Microglia Plasticity During Health and Disease: An Immunological Perspective

Special Issue: Neuroimmunology

Review

Microglia Plasticity During Health and Disease: An Immunological Perspective Anat Shemer,1,z Daniel Erny,2,z Steffen Jung,1,* and Marco Prinz2,3,* Microglia are macrophages of the central nervous system (CNS) that continuously scrutinize their environment for damage. They colonize the cephalic mesenchyme during embryogenesis and actively shape the developing neuronal network by immune-mediated mechanisms. Upon CNS maturation, microglia drastically change phenotype and function. During health, adult microglia contribute to homeostasis, but also the establishment and resolution of inflammatory conditions. Fulfillment of these distinct tasks requires these long-lived cells to accurately adjust to their changing environment. Deciphering microglia responsiveness to divergent stimuli is central to understanding this cell type and for eventual microglia manipulation to potentially reduce disease burden. Here we discuss new aspects of myeloid cell biology in general with special emphasis on the shifting role of microglia during establishment and protection of CNS integrity.

Trends Microglia respond to environmental stimuli, including from surrounding tissue and from systemic inputs. These stimuli impact microglial development and function in both health and disease. The tissue microenvironment, in combination with lineage-specific TFs, shapes the chromatin landscape of microglia and tissue-resident macrophages and drives the selection and usage of enhancers. There is plasticity in the system: adult macrophages can alter their epigenetic landscape when transplanted to a different tissue.

Microglia: A Macrophage Type Captured in the CNS Microglia are the only myeloid cell type located within the healthy CNS parenchyma and are classified as mononuclear phagocytes. This heterogeneous family of cells includes peripheral tissue-specific macrophages, dendritic cells (DCs), and monocytes [1–4] as well as nonparenchymal CNS-associated myeloid cells (meningeal, perivascular, and choroid plexus macrophages) [5,6]. Macrophages are present in most organs and compartments of the body and are, as innate immune cells, fundamental effectors and regulators of immune responses. Moreover, macrophages also critically contribute to the maintenance of tissue homeostasis during development and in adulthood [5,6]. Until recently myeloid cells in the CNS have been defined according to their anatomical localization and surface marker expression. This concept has now been challenged by the results of developmental and high-throughput gene expression profiling studies that took advantage of novel molecular biological tools to unravel microglial origin, fate, and function. In this review we summarize how these results have changed our current view on microglia as CNS-specific immune cells and highlight recent developments in the field, with a focus on immune-related aspects of microglia. Microglia cover the whole CNS parenchyma, albeit with distinct cell densities [7]. Microglia are special with respect to their restricted origin and extreme longevity compared with other tissue macrophages such as liver Kupffer cells, skin Langerhans cells, alveolar macrophages, and red pulp macrophages [3,5]. Pio del Rio-Hortega postulated nearly 100 years ago a mesodermal source of microglia [8,9]. However, conclusions about the exact microglial origin during CNS development were largely based on correlations and remained for decades highly debated.

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http://dx.doi.org/10.1016/j.it.2015.08.003

Disruption of the intestinal microbiota results in significant alterations in microglial maturation and function in both germ-free mice and on antibiotic treatment of adult mice, indicating that microglia are responsive to continuous input from the intestinal system throughout adulthood.

1

Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel 2 Institute of Neuropathology, University of Freiburg, Freiburg, Germany 3 BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany

*Correspondence: [email protected] (S. Jung) and [email protected] (M. Prinz). z These authors contributed equally.

