Neuropilin-1 identifies a subset of highly activated CD8+ T cells during parasitic and viral infections.

Neuropilin-1 (Nrp-1) expression on CD8+ T cells has been identified in tumor-infiltrating lymphocytes and in persistent murine gamma-herpes virus infections, where it interferes with the development of long-lived memory T cell responses. In parasitic and acute viral infections, the role of Nrp-1 expression on CD8+ T cells remains unclear. Here, we demonstrate a strong induction of Nrp-1 expression on CD8+ T cells in Plasmodium berghei ANKA (PbA)-infected mice that correlated with neurological deficits of experimental cerebral malaria (ECM). Likewise, the frequency of Nrp-1+CD8+ T cells was significantly elevated and correlated with liver damage in the acute phase of lymphocytic choriomeningitis virus (LCMV) infection. Transcriptomic and flow cytometric analyses revealed a highly activated phenotype of Nrp-1+CD8+ T cells from infected mice. Correspondingly, in vitro experiments showed rapid induction of Nrp-1 expression on CD8+ T cells after stimulation in conjunction with increased expression of activation-associated molecules. Strikingly, T cell-specific Nrp-1 ablation resulted in reduced numbers of activated T cells in the brain of PbA-infected mice as well as in spleen and liver of LCMV-infected mice and alleviated the severity of ECM and LCMV-induced liver pathology. Mechanistically, we identified reduced blood-brain barrier leakage associated with reduced parasite sequestration in the brain of PbA-infected mice with T cell-specific Nrp-1 deficiency. In conclusion, Nrp-1 expression on CD8+ T cells represents a very early activation marker that exacerbates deleterious CD8+ T cell responses during both, parasitic PbA and acute LCMV infections.

Hypothermia combined with extracellular vesicles from clonally expanded immortalized mesenchymal stromal cells improves neurodevelopmental impairment in neonatal hypoxic-ischemic brain injury.

BACKGROUND: Neonatal encephalopathy following hypoxia-ischemia (HI) is a leading cause of childhood death and morbidity. Hypothermia (HT), the only available but obligatory therapy is limited due to a short therapeutic window and limited efficacy. An adjuvant therapy overcoming limitations of HT is still missing. Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have shown promising therapeutic effects in various brain injury models. Challenges associated with MSCs' heterogeneity and senescence can be mitigated by the use of EVs from clonally expanded immortalized MSCs (ciMSCs). In the present study, we hypothesized that intranasal ciMSC-EV delivery overcomes limitations of HT. METHODS: Nine-day-old C57BL/6 mice were exposed to HI by occlusion of the right common carotid artery followed by 1 h hypoxia (10% oxygen). HT was initiated immediately after insult for 4 h. Control animals were kept at physiological body core temperatures. ciMSC-EVs or vehicle were administered intranasally 1, 3 and 5 days post HI/HT. Neuronal cell loss, inflammatory and regenerative responses were assessed via immunohistochemistry, western blot and real-time PCR 7 days after insult. Long-term neurodevelopmental outcome was evaluated by analyses of cognitive function, activity and anxiety-related behavior 5 weeks after HI/HT. RESULTS: In contrast to HT monotherapy, the additional intranasal therapy with ciMSC-EVs prevented HI-induced cognitive deficits, hyperactivity and alterations of anxiety-related behavior at adolescence. This was preceded by reduction of striatal neuronal loss, decreased endothelial, microglia and astrocyte activation; reduced expression of pro-inflammatory and increased expression of anti-inflammatory cytokines. Furthermore, the combination of HT with intranasal ciMSC-EV delivery promoted regenerative and neurodevelopmental processes, including endothelial proliferation, neurotrophic growth factor expression and oligodendrocyte maturation, which were not altered by HT monotherapy. CONCLUSION: Intranasal delivery of ciMSC-EVs represents a novel adjunct therapy, overcoming limitations of acute HT thereby offering new possibilities for improving long-term outcomes in neonates with HI-induced brain injury.

A report on the International Society for Cell & Gene Therapy 2022 Scientific Signature Series, "Therapeutic advances with native and engineered human extracellular vesicles".

