Department of Immunology, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Victoria, Australia
Correspondence: Professor Jennifer L Wilkinson-Berka, Department of Immunology, Monash University, Alfred Medical Research and Education Precinct (AMREP), Level 5, Alfred Centre, 99 Commercial Road, Melbourne, Victoria 3004, Australia. Email: Jennifer.firstname.lastname@example.org
Microglia are the resident immune cells within the brain and retina, commonly known as the macrophages of the central nervous system (CNS). Microglia survey the surrounding milieu to eliminate invading microbes, clear cellular debris and enforce programmed cell death by removing apoptotic cells. Complementary to their ‘house-keeping’ role, microglia are capable of releasing brain-derived neurotrophic factor (BDNF), as well as various anti-inflammatory cytokines that sustain and support neuronal survival.
Although microglia are essential for maintaining a healthy CNS, paradoxically they may undergo phenotypic changes to influence numerous neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease. Understanding the underlying mechanisms that determine whether microglia are supportive or toxic could elucidate novel and more effective therapeutic targets to treat an array of neurological and retinal diseases.
Although relatively little is known about the influences that evoke phenotypic changes in the microglial population, there is accumulating evidence illustrating an interaction with the renin-angiotensin system (RAS). The angiotensin AT1 and AT2 receptors may have differential roles in mediating the activity of microglia. Understanding the actions of these angiotensin receptors will be important in defining whether microglia are an important therapeutic target for RAS blockade in brain and ocular diseases.
Initially, it was thought that, like neurons, astrocytes and oligodendrocytes, microglia were derived from the neuroectoderm; however, recent evidence demonstrates they share some common monocyte markers and therefore stem from the myeloid cell line.[1, 2] Moreover, in certain situations microglia are completely indistinguishable from bone marrow-derived peripheral macrophages, indicating they are likely to originate from the same precursor cells. However, microglia have retained the ability to divide via mitosis and therefore are not wholly reliant on bone marrow to maintain a constant population.[1, 4] Furthermore, the capacity for self-renewal implies that not all microglia cross the blood–brain barrier (BBB) from the periphery and that there is a population of resident central nervous system (CNS) microglia.
“Microglia stem from the myeloid cell line”
Morphology and activation state
Microglia are a dynamic cell population and exist in various states of activation, which are associated with distinct morphological transformations. In the normal basal state, microglia are ramified with long, star-like projections that extend into the surrounding environment. Although this was thought to be their resting state, it is now clear that microglia are always active, constantly monitoring the local environment by extending and retracting their spindle-like processes. In their ‘ramified phenotype’, microglia can be differentiated from macrophages by assessing the level of leucocyte CD45 receptor expression, which is abundantly expressed in macrophages but only sparsely in microglia.[6-8] However, in response to injury, microglia will retract their spindles and adopt an amoeboid appearance, upregulating the level of CD45 receptor expression in the process and making them indistinguishable from macrophages. The observation that microglia undergo these transformations has led to the suggestion that, similar to macrophages, microglia are able to exist in two polarized states. Recently, Hu et al. showed that, in vitro, when microglia are adjacent to healthy neurons they will assume a protective M2 phenotype. In contrast, when put in the proximity of a damaged neuron, microglia gradually morph into a proinflammatory cell, illustrating their paradoxical nature. Similarly, microglia have been categorized on the basis of superoxide production, with distinct populations being identified and labelled as either high or low superoxide producers and as being detrimental or protective accordingly.
“Microglia are able to exist in two polarized states”
Interaction with other cell types
Microglia exist in a symbiotic relationship with neurons, where microglia sustain neurons and neurons maintain microglia in a non-inflammatory phenotype. In vitro, unpolarized microglia will increase neuronal survival in both normal and hypoxic situations, suggesting that the inherent role of microglia is a supportive one. Moreover, this basal trophic influence of neutral microglia can be further augmented by certain cytokines that cause polarization into an M2 anti-inflammatory phenotype. Conversely, when microglia are pushed into the M1 phenotype by an injured neuron or toxic background, they will become phagocytic and noxious to healthy cells. Interestingly, this codependent relationship is evident during development, such that microglia are able to guide neuronal differentiation down either the glial or neuronal line depending on specific cytokine stimulation. Specifically, when stimulated with interleukin (IL)-14, microglia will evoke gliosis and consequently oligodentrocytes will develop; yet, in the presence of interferon-γ neurogenesis will occur. Moreover, when in an environment not conducive for cell survival, microglia will prevent differentiation altogether.
