Microglia as Central Protagonists in the Chronic Stress Response

Microglia Are Tissue-Resident Macrophages of the Central Nervous System

Microglia are innate immune cells that reside in the CNS that derive from yolk sac precursors^19,20 and maintain their population by self-renewal without major contributions from the hematopoietic system.^21,22 In the steady state, microglia have a ramified morphology with fine, branched processes that radially extend from their soma into the surrounding. Microglia processes are highly motile, constantly scanning the environment for potential threats and making contacts with neighboring cells.^23,24 In a healthy adult CNS, microglia contact neuronal synapses and phagocytose synaptic elements in response to neuronal activity, thereby shaping synaptic connections.^25,-,27 In addition, microglia control neurogenesis by interacting with adult neural precursor cells²⁸ and maintain the oligodendrocyte precursor cell pool.²⁹ On infection and in the diseased brain, microglia change their morphology and phagocytose debris, dead cells, and foreign material. These reactive microglia have shorter, thicker processes and can acquire an ameboid shape.^17,30 Furthermore, they upregulate the expression of ionized calcium-binding adapter molecule 1 (Iba1), CD45, and CD11b and can increase the expression of other surface immune molecules involved in antigen presentation such as major histocompatibility complex Class II (MHC-II), CD80, and CD86.³¹ To fight infection or help with regeneration, microglia secrete a variety of effector molecules. These include not only proinflammatory cytokines and reactive oxygen species (ROS) but also anti-inflammatory factors and growth factors aiding regeneration.^32,-,34 Microglia can be activated by various molecules indicative of infection or cell death, such as proinflammatory cytokines, lipopolysaccharide (LPS), and adenosine triphosphate (ATP).^31,35,36 In addition, microglia carry receptors for neuronal ligands on their surface. Some of these are inhibitory receptors (e.g., CX₃CR1 and CD200R), which bind ligands on the surface of healthy neurons preventing the microglia from acquiring a reactive phenotype.^35,37 Taken together, microglia are important mediators of neuroinflammation, keep the CNS clean from unneeded material, and can contribute to tissue repair and regeneration.

Potential Pathways That Activate Microglia in Chronic Stress

Neurons sense and process environmental information, initiating the body's stress response in the case of potential danger, which prepares the organism to successfully overcome threat. Intriguingly, microglia and other immune cells can be activated during long-lasting stressful situations even in the absence of any physical trigger such as infection.^38,39 A better understanding of the mechanisms underlying microglial activation during chronic stress will be a first important step for the prevention and treatment of stress-induced psychopathologies.

In stressful situations, components of the limbic system such as the amygdala, prefrontal cortex, hippocampus, and hypothalamus are activated.⁴⁰ This causes the activation of the sympathetic nervous system, triggering the release of adrenaline and noradrenaline from the medulla of the adrenal gland into the bloodstream. The 2 catecholamines then mediate the body's reaction to stress known as the fight-or-flight response. In addition, the hypothalamic-pituitary-adrenal (HPA) axis is activated.⁴¹ Neurons in the hypothalamus, the central regulator of the autonomic nervous system, secrete vasopressin and corticotropin-releasing hormone (CRH), which in turn stimulate the secretion of adrenocorticotropic hormone (ACTH) from the pituitary gland.⁴² ACTH then stimulates the adrenal cortex to produce glucocorticoid hormones,⁴² mainly cortisol in humans and corticosterone in rodents (Figure 1). In addition, signaling by members of the CRH family itself might further influence the stress response,⁴³ potentially also by directly affecting immune cells.⁴⁴ Altogether, the experience of stressful situations has 2 main outcomes: (1) increases in systemic levels of adrenaline and noradrenaline and (2) release of glucocorticoid hormones from the adrenal gland into the bloodstream. Catecholamines and glucocorticoids then mediate most of the effects of stress on the immune system.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1 Activation of the Hypothalamic-Pituitary-Adrenal (HPA) Axis During Stress

The activation of brain regions of the limbic system in stressful situations leads to the release of corticotropin-releasing hormone (CRH) from the hypothalamus. CRH then acts on the pituitary gland to stimulate the release of adrenocorticotropic hormone (ACTH) into the blood. ACTH in turn stimulates the adrenal gland to release corticosterone. Furthermore, the activation of the sympathetic nervous system during stress (not shown) triggers the release of noradrenaline and adrenaline from the adrenal gland into the bloodstream.

