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

Abstract

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.

Neuroprotective Effects of microRNA-140-5p on Ischemic Stroke in Mice via its Regulation on the TLR4/NF-κB Axis

“…Then, dual-luciferase reporter assay was employed and veri ed that miR-140-5p bound to TLR4. Most interestingly, TLR4 has been suggested to play the crucial role of in stroke, which could mediate several pathways related to in ammation or migration of neutrophils into the parenchyma that induced by ischemia [41]. Hence, we guessed that TLR4 may be involved in the ischemic stroke affected by miR-140-5p, which was validated by means of RT-qPCR, Western blot, ow cytometry, and TTC staining, as well as TUNEL staining.…”

Systemic Inflammatory Response Syndrome and Outcomes in Ischemic Patients Treated with Endovascular Treatment

“…A generally accepted view is that the amount of collateral-dependent blood ow is one of the dominating determinants of infarct volume after LVO [26]. Therefore, rarefaction of the preexisting collateral circulation, results in more severe tissue injury and augmented neuronal necrosis following arterial occlusion; then, endogenous molecules are released from damaged cells amplifying in ammatory mediator expression and tissue damage by triggering and inducing the activation of Toll-like receptors (TLRs) [27], and some other damage-associated molecular patterns (DAMPs) [28]. Furthermore, collateral remodeling involves endothelial cell activation, leukocyte recruitment, matrix changes and cell proliferation; for patients with poor collateral circulation, the proin ammatory process involving angiogenesis will be more obvious [29].…”

The role of Toll-like receptor signaling pathways in cerebrovascular disorders: the impact of spreading depolarization

“…Inhibition of TLR2 signaling pathway regulates leukocytes and microglial infiltration and the subsequent neuronal death after mild AIS [78,87,104,105]. Inhibition of TLR4 could attenuate the inflammation and H-I damages through blockade of tissue-type plasminogen activator-induced hemorrhagic transformation [106] as well as enhancement of the ratio of alternative neutrophils [15]. It has been shown that TLR4-deficient mice have significantly less tolerance to H-I insults than wild-type mice, possibly via the lesser expression of TNFα, cyclooxygenase-2 (COX-2), and NF-κB [107].…”

“…Accumulating evidence suggest that TLRs play a crucial role in the pathogenesis of AIS [15,16], ICH [17,18], and SAH [19]. TLRs activation following ischemic insults could be pathogenic or neuroprotective, depending on the context.…”