In addition to having an intimate relationship with surroundings neurons, there is also a significant amount of cross-talk between microglia and the adaptive immune system. Microglia express both Class I and II major histocompatibility complex molecules (MHC). In the presence of certain cytokines, these MHC are upregulated, allowing microglia to act as antigen-presenting cells and become an integral component in the immune network.[12, 13] Microglia are also able to modify lymphocyte adherence, migration and activation via the secretion of chemokines and proinflammatory cytokines, such that microglia modulate the activity and function of nearly all peripheral immune cells that cross the BBB. Consequently, their close communication with immune cells, in conjunction with their ability to adopt various phenotypes, enables microglia to tailor their function specifically to the surrounding milieu.
Perivascular microglia have also been described and are viewed to have a key role in the promotion of normal vascularization of the retina (Fig. 1). This has been demonstrated in studies in which a deficiency in microglia leads to disruption of the developing retinal vasculature.[15, 16] Interestingly, intravitreal injection of microglia into microglial-depleted retinas restores physiological retinal vascularization. Rymo et al. suggest that a two-way communication exists between microglia and the growing tips of retinal blood vessels, whereby microglia migrate to the vicinity of sprouting vessels in response to factors expressed on these vessels and subsequently release soluble factors that influence blood vessel growth. Although the nature of these factors is not entirely clear, retinal microglia are known to release a number of pro-angiogenic mediators, including tumour necrosis factor (TNF)-α,[18, 19] IL-1β, reactive oxygen species and cyclo-oxygenase-2. Further studies in this area have identified that microglia participate in the sprouting of developing retinal blood vessels by processes that involve stromal-derived growth factor-1/CXCR4. Of interest is a recent study that determined that resident myeloid cells in the retina (positive for the microglial markers ionized binding adaptor molecule-1 (Iba1) and CD11b) can directly suppress angiogenesis by producing the vascular endothelial growth factor inhibitory receptor Flt1. This effect was shown to depend on myeloid non-canonical Wnt ligands. Similar to findings in the brain, a study by Mendes-Jorge et al. has indicated an important distinction between perivascular microglia and perivascular macrophages. These authors identified that a small population of resident perivascular cells in the retina are different from retinal microglia due to their expression of the macrophage markers BM8 (F4/80) and monocyte/macrophage-2, the absence of labelling for Iba1, constitutive expression of scavenger receptors class A and particular autofluoresence. Perivascular macrophages were suggested to contribute to the maintenance of the blood–retinal barrier (BRB) by scavenging blood-borne proteins and lipids.
Injurious functions of microglia
Although a basal level of microglial activity is essential for maintaining a healthy CNS, overactivation can result in oxidative damage, which is a common feature of many neurological disorders, including Parkinson's disease (PD), Alzheimer's disease (AD), multiple sclerosis (MS) and Huntington's disease. Constantly surveying the local environment, microglia will respond to proinflammatory triggers, such as lipopolysaccharide (LPS) from Gram-negative bacteria and disease pathogens, by assuming a proinflammatory phenotype. During this defensive reaction, microglia acquire the ability to release a host of toxic cytokines, including TNF-α, IL-1β, superoxide and nitric oxide, to destroy any potential threat.[1, 27] Unfortunately, the lack of specificity of this protective armoury often causes damage to surrounding neurons, which has serious consequences if the process becomes unregulated. In a disease setting, invading pathogens are able to recruit the proinflammatory microglia to perpetuate the disease pathology. In AD, microglia cluster around the neurotoxic β-amyloid plaques, exacerbating the oxidative damage to underlying neurons. Similarly, chronic neuroinflammation associated with elevated numbers of activated microglia has been reported in PD and MS. The role of microglia in the pathology of PD and MS is discussed in further detail below when specifically addressing the involvement of the renin–angiotensin system (RAS) in both disease settings.
