Tobias Schwarz

Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration

The identification of NOX4 as a major source of oxidative stress in stroke and demonstration of dramatic protection after stroke in mice by NOX4 deletion or NOX inhibition, opens up new avenues for treatment.

Early detrimental T-cell effects in experimental cerebral ischemia are neither related to adaptive immunity nor thrombus formation

T cells contribute to the pathophysiology of ischemic stroke by yet unknown mechanisms. Mice with transgenic T-cell receptors (TCRs) and mutations in costimulatory molecules were used to define the minimal immunologic requirements for T cell-mediated ischemic brain damage. Stroke was induced in recombination activating gene 1-deficient (RAG1(-/-)) mice devoid of T and B cells, RAG1(-/-) mice reconstituted with B cells or T cells, TCR-transgenic mice bearing 1 single CD8(+) (2C/RAG2, OTI/RAG1 mice) or CD4(+) (OTII/RAG1, 2D2/RAG1 mice) TCR, mice lacking accessory molecules of TCR stimulation (CD28(-/-), PD1(-/-), B7-H1(-/-) mice), or mice deficient in nonclassical T cells (natural killer T [NKT] and gammadelta T cells) by transient middle cerebral artery occlusion (tMCAO). Stroke outcome was assessed at day 1. RAG1(-/-) mice and RAG1(-/-) mice reconstituted with B cells developed significantly smaller brain infarctions compared with controls, but thrombus formation after FeCl(3)-induced vessel injury was unimpaired. In contrast, TCR-transgenic mice and mice lacking costimulatory TCR signals were fully susceptible to tMCAO similar to mice lacking NKT and gammadelta T cells. These findings were corroborated by adoptive transfer experiments. Our data demonstrate that T cells critically contribute to cerebral ischemia, but their detrimental effect neither depends on antigen recognition nor TCR costimulation or thrombus formation.

Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature

We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) are generally regarded as prototypic anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in Rag1(-/-) mice lacking lymphocytes. Mechanistically, Tregs induced microvascular dysfunction in vivo by increased interaction with the ischemic brain endothelium via the LFA-1/ICAM-1 pathway and platelets and these findings were confirmed in vitro. Ablation of Tregs reduced microvascular thrombus formation and improved cerebral reperfusion on stroke, as revealed by ultra-high-field magnetic resonance imaging at 17.6 Tesla. In contrast, established immunoregulatory characteristics of Tregs had no functional relevance. We define herein a novel and unexpected role of Tregs in a primary nonimmunologic disease state.

Deficiency of von Willebrand factor protects mice from ischemic stroke

We recently demonstrated that blockade of the platelet adhesion receptor glycoprotein (GP) Ibalpha protects mice from ischemic stroke. Although von Willebrand factor (VWF) is the major ligand for GPIbalpha, GPIbalpha can engage other counterreceptors on endothelial cells, platelets, and leukocytes (eg, Mac-1 or P-selectin) potentially involved in stroke outcome. To further analyze whether VWF is of particular relevance for stroke development, VWF(-/-) mice underwent 60 minutes of middle cerebral artery occlusion. After 24 hours, VWF(-/-) mice had significantly smaller infarctions (P< .05) and less severe neurologic deficits (P< .01) compared with controls. This effect was sustained after 1 week, and intracranial bleeding was absent in VWF(-/-) mice as revealed by serial magnetic resonance imaging. Hydrodynamic injection of a VWF-encoding plasmid restored the susceptibility for stroke in VWF(-/-) mice. This study indicates that VWF is critically involved in cerebral ischemia. Hence, targeted inhibition of the GPIbalpha-VWF pathway might become a promising therapeutic option.

