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Immunosuppression commonly occurs after a stroke, which is believed to be associated with the increased risk of infectious comorbidities of stroke patients, while the mechanisms underlying post-stroke immunosuppression is yet to be elucidated. In the brains of intracerebral hemorrhage (ICH) patients and murine ICH models, we identified that neuron-derived programmed death-ligand 1 (PD-L1) is reduced in the perihematomal area, associating increased soluble PD-L1 level in the peripheral blood. ICH induced a significant decrease of T and natural killer (NK) cell numbers in the periphery with an upregulation of programed death-1 (PD-1) in these cells. Blocking PD-1 pathway with an anti-PD1 monoclonal antibody prevented the T and NK cell compartment contraction and spleen atrophy post-ICH, with reduced pulmonary bacterial burden and improved neurological outcome. Thus, we here identified that brain-derived PD-L1 as a new mechanism driving post-stroke immunosuppression, and anti-PD1 treatment could be potentially developed to reducing the risk of post-stroke infections.

Stroke accounts for a large proportion of morbidity and mortality burden in China. Moreover, there is a high prevalence of the leading risk factors for stroke, including hypertension and smoking. Understanding the underlying mechanisms and developing effective therapeutic interventions for patients with stroke is a key imperative. The pathophysiology of stroke involves a complex interplay between the immune and inflammatory mechanisms. Focal brain inflammation triggered by neuronal cell death and the release of factors such as damage-associated molecular patterns can further exacerbate neuronal injury; in addition, impairment of the blood-brain barrier, oxidative stress, microvascular dysfunction, and brain edema cause secondary brain injury. Immune cells, including microglia and other infiltrating inflammatory cells, play a key role in triggering focal and global brain inflammation. Anti-inflammatory therapies targeting the aforementioned mechanisms can alleviate primary and secondary brain injury in the aftermath of a stroke. Further experimental and clinical studies are required to explore the beneficial effects of anti-inflammatory drugs in stroke.

BackgroundStroke is a devastating disease, including intracerebral haemorrhage (ICH) and ischaemic stroke. Emerging evidences indicate that systemic inflammatory cascades after stroke contribute to brain damage. However, the direct effects and features of systemic inflammation on brain injury, especially comparing between ischaemic and haemorrhagic stroke, are still obscure.MethodsPertussis toxin (PT) was used to build a pro-inflammatory milieu after ICH and ischaemic stroke in mouse model. The neurodeficits, stroke lesion, immune response and blood–brain barrier (BBB) destruction were assessed.ResultsIn ICH mouse model, PT-induced systemic inflammation exacerbated neurological deficits, and enlarged haemorrhage lesion and perihaematomal oedema. We also found promoted leucocyte infiltration and inflammatory cytokine release into the brain after PT treatment. Moreover, the integrity of the BBB was further disrupted after receiving PT. Furthermore, we demonstrated that PT enhanced brain inflammation and aggravated stroke severity in middle cerebral artery occlusion mouse model.ConclusionsOur results suggest that PT increases inflammatory response that exacerbates brain injury after ICH or ischaemic stroke in mouse model.

Epilepsy is a neurological condition that causes unprovoked, recurrent seizures. Accumulating evidence from clinical and experimental studies indicates that neuroinflammation exacerbates seizure activity.We investigated the transcriptional changes occurring in specific brain domains of a seizure mouse model, using 10× Genomics spatial transcriptomics. Differential gene expression and pathway analysis were applied to investigate potential signaling targets for seizure, including CCL5/CCR5 pathway. Maraviroc, an FDA-approved C-C chemokine receptor 5 (CCR5) antagonist, was used to verify the impact of CCL5/CCR5 signaling in seizure mice.We found distinguished regional transcriptome features in the hippocampus of seizure mice. The hippocampus exhibited unique inflammatory gene signatures, including glia activation, apoptosis, and immune response in seizure mice. Especially, we observed notable expression of C-C chemokine ligand 5 (CCL5) throughout the entire seizure hippocampus. Blockade of CCL5/CCR5 signaling via maraviroc prevented microglia activation and neuron degeneration in seizure mice.This study supports the potential of CCL5/CCR5 signaling for targeting neuroinflammation after seizure.

BackgroundAcute brain ischaemia elicits pronounced inflammation, which aggravates neural injury. However, the mechanisms governing the resolution of acute neuroinflammation remain poorly understood. In contrast to regulatory T and B cells, group 2 innate lymphoid cells (ILC2s) are immunoregulatory cells that can be swiftly mobilised without antigen presentation; whether and how these ILC2s participate in central nervous system inflammation following brain ischaemia is still unknown.MethodsLeveraging brain tissues from patients who had an ischaemic stroke and a mouse model of focal ischaemia, we characterised the presence and cytokine release of brain-infiltrating ILC2s. The impact of ILC2s on neural injury was evaluated through antibody depletion and ILC2 adoptive transfer experiments. Using Rag2−/−γc−/−mice receiving passive transfer of IL-4−/−ILC2s, we further assessed the contribution of interleukin (IL)-4, produced by ILC2s, in ischaemic brain injury.ResultsWe demonstrate that ILC2s accumulate in the areas surrounding the infarct in brain tissues of patients with cerebral ischaemia, as well as in mice subjected to focal cerebral ischaemia. Oligodendrocytes were a major source of IL-33, which contributed to ILC2s mobilisation. Adoptive transfer and expansion of ILC2s reduced brain infarction. Importantly, brain-infiltrating ILC2s reduced the magnitude of stroke injury severity through the production of IL-4.ConclusionsOur findings revealed that brain ischaemia mobilises ILC2s to curb neuroinflammation and brain injury, expanding the current understanding of inflammatory networks following stroke.

