Brain Ischemia Suppresses Immunity in the Periphery and Brain via Different Neurogenic Innervations.

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.

Plasma Reference Ranges for Brain Injury Biomarkers (NfL, NfH, MCP-1, and MMP-9) in Healthy Chinese.

BACKGROUND: Brain injury triggers neuroaxonal injury and neural death, that leads to the development of secondary sequelae. Throughout this process, brain injury factors released into circulation via the injured neurovascular unit are important prognostic parameters. Plasma NfL, NfH, MCP-1, and MMP-9 have been identified as potential indicators in this regard. METHODS: Using a microfluidic ELISA platform, we measured plasma from 273 healthy subjects that underwent quantifications of NfL, NfH, MCP-1, and MMP-9 levels. We investigated the possible associations between biomarkers and basic demographics. RESULTS: The median concentration of plasma NfL was 10.40 (IQR = 6.73 - 16.60) pg/mL, NfH was 70.70 (IQR = 39.75 - 125.50) pg/mL, MCP-1 was 191.0 (IQR = 162.0 - 237.5) pg/mL, and MMP-9 was 169,255 (IQR = 107,657 - 231,276) pg/mL. Among all four biomarkers, plasma NfL and NfH levels were positively correlated with age (r = 0.557, p < 0.001, r = 0.364, p = 0.003). NfL was also correlated with NfH (r = 0.391, p = 0.002). CONCLUSIONS: These data provide a basis for the potential application of a brain-injury biomarker panel in routine clinical practice. It lays a significant foundation in supporting circulating CNS-biomarkers as noninvasive biomarkers for neurological disorders.

Role of Immune and Inflammatory Mechanisms in Stroke: A Review of Current Advances.

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.

miR-1224 contributes to ischemic stroke-mediated natural killer cell dysfunction by targeting Sp1 signaling.

BACKGROUND: Brain 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. METHODS: Using 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. RESULTS: We 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. CONCLUSIONS: These 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.

Astrocyte-derived interleukin-15 exacerbates ischemic brain injury via propagation of cellular immunity.

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.

Acetylcholine-producing NK cells attenuate CNS inflammation via modulation of infiltrating monocytes/macrophages.

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.

Activation of JAK/STAT3 restores NK-cell function and improves immune defense after brain ischemia.

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 Induces Diversified Neuroantigen-Specific T-Cell Responses That Exacerbate Brain Injury.

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.

A translocator protein 18 kDa agonist protects against cerebral ischemia/reperfusion injury.

BACKGROUND: Cerebral ischemia is a leading cause of death and disability with limited treatment options. Although inflammatory and immune responses participate in ischemic brain injury, the molecular regulators of neuroinflammation after ischemia remain to be defined. Translocator protein 18 kDa (TSPO) mainly localized to the mitochondrial outer membrane is predominantly expressed in glia within the central nervous system during inflammatory conditions. This study investigated the effect of a TSPO agonist, etifoxine, on neuroinflammation and brain injury after ischemia/reperfusion. METHODS: We used a mouse model of middle cerebral artery occlusion (MCAO) to examine the therapeutic potential and mechanisms of neuroprotection by etifoxine. RESULTS: TSPO was upregulated in Iba1+ or CD11b+CD45int cells from mice subjected to MCAO and reperfusion. Etifoxine significantly attenuated neurodeficits and infarct volume after MCAO and reperfusion. The attenuation was pronounced in mice subjected to 30, 60, or 90 min MCAO. Etifoxine reduced production of pro-inflammatory factors in the ischemic brain. In addition, etifoxine treatment led to decreased expression of interleukin-1ß, interleukin-6, tumor necrosis factor-α, and inducible nitric oxide synthase by microglia. Notably, the benefit of etifoxine against brain infarction was ablated in mice depleted of microglia using a colony-stimulating factor 1 receptor inhibitor. CONCLUSIONS: These findings indicate that the TSPO agonist, etifoxine, reduces neuroinflammation and brain injury after ischemia/reperfusion. The therapeutic potential of targeting TSPO requires further investigations in ischemic stroke.

Infiltration and persistence of lymphocytes during late-stage cerebral ischemia in middle cerebral artery occlusion and photothrombotic stroke models.

