María I. Cuartero

Neutrophils scan for activated platelets to initiate inflammation

Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with an asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream. The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in the redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thromboinflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and they suggest that the neutrophils’ bipolarity allows the integration of signals present at both the endothelium and the circulation before inflammation proceeds. Blood neutrophil cells sample platelets in the bloodstream before infiltrating surrounding tissue. A two-cell collaboration for inflammation Immune cells called neutrophils are first responders to infection. Neutrophils move within and through blood vessels to get to sites of infection quickly. Sreeramkumar et al. found that mouse neutrophils rely on platelets to help find such sites. Neutrophils extended protrusions into blood vessels. When these protrusions came into contact with platelets, the neutrophils migrated into the surrounding tissue to carry out their inflammatory functions. Preventing these neutrophilplatelet interactions alleviated collateral inflammatory damage to tissues in several injury models in mice. Science, this issue p. 1234

Silent Information Regulator 1 Protects the Brain Against Cerebral Ischemic Damage

Background and Purpose— Sirtuin 1 (SIRT1) is a member of NAD+-dependent protein deacetylases implicated in a wide range of cellular functions and has beneficial properties in pathologies including ischemia/reperfusion processes and neurodegeneration. However, no direct evidence has been reported on the direct implication of SIRT1 in ischemic stroke. The aim of this study was to establish the role of SIRT1 in stroke using an experimental model in mice. Methods— Wild-type and Sirt1−/− mice were subjected to permanent focal ischemia by permanent ligature. In another set of experiments, wild-type mice were treated intraperitoneally with vehicle, activator 3 (SIRT1 activator, 10 mg/kg), or sirtinol (SIRT1 inhibitor, 10 mg/kg) for 10 minutes, 24 hours, and 40 hours after ischemia. Brains were removed 48 hours after ischemia for determining the infarct volume. Neurological outcome was evaluated using the modified neurological severity score. Results— Exposure to middle cerebral artery occlusion increased SIRT1 expression in neurons of the ipsilesional mouse brain cortex. Treatment of mice with activator 3 reduced infarct volume, whereas sirtinol increased ischemic injury. Sirt1−/− mice displayed larger infarct volumes after ischemia than their wild-type counterparts. In addition, SIRT1 inhibition/deletion was concomitant with increased acetylation of p53 and nuclear factor &kgr;B (p65). Conclusions— These results support the idea that SIRT1 plays an important role in neuroprotection against brain ischemia by deacetylation and subsequent inhibition of p53-induced and nuclear factor &kgr;B-induced inflammatory and apoptotic pathways.

N2 Neutrophils, Novel Players in Brain Inflammation After Stroke Modulation by the PPARγ Agonist Rosiglitazone

Background and Purpose— Neutrophils have been traditionally recognized as major mediators of a deleterious inflammatory response in acute ischemic stroke, but their potential as a therapeutic target remains unexplored. Recent evidence indicates that neutrophils may acquire different phenotypes and contribute to resolution of inflammation through the release of anti-inflammatory mediators. Thus, similar to M2 macrophages, neutrophils have been proposed to shift toward an N2 phenotype, a polarization that is peroxisome proliferator-activated receptor-&ggr; dependent in macrophages. We hypothesize that peroxisome proliferator-activated receptor-&ggr; activation with rosiglitazone induces changes in neutrophilic mobilization and phenotype that might influence stroke outcome. Methods— Brain sections and cell suspensions were prepared from mice exposed to permanent distal middle cerebral artery occlusion. Double immunostaining with stereological counting of brain sections and flow-cytometry analysis of brain cell suspensions were performed. Results— Rosiglitazone accelerated neutrophil infiltration to the ischemic core, concomitantly to neuroprotection. Some neutrophils (≈31%) expressed M2 markers, namely Ym1 and CD206 (mannose receptor). After treatment with the peroxisome proliferator-activated receptor-&ggr; agonist rosiglitazone, most neutrophils (≈77%) acquired an N2 phenotype. Interestingly, rosiglitazone increased neutrophil engulfment by microglia/macrophages, a clearance that preferentially affected the N2 subset. Conclusions— We present the first evidence of neutrophil reprogramming toward an N2 phenotype in brain inflammation, which can be modulated by activation of the peroxisome proliferator-activated receptor-&ggr; nuclear receptor. We also show that N2 polarization is associated with an increased neutrophil clearance, thus suggesting that this switch is a crucial event for resolution of inflammation that may participate in neuroprotection.

