Anthony Lyons

CD200 Ligand–Receptor Interaction Modulates Microglial Activation In Vivo and In Vitro: A Role for IL-4

Deficits in cognitive function are associated with neuroinflammatory changes, typified by activation of glial cells and an alteration of the pro- and anti-inflammatory cytokine balance in the brain. Although there is evidence to suggest that activation of microglia is regulated by interaction with other cell types in the brain, the mechanism(s) involved is poorly understood. Here, we provide evidence that interaction between CD200 and its receptor plays a role in modulating microglial activation under conditions of chronic and acute inflammation of the brain. We report that interleukin-4 (IL-4) plays a central role in modulating expression of CD200 and identify a mechanism by which IL-4 directly controls microglial cell activation. Our findings provide the first demonstration of a role for IL-4 in modulating CD200 expression and suggest a mechanism for regulation of microglial activation in the intact CNS under inflammatory conditions.

Fractalkine‐induced activation of the phosphatidylinositol‐3 kinase pathway attentuates microglial activation in vivo and in vitro

Several neurodegenerative disorders are associated with evidence of inflammation, one feature of which is increased activation of microglia, the most likely cellular source of inflammatory cytokines like interleukin‐1β. It is now recognized that interaction of microglia with other cells contributes to maintenance of microglia in a quiescent state and the complementary distribution of the chemokine, fractalkine (CX3CL1) on neurons and its receptor (CX3CR1) on microglia, suggests that this interaction may play a role in modulating microglial activation. Here we demonstrate that both soluble and membrane‐bound fractalkine attenuate lipopolysaccharide‐induced microglial activation in vitro. We also show that fractalkine expression is reduced in the brain of aged rats and this is accompanied by an age‐related increase in microglial activation. Treatment of aged rats with fractalkine attenuates the age‐related increase in microglial activation and the evidence indicates that fractalkine‐induced activation of the phosphatidylinositol‐3 kinase pathway is required to maintain microglia in a quiescent state both in vivo and in vitro.

IL‐4 attenuates the neuroinflammation induced by amyloid‐β in vivo and in vitro

It has been shown that Aβ inhibits long‐term potentiation (LTP) in the rat hippocampus and this is accompanied by an increase in hippocampal concentration of IL‐1β. Aβ also increases microglial activation, which is the likely cell source of IL‐1β. Because IL‐4 attenuates the effects of IL‐1β in hippocampus, and microglial activation is inhibited by minocycline, we assessed the ability of both IL‐4 and minocycline to modulate the effects of Aβ on LTP and IL‐1β concentration. Following treatment with Aβ, IL‐4 or minocycline, rats were assessed for their ability to sustain LTP in perforant path‐granule cell synapses. We report that the Aβ‐induced inhibition of LTP was associated with increases in expression of MHCII, JNK phosphorylation and IL‐1β concentration, and that these changes were attenuated by treatment of rats with IL‐4 and minocycline. We also report that Aβ‐induced increases in expression of MHCII and IL‐1β were similarly attenuated by IL‐4 and minocycline in glial cultures prepared from neonatal rats. These data suggest that glial cell activation and the consequent increase in IL‐1β concentration mediate the inhibitory effect of Aβ on LTP and indicate that IL‐4, by down‐regulating glial cell activation, antagonizes the effects of Aβ.

Long Term Potentiation Is Impaired in Membrane Glycoprotein CD200-deficient Mice A ROLE FOR Toll-LIKE RECEPTOR ACTIVATION

The membrane glycoprotein CD200 is expressed on several cell types, including neurons, whereas expression of its receptor, CD200R, is restricted principally to cells of the myeloid lineage, including microglia. The interaction between CD200 and CD200R maintains microglia and macrophages in a quiescent state; therefore, CD200-deficient mice express an inflammatory phenotype exhibiting increased macrophage or microglial activation in models of arthritis, encephalitis, and uveoretinitis. Here, we report that lipopolysaccharide (LPS) and Pam3CysSerLys4 exerted more profound effects on release of the proinflammatory cytokines, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNFα), in glia prepared from CD200−/− mice compared with wild type mice. This effect is explained by the loss of CD200 on astrocytes, which modulates microglial activation. Expression of Toll-like receptors 4 and 2 (TLR4 and -2) was increased in glia prepared from CD200−/− mice, and the evidence indicates that microglial activation, assessed by the increased numbers of CD11b+ cells that stained positively for both MHCII and CD40, was enhanced in CD200−/− mice compared with wild type mice. These neuroinflammatory changes were associated with impaired long term potentiation (LTP) in CA1 of hippocampal slices prepared from CD200−/− mice. One possible explanation for this is the increase in TNFα in hippocampal tissue prepared from CD200−/− mice because TNFα application inhibited LTP in CA1. Significantly, LPS and Pam3CysSerLys4, at concentrations that did not affect LTP in wild type mice, inhibited LTP in slices prepared from CD200−/− mice, probably due to the accompanying increase in TLR2 and TLR4. Thus, the neuroinflammatory changes that result from CD200 deficiency have a negative impact on synaptic plasticity.

