Microglial signal transduction as a target of alcohol action in the brain

Although alcohol is believed to exert deleterious effects on the nervous system in general, its specific effect on the brain's immune system remains poorly understood. In particular, the effects of alcohol consumption on the immune and inflammatory responses in the central nervous system (CNS) have not been extensively investigated. Here, reviewed is the recent progress on how ethanol influences the signal transduction pathways of the inflammatory activation of brain microglia, which are thought to function as the resident immune defense system of the brain. Microglia are the CNS representatives of macrophages, which have the ability to clean up cellular debris. Microglia participate in neuroinflammation in response to various intrinsic or extrinsic stimuli. It has been recently suggested that microglial signal transduction is one of the main targets of ethanol action in the brain: ethanol exposure selectively modulates the intracellular signal transductions of microglia, rather than globally inhibiting signaling pathways in a nonspecific manner. Deregulation by ethanol of the inflammatory activation signaling of microglia may contribute to the derangement of CNS immune and inflammatory responses.     

The role of microglia in central nervous system immunity and glioma immunology

The central nervous system (CNS) historically has been considered an immune-privileged organ, lacking a lymphatic system and shielded from the circulatory system by the blood-brain barrier. Microglia are an abundant portion of the CNS cell population, comprising 5% to 20% of the total glial cell population, and are as numerous as neurons. A crucial function of microglia is the ability to generate significant innate and adaptive immune responses. Microglia are involved in first line innate immunity of the CNS. Proper antigen presentation is critical in the generation of specific, durable responses by the adaptive immune system, and requires interaction between the T cell receptor and processed antigen peptide presented on major histocompatibility complex (MHC) molecules by the antigen presenting cells (APC). Microglia also have a large regulatory role in CNS immunity. Histopathologic studies of glioma tissue have consistently shown high levels of infiltrating microglia. Microglia are also localized diffusely throughout the tumor, rather than to the areas of necrosis, and phagocytosis of glioma cells or debris by microglia is not observed. Recent evidence indicates that glioma-infiltrating microglia/macrophages might be promoting tumor growth by facilitating immunosuppression of the tumor microenvironment. When activated, microglia can be potent immune effector cells, able to perform a broad range of functions, and they mediate both innate and adaptive responses during CNS injury and disease while remaining quiescent in the steady state. Their versatility in bridging the gap between the immune-privileged CNS and the peripheral immune system, in addition to their significant numbers in gliomas, makes them an attractive candidate in immunotherapy for gliomas. An enhanced understanding of microglia–glioma interaction may provide better methods to manipulate the glioma microenvironment to allow the generation of a specific and durable anti-glioma immunity. The role of microglia in CNS immunity is reviewed, with a focus on key advances made in glioma immunology.     

Microglial repopulation resolves inflammation and promotes brain recovery after injury

Microglia mediate chronic neuroinflammation following central nervous system (CNS) disease or injury, and in doing so, damage the local brain environment by impairing recovery and contributing to disease processes. Microglia are critically dependent on signaling through the colony‐stimulating factor 1 receptor (CSF1R) and can be eliminated via administration of CSF1R inhibitors. Resolving chronic neuroinflammation represents a universal goal for CNS disorders, but long‐term microglial elimination may not be amenable to clinical use. Notably, withdrawal of CSF1R inhibitors stimulates new microglia to fully repopulate the CNS, affording an opportunity to renew this cellular compartment. To that end, we have explored the effects of acute microglial elimination, followed by microglial repopulation, in a mouse model of extensive neuronal loss. Neuronal loss leads to a prolonged neuroinflammatory response, characterized by the presence of swollen microglia expressing CD68 and CD45, as well as elevated levels of cytokines, chemokines, complement, and other inflammatory signals. These collective responses are largely resolved by microglial repopulation. Furthermore, microglial repopulation promotes functional recovery in mice, with elevated plus maze performance matching that of uninjured mice, despite the loss of 80% of hippocampal neurons. Analyses of synaptic surrogates revealed increases in PSD95 and synaptophysin puncta with microglial repopulation, suggesting that these cells sculpt and regulate the synaptic landscape. Thus, our results show that short‐term microglial elimination followed by repopulation may represent a clinically feasible and novel approach to resolve neuroinflammatory events and promote brain recovery.     

