Emmanuel Pinteaux

Genetic Cell Ablation Reveals Clusters of Local Self-Renewing Microglia in the Mammalian Central Nervous System

During early embryogenesis, microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how the tissue-resident macrophages are maintained throughout the organisms lifespan still remains unclear. Here, we describe a system that allows specific, conditional ablation of microglia in adult mice. We found that the microglial compartment was reconstituted within 1 week of depletion. Microglia repopulation relied on CNS-resident cells, independent from bone-marrow-derived precursors. During repopulation, microglia formed clusters of highly proliferative cells that migrated apart once steady state was achieved. Proliferating microglia expressed high amounts of the interleukin-1 receptor (IL-1R), and treatment with an IL-1R antagonist during the repopulation phase impaired microglia proliferation. Hence, microglia have the potential for efficient self-renewal without the contribution of peripheral myeloid cells, and IL-1R signaling participates in this restorative proliferation process.

Interleukin-1-induced neurotoxicity is mediated by glia and requires caspase activation and free radical release.

Interleukin (IL)‐1 expression is induced rapidly in response to diverse CNS insults and is a key mediator of experimentally induced neuronal injury. However, the mechanisms of IL‐1‐induced neurotoxicity are unknown. The aim of the present study was to examine the toxic effects of IL‐1 on rat cortical cell cultures. Treatment with IL‐1β did not affect the viability of pure cortical neurones. However, IL‐1 treatment of cocultures of neurones with glia or purified astrocytes induced caspase activation resulting in neuronal death. Neuronal cell death induced by IL‐1 was prevented by pre‐treatment with the IL‐1 receptor antagonist, the broad spectrum caspase inhibitor Boc‐Asp‐(OMe)‐CH2F or the antioxidant α‐tocopherol. The NMDA receptor antagonist dizolcipine (MK‐801) attenuated cell death induced by low doses of IL‐1β but the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptor antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo(F)quinoxaline (NBQX) had no effect. Inhibition of inducible nitric oxide synthase with N(ω)‐nitro‐l‐arginine methyl ester had no effect on neuronal cell death induced by IL‐1β. Thus, IL‐1 activates the IL‐1 type 1 receptor in astrocytes to induce caspase‐dependent neuronal death, which is dependent on the release of free radicals and may contribute to neuronal cell death in CNS diseases.

Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells

Interleukin (IL)‐1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL‐1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL‐1 receptors [IL‐1 type‐I receptor (IL‐1RI), IL‐1 type‐II receptor (IL‐1RII) and IL‐1 receptor accessory protein (IL‐1RAcP)] on microglia. In the present study we investigated whether microglia express IL‐1 receptors and whether they present target or modulatory properties for IL‐1 actions. RT–PCR analysis demonstrated lower expression of IL‐1RI and higher expression of IL‐1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL‐1RI, IL‐1RII and IL‐1RAcP mRNAs, induced the release of IL‐1β, IL‐6 and prostaglandin‐E2 (PGE2), and activated nuclear factor κB (NF‐κB) and the mitogen‐activated protein kinases (MAPKs) p38, and extracellular signal‐regulated protein kinase (ERK1/2), but not c‐Jun N‐terminal kinase (JNK) in microglial cultures. In comparison, IL‐1β induced the release of PGE2, IL‐6 and activated NF‐κB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL‐1β also failed to affect LPS‐primed microglial cells. Interestingly, a neutralizing antibody to IL‐1RII significantly increased the concentration of IL‐1β in the medium of LPS‐treated microglia and exacerbated the IL‐1β‐induced IL‐6 release in mixed glia, providing the first evidence that microglial IL‐1RII regulates IL‐1β actions by binding excess levels of this cytokine during brain inflammation.

Neuroprotective actions of endogenous interleukin-1 receptor antagonist (IL-1ra) are mediated by glia.

The pro‐inflammatory cytokine interleukin‐1 (IL‐1), contributes to neuronal inflammation and cell death induced by ischemia, excitotoxicity, or trauma, while administration of IL‐1 receptor antagonist (IL‐1ra) reduces neuronal injury. The aim of the present study was to test the hypothesis that endogenous IL‐1ra is neuroprotective in vivo and in vitro, and to identify its mechanism of actions. Mice lacking IL‐1ra (IL‐1ra knock‐out (KO]) exhibited a dramatic increase in neuronal injury (3.6‐fold increase in infarct size) induced by transient cerebral ischemia compared to wild‐type (WT) animals. Basal cell death of cultured cortical neurons from WT and IL‐1ra KO was identical, and treatment with NMDA or AMPA (20 μM) increased cell death to the same extent in WT and IL‐1ra KO neurons. However, basal and NMDA‐ or AMPA‐induced cells death was significantly higher in glial–neuronal co‐cultures from IL‐1ra KO than from WT mice. We further showed that pure microglial cultures, but not pure astrocytes cultures, released IL‐1ra in response to treatment with conditioned medium from NMDA‐ or AMPA‐treated primary neurons. These results demonstrate that endogenous IL‐1ra produced by microglia is neuroprotective in cerebral ischemia or excitotoxicity.