Evidence from an avian model indicated early CNS immigration of embryonic mesodermal precursors originating from the yolk sac [10,11]. These findings were supported by the detection of microglia within the neuroectodermal parenchyma of developing rodents as early as embryonic day E9.5 [12]. Importantly, in the rat macrophage progenitors were observed to primarily migrate into the developing CNS, followed by liver and lung [13]. Also, murine microglial progenitors were suggested to originate from the yolk sac, but there had as yet been no confirmation [12,13]. It was discovered that macrophage progenitor populations in the yolk sac exhibited both erythroid and myeloid differentiation potential [14] and these cells became the focus of attention as potential microglial progenitors. More recently, researchers tackled this topic using elegant fate-mapping approaches. First, in 2010, it was uncovered that under homeostatic conditions it is immature yolk sac precursors that give rise to the microglial population, rather than adult bone marrow (BM)-derived precursors [15]. Shortly after, in a more detailed study, it was shown that adult microglia originate from noncommitted c-Kit+ F4/ 80 CX3CR1 erythromyeloid progenitors during primitive hematopoiesis (via c-Kitlow yolk sac populations and immature c-Kit F4/80high CX3CR1+ macrophage populations, termed A1 and A2, respectively) [16] Figure 1 Key Figure. At later stages, when fetal liver-derived monocytes are released to the circulation and definitive hematopoiesis yields myeloid cells, it assumed that the CNS is uncoupled from the periphery by the closing blood–brain barrier (BBB) [17]. The forming CNS is thus established as a defined and restricted environment excluding any substantial immigration of peripheral myeloid cells [3,5,17]. Of note, transcription factors (TFs) such as Runt-related transcription factor 1 (RUNX1), which is essential for the ramification process [18], PU.1, which plays a substantial role in the transition from EMP to the A1 state, and interferon regulatory factor 8 (IRF8), which is crucial for differentiation from the A1 to A2 state, are required regulators of the differentiation of microglia during murine embryonic development, whereas other myeloid TFs, like inhibitor of DNA binding 2 (ID2), basic leucine zipper transcriptional factor ATF-like 3 (BATF3), and Krüppel-like factor 4 (KLF4), are redundant [15,16]. IRF8 is a heterodimeric partner of PU.1 and microglial numbers are strongly reduced in adulthood in animals lacking IRF8 or PU.1 [16,19]. Moreover, IRF8 was reported to be essential for transforming microglia into a reactive phenotype [20]. Notably, microglial development and maturation were also shown to be independent of the central TF MYB, in marked contrast to blood monocytes and tissue macrophages raised from the fetal liver [21]. Specifically, MYB-deficient mice, which do not generate hematopoietic stem cells (HSCs), harbored microglial compartments like their wild-type littermates [21]. Microglia share initial origin from the yolk sac with other tissue macrophages [21]. In contrast to most of the latter, which include Langerhans cells [22] and cardiac macrophages [1,23], yolk sac-derived microglia are, however, not replaced by fetal liver or BM-derived cells. Results of parabiosis experiments, which involve surgical fusion of the blood circulations of two animals, have clearly demonstrated that BM-derived cells do not contribute to the resident microglial pool under healthy conditions [24]. By contrast, sole conditioning of the BBB (e.g., by g-irradiation, under certain pathological conditions) allows immigration of BM-derived cells into the primed CNS leading to de novo generation of BM-derived CNS macrophages [25]. Approaches such as fate mapping, parabiosis, and BM engraftment have allowed the definition of important features of microglia that distinguish them from other tissue macrophages: microglia are long lived in situ and able to self-renew. Moreover importantly, the experiments suggest that there is no significant replacement of microglia by peripheral hematopoietic cells from the blood circulation during a life of a mouse. Now that we have a better understanding of the origins of microglia, how does this inform our understanding of the functions of these cells in health and disease? Furthermore, are these insights informative in terms of diseases associated with microglial function?

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Key Figure (A)

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Figure 1. (A) Ontogeny of microglia and crucial molecules involved. Microglia derive from F4/80 CX3CR1 c-kit+ erythromyeloid precursors (EMPs) present in the yolk sac. During maturation microglia require signals via the colony-stimulating factor 1 receptor (CSF-1R), Runt-related transcription factor (RUNX) 1, PU.1, and interferon regulatory factor (IRF) 8. Adult F4/80lo CX3CR1+ c-kit CSF-1R+ microglia are dependent on survival factors such as colony-stimulating factor (CSF) 1, interleukin (IL)34, fractalkine (CX3CL1), and bacterially derived short-chain fatty acids (SCFAs). (B) Function of microglia in the adult central nervous system (CNS) during homeostasis. Microglia survey mature neurons and mediate synapse formation and nourish neurons by producing brain-derived neurotrophic factor (BDNF). Further, microglia constantly remove myelin debris by phagocytosis. (C) Supposed microglial function during autoimmune inflammation as depicted in the mouse model experimental autoimmune encephalomyelitis (EAE). Leakage through the blood–brain barrier (BBB) allows the disease-associated entry of circulating C-C chemokine receptor type (CCR) 2 expressing Ly-6Chi monocytes into the CNS that become monocyte-derived macrophages (moMFs) producing tumor necrosis factor alpha (TNF/) or IL-4, IL-6, IL-10, IL-12, or IL-23, respectively. The latter cytokine is also produced by activated amoeboid microglia and primes invading T helper (Th) cells that become encephalitogenic on interaction with the T cell receptor (TCR) and MHC class II molecules on local antigen-presenting cells (APCs). Activated microglia produce C-C motif chemokine (CCL) 2, which might induce recruitment of monocytes. This scenario induces damage to the myelin sheaths around neuronal axons.

