Role of neutrophil TLR4 in experimental acute stroke

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Medicina, Departamento de Farmacología y Toxicología, leída el 10/12/2020El sistema inmune es capaz de provocar una respuesta contra un estímulo de tipo no patógeno, lo que se denomina inflamación estéril. Las moléculas endógenas que se liberan cuando un tejido se daña pueden iniciar una respuesta inflamatoria mediante la activación de receptores como el TLR4 (del inglés, Toll-like receptor 4). Este receptor se expresa en los neutrófilos, además de en otras células circulantes. Los neutrófilos constituyen la primera línea de defensa del sistema inmune innato ya que son las primeras células en responder cuando se produce un proceso de inflamación aguda. En la última década, los neutrófilos han sido reconsiderados como células complejas capaces de llevar a cabo un amplio conjunto de funciones especializadas, así como efectores de la respuesta inmune innata. La unión de DAMPs (patrones moleculares asociados a daño, del inglés, damage-associated molecular patterns) al TLR4 neutrofílico activa al neutrófilo. Una vez activados, los neutrófilos se adhieren al endotelio y comienzan el proceso de infiltración que culmina con la entrada en el tejido dañado, donde el neutrófilo es capaz de ejecutar distintas funciones como la fagocitosis, el estallido respiratorio, la degranulación o la NETosis...The immune system can elicit responses towards non-pathogen stimulus, which is called sterile inflammation. Endogenous molecules released when the tissue is damaged can initiate the inflammatory response by activating receptors such as TLR4. This receptor is expressed, among other circulating cells, in neutrophils. Neutrophils are the front-line defensive cells of the innate immune system being the first responders during acute inflammation. In the last decade, neutrophils have been reconsidered as complex cells capable of a significant array of specialized functions and as effectors of the innate immune response. The binding of DAMPs to the neutrophilic TLR4 activates the neutrophil. When activated neutrophils adhere to the endothelium and start the infiltration process which culminates in the infiltration into the damaged tissue where the neutrophil is able to execute functions such as phagocytosis, oxidative burst, degranulation or NETosis...Fac. de MedicinaTRUEunpu

Lentes de contacto como dispositivo de liberación controlada de principios activos

La vía tópica ocular es una de las vías de administración de principios activos más compleja. En la actualidad, hay una gran demanda en el mercado oftalmológico farmacéutico de nuevas y más eficaces formas de administracion de fármacos. La mayoría de ellos se administran actualmente mediantes gotas oftálmicas, con las que solo entre el 1 y el 7% del princpio activo es absorbido. Además, representan un problema para la adherencia al tratamiento ya que requieren una pauta de administracion constante. En este contexto, las lentes de contacto como sistema de liberación de fármacos se esgrimen como un sistema alternativo de administración de fármacos por vía tópica ocular, ya que son capaces de aumentar la biodisponibilidad del fármaco y de simplificar la pauta de adminsitración. Diferentes estrategias y sus respectivos requerimientos físicos para la incorporación del fármaco en la lente, como lentes embebidas en princpio activo, barreras de vitamina E, impresión molecular e incorporación de nanopartículas están siendo desarrolladas por numerosos grupos de investigación en la actualidad. En este trabajo se lleva a cabo una revisión de las diferentes técnicas utilizadas para la liberación de principios activos a través de las lentes de contacto y de su potencial aplicación en el tratamiento de distintas patologías oculares

Neutrophil Extracellular Trap Targeting Protects Against Ischemic Damage After Fibrin-Rich Thrombotic Stroke Despite Non-Reperfusion.

