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Astrocytes are active players in cerebral innate immunity
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TRENDS in Immunology
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Astrocytes are active players in cerebral innate immunity Cinthia Farina1, Francesca Aloisi2 and Edgar Meinl3,4 1
Neuroimmunology and Neuromuscular Disorders Unit, National Neurological Institute Carlo Besta, 20133 Milan, Italy Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita`, 00161 Rome, Italy 3 Institute of Clinical Neuroimmunology, Ludwig-Maximilians University, 81377 Munich, Germany 4 Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, 82152 Martinsried-Planegg, Germany 2
Innate immunity is a constitutive component of the central nervous system (CNS) and relies strongly on resident myeloid cells, the microglia. However, evidence is emerging that the most abundant glial cell population of the CNS, the astrocyte, participates in the local innate immune response triggered by a variety of insults. Astrocytes display an array of receptors involved in innate immunity, including Toll-like receptors, nucleotide-binding oligomerization domains, double-stranded RNAdependent protein kinase, scavenger receptors, mannose receptor and components of the complement system. Following activation, astrocytes are endowed with the ability to secrete soluble mediators, such as CXCL10, CCL2, interleukin-6 and BAFF, which have an impact on both innate and adaptive immune responses. The role of astrocytes in inflammation and tissue repair is elaborated by recent in vivo studies employing cell-type specific gene targeting. Introduction The central nervous system (CNS) has been commonly regarded as an immune-privileged site [1]. However, important studies published during the past ten years indicate that the CNS can offer the setting for innate immune responses [2]. This might reflect the ability of the CNS to fight infections despite its immune-privileged state. The recognition of infectious non-self is mediated by a limited number of germline-encoded pattern-recognition receptors (PRRs) and triggers rapid responses. Some PRRs can also recognize endogenous ‘danger signals’ that alert the immune system to cell damage, independently from the context of infection. Consistently, the activation of innate immune pathways occurs not only in infectious CNS diseases but also after brain injury and ischemia, and in autoimmune and neurodegenerative disorders of the CNS (Table 1), indicating that the relevance of these pathways extends beyond antimicrobial defense. To date, the neurotoxic and neuroprotective roles of innate immune reactions in non-infectious CNS diseases are an intensively investigated and debated issue. The expression of PRRs is found in various immune and non-immune cell types of the CNS, similarly to peripheral tissues. Microglia and astrocytes are the main CNS-resident cell types for which consistent data have been published Corresponding author: Farina, C. ([email protected]). Available online 2 February 2007. www.sciencedirect.com
(Table 2). Microglia are myeloid lineage cells and are considered ‘the CNS professional macrophages’, owing to their phenotype and reactivity following injury and inflammation [3]. For this reason, most of the studies on innate immune responses in the CNS have focused on microglia, which express a wide range of PRRs (Table 2). The contribution of other cell types to these processes has often been neglected. However, recent evidence suggests that astrocytes have a complex, dual role in the local regulation of immune reactivity. Astrocytes, the most abundant glial cell population, are of neuroectodermal origin and are essential for brain homeostasis and neuronal function [4] (Box 1). They form the glia limitans around blood vessels restricting the access of immune cells to the CNS parenchyma [5] (see Figure I in Box 2). In contrast to other brain cells, astrocytes are resistant to death receptor-induced apoptosis [6], indicating that they are well equipped to survive inflammatory insults. In this article, we aim to answer the following questions: (i) do astrocytes participate in innate immune reactions? (ii) Which are the activating signals and functional responses involved? The Toll-like receptor system in astrocytes: expression, signaling and biological relevance Toll-like receptors (TLRs) are evolutionarily conserved type I membrane glycoproteins characterized by leucinerich-repeat motifs in the extracellular domain and by a cytoplasmic signaling domain similar to that of the interleukin (IL)-1 receptor [7]. TLR1, TLR2 and TLR6 recognize bacterial lipoproteins; TLR3, TLR7, TLR8 and TLR9 are specific for nucleic acids; TLR5 binds to flagellin, the main constituent of bacterial flagella; TLR4 has a wide spectrum of ligands, including bacterial lipopolysaccharide (LPS) and fungal zymosan. TLR expression has been detected in cells of the innate and adaptive immune system and in non-immune cells. But are TLRs important for pathogen recognition in the CNS, and where are they expressed? Increasing evidence indicates that TLRs have a major role in several inflammatory CNS pathologies (Table 1). For example, genetic polymorphisms in TLR4 have been associated with increased susceptibility of humans to meningococcal infection [8]. In animal models of infectious CNS diseases, TLR2 deficiency can lead to opposite outcomes, such as increased susceptibility to Streptococcus pneumoniae
1471-4906/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2007.01.005
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Table 1. Innate immune pathways involved in CNS pathologies PRR system TLR
Scavenger receptors
Complement
CNS disease Neisseria meningitides meningitis Streptococcus pneumoniae meningitis Listeria monocytogenes meningitis Herpes simplex virus encephalitis West Nile virus encephalitis Multiple sclerosis or experimental autoimmune encephalitis Nerve injury Ischemia Alzheimer’s disease N. meningitides meningitis Nerve injury Ischemia Alzheimer’s disease Parkinson’s disease Huntington’s disease Pick’s disease Multiple sclerosis or experimental autoimmune encephalitis Rasmussen’s encephalitis Prion disease N. meningitides meningitis HIV encephalitis Epstein-Barr virus encephalitis Measles virus encephalitis West Nile virus encephalitis Nerve injury Ischemia
Refs [8] [9] [9] [11] [77] [22,78] [79] [80] [48,81] [50] [82] [83] [40,84] [85] [40] [40] [86,87] [88] [85] [8,44,89] [89] [89] [89] [46,47] [90] [91]
meningitis [9,10] and protection from herpes simplex virus encephalitis [11]. However, in these models, owing to PRR expression on peripheral immune cells and on brain-resident cells, it is difficult to distinguish the contributions to infection control of each cell type. Under physiological conditions, basal expression of TLR2 and TLR4 was demonstrated in the meninges, choroid plexus and circumventricular organs of the brain, namely in CNS areas that lack a blood–brain barrier (BBB) and are more exposed to invading pathogens [12– 15]. More recently, low basal levels of TLR4 expression were shown in microglia in vivo [15]. Accordingly, the systemic administration of LPS results in rapid upregulation of TLR2 in microglia and generates an innate inflammatory response that is readily detected and more prominent in BBB-free areas of the CNS but also extends into the brain parenchyma [12–14].
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Box 1. Features and functions of astrocytes Features Most abundant glial cell in the CNS Neuroectodermal origin Largely resistant to death receptor (Fas, TRAIL)-mediated apoptosis Functions Metabolic support of neurons; glycogen storage and export of lactate Uptake of neurotransmitters, including glutamate Production of neurotrophic factors Ion homeostasis (i.e. potassium uptake) Blood–brain barrier induction and maintenance Scar formation and tissue repair Regulation of immune responses in the CNS
The brain displays basal expression of TLR3, which is detected on glial fibrillary acidic protein (GFAP)-positive astrocytes in the hippocampus and striatum [16]. Several recent in vitro studies have shown that TLR3 is the predominant TLR expressed in astrocytes. In two studies, the complete human TLR repertoire in human fetal astrocytes was analyzed by quantitative polymerase chain reaction and indicated TLR3 as the only TLR with consistent expression in the resting state [17,18] and with upregulated levels following treatment with inflammatory cytokines such as IL-1b, interferon (IFN)b and IFNg [17]. The analysis of cultured human adult astrocytes showed basal levels of TLR2, TLR3 and TLR4; however, exposure to inflammatory cytokines strongly induced TLR3, while having no effect on TLR4 and downregulating TLR2 [19]. Similarly, cultured mouse and rat astrocytes displayed high constitutive levels of TLR3 [20,21]. TLR3 is commonly found in intracellular compartments such as endosomes, and signaling requires ligand internalization. Interestingly, in human astrocytes, TLR3 was detected not only intracellularly [18] but also on the cell membrane [18,22], and its protein expression was induced following ligand binding [18]. TLR2, TLR4, TLR5 and TLR9 have been described in human astrocyte-enriched cultures [19,20, 22,23] (Table 2); however, expression of these TLRs on astrocytes in vivo has
Table 2. PRRs in astrocytes and microgliaa PRR TLR
Astrocytes TLR2 [22,23], TLR3 [16], TLR4 [20,23], TLR5 [20,23], TLR9 [20,23]
CD14 NOD PKR Scavenger receptors
Not expressed NOD1, NOD2 [36] Expressed [35] SR-BI [94], SR-MARCO [51], RAGE [95], SRCL [53]
Mannose receptor Complement factors
Expressed [38] C1q, C1r, C1s, C4, C2, C3, factor B, factor D, C5, C6, C7, C8, C9 [40] CR1, CR2, C3aR, C5aR [40]
Complement receptors Complement inhibitors a
C1-INH, DAF/CD55, MCP/CD46, CD59, factor H, factor I, S protein, clusterin [40]
Microglia TLR1 [24], TLR2 [13], TLR3 [24], TLR4 [15], TLR5 [24], TLR6 [24], TLR7 [24], TLR8 [24], TLR9 [24] Expressed [92] Unknown Expressed [93] SR-A [48], SR-BI [48], SR-MARCO [51], RAGE [51], CD36 [48], SRCL [53] Expressed [3] C1q, C3, C4 [40] CR1, CR3/CD11b, CR4/CD11c, C3aR, C5aR, C1qRp [40] C1-INH, CD59, clusterin [40]
Blue text indicates in vitro evidence (not yet supported by in vivo studies); red text indicates in vivo evidence (mostly supported by in vitro studies not cited in the table).