BACKGROUND: Evidence suggests that brain infiltration of lymphocytes contributes to acute neural injury after cerebral ischemia. However, the spatio-temporal dynamics of brain-infiltrating lymphocytes during the late stage after cerebral ischemia remains unclear. METHODS: C57BL/6 (B6) mice were subjected to sham, photothrombosis, or 60-min transient middle cerebral artery occlusion (MCAO) procedures. Infarct volume, neurodeficits, production of reactive oxygen species (ROS) and inflammatory factors, brain-infiltrating lymphocytes, and their activation as well as pro-inflammatory cytokine IFN-γ production were assessed. Brain-infiltrating lymphocytes were also measured in tissue sections from post-mortem patients after ischemic stroke by immunostaining. RESULTS: In mice subjected to transient MCAO or photothrombotic stroke, we found that lymphocyte infiltration persists in the ischemic brain until at least day 14 after surgery, during which brain infarct volume significantly diminished. These brain-infiltrating lymphocytes express activation marker CD69 and produce proinflammatory cytokines such as IFN-γ, accompanied with a sustained increase of reactive oxygen species (ROS) and inflammatory cytokines release in the brain. In addition, brain-infiltrating lymphocytes were observed in post-mortem brain sections from patients during the late stage of ischemic stroke. CONCLUSION: Our results demonstrate that brain-infiltration of lymphocytes persists after the acute stage of cerebral ischemia, facilitating future advanced studies to reveal the precise role of lymphocytes during late stage of stroke.

Glatiramer acetate ameliorates experimental autoimmune neuritis.

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.

Potential Protein Signatures for Recurrence Prediction of Ischemic Stroke.

BACKGROUND: Acute ischemic stroke is a major cause of mortality and disability worldwide, with approximately 7.4% to 7.7% recurrence within the first 3 months. This study aimed to identify potential biomarkers for predicting stroke recurrence. METHODS AND RESULTS: We conducted a nested case-control study using a hospital-based cohort from the Third China National Stroke Registry selecting 214 age- and sex-matched patients with ischemic stroke with hypertension and no history of diabetes or heart disease. Using data-independent acquisition for discovery and multiple reaction monitoring for quantitative validation, we identified 26 differentially expressed proteins in large-artery atherosclerosis (Causative Classification of Ischemic Stroke [CCS]1), 16 in small-artery occlusion (CCS3), and 25 in undetermined causes (CCS5) among patients with recurrent stroke. In the CCS1 and CCS3 subgroups, differentially expressed proteins were associated with platelet aggregation, neuronal death/cerebroprotection, and immune response, whereas differentially expressed proteins in the CCS5 subgroup were linked to altered metabolic functions. Validated recurrence predictors included proteins associated with neutrophil activity and vascular inflammation (TAGLN2 [transgelin 2], ITGAM [integrin subunit α M]/TAGLN2 ratio, ITGAM/MYL9 [myosin light chain 9] ratio, TAGLN2/RSU1 [Ras suppressor protein 1] ratio) in the CCS3 subgroup and proteins associated with endothelial plasticity and blood-brain barrier integrity (ITGAM/MYL9 ratio and COL1A2 [collagen type I α 2 chain]/MYL9 ratio) in the CCS3 and CCS5 subgroups, respectively. CONCLUSIONS: These findings provide a foundation for developing a blood-based biomarker panel, using causative classifications, which may be used in routine clinical practice to predict stroke recurrence.

APOE from patient-derived astrocytic extracellular vesicles alleviates neuromyelitis optica spectrum disorder in a mouse model.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune astrocytopathy of the central nervous system, mediated by antibodies against aquaporin-4 water channel protein (AQP4-Abs), resulting in damage of astrocytes with subsequent demyelination and axonal damage. Extracellular communication through astrocyte-derived extracellular vesicles (ADEVs) has received growing interest in association with astrocytopathies. However, to what extent ADEVs contribute to NMOSD pathogenesis remains unclear. Here, through proteomic screening of patient-derived ADEVs, we observed an increase in apolipoprotein E (APOE)-rich ADEVs in patients with AQP4-Abs-positive NMOSD. Intracerebral injection of the APOE-mimetic peptide APOE130-149 attenuated microglial reactivity, neuroinflammation, and brain lesions in a mouse model of NMOSD. The protective effect of APOE in NMOSD pathogenesis was further established by the exacerbated lesion volume in APOE-deficient mice, which could be rescued by exogenous APOE administration. Genetic knockdown of the APOE receptor lipoprotein receptor-related protein 1 (LRP1) could block the restorative effects of APOE130-149 administration. The transfusion ADEVs derived from patients with NMOSD and healthy controls also alleviated astrocyte loss, reactive microgliosis, and demyelination in NMOSD mice. The slightly larger beneficial effect of patient-derived ADEVs as compared to ADEVs from healthy controls was further augmented in APOE-/- mice. These results indicate that APOE from astrocyte-derived extracellular vesicles could mediate disease-modifying astrocyte-microglia cross-talk in NMOSD.

Colchicine in patients with acute ischaemic stroke or transient ischaemic attack (CHANCE-3): multicentre, double blind, randomised, placebo controlled trial.