The International Society for Cell & Gene Therapy Scientific Signature Series event "Therapeutic Advances With Native and Engineered Human EVs" took place as part of the International Society for Cell & Gene Therapy 2022 Annual Meeting, held from May 4 to 7, 2022, in San Francisco, California, USA. This was the first signature series event on extracellular vesicles (EVs) and a timely reflection of the growing interest in EVs, including both native and engineered human EVs, for therapeutic applications. The event successfully gathered academic and industrial key opinion leaders to discuss the current state of the art in developing and understanding native and engineered EVs and applying our knowledge toward advancing EV therapeutics. Latest advancements in understanding the mechanisms by which native and engineered EVs exert their therapeutic effects against different diseases in animal models were presented, with some diseases such as psoriasis and osteoarthritis already reaching clinical testing of EVs. The discussion also covered various aspects relevant to advancing the clinical translation of EV therapies, including EV preparation, manufacturing, consistency, site(s) of action, route(s) of administration, and luminal cargo delivery of RNA and other compounds.

Regulatory T Cells Contribute to Sexual Dimorphism in Neonatal Hypoxic-Ischemic Brain Injury.

BACKGROUND AND PURPOSE: Neonatal encephalopathy caused by hypoxia-ischemia (HI) is a major cause of death and disability in newborns. Clinical and experimental studies suggest a sexual dimorphism in HI-induced brain injury and therapy responses. A major hallmark of HI pathophysiology is the infiltration of peripheral immune cells into the injured brain. However, the specific role of regulatory T cells (Tregs) in neonatal HI is still unknown. METHODS: Nine-day-old mice were exposed to HI by ligation of the right common carotid artery followed by 1 hour hypoxia (10% oxygen). Using immunohistochemistry, flow cytometry, and microarray analyses, Tregs were investigated in the brain, spleen, and blood 24 hours post HI. The functional role of Tregs was evaluated by acute Treg depletion in depletion of regulatory T cells transgenic mice. Brain injury, neuroinflammatory responses, and vascular injury were analyzed via immunohistochemistry and Western blot 48 hours and 7 days after HI. Functional outcome was assessed 3 days and 5 weeks after HI. RESULTS: Female mice revealed an increased cerebral Treg infiltration, coinciding with elevated chemokine receptor expression. Treg depletion in females aggravated HI-induced brain tissue injury, short-term motor deficits, and long-term deficits in exploratory activity, paralleled by an increased microglia and endothelial activation and leukocyte infiltration. Treg depletion in male mice reduced HI-induced brain injury, short-term motor, and long-term cognitive deficits, associated with reduced vascular injury. Ex vivo isolated female Tregs displayed an increased immunosuppressive activity on effector T cell proliferation and an increased gene enrichment in pathways related to enhanced Treg activity. CONCLUSIONS: Tregs from neonatal female mice provide endogenous neuroprotection, whereas Tregs from male mice increase secondary neurodegeneration. As potential mechanisms, we identified intrinsic transcriptional differences associated with enhanced anti-inflammatory activity of female Tregs. Our study emphasizes the urgent need for sex-stratified clinical and preclinical analyses.

Postischemic Neuroprotection Associated With Anti-Inflammatory Effects by Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles in Aged Mice.

BACKGROUND AND PURPOSE: Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce ischemic neuroprotection in mice by modulating the brain infiltration of leukocytes and, specifically polymorphonuclear neutrophils. So far, effects of MSC-sEVs were only studied in young ischemic rodents. We herein examined the effects of MSC-sEVs in aged mice. METHODS: Male and female C57Bl6/j mice (8-10 weeks or 15-24 months) were exposed to transient intraluminal middle cerebral artery occlusion. Vehicle or sEVs (equivalent of 2×106 MSCs) were intravenously administered. Neurological deficits, ischemic injury, blood-brain barrier integrity, brain leukocyte infiltration, and blood leukocyte responses were evaluated over up to 7 days. RESULTS: MSC-sEV delivery reduced neurological deficits, infarct volume, brain edema, and neuronal injury in young and aged mice of both sexes, when delivered immediately postreperfusion or with 6 hours delay. MSC-sEVs decreased leukocyte and specifically polymorphonuclear neutrophil, monocyte, and macrophage infiltrates in ischemic brains of aged mice. In peripheral blood, the number of monocytes and activated T cells was significantly reduced by MSC-sEVs. CONCLUSIONS: MSC-sEVs induce postischemic neuroprotection and anti-inflammation in aged mice.