BackgroundBrain ischemia compromises natural killer (NK) cell-mediated immune defenses by acting on neurogenic and intracellular pathways. Less is known about the posttranscriptional mechanisms that regulate NK cell activation and cytotoxicity after ischemic stroke.MethodsUsing a NanoString nCounter® miRNA array panel, we explored the microRNA (miRNA) profile of splenic NK cells in mice subjected to middle cerebral artery occlusion. Differential gene expression and function/pathway analysis were applied to investigate the main functions of predicted miRNA target genes. miR-1224 inhibitor/mimics transfection and passive transfer of NK cells were performed to confirm the impact of miR-1224 in NK cells after brain ischemia.ResultsWe observed striking dysregulation of several miRNAs in response to ischemia. Among those miRNAs, miR-1224 markedly increased 3 days after ischemic stroke. Transfection of miR-1224 mimics into NK cells resulted in suppression of NK cell activity, while an miR-1224 inhibitor enhanced NK cell activity and cytotoxicity, especially in the periphery. Passive transfer of NK cells treated with an miR-1224 inhibitor prevented the accumulation of a bacterial burden in the lungs after ischemic stroke, suggesting an enhanced immune defense of NK cells. The transcription factor Sp1, which controls cytokine/chemokine release by NK cells at the transcriptional level, is a predicted target of miR-1224. The inhibitory effect of miR-1224 on NK cell activity was blocked in Sp1 knockout mice.ConclusionsThese findings indicate that miR-1224 may serve as a negative regulator of NK cell activation in an Sp1-dependent manner; this mechanism may be a novel target to prevent poststroke infection specifically in the periphery and preserve immune defense in the brain.

Normal aging is accompanied by escalating systemic inflammation. Yet the potential impact of immune homeostasis on neurogenesis and cognitive decline during brain aging have not been previously addressed. Here we report that natural killer (NK) cells of the innate immune system reside in the dentate gyrus neurogenic niche of aged brains in humans and mice. In situ expansion of these cells contributes to their abundance, which dramatically exceeds that of other immune subsets. Neuroblasts within the aged dentate gyrus display a senescence-associated secretory phenotype and reinforce NK cell activities and surveillance functions, which result in NK cell elimination of aged neuroblasts. Genetic or antibody-mediated depletion of NK cells leads to sustained improvements in neurogenesis and cognitive function during normal aging. These results demonstrate that NK cell accumulation in the aging brain impairs neurogenesis, which may serve as a therapeutic target to improve cognition in the aged population.

Background: Stroke is a devastating and debilitating disease and is a leading cause of death worldwide, including intracerebral hemorrhage (ICH) and ischemic stroke. Emerging evidence indicates that inflammatory cascades after hemorrhagic or ischemic stroke makes a great contribution to brain damage, mainly are involved in neuronal cell death, blood-brain-barrier (BBB) destruction and development of vasogenic edema. However, the features and direct effect of brain inflammation following stroke is still unknown. Methods: We adopted the ICH model by injection of collagenase and used a mouse model of transient cerebral ischemia and reperfusion. And pertussis toxin was used to create a pro-inflammatory milieu. Neurodeficits, lesion volume, production of reactive oxygen species (ROS) and inflammatory factors, brain-infiltrating leukocytes and blood-brain-barrier (BBB) destruction were assessed in mice model treated with pertussis toxin or vehicle.Results: Adopting collagenase induced intracerebral hemorrhage mouse model, we show that pertussis toxin-induced systemic inflammation exacerbated neurological deficits, enlarged lesion size and brain perihematomal edema after intracerebral hemorrhage. Pertussis toxin promoted leukocyte infiltration and inflammatory cytokine release in the brain. Moreover, the integrity of the BBB was further disrupted after receiving pertussis toxin in ICH mice. Furthermore, we demonstrated that pertussis toxin increased stroke severity and enhanced brain inflammation in middle cerebral artery occlusion (MCAO) mouse model. Conclusion: Our results suggest that pertussis toxin increases inflammatory response that exacerbates brain injury after intracerebral hemorrhage or ischemic stroke in mouse model.

Normal aging is accompanied by escalating systemic inflammation. Yet the potential impact of immune homeostasis on neurogenesis and cognitive decline during brain aging have not been previously addressed. Here we report that natural killer (NK) cells of the innate immune system reside in the dentate gyrus neurogenic niche of aged brains in humans and mice. In situ expansion of these cells contributes to their abundance, which dramatically exceeds that of other immune subsets. Neuroblasts within the aged dentate gyrus display a senescence-associated secretory phenotype and reinforce NK cell activities and surveillance functions, which result in NK cell elimination of aged neuroblasts. Genetic or antibody-mediated depletion of NK cells leads to sustained improvements in neurogenesis and cognitive function during normal aging. These results demonstrate that NK cell accumulation in the aging brain impairs neurogenesis, which may serve as a therapeutic target to improve cognition in the aged population.