Microglia can also injure the retina by releasing proinflammatory and pro-angiogenic factors in diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Age-related macular degeneration is a major cause of irreversible blindness in people over 50 years of age. In AMD, microglia can have a protective role by migrating to the area of the retina between the retinal pigment epithelium (RPE) and photoreceptors (subretinal space). Here, microglia engulf waste material, including lipofuscin, a constit-uent of drusen, which is involved in the atrophy of the RPE and the development of AMD. The CX3C chemokine receptor 1 (CX3CR1) and monocyte chemoattractant protein-1 (MCP-1) are likely to be involved in these actions of microglia.[30, 31] Conversely, microglia may initiate pathology, with studies in mice deficient in CX3CR1 and MCP-1 showing the accumulation of microglia and drusen in the subretinal space, degeneration of the RPE, choroidal neovascularization and atrophy of photoreceptors.[30, 31] Diabetic retinopathy is the major cause of vision impairment and loss in people of working age. It develops over one to two decades and involves progressive damage to the retinal microvasculature, neurons and glia. Microglia become activated in DR[19, 33] and, as for the aforementioned brain disorders, are considered to elicit a neuroinflammatory state in the retina. For example, the upregulation of MCP-1 in retinal neurons was reported to activate retinal microglia, an effect that was exacerbated by exposure to advanced glycation end-products. Of interest is a report that minocycline, an anti-inflammatory and neuroprotective agent, repressed the release of cytotoxins from activated microglia and significantly reduced caspase 3 activity within the retina. The injurious actions of activated microglia on retinal neurons extend to ROP, a major cause of vision loss in preterm infants that occurs following exposure to a varying oxygen environment.[18, 35]
“Microglial overactivation is a feature of various CNS disorders”
In addition to roles in neuroinflammation, microglia may contribute to pathological neovascularization in the brain and retina by releasing angiogenic factors that work in concert with vascular endothelial growth factor to stimulate endothelial cell sprouting.[17, 36, 37] For example, in individuals with DR, microglia accumulate around regions of vascular damage, including dilated veins, microaneurysms, intraretinal haemorrhages, cotton-wool spots and the optic nerve, as well as retinal and vitreal neovascularization. Brain disorders such as PD, AD and glioblastoma are also considered to be pro-angiogenic states and are associated with the increased proliferation of microglia. The precise mechanisms by which microglia influence pathological neovascularization in the brain and retina remain to be fully understood.
Protective function of microglia
Although microglia are often classified as a defensive immune cell, they are also a vital component in the endogenous reparative pathway. Elimination of microglia via intracerebroventricular injection of liposome-encapsulated clondrate will exacerbate the degree of tissue damage after stroke, demonstrating a blunting of the recovery processes. Microglia have been shown to potentiate the release of brain derived neurotrophic factor (BDNF) in response to damage, which accelerates wound healing and is likely to be one of the mechanisms by which microglia reduce injury.[1, 43, 44] Indeed, in response to head trauma, exogenously administered microglia display a specific affinity for damaged tissue, migrating directly to the injured area to reduce the extent of neuronal loss. In addition, in both stroke and traumatic head injury, microglia release anti-inflammatory cytokines, such as IL-10 and transforming growth factor-β, which are likely to facilitate the reparative actions of BDNF.[45, 46] More recently, it has also become apparent that the classical injurious cytokines TNF-α and IL-1β seem to be necessary to trigger a series of events required for neuronal repair, such that mice lacking these cytokines exhibit impaired remyelination.[47-49] The phagocytic removal of cellular debris, facilitated by potentially noxious cytokines, is required before regeneration can occur.
