Paul Heuschling

Characterization of the microglial phenotype under specific pro-inflammatory and anti-inflammatory conditions: Effects of oligomeric and fibrillar amyloid-β

M1 and M2 are the extremes of the differentiation spectrum of activated macrophages. Since microglia are members of the same cell lineage, we have characterized their transcription profile and their phagocytic activity under different conditions. LPS or IFN-gamma induce a M1-like phenotype, while IL-10 or IL-4 differentiate microglia towards a M2-deactivated or M2-alternatively-activated phenotype respectively. These differentiation processes also affect the Notch pathway. In order to study the polarization induced by Abeta, microglia was stimulated with different forms of the peptide. The oligomeric Abeta is a stronger M1-inductor than the fibrillar form. Moreover, a cytokine-induced anti-inflammatory environment reduces the microglial reactivity towards oligomeric Abeta.

NLRP3 Inflammasome Is Expressed and Functional in Mouse Brain Microglia but Not in Astrocytes.

Neuroinflammation is the local reaction of the brain to infection, trauma, toxic molecules or protein aggregates. The brain resident macrophages, microglia, are able to trigger an appropriate response involving secretion of cytokines and chemokines, resulting in the activation of astrocytes and recruitment of peripheral immune cells. IL-1β plays an important role in this response; yet its production and mode of action in the brain are not fully understood and its precise implication in neurodegenerative diseases needs further characterization. Our results indicate that the capacity to form a functional NLRP3 inflammasome and secretion of IL-1β is limited to the microglial compartment in the mouse brain. We were not able to observe IL-1β secretion from astrocytes, nor do they express all NLRP3 inflammasome components. Microglia were able to produce IL-1β in response to different classical inflammasome activators, such as ATP, Nigericin or Alum. Similarly, microglia secreted IL-18 and IL-1α, two other inflammasome-linked pro-inflammatory factors. Cell stimulation with α-synuclein, a neurodegenerative disease-related peptide, did not result in the release of active IL-1β by microglia, despite a weak pro-inflammatory effect. Amyloid-β peptides were able to activate the NLRP3 inflammasome in microglia and IL-1β secretion occurred in a P2X7 receptor-independent manner. Thus microglia-dependent inflammasome activation can play an important role in the brain and especially in neuroinflammatory conditions.

An in vitro model for the study of microglia-induced neurodegeneration: involvement of nitric oxide and tumor necrosis factor-α

The precise function of activated microglia and their secretory products remains controversial. In order to assess the role of microglial secretion products, we established an in vitro model of an inflammatory reaction in the brain by co-culturing microglial and neuronal cell lines. Upon stimulation with interferon-gamma and lipopolysaccharides, the microglial cells adopted an activated phenotype and secreted tumor necrosis factor-alpha (TNF-alpha), prostaglandin E(2) and nitric oxide (NO). Neuronal degeneration was quantified by measuring the concentrations of microtubule associated protein tau and neuron specific enolase, which are also used as diagnostic tool in Alzheimers disease, in supernatants. In activated contact co-cultures, the levels of these neuronal markers were significantly raised compared to non-activated co-cultures. NO-synthase inhibitors significantly diminished the rise of tau in activated co-cultures, while indomethacin, superoxide dismutase, or a neutralizing TNF-alpha antibody did not. When a chemical NO-donor or TNF-alpha were added to pure neuronal cultures, cell viability was significantly reduced. TNF-alpha increased neuronal sensitivity towards NO. There were indications that a part of the cells died by apoptosis. This model demonstrates a neurotoxic role for NO in microglia-induced neurodegeneration and provides a valuable in vitro tool for the study of microglia-neuron interactions during inflammation in the brain.

Notch signaling modulates the activation of microglial cells

The Notch signaling pathway plays a crucial role in specifying cellular fate in metazoan development by regulating communication between adjacent cells. Correlative studies suggested an involvement of Notch in hematopoietic cell development. Here, we report that the Notch pathway is expressed and active in microglial cells. During inflammatory activation, the transcription of the Notch down‐stream effector Hes1 is downregulated. When Notch1 transcription in microglia is inhibited, an upregulation of the expression of pro‐inflammatory cytokines is observed. Notch stimulation in activated microglia, using a soluble form of its ligand Jagged1, induces a decrease in pro‐inflammatory cytokines secretion and nitric oxide production as well as an increase in phagocytic activity. Notch‐stimulation is accompanied by an increase in the rate of STAT3 phosphorylation and nuclear translocation. Our results show that the Notch pathway plays an important role in the control of inflammatory reactions in the CNS.

Jagged1 regulates the activation of astrocytes via modulation of NFκB and JAK/STAT/SOCS pathways

The Notch pathway is implicated in many aspects of the central nervous system (CNS) development and functions. Recently, we and others identified the Notch pathway to be involved in inflammatory events of the CNS. To understand the implication of this pathway on astrocytes, we have studied the Jagged‐Notch‐Hes pathway under inflammatory conditions. LPS exposure induced an upregulation of Jagged1 expression on cultured astrocytes. To address the role of Jagged1 in the modulation of inflammation, we used a siRNA mediated silencing of Jagged1 (siRNA J1). Jagged1 inhibition induced important variations on the Notch pathway components like Hes1, Hes5, Notch3, and RBP‐Jκ. siRNA J1 repressed the mRNA expression of genes known as hallmarks of the gliosis like GFAP and endothelin(B) receptor. On activated astrocytes, the inhibition of Jagged1 had antiinflammatory effects and resulted in a decrease of LPS‐induced proinflammatory cytokines (IL1β, IL1α, and TNFα) as well as the iNOS expression. The inhibition of Jagged1 induced a modulation of the JAK/STAT/SOCS signaling pathway. Most interestingly, the siRNA J1 decreased the LPS‐induced translocation of NFκB p65 and this could be correlated to the phosphorylation of IκBα. These results suggest that during inflammatory and gliotic events of the CNS, Jagged1/Notch signaling sustains the inflammation mainly through NFκB and in part through JAK/STAT/SOCS signaling pathways.

