Search

Abstract Background Using a live imaging approach, we have previously shown that microglia activation after stroke is characterized by marked and long-term induction of the Toll-like receptor (TLR) 2 biophotonic signals. However, the role of TLR2 (and potentially other TLRs) beyond the acute innate immune response and as early neuroprotection against ischemic injury is not well understood. Methods TLR2−/− mice were subjected to transient middle cerebral artery occlusion followed by different reperfusion times. Analyses assessing microglial activation profile/innate immune response were performed using in situ hybridization, immunohistochemistry analysis, flow cytometry and inflammatory cytokine array. The effects of the TLR2 deficiency on the evolution of ischemic brain injury were analyzed using a cresyl violet staining of brain sections with appropriate lesion size estimation. Results Here we report that TLR2 deficiency markedly affects post-stroke immune response resulting in delayed exacerbation of the ischemic injury. The temporal analysis of the microglia/macrophage activation profiles in TLR2−/− mice and age-matched controls revealed reduced microglia/macrophage activation after stroke, reduced capacity of resident microglia to proliferate as well as decreased levels of monocyte chemotactic protein-1 (MCP-1) and consequently lower levels of CD45high/CD11b+ expressing cells as shown by flow cytometry analysis. Importantly, although acute ischemic lesions (24 to 72 h) were smaller in TLR2−/− mice, the observed alterations in innate immune response were more pronounced at later time points (at day 7) after initial stroke, which finally resulted in delayed exacerbation of ischemic lesion leading to larger chronic infarctions as compared with wild-type mice. Moreover, our results revealed that TLR2 deficiency is associated with significant decrease in the levels of neurotrophic/anti-apoptotic factor Insulin-like growth factor-1 (IGF-1), expressed by microglia in the areas both in and around ischemic lesion. Conclusion Our results clearly suggest that optimal and timely microglial activation/innate immune response is needed to limit neuronal damage after stroke.

Innate immune response in neonatal brain is associated with a robust microglial activation and induction of Toll-like Receptors (TLRs). To date, the role of the scavenger receptor CD36 in TLRs modulation, particularly TLR2 signaling, has been well established in adult brain. However, the crosstalk between TLR4, TLR2 and CD36 and its immunogenic influence in the neonatal brain remains unclear. In this study, using a CD36 blocking antibody (anti-CD36) at post-natal day 8, we evaluated the response of neonates to systemic endotoxin (lipopolysaccharide; LPS) challenge. We visualized the TLR2 response by bioluminescence imaging using the transgenic mouse model bearing the dual reporter system luciferase/green fluorescent protein under transcriptional control of a murine TLR2 promoter. The anti-CD36 treatment modified the LPS induced inflammatory profile in neonatal brains, causing a significant decrease in inflammatory cytokine levels and the TLR2 and TLR3 mediated signalling.The interferon regulatory factor 3 (IRF3) pathway remained unaffected. Treatment of the LPS-challenged human immature microglia with anti-CD36 induced a marked decrease in TLR2/TLR3 expression levels while TLR4 and IRF3 expression was not affected, suggesting the shared CD36 regulatory mechanisms in human and mouse microglia. Collectively, our results indicate that blocking CD36 alters LPS-induced inflammatory profile of mouse and human microglia, suggesting its role in fine-tuning of neuroinflammation., (© 2023. The Author(s).)