Objectives: To screen miRNA profile of peripheral NK cells in ischemic stroke mouse model and investigate a most promising candidate (miR-1224) for post-transcriptional regulation of NK cell function after ischemic stroke. Methods: Mice were subjected to a 60 min focal cerebral ischemia produced by transient intraluminal occlusion of MCAO. For NK cell isolation, cell suspensions from the spleens after reperfusion were enriched for NK cells using magnetic-bead sorting system after staining with anti-NK1.1 microbeads. The nCounter Mouse miRNA array was used to analyze miRNA expression profile in splenic NK cells over the time course of experimental ischemic stroke. Based on the miRNA data, we further in vitro modulated miR-1224 in NK cells using mimics or inhibitor, then injected i.v into Rag2-/-γc-/- recipient mice. Neurological function score was compared and spontaneous infection was assessed by pulmonary bacteria colony culture, and changes in potential signaling pathway (SP1/TNF-α) were verified by rt-PCR and western blot. Results: Through miRNA expression profile analysis, we have identified significant changes at each time point in peripheral NK cells after cerebral ischemia. Among all screened miRNA, miR-1224 remarkably increased in MCAO group, which was verified by PCR. Then isolated NK cells treated with mimics or inhibitors, were transferred to Rag2-/-γc-/- recipient mice. Compared with WT mice, Rag2-/-γc-/- mice with miR-1224 inhibitor exhibited increased NK cell number, enhanced NK cell activation/cytotoxicity feature, as well as better neurological behaviors and reduced pulmonary infection after MCAO. Moreover, compared with the control group, NK cells with miR-1224 inhibitor showed significantly increased SP1 gene and protein phosphorylation. As SP1 gene is one of the potential targets of miR-1224, this study suggests that miR-1224 may regulate NK cell function after MCAO, which is associated with SP1 pathway. Conclusion: The miRNA profiling of splenic NK cells provided insight into the functional mechanism and signaling pathways underlying the distinct organ-specific NK cell properties, which will contribute to the better understanding of NK cell mediated immune-response in relation to different stages of stroke.

Supplemental Digital Content is available in the text., Background and Purpose— Autoimmune responses can occur when antigens from the central nervous system are presented to lymphocytes in the periphery or central nervous system in several neurological diseases. However, whether autoimmune responses emerge after brain ischemia and their impact on clinical outcomes remains controversial. We hypothesized that brain ischemia facilitates the genesis of autoimmunity and aggravates ischemic brain injury. Methods— Using a mouse strain that harbors a transgenic T-cell receptor to a central nervous system antigen, MOG35-55 (myelin oligodendrocyte glycoprotein) epitope (2D2), we determined the anatomic location and involvement of antigen-presenting cells in the development of T-cell reactivity after brain ischemia and how T-cell reactivity impacts stroke outcome. Transient middle cerebral artery occlusion and photothrombotic stroke models were used in this study. We also quantified the presence and status of T cells from brain slices of ischemic patients. Results— By coupling transfer of labeled MOG35-55-specific (2D2) T cells with tetramer tracking, we show an expansion in reactivity of 2D2 T cells to MOG91-108 and MOG103-125 in transient middle cerebral artery occlusion and photothrombotic stroke models. This reactivity and T-cell activation first occur locally in the brain after ischemia. Also, microglia act as antigen-presenting cells that effectively present MOG antigens, and depletion of microglia ablates expansion of 2D2 reactive T cells. Notably, the adoptive transfer of neuroantigen-experienced 2D2 T cells exacerbates Th1/Th17 responses and brain injury. Finally, T-cell activation and MOG-specific T cells are present in the brain of patients with ischemic stroke. Conclusions— Our findings suggest that brain ischemia activates and diversifies T-cell responses locally, which exacerbates ischemic brain injury.

Stroke-induced immune suppression predisposes the host to infections and can contribute to high morbidity and mortality in stroke patients. Because ischemic stroke has a profound effect on the systemic immune response, which may explain the increased susceptibility of stroke patients to infection, an urgent need persists for a better understanding of mechanisms associated with immune suppression; new and effective treatments for stroke can then be identified. NK cells play a key role in early host defense against pathogens by killing infected cells and/or producing cytokines such as IFN-γ. Because the phenotype and function of peripheral NK cells have been widely investigated in ischemic stroke, nCounter Inflammation Gene Array Analysis was used to build immune-related gene profiles of NK cells to comprehensively analyze the molecular signature of NK cells after ischemic brain injury. We observed distinct gene expression profiles reflecting different splenic NK-cell phenotypes and functional properties across the time course of transient middle cerebral artery occlusion (MCAO). Based on gene expression and pathway-network analysis, lower expression levels of signal transducer and activator of transcription-3 (STAT3) were observed in animals with MCAO compared with sham control animals. Genetic activation of STAT3 through the introduction of STAT3 clustered regularly interspaced short palindromic repeats (CRISPR) plasmid prevented the loss of NK-cell–derived IFN-γ production after MCAO, together with reduced bacterial burden and mortality. Our data suggest that brain ischemia impairs NK-cell–mediated immune defense in the periphery, at least in part through the JAK-STAT3 pathway, which can be readdressed by modulating STAT3 activation status.—Jin, W.-N., Ducruet, A. F., Liu, Q., Shi, S. X.-Y., Waters, M., Zou, M., Sheth, K. N., Gonzales, R., Shi, F.-D. Activation of JAK/STAT3 restores NK-cell function and improves immune defense after brain ischemia.