IL‐1β signalling in glial cells in wildtype and IL‐1RI deficient mice

Interleukin‐1 (IL‐1) has been implicated in neurodegeneration and in central nervous system (CNS)‐mediated host defence responses to inflammation. All actions of IL‐1 identified to date appear to be mediated through its only known functional type I receptor (IL‐1RI). However, our recent evidence suggests that some actions of IL‐1 in the brain may be IL‐1RI independent, suggesting the involvement of a new, hitherto unknown functional receptor for IL‐1. The objective of the present study was to determine if primary mixed glial cells express additional functional IL‐1 receptors by studying the signalling mechanisms responsible for the pro‐inflammatory actions of IL‐1β in cultures derived from IL‐1RI−/− and wildtype mice, and to characterize the functional importance of IL‐1 signalling pathways in glia. IL‐1β induced marked release of IL‐6 and prostaglandin‐E2 (PGE2) in the culture medium, and activated nuclear factor‐kappa B (NFκB) and the mitogen‐activated protein kinases (MAPK) p38, c‐Jun N‐terminal kinase (JNK) and the extracellular signal‐regulated protein kinase (ERK1/2) in cells from wildtype mice. These responses were dependent on IL‐1RI, since cells isolated from IL‐1R1−/− mice did not demonstrate any of these responses. In wildtype mice, inhibition of p38 or ERK1/2 MAPKs significantly reduced IL‐1β induced IL‐6 release, whilst the NFκB inhibitor caffeic acid phenethyl ester (CAPE) modulated IL‐1 induced IL‐6 release by action on NFκB and MAPKs pathways. These data demonstrate that IL‐1RI is essential for IL‐1β signalling in cultured mixed glial cells. Thus IL‐1 actions observed in IL‐1RI−/− mice in vivo may occur via an alternative pathway and/or via different CNS cells.

Neuronal toll-like receptor 4 signaling induces brain endothelial activation and neutrophil transmigration in vitro

BackgroundThe innate immune response in the brain is initiated by pathogen-associated molecular patterns (PAMPS) or danger-associated molecular patterns (DAMPS) produced in response to central nervous system (CNS) infection or injury. These molecules activate members of the Toll-like receptor (TLR) family, of which TLR4 is the receptor for bacterial lipopolysaccharide (LPS). Although neurons have been reported to express TLR4, the function of TLR4 activation in neurons remains unknown.MethodsTLR4 mRNA expression in primary mouse glial and neuronal cultures was assessed by RT-PCR. Mouse mixed glial, neuronal or endothelial cell cultures were treated with LPS in the absence or the presence of a TLR4 specific antagonist (VIPER) or a specific JNK inhibitor (SP600125). Expression of inflammatory mediators was assayed by cytometric bead array (CBA) and ELISA. Activation of extracellular-signal regulated kinase 1/2 (ERK1/2), p38, c-Jun-N-terminal kinase (JNK) and c-Jun was assessed by Western blot. The effect of conditioned media of untreated- versus LPS-treated glial or neuronal cultures on endothelial activation was assessed by neutrophil transmigration assay, and immunocytochemistry and ELISA were used to measure expression of intercellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1).ResultsLPS induces strong release of the chemokines RANTES and CXCL1 (KC), tumor necrosis factor-α (TNFα) and IL-6 in primary mouse neuronal cultures. In contrast, LPS induced release of IL-1α, IL-1β and granulocyte-colony stimulating factor (G-CSF) in mixed glial, but not in neuronal cultures. LPS-induced neuronal KC expression and release were completely blocked by VIPER. In glial cultures, LPS induced activation of ERK1/2, p38 and JNK. In contrast, in neuronal cultures, LPS activated JNK but not ERK1/2 or p38, and the specific JNK inhibitor SP600125 significantly blocked LPS-induced KC expression and release. Finally, conditioned medium of LPS-treated neuronal cultures induced strong expression of ICAM-1 and VCAM-1 on endothelial cells, and induced infiltration of neutrophils across the endothelial monolayer, which was inhibited by VIPER.ConclusionThese data demonstrate for the first time that neurons can play a role as key sensors of infection to initiate CNS inflammation.