Rational modulation of the innate immune system for neuroprotection in ischemic stroke

The innate immune system plays a dualistic role in the evolution of ischemic brain damage and has also been implicated in ischemic tolerance produced by different conditioning stimuli. Early after ischemia, perivascular astrocytes release cytokines and activate metalloproteases (MMPs) that contribute to blood–brain barrier (BBB) disruption and vasogenic oedema; whereas at later stages, they provide extracellular glutamate uptake, BBB regeneration and neurotrophic factors release. Similarly, early activation of microglia contributes to ischemic brain injury via the production of inflammatory cytokines, including tumor necrosis factor (TNF) and interleukin (IL)-1, reactive oxygen and nitrogen species and proteases. Nevertheless, microglia also contributes to the resolution of inflammation, by releasing IL-10 and tumor growth factor (TGF)-β, and to the late reparative processes by phagocytic activity and growth factors production. Indeed, after ischemia, microglia/macrophages differentiate toward several phenotypes: the M1 pro-inflammatory phenotype is classically activated via toll-like receptors or interferon-γ, whereas M2 phenotypes are alternatively activated by regulatory mediators, such as ILs 4, 10, 13, or TGF-β. Thus, immune cells exert a dualistic role on the evolution of ischemic brain damage, since the classic phenotypes promote injury, whereas alternatively activated M2 macrophages or N2 neutrophils prompt tissue remodeling and repair. Moreover, a subdued activation of the immune system has been involved in ischemic tolerance, since different preconditioning stimuli act via modulation of inflammatory mediators, including toll-like receptors and cytokine signaling pathways. This further underscores that the immuno-modulatory approach for the treatment of ischemic stroke should be aimed at blocking the detrimental effects, while promoting the beneficial responses of the immune reaction.

Rosiglitazone-induced CD36 up-regulation resolves inflammation by PPARγ and 5-LO-dependent pathways

PPARγ‐achieved neuroprotection in experimental stroke has been explained by the inhibition of inflammatory genes, an action in which 5‐LO, Alox5, is involved. In addition, PPARγ is known to promote the expression of CD36, a scavenger receptor that binds lipoproteins and mediates bacterial recognition and also phagocytosis. As phagocytic clearance of neutrophils is a requisite for resolution of the inflammatory response, PPARγ‐induced CD36 expression might help to limit inflammatory tissue injury in stroke, an effect in which 5‐LO might also be involved. Homogenates, sections, and cellular suspensions were prepared from brains of WT and Alox5−/− mice exposed to distal pMCAO. BMMs were obtained from Lys‐M Cre+ PPARγf/f and Lys‐M Cre− PPARγf/f mice. Stereological counting of double‐immunofluorescence‐labeled brain sections and FACS analysis of cell suspensions was performed. In vivo and in vitro phagocytosis of neutrophils by microglia/macrophages was analyzed. PPARγ activation with RSG induced CD36 expression in resident microglia. This process was mediated by the 5‐LO gene, which is induced in neurons by PPARγ activation and at least by one of its products—LXA4—which induced CD36 independently of PPARγ. Moreover, CD36 expression helped resolution of inflammation through phagocytosis, concomitantly to neuroprotection. Based on these findings, in addition to a direct modulation by PPARγ, we propose in brain a paracrine model by which products generated by neuronal 5‐LO, such as LXA4, increase the microglial expression of CD36 and promote tissue repair in pathologies with an inflammatory component, such as stroke.