Peripheral immune cells and perinatal brain injury: a double-edged sword?

Perinatal brain injury is the leading cause of neurological mortality and morbidity in childhood ranging from motor and cognitive impairment to behavioural and neuropsychiatric disorders. Various noxious stimuli, including perinatal inflammation, chronic and acute hypoxia, hyperoxia, stress and drug exposure contribute to the pathogenesis. Among a variety of pathological phenomena, the unique developing immune system plays an important role in the understanding of mechanisms of injury to the immature brain. Neuroinflammation following a perinatal insult largely contributes to evolution of damage to resident brain cells, but may also be beneficial for repair activities. The present review will focus on the role of peripheral immune cells and discuss processes involved in neuroinflammation under two frequent perinatal conditions, systemic infection/inflammation associated with encephalopathy of prematurity (EoP) and hypoxia/ischaemia in the context of neonatal encephalopathy (NE) and stroke at term. Different immune cell subsets in perinatal brain injury including their infiltration routes will be reviewed and critical aspects such as sex differences and maturational stage will be discussed. Interactions with existing regenerative therapies such as stem cells and also potentials to develop novel immunomodulatory targets are considered. IMPACT: Comprehensive summary of current knowledge on the role of different immune cell subsets in perinatal brain injury including discussion of critical aspects to be considered for development of immunomodulatory therapies.

Hypothermia modulates myeloid cell polarization in neonatal hypoxic-ischemic brain injury.

BACKGROUND: Neonatal encephalopathy due to hypoxia-ischemia (HI) is a leading cause of death and disability in term newborns. Therapeutic hypothermia (HT) is the only recommended therapy. However, 30% still suffer from neurological deficits. Inflammation is a major hallmark of HI pathophysiology with myeloid cells being key players, participating either in progression or in resolution of injury-induced inflammation. In the present study, we investigated the impact of HT on the temporal and spatial dynamics of microglia/macrophage polarization after neonatal HI in newborn mice. METHODS: Nine-day-old C57BL/6 mice were exposed to HI through occlusion of the right common carotid artery followed by 1 h hypoxia. Immediately after HI, animals were cooled for 4 h or kept at physiological body core temperature. Analyses were performed at 1, 3 and 7 days post HI. Brain injury, neuronal cell loss, apoptosis and microglia activation were assessed by immunohistochemistry. A broad set of typical genes associated with classical (M1) and alternative (M2) myeloid cell activation was analyzed by real time PCR in ex vivo isolated CD11b+ microglia/macrophages. Purity and composition of isolated cells was determined by flow cytometry. RESULTS: Immediate HT significantly reduced HI-induced brain injury and neuronal loss 7 days post HI, whereas only mild non-significant protection from HI-induced apoptosis and neuronal loss were observed 1 and 3 days after HI. Microglia activation, i.e., Iba-1 immunoreactivity peaked 3 days after HI and was not modulated by HT. However, ex vivo isolated CD11b+ cells revealed a strong upregulation of the majority of M1 but also M2 marker genes at day 1, which was significantly reduced by HT and rapidly declined at day 3. HI induced a significant increase in the frequency of peripheral macrophages in sorted CD11b+ cells at day 1, which deteriorated until day 7 and was significantly decreased by HT. CONCLUSION: Our data demonstrate that HT-induced neuroprotection is preceded by acute suppression of HI-induced upregulation of inflammatory genes in myeloid cells and decreased infiltration of peripheral macrophages, both representing potential important effector mechanisms of HT.