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Box 2. Mediators of astrocyte function 1. Cytokines: IL-6, IFNb, TGFb, GM-CSF, BAFF, IL-1b and TNF
Increased BBB permeability Endothelial cell activation Microglial and monocytic activation, differentiation and proliferation Astrocyte activation B cell survival and differentiation Immunosuppression Release of neuroprotective mediators
2. Chemokines: CCL2, CCL5, CCL20, CXCL10, CXCL12, CXCL1, CXCL2 and CX3CL1 Recruitment of monocytes and macrophages, dendritic cells, T and B lymphocytes, and neutrophils Regulation of myelination and microglial activity Astrocyte proliferation and survival Migration of microglia and neural progenitors 3. Neurotrophic factors: NGF, CNTF, BDNF, VEGF, IGF1 and LIF
Neuronal survival, differentiation, function and regeneration Oligodendrocyte survival Remyelination Neurogenesis
Mediators released by astrocytes (bold text) following pathogen recognition trigger two types of events (Figure I): (i) they activate
neighboring cells and amplify the local, initial innate immune response further; and (ii) they modify BBB permeability and attract immune cells from the blood circulation into the neural tissue, thus supporting an adaptive immune response. The balance between inflammatory (tissue damaging; red text) and immunosuppressive (tissue regenerating; green text) pathways is fundamental for controlled reactions to CNS trauma. Dysregulation of these pathways might lead to pathogenic chronic neuroinflammation and neurodegeneration. Astrocytes themselves can be the direct target of such factors. Astrocytic responses to chemokines involve chemotaxis, cell proliferation and survival. Additionally, chemokines, such as CXCL12 and CCL5, induce glutamate release and cytokine and chemokine synthesis, indicating a role for this class of molecules in regulating glia–glia and glia–neuron communication [72,96]. Among cytokines, TNF, IFNg and IL-1 are the main astrocytic activators. Gene profiling studies in astrocytes exposed to inflammatory cytokines in vitro showed profound regulations in genes involved in several cellular pathways, such as innate and adaptive immunity, antigen presentation, apoptosis, leukocyte migration and BBB permeability [97–99]. Astrocyte-derived factors, such as GM-CSF, IL-6, CCL2 and CCL5 regulate microglial migration, activation and proliferation. Some glia-derived soluble mediators, including CCL2, CCL5, CXCL10, CXCL12 and BAFF, are also responsible for triggering adaptive immunity in the inflamed CNS.
Figure I. Astrocyte activation generates waves of innate and adaptive immunity. The astrocyte has a strategic location, being in close contact with CNS-resident cells (neurons, microglia, oligodendrocytes and other astrocytes) and with blood vessels. The astrocyte-derived glia limitans (dark green) forms a barrier between the brain parenchyma and the vascular system. The perivascular space, a site of immune cell accumulation in CNS inflammation, is delimited by the endothelial basement membrane (dark pink) and the glia limitans [5]. Following PRR engagement, astrocytes secrete factors that target bystander brain cells and promote changes in the permeability of the BBB, resulting in the recruitment of professional immune cells into the CNS parenchyma. This leads to an amplification of the initial innate immune reaction, which can result in the elimination of the insult, with restoration of tissue integrity or scar formation. Relative sizes of the distinct cell types reflect in situ observations. T and B lymphocytes, macrophages and microglia have the same color to indicate their common hematopoietic origin.
not yet been demonstrated. By contrast, similarly to monocytes and macrophages, microglia (the professional immune cells of the brain) display a broader repertoire of TLRs in vitro [18,24] (Table 2). Human astrocytes express mRNAs for the adaptor molecules TIRAP (also known as Mal), TICAM (also known as TRIF), MyD88 and the shorter splice variant MyD88S [17]. The MyD88-dependent pathway involves www.sciencedirect.com
the sequential activation of IL-1 receptor-associated kinase (IRAK) proteins, tumor necrosis factor (TNF) receptor-associated factor (TRAF)6 and mitogen-activated protein (MAP) kinases down to activator protein (AP)-1 and nuclear factor (NF)-kB transcription factors. The TRIF-dependent pathway activates NF-kB on one side through receptor-interacting protein (Rip)1 and interferon regulatory factor (IRF)3 and IRF7 on the other side through
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Tank-binding kinase (TBK)1 and phosphatidylinositol 3-kinase (PI3K), resulting in the expression of IFN-inducible genes [7]. Double-stranded (ds)RNA-triggered biological effects are mediated largely by TLR3. Injecting poly(I:C), a synthetic analog of dsRNA, into the brain triggered microglial and astrocytic activation in wild-type mice but not in TLR3 knockout (KO) mice [25], indicating the important role of TLR3 in the CNS response to viral infection. In cultured astrocytes, poly(I:C) induces the expression of several cytokines [TNF, IL-6, IFNb, granulocyte–macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)b] and chemokines (CCL2, CCL5, CCL20, CXCL8 and CXCL10) [16–19]. The astrocytic response to dsRNA involves inducible nitric oxide synthase (iNOS) upregulation with nitric oxide production [20,21,26], a decrease in glutamate uptake [21], and downregulation of connexin 43 with a disturbance of gap junction-mediated intercellular communication [27]. This suggests that astrocytes contribute to tissue damage and neurotoxicity. Similarly, in vitro infection of mouse astrocytes with Theiler’s murine encephalomyelitis virus (TMEV) triggered an initial inflammatory response (CCL2 and CXCL10 secretion) through TLR3 [28]. However, viral replication was equal in wild-type and TLR3 KO astrocytes, indicating that TLR3-mediated activation is insufficient to block TMEV replication [28]. Gene profiling of poly(I:C)-treated adult human astrocytes indicated the induction of an antiviral program [29] and of growth, differentiation and neuroprotective mediators, including GM-CSF, vascular endothelial growth factor (VEGF)-C, neurotrophin (NT)-4 and ciliary neurotrophic factor (CNTF), suggesting that TLR stimulation in astrocytes also activates tissue repair pathways [19]. In summary, astrocytes express TLR3 in vivo and in vitro, and react to TLR3 ligands by producing mediators that promote, on the one hand, the local inflammatory response and, on the other hand, tissue repair. Because recent studies indicate that mRNA from necrotic cells functions as an endogenous ligand for TLR3 [30], the possibility that neural tissue damage activates astrocytes is intriguing and deserves further investigation. Astrocytes express PKR, NOD proteins, scavenger receptors, mannose receptor and components of the complement system PRRs other than TLRs are involved in innate immune responses. TLR3 is not the only weapon in antiviral immunity, because TLR3 KO mice were able to mount a normal peripheral immune response to lymphocytic choriomeningitis virus, vesicular stomatitis virus (VSV) and murine cytomegalovirus [31]. Following intracerebral inoculation of reovirus, a dsRNA virus, wild-type and TLR3 KO mice showed similar patterns of survival, viral titer and neuropathology [31]. Sendai virus was able to activate TLR3negative cells and to induce interferon-stimulated and interferon-related genes [32]. Indeed, many other PRRs that mediate RNA recognition exist [33] and each might recognize distinct dsRNA structures [34]. One of these PRRs is dsRNA-dependent protein kinase (PKR). Its actiwww.sciencedirect.com
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vation leads to the phosphorylation of the a subunit of the eukaryotic initiation factor 2 resulting in the blockade of viral translation and subsequent shutdown of protein synthesis in virus-infected cells. Although no data are yet available on the distribution of PKR in the CNS, recent studies showed that PKR is involved in the dsRNA-induced response in cultured astrocytes [16,21,35]. Nucleotide-binding oligomerization domain (NOD)1 and NOD2 proteins, intracellular proteins that recognize distinct motifs of bacterial peptidoglycan, have been described in cultured mouse astrocytes, and NOD2 was found to mediate IL-6 secretion in response to its specific ligand [36]. Expression of the mannose receptor (MR) has also been demonstrated in the CNS [37,38]. MR enables recognition of mannosylated ligands of endogenous or microbial origin, resulting in receptor-mediated endocytosis and enhanced microbicidal activity. Both cultured astrocytes and microglia express MR protein on the cell surface and in intracellular pools [37]. MR was identified as the receptor responsible for CD4-independent HIV-1 entry into astrocytes [38]. This interaction triggered intracellular signaling leading to matrix metalloproteinase 2 production [39]. Complement is another important component of innate immunity. It comprises soluble and surface proteins expressed in almost every cell type. Recent studies have highlighted the role of complement in the normal and pathological CNS. Notably, brain cells express a full complement system to kill pathogens but are somewhat protected from complement-induced lysis because of the expression of complement inhibitors [40,41]. Reactive astrocytes can produce most of the complement factors after activation and express in vivo complement receptors and complement-regulatory proteins [40,42] (Table 2). Interestingly, pathogens can use some of these factors to promote infection. For example, Epstein–Barr virus binds to CR2, whereas CD46 serves as a receptor for human herpesvirus 6, measles virus and Neisseria meningitidis [43,44]. Although these pathogens were shown to infect astrocytes in vitro, formal in vivo evidence is still required. Studies of human pathological samples and animal models of CNS infection have, in some instances, demonstrated the contribution of complement to anti-viral immunity [45–47] (Table 1). Furthermore, genetic polymorphisms in genes encoding complement components have been associated with N. meningitidis infection [8] (Table 1). Scavenger receptors (SRs), initially described as high-affinity receptors on macrophages for acetylated low-density lipoproteins, comprise several receptor classes, each including numerous members [48,49]. SRs have a role in the binding and internalization of many unrelated ligands, such as fibrillar b-amyloid, lipids, glycated collagen and apoptotic cells, and, therefore, are important for tissue homeostasis. Astrocytes were shown to express various SRs in situ using immunohistochemistry (Table 2). SR-MARCO is important for host defense, because it can bind to Gram-positive and Gram-negative bacteria. Interestingly, SR-MARCO is involved in the recognition and uptake of N. meningitidis [50], an important cause of