Disease progression after bone marrow transplantation in a model of multiple sclerosis is associated with chronic microglial and glial progenitor response

Multiple sclerosis (MS), the most common nontraumatic cause of neurologic disability in young adults in economically developed countries, is characterized by inflammation, gliosis, demyelination, and neuronal degeneration in the CNS. Bone marrow transplantation (BMT) can suppress inflammatory disease in a majority of patients with MS but retards clinical progression only in patients treated in the early stages of the disease. Here, we applied BMT in a mouse model of neuroinflammation, experimental autoimmune encephalomyelitis (EAE), and investigated the kinetics of reconstitution of the immune system in the periphery and in the CNS using bone marrow cells isolated from syngeneic donors constitutively expressing green fluorescent protein. This approach allowed us to dissect the contribution of donor cells to the turnover of resident microglia and to the pathogenesis of observed disease relapses after BMT. BMT effectively blocked or delayed EAE development when mice were treated early in the course of the disease but was without effect in mice with chronic disease. We found that there is minimal overall replacement of host microglia with donor cells in the CNS and that newly transplanted cells do not appear to contribute to disease progression. In contrast, EAE relapses are accompanied by the robust activation of endogenous microglial and macroglial cells, which further involves the maturation of endogenous Olig2 glial progenitor cells into reactive astrocytes through the cytoplasmic translocation of Olig2 and the expression of CD44 on the cellular membrane. The observed maturation of large numbers of reactive astrocytes from glial progenitors and the chronic activation of host microglial cells have relevance for our understanding of the resident glial response to inflammatory injury in the CNS. Our data indicate that reactivation of a local inflammatory process after BMT is sustained predominantly by endogenous microglia/macrophages.     

Role of glial cells in innate immunity and their role in CNS demyelination

The adaptive and innate arms of the immune system are the two pillars of host defense against environmental pathogens. Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS which is considered to be autoimmune and is thought to result from breakdown in the usual checks and balances of the adaptive immune response. The major pathological outcome of the disease is "the MS plaque" a unique feature of CNS demyelination characterized by the destruction of oligodendrocytes with loss of myelin and underlying axons. The MS plaque is not seen in other inflammatory disorders of the CNS. The prevailing opinion suggests that MS is mediated by the activation of an adaptive immune response which targets neural antigens. Currently, the role of an innate immune in the development of the lesions in MS has remained unclear. We explore the potential cellular elements of the innate immune system and in particular glial cells, which are likely candidates in inducing the specific pathological picture that is evident in MS. Activated microglia and the release of molecules which are detrimental to oligodendrocyte have been suggested as mechanisms by which innate immunity causes demyelination in MS. However a microglia/macrophage centric model does not explain the specificity of lesion development in MS. We propose that activation pathways of receptors of the innate immune system present on oligodendrocytes and astrocytes rather than microglia are central to the pathogenesis of demyelination seen in MS.     

Microglia and multiple sclerosis

Microglia participate in all phases of the multiple sclerosis (MS) disease process. As members of the innate immune system, these cells have evolved to respond to stranger/danger signals; such a response within the central nervous system (CNS) environment has the potential to induce an acute inflammatory response. Engagement of Toll-like receptors (TLRs), a major family of pattern-recognition receptors (PRRs), provides an important mechanism whereby microglia can interact with both exogenous and endogenous ligands within the CNS. Such interactions modulate the capacity of microglia to present antigens to cells of the adaptive immune system and thus contribute to the initiation and propagation of the more sophisticated antigen-directed responses. This inflammatory response introduces the potential for bidirectional feedback between CNS resident and infiltrating systemic cells. Such interactions acquire particular relevance in the era of therapeutics for MS because the infiltrating cells can be subjected to systemic immunomodulatory therapies known to change their functional properties. Phagocytosis by microglia/macrophages is a hallmark of the MS lesion; however, the extent of tissue damage and the type of cell death will dictate subsequent innate responses. Microglia/macrophages are armed with a battery of effector molecules, such as reactive nitrogen species, that may contribute to CNS tissue injury, specifically to the injury of oligodendrocytes that is associated with MS. A therapeutic challenge is to modulate the dynamic properties of microglia/macrophages so as to limit potentially damaging innate responses, to protect the CNS from injury, and to promote local recovery.     

