IL-10 deficiency exacerbates the brain inflammatory response to permanent ischemia without preventing resolution of the lesion

El pdf del artículo es la versión post-print.Stroke induces inflammation that can aggravate brain damage. This work examines whether interleukin-10 (IL-10) deficiency exacerbates inflammation and worsens the outcome of permanent middle cerebral artery occlusion (pMCAO). Expression of IL-10 and IL-10 receptor (IL-10R) increased after ischemia. From day 4, reactive astrocytes showed strong IL-10R immunoreactivity. Interleukin-10 knockout (IL-10 KO) mice kept in conventional housing showed more mortality after pMCAO than the wild type (WT). This effect was associated with the presence of signs of colitis in the IL-10 KO mice, suggesting that ongoing systemic inflammation was a confounding factor. In a pathogen-free environment, IL-10 deficiency slightly increased infarct volume and neurologic deficits. Induction of proinflammatory molecules in the IL-10 KO brain was similar to that in the WT 6 hours after ischemia, but was higher at day 4, while differences decreased at day 7. Deficiency of IL-10 promoted the presence of more mature phagocytic cells in the ischemic tissue, and enhanced the expression of M2 markers and the T-cell inhibitory molecule CTLA-4. These findings agree with a role of IL-10 in attenuating local inflammatory reactions, but do not support an essential function of IL-10 in lesion resolution. Upregulation of alternative immunosuppressive molecules after brain ischemia can compensate, at least in part, the absence of IL-10. © 2013 ISCBFM.Work supported by the Spanish Ministry of Economy (SAF2011-30492), and the European Community (FP7, grant agreements: n°201024 ARISE and n°278850 InMiND), and the ERANET-NEURON project (PRI-PIMNEU-2011-1342). IPP and EBT had PhD fellowships from the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) of the Generalitat de Catalunya and the FPU program of the Spanish Ministry of Economy, respectively.Peer Reviewe

Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E2 (PGE2) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE2 and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE2 production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE2, and suggest that the coordinated down-regulation of COX-1 facilitates PGE2 production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation

CNS-border associated macrophages respond to acute ischemic stroke attracting granulocytes and promoting vascular leakage

The central nervous system (CNS) contains several types of immune cells located in specific anatomic compartments. Macrophages reside at the CNS borders surrounding the brain vessels, in leptomeningeal spaces and the choroid plexus, where they interact with the vasculature and play immunological surveillance and scavenging functions. We investigated the phenotypic changes and role of these macrophages in response to acute ischemic stroke. Given that CD163 expression is a hallmark of perivascular and meningeal macrophages in the rat and human brain, we isolated CD163+ brain macrophages by fluorescence activated cell sorting. We obtained CD163+ cells from control rats and 16 h following transient middle cerebral artery occlusion, after verifying that infiltration of CD163+ peripheral myeloid cells is negligible at this acute time point. Transcriptome analysis of the sorted CD163+ cells identified ischemia-induced upregulation of the hypoxia inducible factor-1 pathway and induction of genes encoding for extracellular matrix components and leukocyte chemoattractants, amongst others. Using a cell depletion strategy, we found that CNS border-associated macrophages participate in granulocyte recruitment, promote the expression of vascular endothelial growth factor (VEGF), increase the permeability of pial and cortical blood vessels, and contribute to neurological dysfunction in the acute phase of ischemia/reperfusion. We detected VEGF expression surrounding blood vessels and in some CD163+ perivascular macrophages in the brain tissue of ischemic stroke patients deceased one day after stroke onset. These findings show ischemia-induced reprogramming of the gene expression profile of CD163+ macrophages that has a rapid impact on leukocyte chemotaxis and blood-brain barrier integrity, and promotes neurological impairment in the acute phase of stroke

Estudio de la lesión por reperfusión en la isquemia cerebral experimental y su tratamiento