Microglial Responses to Adrenergic Signaling

Inputs from stress-responsive brain regions such as the amygdala and the hypothalamus also activate the locus coeruleus, the main site of noradrenaline production within the brain.⁴⁵ The catecholaminergic axons from the locus coeruleus innervate many brain regions, resulting in a release of noradrenaline throughout the brain in stressful situations.⁴⁵ In vitro, stimulation of microglial adrenergic receptors has anti-inflammatory effects because it reduces the production of proinflammatory cytokines, superoxide anions, and nitric oxide by microglia.^46,-,50 By contrast, the stimulation of β-adrenergic receptors during stressful situations in vivo enhances neuroinflammation. Activation of β-adrenergic receptors elevates IL-1β levels in the brain,⁵¹ while blocking the receptor prevents stress-induced increases in brain IL-1β.^51,52 Similarly, blockade of β-adrenergic signaling inhibits the increase in microglial Iba1 expression observed in mice exposed to chronic social defeat (CSD; Tables 1 and 2) stress³⁹ (Figure 2A).

View this table:

• View inline
• View popup
• Download powerpoint

Table 1

Rodent Models of Chronic Stress

View this table:

• View inline
• View popup
• Download powerpoint

Table 2

Protocols to Generate Chronic Stress in Rodents

• Download figure
• Open in new tab
• Download powerpoint

Figure 2 Microglial Responses to Noradrenaline and to Different Glucocorticoid Concentrations

(A) In stressful situations, noradrenaline is distributed throughout the brain by neurons in the locus coeruleus and can bind to β-adrenergic receptors (β-AR). β-adrenergic signaling during stress then contributes to neuroinflammation with increased Iba1 expression in microglia and increased IL-1β levels in the brain. (B) Glucocorticoids released during the stress response can bind to 2 different types of receptors, glucocorticoid receptors (GR) and mineralocorticoid receptors (MR), which are expressed in a variety of cell types including microglia. Because of different receptor affinities, glucocorticoids present at a high concentration preferentially activate GRs. GR activation in microglia in steady state has anti-inflammatory effects and reduces cytokine production. However, at the same time, high glucocorticoid concentrations also prime microglia to subsequent stimuli. Glucocorticoids present at low concentrations preferentially activate MRs leading to increased proinflammatory cytokine production and phagocytosis by microglia.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3 Consequences of Microglia Activation During Chronic Stress

Exposure to chronic stress activates stress-responsive brain regions, the sympathetic nervous system, and the HPA axis. As a result, corticosterone, noradrenaline, and adrenaline are released from the adrenal gland into the circulation and the immune system is activated. Microglia in stress-responsive brain regions upregulate the expression of several markers associated with their activation, including Iba1, CD11b, CD86, TLR4, CD14, and CD68. Furthermore, stress-activated microglia release the proinflammatory cytokines IL-1β and CCL2, enhance ROS production in the brain, and directly phagocytose neuronal elements, thereby modulating the response to chronic stress. CCL2 = chemokine (C-C motif) ligand 2; IL = interleukin-1; HPA = hypothalamic-pituitary-adrenal; ROS = reactive oxygen species.

Although these studies implicate β-adrenergic signaling in inducing proinflammatory responses in microglia after stress, it is not yet clear whether the microglia themselves directly respond to noradrenaline during the stress response. β-adrenergic signaling also has various effects on the activation state of the peripheral immune system during chronic stress^4,5,53 and is required for neuronal activation in CSD stress.³⁹ In the studies investigating the effects of β-adrenergic receptor stimulation on microglia, the receptor activity was usually blocked in the whole organism, making it difficult to differentiate between direct effects of this blockade on microglia and secondary effects caused by other cell types. Therefore, it would be interesting to study microglial stress responses in transgenic mice lacking β-adrenergic receptors specifically in microglia, ideally in a system that allows timed deletion of the receptor in adult mice shortly before the stress exposure. Furthermore, most studies investigating the role of β-adrenergic signaling during stress only used models of acute stress; data on noradrenergic signaling in the CNS during chronic stress are scarce.