The Microglial Signature and its Modulation Recent profiling studies have revealed that microglia – located in the CNS separated by the BBB – differ markedly from other tissue macrophage populations with respect to their gene expression profile and chromatin state [26–28]. Novel myeloid-specific gene targeting techniques

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exploiting the promoter of CX3C chemokine receptor 1 (CX3CR1) (also known as the fractalkine receptor) have opened new possibilities to investigate the kinetics of myeloid cell turnover in the steady state as well as pathological conditions [29,30] (for a review see [31]). Microglia were shown to display a unique expression profile that discriminates them from their peripheral macrophage relatives [26,28,32,33]. Importantly, more recent studies revealed the underlying distinct tissue-specific enhancer usage and chromatin states of different tissue-specific macrophages [27,28]. Notably, these studies suggest that these molecular genetic imprints are shaped in part during development but probably more prominently by the local tissue environment. Furthermore, these pioneering studies indicate that enhancer usage remains plastic and might be reprogrammable by changing the surrounding environment [28]. A recent study has highlighted the host microbiota as an important environmental factor that shapes microglia under steady-state conditions. Thus, products of intestinal commensals were shown to provide stimuli that affect microglial maintenance and function in mice [34]. Housing mice under completely sterile conditions (also termed ‘germ free’) led to a pronounced immature phenotype of microglia, including upregulation of several surface proteins (e.g., CSF-1R, F4/80, CD31) that are consistently downregulated in mature adult microglia [16]. Moreover, decreased expression of numerous activation markers (e.g., Il-1a, Stat1, Jak3, B2m, Trim30a) and increased mRNA levels of inhibitors of transcription such as Nfkbia (which encodes IkB/) were found. Furthermore, the crucial microglial transcription and survival factor Sfpi1, encoding PU.1, was significantly upregulated in microglia from germ-free mice. In line with altered expression of genes regulating the cell cycle (such as Ddit4, Iqgap, Cdk9, Ccnd3, and Bcl2), microglial numbers were found to be increased in various brain regions under germ-free conditions, suggesting that physiological reduction of microglial number is affected during early postnatal development [35]. Interestingly, this finding contrasts with BM-derived hematopoietic cells such as neutrophils and monocytes, which are generally found in the absence of a microbiome [36]. Notably, these alterations were found to be extremely plastic in adult mice. If the gut bacteria of conventionally housed specific pathogen-free (SPF) mice were eradicated by treatment with antibiotics, microglia gained the immature status. Vice versa, recolonization of mice harboring a reduced flora (tricolonized ASF mice) with a complex microbiota led to microglial ‘maturation’, indicating great plasticity of the gut–microglial connection. Remarkably, short-chain fatty acids (SCFAs), microbiota-derived bacterial fermentation products, were determined to indirectly mediate microglial homeostasis independent of TLR signaling [34]. Several molecules are known to be essential for proper microglial function and development in addition to the aforementioned TFs PU.1 and IRF8. Colony-stimulating factor 1 receptor (CSF1R) (also known as macrophage colony-stimulating factor receptor or CD115), encoded by the c-fms gene, is an integral tyrosine kinase transmembrane receptor and highly expressed on the microglial surface. CSF-1R is essential for the proliferation, maturation, function, and survival of microglia [37], as for other mononuclear phagocytes [38]. Accordingly, microglial numbers are significantly reduced in CSF-1R knockout mice [15,39]. Interestingly, however, lack of CSF1 did not affect microglial density to the same extent, suggesting at least one more additional ligand. A second CSF-1R ligand, a cytokine called interleukin-34 (IL-34) was described [40] and found to be expressed in the postnatal mouse brain, mainly by neurons [41]. Although microglia and their yolk sac progenitors were shown to develop independently of IL-34, they rely on the factor for their maintenance in the postnatal brain [42,43]. Thus, IL-34-deficient mice harbor decreased microglial numbers in certain brain compartments [42,43]. Another factor that was proposed to play a major role in microglial development is transforming growth factor beta (TGF-b). TGF-b1 was shown to imprint a characteristic molecular microglial signature in in vitro cultures [27,32]. Moreover, microglia were reported to be reduced in the CNS of TGF-b1-deficient mice [32]. Additional intrinsic and extrinsic factors are likely to define microglial expression profiles and are critical for microglial functions during health and disease [37].