[spa] El ictus es un fenómeno agudo que causa una interrupción del aporte sanguíneo al cerebro, causando muerte de las células por la falta de oxígeno. El ictus es la segunda causa de muerte para personas mayores de 60 años y la causa más frecuente de discapacidad permanente. Debido al creciente envejecimiento de la población y al gran impacto que tiene el ictus, existe una gran necesidad de investigar sobre esta enfermedad, los factores de riesgo, sus consecuencias y por supuesto, nuevos tratamientos. El principal objetivo del tratamiento del ictus es el retorno del FSC al tejido. Aunque la reperfusión es indispensable para salvar el tejido cerebral hipoperfundido, esta puede tener efectos nocivos que se conocen como daño por reperfusión. Diversos estudios han demostrado las alteraciones provocadas por la reperfusión, como el aumento de la producción de ROS y RNS, aumento de expresión de moléculas de adhesión intercelular, inflamación, disminución de la función y ruptura de la BHE, transformación hemorrágica del infarto y desarrollo de un fenotipo procoagulante/protrombótico de la pared vascular. Esta tesis se ha enfocado en el estudio de dos principales consecuencias del daño por reperfusión como son el estrés oxidativo y la transformación hemorrágica (TH) del infarto en modelos animales. Sin embargo, los seres humanos que sufren un ictus suelen presentar enfermedades concomitantes que exacerban el daño por isquemia/reperfusión y es importante que en los modelos animales también se evalúen estos factores de riesgo o situaciones clínicas relacionadas con el ictus isquémico humano. Teniendo en cuenta estas circunstancias, este trabajo de tesis se ha centrado en el estudio del estrés oxidativo aumentado por hiperglucemia y en la trasformación hemorrágica del infarto en condiciones de linfopenia. Así mismo, estudiamos la posibilidad de modular el estrés oxidativo y la respuesta inflamatoria como métodos para potenciar el tratamiento del ictus y minimizar los daños por reperfusión. En el primer trabajo mostramos que los ratones a los que se les indujo hiperglucemia previamente a la I/R podían beneficiarse del tratamiento con el antioxidante natural ácido úrico (AU). Este estudio ha demostrado que las altas concentraciones sanguíneas de glucosa exacerban el daño por isquemia/reperfusión. Y que el AU puede contribuir a reducir el volumen de infarto en los ratones hiperglucémicos a través de su efecto antioxidante con una gran eficacia en la limpieza de radicales libres derivados del peroxinitrito y sus efectos vasculoprotectores limitando la toxicidad de la glucosa y la muerte celular secundaria. El segundo trabajo se centra en el estudio de las células T y su relación con la TH. Este estudio demuestra que los linfocitos T reducen el riesgo de TH. Confirma la relevancia del volumen del infarto como un factor crítico que incrementa el riesgo de TH. A su vez, los resultados de este estudio resaltan la comunicación bidireccional que existe entre las células T y las plaquetas creando un puente entre la inmunidad y la hemostasia para prevenir la TH tras un ictus isquémico agudo. Estos resultados pueden determinar nuevas dianas celulares y moleculares para la prevención de la TH secundaria al ictus isquémico. En el tercer trabajo, hemos intentado reducir la TH mediante un tratamiento farmacológico con el inmunomodulador Fingolimod. Los resultados de este estudio muestran una activación de la vía de señalización S1P en el cerebro después de la I/R, esta activación sugiere que los fármacos moduladores del receptor S1P pueden desempeñar un papel en el parénquima independiente de los efectos del fármaco sobre el sistema inmune. También demostró que el Fingolimod puede atenuar la TH del infarto cerebral por una vía independiente de los linfocitos, pero el tratamiento no es efectivo en condiciones de trombocitopenia.[eng] Stroke is an acute phenomenon caused by the interruption of the blood supply to the brain. Stroke is one of the leading causes of death and permanent disability in the world. Due to the huge impact of stroke in our society, there is a great need to investigate this disease, the risk factors, their consequences and, of course, new treatments. The main objective of stroke treatment is the return of cerebral blood flow to the brain tissue. Although reperfusion is essential to save hypoperfused brain tissue, it may have unwanted complications that are known as reperfusion damage. This thesis has focused on the study of two negative consequences of reperfusion, such as oxidative stress and hemorrhagic transformation (HT) of infarction in animal models. Likewise, we studied the possibility of modulating oxidative stress and the inflammatory response in order to minimize reperfusion damage. In the first work we showed that mice that had hyperglycemia induced before ischemia/reperfusion could benefit from treatment with uric acid (AU) through its antioxidant effect, limiting glucose toxicity and secondary cell death. The second work focuses on the study of T cells and their relationship with HT. The results of this study highlight the bidirectional communication that exists between T cells and platelets, creating a bridge between immunity and hemostasis to prevent HT after an acute ischemic stroke. These results can determine new cellular and molecular targets for the prevention of HT secondary to ischemic stroke. In the third work, we have tried to reduce HT through a pharmacological treatment with Fingolimod. It also showed that Fingolimod can attenuate the HT of cerebral infarction through a lymphocyte-independent pathway, but the treatment is not effective in thrombocytopenia conditions. Overall this thesis identifies cellular and molecular mechanisms underlying complications of reperfusion therapies following ischemic stroke, and suggests pharmacological therapies to increase the benefits of reperfusion treatments