Neutrophil dynamics, plasticity and function in acute neurodegeneration following neonatal hypoxia-ischemia.

Neonatal encephalopathy following hypoxia-ischemia (HI) is a major cause of long-term morbidity and mortality in children. Even though HI-induced neuroinflammation, involving infiltration of peripheral immune cells into the CNS has been associated with disease pathogenesis, the specific role of neutrophils is highly debated. Due to immaturity of the neonatal immune system, it has been assumed that neutrophils are less clinically relevant in neonatal HI-induced brain injury. In the present study, we demonstrate that neutrophils are rapidly activated in the neonatal brain after exposure to experimental HI, revealed by an enhanced proportion of CD86+ cells and an increased expression of CD11b compared to splenic and blood neutrophils. Furthermore, production of reactive oxygen species and the proportion of hyperactivated/aged (CXCR4+CD62L-) cells was enhanced in brain compared to peripheral neutrophils. Delayed neutrophil depletion, initiated 12 h after HI resulted in reduced cellular neurodegeneration, associated with reduced micro- and astroglial activation. In the present study, we uncovered a new complex switch of the phenotype in brain neutrophils, which may offer new possibilities for the development of selective therapeutic approaches by modulation of neutrophils in the early post-hypoxic disease phase.

White Matter Brain Development after Exposure to Circulating Cell-Free Hemoglobin and Hyperoxia in a Rat Pup Model.

Neonates born with critical congenital heart defects are at risk of diffuse white matter injuries and neurodevelopmental impairments. This study aimed to determine the impact of circulating cell-free hemoglobin and hyperoxia, both present during cardiopulmonary bypass circulation, on white matter brain development. Postnatal day 6 rat pups were injected intraperitoneally with cell-free Hb or vehicle and exposed to hyperoxia (fiO2 = 0.8) or normoxia (fiO2 = 0.21) for 24 h. We evaluated apoptosis, myelination, and oligodendrocyte maturation with immunohistochemistry, gene and protein analyses, and in vivo diffusion tensor magnetic resonance imaging (MRI). Consistent with previous studies, we found an increase in apoptosis of oligodendrocytes as determined by TUNEL+ staining in Olig2+ cells in white matter, cortex, thalamus, and hippocampus following exposure to hyperoxia with no additional effect of cell-free Hb. A transient increase in the mRNA expression of intercellular adhesion molecule 1 at 6 h was observed following combined exposure to cell-free Hb and hyperoxia. No indications of oligodendrocyte maturational delay or hypomyelination were observed after either insult, delivered separately or combined, as determined by immunohistochemistry, Western blot, and diffusion tensor MRI. In our model, exposure to circulatory cell-free Hb, with or without concomitant hyperoxia, did not significantly alter brain white matter development.

In vivo imaging of partially reversible th17 cell-induced neuronal dysfunction in the course of encephalomyelitis.

Neuronal damage in autoimmune neuroinflammation is the correlate for long-term disability in multiple sclerosis (MS) patients. Here, we investigated the role of immune cells in neuronal damage processes in animal models of MS by monitoring experimental autoimmune encephalomyelitis (EAE) by using two-photon microscopy of living anaesthetized mice. In the brainstem, we detected sustained interaction between immune and neuronal cells, particularly during disease peak. Direct interaction of myelin oligodendrocyte glycoprotein (MOG)-specific Th17 and neuronal cells in demyelinating lesions was associated with extensive axonal damage. By combining confocal, electron, and intravital microscopy, we showed that these contacts remarkably resembled immune synapses or kinapses, albeit with the absence of potential T cell receptor engagement. Th17 cells induced severe, localized, and partially reversible fluctuation in neuronal intracellular Ca(2+) concentration as an early sign of neuronal damage. These results highlight the central role of the Th17 cell effector phenotype for neuronal dysfunction in chronic neuroinflammation.

Adverse neuropsychiatric development following perinatal brain injury: from a preclinical perspective.