Microglial activation depends on beta‐amyloid conformation: role of the formylpeptide receptor 2

J. Neurochem. (2010) 114, 576–586.

Stimulation of Endothelin B Receptor Modulates the Inflammatory Activation of Rat Astrocytes

Abstract: Inside the brain tissue, endothelins play numerous important biological roles. One of the targets, astrocytes, predominantly display endothelin receptor subtype B (ETB). On cultured primary rat astroglial cells, we analyzed the effect of IRL 1620, a selective ETB receptor agonist, on the production of nitric oxide (NO) and the synthesis of interleukin (IL)‐6 and tumor necrosis factor (TNF)‐α. We performed these experiments in the presence or absence of interferon‐γ (IFN‐γ) and/or lipopolysaccharide (LPS). IRL 1620 decreases NO production under basal conditions and after IFN‐γ stimulation. However, during LPS‐induced NO production, IRL 1620 enhances this release. The basal IL‐6 secretion and especially the LPS‐induced synthesis are enhanced by the IRL 1620 stimulation. The LPS‐dependent TNF‐α production is increased by the ETB stimulation. The IRL 1620‐induced decrease of basal NO production is not dependent on Ca2+ entry or on phospholipase C (PLC) activation, as shown by the use of LaCl3 and U73122, respectively. In the presence of LPS, the IRL 1620 potentiation of NO production is inhibited by LaCl3 and U73122. The IRL 1620‐induced increase of IL‐6 is dependent on PLC activation. These results suggest that endothelins can have dual effects depending on the costimulatory factors present. Endothelins thus have important immunomodulatory functions in the brain.

Nitric oxide modulates y-interferon-induced MHC class II antigen expression on rat astrocytes

Brain astroglial cells can be brought in vivo and in vitro to express an immunocompetent cell-like phenotype. We investigated the effect of the NO. releasing compound sodium nitroprusside (SNP) on Ia expression in rat astrocyte cultures. SNP down-regulates, in a concentration-dependent manner, the gamma-interferon-induced Ia expression. Inhibition of the NO. synthesis attenuates the glutamate mediated down-regulation of class II expression. Our results show that NO. is implicated in the immunomodulatory reactions in the brain parenchyma.

Cultured astrocytes express regional heterogeneity of the immunoreactive phenotype under basal conditions and after γ-IFN induction

Cerebral astrocytes are known to show a region-specific phenotype, concerning the expression of several receptors and the synthesis of secreted substances. In order to find out whether this heterogeneity also exists for the immunological activation, we studied several parameters that are known to characterize activated astroglia on cultured primary rat astrocytes originating from cortex, hippocampus, striatum, septum and brain stem: major histocompatibility complex (MHC) class II and intercellular adhesion molecule (ICAM)-1 expression, nitric oxide (NO) production and interleukin-6 (IL-6) synthesis. Unstimulated cultures show a baseline expression of MHC class II molecules that differs from one region to another, hippocampus and brain stem showing the highest values. These differences are strongly enhanced after a 48-h incubation with gamma-interferon (gamma-IFN). NO production is also induced by a 72-h incubation with gamma-IFN, showing similar patterns of regional specialization. The baseline expressions of ICAM-1 and IL-6 also show major regional differences, with the brain stem and the striatum showing elevated values for ICAM-1, and the septum and the brain stem producing the largest amounts of IL-6. The expressions of ICAM-1 and IL-6 are not affected by an incubation with gamma-IFN. Our results demonstrate that the immunological activities of astroglial cells show regional heterogeneities. This specialization may be implicated in the pathophysiological pathways of several neurodegenerative disorders.

Tocopherol derivative TFA-12 promotes myelin repair in experimental models of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is associated with demyelination and axonal loss, resulting in severe neurological handicap. Current MS therapies mostly target neuroinflammation but have only a little impact on CNS myelin repair. Progress toward treatments that enhance remyelination would therefore represent major advances in MS treatment. Here, we examined the ability of TFA-12, a new synthetic compound belonging to tocopherol long-chain fatty alcohols, to promote oligodendrocyte regeneration and remyelination in experimental models of MS. We showed that TFA-12 significantly ameliorates neurological deficit and severity of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) in mice. Histological evaluation of mouse EAE spinal cords showed that TFA-12 treatment reduces inflammation, astrogliosis, and myelin loss. Additionally, we demonstrated that TFA-12 accelerates remyelination of focal demyelinated lesions induced by lysolecithin injections. We also found that this compound induces the differentiation of oligodendrocyte precursor cells into mature oligodendrocytes through the inhibition of the Notch/Jagged1 signaling pathway. Altogether, our data provide important proof of principle indicating that TFA-12 could be a potential therapeutic compound for myelin repair in MS.