Combined treatment with recombinant tissue plasminogen activator and dexamethasone phosphate-containing liposomes improves neurological outcome and restricts lesion progression after embolic stroke in rats

Variable efficacies have been reported for glucocorticoid drugs as anti-inflammatory treatment after stroke. We applied an alternative drug delivery strategy, by injection of dexamethasone phosphate-containing liposomes in combination with recombinant tissue plasminogen activator (rtPA), in an experimental stroke model, and tested the hypothesis that this approach improves behavioral recovery and reduces lesion growth. Rats were subjected to right middle cerebral artery occlusion with a blood clot. After 2 h, animals were intravenously injected with rtPA plus empty long-circulating liposomes (LCL), free dexamethasone phosphate (DXP), or DXP-containing LCL (LCL-DXP). Neurological status was evaluated with different behavioral tests up to 7 days after stroke. Lesion development was assessed by magnetic resonance imaging of tissue and perfusion parameters from 0-2 h until 7 days after stroke. Expression of brain inflammatory markers was measured with RT-PCR at post-stroke day 7. Treatment with rtPA plus LCL-DXP resulted in significantly improved behavioral outcome as compared to treatment with rtPA plus empty LCL or free DXP. Acute and final brain lesion sizes were comparable between treatment groups; however a predictive algorithm revealed a significantly larger salvaged tissue area after treatment with LCL-DXP. We conclude that delivery of dexamethasone phosphate via LCL in combination with rtPA-induced thrombolysis can significantly improve outcome after stroke. Furthermore, magnetic resonance imaging-based predictive algorithms provide a sensitive means to measure treatment effects on lesion development.The research leading to these results was funded by the Netherlands Heart Foundation (2005B156), The Netherlands Organization for Scientific Research (NWO-Vidi 917.76.351), and the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreements nº 201024 and nº 202213 (European Stroke Network) and MediTrans.Peer Reviewe

Interleukin-13 immune gene therapy prevents CNS inflammation and demyelination via alternative activation of microglia and macrophages

Detrimental inflammatory responses in the central nervous system are a hallmark of various brain injuries and diseases. With this study we provide evidence that lentiviral vector-mediated expression of the immune-modulating cytokine interleukin 13 (IL-13) induces an alternative activation program in both microglia and macrophages conferring protection against severe oligodendrocyte loss and demyelination in the cuprizone mouse model for multiple sclerosis (MS). First, IL-13 mediated modulation of cuprizone induced lesions was monitored using T-weighted magnetic resonance imaging and magnetization transfer imaging, and further correlated with quantitative histological analyses for inflammatory cell influx, oligodendrocyte death, and demyelination. Second, following IL-13 immune gene therapy in cuprizone-treated eGFP bone marrow chimeric mice, we provide evidence that IL-13 directs the polarization of both brain-resident microglia and infiltrating macrophages towards an alternatively activated phenotype, thereby promoting the conversion of a pro-inflammatory environment toward an anti-inflammatory environment, as further evidenced by gene expression analyses. Finally, we show that IL-13 immune gene therapy is also able to limit lesion severity in a pre-existing inflammatory environment. In conclusion, these results highlight the potential of IL-13 to modulate microglia/macrophage responses and to improve disease outcome in a mouse model for MS. GLIA 2016;64:2181–2200.Peer Reviewe

Brain ischemia induces an INF-mediated response in microglia of mice and humans.