Growing evidence suggests that galectin-3 (Gal-3) is instrumental in orchestrating innate immune response and microglia activation following different brain pathologies. However, its role remains controversial. We recently showed that a readily available natural product glucosamine may act as a strong modulator of Gal-3. Glucosamine is a naturally occurring sugar and a precursor in the synthesis of glycosylated proteins. It is often used as a supplement to treat symptoms of various inflammatory conditions. Our recent work suggests that by increasing the synthesis and availability of Gal-3 ligands and/or by regulating its expression levels, glucosamine may significantly modulate Gal-3 signaling. Because evidence suggests that Gal-3 might be differentially regulated after ischemic injury in the brains of female mice, here we examined and compared the immunomodulatory potential of glucosamine in male and female stroke. The mice were subjected to transient middle cerebral artery occlusion (MCAO), followed by different reperfusion periods. The short-term 5 days treatment with glucosamine (150 ​mg/kg i.p.) was initiated 2 ​hrs after stroke. To visualize the effects of glucosamine treatment on post-stroke inflammation, we took advantage of a transgenic mouse model bearing the dual reporter system luciferase/GFP under transcriptional control of a murine TLR2 promoter (TLR2-luc-GFP) allowing in vivo bioluminescence imaging of innate immune response and microglial activation. We report that after stroke, both, male and female mice strongly up-regulate the TLR2 bioluminescence signals from activated microglia, however, the observed in vivo immunomodulatory effects of glucosamine after stroke were sex-dependent. Analysis of cytokine profiles at protein level, in glucosamine-treated male mice 72hsr after stroke, revealed down regulation of pro-inflammatory cytokines, an increase in levels of anti-inflammatory cytokines including IL-4, IL13 and colony stimulating factors MCFC and GM-CSF and a significant decrease in the size of ischemic lesion in male mice. Conversely, in female mice glucosamine markedly increases the pro-inflammatory signaling and exacerbates ischemic injury. Analysis of the downstream signaling target of glucosamine/Gal-3 revealed that glucosamine administration restored PPAR-γ activity in male but not in female mice 3 days following MCAO. Together, our results suggest that glucosamine acts as a fine tuner of post-ischemic inflammation in a sex dependent-manner and may have therapeutic potential after stroke in males. Based on our results propose that targeting immune system after stroke may require adapted sex-specific therapeutic approaches., Competing Interests: All coauthors have seen and agreed with the content of the manuscript. The material in the manuscript has not been published and is not being considered for publication elsewhere in whole or in part in any language except as an abstract. The Authors disclose no financial and/or conflict of interest., (© 2020 The Authors.)

Uncontrolled microglial activation may lead to the development of inflammation-induced brain damage. Here, we uncover a ribosome-based mechanism/checkpoint involved in control of the innate immune response and microglial activation. Using an in vivo model system for analysis of the dynamic translational state of microglial ribosomes, with mRNAs as input and newly synthesized peptides as an output, we find a marked dissociation of microglia mRNA and protein networks following innate immune challenge. Highly upregulated and ribosome-associated mRNAs were not translated, resulting in two distinct microglial molecular signatures, a highly specialized pro-inflammatory mRNA signature and an immunomodulatory/homeostatic protein signature. We find that this is due to specific translational suppression of highly expressed mRNAs through a 3' UTR-mediated mechanism involving the RNA-binding protein SRSF3. This discovery suggests avenues for therapeutic modulation of innate immune response in resident microglia., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Activation of microglial cells in response to brain injury and/or immune stimuli is associated with a marked induction of Toll-like receptors (TLRs). While in adult brain, the contribution of individual TLRs, including TLR2, in pathophysiological cascades has been well established, their role and spatial and temporal induction patterns in immature brain are far less understood. To examine whether infectious stimuli and sterile inflammatory stimuli trigger distinct TLR2-mediated innate immune responses, we used three models in postnatal day 9 (P9) mice, a model of infection induced by systemic endotoxin injection and two models of sterile inflammation, intra-cortical IL-1β injection and transient middle cerebral artery occlusion (tMCAO). We took advantage of a transgenic mouse model bearing the dual reporter system luciferase/GFP under transcriptional control of a murine TLR2 promoter (TLR2-luc-GFP) to visualize the TLR2 response in the living neonatal brain and then determined neuroinflammation, microglial activation and leukocyte infiltration. We show that in physiological postnatal brain development the in vivo TLR2-luc signal undergoes a marked ∼30-fold decline and temporal-spatial changes during the second and third postnatal weeks. We then show that while endotoxin robustly induces the in vivo TLR2-luc signal in the living brain and increases levels of several inflammatory cytokines and chemokines, the in vivo TLR2-luc signal is reduced after both IL-1β and tMCAO and the inflammatory response is muted. Immunofluorescence revealed that microglial cells are the predominant source of TLR2 production during postnatal brain development and in all three neonatal models studied. Flow cytometry revealed developmental changes in CD11b + /CD45 + and CD11b + /Ly6C + cell populations, involvement of cells of the monocyte lineage, but lack of Ly6G + neutrophils or CD3 + cells in acutely injured neonatal brains. Cumulatively, our results suggest distinct TLR2 induction patterns following PAMP and DAMP - mediated inflammation in immature brain., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Estrogens are known to exert neuroprotective and immuneomodulatory effects after stroke. However, at present, little is known about the role of estrogens and its receptors in postischemic inflammation after menopause. Here, we provide important in vivo evidence of a distinct shift in microglial phenotypes in the model of postmenopause brain. Using a model-system for live imaging of microglial activation in the context of chronic estrogen- and ERα-deficiency associated with aging, we observed a marked deregulation of the TLR2 signals and/or microglial activation in ovariectomized and/or ERα knockout mice. Further analysis revealed a 5.7-fold increase in IL-6, a 4.7-fold increase in phospho-Stat3 levels suggesting an overactivation of JAK/STAT3 pathway and significantly larger infarction in ERα knockouts chronically deprived of estrogen. Taken together, our results suggest that in the experimental model of menopause and/or aging, ERα mediates innate immune responses and/or microglial activation, and ischemia-induced production of IL-6. Based on our results, we propose that the loss of functional ERα may lead to deregulation of postischemic inflammatory responses and increased vulnerability to ischemic injury in aging female brains., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Growing evidence suggests that galectin-3 is involved in fine tuning of the inflammatory responses at the periphery, however, its role in injured brain is far less clear. Our previous work demonstrated upregulation and coexpression of galectin-3 and IGF-1 in a subset of activated/proliferating microglial cells after stroke. Here, we tested the hypothesis that galectin-3 plays a pivotal role in mediating injury-induced microglial activation and proliferation. By using a galectin-3 knock-out mouse (Gal-3KO), we demonstrated that targeted disruption of the galectin-3 gene significantly alters microglia activation and induces ∼4-fold decrease in microglia proliferation. Defective microglia activation/proliferation was further associated with significant increase in the size of ischemic lesion, ∼2-fold increase in the number of apoptotic neurons, and a marked deregulation of the IGF-1 levels. Next, our results revealed that contrary to WT cells, the Gal3-KO microglia failed to proliferate in response to IGF-1. Moreover, the IGF-1-mediated mitogenic microglia response was reduced by N-glycosylation inhibitor tunicamycine while coimmunoprecipitation experiments revealed galectin-3 binding to IGF-receptor 1 (R1), thus suggesting that interaction of galectin-3 with the N-linked glycans of receptors for growth factors is involved in IGF-R1 signaling. While the canonical IGF-1 signaling pathways were not affected, we observed an overexpression of IL-6 and SOCS3, suggesting an overactivation of JAK/STAT3, a shared signaling pathway for IGF-1/IL-6. Together, our findings suggest that galectin-3 is required for resident microglia activation and proliferation in response to ischemic injury.