OBJECTIVES: To assess the efficacy and safety of colchicine versus placebo on reducing the risk of subsequent stroke after high risk non-cardioembolic ischaemic stroke or transient ischaemic attack within the first three months of symptom onset (CHANCE-3). DESIGN: Multicentre, double blind, randomised, placebo controlled trial. SETTING: 244 hospitals in China between 11 August 2022 and 13 April 2023. PARTICIPANTS: 8343 patients aged 40 years of age or older with a minor-to-moderate ischaemic stroke or transient ischaemic attack and a high sensitivity C-reactive protein ≥2 mg/L were enrolled. INTERVENTIONS: Patients were randomly assigned 1:1 within 24 h of symptom onset to receive colchicine (0.5 mg twice daily on days 1-3, followed by 0.5 mg daily thereafter) or placebo for 90 days. MAIN OUTCOME MEASURES: The primary efficacy outcome was any new stroke within 90 days after randomisation. The primary safety outcome was any serious adverse event during the treatment period. All efficacy and safety analyses were by intention to treat. RESULTS: 4176 patients were assigned to the colchicine group and 4167 were assigned to the placebo group. Stroke occurred within 90 days in 264 patients (6.3%) in the colchicine group and 270 patients (6.5%) in the placebo group (hazard ratio 0.98 (95% confidence interval 0.83 to 1.16); P=0.79). Any serious adverse event was observed in 91 (2.2%) patients in the colchicine group and 88 (2.1%) in the placebo group (P=0.83). CONCLUSIONS: The study did not provide evidence that low-dose colchicine could reduce the risk of subsequent stroke within 90 days as compared with placebo among patients with acute non-cardioembolic minor-to-moderate ischaemic stroke or transient ischaemic attack and a high sensitivity C-reactive protein ≥2 mg/L. TRIAL REGISTRATION: ClinicalTrials.gov, NCT05439356.

Neutrophil gelatinase-associated lipocalin regulates intracellular accumulation of Rh123 in cancer cells.

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.

Targeting CCL5 signaling attenuates neuroinflammation after seizure.

BACKGROUND: Epilepsy is a neurological condition that causes unprovoked, recurrent seizures. Accumulating evidence from clinical and experimental studies indicates that neuroinflammation exacerbates seizure activity. METHODS: 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. RESULTS: 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. CONCLUSIONS: This study supports the potential of CCL5/CCR5 signaling for targeting neuroinflammation after seizure.

CD4+ T cells aggravate hemorrhagic brain injury.

Leukocyte infiltration accelerates brain injury following intracerebral hemorrhage (ICH). Yet, the involvement of T lymphocytes in this process has not been fully elucidated. Here, we report that CD4+ T cells accumulate in the perihematomal regions in the brains of patients with ICH and ICH mouse models. T cells activation in the ICH brain is concurrent with the course of perihematomal edema (PHE) development, and depletion of CD4+ T cells reduced PHE volumes and improved neurological deficits in ICH mice. Single-cell transcriptomic analysis revealed that brain-infiltrating T cells exhibited enhanced proinflammatory and proapoptotic signatures. Consequently, CD4+ T cells disrupt the blood-brain barrier integrity and promote PHE progression through interleukin-17 release; furthermore, the TRAIL-expressing CD4+ T cells engage DR5 to trigger endothelial death. Recognition of T cell contribution to ICH-induced neural injury is instrumental for designing immunomodulatory therapies for this dreadful disease.

Group 2 innate lymphoid cells resolve neuroinflammation following cerebral ischaemia.

BACKGROUND: Acute 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. METHODS: Leveraging 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. RESULTS: We 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. CONCLUSIONS: Our findings revealed that brain ischaemia mobilises ILC2s to curb neuroinflammation and brain injury, expanding the current understanding of inflammatory networks following stroke.

Nuclear accumulation of calcineurin B homologous protein 2 (CHP2) results in enhanced proliferation of tumor cells.

The interaction between calcineurin B homologous protein 2 (CHP2) and Na(+) /H(+) exchanger 1 (NHE1), two membrane proteins, is essential for protecting cells from serum deprivation-induced death. Although four putative EF-hands in CHP2 had been predicted for years, Ca²(+) -binding activities of these motifs have not been tested yet, their role in this process remain poorly understood. To identify Ca²(+) -binding motifs required for the stable formation of CHP2/NHE1 complexes, we developed a mutagenesis-based assay in PS120 cells. We found that (45) Ca²(+) bond to two EF-hand motifs (EF3 and 4) of CHP2 proteins with high affinity. Complex formation between CHP2 and the CHP2 binding domain of NHE1 resulted in a marked increase in the Ca²(+) -binding affinity of CHP2. Co-immunoprecipitation and distribution of GFP-tagged CHP2-EF3m/4m also indicated that Ca²(+) affected the membrane location of CHP2 to interact with NHE1. The C-terminal region of CHP2 contains a nuclear export sequence (NES). When the six leucines of NES were mutated to alanines, the resulting CHP2 protein was predominantly localized to the nucleus. Furthermore, mutation of the NES resulted in enhanced proliferation and oncogenic potential of HeLa cells. Together, these results show that calcium and NES control the subcellular distribution of CHP2 and then distinctively regulate cell proliferation.