Brain ischemia inhibits immune function systemically, with resulting infectious complications. Whether in stroke different immune alterations occur in brain and periphery and whether analogous mechanisms operate in these compartments remains unclear. Here we show that in patients with ischemic stroke and in mice subjected to middle cerebral artery occlusion, natural killer (NK) cells display remarkably distinct temporal and transcriptome profiles in the brain as compared to the periphery. The activation of catecholaminergic and hypothalamic-pituitary-adrenal axis leads to splenic atrophy and contraction of NK cell numbers in the periphery through a modulated expression of SOCS3, whereas cholinergic innervation-mediated suppression of NK cell responses in the brain involves RUNX3. Importantly, pharmacological or genetic ablation of innervation preserved NK cell function and restrained post-stroke infection. Thus, brain ischemia compromises NK cell-mediated immune defenses through mechanisms that differ in the brain versus the periphery, and targeted inhibition of neurogenic innervation limits post-stroke infection.

Brain ischemia elicits microglial activation and microglia survival depend on signaling through colony-stimulating factor 1 receptor (CSF1R). Although depletion of microglia has been linked to worse stroke outcomes, it remains unclear to what extent and by what mechanisms activated microglia influence ischemia-induced inflammation and injury in the brain. Using a mouse model of transient focal cerebral ischemia and reperfusion, we demonstrated that depletion of microglia via administration of the dual CSF1R/c-Kit inhibitor PLX3397 exacerbates neurodeficits and brain infarction. Depletion of microglia augmented the production of inflammatory mediators, leukocyte infiltration, and cell death during brain ischemia. Of note, microglial depletion-induced exacerbation of stroke severity did not solely depend on lymphocytes and monocytes. Importantly, depletion of microglia dramatically augmented the production of inflammatory mediators by astrocytes after brain ischemia . In vitro studies reveal that microglia restricted ischemia-induced astrocyte response and provided neuroprotective effects. Our findings suggest that neuroprotective effects of microglia may result, in part, from its inhibitory action on astrocyte response after ischemia.

Colivelin is a neuroprotective humanin family peptide with potent long-term capacity against Aβ deposition, neuronal apoptosis, and synaptic plasticity deficits in neurodegenerative disease. We seek to investigate whether this effect of Colivelin also govern ischemic brain injury, and potential mechanism underlying the Colivelin-mediated action on ischemic neurons. We adopted 60 min induction of transient focal cerebral ischemia and reperfusion in mice. We found that relative to mice receiving vehicle, Colivelin administration decreased the neurological deficits and infarct lesion induced by brain ischemia. Colivelin inhibited axonal damage and neuronal death in brain tissue, which was associated with elevated anti-apoptotic gene expression in ischemic neurons as well as increased axonal growth up until two-weeks post-stroke. Moreover, Colivelin activated STAT3 signaling, which may partially contribute to its beneficial effect against neuronal death and axon growth. In conclusion, Colivelin induce anti-apoptotic genes up-regulation, and activate JAK/STAT3 signaling after ischemic stroke, which may contribute to its effects of rescuing ischemic neuronal death and axonal remodeling. This study may justify further works to examine Colivelin as a single or adjunct therapy in ischemic stroke.

Background: Stroke-induced immune suppression predisposes the host to infections, and can contribute to high morbidity and mortality in stroke patients worldwide. Since ischemic stroke has a profound effect on the systemic immune response which may explain the increased susceptibility of stroke patients to infection, an urgent need persists for a better understanding of mechanisms associated with immune suppression so that new and effective treatments for stroke can be identified. Natural killer (NK) cells play a key role in early host defense against pathogens by killing infected cells and/or producing cytokines such as interferon-γ. As the phenotype and function of peripheral NK cells have been widely investigated in ischemic stroke, in this study we comprehensively analyzed molecular signature of NK cells after ischemic brain injury. Methods: The nCounter Mouse Inflammation gene expression kit containing 561 inflammation related genes was used to analyze gene expression changes in splenic NK cells over the time course of experimental ischemic stroke. In order to investigate the effect of STAT3 activation on NK cell phenotype and function after stroke, splenic NK cells isolated from wildtype C57 mice targeting STAT3 was established by introducing STAT3 CRISPR activation plasmid. Results: We observed that different splenic NK cell phenotypes and functional properties across the time course of MCAO was reflected in distinct gene expression profiles. Functional analysis revealed that the most overrepresented NK cell biologic processes are those related to NK cell mediated immune response and functions. Based on gene expression and pathway-network analysis, we observed that STAT3 exhibited lower expression levels after transient middle cerebral artery occlusion (MCAO) compared with sham controls. Genetic activation of STAT3 by introducing STAT3 CRISPR plasmid prevented the loss of NK cell derived interferon-γ production after MCAO, together with reduced bacteria burden and mortality. Conclusion: Our data suggest that brain ischemia impairs NK cell-mediated immune defense in periphery at least in part through JAK-STAT3 pathway, which can be re-addressed by modulating STAT3 activation status.