Neutrophil Cerebrovascular Transmigration Triggers Rapid Neurotoxicity through Release of Proteases Associated with Decondensed DNA

Cerebrovascular inflammation contributes to diverse CNS disorders through mechanisms that are incompletely understood. The recruitment of neutrophils to the brain can contribute to neurotoxicity, particularly during acute brain injuries, such as cerebral ischemia, trauma, and seizures. However, the regulatory and effector mechanisms that underlie neutrophil-mediated neurotoxicity are poorly understood. In this study, we show that mouse neutrophils are not inherently toxic to neurons but that transendothelial migration across IL-1–stimulated brain endothelium triggers neutrophils to acquire a neurotoxic phenotype that causes the rapid death of cultured neurons. Neurotoxicity was induced by the addition of transmigrated neutrophils or conditioned medium, taken from transmigrated neutrophils, to neurons and was partially mediated by excitotoxic mechanisms and soluble proteins. Transmigrated neutrophils also released decondensed DNA associated with proteases, which are known as neutrophil extracellular traps. The blockade of histone–DNA complexes attenuated transmigrated neutrophil-induced neuronal death, whereas the inhibition of key neutrophil proteases in the presence of transmigrated neutrophils rescued neuronal viability. We also show that neutrophil recruitment in the brain is IL-1 dependent, and release of proteases and decondensed DNA from recruited neutrophils in the brain occurs in several in vivo experimental models of neuroinflammation. These data reveal new regulatory and effector mechanisms of neutrophil-mediated neurotoxicity (i.e., the release of proteases and decondensed DNA triggered by phenotypic transformation during cerebrovascular transmigration). Such mechanisms have important implications for neuroinflammatory disorders, notably in the development of antileukocyte therapies.

Leptin induces interleukin-1β release from rat microglial cells through a caspase 1 independent mechanism

Leptin regulates energy balance by suppressing appetite and increasing energy expenditure through actions in the hypothalamus. Recently we demonstrated that the effects of leptin are, at least in part, mediated by the release of interleukin (IL)‐1β in the brain. Microglia constitute the major source of IL‐1β in the brain but it is not known whether these cells express leptin receptors, or respond to leptin to produce IL‐1β. Using RT‐PCR and immunocytochemistry, we demonstrate that primary rat microglial cells express the short (non‐signalling) and long (signalling) isoforms of the leptin receptors (Ob‐R)s. Immunoassays performed on cell medium collected 24 h after leptin treatment (0.01–10 μg/mL) demonstrated a dose‐dependent production and release of IL‐1β and its endogenously occurring receptor antagonist IL‐1RA. In addition leptin‐induced IL‐1β release occurs via a signal transducer and activator of transcription 3 (STAT3)‐dependent mechanism. Western blot analysis demonstrated that leptin induced the synthesis of pro‐IL‐1β in microglial cells and the release of mature 17 kDa isoform into the culture medium. Leptin‐induced IL‐1β release was neither inhibited by the pan‐caspase inhibitor BOC‐D‐FMK, nor by the caspase 1 inhibitor Ac‐YVAD‐CHO indicating that IL‐1 cleavage is independent of caspase activity. These results confirm our earlier observations in vivo and demonstrate that microglia are an important source of IL‐1β in the brain in response to leptin.

Transport of interleukin‐1 across cerebromicrovascular endothelial cells

Background and purpose:  The inflammatory cytokine interleukin‐1 (IL‐1) has profound actions in the brain, causing neuronal cell death and exacerbating brain damage. While circulating levels are normally low, IL‐1 can be produced on the vascular side of the brain endothelium, and within the brain. The naturally occurring IL‐1 receptor antagonist has been administered peripherally in a Phase II trial in acute stroke patients; understanding how IL‐1 and IL‐1 receptor antagonist penetrate the brain is, therefore, of considerable importance.

Neuroprotective effects of the synthetic cannabinoid HU-210 in primary cortical neurons are mediated by phosphatidylinositol 3-kinase/AKT signaling.

Cannabinoids (CBs) are neuroprotective in vivo and in vitro, but the mechanisms of their actions are unknown. The aim of this study was to elucidate the signaling pathways that mediate the protective effect of CBs on primary cultured neurons. The neurotoxin S-AMPA induced significant death of rat primary cortical neurons, which was inhibited by the CB agonist HU-210. Antagonists selective for CB(1) or CB(2) receptors (AM 281 or AM 630, respectively) reversed the neuroprotective effect of HU-210 on S-AMPA-induced cell death. HU-210 triggered activation of AKT, but not activation of the ERK1/2, JNK or p38 signaling pathways. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors LY294002 and wortmannin prevented phosphorylation of AKT in response to HU-210, and reversed the neuroprotective effect of HU-210 on S-AMPA-induced excitotoxicity. Thus the PI 3-K/AKT signaling pathway mediates the neuroprotective effect of exogenous cannabinoids such as HU-210 in primary CNS neurons.