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Microglial Function during Homeostasis For a decade it has been established that microglia perform tissue surveillance under healthy conditions, in addition to their well-known roles in defense against bacterial and viral CNS infection, injury, and neurodegenerative and autoimmune diseases [6,44,45]. Using CX3CR1GFP mice [46], Nimmerjahn and colleagues showed with two-photon in vivo imaging that microglia in the healthy adult brain constantly explore their environment by virtue of highly dynamic processes, but without cell body movement [47]. Moreover, microglia rapidly respond by redirection of these extensions to sites of injury [47]. Activation of microglia is at least in part mediated by extracellular ATP, which is secreted by other brain cells (e.g., after brain injury) and is critical for the rapid recruitment of microglia to injury sites as well as for the release of neurotrophic factors [48]. This nucleoside triphosphate is recognized by G-protein-coupled purinergic receptors. Accordingly, several ionotropic (P2X4 and P2X7) and metabotropic (P2Y1, P2Y2, and P2Y12) purinergic receptors are found to be expressed on the microglial surface [49,50]. Interestingly, the processes of neighboring microglia usually do not contact each other under steady-state conditions, indicating a territorially restricted distribution. Rather, during sampling microglial processes are frequently in transient direct contact with adjacent neurons and synaptic spines [51]. Microglia were found to be crucial for the elimination of redundant dendritic spines by phagocytosis during brain development [52]. This so-called ‘synaptic pruning’ is necessary for the proper formation and maturation of neural circuits [52]. Synaptic pruning is thought to involve CX3CL1 expressed by neurons during synapse maturation and its receptor CX3CR1 on microglia [52]. Interestingly, the CX3C axis seems to also be involved in the correct wiring forebrain circuits [53]. The mechanisms that underlie microglial–synapse or microglial–axonal interactions remain unsolved. Interestingly, the synaptic modifications conducted by microglia were shown to be dependent on complement factors like complement receptor 3 (CR3) [also known as CD11b/CD18 or macrophage 1 antigen (Mac-1)] [54,55] (for a detailed review, see [56] or the review by Beth Stevens and Brian McVicar in this issue). Brain function requires a dynamic and flexible architecture of communicating neurons. Sensory input is handled through synaptic plasticity. Intriguingly, it was suggested that the maintenance and modification of synapses is linked to viable microglia. Specifically, ablation of microglia in adult mice using a diphtheria toxin-based strategy was shown to affect learning-dependent synapse formation [57]. Furthermore, genetic depletion of brain-derived neurotrophic factor (BDNF) from microglia using CX3CR1CreER–BDNFfl/fl mice largely mirrored this phenotype, suggesting that BDNF production by microglia might be a crucial factor for synaptic remodeling associated with learning and memory [57,58].

Microglial Function during Disease Under pathological conditions, microglia encounter another changed environment distinct from the challenges associated with CNS development and adult homeostasis. Pathology means altered stimuli and microglia respond to these alterations by activation and potential redirection of their phagocytic activity from synaptic pruning to the clearance of hazardous factors [45,54]. As a major part of the innate CNS immune system, microglia are believed to prominently contribute to CNS pathologies, including their establishment, perpetuation, and resolution. Long-lasting chronic inflammation was proposed to drive the physiological functions of microglia off balance. For instance, microglia were described in vitro to clear and digest amyloid beta (Ab) plaques, a main feature of Alzheimer's disease pathology [59]. In vivo, however, microglial clearance of amyloid is thought to be impaired by chronic Ab exposure [60]. Interestingly, inhibition of IL-12/IL-23 signaling was shown to reduce Alzheimer's disease-like pathology and cognitive decline. Thus, BM chimeras, in which expression of the proinflammatory cytokines IL12 and IL-23 is restricted to peripheral immune cells, harbor a reduced Ab plaque load [61]. Microglia are also assumed to have a detrimental effect in Parkinson's disease (PD); thus,