Perinatal brain injury is a leading cause of death and disability in young children. Recent advances in obstetrics, reproductive medicine and neonatal intensive care have resulted in significantly higher survival rates of preterm or sick born neonates, at the price of increased prevalence of neurological, behavioural and psychiatric problems in later life. Therefore, the current focus of experimental research shifts from immediate injury processes to the consequences for brain function in later life. The aetiology of perinatal brain injury is multi-factorial involving maternal and also labour-associated factors, including not only placental insufficiency and hypoxia-ischaemia but also exposure to high oxygen concentrations, maternal infection yielding excess inflammation, genetic factors and stress as important players, all of them associated with adverse long-term neurological outcome. Several animal models addressing these noxious stimuli have been established in the past to unravel the underlying molecular and cellular mechanisms of altered brain development. In spite of substantial efforts to investigate short-term consequences, preclinical evaluation of the long-term sequelae for the development of cognitive and neuropsychiatric disorders have rarely been addressed. This review will summarise and discuss not only current evidence but also requirements for experimental research providing a causal link between insults to the developing brain and long-lasting neurodevelopmental disorders.

Interaction between hypothermia and delayed mesenchymal stem cell therapy in neonatal hypoxic-ischemic brain injury.

Acute hypothermia treatment (HT) is the only clinically established intervention following neonatal hypoxic-ischemic brain injury. However, almost half of all cooled infants still die or suffer from long-lasting neurological impairments. Regenerative therapies, such as mesenchymal stem cells (MSC) appear promising as adjuvant therapy. In the present study, we hypothesized that HT combined with delayed MSC therapy results in augmented protection, improving long-term neurological outcome. Postnatal day 9 (P9) C57BL/6 mice were exposed to hypoxia-ischemia followed by 4 h HT. Murine bone marrow-derived MSC (1 × 106 cells/animal) were administered intranasally at P12. Cytokine and growth factor levels were assessed by ELISA and Luminex® multiplex assay 24 h following MSC delivery. One week after HI, tissue injury and neuroinflammatory responses were determined by immunohistochemistry and western blot. Long-term motor-cognitive outcome was assessed 5 weeks post injury. MSC responses to the brains' environment were evaluated by gene expression analysis in MSC, co-cultured with brain homogenates isolated at P12. Both, MSC and HT improved motor deficits, while cognitive function could only be restored by MSC. Compared to each single therapy, combined treatment led to increased long-lasting motor-cognitive deficits and exacerbated brain injury, accompanied by enhanced endothelial activation and peripheral immune cell infiltration. MSC co-cultured with brain extracts of HT-treated animals revealed increased pro-inflammatory cytokine and decreased growth factor expression. In vivo protein analysis showed higher pro-inflammatory cytokine levels after combined treatment compared to single therapy. Furthermore, HI-induced increase in growth factors was normalized to control levels by HT and MSC single therapy, while the combination induced a further decline below control levels. Our results suggest that alteration of the brains' microenvironment by acute HT modulates MSC function resulting in a pro-inflammatory environment combined with alteration of the homeostatic growth factor milieu in the neonatal hypoxic-ischemic brain. This study delineates potential unexpected side effects of cell-based therapies as add-on therapy for acute hypothermia treatment.

Sildenafil Enhances Quantity of Immature Neurons and Promotes Functional Recovery in the Developing Ischemic Mouse Brain.