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bacterial meningitis; therefore, its expression in astrocytes [51] might be an additional mechanism through which CNS-resident cells can fight bacterial infections. The other SRs present on astrocytes recognize endogenous signals. For example, SR-BI exerts its scavenger activity through binding to low-density lipoproteins, apoptotic cells and anionic phospholipids. SR-BI, RAGE and SRCL bind to and mediate the internalization of b-amyloid peptides and fibrils [48,52,53]. These observations, together with the finding that astrocytes associate with and degrade b-amyloid deposits in situ [54], highlight an important role for astrocytes in counteracting neurodegeneration. A deficit in this function could have direct implications in Alzheimer’s disease. Astrocytes are important regulators of neuroinflammation In the previous sections, we have seen that astrocytes carry a series of PRRs, which are important for the primary recognition of infectious agents and of endogenous danger signals. Factors produced by activated astrocytes (and microglia) target neighboring cells and promote leukocyte recruitment, resulting in the local amplification of inflammatory responses (Box 2). For example, following stimulation with inflammatory cytokines, astrocytes in vitro produce high amounts of the B cell survival factor BAFF (B cell-activating factor of the TNF family) [55]. Notably, BAFF is expressed in astrocytes in the normal human CNS and is strongly upregulated in activated astrocytes in the demyelinated lesions of multiple sclerosis (MS), a chronic inflammatory disease of the CNS [55]. BAFF secretion could be relevant in sustaining intrathecal B cell responses in autoimmune and infectious diseases of the CNS. Accordingly, BAFF expression is readily induced in the CNS by neurotropic viruses and correlates with the recruitment of antibody-secreting cells [56]. The central role of astrocytes in regulating neuroinflammation was demonstrated recently in vivo [57,58]. Transgenic mice were generated in which NFkB, a fundamental transcription factor family in innate responses, was selectively inactivated in astrocytes [57]. These mice displayed normal spinal cord architecture and cellular turnover; however, functional recovery after injury was increased dramatically, and lesion volume and white matter injury was reduced. These observations correlated with the drop in leukocyte recruitment into the lesioned area owing to the reduced NF-kB-dependent expression of CXCL10 and CCL2 [57]. Similarly, blockading the NF-kB pathway in neuroectodermal cells of the CNS (including neurons, astrocytes and oligodendrocytes) led to a consistent decrease in proinflammatory gene expression during experimental autoimmune encephalomyelitis and clinical– pathological amelioration [58]. In summary, the NF-kB pathway in astrocytes is a key regulator of inflammation in the CNS, and its inhibition has beneficial effects on tissue regeneration. Astrocytes contribute to neuroprotective innate inflammation Innate immune responses are not necessarily detrimental for the cerebral tissue. The innate immune system also www.sciencedirect.com
performs the task of restricting the lesioned area, removing the insult and restoring tissue homeostasis. A major, therapeutically relevant question is: what role do astrocytes have in the innate neuroprotective immune response? A hallmark of CNS injury, whatever its origin (i.e. infectious, autoimmune, mechanical or toxic) is the formation of scar tissue composed of activated (‘reactive’) astrocytes and microglia. An inflammatory response occurs within this clearly demarcated area. This phenomenon is commonly regarded as harmful and responsible for the restriction of axonal regrowth within the lesion. However, several studies indicate that activated astrocytes (in addition to activated microglia) and their secreted products might exert neuroprotective actions. Subsequent to trauma, astrocytes proliferate, accumulate glycogen and undergo fibrosis by accumulating intermediate filaments, expressed as an increase in GFAP. Transgenic mice expressing herpes simplex virus-thymidine kinase (HSV-TK) under the GFAP promoter were generated. HSV-TK-expressing cells metabolize the antiviral drug ganciclovir (GCV) to toxic nucleotide analogs, leading preferentially to the death of proliferating cells. In uninjured GFAP-TK mice, astrocyte proliferation is rare; therefore, GCV administration does not kill resting astrocytes. After CNS insults, local scar formation occurs and astrocytes proliferate, resulting in the loss of these cells in GCV-treated GFAP-TK mice [59,60]. This experimental model unraveled a fundamental neuroprotective role for reactive astrocytes in: (i) demarcating the damaged area and limiting leukocyte extravasation, because the ablation of reactive astrocytes resulted in improper scar formation and more extensive and long-lasting inflammation; (ii) promoting BBB repair, because in GCV-treated transgenic animals, immunoglobulin entry into the brain parenchyma persisted after injury; and (iii) sustaining neuronal survival, because enhanced neuronal degeneration occurred after astrocyte loss. Several cytokines, including IL-1 and IL-6, have been implicated in the induction and modulation of reactive astrogliosis and pathological inflammatory responses. However, the same factors in different settings might also behave as mediators of neuroprotection and remyelination. For example, in various injury models, IL-1b and IL-6 KO mice displayed delayed astrocyte activation and increased BBB permeability, indicating that cytokine-induced astrogliosis following trauma is important to restore the integrity of the BBB and to repair the lesion [61–65]. Interestingly, IL-1b KO mice failed to upregulate the neurotrophic factors CNTF [61] and insulin-like growth factor (IGF)1 [63] following CNS trauma, indicating that the initial IL-1b-dependent inflammatory response mediates the release of these neurotrophic factors in the injured CNS. In wild-type mice, both microglia [61,63] and astrocytes [63] express IL-1b, whereas mainly astrocytes were found to be positive for IGF1 in a demyelination model [63]. Several in vitro data demonstrate that cytokines such as IL-1, IL-6 and TNF support the production of neuroprotective mediators [66]. In this regard, the observation that TLR engagement on cultured astrocytes triggers the production of neurotrophic factors supports a role for
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these cells in the neuroprotective innate immune response [19]. A Janus-like action is displayed also by astrocyte-derived chemokines. CCL2 and CXCL12 not only promote the recruitment of inflammatory immune cells into the CNS parenchyma but also have a role in the migration of neural progenitors in the developing brain [67] and towards areas of brain injury [68–70]. CCL2 is readily upregulated in various pathological conditions in microglia and astrocytes [71,72], and increased CXCL12 immunoreactivity was detected in astrocytes in MS lesions [73,74]. CXCL1 was seen on reactive astrocytes in MS lesions in the vicinity of CXCR2positive oligodendrocytes, suggesting a role for this chemokine in remyelination [75]. Another chemokine constitutively present in the CNS is CX3CL1, which is produced by astrocytes and neurons. CX3CL1 is an important inhibitor of microglial toxicity, as demonstrated in three different in vivo models [76]. Therefore, the production of particular chemokines might reflect the attempt of reactive astrocytes to promote regeneration in the lesioned CNS. Concluding remarks Research in the field of innate immunity in the CNS has developed during the past decade and has concentrated mainly on the reactivity of the professional resident immune cells, the microglia. However, based on current evidence, we can definitely consider the astrocyte as the innate-immunecompetent cell, because it bears several PRRs involved in the primary recognition of microbial agents or of endogenous danger signals. This cell type reacts with a unique activation program that modulates or amplifies the local inflammatory reaction. In vivo studies have revealed Janus-like features of astrocytes. On the one hand, they can promote inflammation through NF-kB-dependent pathways; on the other hand, proliferating reactive astrocytes confine lesions and restore brain homeostasis. Increasing knowledge on the involvement of astrocytes in shaping the innate immune response can provide new insights into the intrinsic capacity of the CNS tissue to face pathogenic insults and can aid in the identification of molecular targets for therapeutic interventions in a variety of neuroinflammatory and neurodegenerative diseases. Acknowledgements Our original work was supported by the Deutsche Forschungsgemeinschaft (SFB 571), the Gemeinnu¨tzige Hertie-Foundation, the Deutsche Multiple Sklerose Gesellschaft, the Verein zur Therapieforschung fu¨r MS-Kranke, and the Italian Ministry of Health. The Institute for Clinical Neuroimmunology is supported by the Hermann and Lilly Schilling Foundation.