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lipopolysaccharide (LPS) treatment was shown to result in an inflammatory microglial response followed by the death of dopaminergic neurons, a major hallmark of PD [62]. Many of the studies addressing in vivo microglial functions have focused on the contributions of these cells to pathologies associated with demyelination, including chemically induced demyelination and experimental autoimmune encephalomyelitis (EAE). Cuprizone, a copper chelator, is thought to disrupt metabolic processes specifically in oligodendrocytes, leading to their death [63]. Oligodendrocyte apoptosis results in demyelination, which is most pronounced in the largest white matter commissure of the brain, the corpus callosum. Importantly, the BBB seems to remain intact in this model and no recruitment of peripheral cells to the CNS is observed [25]. However, this form of primary demyelination is reversible, as mice that are returned to a cuprizone-free diet remyelinate their axons within 2 weeks [64]. Remyelination requires prior clearance of myelin debris by microglia. A microglial phenotype supportive of remyelination in the cuprizone model was described that is characterized by elevated expression of genes associated with phagocytosis [65]. In line with these findings, CX3CR1-deficient microglia, which display impaired phagocytic activity, were also reported to fail to support remyelination [66]. Collectively, these studies support a beneficial role of microglia in this demyelination model and their requirement for resolution, highlighting the importance of these professional phagocytes in re-establishing health [65]. EAE is the most commonly used animal model for multiple sclerosis (MS), sharing with the latter an autoimmune etiology, although the merits of EAE as a MS model remain debated [67]. EAE is induced by peripheral immunization with myelin peptides in an adjuvant context and is, in contrast to the cuprizone model, characterized by prominent BBB disruption associated with massive immune infiltration of the CNS. This includes myelin-specific T lymphocytes, monocytes, and DCs [68]. EAE thus causes a far more complex environment, in which resident microglia not only interact with injured neurons and macroglia but are also faced with peripheral invaders. The study of the contributions of microglia to the perpetuation and resolution of inflammatory CNS reactions remains a major challenge, particularly with respect to the assignment of distinct molecular contribution to disease pathophysiology. Discrimination of resident microglia and monocyte-derived macrophages (moMFs) is difficult, as these two populations share many surface markers and potentially can engage in similar effector functions. Results of experiments involving genetic manipulation of microglia using the LysMCre system and ablation studies using herpes simplex virus thymidine kinase (HSV-TK) expression driven by the CD11b promoter implied a detrimental role of microglia in EAE development [31,69,70]. However, these strategies also ‘hit’ macrophages and other myeloid cells and thus do not allow definitive conclusions on microglia-specific roles in these settings. Goldmann and colleagues recently introduced a novel Cre–LoxP-based system that specifically targets microglia but spares moMFs [29,30]. CX3CR1CreER animals harbor a transgene that encodes a Cre recombinase fused to a mutant estrogen ligand-binding domain and is sequestered in the cytoplasm; nuclear translocation and thus activation of the CreER molecule requires the estrogen antagonist tamoxifen (TAM) [30]. Following TAM treatment, rearrangements occur not only in microglia but also in other, peripheral CX3CR1+ cells, including monocytes [30]. However, since the latter are continuously replaced by BM-derived cells [30,71], rearrangements in these cellular compartments are only transient while long-lived CX3CR1+ cells, such as microglia, remain permanently modified [72]. Interestingly, mice harboring specific microglial ablation of MAP3K, TGF-b-activated kinase 1 (TAK1), a ‘master kinase’ critical for activation [73], are relatively protected from EAE [29]. Of note, TAK1-deficient microglia seem unaffected in the steady state. However, during challenge these cells fail to acquire the typical activated amoeboid morphology and display significantly decreased production of the proinflammatory cytokine IL-1b and the CCL2 chemokine. In addition, immunized CX3CR1CreER:TAK1fl/fl mice exhibit reduced immune CNS infiltration and demyelination [29]. These data support the hypothesis of significant microglia; contributions to EAE induction.