Background: Autoimmune responses can occur when antigens from the central nervous system (CNS) are presented to lymphocytes in the periphery or CNS. However, whether autoimmune responses emerge after brain ischemia remains controversial. Similarly, it is debated whether genesis of autoimmune responses can impact outcome of ischemic stroke. We hypothesized that that brain ischemia can diversify T cell responses against CNS antigens, and that expanded CNS antigen-specific T cells can promote ischemic brain injury. Methods: We quantified the presence and status of T cells from brain slices of ischemic patients. Using a mouse strain that harbor a transgenic T cell receptor (TCR) to a CNS antigen, the myelin oligodendrocyte glycoprotein (MOG 35-55 ) epitope (2D2), we determined the anatomic location and involvement of antigen presentation cells in the development of T cell reactivity after brain ischemia and how T cell reactivity impacts stroke outcome. Transient middle cerebral artery occlusion (MCAO) and photothrombotic stroke models were used in this study. Results: By coupling transfer of labeled myelin oligodendrocyte glycoprotein (MOG) 35-55 -specific (2D2) T cells with tetramer tracking, we show an expansion in reactivity of 2D2 T cells to MOG 97-108 and MOG 103-125 in transient MCAO and photothrombotic stroke models. This reactivity and T cell activation occur in the brain and not in the peripheral lymphoid organs after ischemia. Also, microglia act as antigen-presenting cells (APCs) that effectively present MOG antigen, and depletion of microglia ablates expansion of 2D2 reactive T cells. Notably, the adoptive transfer of neuroantigen-experienced 2D2 T cells exacerbates Th1/Th17 responses and brain injury. Finally, T cell activation and MOG-specific T cells can be seen in the brain of ischemic stroke patients. Conclusion: Our findings suggest that brain ischemia activates and diversifies T cell responses locally, which exacerbates ischemic brain injury.

The nonneural cholinergic system of immune cells is pivotal for the maintenance of immunological homeostasis. Here we demonstrate the expression of choline acetyltransferase (ChAT) and cholinergic enzymes in murine natural killer (NK) cells. The capacity for acetylcholine synthesis by NK cells increased markedly under inflammatory conditions such as experimental autoimmune encephalomyelitis (EAE), in which ChAT expression escalated along with the maturation of NK cells. ChAT+ and ChAT- NK cells displayed distinctive features in terms of cytotoxicity and chemokine/cytokine production. Transfer of ChAT+ NK cells into the cerebral ventricles of CX3CR1-/- mice reduced brain and spinal cord damage after EAE induction, and decreased the numbers of CNS-infiltrating CCR2+Ly6Chi monocytes. ChAT+ NK cells killed CCR2+Ly6Chi monocytes directly via the disruption of tolerance and inhibited the production of proinflammatory cytokines. Interestingly, ChAT+ NK cells and CCR2+Ly6Chi monocytes formed immune synapses; moreover, the impact of ChAT+ NK cells was mediated by α7-nicotinic acetylcholine receptors. Finally, the NK cell cholinergic system up-regulated in response to autoimmune activation in multiple sclerosis, perhaps reflecting the severity of disease. Therefore, this study extends our understanding of the nonneural cholinergic system and the protective immune effect of acetylcholine-producing NK cells in autoimmune diseases.

Astrocytes are believed to bridge interactions between infiltrating lymphocytes and neurons during brain ischemia, but the mechanisms for this action are poorly understood. Here we found that interleukin-15 (IL-15) is dramatically up-regulated in astrocytes of postmortem brain tissues from patients with ischemic stroke and in a mouse model of transient focal brain ischemia. We generated a glial fibrillary acidic protein (GFAP) promoter-controlled IL-15-expressing transgenic mouse (GFAP-IL-15tg) line and found enlarged brain infarcts, exacerbated neurodeficits after the induction of brain ischemia. In addition, knockdown of IL-15 in astrocytes attenuated ischemic brain injury. Interestingly, the accumulation of CD8+ T and natural killer (NK) cells was augmented in these GFAP-IL-15tg mice after brain ischemia. Of note, depletion of CD8+ T or NK cells attenuated ischemic brain injury in GFAP-IL-15tg mice. Furthermore, knockdown of the IL-15 receptor α or blockade of cell-to-cell contact diminished the activation and effector function of CD8+ T and NK cells in GFAP-IL-15tg mice, suggesting that astrocytic IL-15 is delivered in trans to target cells. Collectively, these findings indicate that astrocytic IL-15 could aggravate postischemic brain damage via propagation of CD8+ T and NK cell-mediated immunity.