“Microglia potentiate the release of BDNF and anti-inflammatory cytokines”
Apart from these anti-inflammatory and neuroprotective effects, microglia also play a critical role in normal developmental angiogenesis and vascular repair in both the brain and retina. Microglia migrate into the brain and have been shown to shape and prune the developing vasculature and are found at sites of vascular repair. As discussed above, a similar situation occurs in the developing retina, where intravitreal injection of microglia promotes repair of the damaged retinal microvasculature. Furthermore, an excellent study by Ritter et al. showed that the intravitreal administration of bone marrow-derived myeloid progenitor cells during ROP caused the migration of these cells to the damaged retina, where they differentiated into microglia to enhance vascular repair. Overall, it is clear that microglia have a dual life, contributing in certain situations to neurovascular pathology and also playing a key role as a line of defence against tissue damage.
Introduction to the RAS
The RAS is a hormonal cascade that has multiple roles throughout the body, including maintaining cardiovascular, renal and adrenal function. Furthermore, the influence of the RAS extends to effects as diverse as proliferation, differentiation, regeneration and apoptosis. Angiotensin (Ang) II, an octapeptide hormone, is the main effector peptide in the RAS and can activate numerous signalling cascades by binding to various cell surface receptors. Although there are several AngII receptors, the two predominant G-protein-coupled receptors are known as the AT1 and AT2 receptors (AT1R and AT2R, respectively). The dominant AT1R is associated with most of the biological and pathological actions of AngII and usually masks the influence of the AT2R. The AT2R is highly expressed in fetal tissue, yet is only expressed at low levels in adult tissue.[51, 52]
Central and ocular RAS
In contrast with peripheral organs, where angiotensin peptide production is mainly the result of kidney-derived renin acting on liver-derived angiotensinogen, angiotensin peptide production in the CNS is dependent on locally synthesised RAS components. Angiotensinogen is synthesised and secreted by glia.[54, 55] However, renin is relatively absent in the brain and the enzymes responsible for the cleavage of angiotensinogen to produce angiotensin peptides remain to be defined. Furthermore, the major AngII receptor subtypes (AT1R and AT2R) have been identified in the brain.[56, 57] In healthy conditions, the AT1R is the most abundant, being highly expressed in areas involved with cardiovascular control and fluid and electrolyte homeostasis. Central administration of AngII will cause an increase in blood pressure, fluid intake and vasopressin release, all of which are mediated via the AT1R. In certain disease settings of the CNS, the AT2R is upregulated, which has led to the speculation that it may be involved in the body's endogenous reparative pathway.[59, 60]
All components of the RAS have been identified in the retina in either vascular, neuronal or glial components and in the RPE. The RAS is known to play an important role in the development of AMD, DR and ROP.[61-66] Briefly, blockade of the RAS at the level of the AT1R and angiotensin-converting enzyme can improve neurovascular pathology in these diseases. Despite this information, it is only recently that components of the RAS have been localized to retinal microglia. Primary cultures of rat retinal microglia express the AT1R, the (pro)renin receptor, the mineralocorticoid receptor (MR) and aldosterone synthase.[61, 67] However, it has not been determined whether other components of the RAS, including the AT2R, are present in retinal microglia.
Although yet to be studied in detail, there is evidence that pharmacological manipulation of the RAS with AT1R antagonists influences the activity of microglia within the brain and retina (see below). For this to occur, these agents must cross the BBB and BRB, which are structurally similar, and act to prevent the entry of toxic and damaging factors into brain and ocular tissues. Angiotensin receptor blockers (ARBs) differ in their tissue penetration and therefore not all may necessarily cross the BBB and BRB under normal conditions. However, certain ARBs, such as candesartan and telmisartan, have been shown to effectively inhibit centrally mediated AngII responses when administered orally, which illustrates the ability of these compounds to cross the BBB. In disease, both barriers may be compromised to allow RAS blockers to more efficiently reach target cells within the brain and retina. Certainly in retinal diseases such as ROP, DR and wet AMD, vascular leakage from the BRB is a characteristic feature and therefore RAS blockers are highly likely to reach angiotensin receptors located in retinal tissue. Likewise, various diseases of the CNS, including stroke, AD and MS, cause a similar breakdown in BBB integrity that will also render the central angiotensin receptors accessible to previously impenetrable compounds.