A pivotal role for interleukin-4 in atorvastatin-associated neuroprotection in rat brain.

Inflammatory changes, characterized by an increase in pro-inflammatory cytokine production and up-regulation of the corresponding signaling pathways, have been described in the brains of aged rats and rats treated with the potent immune modulatory molecule lipopolysaccharide (LPS). These changes have been coupled with a deficit in long-term potentiation (LTP) in hippocampus. The evidence suggests that anti-inflammatory agents, which attenuate the LPS-induced and age-associated increase in hippocampal interleukin-1β (IL-1β) concentration, lead to restoration of LTP. Here we report that atorvastatin, a member of the family of agents that act as inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, exerts powerful anti-inflammatory effects in brain and that these effects are mediated by IL-4 and independent of its cholesterol-lowering actions. Treatment of rats with atorvastatin increased IL-4 concentration in hippocampal tissue prepared from LPS-treated and aged rats and abrogated the age-related and LPS-induced increases in pro-inflammatory cytokines, interferon-γ (IFNγ) and IL-1β, and the accompanying deficit in LTP. The effect of atorvastatin on the LPS-induced increases in IFNγ and IL-1β was absent in tissue prepared from IL-4–/– mice. The increase in IL-1β in LPS-treated and aged rats is associated with increased microglial activation, assessed by analysis of major histocompatibility complex II expression, and the evidence suggests that IFNγ may trigger this activation. We propose that the primary effect of atorvastatin is to increase IL-4, which antagonizes the effects of IFNγ, the associated increase in microglial activation, and the subsequent cascade of events.

A novel anti-inflammatory role of NCAM-derived mimetic peptide, FGL.

Age-related cognitive deficits in hippocampus are correlated with neuroinflammatory changes, typified by increased pro-inflammatory cytokine production and microglial activation. We provide evidence that the neural cell adhesion molecule (NCAM)-derived mimetic peptide, FG loop (FGL), acts as a novel anti-inflammatory agent. Administration of FGL to aged rats attenuated the increased expression of markers of activated microglia, the increase in pro-inflammatory interleukin-1beta (IL-1beta) and the impairment in long-term potentiation (LTP). We report that the age-related increase in microglial activation was accompanied by decreased expression of neuronal CD200, and suggest that the proclivity of FGL to suppress microglial activation is due to its stimulatory effect on neuronal CD200. We demonstrate that FGL enhanced interleukin-4 (IL-4) release from glial cells and IL-4 in turn enhanced neuronal CD200 in vitro. We provide evidence that the increase in CD200 is reliant on IL-4-induced extracellular signal-regulated kinase (ERK) signal transduction. These findings provide the first evidence of a role for FGL as an anti-inflammatory agent and identify a mechanism by which FGL controls microglial activation.

Decreased neuronal CD200 expression in IL-4-deficient mice results in increased neuroinflammation in response to lipopolysaccharide

Maintenance of the balance between pro- and anti-inflammatory cytokines in the brain, which is affected by the activation state of microglia, is important for maintenance of neuronal function. Evidence has suggested that IL-4 plays an important neuromodulatory role and has the ability to decrease lipopolysaccharide-induced microglial activation and the production of IL-1beta. We have also demonstrated that CD200-CD200R interaction is involved in immune homeostasis in the brain. Here, we investigated the anti-inflammatory role of IL-4 and, using in vitro and in vivo analysis, established that the effect of lipopolysaccharide was more profound in IL-4(-/-), compared with wildtype, mice. Intraperitoneal injection of lipopolysaccharide exerted a greater inhibitory effect on exploratory behaviour in IL-4(-/-), compared with wildtype, mice and this was associated with evidence of microglial activation. We demonstrate that the increase in microglial activation is inversely related to CD200 expression. Furthermore, CD200 was decreased in neurons prepared from IL-4(-/-) mice, whereas stimulation with IL-4 enhanced CD200 expression. Importantly, neurons prepared from wildtype, but not from IL-4(-/-), mice attenuated the lipopolysaccharide-induced increase in pro-inflammatory cytokine production by glia. These findings suggest that the neuromodulatory effect of IL-4, and in particular its capacity to maintain microglia in a quiescent state, may result from its ability to upregulate CD200 expression on neurons.