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32 Elco, C.P. et al. (2005) Analysis of genes induced by Sendai virus infection of mutant cell lines reveals essential roles of interferon regulatory factor 3, NF-kB, and interferon but not Toll-like receptor 3. J. Virol. 79, 3920–3929 33 Saunders, L.R. and Barber, G.N. (2003) The dsRNA binding protein family: critical roles, diverse cellular functions. FASEB J. 17, 961– 983 34 Okahira, S. et al. (2005) Interferon-b induction through Toll-like receptor 3 depends on double-stranded RNA structure. DNA Cell Biol. 24, 614–623 35 Ong, C.L. et al. (2005) Low TRBP levels support an innate human immunodeficiency virus type 1 resistance in astrocytes by enhancing the PKR antiviral response. J. Virol. 79, 12763–12772 36 Sterka, D., Jr et al. (2006) Functional expression of NOD2, a novel pattern recognition receptor for bacterial motifs, in primary murine astrocytes. Glia 53, 322–330 37 Burudi, E.M. et al. (1999) Identification and functional characterization of the mannose receptor in astrocytes. Glia 25, 44–55 38 Liu, Y. et al. (2004) CD4-independent infection of astrocytes by human immunodeficiency virus type 1: requirement for the human mannose receptor. J. Virol. 78, 4120–4133 39 Lopez-Herrera, A. et al. (2005) HIV-1 interaction with human mannose receptor (hMR) induces production of matrix metalloproteinase 2 (MMP2) through hMR-mediated intracellular signaling in astrocytes. Biochim. Biophys. Acta 1741, 55–64 40 Gasque, P. et al. (2000) Complement components of the innate immune system in health and disease in the CNS. Immunopharmacology 49, 171–186 41 Hauwel, M. et al. (2005) Innate (inherent) control of brain infection, brain inflammation and brain repair: the role of microglia, astrocytes, ‘protective’ glial stem cells and stromal ependymal cells. Brain Res. Brain Res. Rev. 48, 220–233 42 Ohlsson, M. et al. (2003) Complement activation following optic nerve crush in the adult rat. J. Neurotrauma 20, 895–904 43 Cattaneo, R. (2004) Four viruses, two bacteria, and one receptor: membrane cofactor protein (CD46) as pathogens’ magnet. J. Virol. 78, 4385–4388 44 Gill, D.B. and Atkinson, J.P. (2004) CD46 in Neisseria pathogenesis. Trends Mol. Med. 10, 459–465 45 Ochsenbein, A.F. et al. (1999) Protective T cell-independent antiviral antibody responses are dependent on complement. J. Exp. Med. 190, 1165–1174 46 Mehlhop, E. et al. (2005) Complement activation is required for induction of a protective antibody response against West Nile virus infection. J. Virol. 79, 7466–7477 47 Mehlhop, E. and Diamond, M.S. (2006) Protective immune responses against West Nile virus are primed by distinct complement activation pathways. J. Exp. Med. 203, 1371–1381 48 Husemann, J. et al. (2002) Scavenger receptors in neurobiology and neuropathology: their role on microglia and other cells of the nervous system. Glia 40, 195–205 49 Murphy, J.E. et al. (2005) Biochemistry and cell biology of mammalian scavenger receptors. Atherosclerosis 182, 1–15 50 Mukhopadhyay, S. et al. (2006) MARCO, an innate activation marker of macrophages, is a class A scavenger receptor for Neisseria meningitides. Eur. J. Immunol. 36, 940–949 51 Alarcon, R. et al. (2005) Expression of scavenger receptors in glial cells. Comparing the adhesion of astrocytes and microglia from neonatal rats to surface-bound b-amyloid. J. Biol. Chem. 280, 30406–30415 52 Verdier, Y. et al. (2004) Amyloid b-peptide interactions with neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer’s disease. J. Pept. Sci. 10, 229–248 53 Nakamura, K. et al. (2006) Possible role of scavenger receptor SRCL in the clearance of amyloid-b in Alzheimer’s disease. J. Neurosci. Res. 84, 874–890 54 Wyss-Coray, T. et al. (2003) Adult mouse astrocytes degrade amyloid-b in vitro and in situ. Nat. Med. 9, 453–457 55 Krumbholz, M. et al. (2005) BAFF is produced by astrocytes and upregulated in multiple sclerosis lesions and primary central nervous system lymphoma. J. Exp. Med. 201, 195–200 56 Tschen, S.I. et al. (2006) CNS viral infection diverts homing of antibody-secreting cells from lymphoid organs to the CNS. Eur. J. Immunol. 36, 603–612 www.sciencedirect.com
57 Brambilla, R. et al. (2005) Inhibition of astroglial nuclear factor kB reduces inflammation and improves functional recovery after spinal cord injury. J. Exp. Med. 202, 145–156 58 van Loo, G. et al. (2006) Inhibition of transcription factor NF-kB in the central nervous system ameliorates autoimmune encephalomyelitis in mice. Nat. Immunol. 7, 954–961 59 Bush, T.G. et al. (1999) Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice. Neuron 23, 297–308 60 Faulkner, J.R. et al. (2004) Reactive astrocytes protect tissue and preserve function after spinal cord injury. J. Neurosci. 24, 2143–2155 61 Herx, L.M. et al. (2000) Central nervous system-initiated inflammation and neurotrophism in trauma: IL-1b is required for the production of ciliary neurotrophic factor. J. Immunol. 165, 2232–2239 62 Herx, L.M. and Yong, V.W. (2001) Interleukin-1b is required for the early evolution of reactive astrogliosis following CNS lesion. J. Neuropathol. Exp. Neurol. 60, 961–971 63 Mason, J.L. et al. (2001) Interleukin-1b promotes repair of the CNS. J. Neurosci. 21, 7046–7052 64 Penkowa, M. et al. (1999) Strongly compromised inflammatory response to brain injury in interleukin-6-deficient mice. Glia 25, 343–357 65 Swartz, K.R. et al. (2001) Interleukin-6 promotes post-traumatic healing in the central nervous system. Brain Res. 896, 86–95 66 Liberto, C.M. et al. (2004) Pro-regenerative properties of cytokineactivated astrocytes. J. Neurochem. 89, 1092–1100 67 Tran, P.B. and Miller, R.J. (2003) Chemokine receptors: signposts to brain development and disease. Nat. Rev. Neurosci. 