Motor pools comprise a heterogeneous population of motor neurons that innervate distinct intramuscular targets. While the organization of motor neurons into motor pools has been well described, the time course and mechanism of motor pool diversification into functionally distinct classes remains unclear. γ-Motor neurons (γ-MNs) and α-motor neurons (α-MNs) differ in size, molecular identity, synaptic input and peripheral target. While α-MNs innervate extrafusal skeletal muscle fibers to mediate muscle contraction, γ-MNs innervate intrafusal fibers of the muscle spindle, and regulate sensitivity of the muscle spindle in response to stretch. In this study, we find that the secreted signaling molecule Wnt7a is selectively expressed in γ-MNs in the mouse spinal cord by embryonic day 17.5 and continues to molecularly distinguish γ-from α-MNs into the third postnatal week. Our data demonstrate that Wnt7a is the earliest known γ-MN marker, supporting a model of developmental divergence between α- and γ-MNs at embryonic stages. Furthermore, using Wnt7a expression as an early marker of γ-MN identity, we demonstrate a previously unknown dependence of γ-MNs on a muscle spindle-derived, GDNF-independent signal during the first postnatal week.

Background and Purpose: Consumption of fish has been shown to reduce risk of coronary heart disease and, possibly, of ischemic stroke. Because docosahexaenoic acid (DHA) is the most likely neuroactive component within fish oil, we hypothesized that exposing mice to a DHA-enriched diet may reduce inflammation and protect neurons against ischemic injury., Methods: To visualize the effects of DHA on neuroinflammation after stroke, TLR2-fluc-GFP transgenic mice were exposed to either a control diet, a diet depleted in n-3 polyunsaturated fatty acid, or a diet enriched in DHA during 3 months. Real-time biophotonic/bioluminescence imaging of the TLR2 response was performed before and after middle cerebral artery occlusion, whereas cytokines concentrations and stroke area analyses were performed at 3 and 7 days after middle cerebral artery occlusion, respectively., Results: We show that a 3-month DHA treatment prevented microglial activation after ischemic injury, reduced the ischemic lesion size, and increased levels of the antiapoptotic molecule Bcl-2 in the brain. Additional analysis revealed a significant decrease in the levels of COX2 and IL-1β, but not in other proinflammatory cytokines. Importantly, long-term DHA supplementation significantly changed the n-3:n-6 polyunsaturated fatty acid ratio in the brain., Conclusions: Collectively, these data indicate that diet-induced accumulation of DHA in the brain protects against postischemic inflammation and injury. Because DHA is widely available at low cost and has an excellent safety profile, our data suggest that increased DHA intake may provide protection against acute immune response/brain damage in ischemic stroke.