Stroke is one of the most prevalent diseases worldwide caused primarily by a thrombotic vascular occlusion that leads to cell death. To date, t-PA (tissue-type plasminogen activator) is the only thrombolytic therapy approved which targets fibrin as the main component of ischemic stroke thrombi. However, due to its highly restrictive criteria, t-PA is only administrated to less than 10% of all stroke patients. Furthermore, the research in neuroprotective agents has been extensive with no translational results from medical research to clinical practice up to now. Since we first described the key role of NETs (Neutrophil Extracellular Traps) in platelet-rich thrombosis, we asked, first, whether NETs participate in fibrin-rich thrombosis and, second, if NETs modulation could prevent neurological damage after stroke. To this goal, we have used the thromboembolic in situ stroke model which produces fibrin-rich thrombotic occlusion, and the permanent occlusion of the middle cerebral artery by ligature. Our results demonstrate that NETs do not have a predominant role in fibrin-rich thrombosis and, therefore, DNase-I lacks lytic effects on fibrin-rich thrombosis. Importantly, we have also found that NETs exert a deleterious effect in the acute phase of stroke in a platelet-TLR4 dependent manner and, subsequently, that its pharmacological modulation has a neuroprotective effect. Therefore, our data strongly support that the pharmacological modulation of NETs in the acute phase of stroke, could be a promising strategy to repair the brain damage in ischemic disease, independently of the type of thrombosis involved.This work was supported by grants from Instituto de Salud Carlos III and co-financed by the European Development Regional Fund “A Way to Achieve Europe” PI20/00535 and RETICS RD16/0019/ 0009 (IL), from Regional Madrid Government B2017/BMD- 3688 (IL), from Spanish Ministry of Science and Innovation PID2019- 106581RB-I00 (MÁM), from Leducq Foundation for Cardiovascular Research TNE-19CVD01 (MÁM), from Fundación La Caixa HR17_00527 (MM). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Role of TLR4 in Neutrophil Dynamics and Functions: Contribution to Stroke Pathophysiology.

BACKGROUND AND PURPOSE The immune response subsequent to an ischemic stroke is a crucial factor in its physiopathology and outcome. It is known that TLR4 is implicated in brain damage and inflammation after stroke and that TLR4 absence induces neutrophil reprogramming toward a protective phenotype in brain ischemia, but the mechanisms remain unknown. We therefore asked how the lack of TLR4 modifies neutrophil function and their contribution to the inflammatory process. METHODS In order to assess the role of the neutrophilic TLR4 after stroke, mice that do not express TLR4 in myeloid cells (TLR4loxP/Lyz-cre) and its respective controls (TLR4loxP/loxP) were used. Focal cerebral ischemia was induced by occlusion of the middle cerebral artery and infarct size was measured by MRI. A combination of flow cytometry and confocal microscopy was used to assess different neutrophil characteristics (circadian fluctuation, cell surface markers, cell complexity) and functions (apoptosis, microglia engulfment, phagocytosis, NETosis, oxidative burst) in both genotypes. RESULTS As previously demonstrated, mice with TLR4 lacking-neutrophils had smaller infarct volumes than control mice. Our results show that the absence of TLR4 keeps neutrophils in a steady youth status that is dysregulated, at least in part, after an ischemic insult, preventing neutrophils from their normal circadian fluctuation. TLR4-lacking neutrophils showed a higher phagocytic activity in the basal state, they were preferentially engulfed by the microglia after stroke, and they produced less radical oxygen species (ROS) in the first stage of the inflammatory process. CONCLUSIONS TLR4 is specifically involved in neutrophil dynamics under physiological conditions as well as in stroke-induced tissue damage. This research contributes to the idea that TLR4, especially when targeted in specific cell types, is a potential target for neuroprotective strategies.This work was supported by grants from Instituto de Salud Carlos III and co-financed by the European Development Regional Fund “A Way to Achieve Europe” PI20/00535 and RETICS RD16/0019/ 0009 (IL), from Regional Madrid Government B2017/BMD- 3688 (IL), from Spanish Ministry of Science and Innovation PID2019- 106581RB-I00 (MM), from Leducq Foundation for Cardiovascular Research TNE-19CVD01 (MM), from Fundació n La Caixa HR17_00527 (MM). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation.S

Abolition of aberrant neurogenesis ameliorates cognitive impairment after stroke in mice