Dual polarization of microglia isolated from mixed glial cell cultures

Microglia are versatile immune effector cells of the CNS and are sensitive to various stimuli. The different methods used to isolate microglia may affect some of their characteristics, such as their polarization state. The influence of cell sorting methods on the polarization state of microglia has never been studied. Mixed glial culture system (MGCS) and magnetic activated cell sorting (MACS) are two methods that are commonly used to purify microglia. This study compares the immunological states between microglia isolated by MGCS and microglia isolated by MACS. We show that microglia isolated by MGCS exhibit a stronger immune‐activated state than microglia isolated by MACS. They present an elevated phagocytic ability and high levels of markers associated with classical activation (M1) and alternative activation (M2). In addition, high levels of M1‐type and M2‐type chemokine (C‐C motif) ligand 2 and transforming growth factor‐β1 were detected in the culture medium of mixed glial cells. Our results show that microglia isolated by MGCS are in an immune‐activated state, whereas microglia isolated by MACS appear to be closer to their primary in vivo state. Therefore, the immune status of microglia, depending on the protocol used to purify them, should be carefully considered in neuropathology research. © 2015 Wiley Periodicals, Inc.     

Neuroinflammation in the central nervous system: Symphony of glial cells

Neuroinflammation in the central nervous system (CNS) is an important subject of neuroimmunological research. Emerging evidence suggests that neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases and CNS injury. Neuroinflammation is a complex and well-orchestrated process by various groups of glial cells in CNS and peripheral immune cells. The cross-talks between various groups of glial cells in CNS neuroinflammation is an extremely complex and dynamic process which resembles a well-orchestrated symphony. However, the understanding of how glial cells interact with each other to shape the distinctive immune responses of the CNS remains limited. In this review, we will discuss the joint actions of glial cells in three phases of neuroinflammation, including initiation, progression, and prognosis, the three movements of the symphony, as the role of each type of glial cells in neuroinflammation depends on the nature of inflammatory cues and specific course of diseases. This perspective of glial cells in neuroinflammation might provide helpful clues to the development of the early diagnosis and therapeutic intervention of the various CNS diseases.     

Feeding the beast: Can microglia in the senescent brain be regulated by diet?

Microglial cells, resident macrophages in the central nervous system (CNS), are relatively quiescent but can respond to signals from the peripheral immune system and induce neuroinflammation. In aging, microglia tend to transition to the M1 pro-inflammatory state and become hypersensitive to messages emerging from immune-to-brain signaling pathways. Thus, whereas in younger individuals where microglia respond to signals from the peripheral immune system and induce a well-controlled neuroinflammatory response that is adaptive (e.g., when well controlled, fever and sickness behavior facilitate recovery from infection), in older individuals with an infection, microglia overreact and produce excessive levels of inflammatory cytokines causing behavioral pathology including cognitive dysfunction. Importantly, recent studies indicate a number of naturally occurring bioactive compounds present in certain foods have anti-inflammatory properties and are capable of mitigating brain microglial cells. These include, e.g., flavonoid and non-flavonoid compounds in fruits and vegetables, and n-3 polyunsaturated fatty acids (PUFA) in oily fish. Thus, dietary bioactives have potential to restore the population of microglial cells in the senescent brain to a more quiescent state. The pragmatic concept to constrain microglia through dietary intervention is significant because neuroinflammation and cognitive deficits are co-morbid factors in many chronic inflammatory diseases. Controlling microglial cell reactivity has important consequences for preserving adult neurogenesis, neuronal structure and function, and cognition.     

