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.

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-γ dependent in macrophages. We hypothesize that peroxisome proliferator-activated receptor-γ 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-γ 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-γ 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.

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 κB (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 κB-induced inflammatory and apoptotic pathways.

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.

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.