Toll-like receptor 4 modulates cell migration and cortical neurogenesis after focal cerebral ischemia

Toll‐like receptor 4 (TLR4) mediates brain damage after stroke. Now our objective is to determine TLR4 involvement in stroke‐induced neurogenesis. Stroke was induced by permanent middle cerebral artery occlusion in wild‐type and TLR4‐deficient mice. Stereological and densitometric analysis of immunofluorescence‐labeled brain sections and FACS analysis of cell suspensions were performed. Our results show that subventricular zone (SVZ) cell proliferation after stroke depends on infarct size. Second, when comparing brains with similar lesions, TLR4 attenuated SVZ proliferation, as shown by a decrease in prominin‐1+/EGFR+/nestin‐ cells (type‐C cells) at 1‐2 d, and in BrdU+ cells at 7 d, in TLR4+/+ vs. TLR4‐/‐mice. Interestingly, 7 d after the infarct, neuroblasts in TLR4+ mice migrated farther distances, reaching areas closer to the lesion than those in TLR4‐deficient mice. However, at 14 d, TLR4‐deficient mice presented a higher number of neuroblasts in all migratory zones than the TLR4+/+ counterparts, which suggests that TLR4 deficiency delays neuroblast migration. Consistently, TLR4+/+ mice showed an increased number of interneurons (NeuN+/BrdU+/GAD67+ cells) in peri‐infarct cortex 14‐28 d after stroke. Our data indicate that, despite a negative effect on SVZ cell proliferation, TLR4 plays an important role in stroke‐induced neurogenesis by promoting neuroblasts migration and increasing the number of new cortical neurons after stroke.—Moraga, A., Pradillo, J. M., Cuartero, M. I., Hernández‐Jiménez, M., Oses, M., Moro, M. A., Lizasoain, I., Toll‐like receptor 4 modulates cell migration and cortical neurogenesis after focal cerebral ischemia. FASEB J. 28, 4710–4718 (2014). www.fasebj.org

L-Kynurenine/Aryl Hydrocarbon Receptor Pathway Mediates Brain Damage After Experimental Stroke

Background— Aryl hydrocarbon receptor (AhR) is a transcription factor that belongs to the basic helix-loop-helix PAS (Per-Arnt-Sim homology domain) family known to mediate the toxic and carcinogenic effects of xenobiotics. Interestingly, AhR is widely expressed in the central nervous system, but its physiological and pathological roles are still unclear. Methods and Results— To define the role of AhR in stroke, we used middle cerebral artery occlusion in mice and oxygen-glucose deprivation in rat cortical neurons. The results presented here show that the ischemic insult increases total and nuclear AhR levels and AhR transcriptional activity in neurons in vivo and in vitro. We also show that AhR has a causal role in acute ischemic damage because pharmacological or genetic loss-of-function approaches result in neuroprotection. Inhibition of cAMP response element-binding protein–dependent signaling may participate in the deleterious actions of AhR. Finally, we have also found that L-kynurenine, a tryptophan metabolite with AhR agonistic properties, is an endogenous ligand that mediates AhR activation in the brain after middle cerebral artery occlusion. Conclusions— Our data demonstrate that an L-kynurenine/AhR pathway mediates acute brain damage after stroke and open new possibilities for the diagnosis and treatment of this pathology.

Cannabinoid Type-2 Receptor Drives Neurogenesis and Improves Functional Outcome After Stroke

Background and Purpose— Stroke is a leading cause of adult disability characterized by physical, cognitive, and emotional disturbances. Unfortunately, pharmacological options are scarce. The cannabinoid type-2 receptor (CB2R) is neuroprotective in acute experimental stroke by anti-inflammatory mechanisms. However, its role in chronic stroke is still unknown. Methods— Stroke was induced by permanent middle cerebral artery occlusion in mice; CB2R modulation was assessed by administering the CB2R agonist JWH133 ((6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) or the CB2R antagonist SR144528 (N-[(1S)-endo-1,3,3-trimethylbicyclo-[2.2.1]-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) once daily from day 3 to the end of the experiment or by CB2R genetic deletion. Analysis of immunofluorescence-labeled brain sections, 5-bromo-2´-deoxyuridine (BrdU) staining, fluorescence-activated cell sorter analysis of brain cell suspensions, and behavioral tests were performed. Results— SR144528 decreased neuroblast migration toward the boundary of the infarct area when compared with vehicle-treated mice 14 days after middle cerebral artery occlusion. Consistently, mice on this pharmacological treatment, like mice with CB2R genetic deletion, displayed a lower number of new neurons (NeuN+/BrdU+ cells) in peri-infarct cortex 28 days after stroke when compared with vehicle-treated group, an effect accompanied by a worse sensorimotor performance in behavioral tests. The CB2R agonist did not affect neurogenesis or outcome in vivo, but increased the migration of neural progenitor cells in vitro; the CB2R antagonist alone did not affect in vitro migration. Conclusions— Our data support that CB2R is fundamental for driving neuroblast migration and suggest that an endocannabinoid tone is required for poststroke neurogenesis by promoting neuroblast migration toward the injured brain tissue, increasing the number of new cortical neurons and, conceivably, enhancing motor functional recovery after stroke.