4, 444–455 68 Imitola, J. et al. (2004) Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1a/CXC chemokine receptor 4 pathway. Proc. Natl. Acad. Sci. U. S. A. 101, 18117– 18122 69 Pluchino, S. et al. (2005) Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism. Nature 436, 266–271 70 Belmadani, A. et al. (2006) Chemokines regulate the migration of neural progenitors to sites of neuroinflammation. J. Neurosci. 26, 3182–3191 71 Babcock, A.A. et al. (2003) Chemokine expression by glial cells directs leukocytes to sites of axonal injury in the CNS. J. Neurosci. 23, 7922– 7930 72 Ambrosini, E. and Aloisi, F. (2004) Chemokines and glial cells: a complex network in the central nervous system. Neurochem. Res. 29, 1017–1038 73 Ambrosini, E. et al. (2005) Astrocytes produce dendritic cell-attracting chemokines in vitro and in multiple sclerosis lesions. J. Neuropathol. Exp. Neurol. 64, 706–715 74 Krumbholz, M. et al. (2006) Chemokines in multiple sclerosis: CXCL12 and CXCL13 up-regulation is differentially linked to CNS immune cell recruitment. Brain 129, 200–211 75 Omari, K.M. et al. (2006) Role for CXCR2 and CXCL1 on glia in multiple sclerosis. Glia 53, 24–31 76 Cardona, A.E. et al. (2006) Control of microglial neurotoxicity by the fractalkine receptor. Nat. Neurosci. 9, 917–924 77 Wang, T. et al. (2004) Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis. Nat. Med. 10, 1366– 1373 78 Prinz, M. et al. (2006) Innate immunity mediated by TLR9 modulates pathogenicity in an animal model of multiple sclerosis. J. Clin. Invest. 116, 456–464 79 Tanga, F.Y. et al. (2005) The CNS role of Toll-like receptor 4 in innate neuroimmunity and painful neuropathy. Proc. Natl. Acad. Sci. U. S. A. 102, 5856–5861 80 Kariko, K. et al. (2004) Inhibition of Toll-like receptor and cytokine signaling – a unifying theme in ischemic tolerance. J. Cereb. Blood Flow Metab. 24, 1288–1304 81 Ding, Q. and Keller, J.N. (2005) Evaluation of rage isoforms, ligands, and signaling in the brain. Biochim. Biophys. Acta 1746, 18–27 82 Eto, M. et al. (2003) The role of CD36 in peripheral nerve remyelination after crush injury. Eur. J. Neurosci. 17, 2659–2666 83 Cho, S. et al. (2005) The class B scavenger receptor CD36 mediates free radical production and tissue injury in cerebral ischemia. J. Neurosci. 25, 2504–2512
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84 McGeer, P.L. and McGeer, E.G. (2002) The possible role of complement activation in Alzheimer disease. Trends Mol. Med. 8, 519–523 85 Bonifati, D.M. and Kishore, U. (2007) Role of complement in neurodegeneration and neuroinflammation. Mol. Immunol. 44, 999– 1010 86 Storch, M.K. et al. (1998) Multiple sclerosis: in situ evidence for antibody- and complement-mediated demyelination. Ann. Neurol. 43, 465–471 87 Boos, L. et al. (2004) Deletion of the complement anaphylatoxin C3a receptor attenuates, whereas ectopic expression of C3a in the brain exacerbates, experimental autoimmune encephalomyelitis. J. Immunol. 173, 4708–4714 88 Whitney, K.D. et al. (1999) Immunoglobulin G and complement immunoreactivity in the cerebral cortex of patients with Rasmussen’s encephalitis. Neurology 53, 699–708 89 Speth, C. et al. (2002) Neuroinvasion by pathogens: a key role of the complement system. Mol. Immunol. 38, 669–679 90 de Jonge, R.R. et al. (2004) Expression of complement components in the peripheral nervous system. Hum. Mol. Genet. 13, 295– 302 91 Rahpeymai, Y. et al. (2006) Complement: a novel factor in basal and ischemia-induced neurogenesis. EMBO J. 25, 1364–1374
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92 Nadeau, S. and Rivest, S. (2000) Role of microglial-derived tumor necrosis factor in mediating CD14 transcription and nuclear factor kB activity in the brain during endotoxemia. J. Neurosci. 20, 3456–3468 93 Lee, J.H. et al. (2005) Double-stranded RNA-activated protein kinase is required for the LPS-induced activation of STAT1 inflammatory signaling in rat brain glial cells. Glia 50, 66–79 94 Husemann, J. and Silverstein, S.C. (2001) Expression of scavenger receptor class B, type I, by astrocytes and vascular smooth muscle cells in normal adult mouse and human brain and in Alzheimer’s disease brain. Am. J. Pathol. 158, 825–832 95 Sasaki, N. et al. (2001) Immunohistochemical distribution of the receptor for advanced glycation end products in neurons and astrocytes in Alzheimer’s disease. Brain Res. 888, 256–262 96 Dorf, M.E. et al. (2000) Astrocytes express functional chemokine receptors. J. Neuroimmunol. 111, 109–121 97 John, G.R. et al. (2002) Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation. Nat. Med. 8, 1115–1121 98 John, G.R. et al. (2005) IL-1-regulated responses in astrocytes: relevance to injury and recovery. Glia 49, 161–176 99 Rivieccio, M.A. et al. (2005) The cytokine IL-1b activates IFN response factor 3 in human fetal astrocytes in culture. J. Immunol. 174, 3719–3726
Endeavour The quarterly magazine for the history and philosophy of science. You can access Endeavour online on ScienceDirect, where you’ll find book reviews, editorial comment and a collection of beautifully illustrated articles on the history of science. Featuring: Information revolution: William Chambers, the publishing pioneer by A. Fyfe Does history count? by K. Anderson Waking up to shell shock: psychiatry in the US military during World War II by H. Pols Deserts on the sea floor: Edward Forbes and his azoic hypothesis for a lifeless deep ocean by T.R. Anderson and T. Rice ‘Higher, always higher’: technology, the military and aviation medicine during the age of the two world wars by C. Kehrt Bully for Apatosaurus by P. Brinkman Coming soon: Environmentalism out of the Industrial Revolution by C. Macleod Pandemic in print: the spread of influenza in the Fin de Sie`cle by J. Mussell Earthquake theories in the early modern period by F. Willmoth Science in fiction - attempts to make a science out of literary criticism by J. Adams The birth of botanical Drosophila by S. Leonelli And much, much more. . .