Poststroke cognitive impairment is considered one of the main complications during the chronic phase of ischemic stroke. In the adult brain, the hippocampus regulates both encoding and retrieval of new information through adult neurogenesis. Nevertheless, the lack of predictive models and studies based on the forgetting processes hinders the understanding of memory alterations after stroke. Our aim was to explore whether poststroke neurogenesis participates in the development of long-term memory impairment. Here, we show a hippocampal neurogenesis burst that persisted 1 month after stroke and that correlated with an impaired contextual and spatial memory performance. Furthermore, we demonstrate that the enhancement of hippocampal neurogenesis after stroke by physical activity or memantine treatment weakened existing memories. More importantly, stroke-induced newborn neurons promoted an aberrant hippocampal circuitry remodeling with differential features at ipsi- and contralesional levels. Strikingly, inhibition of stroke-induced hippocampal neurogenesis by temozolomide treatment or using a genetic approach (Nestin-CreERT2/NSE-DTA mice) impeded the forgetting of old memories. These results suggest that hippocampal neurogenesis modulation could be considered as a potential approach for treatment of poststroke cognitive impairment.Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2015-68632-R to MAM), the Instituto de Salud Carlos III (ISCIII) (FIS PI17/01601 to IL), and ISCIII cofinanced by the Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa” RETICS (RD12/0014/0003 to IL), and the Canadian Institutes of Health Research (FDN143227to PWF

Role of TLR4 (toll-like receptor 4) in N1/N2 neutrophil programming after stroke

11p.-5 fig.Background and Purpose—After stroke, the population of infiltrated neutrophils in the brain is heterogeneous, including a population of alternative neutrophils (N2) that express M2 phenotype markers. We explored the role of TLR4 (toll-likereceptor 4) on neutrophil infiltration and polarization in this setting.Methods—Focal cerebral ischemia was induced by occlusion of the middle cerebral artery occlusion in TLR4-KO and WT (wild type) mice. Infarct size was measured by Nissl staining and magnetic resonance imaging. Leukocyte infiltration was quantified 48 hours after middle cerebral artery occlusion by immunofluorescence and flow cytometry. To elucidate mechanisms underlying TLR4-mediated N2 phenotype, a cDNA microarray analysis was performed in neutrophils isolated from blood 48 hours after stroke in WT and TLR4-KO mice.Results—As demonstrated previously, TLR4-deficient mice presented lesser infarct volumes than WT mice. TLR4-deficient mice showed higher density of infiltrated neutrophils 48 hours after stroke compared with WT mice, concomitantly to neuroprotection. Furthermore, cytometric and stereological analyses revealed an increased number of N2 neutrophils(YM1+ cells) into the ischemic core in TLR4-deficient mice, suggesting a protective effect of this neutrophil subset that was corroborated by depleting peripheral neutrophils or using mice with TLR4 genetically ablated in the myeloid lineage.Finally, cDNA microarray analysis in neutrophils, confirmed by quantitative polymerase chain reaction, showed that TLR4 modulates several pathways associated with ischemia-induced inflammation, migration of neutrophils into the parenchyma,and their functional priming, which might explain the opposite effect on outcome of the different neutrophil subsets.Conclusions—TLR4 deficiency increased the levels of alternative neutrophils (N2)—an effect associated with neuroprotection after stroke—supporting that modulation of neutrophil polarization is a major target of TLR4 and highlighting the crucial role of TLR4 at the peripheral level after stroke.This work was supported by grants from Instituto de Salud Carlos III and cofinanced by the European Development Regional Fund “A way to achieve Europe” (PI17/01601 and RETICS RD16/0019/0009;Dr Lizasoain), from Regional Madrid Government B2017/BMD-3688 (Dr Lizasoain) and from Spanish Ministry of Economy and Competitiveness SAF2015-68632-R (Dr Moro).Peer reviewe

Endothelial cells regulate astrocyte to neural progenitor cell trans-differentiation in a mouse model of stroke.