Glatiramer acetate (GA) is one of the first-line disease-modifying medications that have been approved for the treatment of multiple sclerosis via immune modulatory mechanisms. However, it remains unclear whether the immunomodulation effect of GA is central nervous system (CNS) antigen specific. Here, we explored the mechanism of action of GA by subcutaneously injecting GA in experimental autoimmune neuritis (EAN) rats, an animal model for Guillain-Barré syndrome (GBS). Clinical, electrophysiological and histological findings showed that neurological deficits, demyelination and axonal injury of sciatic nerves were all significantly attenuated in Lewis rats when GA was administered before immunization with peripheral nervous system antigen P0. Our results further demonstrated that GA treatment inhibited either P0 or myelin basic protein (MBP) (CNS antigen)-stimulated auto-immune T-cell proliferation in vitro. GA administrated at 10 days after induction of EAN when neurological sign became apparent also ameliorated the severity of disease, inhibited T-cell response to P0 and MBP and induced shift of proinflammatory and immune modulatory cytokines. Collectively, our findings suggested that GA attenuated neurological deficits in EAN rats and that the immune modulatory mechanisms of GA were not CNS antigen specific.

Macrophage migration inhibitory factor (MIF) is a key cytokine/chemokine in the activation and recruitment of inflammatory T lymphocytes known to exacerbate experimental stroke severity. MIF effects are mediated through its primary cellular receptor, CD74, the MHC class II invariant chain present on all class II expressing cells, including monocytes, macrophages and dendritic cells (DC). We demonstrated previously that partial MHC class II/peptide constructs (pMHC) can effectively treat mice with experimental stroke, in part through their ability to competitively inhibit MIF/CD74 interactions and downstream signaling. However, the role of MIF and CD74 in human ischemic stroke is not yet well established. To evaluate the therapeutic potential for pMHC, we assessed MIF and CD74 expression levels and their association with disease outcome in subjects with ischemic stroke. MIF levels were assessed in blood plasma by ELISA and CD74 expression was quantified by flow cytometry and qRT-PCR in peripheral blood mononuclear cells (PBMCs) obtained from subjects with ischemic stroke and age and sex-matched healthy controls (HC). MIF levels were increased in plasma and the number of CD74+ cells and CD74 mRNA expression levels were significantly increased in PBMC of subjects with ischemic stroke versus HC, mainly on CD4+ T cells, monocytes and DC. Greater increases of CD74+ cells were seen in subjects with cortical vs. subcortical infarcts and the number of CD74+ cells in blood correlated strongly with infarct size and neurological outcomes. However, differences in MIF and CD74 expression were not affected by age, gender or lesion laterality. Increased CD74 expression levels may serve as a useful biomarker for worse stroke severity and predicted outcomes in subjects with ischemic stroke and provide a rationale for potential future treatment with pMHC constructs.

Microglia are the first responders to intracerebral hemorrhage, but their precise role in intracerebral hemorrhage remains to be defined. Microglia are the only type of brain cells expressing the colony-stimulating factor 1 receptor, a key regulator for myeloid lineage cells. Here, we determined the effects of a colony-stimulating factor 1 receptor inhibitor (PLX3397) on microglia and the outcome in the context of experimental mouse intracerebral hemorrhage. We show that PLX3397 effectively depleted microglia, and the depletion of microglia was sustained after intracerebral hemorrhage. Importantly, colony-stimulating factor 1 receptor inhibition attenuated neurodeficits and brain edema in two experimental models of intracerebral hemorrhage induced by injection of collagenase or autologous blood. The benefit of colony-stimulating factor 1 receptor inhibition was associated with reduced leukocyte infiltration in the brain and improved blood–brain barrier integrity after intracerebral hemorrhage, and each observation was independent of lesion size or hematoma volume. These results demonstrate that suppression of colony-stimulating factor 1 receptor signaling ablates microglia and confers protection after intracerebral hemorrhage.

The effect of hypoxia on the differentiation of chronic myeloid leukaemic K562 cells were studied, as was the role of the NHE1 (Na+/H+ exchanger 1). Hypoxia induced differentiation of K562 cells as seen by modifications in their morphological features, up-regulation of C/EBPα (CCAAT/enhancer-binding protein α), and marked IL-8 (interleukin-8) release. Inhibition of NHE1 under hypoxia additionally enhanced the level of C/EBPα and further promoted leukaemic cells differentiation. Pharmacological inhibition of p38 MAPK (mitogen-activated protein kinase) also significantly suppressed C/EBPα expression under hypoxia conditions after NHE1 inhibition. These results indicate the enhancement of hypoxia-induced K562 differentiation by NHE1 inhibition, which may be due to up-regulation of C/EBPα via p38 MAPK signalling pathway, which suggests a possible therapeutic target of NHE1 under hypoxia microenvironment in the treatment of leukaemic diseases.

This study focused on the distribution and pasting properties of starches in sweet potato (Ipomoea batatas L.) tuber. A fresh sweet potato tuber was cut into six equivalent parts. The extractable contents and pasting properties of starches from different parts of fresh sweet potato tuber were determined. The results showed that the extractable content of starches from the Middle section of sweet potato tuber is lower than that from the head and the end section of sweet potato tuber. The extractable content of starches from the inner-end part and the outer-Middle part of sweet potato tuber are 16.2% and 10.3% respectively. The amylose content of starches is relatively higher in the Middle section and outer layer of sweet potato tuber. The amylose content of starches from the outer-Middle part and the inner-end part of sweet potato tuber are 29.5% and 26.7% respectively. Pasting properties of starches from different parts of sweet potato tubers are different from each other. Among them, the starches from inner-end part of sweet potato tuber have the highest viscosities and a lower breakdown.