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Astrocytes are active players in cerebral innate immunity Cinthia Farina1, Francesca Aloisi2 and Edgar Meinl3,4 1
Neuroimmunology and Neuromuscular Disorders Unit, National Neurological Institute Carlo Besta, 20133 Milan, Italy Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita`, 00161 Rome, Italy 3 Institute of Clinical Neuroimmunology, Ludwig-Maximilians University, 81377 Munich, Germany 4 Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, 82152 Martinsried-Planegg, Germany 2
Innate immunity is a constitutive component of the central nervous system (CNS) and relies strongly on resident myeloid cells, the microglia. However, evidence is emerging that the most abundant glial cell population of the CNS, the astrocyte, participates in the local innate immune response triggered by a variety of insults. Astrocytes display an array of receptors involved in innate immunity, including Toll-like receptors, nucleotide-binding oligomerization domains, double-stranded RNAdependent protein kinase, scavenger receptors, mannose receptor and components of the complement system. Following activation, astrocytes are endowed with the ability to secrete soluble mediators, such as CXCL10, CCL2, interleukin-6 and BAFF, which have an impact on both innate and adaptive immune responses. The role of astrocytes in inflammation and tissue repair is elaborated by recent in vivo studies employing cell-type specific gene targeting. Introduction The central nervous system (CNS) has been commonly regarded as an immune-privileged site [1]. However, important studies published during the past ten years indicate that the CNS can offer the setting for innate immune responses [2]. This might reflect the ability of the CNS to fight infections despite its immune-privileged state. The recognition of infectious non-self is mediated by a limited number of germline-encoded pattern-recognition receptors (PRRs) and triggers rapid responses. Some PRRs can also recognize endogenous ‘danger signals’ that alert the immune system to cell damage, independently from the context of infection. Consistently, the activation of innate immune pathways occurs not only in infectious CNS diseases but also after brain injury and ischemia, and in autoimmune and neurodegenerative disorders of the CNS (Table 1), indicating that the relevance of these pathways extends beyond antimicrobial defense. To date, the neurotoxic and neuroprotective roles of innate immune reactions in non-infectious CNS diseases are an intensively investigated and debated issue. The expression of PRRs is found in various immune and non-immune cell types of the CNS, similarly to peripheral tissues. Microglia and astrocytes are the main CNS-resident cell types for which consistent data have been published Corresponding author: Farina, C. ([email protected]). Available online 2 February 2007. www.sciencedirect.com
(Table 2). Microglia are myeloid lineage cells and are considered ‘the CNS professional macrophages’, owing to their phenotype and reactivity following injury and inflammation [3]. For this reason, most of the studies on innate immune responses in the CNS have focused on microglia, which express a wide range of PRRs (Table 2). The contribution of other cell types to these processes has often been neglected. However, recent evidence suggests that astrocytes have a complex, dual role in the local regulation of immune reactivity. Astrocytes, the most abundant glial cell population, are of neuroectodermal origin and are essential for brain homeostasis and neuronal function [4] (Box 1). They form the glia limitans around blood vessels restricting the access of immune cells to the CNS parenchyma [5] (see Figure I in Box 2). In contrast to other brain cells, astrocytes are resistant to death receptor-induced apoptosis [6], indicating that they are well equipped to survive inflammatory insults. In this article, we aim to answer the following questions: (i) do astrocytes participate in innate immune reactions? (ii) Which are the activating signals and functional responses involved? The Toll-like receptor system in astrocytes: expression, signaling and biological relevance Toll-like receptors (TLRs) are evolutionarily conserved type I membrane glycoproteins characterized by leucinerich-repeat motifs in the extracellular domain and by a cytoplasmic signaling domain similar to that of the interleukin (IL)-1 receptor [7]. TLR1, TLR2 and TLR6 recognize bacterial lipoproteins; TLR3, TLR7, TLR8 and TLR9 are specific for nucleic acids; TLR5 binds to flagellin, the main constituent of bacterial flagella; TLR4 has a wide spectrum of ligands, including bacterial lipopolysaccharide (LPS) and fungal zymosan. TLR expression has been detected in cells of the innate and adaptive immune system and in non-immune cells. But are TLRs important for pathogen recognition in the CNS, and where are they expressed? Increasing evidence indicates that TLRs have a major role in several inflammatory CNS pathologies (Table 1). For example, genetic polymorphisms in TLR4 have been associated with increased susceptibility of humans to meningococcal infection [8]. In animal models of infectious CNS diseases, TLR2 deficiency can lead to opposite outcomes, such as increased susceptibility to Streptococcus pneumoniae
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Table 1. Innate immune pathways involved in CNS pathologies PRR system TLR
Scavenger receptors
Complement
CNS disease Neisseria meningitides meningitis Streptococcus pneumoniae meningitis Listeria monocytogenes meningitis Herpes simplex virus encephalitis West Nile virus encephalitis Multiple sclerosis or experimental autoimmune encephalitis Nerve injury Ischemia Alzheimer’s disease N. meningitides meningitis Nerve injury Ischemia Alzheimer’s disease Parkinson’s disease Huntington’s disease Pick’s disease Multiple sclerosis or experimental autoimmune encephalitis Rasmussen’s encephalitis Prion disease N. meningitides meningitis HIV encephalitis Epstein-Barr virus encephalitis Measles virus encephalitis West Nile virus encephalitis Nerve injury Ischemia
Refs [8] [9] [9] [11] [77] [22,78] [79] [80] [48,81] [50] [82] [83] [40,84] [85] [40] [40] [86,87] [88] [85] [8,44,89] [89] [89] [89] [46,47] [90] [91]
meningitis [9,10] and protection from herpes simplex virus encephalitis [11]. However, in these models, owing to PRR expression on peripheral immune cells and on brain-resident cells, it is difficult to distinguish the contributions to infection control of each cell type. Under physiological conditions, basal expression of TLR2 and TLR4 was demonstrated in the meninges, choroid plexus and circumventricular organs of the brain, namely in CNS areas that lack a blood–brain barrier (BBB) and are more exposed to invading pathogens [12– 15]. More recently, low basal levels of TLR4 expression were shown in microglia in vivo [15]. Accordingly, the systemic administration of LPS results in rapid upregulation of TLR2 in microglia and generates an innate inflammatory response that is readily detected and more prominent in BBB-free areas of the CNS but also extends into the brain parenchyma [12–14].
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Box 1. Features and functions of astrocytes Features Most abundant glial cell in the CNS Neuroectodermal origin Largely resistant to death receptor (Fas, TRAIL)-mediated apoptosis Functions Metabolic support of neurons; glycogen storage and export of lactate Uptake of neurotransmitters, including glutamate Production of neurotrophic factors Ion homeostasis (i.e. potassium uptake) Blood–brain barrier induction and maintenance Scar formation and tissue repair Regulation of immune responses in the CNS
The brain displays basal expression of TLR3, which is detected on glial fibrillary acidic protein (GFAP)-positive astrocytes in the hippocampus and striatum [16]. Several recent in vitro studies have shown that TLR3 is the predominant TLR expressed in astrocytes. In two studies, the complete human TLR repertoire in human fetal astrocytes was analyzed by quantitative polymerase chain reaction and indicated TLR3 as the only TLR with consistent expression in the resting state [17,18] and with upregulated levels following treatment with inflammatory cytokines such as IL-1b, interferon (IFN)b and IFNg [17]. The analysis of cultured human adult astrocytes showed basal levels of TLR2, TLR3 and TLR4; however, exposure to inflammatory cytokines strongly induced TLR3, while having no effect on TLR4 and downregulating TLR2 [19]. Similarly, cultured mouse and rat astrocytes displayed high constitutive levels of TLR3 [20,21]. TLR3 is commonly found in intracellular compartments such as endosomes, and signaling requires ligand internalization. Interestingly, in human astrocytes, TLR3 was detected not only intracellularly [18] but also on the cell membrane [18,22], and its protein expression was induced following ligand binding [18]. TLR2, TLR4, TLR5 and TLR9 have been described in human astrocyte-enriched cultures [19,20, 22,23] (Table 2); however, expression of these TLRs on astrocytes in vivo has
Table 2. PRRs in astrocytes and microgliaa PRR TLR
Astrocytes TLR2 [22,23], TLR3 [16], TLR4 [20,23], TLR5 [20,23], TLR9 [20,23]
CD14 NOD PKR Scavenger receptors
Not expressed NOD1, NOD2 [36] Expressed [35] SR-BI [94], SR-MARCO [51], RAGE [95], SRCL [53]
Mannose receptor Complement factors
Expressed [38] C1q, C1r, C1s, C4, C2, C3, factor B, factor D, C5, C6, C7, C8, C9 [40] CR1, CR2, C3aR, C5aR [40]
Complement receptors Complement inhibitors a
C1-INH, DAF/CD55, MCP/CD46, CD59, factor H, factor I, S protein, clusterin [40]
Microglia TLR1 [24], TLR2 [13], TLR3 [24], TLR4 [15], TLR5 [24], TLR6 [24], TLR7 [24], TLR8 [24], TLR9 [24] Expressed [92] Unknown Expressed [93] SR-A [48], SR-BI [48], SR-MARCO [51], RAGE [51], CD36 [48], SRCL [53] Expressed [3] C1q, C3, C4 [40] CR1, CR3/CD11b, CR4/CD11c, C3aR, C5aR, C1qRp [40] C1-INH, CD59, clusterin [40]
Blue text indicates in vitro evidence (not yet supported by in vivo studies); red text indicates in vivo evidence (mostly supported by in vitro studies not cited in the table).