Microglia inflammatory responses are controlled by an intrinsic circadian clock

The circadian system regulates many physiological functions including inflammatory responses. For example, mortality caused by lipopolysaccharide (LPS) injection varies depending on the time of immunostimulation in mammals. The effects of more subtle challenges on the immune system and cellular mechanisms underlying circadian differences in neuroinflammatory responses are not well understood. Here we show that adult male Sprague–Dawley rats injected with a sub-septic dose of LPS during the light phase displayed elevated sickness behaviors and hippocampal cytokine production compared to rats injected during the dark phase. Microglia are the primary central nervous system (CNS) immune cell type and may mediate diurnal differences in sickness response, thus we explored whether microglia demonstrate temporal variations in inflammatory factors. Hippocampal microglia isolated from adult rats rhythmically expressed inflammatory factors and circadian clock genes. Microglia displayed robust rhythms of TNFα, IL1β and IL6 mRNA, with peak cytokine gene expression occurring during the middle of the light phase. Microglia isolated during the light phase were also more reactive to immune stimulation; such that, ex vivo LPS treatment induced an exaggerated cytokine response in light phase-isolated microglia. Treating microglia with corticosterone ex vivo induced expression of the circadian clock gene Per1. However, microglia isolated from adrenalectomized rats maintained temporal differences in clock and inflammatory gene expression. This suggests circadian clock gene expression in microglia is entrained by, but oscillates in the absence of, glucocorticoids. Taken together, these findings demonstrate that microglia possess a circadian clock that influences inflammatory responses. These results indicate time-of-day is an important factor to consider when planning inflammatory interventions such as surgeries or immunotherapies.     

Euflammation attenuates peripheral inflammation-induced neuroinflammation and mitigates immune-to-brain signaling

Peripheral inflammation can trigger a number of neuroinflammatory events in the CNS, such as activation of microglia and increases of proinflammatory cytokines. We have previously identified an interesting phenomenon, termed “euflammation”, which can be induced by repeated subthreshold infectious challenges. Euflammation causes innate immune alterations without overt neuroimmune activation. In the current study, we examined the protective effect of euflammation against peripheral inflammation-induced neuroinflammation and the underlying mechanisms. When Escherichia coli or lipopolysaccharide (LPS) was injected inside or outside the euflammation induction locus (EIL), sickness behavior, global microglial activation, proinflammatory cytokine production in the brain, expression of endothelial cyclooxygenase II and induction of c-fos expression in the paraventricular nucleus of the hypothalamus were all attenuated in the euflammatory mice compared with those in the control unprimed mice. Euflammation also modulated innate immunity outside the EIL by upregulating receptors for pathogen-associated molecular patterns in spleen cells. In addition, euflammation attenuated CNS activation in response to an intra-airpouch (outside the EIL) injection of LPS without suppressing the cytokine expression in the airpouch. Collectively, our study demonstrates that signaling of peripheral inflammation to the CNS is modulated dynamically by peripheral inflammatory kinetics. Specifically, euflammation can offer effective protection against both bacterial infection and endotoxin induced neuroinflammation.     

Review: Microglia of the aged brain: primed to be activated and resistant to regulation

Innate immunity within the central nervous system (CNS) is primarily provided by resident microglia. Microglia are pivotal in immune surveillance and also facilitate the co‐ordinated responses between the immune system and the brain. For example, microglia interpret and propagate inflammatory signals that are initiated in the periphery. This transient microglial activation helps mount the appropriate physiological and behavioural response following peripheral infection. With normal ageing, however, microglia develop a more inflammatory phenotype. For instance, in several models of ageing there are increased pro‐inflammatory cytokines in the brain and increased expression of inflammatory receptors on microglia. This increased inflammatory status of microglia with ageing is referred to as primed, reactive or sensitized. A modest increase in the inflammatory profile of the CNS and altered microglial function in ageing has behavioural and cognitive consequences. Nonetheless, there are major differences in microglial biology between young and old age when the immune system is challenged and microglia are activated. In this context, microglial activation is amplified and prolonged in the aged brain compared with adults. The cause of this amplified microglial activation may be related to impairments in several key regulatory systems with age that make it more difficult to resolve microglial activation. The consequences of impaired regulation and microglial hyper‐activation following immune challenge are exaggerated neuroinflammation, sickness behaviour, depressive‐like behaviour and cognitive deficits. Therefore the purpose of this review is to discuss the current understanding of age‐associated microglial priming, consequences of priming and reactivity, and the impairments in regulatory systems that may underlie these age‐related deficits.     