To investigate the effect of inhibition of Na(+)/H(+) exchanger isoform1 (NHE1) on K562 leukemia-driven angiogenesis, the selective NHE1 inhibitor cariporide was used. Cariporide treatment of K562 resulted in a decrease in pHi and down-regulation of VEGF secretion. The proliferation, migration and in vitro tube formation of human umbilical vein endothelial cells was decreased in cariporide treated K562 condition medium (CM) while VEGF supplement could partially restore the inhibitory effect. Subcutaneous injection of nude mice with cariporide inhibited K562 tumor growth with a reduction of the density of microvessels compared to the control group.

Na⁺/H⁺ exchanger 1 (NHE1), an important regulator of intracellular pH (pH(i)) and extracellular pH (pH(e)), has been shown to play a key role in breast cancer metastasis. However, the exact mechanism by which NHE1 mediates breast cancer metastasis is not yet well known. We showed here that inhibition of NHE1 activity, with specific inhibitor Cariporide, could suppress MDA-MB-231 cells invasion as well as the activity and expression of MT1-MMP. Overexpression of MT1-MMP resulted in a distinguished increase in MDA-MB-231 cells invasiveness, but treatment with Cariporide reversed the MT1-MMP-mediated enhanced invasiveness. To explore the role of MAPK signaling pathways in NHE1-mediated breast cancer metastasis, we compared the difference of constitutively phosphorylated ERK1/2, p38 MAPK and JNK in non-invasive MCF-7 cells and invasive MDA-MB-231 cells. Interestingly, we found that the phosphorylation levels of ERK1/2 and p38 MAPK in MDA-MB-231 cells were higher than in MCF-7 cells, but both MCF-7 cells and MDA-MB-231 cells expressed similar constitutively phosphorylated JNK. Treating MDA-MB-231 cells with Cariporide led to decreased phosphorylation level of both p38 MAPK and ERK1/2 in a time-dependent manner, but JNK activity was not influenced. Supplementation with MAPK inhibitor (MEK inhibitor PD98059, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125) or Cariporide all exhibited significant depression of MDA-MB-231 cells invasion and MT1-MMP expression. Furthermore, we co-treated MDA-MB-231 cells with MAPK inhibitor and Cariporide. The result showed that Cariporide synergistically suppressed invasion and MT1-MMP expression with MEK inhibitor and p38 MAPK inhibitor, but not be synergistic with the JNK inhibitor. These findings suggest that NHE1 mediates MDA-MB-231 cells invasion partly through regulating MT1-MMP in ERK1/2 and p38 MAPK signaling pathways dependent manner.

Recovery from organ-specific autoimmune diseases largely relies on the mobilization of endogenous repair mechanisms and local factors that control them. Natural killer (NK) cells are swiftly mobilized to organs targeted by autoimmunity and typically undergo numerical contraction when inflammation wanes. We report the unexpected finding that NK cells are retained in the brain subventricular zone (SVZ) during the chronic phase of multiple sclerosis in humans and its animal model in mice. These NK cells were found preferentially in close proximity to SVZ neural stem cells (NSCs) that produce interleukin-15 and sustain functionally competent NK cells. Moreover, NK cells limited the reparative capacity of NSCs following brain inflammation. These findings reveal that reciprocal interactions between NSCs and NK cells regulate neurorepair.

Recent studies have demonstrated that lymphocytes play a key role in ischemic brain injury. However, there is still a lack of viable approaches to non-invasively track infiltrating lymphocytes and reveal their key spatiotemporal events in the inflamed central nervous system (CNS). Here we describe an in vivo imaging approach for sequential monitoring of brain-infiltrating CD4+ T cells in experimental ischemic stroke. We show that magnetic resonance imaging (MRI) or Xenogen imaging combined with labeling of SPIO-Molday ION Rhodamine-B (MIRB) can be used to monitor the dynamics of CD4+ T cells in a passive transfer model. MIRB-labeled CD4+ T cells can be longitudinally visualized in the mouse brain and peripheral organs such as the spleen and liver after cerebral ischemia. Immunostaining of tissue sections showed similar kinetics of MIRB-labeled CD4+ T cells when compared with in vivo observations. Our results demonstrated the use of MIRB coupled with in vivo imaging as a valid method to track CD4+ T cells in ischemic brain injury. This approach will facilitate future investigations to identify the dynamics and key spatiotemporal events for brain-infiltrating lymphocytes in CNS inflammatory diseases.

To investigate the relationship between natural killer (NK) cells and traumatic brain injury (TBI), we tracked an established phenotype of circulating NK cells at several time points in patients with different grades of TBI. In serial peripheral blood samples, NK cells were prospectively measured by flow cytometry of CD3(-) CD56(+) lymphocytes. Compared to healthy controls, TBI patients had reductions in both the percentage and the absolute number of NK cells. Furthermore, the magnitude of NK cell reduction correlated with the degree of TBI severity at several time points. That is, NK cell population size was independently associated with lower Glasgow Coma Scale scores. In addition, at some time points, a positive correlation was found between the NK cell counts and Glasgow Outcome Scale scores. Our results indicate that TBI induces a reduction in the number of NK cells, and the magnitude of the reduction appears to parallel the severity of TBI.