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Recently, the dissection of resident microglial and moMF contributions to the disease was approached using high-resolution electron microscopy techniques in CCR2RFP/+:CX3CR1GFP/+ double-reporter mice [74]. Interestingly, gene expression profiling revealed that, compared with moMFs, microglia display a rather inert signature at the onset of clinical EAE symptoms. In accordance, microglia were reported by others to express lower levels of activation markers and some inflammatory cytokines in EAE, relatively to monocyte infiltrates [75]. However, the exact molecular contributions of microglia in EAE, be they antigen presentation or secretion or production of proinflammatory agents and chemokines perhaps in the later stages of the disease, remain to be determined. Progress in this field should profit from the CX3CR1CreER system combined with the respective ‘floxed’ alleles. Microglial activation was previously suggested to play a major role in EAE development and progression [69,70,76]. On sensing of injury, microglia undergo rapid activation and are believed to respond by increased phagocytosis and production of cytokines and chemokines as well as, potentially, antigen presentation [45]. Presentation of antigen in the MHC class II (MHCII) context is critical for EAE pathophysiology, as it drives the expansion of myelinspecific CD4+ T cells [68]. After their initial activation and polarization toward T helper 17 (Th17) cells in the peripheral lymph nodes by antigen-presenting cells (APCs) [77,78], these pathogenic T lymphocytes invade the CNS. There they are thought to require local APCs for re-stimulation, culminating in detrimental CNS inflammation and eventually demyelination [78,79]. The exact identity of these CNS-resident APCs remains to be revealed; microglia, infiltrating moMFs, and DCs are prime candidates. Involvement of DCs in the activation process was demonstrated in several studies [80–82]. However, depletion of CD11chi classical DCs and plasmacytoid DCs using the CD11c–DTR or CD11c–DTA system [83,84] in combination with anti-PDCA administration did not impair EAE development [85]. As opposed to DCs and other macrophages, murine resident microglia were demonstrated to express surface MHCII on activation but not in the steady state. Microglia and macrophages were shown in vitro to present myelin antigens, although they required a higher APC/T cell ratio than DCs for efficient T cell stimulation [80]. However, to date neither microglia nor macrophages have been definitively shown to contribute to EAE development by antigen presentation. Moreover, productive T cell activation requires not only antigen presentation but also costimulatory signals. Although microglia rapidly upregulate MHCII at disease onset, expression of molecules such as CD80 and CD86 is rather low [86]. As T cell receptor binding of MHCII in the absence of costimulation results in T cell apoptosis or anergy rather than activation [78,86,87], microglia may play a tolerogenic role similar to immature DCs [85,87]. Of note, microglia also elevate MHCII expression in the cuprizone model, which does not involve T cell infiltration [88–90]. This might imply MHCII contributions to microglial function other than in antigen presentation [91]. While evidence for APC involvement of microglia or infiltrating macrophages in EAE is missing, imaging of perivascular/meningeal macrophages has shown that these cells are capable of interacting with and presenting antigen to adoptively transferred pathogenic T cells at the CNS borders [92]. This might suggest a role of CNS-resident phagocytes other than microglia in the re-stimulation event. Phagocytosis remains one of the main established functional features of microglia in development, homeostasis, and pathology. During EAE, microglia were shown to express triggering receptor expressed on myeloid cells 2 (TREM-2), a critical activator of phagocytic activity. TREM2-deficient microglia harbor in vitro the reduced phagocytic capacity of dying neurons and increased production of proinflammatory molecules, whereas microglia overexpressing TREM-2 display increased phagocytosis and reduced inflammation [93]. Microglial clearance of myelin debris is critical for remyelination following a cuprizone challenge [66]. In EAE, phagocytosis can be both beneficial and harmful. Microglia were demonstrated to clear debris at the onset of clinical symptoms and to express an overall inert phenotype relative to infiltrating moMFs. The