Trabajo presentado en el 19th National Meeting of the Spanish Society of Neuroscience, celebrado en Lleida (España), del 3 al 5 de noviembre de 2021Brain ischemia causes necrotic cell death, microglia reactivity and blood-brain barrier breakdown. Danger signals ¿ including nuclear proteins, nucleic acids and heat-shock proteins ¿ are released from injured cells and trigger immune responses by activating pattern recognition receptors (PRRs). Microglial cells are equipped with PRRs, can sense danger signals in the environment and induce inflammation. Our aim was to investigate the inflammatory response of microglia to ischemia in mice and humans. We induced cerebral ischemia in mice by 45-min middle cerebral artery occlusion followed by reperfusion. We compared the transcriptomic profile of microglia isolated from brain tissue of control and ischemic mice using fluorescence activated cell sorting followed by RNA-Seq. Enrichment analysis showed a strong anti-viral response induced by ischemia in microglia, highlighted by upregulation of type-I interferons (IFNs) including Ifnb and many IFN-stimulated genes (ISG). Accordingly, in whole brain tissue, ischemia increased Ifnb, Ifna7 and Ifna9 mRNA expression, as well as many ISG, including Dhx58, Cxcl10, Irf7, and Irg15. A time-course study in brain tissue samples obtained between 1h and 7 days post-ischemia showed ISG expression increase from about 16h post-ischemia, reaching a plateau at 4-7 days. We also detected enhanced expression of ISG in post-mortem human brain tissue of ischemic stroke patients. To find out the contribution of microglia to gene expression in whole brain tissue, we depleted microglia in mice with a CSF1R inhibitor (PLX5622). Ischemia-induced expression of Ifnb, Ifna7, or Ifna9 mRNA was not reduced after microglia depletion. This implies that cells other than microglia are the main source of IFNs in the injured brain. In contrast, we found that microglia depletion reduced cerebral ISG expression. The results show that type I IFNs generated after ischemia in cells other than microglia activate type I IFN receptors in microglia, inducing ISG expression and triggering a specific transcriptional program.Peer reviewe

In vivo imaging of induction of heat-shock protein-70 gene expression with fluorescence reflectance imaging and intravital confocal microscopy following brain ischaemic in reporter mice

El pdf del artículo es la versión post-print.-- et al.Purpose: Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice. Methods: A genomic DNA sequence of the Hspa1b promoter was used to generate an Hsp70-mPlum far-red fluorescence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum transgenic mice were generated. Focal brain ischaemia was induced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window. Results: Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1 day after induction of ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction. Conclusion: This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core. © 2012 Springer-Verlag Berlin Heidelberg.Financed in part by the European Community (Diagnostic Molecular Imaging Project, DIMI, EC-FP6-project LSHB-CT-2005-512146 and FP7/2007-2013 HEALTH, grant agreement no. 201024-ARISE), and the Spanish Ministry of Economy (SAF2008-04515-CO2-01/02 and SAF2011-30492). X. de la Rosa has a FPI PhD Fellowship from the Spanish Ministry of Economy.Peer Reviewe

IL-4 is induced in the brain after ischemia and it down-regulates the inflammatory profile of microglia

Trabajo presentado en la VI Reunión de la Red Glial Española, celebrada en Oviedo, en septiembre de 2013Peer Reviewe

Results of a preclinical randomized controlled multicenter trial (pRCT): Anti-CD49d treatment for acute brain ischemia

Gemma Llovera et al.Numerous treatments have been reported to provide a beneficial outcome in experimental animal stroke models; however, these treatments (with the exception of tissue plasminogen activator) have failed in clinical trials. To improve the translation of treatment efficacy from bench to bedside, we have performed a preclinical randomized controlled multicenter trial (pRCT) to test a potential stroke therapy under circumstances closer to the design and rigor of a clinical randomized control trial. Anti-CD49d antibodies,which inhibit the migration of leukocytes into the brain, were previously investigated in experimental strokemodels by individual laboratories. Despite the conflicting results from four positive and one inconclusive preclinical studies, a clinical trial was initiated. To confirm the preclinical results and to test the feasibility of conducting a pRCT, six independent European research centers investigated the efficacy of anti-CD49d antibodies in two distinct mouse models of stroke in a centrally coordinated, randomized, and blinded approach. The results pooled from all research centers revealed that treatment with CD49d-specific antibodies significantly reduced both leukocyte invasion and infarct volume after the permanent distal occlusion of the middle cerebral artery, which causes a small cortical infarction. In contrast, anti-CD49d treatment did not reduce lesion size or affect leukocyte invasion after transient proximal occlusion of the middle cerebral artery, which induces large lesions. These results suggest that the benefits of immune-targeted approachesmay depend on infarct severity and localization. This study supports the feasibility of performing pRCTs.Peer Reviewe