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.

The intranasal entry of biological and artificial nanoparticles can induce inflammatory responses both locally and more widely in surrounding tissues. The aim of this study was to assess the microglia activation induced by nanoparticles with different surfaces in (i) a transgenic mouse (Toll-like receptor (TLR)-2-luciferase (Luc) reporter) which allowed the biophotonic imaging of microglial activation/innate immune response after intranasal delivery of nanoparticles and (ii) in microglial dispersed cells in vitro. Cadmium selenide nanoparticles (quantum dots, QD), surface-exchanged with lipopolysaccharide (LPS) to form micelles, were tested to assess microglia activation and lipid droplet formation in both model systems. In vivo imaging revealed a robust increase in the extent of microglial activation/TLR2 response, initially in the olfactory bulb, but also in other more caudal brain regions. The increased TLR2 expression was complemented with enhanced CD68 expression in activated microglia in the same regions. Intense in vitro microglial activation by LPS-QD micelles was accompanied by a significant enhancement of nitric oxide production and formation of large lipid droplets, suggesting the possibility of this organelle acting as an inflammatory biomarker in response to nanoparticles, and not simply as a storage site in fat tissues.

Given the emergence of nanotherapeutics and nanodiagnostics as key tools in today's medicine, it has become of critical importance to define precisely the interactions of nanomaterials with biological systems and to characterize the resulting cellular response. We report here the interactions of microglia and neurons with gold nanoparticles (GNPs) of three morphologies, spheres, rods, and urchins, coated with poly(ethylene glycol) (PEG) or cetyl trimethylammonium bromide (CTAB). Microglia are the resident immune cells of the brain, primarily involved in surveillance, macrophagy, and production of cytokines and trophic factors. Analysis by dark-field microscopy and by two-photon-induced luminescence (TPL) indicates that the exposure of neural cells to GNPs resulted in (i) GNP internalization by both microglial cells and primary hippocampal neurons, as revealed by dark-field microscopy and by two-photon-induced luminescence (TPL), (ii) transient toll-like receptor 2 (TLR-2) up-regulation in the olfactory bulb, after intranasal administration in transgenic mice, in vivo, in real time, and (iii) differential up-regulation in vitro of TLR-2 together with interleukin 1 alpha (IL-1alpha), granulocyte macrophage colony-stimulating factor (GM-CSF) and nitric oxide (NO) in microglia. The study demonstrates that GNP morphology and surface chemistry strongly influence the microglial activation status and suggests that interactions between GNPs and microglia can be differentially regulated by tuning GNP nanogeometry.

Macrophage colony stimulating factor (M-CSF) is a cytokine that regulates the survival, proliferation and maturation of microglial cells. Administration of M-CSF can promote neuronal survival in various models of central nervous system (CNS) injury. Here, in an attempt to induce a neuroprotective microglial cell phenotype and enhance motor neuron survival, mutant SOD1(G37R) transgenic mice were treated, weekly, with M-CSF starting at onset of disease. Unexpectedly, M-CSF accelerated disease progression in SOD1(G37R) mouse model of ALS. The shortened survival of M-CSF-treated animals was associated with diminished muscle innervation and enhanced adoption of a macrophage-like phenotype by microglial cells characterised by the upregulation of pro-inflammatory cytokines TNF-alpha and IL-1 beta and of the phagocytic marker CD68.

With an incidence of approximately 350 in 100,000, stroke is the third leading cause of death and a major cause of disability in industrialized countries. At present, although progress has been made in understanding the molecular pathways that lead to ischemic cell death, the current clinical treatments remain poorly effective. There is mounting evidence that inflammation plays an important role in cerebral ischemia. Experimentally and clinically, brain response to ischemic injury is associated with an acute and prolonged inflammatory process characterized by the activation of resident glial cells, production of inflammatory cytokines as well as leukocyte and monocyte infiltration in the brain, events that may contribute to ischemic brain injury and affect brain recovery and plasticity. However, whether the post-ischemic inflammatory response is deleterious or beneficial to brain recovery is presently a matter of debate and controversies. Here, we summarize the current knowledge on the molecular mechanisms underlying post-ischemic neuronal plasticity and the potential role of inflammation in regenerative processes and functional recovery after stroke. Furthermore, because of the dynamic nature of the brain inflammatory response, we highlight the importance of the development of novel experimental approaches such as real-time imaging. Finally, we discuss the novel transgenic reporter mice models that have allowed us to visualize and to analyze the processes such as neuroinflammation and neuronal repair from the ischemic brains of live animals.