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Box 2. Mediators of astrocyte function 1. Cytokines: IL-6, IFNb, TGFb, GM-CSF, BAFF, IL-1b and TNF
Increased BBB permeability Endothelial cell activation Microglial and monocytic activation, differentiation and proliferation Astrocyte activation B cell survival and differentiation Immunosuppression Release of neuroprotective mediators
2. Chemokines: CCL2, CCL5, CCL20, CXCL10, CXCL12, CXCL1, CXCL2 and CX3CL1 Recruitment of monocytes and macrophages, dendritic cells, T and B lymphocytes, and neutrophils Regulation of myelination and microglial activity Astrocyte proliferation and survival Migration of microglia and neural progenitors 3. Neurotrophic factors: NGF, CNTF, BDNF, VEGF, IGF1 and LIF
Neuronal survival, differentiation, function and regeneration Oligodendrocyte survival Remyelination Neurogenesis
Mediators released by astrocytes (bold text) following pathogen recognition trigger two types of events (Figure I): (i) they activate
neighboring cells and amplify the local, initial innate immune response further; and (ii) they modify BBB permeability and attract immune cells from the blood circulation into the neural tissue, thus supporting an adaptive immune response. The balance between inflammatory (tissue damaging; red text) and immunosuppressive (tissue regenerating; green text) pathways is fundamental for controlled reactions to CNS trauma. Dysregulation of these pathways might lead to pathogenic chronic neuroinflammation and neurodegeneration. Astrocytes themselves can be the direct target of such factors. Astrocytic responses to chemokines involve chemotaxis, cell proliferation and survival. Additionally, chemokines, such as CXCL12 and CCL5, induce glutamate release and cytokine and chemokine synthesis, indicating a role for this class of molecules in regulating glia–glia and glia–neuron communication [72,96]. Among cytokines, TNF, IFNg and IL-1 are the main astrocytic activators. Gene profiling studies in astrocytes exposed to inflammatory cytokines in vitro showed profound regulations in genes involved in several cellular pathways, such as innate and adaptive immunity, antigen presentation, apoptosis, leukocyte migration and BBB permeability [97–99]. Astrocyte-derived factors, such as GM-CSF, IL-6, CCL2 and CCL5 regulate microglial migration, activation and proliferation. Some glia-derived soluble mediators, including CCL2, CCL5, CXCL10, CXCL12 and BAFF, are also responsible for triggering adaptive immunity in the inflamed CNS.
Figure I. Astrocyte activation generates waves of innate and adaptive immunity. The astrocyte has a strategic location, being in close contact with CNS-resident cells (neurons, microglia, oligodendrocytes and other astrocytes) and with blood vessels. The astrocyte-derived glia limitans (dark green) forms a barrier between the brain parenchyma and the vascular system. The perivascular space, a site of immune cell accumulation in CNS inflammation, is delimited by the endothelial basement membrane (dark pink) and the glia limitans [5]. Following PRR engagement, astrocytes secrete factors that target bystander brain cells and promote changes in the permeability of the BBB, resulting in the recruitment of professional immune cells into the CNS parenchyma. This leads to an amplification of the initial innate immune reaction, which can result in the elimination of the insult, with restoration of tissue integrity or scar formation. Relative sizes of the distinct cell types reflect in situ observations. T and B lymphocytes, macrophages and microglia have the same color to indicate their common hematopoietic origin.
not yet been demonstrated. By contrast, similarly to monocytes and macrophages, microglia (the professional immune cells of the brain) display a broader repertoire of TLRs in vitro [18,24] (Table 2). Human astrocytes express mRNAs for the adaptor molecules TIRAP (also known as Mal), TICAM (also known as TRIF), MyD88 and the shorter splice variant MyD88S [17]. The MyD88-dependent pathway involves www.sciencedirect.com
the sequential activation of IL-1 receptor-associated kinase (IRAK) proteins, tumor necrosis factor (TNF) receptor-associated factor (TRAF)6 and mitogen-activated protein (MAP) kinases down to activator protein (AP)-1 and nuclear factor (NF)-kB transcription factors. The TRIF-dependent pathway activates NF-kB on one side through receptor-interacting protein (Rip)1 and interferon regulatory factor (IRF)3 and IRF7 on the other side through
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Tank-binding kinase (TBK)1 and phosphatidylinositol 3-kinase (PI3K), resulting in the expression of IFN-inducible genes [7]. Double-stranded (ds)RNA-triggered biological effects are mediated largely by TLR3. Injecting poly(I:C), a synthetic analog of dsRNA, into the brain triggered microglial and astrocytic activation in wild-type mice but not in TLR3 knockout (KO) mice [25], indicating the important role of TLR3 in the CNS response to viral infection. In cultured astrocytes, poly(I:C) induces the expression of several cytokines [TNF, IL-6, IFNb, granulocyte–macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)b] and chemokines (CCL2, CCL5, CCL20, CXCL8 and CXCL10) [16–19]. The astrocytic response to dsRNA involves inducible nitric oxide synthase (iNOS) upregulation with nitric oxide production [20,21,26], a decrease in glutamate uptake [21], and downregulation of connexin 43 with a disturbance of gap junction-mediated intercellular communication [27]. This suggests that astrocytes contribute to tissue damage and neurotoxicity. Similarly, in vitro infection of mouse astrocytes with Theiler’s murine encephalomyelitis virus (TMEV) triggered an initial inflammatory response (CCL2 and CXCL10 secretion) through TLR3 [28]. However, viral replication was equal in wild-type and TLR3 KO astrocytes, indicating that TLR3-mediated activation is insufficient to block TMEV replication [28]. Gene profiling of poly(I:C)-treated adult human astrocytes indicated the induction of an antiviral program [29] and of growth, differentiation and neuroprotective mediators, including GM-CSF, vascular endothelial growth factor (VEGF)-C, neurotrophin (NT)-4 and ciliary neurotrophic factor (CNTF), suggesting that TLR stimulation in astrocytes also activates tissue repair pathways [19]. In summary, astrocytes express TLR3 in vivo and in vitro, and react to TLR3 ligands by producing mediators that promote, on the one hand, the local inflammatory response and, on the other hand, tissue repair. Because recent studies indicate that mRNA from necrotic cells functions as an endogenous ligand for TLR3 [30], the possibility that neural tissue damage activates astrocytes is intriguing and deserves further investigation. Astrocytes express PKR, NOD proteins, scavenger receptors, mannose receptor and components of the complement system PRRs other than TLRs are involved in innate immune responses. TLR3 is not the only weapon in antiviral immunity, because TLR3 KO mice were able to mount a normal peripheral immune response to lymphocytic choriomeningitis virus, vesicular stomatitis virus (VSV) and murine cytomegalovirus [31]. Following intracerebral inoculation of reovirus, a dsRNA virus, wild-type and TLR3 KO mice showed similar patterns of survival, viral titer and neuropathology [31]. Sendai virus was able to activate TLR3negative cells and to induce interferon-stimulated and interferon-related genes [32]. Indeed, many other PRRs that mediate RNA recognition exist [33] and each might recognize distinct dsRNA structures [34]. One of these PRRs is dsRNA-dependent protein kinase (PKR). Its actiwww.sciencedirect.com
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vation leads to the phosphorylation of the a subunit of the eukaryotic initiation factor 2 resulting in the blockade of viral translation and subsequent shutdown of protein synthesis in virus-infected cells. Although no data are yet available on the distribution of PKR in the CNS, recent studies showed that PKR is involved in the dsRNA-induced response in cultured astrocytes [16,21,35]. Nucleotide-binding oligomerization domain (NOD)1 and NOD2 proteins, intracellular proteins that recognize distinct motifs of bacterial peptidoglycan, have been described in cultured mouse astrocytes, and NOD2 was found to mediate IL-6 secretion in response to its specific ligand [36]. Expression of the mannose receptor (MR) has also been demonstrated in the CNS [37,38]. MR enables recognition of mannosylated ligands of endogenous or microbial origin, resulting in receptor-mediated endocytosis and enhanced microbicidal activity. Both cultured astrocytes and microglia express MR protein on the cell surface and in intracellular pools [37]. MR was identified as the receptor responsible for CD4-independent HIV-1 entry into astrocytes [38]. This interaction triggered intracellular signaling leading to matrix metalloproteinase 2 production [39]. Complement is another important component of innate immunity. It comprises soluble and surface proteins expressed in almost every cell type. Recent studies have highlighted the role of complement in the normal and pathological CNS. Notably, brain cells express a full complement system to kill pathogens but are somewhat protected from complement-induced lysis because of the expression of complement inhibitors [40,41]. Reactive astrocytes can produce most of the complement factors after activation and express in vivo complement receptors and complement-regulatory proteins [40,42] (Table 2). Interestingly, pathogens can use some of these factors to promote infection. For example, Epstein–Barr virus binds to CR2, whereas CD46 serves as a receptor for human herpesvirus 6, measles virus and Neisseria meningitidis [43,44]. Although these pathogens were shown to infect astrocytes in vitro, formal in vivo evidence is still required. Studies of human pathological samples and animal models of CNS infection have, in some instances, demonstrated the contribution of complement to anti-viral immunity [45–47] (Table 1). Furthermore, genetic polymorphisms in genes encoding complement components have been associated with N. meningitidis infection [8] (Table 1). Scavenger receptors (SRs), initially described as high-affinity receptors on macrophages for acetylated low-density lipoproteins, comprise several receptor classes, each including numerous members [48,49]. SRs have a role in the binding and internalization of many unrelated ligands, such as fibrillar b-amyloid, lipids, glycated collagen and apoptotic cells, and, therefore, are important for tissue homeostasis. Astrocytes were shown to express various SRs in situ using immunohistochemistry (Table 2). SR-MARCO is important for host defense, because it can bind to Gram-positive and Gram-negative bacteria. Interestingly, SR-MARCO is involved in the recognition and uptake of N. meningitidis [50], an important cause of