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latter, in contrast, accumulate at the nodes of Ranvier and were shown to strip axons of their coverage; moMFs therefore seem to actively engage in demyelination while simultaneously secreting toxic factors [74]. EAE-associated activated microglia also prominently secrete cytokines, including both pro- and anti-inflammatory factors, as well as chemokines. The most extensively studied proinflammatory agents are tumor necrosis factor alpha (TNF/), IL-6, IL-12, and IL-23, which were shown to have detrimental effects on disease progression [45]. In particular, IL-23, the most recently identified IL-12 family member, has received major attention. Mice lacking the unique subunit of IL- 23 p19 were shown to be protected from EAE [94] while mice lacking p35, which is part of IL-12 but not IL-23, are susceptible [95,96]. IL-23 is required for the terminal differentiation and survival of pathogenic Th17 cells [77,97–99], the main drivers of CNS autoimmunity in this model. Both microglia and infiltrating moMFs were demonstrated to express IL-23 in MS plaques [94,100]. BM chimeras, in which only peripheral immune cells and not CNS-resident cells express IL-23, display a somewhat milder disease, indicating the significance of local CNS production of this inflammatory agent [101]. However, when grafted into IL-23-deficient mice, myelin-specific Th17 cells cause disease. Once encephalitogenic lymphocytes are generated in the periphery, EAE thus can progress even in the absence of CNS-derived IL-23 [102]; IL-23 production by resident microglia might hence be obsolete in this model. Notably, the abovementioned conflicting results might largely be due to our limited understanding of the differential contributions of microglia and moMFs, as discussed above. Microglia are also potent secretors of anti-inflammatory cytokines, including IL-4 and IL-10, which are beneficial for the disease outcome. Both IL-4- and IL-10-deficient mice display aggravated EAE courses [103,104]. Microglia were suggested to be the main IL-4 source, as lack of CNS but not peripheral IL-4 leads to exacerbated symptoms [105]. Specifically, IL-4 was shown to enhance oligodendrogenesis in EAE and to reverse the apoptotic effect of interferon gamma (IFNg) [106]. However, astrocytes also express IL-10 [107]. Microglia are also chief producers of chemokines inducing and guiding cell migration, such as CCL2, CCL3, CCL4, CCL5, CCL12, and CCL22, all of which are thought to play a role in the EAE course [108]. The role of CCL2, expression of which is induced by TNF/, IL-1b, and IFNg during CNS inflammation [109], is especially intriguing. Besides disrupting BBB integrity [110], CCL2 also induces the infiltration of Ly6Chi monocytes into the CNS, which express the corresponding receptor CCR2. Ly6Chi CCR2+ cells are crucial for the effector phase of the disease and invade the CNS in a CCR2-dependent manner [111]. The question remains: which CNS-resident cell is the critical source of CCL2? Besides microglia, CCL2 was also reported to be produced by astrocytes, endothelial cells, and neurons. Endothelial- and astroglia-derived CCL2 was shown to play a role in various stages of disease, although conditional ablation in these cells did not lead to disease resistance [112]. It would be an interesting twist if microglia recruit monocytes and thus call in additional macrophages with new functions. However, whether microglial CCL2 is critical for the induction of CNS autoimmunity will have to be studied by specific genetic manipulation of microglia.

Outstanding Questions What determines the recruitment to the developing brain of myeloid precursors in the yolk sac and their becoming microglia? Are there microglial sources in the developing embryo other than the yolk sac? What is the role of the BBB in the establishment of the microglial pool during development? How plastic are microglia at defined time points in development? What is the lifespan of microglia? What is the basis of microglial heterogeneity in the CNS? How is microglial activation and maturation achieved under steady-state conditions? What is the fate of microglia following disease? What determines microglial quiescence, stimulation, and cell division? How can disease-associated SNPs shape microglial function? How is microglial dysfunction achieved? How do microglia communicate with each other and with their neighboring cells in the CNS? Given that most our insights are derived from mouse models with limited lifespans, do HSC-derived cells contribute to the human brain macrophage compartment with progressive time and aging? If so, do HSC-derived brain macrophages and microglia differ in function?

Concluding Remarks Microglia are a unique population of long-lived macrophages that are intimately associated with CNS development and homeostasis in the adult. Despite major recent advances in our understanding of these cells, their specific molecular contributions to brain function and details of their crosstalk with neurons and astrocytes remain largely to be defined (see Outstanding Questions). The uniqueness of microglia is established by environmental signals and much of their key signature is lost when the cells are taken in culture [27,32]. In-depth understanding of microglial functions, including the heterogeneity of these cells in brain regions as distinct as the

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cerebellum, cortex and spinal cord, and aging-related alterations hence require their study in context, using intravital imaging and conditional targeted mutagenesis. Moreover, microglial studies are an example par excellence of the need for interdisciplinary research efforts by neurobiologists and immunologists, which only when intimately joined will unravel the full spectrum of the biology of these exciting cells. References 1.

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