BACKGROUND: Hypoxic-ischemic (HI) injury to the developing brain occurs in 1 out of 1,000 live births and remains a major cause of significant morbidity and mortality. A large number of survivors suffer from long-term sequelae including seizures and neurological deficits. However, the pathophysiological mechanisms of recovery after HI insult are not clearly understood, and preventive measures or clinical treatments are nonexistent or not sufficiently effective in the clinical setting. Sildenafil as a specific phosphodiesterase 5 inhibitor leads to increased levels of the second messenger cyclic guanosine monophosphate (cGMP) and promotes functional recovery and neurogenesis after ischemic injury to the adult brain. OBJECTIVE: Here, we investigated the effect of sildenafil treatment on activation of intracellular signaling pathways, histological and neurogenic response including functional recovery after an ischemic insult to the developing brain. DESIGN/METHODS: Nine-day-old C57BL/6 mice were subjected either to sham operation or underwent ligation of the right common carotid artery followed by hypoxia (8%) for 60 min. Animals were either administered sildenafil (10 mg/kg, i.p.) or vehicle 2 h after hypoxia. A subgroup of animals received multiple injections of 10 mg/kg daily on 5 consecutive days. Pups were either perfusion fixed at postnatal days 14 or 47 for immunohistochemical analysis, or brains were dissected 2, 6, 12, and 24 h after the end of hypoxia and analyzed for cGMP, pAkt, pGSK-3ß, and ß-catenin by means of ELISA or immunoblotting. In addition, behavioral studies using the wire hang test and elevated plus maze were conducted 21 and 38 days after HI injury. RESULTS: Based on cresyl violet staining, single or multiple sildenafil injections did not reveal any differences in injury scoring compared to sham animals. However, cerebral levels of cGMP were altered after sildenafil therapy. Treatment significantly increased numbers of immature neurons, as indicated by doublecortin immunoreactivity in the ipsilateral subventricular zone and striatum. In addition, animals treated with sildenafil after HI insult demonstrated improved functional recovery. pAkt, pGSK-3ß, and ß-catenin levels vary after HI injury but additional sildenafil treatment had no impact on protein expression compared to the level of sham controls. CONCLUSIONS: Here, we report that treatment with sildenafil after HI insult did not improve histological brain injury scores. Nevertheless, our results suggest involvement of the cGMP and PI3K/Akt/GSK-3ß signaling pathway with promotion of a neurogenic response and reduction of neurological deficits. In summary, sildenafil may have a role in promoting recovery from HI injury in the developing brain.

Dendritic cells tip the balance towards induction of regulatory T cells upon priming in experimental autoimmune encephalomyelitis.

Counter-balancing regulatory mechanisms, such as the induction of regulatory T cells (Treg), limit the effects of autoimmune attack in neuroinflammation. However, the role of dendritic cells (DCs) as the most powerful antigen-presenting cells, which are intriguing therapeutic targets in this context, is not fully understood. Here, we demonstrate that conditional ablation of DCs during the priming phase of myelin-specific T cells in experimental autoimmune encephalomyelitis (EAE) selectively aborts inducible Treg (iTreg) induction, whereas generation of T helper (Th)1/17 cells is unaltered. DCs facilitate iTreg induction by creating a milieu with high levels of interleukin (IL)-2 due to a strong proliferative response. In the absence of DCs, B220+ B cells take over priming of Th17 cells in the place of antigen-presenting cells (APCs), but not the induction of iTreg, thus leading to unregulated, severe autoimmunity.

Mesenchymal stem cell-derived extracellular vesicles ameliorate inflammation-induced preterm brain injury.

OBJECTIVE: Preterm brain injury is a major cause of disability in later life, and may result in motor, cognitive and behavioural impairment for which no treatment is currently available. The aetiology is considered as multifactorial, and one underlying key player is inflammation leading to white and grey matter injury. Extracellular vesicles secreted by mesenchymal stem/stromal cells (MSC-EVs) have shown therapeutic potential in regenerative medicine. Here, we investigated the effects of MSC-EV treatment on brain microstructure and maturation, inflammatory processes and long-time outcome in a rodent model of inflammation-induced brain injury. METHODS: 3-Day-old Wistar rats (P3) were intraperitoneally injected with 0.25mg/kg lipopolysaccharide or saline and treated with two repetitive doses of 1×108 cell equivalents of MSC-EVs per kg bodyweight. Cellular degeneration and reactive gliosis at P5 and myelination at P11 were evaluated by immunohistochemistry and western blot. Long-term cognitive and motor function was assessed by behavioural testing. Diffusion tensor imaging at P125 evaluated long-term microstructural white matter alterations. RESULTS: MSC-EV treatment significantly ameliorated inflammation-induced neuronal cellular degeneration reduced microgliosis and prevented reactive astrogliosis. Short-term myelination deficits and long-term microstructural abnormalities of the white matter were restored by MSC-EV administration. Morphological effects of MSC-EV treatment resulted in improved long-lasting cognitive functions INTERPRETATION: MSC-EVs ameliorate inflammation-induced cellular damage in a rat model of preterm brain injury. MSC-EVs may serve as a novel therapeutic option by prevention of neuronal cell death, restoration of white matter microstructure, reduction of gliosis and long-term functional improvement.