Chronic Stress Influences Microglia Phenotype and Number in Stress-Responsive Brain Regions

Exposure to chronic stress increases the expression of Iba1 in stress-responsive brain regions in repeated restraint stress (Tables 1 and 2),^16,e10,e11 CUS^e12, and CSD stress,^39,e13 demonstrating microglial activation in these models. It is of interest that Iba1 expression was significantly lower in the prefrontal cortex of mice resilient to CSD stress than in stress-susceptible and naïve mice,^e4 which points toward a role for microglial activation in rendering mice susceptible or resilient to stress. In addition, CD11b expression was increased in the brains of mice subjected to CSD or CUS,^e14 which is an additional sign of microglial activation.³¹ By contrast, chronic stress does not induce microglial expression of MHC-II,^16,39,e10 a molecule required for antigen presentation,^e15 which is expressed by microglia during neuroinflammation and CNS disease.³¹ This observation is not too surprising, given that stress is a sterile inducer of inflammation. However, microglial expression of CD86, another molecule required for T-cell activation,^e16 was increased by CSD stress.³⁹ Similarly, the percentage of TLR4-expressingand CD14-expressing microglia was increased after CSD stress.^39,e13 The expression of CD68, a lysosomal protein expressed by monocytes and macrophages, is low during homeostasis, but is upregulated in phagocytically active microglia.³¹ CD68 expression is increased in stress-susceptible mice shortly after CSD stress,^e4 indicating that microglia are more phagocytically active during chronic stress. Taken together, these findings suggest that in brain regions associated with threat appraisal and emotional responses, chronic stress activates microglia.

In addition to influencing the phenotype of individual microglia, chronic stress affects microglial proliferation and programmed cell death, resulting in alterations in microglia numbers in stressed mice.^16,e17-e19 The proliferation of microglia was detected after 2–4 days of repeated restraint, social defeat, or unpredictable stress.^e17-e19 This proliferation is associated with increased numbers of microglia in stress-responsive brain regions such as the dentate gyrus of the hippocampus, the prefrontal cortex, and the amygdala.^e18,e19 Shortly after the onset of proliferation, a fraction of microglia undergoes apoptosis,^e17,e19 leading to a decline in microglia numbers. After exposure to stress for several weeks, microglia numbers were significantly decreased in certain brain regions in the different stress models.^e19,e20 The functional consequences of these changes in microglia phenotype and numbers are only partially known and will be discussed in the remaining sections of this article.

Chronic Stress Induces Long-Lasting Priming of Microglia

Chronic stress induces an increased expression of proinflammatory mediators in microglia.^{10,-,12,39,e1,e13,e21-e23} It is of interest that these stress-activated microglia also show an exaggerated inflammatory response to subsequent proinflammatory stimuli such as LPS both in vitro and in vivo.^e13 This reinforces the idea that exposure to chronic stress induces a functional activation of microglia because they are more responsive to subsequent other immune stimuli. The primed (activated) microglial state is maintained as long as 24 days after the last stress and involves an increased expression of many immune-related genes and pathways such as IL-1β, IL-6, and CD14.^8,e22 Glucocorticoids are one possible mediators of stress-induced microglia priming. Increases in the proinflammatory response of microglia to LPS after acute tail shock stress were prevented by pretreating rats with a glucocorticoid receptor antagonist or by the removal of their adrenal glands before the tail shocks.^e24

It is of importance that chronic stress does not only prime microglia for following LPS stimulation but also for subsequent acute stress exposure, indicating that the microglia response to acute stress might resemble their responses to immune stimulation by LPS. While 1 session of social defeat (acute stress) did not increase the expression of proinflammatory mediators in microglia of naïve mice, it increased the expression of proinflammatory mediators in mice subjected to CSD 24 days earlier.⁸ This priming by CSD stress is also reflected in the behavior of the animals because acute stress was only sufficient to increase anxiety and social avoidance in mice that had been previously subjected to CSD.⁸ To prove that microglia are involved in the increased susceptibility of CSD mice to acute stress, microglia were depleted using an inhibitor of the colony-stimulating factor 1 receptor (CSF1R), which microglia require for survival.^e22,e25 This experiment showed that CSD mice were only more susceptible to acute defeat than unstressed controls when microglia were present during the acute defeat session; the priming effect of chronic stress on the susceptibility to anxiety was erased in mice lacking microglia during the acute defeat.^e22 Surprisingly, the priming effect of chronic stress was observed in mice lacking microglia during the CSD itself. In mice lacking microglia during the CSD, but not during the acute defeat (because of repopulation), the acute defeat was sufficient to induce anxiety.^e22 Certainly, this rather unexpected observation needs to be further confirmed, ideally also by assessing anxious behavior in more than 1 test. Nevertheless, these findings raise the following questions: (1) How is stress sensed in the absence of microglia? (2) Which persistent changes apart from microglial activation occur in the brain after exposure to chronic stress? (3) How can these changes induce a stress-sensitized microglia state once the microglia repopulate? and (4) Is it possible that residual microglia remaining after depletion can transfer the stress-sensitized phenotype to the cells they give rise to during repopulation? Intriguingly, this observation that microglia are primed by CSD stress although they were depleted was also made in a second study. Microglia were depleted with a CSF1R inhibitor during the CSD and allowed to repopulate after the last defeat session.^e26 Even without a subsequent acute stressor, microglia repopulation alone was sufficient to induce anxiety and social avoidance, while behavioral disturbances were absent in animals in which CSF1R inhibitor administration was continued after CSD had ended.^e26 This study therefore further supports the notion that either the microglia that survived the depletion or a surrounding CNS component was altered by CSD stress in a way that enabled them to reprogram the repopulating microglia. Alternatively, it is possible that the process of microglial depletion itself, which is associated with a cytokine storm,^e2 induces changes in the CNS that then reprogram the repopulating microglia, leading to a hypersensitization of the CNS to stress.

