based therapy to improve the efficacy and safety of thro...

The results presented here show that increased hepcidin expression caused by inflammation prevents brain iron efflux via inhibition of the intracellular iron efflux of brain microvascular endothelial cells entering into circulation and aggravating oxidative brain injury and cognition impairment, which identifies a mechanistic target for muting inflammation to promote brain iron efflux and to attenuate oxidative brain injury after ICH.

The repair of white matter damage is of paramount importance for functional recovery after brain injuries. Here, we report that interleukin-4 (IL-4) promotes oligodendrocyte regeneration and remyelination. IL-4 receptor expression was detected in a variety of glial cells after ischemic brain injury, including oligodendrocyte lineage cells. IL-4 deficiency in knockout mice resulted in greater deterioration of white matter over 14 d after stroke. Consistent with these findings, intranasal delivery of IL-4 nanoparticles after stroke improved white matter integrity and attenuated long-term sensorimotor and cognitive deficits in wild-type mice, as revealed by histological immunostaining, electron microscopy, diffusion tensor imaging, and electrophysiology. The selective effect of IL-4 on remyelination was verified in an ex vivo organotypic model of demyelination. By leveraging primary oligodendrocyte progenitor cells (OPCs), microglia-depleted mice, and conditional OPC-specific peroxisome proliferator-activated receptor gamma (PPARγ) knockout mice, we discovered a direct salutary effect of IL-4 on oligodendrocyte differentiation that was mediated by the PPARγ axis. Our findings reveal a new regenerative role of IL-4 in the central nervous system (CNS), which lies beyond its known immunoregulatory functions on microglia/macrophages or peripheral lymphocytes. Therefore, intranasal IL-4 delivery may represent a novel therapeutic strategy to improve white matter integrity in stroke and other brain injuries.

Regulatory T cells (Tregs) are known to protect against ischemic stroke. However, the low frequency of Tregs restricts their clinical utility. This study investigated whether expanding the number of Tregs in vivo with the IL-2/IL-2 antibody complex (IL-2/IL-2Ab) could improve stroke outcomes and further elaborated the mechanisms of protection in male mice. C57BL/6 mice received IL-2/IL-2Ab or isotype IgG (IsoAb) intraperitoneally for 3 d before (pretreatment) or starting 2 h after (posttreatment) 60 min middle cerebral artery occlusion (MCAO). IL-2/IL-2Ab selectively increased the number of Tregs in the blood, spleen, and lymph nodes. The IL-2/IL-2Ab treatment significantly reduced infarct volume, inhibited neuroinflammation, and improved sensorimotor functions, as manifested by rotarod test and foot fault test, compared with IsoAb-treated stroke mice. Treg depletion was then achieved by diphtheria toxin (DT) injection into transgenic mice expressing the DT receptor under the control of the Foxp3 promoter (DTR mice). The depletion of Tregs completely eliminated IL-2/IL-2Ab-afforded neuroprotection. Interestingly, adoptive transfer of Tregs collected from IL-2/IL-2Ab-treated mice demonstrated more potent neuroprotection than an equal number of Tregs prepared from IsoAb-treated mice, suggesting that IL-2/IL-2Ab not only elevated Treg numbers, but also boosted their functions. Mechanistically, IL-2/IL-2Ab promoted the expression of CD39 and CD73 in expanded Tregs. CD73 deficiency diminished the protective effect of IL-2/IL-2Ab-stimulated Tregs in stroke mice. The results show that IL-2/IL-2Ab expands Tregs in vivo and boosts their immunomodulatory function. The activation of CD39/CD73 signaling in Tregs may participate as a potential mechanism underlying IL-2/IL-2Ab-afforded neuroprotection against ischemic brain injury.

BackgroundThe bumetanide (BMT)-sensitive Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) maintains cell volume homeostasis by increasing intracellular K+ and Cl- content via regulatory volume increase (RVI). Expression levels of NKCC1 positively correlate with the histological grade and severity of gliomas, the most common primary adult brain tumors, and up-regulated NKCC1 activity facilitates glioma cell migration and apoptotic resistance to the chemotherapeutic drug temozolomide (TMZ). However, the cellular mechanisms underlying NKCC1 functional up-regulation in glioma and in response to TMZ administration remain unknown.MethodsExpression of NKCC1 and its upstream kinases With-No-K (Lysine) kinase 1 (WNK1) and oxidative stress-responsive kinase-1 (OSR1) in different human glioma cell lines and glioma specimens were detected by western blotting and immunostaining. Live cell imaging and microchemotaxis assay were applied to record glioma cell movements under different treatment conditions. Fluorescence indicators were utilized to measure cell volume, intracellular K+ and Cl- content to reflect the activity of NKCC1 on ion transportation. Small interfering RNA (siRNA)-mediated knockdown of WNK1 or OSR1 was used to explore their roles in regulation of NKCC1 activity in glioma cells. Results of different treatment groups were compared by one-way ANOVA using the Bonferroni post-hoc test in the case of multiple comparisons.ResultsWe show that compared to human neural stem cells and astrocytes, human glioma cells exhibit robust increases in the activation and phosphorylation of NKCC1 and its two upstream regulatory kinases, WNK1 and OSR1. siRNA-mediated knockdown of WNK1 or OSR1 reduces intracellular K+ and Cl- content and RVI in glioma cells by abolishing NKCC1 regulatory phospho-activation. Unexpectedly, TMZ activates the WNK1/OSR1/NKCC1 signaling pathway and enhances glioma migration. Pharmacological inhibition of NKCC1 with its potent inhibitor BMT or siRNA knockdown of WNK1 or OSR1 significantly decreases glioma cell migration after TMZ treatment.ConclusionTogether, our data show a novel role for the WNK1/OSR1/NKCC1 pathway in basal and TMZ-induced glioma migration, and suggest that glioma treatment with TMZ might be improved by drugs that inhibit elements of the WNK1/OSR1/NKCC1 signaling pathway.