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TLR4-activated microglia require IFN-? to induce severe neuronal dysfunction and death in situ.

ABSTRACT: Microglia (tissue-resident macrophages) represent the main cell type of the innate immune system in the CNS; however, the mechanisms that control the activation of microglia are widely unknown. We systematically explored microglial activation and functional microglia-neuron interactions in organotypic hippocampal slice cultures, i.e., postnatal cortical tissue that lacks adaptive immunity. We applied electrophysiological recordings of local field potential and extracellular K(+) concentration, immunohistochemistry, design-based stereology, morphometry, Sholl analysis, and biochemical analyses. We show that chronic activation with either bacterial lipopolysaccharide through Toll-like receptor 4 (TLR4) or leukocyte cytokine IFN-? induces reactive phenotypes in microglia associated with morphological changes, population expansion, CD11b and CD68 up-regulation, and proinflammatory cytokine (IL-1?, TNF-?, IL-6) and nitric oxide (NO) release. Notably, these reactive phenotypes only moderately alter intrinsic neuronal excitability and gamma oscillations (30-100 Hz), which emerge from precise synaptic communication of glutamatergic pyramidal cells and fast-spiking, parvalbumin-positive GABAergic interneurons, in local hippocampal networks. Short-term synaptic plasticity and extracellular potassium homeostasis during neural excitation, also reflecting astrocyte function, are unaffected. In contrast, the coactivation of TLR4 and IFN-? receptors results in neuronal dysfunction and death, caused mainly by enhanced microglial inducible nitric oxide synthase (iNOS) expression and NO release, because iNOS inhibition is neuroprotective. Thus, activation of TLR4 in microglia in situ requires concomitant IFN-? receptor signaling from peripheral immune cells, such as T helper type 1 and natural killer cells, to unleash neurotoxicity and inflammation-induced neurodegeneration. Our findings provide crucial mechanistic insight into the complex process of microglia activation, with relevance to several neurologic and psychiatric disorders.

SUBMITTER: Papageorgiou IE

PROVIDER: S-EPMC4711883 | biostudies-other | 2016 Jan

REPOSITORIES: biostudies-other

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Project description:BackgroundInterleukin-17 (IL-17) acts as a key regulator in central nervous system (CNS) inflammation. ?? T cells are an important innate source of IL-17. Both IL-17+ ?? T cells and microglia, the major resident immune cells of the brain, are involved in various CNS disorders such as multiple sclerosis and stroke. Also, activation of Toll-like receptor (TLR) signaling pathways contributes to CNS damage. However, the mechanisms underlying the regulation and interaction of these cellular and molecular components remain unclear.ObjectiveIn this study, we investigated the crosstalk between ?? T cells and microglia activated by TLRs in the context of neuronal damage. To this end, co-cultures of IL-17+ ?? T cells, neurons, and microglia were analyzed by immunocytochemistry, flow cytometry, ELISA and multiplex immunoassays.ResultsWe report here that IL-17+ ?? T cells but not naïve ?? T cells induce a dose- and time-dependent decrease of neuronal viability in vitro. While direct stimulation of ?? T cells with various TLR ligands did not result in up-regulation of CD69, CD25, or in IL-17 secretion, supernatants of microglia stimulated by ligands specific for TLR2, TLR4, TLR7, or TLR9 induced activation of ?? T cells through IL-1? and IL-23, as indicated by up-regulation of CD69 and CD25 and by secretion of vast amounts of IL-17. This effect was dependent on the TLR adaptor myeloid differentiation primary response gene 88 (MyD88) expressed by both ?? T cells and microglia, but did not require the expression of TLRs by ?? T cells. Similarly to cytokine-primed IL-17+ ?? T cells, IL-17+ ?? T cells induced by supernatants derived from TLR-activated microglia also caused neurotoxicity in vitro. While these neurotoxic effects required stimulation of TLR2, TLR4, or TLR9 in microglia, neuronal injury mediated by bone marrow-derived macrophages did not require TLR signaling. Neurotoxicity mediated by IL-17+ ?? T cells required a direct cell-cell contact between T cells and neurons.ConclusionTaken together, these results point to a crucial role for microglia activated through TLRs in polarization of ?? T cells towards neurotoxic IL-17+ ?? T cells.