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bacterial meningitis; therefore, its expression in astrocytes [51] might be an additional mechanism through which CNS-resident cells can fight bacterial infections. The other SRs present on astrocytes recognize endogenous signals. For example, SR-BI exerts its scavenger activity through binding to low-density lipoproteins, apoptotic cells and anionic phospholipids. SR-BI, RAGE and SRCL bind to and mediate the internalization of b-amyloid peptides and fibrils [48,52,53]. These observations, together with the finding that astrocytes associate with and degrade b-amyloid deposits in situ [54], highlight an important role for astrocytes in counteracting neurodegeneration. A deficit in this function could have direct implications in Alzheimer’s disease. Astrocytes are important regulators of neuroinflammation In the previous sections, we have seen that astrocytes carry a series of PRRs, which are important for the primary recognition of infectious agents and of endogenous danger signals. Factors produced by activated astrocytes (and microglia) target neighboring cells and promote leukocyte recruitment, resulting in the local amplification of inflammatory responses (Box 2). For example, following stimulation with inflammatory cytokines, astrocytes in vitro produce high amounts of the B cell survival factor BAFF (B cell-activating factor of the TNF family) [55]. Notably, BAFF is expressed in astrocytes in the normal human CNS and is strongly upregulated in activated astrocytes in the demyelinated lesions of multiple sclerosis (MS), a chronic inflammatory disease of the CNS [55]. BAFF secretion could be relevant in sustaining intrathecal B cell responses in autoimmune and infectious diseases of the CNS. Accordingly, BAFF expression is readily induced in the CNS by neurotropic viruses and correlates with the recruitment of antibody-secreting cells [56]. The central role of astrocytes in regulating neuroinflammation was demonstrated recently in vivo [57,58]. Transgenic mice were generated in which NFkB, a fundamental transcription factor family in innate responses, was selectively inactivated in astrocytes [57]. These mice displayed normal spinal cord architecture and cellular turnover; however, functional recovery after injury was increased dramatically, and lesion volume and white matter injury was reduced. These observations correlated with the drop in leukocyte recruitment into the lesioned area owing to the reduced NF-kB-dependent expression of CXCL10 and CCL2 [57]. Similarly, blockading the NF-kB pathway in neuroectodermal cells of the CNS (including neurons, astrocytes and oligodendrocytes) led to a consistent decrease in proinflammatory gene expression during experimental autoimmune encephalomyelitis and clinical– pathological amelioration [58]. In summary, the NF-kB pathway in astrocytes is a key regulator of inflammation in the CNS, and its inhibition has beneficial effects on tissue regeneration. Astrocytes contribute to neuroprotective innate inflammation Innate immune responses are not necessarily detrimental for the cerebral tissue. The innate immune system also www.sciencedirect.com
performs the task of restricting the lesioned area, removing the insult and restoring tissue homeostasis. A major, therapeutically relevant question is: what role do astrocytes have in the innate neuroprotective immune response? A hallmark of CNS injury, whatever its origin (i.e. infectious, autoimmune, mechanical or toxic) is the formation of scar tissue composed of activated (‘reactive’) astrocytes and microglia. An inflammatory response occurs within this clearly demarcated area. This phenomenon is commonly regarded as harmful and responsible for the restriction of axonal regrowth within the lesion. However, several studies indicate that activated astrocytes (in addition to activated microglia) and their secreted products might exert neuroprotective actions. Subsequent to trauma, astrocytes proliferate, accumulate glycogen and undergo fibrosis by accumulating intermediate filaments, expressed as an increase in GFAP. Transgenic mice expressing herpes simplex virus-thymidine kinase (HSV-TK) under the GFAP promoter were generated. HSV-TK-expressing cells metabolize the antiviral drug ganciclovir (GCV) to toxic nucleotide analogs, leading preferentially to the death of proliferating cells. In uninjured GFAP-TK mice, astrocyte proliferation is rare; therefore, GCV administration does not kill resting astrocytes. After CNS insults, local scar formation occurs and astrocytes proliferate, resulting in the loss of these cells in GCV-treated GFAP-TK mice [59,60]. This experimental model unraveled a fundamental neuroprotective role for reactive astrocytes in: (i) demarcating the damaged area and limiting leukocyte extravasation, because the ablation of reactive astrocytes resulted in improper scar formation and more extensive and long-lasting inflammation; (ii) promoting BBB repair, because in GCV-treated transgenic animals, immunoglobulin entry into the brain parenchyma persisted after injury; and (iii) sustaining neuronal survival, because enhanced neuronal degeneration occurred after astrocyte loss. Several cytokines, including IL-1 and IL-6, have been implicated in the induction and modulation of reactive astrogliosis and pathological inflammatory responses. However, the same factors in different settings might also behave as mediators of neuroprotection and remyelination. For example, in various injury models, IL-1b and IL-6 KO mice displayed delayed astrocyte activation and increased BBB permeability, indicating that cytokine-induced astrogliosis following trauma is important to restore the integrity of the BBB and to repair the lesion [61–65]. Interestingly, IL-1b KO mice failed to upregulate the neurotrophic factors CNTF [61] and insulin-like growth factor (IGF)1 [63] following CNS trauma, indicating that the initial IL-1b-dependent inflammatory response mediates the release of these neurotrophic factors in the injured CNS. In wild-type mice, both microglia [61,63] and astrocytes [63] express IL-1b, whereas mainly astrocytes were found to be positive for IGF1 in a demyelination model [63]. Several in vitro data demonstrate that cytokines such as IL-1, IL-6 and TNF support the production of neuroprotective mediators [66]. In this regard, the observation that TLR engagement on cultured astrocytes triggers the production of neurotrophic factors supports a role for
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these cells in the neuroprotective innate immune response [19]. A Janus-like action is displayed also by astrocyte-derived chemokines. CCL2 and CXCL12 not only promote the recruitment of inflammatory immune cells into the CNS parenchyma but also have a role in the migration of neural progenitors in the developing brain [67] and towards areas of brain injury [68–70]. CCL2 is readily upregulated in various pathological conditions in microglia and astrocytes [71,72], and increased CXCL12 immunoreactivity was detected in astrocytes in MS lesions [73,74]. CXCL1 was seen on reactive astrocytes in MS lesions in the vicinity of CXCR2positive oligodendrocytes, suggesting a role for this chemokine in remyelination [75]. Another chemokine constitutively present in the CNS is CX3CL1, which is produced by astrocytes and neurons. CX3CL1 is an important inhibitor of microglial toxicity, as demonstrated in three different in vivo models [76]. Therefore, the production of particular chemokines might reflect the attempt of reactive astrocytes to promote regeneration in the lesioned CNS. Concluding remarks Research in the field of innate immunity in the CNS has developed during the past decade and has concentrated mainly on the reactivity of the professional resident immune cells, the microglia. However, based on current evidence, we can definitely consider the astrocyte as the innate-immunecompetent cell, because it bears several PRRs involved in the primary recognition of microbial agents or of endogenous danger signals. This cell type reacts with a unique activation program that modulates or amplifies the local inflammatory reaction. In vivo studies have revealed Janus-like features of astrocytes. On the one hand, they can promote inflammation through NF-kB-dependent pathways; on the other hand, proliferating reactive astrocytes confine lesions and restore brain homeostasis. Increasing knowledge on the involvement of astrocytes in shaping the innate immune response can provide new insights into the intrinsic capacity of the CNS tissue to face pathogenic insults and can aid in the identification of molecular targets for therapeutic interventions in a variety of neuroinflammatory and neurodegenerative diseases. Acknowledgements Our original work was supported by the Deutsche Forschungsgemeinschaft (SFB 571), the Gemeinnu¨tzige Hertie-Foundation, the Deutsche Multiple Sklerose Gesellschaft, the Verein zur Therapieforschung fu¨r MS-Kranke, and the Italian Ministry of Health. The Institute for Clinical Neuroimmunology is supported by the Hermann and Lilly Schilling Foundation.
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