Inhibition of bradykinin receptor B1 protects mice from focal brain injury by reducing blood–brain barrier leakage and inflammation

Kinins are proinflammatory and vasoactive peptides that are released during tissue damage and may contribute to neuronal degeneration, inflammation, and edema formation after brain injury by acting on discrete bradykinin receptors, B1R and B2R. We studied the expression of B1R and B2R and the effect of their inhibition on lesion size, blood–brain barrier (BBB) disruption, and inflammatory processes after a focal cryolesion of the right parietal cortex in mice. B1R and B2R gene transcripts were significantly induced in the lesioned hemispheres of wild-type mice (P<0.05). The volume of the cortical lesions and neuronal damage at 24 h after injury in B1R −/− mice were significantly smaller than in wild-type controls (2.5±2.6 versus 11.5±3.9 mm3, P<0.001). Treatment with the B1R antagonist R-715 1 h after lesion induction likewise reduced lesion volume in wild-type mice (2.6±1.4 versus 12.2±6.1 mm3, P<0.001). This was accompanied by a remarkable reduction of BBB disruption and tissue inflammation. In contrast, genetic deletion or pharmacological inhibition of B2R had no significant impact on lesion formation or the development of brain edema. We conclude that B1R inhibition may offer a novel therapeutic strategy after acute brain injuries.

Glucocorticoid Insensitivity at the Hypoxic Blood–Brain Barrier Can Be Reversed by Inhibition of the Proteasome

Background and Purpose— Glucocorticoids potently stabilize the blood–brain barrier and ameliorate tissue edema in certain neoplastic and inflammatory disorders of the central nervous system, but they are largely ineffective in patients with acute ischemic stroke. The reasons for this discrepancy are unresolved. Methods— To address the molecular basis for the paradox unresponsiveness of the blood–brain barrier during hypoxia, we used murine brain microvascular endothelial cells exposed to O2/glucose deprivation as an in vitro model. In an in vivo approach, mice were subjected to transient middle cerebral artery occlusion to induce brain infarctions. Blood–brain barrier damage and edema formation were chosen as surrogate markers of glucocorticoid sensitivity in the presence or absence of proteasome inhibitors. Results— O2/glucose deprivation reduced the expression of tight junction proteins and transendothelial resistance in murine brain microvascular endothelial cells in vitro. Dexamethasone treatment failed to reverse these effects during hypoxia. Proteasome-dependent degradation of the glucocorticoid receptor impaired glucocorticoid receptor transactivation thereby preventing physiological glucocorticoid activity. Inhibition of the proteasome, however, fully restored the blood–brain barrier stabilizing properties of glucocorticoid during O2/glucose deprivation. Importantly, mice treated with the proteasome inhibitor Bortezomib in combination with steroids several hours after stroke developed significantly less brain edema and functional deficits, whereas respective monotherapies were ineffective. Conclusions— We for the first time show that inhibition of the proteasome can overcome glucocorticoid resistance at the hypoxic blood–brain barrier. Hence, combined treatment strategies may help to combat stroke-induced brain edema formation in the future and prevent secondary clinical deterioration.

Attenuated TLR4/MAPK signaling in monocytes from patients with CRMO results in impaired IL-10 expression.

Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder of unknown origin. We previously demonstrated that monocytes from CRMO patients fail to express the immune-modulatory cytokine interleukin-10 (IL-10) in a chromatin dependent manner. Here, we demonstrate that attenuated extracellular-signal regulated kinase (ERK)1 and 2 signaling in response to TLR4 activation results in failure to induce IL-10 expression in monocytes from CRMO patients. Attenuated ERK1/2 activation results in 1) reduced levels of Sp-1, a transcription factor that induces IL-10 expression in monocytes, and 2) impaired H3S10 phosphorylation of the IL10 promoter, an activating epigenetic mark. The pro-inflammatory cytokines tumor necrosis factor (TNF)α and IL-6 are not negatively affected, resulting in an imbalance towards pro-inflammatory cytokines. Thus, impaired ERK1/2 signaling with subsequently reduced Sp-1 expression and H3S10 phosphorylation of the IL10 promoter may centrally contribute to the pathophysiology of CRMO.

