Homoarginine- and Creatine-Dependent Gene Regulation in Murine Brains with l-Arginine:Glycine Amidinotransferase Deficiency.

l-arginine:glycine amidinotransferase (AGAT) and its metabolites homoarginine (hArg) and creatine have been linked to stroke pathology in both human and mouse studies. However, a comprehensive understanding of the underlying molecular mechanism is lacking. To investigate transcriptional changes in cerebral AGAT metabolism, we applied a transcriptome analysis in brains of wild-type (WT) mice compared to untreated AGAT-deficient (AGAT-/-) mice and AGAT-/- mice with creatine or hArg supplementation. We identified significantly regulated genes between AGAT-/- and WT mice in two independent cohorts of mice which can be linked to amino acid metabolism (Ivd, Lcmt2), creatine metabolism (Slc6a8), cerebral myelination (Bcas1) and neuronal excitability (Kcnip3). While Ivd and Kcnip3 showed regulation by hArg supplementation, Bcas1 and Slc6a8 were creatine dependent. Additional regulated genes such as Pla2g4e and Exd1 need further evaluation of their influence on cerebral function. Experimental stroke models showed a significant regulation of Bcas1 and Slc6a8. Together, these results reveal that AGAT deficiency, hArg and creatine regulate gene expression in the brain, which may be critical in stroke pathology.

IL-23 (Interleukin-23)-Producing Conventional Dendritic Cells Control the Detrimental IL-17 (Interleukin-17) Response in Stroke.

BACKGROUND AND PURPOSE: Inflammatory mechanisms can exacerbate ischemic tissue damage and worsen clinical outcome in patients with stroke. Both αß and γδ T cells are established mediators of tissue damage in stroke, and the role of dendritic cells (DCs) in inducing the early events of T cell activation and differentiation in stroke is not well understood. METHODS: In a murine model of experimental stroke, we defined the immune phenotype of infiltrating DC subsets based on flow cytometry of surface markers, the expression of ontogenetic markers, and cytokine levels. We used conditional DC depletion, bone marrow chimeric mice, and IL-23 (interleukin-23) receptor-deficient mice to further explore the functional role of DCs. RESULTS: We show that the ischemic brain was rapidly infiltrated by IRF4+/CD172a+ conventional type 2 DCs and that conventional type 2 DCs were the most abundant subset in comparison with all other DC subsets. Twenty-four hours after ischemia onset, conventional type 2 DCs became the major source of IL-23, promoting neutrophil infiltration by induction of IL-17 (interleukin-17) in γδ T cells. Functionally, the depletion of CD11c+ cells or the genetic disruption of the IL-23 signaling abrogated both IL-17 production in γδ T cells and neutrophil infiltration. Interruption of the IL-23/IL-17 cascade decreased infarct size and improved neurological outcome after stroke. CONCLUSIONS: Our results suggest a central role for interferon regulatory factor 4-positive IL-23-producing conventional DCs in the IL-17-dependent secondary tissue damage in stroke.

CCR6 (CC Chemokine Receptor 6) Is Essential for the Migration of Detrimental Natural Interleukin-17-Producing γδ T Cells in Stroke.

BACKGROUND AND PURPOSE: Immune-mediated tissue damage after stroke evolves within the first days, and lymphocytes contribute to the secondary injury. Our goal was to identify T-cell subpopulations, which trigger the immune response. METHODS: In a model of experimental stroke, we analyzed the immune phenotype of interleukin-17 (IL-17)-producing γδ T cells and explored the therapeutic potential of neutralizing anti-IL-17 antibodies in combination with mild therapeutic hypothermia. RESULTS: We show that brain-infiltrating IL-17-positive γδ T cells expressed the Vγ6 segment of the γδ T cells receptor and were largely positive for the chemokine receptor CCR6 (CC chemokine receptor 6), which is a characteristic for natural IL-17-producing γδ T cells. These innate lymphocytes are established as major initial IL-17 producers in acute infections. Genetic deficiency in Ccr6 was associated with diminished infiltration of natural IL-17-producing γδ T cells and a significantly improved neurological outcome. In the ischemic brain, IL-17 together with tumor necrosis factor-α triggered the expression of CXC chemokines and neutrophil infiltration. Therapeutic targeting of synergistic IL-17 and tumor necrosis factor-α pathways by IL-17 neutralization and therapeutic hypothermia resulted in additional protective effects in comparison to an anti-IL-17 antibody treatment or therapeutic hypothermia alone. CONCLUSIONS: Brain-infiltrating IL-17-producing γδ T cells belong to the subset of natural IL-17-producing γδ T cells. In stroke, these previously unrecognized innate lymphocytes trigger a highly conserved immune reaction, which is known from host responses toward pathogens. We demonstrate that therapeutic approaches targeting synergistic IL-17 and tumor necrosis factor-α pathways in parallel offer additional neuroprotection in stroke.

Transient receptor potential melastatin subfamily member 2 cation channel regulates detrimental immune cell invasion in ischemic stroke.