Project description:Background & aimsIn cirrhosis, astrocytic swelling is believed to be the principal mechanism of ammonia neurotoxicity leading to hepatic encephalopathy (HE). The role of neuronal dysfunction in HE is not clear. We aimed to explore the impact of hyperammonaemia on mitochondrial function in primary co-cultures of neurons and astrocytes and in acute brain slices of cirrhotic rats using live cell imaging.MethodsTo primary cocultures of astrocytes and neurons, low concentrations (1 and 5 μM) of NH4Cl were applied. In rats with bile duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonaemia and minimal HE, acute brain slices were studied. One group of BDL rats was treated twice daily with the ammonia scavenger ornithine phenylacetate (OP; 0.3 g/kg). Fluorescence measurements of changes in mitochondrial membrane potential (Δψm), cytosolic and mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LP) rates, and cell viability were performed using confocal microscopy.ResultsNeuronal cultures treated with NH4Cl exhibited mitochondrial dysfunction, ROS overproduction, and reduced cell viability (27.8 ± 2.3% and 41.5 ± 3.7%, respectively) compared with untreated cultures (15.7 ± 1.0%, both p <0.0001). BDL led to increased cerebral LP (p = 0.0003) and cytosolic ROS generation (p <0.0001), which was restored by OP (both p <0.0001). Mitochondrial function was severely compromised in BDL, resulting in hyperpolarisation of Δψm with consequent overconsumption of adenosine triphosphate and augmentation of mitochondrial ROS production. Administration of OP restored Δψm. In BDL animals, neuronal loss was observed in hippocampal areas, which was partially prevented by OP.ConclusionsOur results elucidate that low-grade hyperammonaemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonaemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development.Lay summaryThe impact of hyperammonaemia, a common finding in patients with liver cirrhosis, on brain mitochondrial function was investigated in this study. The results show that ammonia in concentrations commonly seen in patients induces severe mitochondrial dysfunction, overproduction of damaging oxygen molecules, and profound injury and death of neurons in rat brain cells. These findings point towards a novel mechanism of ammonia-induced brain injury in liver failure and potential novel therapeutic targets.

Project description:Traumatic brain injury (TBI) can lead to significant neuropsychiatric problems and neurodegenerative pathologies, which develop and persist years after injury. Neuroinflammatory processes evolve over this same period. Therefore, we aimed to determine the contribution of microglia to neuropathology at acute [1 d postinjury (dpi)], subacute (7 dpi), and chronic (30 dpi) time points. Microglia were depleted with PLX5622, a CSF1R antagonist, before midline fluid percussion injury (FPI) in male mice and cortical neuropathology/inflammation was assessed using a neuropathology mRNA panel. Gene expression associated with inflammation and neuropathology were robustly increased acutely after injury (1 dpi) and the majority of this expression was microglia independent. At 7 and 30 dpi, however, microglial depletion reversed TBI-related expression of genes associated with inflammation, interferon signaling, and neuropathology. Myriad suppressed genes at subacute and chronic endpoints were attributed to neurons. To understand the relationship between microglia, neurons, and other glia, single-cell RNA sequencing was completed 7 dpi, a critical time point in the evolution from acute to chronic pathogenesis. Cortical microglia exhibited distinct TBI-associated clustering with increased type-1 interferon and neurodegenerative/damage-related genes. In cortical neurons, genes associated with dopamine signaling, long-term potentiation, calcium signaling, and synaptogenesis were suppressed. Microglial depletion reversed the majority of these neuronal alterations. Furthermore, there was reduced cortical dendritic complexity 7 dpi, reduced neuronal connectively 30 dpi, and cognitive impairment 30 dpi. All of these TBI-associated functional and behavioral impairments were prevented by microglial depletion. Collectively, these studies indicate that microglia promote persistent neuropathology and long-term functional impairments in neuronal homeostasis after TBI.SIGNIFICANCE STATEMENT Millions of traumatic brain injuries (TBIs) occur in the United States alone each year. Survivors face elevated rates of cognitive and psychiatric complications long after the inciting injury. Recent studies of human brain injury link chronic neuroinflammation to adverse neurologic outcomes, suggesting that evolving inflammatory processes may be an opportunity for intervention. Here, we eliminate microglia to compare the effects of diffuse TBI on neurons in the presence and absence of microglia and microglia-mediated inflammation. In the absence of microglia, neurons do not undergo TBI-induced changes in gene transcription or structure. Microglial elimination prevented TBI-induced cognitive changes 30 d postinjury (dpi). Therefore, microglia have a critical role in disrupting neuronal homeostasis after TBI, particularly at subacute and chronic timepoints.

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TLR4-activated microglia require IFN-? to induce severe neuronal dysfunction and death in situ.

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