In conclusion, chronic stress induces a long-lasting priming of microglia similar to as it occurs in neurodegenerative diseases and systemic inflammation.^e27 However, some of the effects of stress on the CNS only seem to influence microglia secondarily because microglia are also primed by chronic stress if they are depleted during the stress exposure. This suggests that stress also induces other, so far unknown, changes in the CNS that in turn can induce microglia priming. Primed microglia are more susceptible to different types of secondary challenges, leading to exaggerated responses to immune stimuli or minor stress events, which otherwise would not have elicited any detrimental response.

The Role of Microglia in Stress-Induced Behavioral Disturbances

Microglia are activated during chronic stress and change their phenotype and functional properties. Studies in which microglial activation was blocked with the antibiotic minocycline or in which microglia were depleted during chronic stress demonstrate that microglia play a key role in the induction of behavioral disturbances such as anxiety, anhedonia, and despair.^{e1,e19,e20,e28,e29} Considering this critical role of microglia in stress-induced psychopathologies, a better understanding of how microglial activation leads to the establishment of behavioral disturbances after stress will improve our knowledge of how stress can cause psychopathologies.

Increased brain levels of the proinflammatory cytokine IL-1β are commonly observed after chronic stress. Although brain-infiltrating monocytes could be a potential source of this cytokine, they do not seem to be the main producer of brain IL-1β during CSD stress because IL-1β mRNA is also increased in CC chemokine receptor 2 (CCR2)-deficient mice in which the recruitment of monocytes into the brain is blocked.⁹ Furthermore, several studies show that IL-1β mRNA levels are increased in brain isolates highly enriched for microglia obtained from mice subjected to CSD,^{10,12,39,e13,e21,e22} further suggesting microglia as the major source of IL-1β during chronic stress. If and how microglia-derived IL-1β induces behavioral disturbances after stress remains to be revealed. In general, IL-1 signaling is a key regulator of the response to chronic stress^e30 because mice deficient in IL-1R1 do not develop anxiety after CSD.^10,39,e4 If IL-1β acts in the periphery, within the blood-brain barrier, inside the CNS, or in all these compartments to induce behavioral disturbances remains unclear. One study found that endothelial cells need to respond to IL-1β for the development of anxiety after CSD.¹⁰ However, given the variety of IL-1β target cells and functions, it seems reasonable that its actions on more than 1 cell type are involved in the response to chronic stress.

In addition to IL-1β, microglial expression of chemokine (C-C motif) ligand 2 (CCL2) is increased during chronic stress.^{10,-,12,e1,e23} The main cell type constitutively expressing CCR2, the receptor for CCL2, is Ly6C^hi monocytes.^e31 It is therefore likely that CCL2 secreted by microglia recruits monocytes to the CNS during CSD; indeed, monocyte recruitment is absent when microglial activation is blocked or when microglia were depleted during CSD.^e1 Cytokines released in the CNS are not only able to recruit peripheral immune cells but have diverse effects on various cell types and processes. For example, proinflammatory cytokines can modulate neurotransmitter systems,^e32 cause neurotoxicity,^e33 inhibit neuronal plasticity,^e33 and modulate neuronal excitability.^e34 Furthermore, cytokines can modulate the metabolic profile of astrocytes^e35 and activate astrocytes, leading to astrogliosis.^e36 Therefore, increases in cytokine production by microglia could be a critical driver of behavioral disturbances after chronic stress. Microglia also secrete other soluble factors, such as brain-derived neurotrophic factor (BDNF), which can influence surrounding CNS cells. A subset of microglia promoted adult hippocampal neurogenesis and stress resistance in a model of CUS, potentially through the secretion of BDNF.^e37