Glial Cells Shape Pathology and Repair After Spinal Cord Injury

Glial cell types were classified less than 100 years ago by del Rio-Hortega. For instance, he correctly surmised that microglia in pathologic central nervous system (CNS) were “voracious monsters” that helped clean the tissue. Although these historical predictions were remarkably accurate, innovative technologies have revealed novel molecular, cellular, and dynamic physiologic aspects of CNS glia. In this review, we integrate recent findings regarding the roles of glia and glial interactions in healthy and injured spinal cord. The three major glial cell types are considered in healthy CNS and after spinal cord injury (SCI). Astrocytes, which in the healthy CNS regulate neurotransmitter and neurovascular dynamics, respond to SCI by becoming reactive and forming a glial scar that limits pathology and plasticity. Microglia, which in the healthy CNS scan for infection/damage, respond to SCI by promoting axon growth and remyelination—but also with hyperactivation and cytotoxic effects. Oligodendrocytes and their precursors, which in healthy tissue speed axon conduction and support axonal function, respond to SCI by differentiating and producing myelin, but are susceptible to death. Thus, post-SCI responses of each glial cell can simultaneously stimulate and stifle repair. Interestingly, potential therapies could also target interactions between these cells. Astrocyte–microglia cross-talk creates a feed-forward loop, so shifting the response of either cell could amplify repair. Astrocytes, microglia, and oligodendrocytes/precursors also influence post-SCI cell survival, differentiation, and remyelination, as well as axon sparing. Therefore, optimizing post-SCI responses of glial cells—and interactions between these CNS cells—could benefit neuroprotection, axon plasticity, and functional recovery.     

Microglia: Intrinsic immuneffector cell of the brain

Microglia form a regularly spaced network of resident glial cells throughout the central nervous system (CNS). They are morphologically, immunophenotypically and functionally related to cells of the monocyte/macrophage lineage. In the ultimate vicinity of the blood-brain barrier two specialized subsets of macrophages/microglia can be distinguished: firstly, perivascular cells which are enclosed within the basal lamina and secondly juxtavascular microglia which make direct contact with the parenchymal side of the CNS vascular basal lamina but represent true intraparenchymal resident microglia. Bone marrow chimera experiments indicate that a high percentage of the perivascular cells undergoes replacement with bone marrow-derived cells. In contrast, juxtavascular microglia like other resident microglia form a highly stable pool of CNS cells with extremely little turnover with the bone marrow compartment. Both the perivascular cells and the juxtavascular microglia play an important role in initiating and maintaining CNS autoimmune injury due to their strategic localization at a site close to the blood-brain barrier, their rapid inducibility for MHC class II antigens and their potential scavenger role as phagocytic cells. The constantly replaced pool of perivascular cells probably represents an entry route by which HIV gets access to the brain. Microglia are the first cell type to respond to several types of CNS injury. Microglial activation involves a stereotypic pattern of cellular responses, such as proliferation, increased or de-novo expression of immunomolecules, recruitment to the site of injury and functional changes, e.g., the release of cytotoxic and/or inflammatory mediators. In addition, microglia have a strong antigen presenting function and a pronounced cytotoxic function. Microglial activation is a graded response, i.e., microglia only transform into intrinsic brain phagocytes under conditions of neuronal and or synaptic/terminal degeneration. In T-cell-mediated autoimmune injury of the nervous system, microglial activation follows these lines and occurs at an early stage of disease development. In experimental autoimmune encephalomyelitis (EAE), microglia proliferate vigorously, show a strong expression of MHC class I and II antigens, cell adhesion molecules, release of reactive oxygen intermediates and inflammatory cytokines and transform into phagocytic cells. Due to their pronounced antigen presenting function in vitro, activated microglia rather than astrocytes or endothelial cells are the candidates as intrinsic antigen presenting cel of the brain. In contrast to microglia, astrocytes react with a delay, appear to encase morphologically the inflammatory lesion and may be instrumental in downregulating the T-cell-mediated immune injury by inducing T-cell apoptosis. In experimental autoimmune neuritis (EAN), microglial activation occurs also rapidly and operates at long distance, suggesting the involvement of remote and fast signaling mechanisms. During autoimmune inflammation of the nervous system microglia release as well as respond to several cytokines, including IL-1, IL-6, TNF-α, IFN-γ, TNF-α and TGFβ which are instrumental in astrocyte activation, induction of cell adhesion molecule expression, recruitment of T-cells into the lesion, but also in down-regulating disease progression at later stages. In addition to the synthesis of inflammatory cytokines, microglia act as cytotoxic effector cells by releasing other harmful substances such as proteases, reactive oxygen intermediates and nitric oxide. New therapeutic strategies to reduce the extent of tissue damage in autoimmune diseases of the nervous system will therefore aim at interfering with microglial cytotoxicity in the early, still potentially reversible stage of tissue damage. In summary, the CNS harbours a network of potential immunoeffector cells, i.e., the microglia, which show a graded response to CNS injury and function as a sensor to threats in the nervous system.     

