N2 Neutrophils, Novel Players in Brain Inflammation After Stroke

Abstract

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. (Stroke. 2013;44:3498-3508.)

Neutrophil recruitment to the brain in mouse and human ischemic stroke

“…However, increased numbers of neutrophils have been detected after permanent brain ischemia and infiltration into the parenchyma was found to begin 12 h after the onset of the arterial occlusion [8,22,52]. Remarkably, increasing data attest that neutrophils cross talk and shape the maturation and effector functions of other leukocytes, show diverse phenotypes [12,19], and could play an important role in several pathological conditions [31,36,40]. In this study, we investigated the route of entry of neutrophils to the brain while the circulation remained arrested in experimental models of permanent occlusion of the middle cerebral artery (MCAo) in mice.…”

Neuroprotective Properties of a Macrolide Antibiotic in a Mouse Model of Middle Cerebral Artery Occlusion: Characterization of the Immunomodulatory Effects and Validation of the Efficacy of Intravenous Administration

“…[7][8][9][10][11] Intriguingly, although neutrophils have classically been regarded as detrimental, being correlated with poor histological and functional outcomes after stroke, [12][13][14][15][16] recent findings suggest that brain recruitment of N2 phenotypes may provide beneficial effects. 17,18 Thus, a strategic approach to rescue ischemic brain injury would consist in promoting polarization shifts toward M2 or N2 phenotypes, while reducing activation of pro-inflammatory phenotypes during the acute phase after stroke. Following this aim, few drugs have to date proved successful in preclinical settings and most of these studies focused on repurposing existing drugs.…”

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

“…This method provides an accurate tool to estimate total neutrophil numbers in the ischemic brain at different times after ligation. Although it has not been shown in this study, this protocol can also be used for estimation of the different neutrophil subpopulations after ischemia 21 and for an accurate quantification of any other cell population found in the ischemic brain like other infiltrated leukocytes (monocytes/macrophages) and also for estimating surviving neurons or even for neurogenesis quantification after stroke. The most important step for an accurate estimation in the desired area is the selection of the appropriate parameters, as the ones shown in the Table 3 for neutrophil quantification in the ischemic area.…”