Iván Ballesteros

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.

Synthesis of Lipoxin A4 by 5-Lipoxygenase Mediates PPARγ-Dependent, Neuroprotective Effects of Rosiglitazone in Experimental Stroke

Peroxisome proliferator-activated receptors gamma (PPARγ) are nuclear receptors with essential roles as transcriptional regulators of glucose and lipid homeostasis. PPARγ are also potent anti-inflammatory receptors, a property that contributes to the neuroprotective effects of PPARγ agonists in experimental stroke. The mechanism of these beneficial actions, however, is not fully elucidated. Therefore, we have explored further the actions of the PPARγ agonist rosiglitazone in experimental stroke induced by permanent middle cerebral artery occlusion (MCAO) in rodents. Rosiglitazone induced brain 5-lipoxygenase (5-LO) expression in ischemic rat brain, concomitantly with neuroprotection. Rosiglitazone also increased cerebral lipoxin A4 (LXA4) levels and inhibited MCAO-induced production of leukotriene B4 (LTB4). Furthermore, pharmacological inhibition and/or genetic deletion of 5-LO inhibited rosiglitazone-induced neuroprotection and downregulation of inflammatory gene expression, LXA4 synthesis and PPARγ transcriptional activity in rodents. Finally, LXA4 caused neuroprotection, which was partly inhibited by the PPARγ antagonist T0070907, and increased PPARγ transcriptional activity in isolated nuclei, showing for the first time that LXA4 has PPARγ agonistic actions. Altogether, our data illustrate that some effects of rosiglitazone are attributable to de novo synthesis of 5-LO, able to induce a switch from the synthesis of proinflammatory LTB4 to the synthesis of the proresolving LXA4. Our study suggests novel lines of study such as the interest of lipoxin-like anti-inflammatory drugs or the use of these molecules as prognostic and/or diagnostic markers for pathologies in which inflammation is involved, such as stroke.

Activation of Liver X Receptors Promotes Neuroprotection and Reduces Brain Inflammation in Experimental Stroke

Background— The liver X receptors (LXRs) belong to the nuclear receptor superfamily and act as transcriptional regulators of cholesterol metabolism in several tissues. Recent work also has identified LXRs as potent antiinflammatory molecules in macrophages and other immune cells. Combined changes in lipid and inflammatory profiles are likely mediating the protective role of LXRs in models of chronic injury like atherosclerosis. These beneficial actions, however, have not been illustrated in other models of acute injury such as stroke in which inflammation is an important pathophysiological feature. Methods and Results— We have studied LXR expression and function in the course of experimental stroke caused by permanent middle cerebral artery occlusion in rats and mice. Here, we show that administration of the synthetic LXR agonists GW3965 or TO901317 after the ischemic occlusion improves stroke outcome as shown by decreased infarct volume area and better neurological scores in rats. Neuroprotection observed with LXR agonists correlated with decreased expression of proinflammatory genes in the brain and with reduced nuclear factor-&kgr;B transcriptional activity. Loss of function studies using LXR&agr;,&bgr;−/− mice demonstrated that the effect of LXR agonists is receptor specific. Interestingly, infarcted brain area and inflammatory signaling were significantly extended in LXR&agr;,&bgr;−/− mice compared with control animals, indicating that endogenous LXR signaling mediates neuroprotection in this setting. Conclusion— This work highlights the transcriptional action of LXR as a protective pathway in brain injury and the potential use of LXR agonists as therapeutic agents in stroke.

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.

miRNA expression is modulated over time after focal ischaemia: up-regulation of miR–347 promotes neuronal apoptosis

Despite the large number of molecules reported as being over‐expressed after ischaemia, little is known regarding their regulation. miRNAs are potent post‐transcriptional regulators of gene expression, and reports have shown differentially miRNA expression in response to focal cerebral ischaemia. The present study analysed miRNA expression from acute to late phases of ischaemia to identify specific ischaemia‐related miRNAs, elucidate their role, and identify potential targets involved in stroke pathophysiology. Of 112 miRNAs, 32 showed significant changes and different expression profiles. In addition to the previously reported differentially expressed miRNAs, new ischaemia‐regulated miRNAs have been found, including miR‐347. Forty‐seven genes involved in brain functions or related to ischaemia are predicted to be potential targets of the differentially expressed miRNAs after middle cerebral artery occlusion. Analysis of four of these targets (Acsl4, Arf3, Btg2 and Dpysl5) showed them to be differentially regulated by ischaemia at the transcriptional or post‐transcriptional level. Acsl4, Bnip3l and Phyhip, potential targets of miR‐347, were up‐regulated after miR‐347 over‐expression, inducing neuronal apoptotic death. Our findings suggest that miR‐347 plays an important role in regulating neuronal cell death, identify Acsl4 as a new protein requiring study in ischaemia, and provide an important resource for future functional studies of miRNAs after ischaemia.

Aging increases microglial proliferation, delays cell migration, and decreases cortical neurogenesis after focal cerebral ischemia.

BackgroundAging is not just a risk factor of stroke, but it has also been associated with poor recovery. It is known that stroke-induced neurogenesis is reduced but maintained in the aged brain. However, there is no consensus on how neurogenesis is affected after stroke in aged animals. Our objective is to determine the role of aging on the process of neurogenesis after stroke.MethodsWe have studied neurogenesis by analyzing proliferation, migration, and formation of new neurons, as well as inflammatory parameters, in a model of cerebral ischemia induced by permanent occlusion of the middle cerebral artery in young- (2 to 3 months) and middle-aged mice (13 to 14 months).ResultsAging increased both microglial proliferation, as shown by a higher number of BrdU+ cells and BrdU/Iba1+ cells in the ischemic boundary and neutrophil infiltration. Interestingly, aging increased the number of M1 monocytes and N1 neutrophils, consistent with pro-inflammatory phenotypes when compared with the alternative M2 and N2 phenotypes. Aging also inhibited (subventricular zone) SVZ cell proliferation by decreasing both the number of astrocyte-like type-B (prominin-1+/epidermal growth factor receptor (EGFR)+/nestin+/glial fibrillary acidic protein (GFAP)+ cells) and type-C cells (prominin-1+/EGFR+/nestin−/Mash1+ cells), and not affecting apoptosis, 1 day after stroke. Aging also inhibited migration of neuroblasts (DCX+ cells), as indicated by an accumulation of neuroblasts at migratory zones 14 days after injury; consistently, aged mice presented a smaller number of differentiated interneurons (NeuN+/BrdU+ and GAD67+ cells) in the peri-infarct cortical area 14 days after stroke.ConclusionsOur data confirm that stroke-induced neurogenesis is maintained but reduced in aged animals. Importantly, we now demonstrate that aging not only inhibits proliferation of specific SVZ cell subtypes but also blocks migration of neuroblasts to the damaged area and decreases the number of new interneurons in the cortical peri-infarct area. Thus, our results highlight the importance of using aged animals for translation to clinical studies.

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.