The HMG-CoA reductase inhibitor, atorvastatin, attenuates the effects of acute administration of amyloid-β1–42 in the rat hippocampus in vivo

One response of the brain to stressors is to increase microglial activation with the consequent production of proinflammatory cytokines like interleukin-1beta (IL-1beta), which has been shown to exert an inhibitory effect on long-term potentiation (LTP) in the hippocampus. It has been consistently shown, particularly in vitro, that amyloid-beta (Abeta) peptides increase activation of microglia, while its inhibitory effect on LTP is well documented, and associated with the Abeta-induced increase in IL-1beta. Here we set out to establish whether the Abeta-induced inhibition of LTP in perforant path-granule cell synapses, was coupled with evidence of microglial activation and to assess whether atorvastatin, which is used primarily in the treatment of hyperlipidaemia but which possesses anti-inflammatory properties, might modulate the effect of Abeta on LTP. We report that intracerebroventricular injection of Abeta increased expression of several markers of microglial activation, and in parallel, inhibited LTP in dentate gyrus. The data show that atorvastatin abrogated the Abeta-induced microglial activation and the associated deficit in LTP. On the basis of the evidence presented, we propose that the action of atorvastatin is mediated by its ability to increase production of the anti-inflammatory cytokine, interleukin-4, which we report mimics several of the actions of atorvastatin in the rat hippocampus.

Classical activation of microglia in CD200-deficient mice is a consequence of blood brain barrier permeability and infiltration of peripheral cells.

The interaction between CD200, expressed on several cell types, and its receptor CD200R, expressed on cells of the myeloid lineage, has been shown to be an important factor in modulating inflammation in macrophage function in several conditions including colitis and arthritis. More recently its modulatory effect on microglial activation has been identified and CD200-deficiency has been associated with increased microglial activation accompanied by increased production of inflammatory cytokines. The response of glia prepared from CD200-deficient mice to stimuli like lipopolysaccharide (LPS) is markedly greater than the response of cells prepared from wildtype mice and, consistent with this, is the recent observation that expression of Toll-like receptor (TLR)4 and signalling through NFκB are increased in microglia prepared from CD200-deficient mice. Here we show that glia from CD200-deficient mice are also more responsive to interferon-γ (IFNγ) which triggers classical activation of microglia. We investigated the effects of CD200-deficiency in vivo and report that there is an increase in expression of several markers of microglial activation including tumor necrosis factor (TNF)-α, which is a hallmark of classically-activated microglia. These changes are accompanied by increased IFNγ, and the evidence suggests that this is produced by infiltrating cells including T cells and macrophages. We propose that these cells enter the brain as a consequence of increased blood brain barrier (BBB) permeability in CD200-deficient mice and that infiltration is assisted by increased expression of the chemokines, monocyte chemotactic protein-1 (MCP-1), IFNγ-induced protein-10 (IP-10) and RANTES. This may have implications in neurodegenerative diseases where BBB permeability is compromised.

TRIL, a functional component of the TLR4 signaling complex, highly expressed in brain

TLR4 is the primary sensor of LPS. In this study, we describe for the first time TLR4 interactor with leucine-rich repeats (TRIL), which is a novel component of the TLR4 complex. TRIL is expressed in a number of tissues, most prominently in the brain but also in the spinal cord, lung, kidney, and ovary. TRIL is composed of a signal sequence, 13 leucine-rich repeats, a fibronectin domain, and a single transmembrane spanning region. TRIL is induced by LPS in the human astrocytoma cell line U373, in murine brain following i.p. injection, and in human PBMC. Endogenous TRIL interacts with TLR4 and this interaction is greatly enhanced following LPS stimulation. TRIL also interacts with the TLR4 ligand LPS. Furthermore, U373 cells stably overexpressing TRIL display enhanced cytokine production in response to LPS. Finally, knockdown of TRIL using small interfering RNA attenuates LPS signaling and cytokine production in cell lines, human PBMC, and primary murine mixed glial cells. These results demonstrate that TRIL is a novel component of the TLR4 complex which may have particular relevance for the functional role of TLR4 in the brain.