Microglia and the RAS
Although it has been established that AngII-mediated AT1R signalling results in the activation of proinflammatory pathways in numerous cell types, including cardiac myocytes, vascular endothelial cells and neurons, the role of the RAS in microglial cell function is not as well understood. In terms of disorders of the CNS, blockade of the AT1R has been found to be beneficial in AD, in which disease progression was slowed in patients treated with an ARB, and depression, in which patients taking an ARB required a lower dose of antidepressant to achieve an efficacious response. These clinical findings are supported by animal studies showing reduced neuronal damage in animals treated with an ARB following experimentally induced AD, PD, traumatic head injury and stroke. Interestingly, reduced microglial activation is a common feature of the therapeutic effect of ARBs in all the aforementioned disease states. Studies directly evaluating the RAS and brain microglia have shown that AngII modulates the production of inflammatory cytokines from microglia via the AT1R.[26, 57, 72] For example, activation of primary microglial cells using LPS causes an upregulation of AT1R expression, which coincides with an elevation in the production of proinflammatory transcription factors nuclear factor-κB and activator protein-1. Moreover, when AT1R signalling is attenuated, the response to LPS is significantly blunted, suggesting that the AT1R is responsible for triggering the inflammation observed previously.[57, 73] In accordance, inflammatory cytokines produced by microglia via the AT1R also appear to play a central role in AngII-induced neurogenic hypertension. Chronic AngII infusion has been shown to activate microglia, specifically in the paraventricular nucleus (PVN), which release various proinflammatory cytokines that elevate blood pressure. However, when microglial activation is prevented using minocycline, which, as mentioned previously has both anti-inflammatory and neuroprotective properties, AngII hypertension is attenuated. Similarly, blockade of the AT1R in the PVN abolishes the elevation in systolic pressure, suggesting that AngII activation of microglia via the AT1R evokes the production of inflammatory mediators, which ultimately cause a rise in peripheral blood pressure.
In the context of neurological disease, blockade of the AT1R has been shown to reduce microglial activity and suppress inflammation, alleviating some of the symptoms of both PD and MS. In a model of PD, where 1-methyl-1–4-phenyl-1,2,3,6-tetrahydropyridine is administered to selectively destroy the striatal dopaminergic neurons, deletion of the AT1R decreases microglial activation and preserves the vulnerable dopaminergic cell population. In agreement, in the experimental autoimmune encephalomyelitis model of MS, AngII has been shown to trigger and sustain chronic neuroinflammation through paracrine signalling, causing microglial release of inflammatory cytokines that gradually demyelinate local axons. The administration of the AT1R antagonist candesartan interrupts the toxic auto-inflammatory cycle, blunts the release of inflammatory mediators from the microglia and delays the onset of MS symptoms. Similarly, candesartan affords protection following traumatic head injury, whereby a single dose significantly reduces the impact of microglial-derived proinflammatory cytokines IL-1β and TNF-α. The attenuation of post-traumatic inflammation correlates with a sparing of neuronal tissue and improvement in behavioural deficit 5 days after injury, highlighting the capacity for AT1R antagonists to modulate microglial activity and potentially be used as therapeutic tools in diseases in which inflammation is a dominant feature, such as PD, MS and head injury.