CCAAT/enhancer binding protein homologous protein (CHOP) expression increases when Na(+)-H(+) exchanger 1 (NHE1) is inhibited. Endoplasmic reticulum (ER) stress has been shown to trigger tumor cell death through CHOP. We therefore hypothesized that NHE1 activity correlates with ER stress and confers pharmaceutical potential to NHE1 inhibitor as an anti-tumor agent. The present study showed that treatment with the NHE1 inhibitor cariporide led to ER stress-induced up-regulation of the death receptor 5 (DR5) which is mediated by CHOP at the transcriptional level. We also determined that ER stress-induced Janus kinase (JNK) activation was responsible for the modulation of CHOP. Combining cariporide with tumor necrosis factor related apoptosis-inducing ligand (TRAIL) led to a significantly enhanced level of apoptosis that was abrogated by siRNA silencing of CHOP. This study provides a potential mechanistic rationale for the use of NHE1 inhibitor in combination with DR5 agonists to induce apoptosis in leukemia.

Summary Aims To investigate the clinical characteristics and sera anti-aquaporin 4 (AQP4) antibody positivity in patients with inflammatory demyelinating disorders (IDDs) of the central nervous system (CNS) in Tianjin, China. Methods We retrospectively evaluated 234 patients with IDDs including neuromyelitis optica (NMO), recurrent optic neuritis (rON), longitudinally extensive transverse myelitis (LETM), clinically isolated syndrome (CIS), and multiple sclerosis (MS) groups. Sera from 217 patients were determined for AQP4-Ab. The clinical characteristics and sera anti-AQP4 positivity were compared. Results The IDDS comprised 63 MS, 51 NMO, 56 LETM, 10 rON, and 54 CIS. Compared with MS, NMO had a higher frequency of occurrence in women, intractable hiccup and nausea (IHN), medullospinal lesion, longitudinally extensive spinal cord lesions (LESCL) and bilateral ON, disease onset at a later age, and worsening residual disability. AQP4-Ab-positive rates were 84.1% and 69% in NMO and NMO spectrum disorders (NMOSD), respectively, whereas it was undetectable in all of the MS sera samples. Conclusions We comprehensively contrast the distinct clinical features of MS, NMO, and NMOSD in our center. A sensitive AQP4-Ab assay is necessary for the early diagnosis of NMOSD in our patients. Neither medullospinal lesion nor IHN is unique in NMO.

This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.

This study was aimed to investigate whether the inhibition of NHE1 activity and intracellular acidification can reverse resistance of leukemia cells to the imatinib and to explore downstream signal molecule networks of BCR/ABL in the cells of chronic myelocytic leukemia (CML) patients. The mRNA and protein expression of P-glycoprotein (Pgp) and the drug accumulation were assayed after acidifying the primary leukemia cells of patients or K562/DOX and K562/G01 cells. The effects of intracellular acidification of primary leukemia cells on the phosphorylation level changes of ERK1/2 and p38 MAPK were analyzed by Western blot. The results showed that the intracellular concentration of drugs in the advanced patients increased and the sensitivity of K562/DOX and K562/G01 cells to imatinib was enhanced after intracellular acidification or treatment with NHE1 inhibitor cariporide. With downregulation of intracellular pH, the phosphorylation of p38 MAPK decreased in advanced patients and the phosphorylation of ERK1/2 increased within 3 min and then decreased after 30 min. SB203580, the specific inhibitor of p38 MAPK, displayed a synergistic effect with the inhibitor of NHE1 to downregulate the mRNA and protein expression of Pgp. It is concluded that the inhibiton of NHE1 can significantly decrease the protein expression of Pgp in K562/DOX and K562/G01 cells, increase the accumulation of Rhodamine123 and doxorubicin in the cells of advanced patients and enhance the sensitivity of cells to imatinib in which the p38 MAPK signal transduction pathways involves.

Multidrug resistance (MDR) is a major problem facing patients with cancer. Although Neutrophil gelatinase-associated lipocalin (NGAL) is highly expressed in various cancers, the possible role of NGAL in MDR is still obscure. In this article, we evaluated the effect of NGAL on Rh123 accumulation in cancer cells. NGAL was first down-regulated by short hairpin RNA-mediated interference. In correlation with the reduced NGAL expression, intracellular Rh123 accumulation was significantly decreased. We finally observed that inhibiting both of the ERK1/2 and p38 MAPK could seriously down-regulate NGAL expression and also decrease the intracellular accumulation of Rh123, indicating that NGAL-mediated Rh123 accumulation is regulated by the phosphorylation of ERK1/2 and p38 MAPK. Pretreatment of MDA-MB-231 with NGAL recombinant protein and antibody had significant effects on the intracellular accumulation of Rh123, whereas little effect was observed in K562 cells treated with the same method, suggesting that NGAL was involved in the regulation of Rh123 accumulation in these two types of cancers, although different pathways. Here we provide new evidence that directly shows the possibility of small chemical substances Rh123 intracellular accumulation that is regulated by NGAL. These results suggest the possibility of NGAL involvement in drug transportation and cancer MDR formation, and indicate the potential of NGAL in cancer therapy.