Fingolimod protects against neonatal white matter damage and long-term cognitive deficits caused by hyperoxia.

Cerebral white matter injury is a leading cause of adverse neurodevelopmental outcome in prematurely born infants involving cognitive deficits in later life. Despite increasing knowledge about the pathophysiology of perinatal brain injury, therapeutic options are limited. In the adult demyelinating disease multiple sclerosis the sphingosine-1-phosphate (S1P) receptor modulating substance fingolimod (FTY720) has beneficial effects. Herein, we evaluated the neuroprotective potential of FTY720 in a neonatal model of oxygen-toxicity, which is associated with hypomyelination and impaired neuro-cognitive outcome. A single dose of FTY720 (1mg/kg) at the onset of neonatal hyperoxia (24h 80% oxygen on postnatal day 6) resulted in improvement of neuro-cognitive development persisting into adulthood. This was associated with reduced microstructural white matter abnormalities 4 months after the insult. In search of the underlying mechanisms potential non-classical (i.e. lymphocyte-independent) pathways were analysed shortly after the insult, comprising modulation of oxidative stress and local inflammatory responses as well as myelination, oligodendrocyte degeneration and maturation. Treatment with FTY720 reduced hyperoxia-induced oxidative stress, microglia activation and associated pro-inflammatory cytokine expression. In vivo and in vitro analyses further revealed that oxygen-induced hypomyelination is restored to control levels, which was accompanied by reduced oligodendrocyte degeneration and enhanced maturation. Furthermore, hyperoxia-induced elevation of S1P receptor 1 (S1P1) protein expression on in vitro cultured oligodendrocyte precursor cells was reduced by activated FTY720 and protection from degeneration is abrogated after selective S1P1 blockade. Finally, FTY720s' classical mode of action (i.e. retention of immune cells within peripheral lymphoid organs) was analysed demonstrating that FTY720 diminished circulating lymphocyte counts independent from hyperoxia. Cerebral immune cell counts remained unchanged by hyperoxia and by FTY720 treatment. Taken together, these results suggest that beneficial effects of FTY720 in neonatal oxygen-induced brain injury may be rather attributed to its anti-oxidative and anti-inflammatory capacity acting in concert with a direct protection of developing oligodendrocytes than to a modulation of peripheral lymphocyte trafficking. Thus, FTY720 might be a potential new therapeutic option for the treatment of neonatal brain injury through reduction of white matter damage.

Role of Neutrophils in Exacerbation of Brain Injury After Focal Cerebral Ischemia in Hyperlipidemic Mice.

BACKGROUND AND PURPOSE: Inflammation-related comorbidities contribute to stroke-induced immune responses and brain damage. We previously showed that hyperlipidemia exacerbates ischemic brain injury, which is associated with elevated peripheral and cerebral granulocyte numbers. Herein, we evaluate the contribution of neutrophils to the exacerbation of ischemic brain injury. METHODS: Wild-type mice fed with a normal chow and ApoE knockout mice fed with a high cholesterol diet were exposed to middle cerebral artery occlusion. CXCR2 was blocked using the selective antagonist SB225002 (2 mg/kg) or neutralizing CXCR2 antiserum. Neutrophils were depleted using an anti-Ly6G antibody. At 72 hours post ischemia, immunohistochemistry, flow cytometry, and real-time polymerase chain reaction were performed to determine cerebral tissue injury and immunologic changes in the blood, bone marrow, and brain. Functional outcome was assessed by accelerated rota rod and tight rope tests at 4, 7, and 14 days post ischemia. RESULTS: CXCR2 antagonization reduced neurological deficits and infarct volumes that were exacerbated in hyperlipidemic ApoE-/- mice. This effect was mimicked by neutrophil depletion. Cerebral neutrophil infiltration and peripheral neutrophilia, which were increased on ischemia in hyperlipidemia, were attenuated by CXCR2 antagonization. This downscaling of neutrophil responses was associated with increased neutrophil apoptosis and reduced levels of CXCR2, inducible nitric oxide synthase, and NADPH oxidase 2 expression on bone marrow neutrophils. CONCLUSIONS: Our data demonstrate a role of neutrophils in the exacerbation of ischemic brain injury induced by hyperlipidemia. Accordingly, CXCR2 blockade, which prevents neutrophil recruitment into the brain, might be an effective option for stroke treatment in patients with hyperlipidemia.