BACKGROUND AND PURPOSE: Brain injury during stroke results in oxidative stress and the release of factors that include extracellular Ca(2+), hydrogen peroxide, adenosine diphosphate ribose, and nicotinic acid adenine dinucleotide phosphate. These alterations of the extracellular milieu change the activity of transient receptor potential melastatin subfamily member 2 (TRPM2), a nonselective cation channel expressed in the central nervous system and the immune system. Our goal was to evaluate the contribution of TRPM2 to the tissue damage after stroke. METHODS: In accordance with current quality guidelines, we independently characterized Trpm2 in a murine ischemic stroke model in 2 different laboratories. RESULTS: Gene deficiency of Trpm2 resulted in significantly improved neurological outcome and decreased infarct size. Besides an already known moderate neuroprotective effect of Trpm2 deficiency in vitro, ischemic brain invasion by neutrophils and macrophages was particularly reduced in Trpm2-deficient mice. Bone marrow chimeric mice revealed that Trpm2 deficiency in the peripheral immune system is responsible for the protective phenotype. Furthermore, experiments with mixed bone marrow chimeras demonstrated that Trpm2 is essential for the migration of neutrophils and, to a lesser extent, also of macrophages into ischemic hemispheres. Notably, the pharmacological TRPM2 inhibitor, N-(p-amylcinnamoyl)anthranilic acid, was equally protective in the stroke model. CONCLUSIONS: Although a neuroprotective effect of TRPM2 in vitro is well known, we can show for the first time that the detrimental role of TRPM2 in stroke primarily depends on its role in activating peripheral immune cells. Targeting TRPM2 systemically represents a promising therapeutic approach for ischemic stroke.

Neutralization of the IL-17 axis diminishes neutrophil invasion and protects from ischemic stroke.

The devastating effect of ischemic stroke is attenuated in mice lacking conventional and unconventional T cells, suggesting that inflammation enhances tissue damage in cerebral ischemia. We explored the functional role of αß and γδ T cells in a murine model of stroke and distinguished 2 different T cell-dependent proinflammatory pathways in ischemia-reperfusion injury. IFN-γ produced by CD4(+) T cells induced TNF-α production in macrophages, whereas IL-17A secreted by γδ T cells led to neutrophil recruitment. The synergistic effect of TNF-α and IL-17A on astrocytes resulted in enhanced secretion of CXCL-1, a neutrophil chemoattractant. Application of an IL-17A-blocking antibody within 3 hours after stroke induction decreased infarct size and improved neurologic outcome in the murine model. In autoptic brain tissue of patients who had a stroke, we detected IL-17A-positive lymphocytes, suggesting that this aspect of the inflammatory cascade is also relevant in the human brain. We propose that selective targeting of IL-17A signaling might provide a new therapeutic option for the treatment of stroke.

Plasma levels of neuron specific enolase quantify the extent of neuronal injury in murine models of ischemic stroke and multiple sclerosis.

OBJECTIVE: We aimed at validating a plasma biomarker for neuronal damage that can be used in acute and chronic models of neurological diseases. METHODS: We investigated two different models, middle cerebral artery occlusion followed by reperfusion and MOG35-55-induced experimental autoimmune encephalomyelitis (EAE). In stroke experiments we measured infarct sizes by magnetic resonance imaging and vital stainings and correlated them with plasma levels of neuron specific enolase (NSE) at different time points after reperfusion. Equally, in EAE experiments, we correlated NSE levels with neurological scores and histopathological damage of axons at different time points. We detected plasma NSE levels by ELISA. RESULTS: Plasma NSE levels correlated significantly with stroke size, EAE score and histopathological damage in EAE. Investigations into the dynamics of neuronal loss over time correlated well with the dynamics of NSE levels. NSE even predicted the onset of EAE, before clinical signs were recordable. CONCLUSIONS: Plasma NSE is a valid and simple experimental biomarker that allows quantifying the degree of neuronal injury in a non-invasive approach.

γδ T cells as early sensors of tissue damage and mediators of secondary neurodegeneration.

Spontaneous or medically induced reperfusion occurs in up to 70% of patients within 24 h after cerebral ischemia. Reperfusion of ischemic brain tissue can augment the inflammatory response that causes additional injury. Recently, T cells have been shown to be an essential part of the post-ischemic tissue damage, and especially IL-17 secreting T cells have been implicated in the pathogenesis of a variety of inflammatory reactions in the brain. After stroke, it seems that the innate γδ T cells are the main IL-17 producing cells and that the γδ T cell activation constitutes an early and mainly damaging immune response in stroke. Effector mechanism of γδ T cell derived IL-17 in the ischemic brain include the induction of metalloproteinases, proinflammatory cytokines and neutrophil attracting chemokines, leading to a further amplification of the detrimental inflammatory response. In this review, we will give an overview on the concepts of γδ T cells and IL-17 in stroke pathophysiology and on their potential importance for human disease conditions.

Deficiency in serine protease inhibitor neuroserpin exacerbates ischemic brain injury by increased postischemic inflammation.

The only approved pharmacological treatment for ischemic stroke is intravenous administration of plasminogen activator (tPA) to re-canalize the occluded cerebral vessel. Not only reperfusion but also tPA itself can induce an inflammatory response. Microglia are the innate immune cells of the central nervous system and the first immune cells to become activated in stroke. Neuroserpin, an endogenous inhibitor of tPA, is up-regulated following cerebral ischemia. To examine neuroserpin-dependent mechanisms of neuroprotection in stroke, we studied neuroserpin deficient (Ns(-/-))mice in an animal model of temporal focal ischemic stroke. Infarct size and neurological outcome were worse in neuroserpin deficient mice even though the fibrinolytic activity in the ischemic brain was increased. The increased infarct size was paralleled by a selective increase in proinflammatory microglia activation in Ns(-/-) mice. Our results show excessive microglial activation in Ns(-/-) mice mediated by an increased activity of tPA. This activation results in a worse outcome further underscoring the potential detrimental proinflammatory effects of tPA.