“AT1R antagonists modulate microglial activity”
Few studies have evaluated the role of the AT1R in retinal microglia. However, there is evidence in a rat model of ROP that blockade of the AT1R with valsartan and inhibition of aldosterone synthase with FAD286 reduces microglial density, which is associated with a reduction in retinal neovascularization and the expression of inflammatory factors. Although that study points towards a role for microglia in the pro-angiogenic effects of AngII and aldosterone, the mechanisms by which this occur have not been fully elucidated. Microglia may also be involved in AngII-mediated retinal inflammation. Rojas et al. reported that intravitreal administration of AngII to Sprague-Dawley rats resulted in elevated numbers of IL-6-positive microglia and a concomitant increase in the expression of IL-6, MCP-1 and intercellular adhesion molecule-1 in the retina. Findings from a study of spontaneously hypertensive rats with type 1 diabetes indicated that CD68 (ED1)-positive cells in retina (presumably microglia) were increased compared with normotensive diabetic rats and could be reduced with RAS blockade. Of interest is a recent study in rats with endotoxin-induced uveitis (EIU), a condition of acute inflammation in the iris and ciliary body of the eye. Resident microglia in these regions are activated in EIU and produce inflammatory factors that are thought to damage these parts of the eye. Bousquet et al., using a rat model of EIU, reported that aldosterone administration reduced the levels of inflammatory factors in the aqueous humor and the number of activated Iba1-positive microglia in the iris and ciliary body. These protective effects of aldosterone were reduced when animals were treated with the MR antagonist spironolactone. These findings appear contradictory to previous reports that aldosterone is pro-inflammatory and MR antagonism is anti-inflammatory in the retina and other tissues.[81, 82] However, the authors suggest that the beneficial effects of aldosterone in EIU may be related to the ability of aldosterone to prevent the downregulation of MR expression. Certainly, the role of aldosterone and the MR in situations of acute and chronic ocular inflammation and the participation of microglia in these processes warrants further investigation.
“AT2R may elicit the protective actions associated with microglia”
The AT2R counterbalances the dominant AT1R and is therefore associated with anti-inflammatory pathways that suppress the release of proinflammatory cytokines in response to noxious stimuli. Certainly this seems to be the case with microglial AT2R-evoked responses, where silencing of AT2R signalling has been found to exacerbate the inflammatory response to LPS in vitro. Furthermore, in monocytes, a well-established microglial progenitor cell, blockade of the AT2R exacerbates the inflammatory response specifically to AngII, illustrating an augmented AT1R response. To gain a better understanding of the functional impact of the AT2R in immune cells, Iwanami et al. selectively deleted the AT2R from bone marrow stromal cells and haematopoietic cells. In animals that were partially lacking the AT2R, they observed poorer stroke outcome and greater inflammation, clearly indicating the involvement of the AT2R mediating the protection associated with cells derived from myeloid precursor. Although relatively little is known about the role the AT2R plays in determining microglial function, it has been shown that central AT2R stimulation increases microglial activation following stroke, which coincides with a reduction in hypoxic damage.[72, 85] Although still in the preliminary stages, evidence suggests that in contrast with the AT1R, the AT2R may be responsible for eliciting some of the protective actions associated with microglia.[72, 84, 85]
Despite evidence that the RAS influences microglial function, there are a number of outstanding questions that need to be addressed before brain and retinal microglia are viewed to be important targets of RAS blockers in disease. These questions include understanding how microglia contribute to neuroinflammation, as well as pathological neovascularization, in various diseases of the CNS and retina. The phenotype of microglia in different stages of these disease states will need to be defined to allow timely and efficient treatment protocols. In addition, direct effects of AT1R and AT2R stimulation and blockade on microglia phenotypes and cytokine profile needs to be firmly established in cell culture to support the in vivo evidence of the RAS–microglia relationship, as already discussed. This information will be important for the application of RAS blockade and particularly AT2R stimulation, which may influence the morphing of microglia between ‘ramified or resting states’ to an activated phenotype.
It is clear that both brain and retinal microglia are not only involved in a number of disease settings, but, paradoxically, they also have the ability to be protective by releasing neurotrophic factors and augment vascular repair (Fig. 2). Understanding the pathways that control this dynamic cell population may be a new avenue for therapeutic intervention for a number of CNS and ocular diseases that currently have limited treatment options. Interestingly, emerging evidence indicates that the RAS could be involved in modulating microglial phenotype and their subsequent actions. This may be relevant to the application of AT1R antagonists for the treatment of disorders in which neuroinflammation is a dominant feature, such as PD and MS. Furthermore, it is becoming apparent that the AT2R should also be considered as a potential target, with preliminary evidence suggesting that it may play a role in the beneficial behaviour of microglia (Fig. 2). This highlights previously unrecognized actions of the RAS that include effects on specific cell populations, such as microglia.
The authors' work reported herein was supported by project grants from the National Health and Medical Research Council (NHMRC) of Australia (APP1007986 and APP1002235). JW-B is an NHMRC Senior Research Fellow.