Very-late-antigen-4 (VLA-4)-mediated brain invasion by neutrophils leads to interactions with microglia, increased ischemic injury and impaired behavior in experimental stroke.

Neuronal injury from ischemic stroke is aggravated by invading peripheral immune cells. Early infiltrates of neutrophil granulocytes and T-cells influence the outcome of stroke. So far, however, neither the timing nor the cellular dynamics of neutrophil entry, its consequences for the invaded brain area, or the relative importance of T-cells has been extensively studied in an intravital setting. Here, we have used intravital two-photon microscopy to document neutrophils and brain-resident microglia in mice after induction of experimental stroke. We demonstrated that neutrophils immediately rolled, firmly adhered, and transmigrated at sites of endothelial activation in stroke-affected brain areas. The ensuing neutrophil invasion was associated with local blood-brain barrier breakdown and infarct formation. Brain-resident microglia recognized both endothelial damage and neutrophil invasion. In a cooperative manner, they formed cytoplasmic processes to physically shield activated endothelia and trap infiltrating neutrophils. Interestingly, the systemic blockade of very-late-antigen-4 immediately and very effectively inhibited the endothelial interaction and brain entry of neutrophils. This treatment thereby strongly reduced the ischemic tissue injury and effectively protected the mice from stroke-associated behavioral impairment. Behavioral preservation was also equally well achieved with the antibody-mediated depletion of myeloid cells or specifically neutrophils. In contrast, T-cell depletion more effectively reduced the infarct volume without improving the behavioral performance. Thus, neutrophil invasion of the ischemic brain is rapid, massive, and a key mediator of functional impairment, while peripheral T-cells promote brain damage. Acutely depleting T-cells and inhibiting brain infiltration of neutrophils might, therefore, be a powerful early stroke treatment.

Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation.

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation.

The role of CD8+ T cells and their local interaction with CD4+ T cells in myelin oligodendrocyte glycoprotein35-55-induced experimental autoimmune encephalomyelitis.

T cells have an essential role in the induction of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Although for CD4(+) T cells it is well established that they contribute to the disease, less is known about the role of CD8(+) T cells. Our aim was to determine the individual contribution of CD4(+) and CD8(+) T cells in myelin oligodendrocyte glycoprotein (MOG)35-55-induced EAE. We investigated MOG35-55-activated CD8(+) T cells to clarify their potential to induce or attenuate EAE. We monitored the behavior of CD8(+) T cells and their interaction with CD4(+) T cells directly at the site of inflammation in the CNS using intravital imaging of the brainstem of EAE-affected living anesthetized mice. We found that mice without CD4(+) T cells did not develop relevant clinical signs of disease, although CD8(+) T cells were present in the CNS of these mice. These CD8(+) T cells displayed reduced motility compared with those in the presence of CD4(+) T cells. In mice that harbored CD4(+) and CD8(+) T cells, we saw a similar extent of clinical signs of EAE as in mice with only CD4(+) T cells. Furthermore, the dynamic motility and viability of CD4(+) T cells were not disturbed by CD8(+) T cells in the lesions of these mice. Therefore, we conclude that in MOG35-55-induced EAE, CD8(+) T cell accumulation in the CNS represents instead an epiphenomenon with no impact on clinical disease or on the effects of CD4(+) T cells, the latter being the true inducers of the disease.