Signaling through JAK2-STAT5 pathway is essential for IL-3-induced activation of microglia

Microglia, the resident macrophage of the brain, mediates immune and inflammatory responses in the central nervous system (CNS). Activation of microglia and secretion of inflammatory cytokines associate with the pathogenesis of CNS diseases, including multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease, prion disease, and AIDS dementia. Microbial pathogens, cytokines, chemokines, and costimulatory molecules are potent inducers of microglial activation in the CNS. Signaling through its receptor, IL-3 induces the activation of JAK-STAT and MAP kinase pathways in microglial cells. In this study, we found that in vitro treatment of EOC-20 microglial cells with tyrphostin AG490 blocked IL-3-induced tyrosine phosphorylation of JAK2, STAT5A, and STAT5B signaling proteins. Stable transfection of EOC-20 cells with a dominant negative JAK2 mutant also blocked IL-3-induced tyrosine phosphorylation of JAK2, STAT5A, and STAT5B in microglia. The blockade of JAK2-STAT5 pathway resulted in a decrease in IL-3-induced proliferation and expression of CD40 and major histocompatibility complex class II molecules in microglia. These findings highlight the fact that JAK2-STAT5 signaling pathway plays a critical role in mediating IL-3-induced activation of microglia.     

Expression of proteinase-activated receptors in mouse microglial cells

Microglia are the resident immune cells of the CNS: they are activated rapidly by CNS damage and perform the function of tissue macrophages. The first steps during microglial activation are currently under intense study, and it is widely believed that substances released from damaged brain tissue can trigger this process. We recently reported that the blood coagulation factor thrombin, which enters the CNS during breakdown of the blood-brain barrier, activates microglial cells. The cellular effects of thrombin and trypsin-like proteases are mediated by proteinase-activated receptors (PARs). Based on our prior data we hypothesized that microglial cells express these receptors. Using RT-PCR and flow cytometry, we report that primary mouse microglial cells, as well as the murine microglial cell lines BV-2 and N9, indeed express PARs, albeit at different levels. Demonstrating multiple PARs on microglia may enhance the attractiveness of PARs as therapeutic targets in neuroinflammatory disorders.     

Murine glia express the immunosuppressive cytokine, interleukin-10, following exposure to Borrelia burgdorferi or Neisseria meningitidis

There is growing appreciation that resident glial cells can initiate and/or regulate inflammation following trauma or infection in the central nervous system (CNS). We have previously demonstrated the ability of microglia and astrocytes, resident glial cells of the CNS, to respond to bacterial pathogens by rapid production of inflammatory mediators. However, inflammation within the brain parenchyma is notably absent during some chronic bacterial infections in humans and nonhuman primates. In the present study, we demonstrate the ability of the immunosuppressive cytokine, interleukin-10 (IL-10), to inhibit inflammatory immune responses of primary microglia and astrocytes to B. burgdorferi and N. meningitidis, two disparate gram negative bacterial species that can cross the blood-brain barrier in humans. Importantly, we demonstrate that these organisms induce the delayed production of significant quantities of IL-10 by both microglia and astrocytes. Furthermore, we demonstrate that such production occurs independent of the actions of bacterial lipopolysaccharide and is secondary to the autocrine or paracrine actions of other glia-derived soluble mediators. The late onset of IL-10 production by resident glia following activation, the previously documented expression of specific receptors for this cytokine on microglia and astrocytes, and the ability of IL-10 to inhibit bacterially induced immune responses by these cells, suggest a mechanism by which resident glial cells can limit potentially damaging inflammation within the CNS in response to invading pathogens, and could explain the suppression of inflammation seen within the brain parenchyma during chronic bacterial infections.