Astrocytes are the principle macroglial brain cells. They are activated by different stressors and brain injuries. Quantum dots (QDs) can cause oxidative stress. This study shows a real-time imaging of primary cortical cultures and assessment of QD-induced activation of astrocytes in the brains of transgenic mice with the luciferase gene driven by the murine astrocyte promoter. This approach may be widely applicable for assessing the astroglia/tissue response to nanoparticles in live animals.

Here we report in vivo evidence of a neuroprotective role of proliferating microglial cells in cerebral ischemia. Using transgenic mice expressing a mutant thymidine kinase form of herpes simplex virus driven by myeloid-specific CD11b promoter and ganciclovir treatment as a tool, we selectively ablated proliferating (Mac-2 positive) microglia after transient middle cerebral artery occlusion. The series of experiments using green fluorescent protein-chimeric mice demonstrated that within the first 72 h after ischemic injury, the Mac-2 marker [unlike Iba1 (ionized calcium-binding adapter molecule 1)] was preferentially expressed by the resident microglia. Selective ablation of proliferating resident microglia was associated with a marked alteration in the temporal dynamics of proinflammatory cytokine expression, a significant increase in the size of infarction associated with a 2.7-fold increase in the number of apoptotic cells, predominantly neurons, and a 1.8-fold decrease in the levels of IGF-1. A double-immunofluorescence analysis revealed a approximately 100% colocalization between IGF-1 positive cells and Mac-2, a marker of activated/proliferating resident microglia. Conversely, stimulation of microglial proliferation after cerebral ischemia by M-CSF (macrophage colony stimulating factor) resulted in a 1.9-fold increase in IGF-1 levels and a significant increase of Mac2+ cells. Our findings suggest that a postischemic proliferation of the resident microglial cells may serve as an important modulator of a brain inflammatory response. More importantly, our results revealed a marked neuroprotective potential of proliferating microglia serving as an endogenous pool of neurotrophic molecules such as IGF-1, which may open new therapeutic avenues in the treatment of stroke and other neurological disorders.

The involvement of genes controlling embryonic processes in the etiology of diseases often escapes attention because of the focus given to their inherent developmental role. Hoxa5 belongs to the Hox gene family encoding transcription factors known for their role in skeletal patterning. Hoxa5 is required for embryonic respiratory tract morphogenesis. We now show that the loss of Hoxa5 function has severe repercussions on postnatal lung development. Hoxa5-/- lungs present an emphysema-like morphology because of impaired alveogenesis. Chronic inflammation characteristics, including goblet cell hyperplasia, mucus hypersecretion, and recruitment of inflammatory cells, were also observed. Altered cell specification during lung morphogenesis triggered goblet cell anomalies. In addition, the defective motility of alveolar myofibroblast precursors in the embryonic lung led to the mispositioning of the alveolar myofibroblasts and to abnormal elastin deposition postnatally. Both goblet cell hyperplasia and elastic fiber abnormalities contributed to the chronic physiopathological features of Hoxa5-/- lungs. They constituted an attractive stimulus to recruit activated macrophages that in turn generated a positive feedback loop that perpetuated macrophage accumulation in the lung. The present work corroborates the notion that altered Hox gene expression may predispose to lung pathologies.

The Hoxa5 mutation is associated with a high perinatal mortality rate caused by a severe obstruction of the laryngotracheal airways, pulmonary dysmorphogenesis, and a decreased production of surfactant proteins. Surviving Hoxa5(-/-) mutant mice also display lung anomalies with deficient alveolar septation and areas of collapsed tissue, thus demonstrating the importance of Hoxa5 throughout lung development and maturation. Here, we address the functional consequences of the Hoxa5 mutation on respiration and chemoreflexes by comparing the breathing pattern of Hoxa5(-/-) mice to that of wild-type animals under resting conditions and during exposure to moderate ventilatory stimuli such as hypoxia and hypercapnia. Resting Hoxa5(-/-) mice present a higher breathing frequency and overall minute ventilation that likely compensate for their reduced lung alveolar surface available for gas exchange and their increased upper airway resistance. When exposed to ventilatory stimuli, Hoxa5(-/-) mice maintain the higher minute ventilation by adapting the tidal volume and/or the breathing frequency. The minute ventilation increase seen during hypoxia was similar for both groups of mice; however, the dynamics of the frequency response was genotype-dependent. The hypercapnic ventilatory response did not differ between genotypes. These findings reveal the strategies allowing survival of Hoxa5(-/-) mice facing morphologic anomalies leading to a significant deficit in gas exchange capacity.