The concept of the neurovascular unit emphasizes the importance of cell-cell signaling between neural, glial, and vascular compartments. In neurogenesis, for example, brain endothelial cells play a key role by supplying trophic support to neural progenitors. Here, we describe a surprising phenomenon where brain endothelial cells may release trans-differentiation signals that convert astrocytes into neural progenitor cells in male mice after stroke. After oxygen-glucose deprivation, brain endothelial cells release microvesicles containing pro-neural factor Ascl1 that enter into astrocytes to induce their trans-differentiation into neural progenitors. In mouse models of focal cerebral ischemia, Ascl1 is upregulated in endothelium prior to astrocytic conversion into neural progenitor cells. Injecting brain endothelial-derived microvesicles amplifies the process of astrocyte trans-differentiation. Endothelial-specific overexpression of Ascl1 increases the local conversion of astrocytes into neural progenitors and improves behavioral recovery. Our findings describe an unexpected vascular-regulated mechanism of neuroplasticity that may open up therapeutic opportunities for improving outcomes after stroke.Supported in part by the Leducq Foundation.S

Specific Features of SVZ Neurogenesis After Cortical Ischemia: a Longitudinal Study

Stroke is a devastating disease with an increasing prevalence. Part of the current development in stroke therapy is focused in the chronic phase, where neurorepair mechanisms such as neurogenesis, are involved. In the adult brain, one of the regions where neurogenesis takes place is the subventricular zone (SVZ) of the lateral ventricles. Given the possibility to develop pharmacological therapies to stimulate this process, we have performed a longitudinal analysis of neurogenesis in a model of cortical ischemia in mice. Our results show an initial decrease of SVZ proliferation at 24 h, followed by a recovery leading to an increase at 14d and a second decrease 28d after stroke. Coinciding with the 24 h proliferation decrease, an increase in the eutopic neuroblast migration towards the olfactory bulb was observed. The analysis of the neuroblast ectopic migration from the SVZ toward the lesion showed an increase in this process from day 14 after the insult. Finally, our data revealed an increased number of new cortical neurons in the peri-infarct cortex 65d after the insult. In summary, we report here critical check-points about post-stroke neurogenesis after cortical infarcts, important for the pharmacological modulation of this process in stroke patients.Depto. de Farmacología y ToxicologíaFac. de MedicinaTRUEpu

Role of TLR4 (Toll-Like Receptor 4) in N1/N2 Neutrophil Programming After Stroke

Background and Purpose- After stroke, the population of infiltrated neutrophils in the brain is heterogeneous, including a population of alternative neutrophils (N2) that express M2 phenotype markers. We explored the role of TLR4 (toll-like receptor 4) on neutrophil infiltration and polarization in this setting. Methods- Focal cerebral ischemia was induced by occlusion of the middle cerebral artery occlusion in TLR4-KO and WT (wild type) mice. Infarct size was measured by Nissl staining and magnetic resonance imaging. Leukocyte infiltration was quantified 48 hours after middle cerebral artery occlusion by immunofluorescence and flow cytometry. To elucidate mechanisms underlying TLR4-mediated N2 phenotype, a cDNA microarray analysis was performed in neutrophils isolated from blood 48 hours after stroke in WT and TLR4-KO mice. Results- As demonstrated previously, TLR4-deficient mice presented lesser infarct volumes than WT mice. TLR4-deficient mice showed higher density of infiltrated neutrophils 48 hours after stroke compared with WT mice, concomitantly to neuroprotection. Furthermore, cytometric and stereological analyses revealed an increased number of N2 neutrophils (YM1+ cells) into the ischemic core in TLR4-deficient mice, suggesting a protective effect of this neutrophil subset that was corroborated by depleting peripheral neutrophils or using mice with TLR4 genetically ablated in the myeloid lineage. Finally, cDNA microarray analysis in neutrophils, confirmed by quantitative polymerase chain reaction, showed that TLR4 modulates several pathways associated with ischemia-induced inflammation, migration of neutrophils into the parenchyma, and their functional priming, which might explain the opposite effect on outcome of the different neutrophil subsets. Conclusions- TLR4 deficiency increased the levels of alternative neutrophils (N2)-an effect associated with neuroprotection after stroke-supporting that modulation of neutrophil polarization is a major target of TLR4 and highlighting the crucial role of TLR4 at the peripheral level after stroke. Visual Overview- An online visual overview is available for this article.Depto. de Farmacología y ToxicologíaFac. de MedicinaTRUEpu