Citicoline (CDP-choline) increases Sirtuin1 expression concomitant to neuroprotection in experimental stroke

CDP‐choline has shown neuroprotective effects in cerebral ischemia. In humans, although a recent trial International Citicoline Trial on Acute Stroke (ICTUS) has shown that global recovery is similar in CDP‐choline and placebo groups, CDP‐choline was shown to be more beneficial in some patients, such as those with moderate stroke severity and not treated with t‐PA. Several mechanisms have been proposed to explain the beneficial actions of CDP‐choline. We have now studied the participation of Sirtuin1 (SIRT1) in the neuroprotective actions of CDP‐choline. Fischer rats and Sirt1−/− mice were subjected to permanent focal ischemia. CDP‐choline (0.2 or 2 g/kg), sirtinol (a SIRT1 inhibitor; 10 mg/kg), and resveratrol (a SIRT1 activator; 2.5 mg/kg) were administered intraperitoneally. Brains were removed 24 and 48 h after ischemia for western blot analysis and infarct volume determination. Treatment with CDP‐choline increased SIRT1 protein levels in brain concomitantly to neuroprotection. Treatment with sirtinol blocked the reduction in infarct volume caused by CDP‐choline, whereas resveratrol elicited a strong synergistic neuroprotective effect with CDP‐choline. CDP‐choline failed to reduce infarct volume in Sirt1−/− mice. Our present results demonstrate a robust effect of CDP‐choline like SIRT1 activator by up‐regulating its expression. Our findings suggest that therapeutic strategies to activate SIRT1 may be useful in the treatment of stroke.

Daidzein has neuroprotective effects through ligand-binding-independent PPARγ activation

Phytoestrogens are a group of plant-derived compounds that include mainly isoflavones like daidzein. Phytoestrogens prevent neuronal damage and improve outcome in experimental stroke; however, the mechanisms of this neuroprotective action have not been fully elucidated. In this context, it has been postulated that phytoestrogens might activate the peroxisome proliferator-activated receptor-γ (PPARγ), which exerts neuroprotective effects in several settings. The aim of this study was to determine whether the phytoestrogen daidzein elicits beneficial actions in neuronal cells by mechanisms involving activation of PPARγ. Our results show that daidzein (0.05-5 μM) decreases cell death induced by exposure to oxygen-glucose deprivation (OGD) from rat cortical neurons and that improves synaptic function, in terms of increased synaptic vesicle recycling at nerve terminals, being both effects inhibited by the PPARγ antagonist T0070907 (1 μM). In addition, this phytoestrogen activated PPARγ in neuronal cultures, as shown by an increase in PPARγ transcriptional activity. Interestingly, these effects were not due to binding to the receptor ligand site, as shown by a TR-FRET PPARγ competitive binding assay. Conversely, daidzein increased PPARγ nuclear protein levels and decreased cytosolic ones, suggesting nuclear translocation. We have used the receptor antagonist (RE) fulvestrant to study the neuroprotective participation of daidzein via estrogen receptor and at least in our model, we have discarded this pathway. These results demonstrate that the phytoestrogen daidzein has cytoprotective properties in neurons, which are due to an increase in PPARγ activity not mediated by direct binding to the receptor ligand-binding domain but likely due to post-translational modifications affecting its subcellular location and not depending to the RE and it is not additive with the agonist rosiglitazone.