Binding of von Willebrand Factor to Collagen and Glycoprotein Ibα, But Not to Glycoprotein IIb/IIIa, Contributes to Ischemic Stroke in Mice—Brief Report

Objective—To unravel crucial von Willebrand factor (VWF) interactions that are detrimental in stroke development. Methods and Results—VWF−/− mice received gene transfer to express mutants of VWF defective either in binding to fibrillar collagen, glycoprotein (GP)Ib&agr; or GPIIb/IIIa, and underwent 60 minutes of transient middle cerebral artery occlusion. In VWF−/− mice reconstituted with VWF mutants defective in binding to collagen or GPIb&agr;, protection against stroke was sustained, whereas VWF lacking the GPIIb/IIIa binding site restored full susceptibility similar to normal VWF. Conclusion—VWF-collagen and VWF-GPIb&agr; (but not VWF-GPIIb/IIIa) interactions are instrumental in thrombus formation after transient middle cerebral artery occlusion, and their inhibition could be a promising target for stroke treatment.

Vaccination with plasmacytoid dendritic cells induces protection against infection with Leishmania major in mice.

DC‐based vaccination against Leishmania major induces a parasite‐specific Th1 response and long‐lasting protective immunity in susceptible mice. Since distinct DC subsets have been proposed to direct the predominant development of either Th1 or Th2 cells, we analyzed the capability of plasmacytoid DC (pDC) to induce protection and elicit a Th1 response against L. major. Pulsing with L. major lysate induced the activation and maturation of semi‐mature murine pDC that had been isolated from the spleen, as indicated by up‐regulation of the co‐stimulatory molecules CD86 and CD80, but did not enhance the level of IFN‐α secretion by pDC. Vaccination of susceptible mice with L. major lysate‐pulsed pDC induced highly effective T cell‐mediated immunity against subsequent infection with L. major parasites. Surprisingly, the protection was not accompanied by a polarized Th1 cytokine profile. Co‐activation of pDC with CpG‐containing oligodeoxynucleotides, which has been shown to be critical for activating the protective potential of myeloid DC, was not required for the protective effect of L. major antigen‐pulsed pDC. These findings demonstrate that antigen‐loaded pDC are able to induce T cell‐mediated protection against a parasite disease and that experimental leishmaniasis is a suitable model to elucidate the mechanisms underlying DC‐based vaccination against infections.

The phosphodiesterase-4 inhibitor rolipram protects from ischemic stroke in mice by reducing blood-brain-barrier damage, inflammation and thrombosis

Blood-brain-barrier (BBB) disruption, inflammation and thrombosis are important steps in the pathophysiology of acute ischemic stroke but are still inaccessible to therapeutic interventions. Rolipram specifically inhibits the enzyme phosphodiesterase (PDE) 4 thereby preventing the inactivation of the intracellular second messenger cyclic adenosine monophosphate (cAMP). Rolipram has been shown to relief inflammation and BBB damage in a variety of neurological disorders. We investigated the therapeutic potential of rolipram in a model of brain ischemia/reperfusion injury in mice. Treatment with 10mg/kg rolipram, but not 2 mg/kg rolipram, 2 h after 60 min of transient middle cerebral artery occlusion (tMCAO) reduced infarct volumes by 50% and significantly improved clinical scores on day 1 compared with vehicle-treated controls. Rolipram maintained BBB function upon stroke as indicated by preserved expression of the tight junction proteins occludin and claudin-5. Accordingly, the formation of vascular brain edema was strongly attenuated in mice receiving rolipram. Moreover, rolipram reduced the invasion of neutrophils as well as the expression of the proinflammatory cytokines IL-1β and TNFα but increased the levels of TGFβ-1. Finally, rolipram exerted antithrombotic effects upon stroke and fewer neurons in the rolipram group underwent apoptosis. Rolipram is a multifaceted antiinflammatory and antithrombotic compound that protects from ischemic neurodegeneration in clinically meaningful settings.