Besides secreting proinflammatory cytokines, microglia can induce the production of ROS to cause behavioral disturbances.^e26 While CSD stress leads to increased ROS activity in the brain, this increase is prevented by microglial depletion.^e26 The administration of the antioxidant N-acetylcysteine or depletion of microglia during the CSD blocks the development of social avoidance in response to stress exposure. Intriguingly, when microglia could repopulate after the end of CSD, this was sufficient to increase ROS activity in the brain and induced a delayed development of social avoidance. To explain those results, the authors offered the following scenario: microglia produce ROS and are necessary to stimulate ROS activity in other brain cells, and this increase in ROS activity is essential for the development of social avoidance after chronic stress. Surprisingly, microglia that repopulate after CSD seem to be reprogrammed to drive ROS production, leading to the development of social avoidance in the recovery phase after CSD.^e26

Microglia are also involved in shaping the CNS environment by phagocytosis. In recent years, it has become clear that microglia can phagocytose synapses in the postnatal CNS, thereby contributing to synaptic pruning.^25,-,27 Induction of CUS in mice increases microglial phagocytosis of neuronal elements such as axon terminals and dendritic spines.^e12,e38,e39 Similarly, microglia isolated from mice subjected to CSD displayed an enhanced phagocytic activity in vitro.^e18 Microglial phagocytosis of neurons is induced by neuronal expression of colony-stimulating factor 1 (CSF1), which binds to CSF1R on microglia.^e12,e39 The expression of both CSF1 and CSF1R mRNA increases in the prefrontal cortex after CUS and shRNA-mediated knockdown of CSF1 1 week before CUS blocks the development of depression-like behavior.^e12,e39 These findings propose that the remodeling of neurons by microglia is another important mechanism contributing to the establishment of behavioral disturbances after chronic stress.

Microglia express high levels of the fractalkine receptor, CX₃CR1, and because neurons are the main source of fractalkine (also known as CX₃CL1), this pathway is a major communication channel between microglia and neurons.^e40 In the healthy CNS, CX₃CL1-CX₃CR1 signaling keeps microglia in a homeostatic state, and its disruption is associated with a proinflammatory microglia phenotype and enhanced phagocytosis.^e41 This enhanced phagocytosis can have both beneficial or detrimental consequences depending on the context and disease model. For instance, the lack of CX₃CR1 reduces the deposition of amyloid-β in models of Alzheimer disease^e42,e43 but enhances neuronal loss in a toxic model of Alzheimer disease and a mouse model of amyotrophic lateral sclerosis.^e44 Lack of CX₃CR1 in the whole body during chronic stress seems beneficial because CX₃CR1-deficient animals are at least partly protected from developing behavioral disturbances after chronic stress.^{9,e38,e45-e47} These studies suggest therefore that the enhanced phagocytosis associated with lack of CX₃CR1 protects the animal from stress-induced neuronal damage. However, because CX₃CR1 is also expressed by other immune cell types, including monocytes, some subsets of natural killer cells, and dendritic cells,^e48,e49 it is not clear whether the hyperactivation of microglia is indeed responsible for the beneficial effect of CX₃CR1 deletion. In addition, the stress-induced recruitment of peripheral monocytes to the brain after CSD is reduced in CX₃CR1-deficient animals,⁹ raising the question of whether the observed resistance to chronic stress on CX₃CR1 deficiency might be, at least partially, due to the deletion of the gene in monocytes rather than in microglia. Therefore, further studies are needed to reveal the mechanisms by which CX₃CR1 deficiency protects from chronic stress, ideally using models in which the deletion of CX₃CR1 can be restricted to adult microglia.

Microglia are not only in contact with neurons but also with other glia cells, and this cross talk influences microglia phenotype and function.^e50 In certain brain regions of stressed rodents, astrocyte density is decreased^e51,e52 and astrocyte morphology is altered.^e52,e53 Depletion of astrocytes induced depressive-like behavior in rats, suggesting that the loss of astrocytes during chronic stress could contribute to the behavioral disturbances.^e51 While the release of ATP by astrocytes was decreased in stressed rodents, external administration of ATP could alleviate stress-induced depressive-like behavior.^e54 On the contrary, extracellular ATP was rapidly increased after the onset of restraint stress, which could trigger inflammasome activation and microglial production of IL-1β.^e55 Taken together, while an astrocyte dysfunction is most likely involved in the pathology of chronic stress, the exact mechanism remains to be revealed. It seems likely that astrocytes exert their effects by directly influencing neighboring cells, such as microglia and neurons.^e56

Differences in Microglia Transcriptome Between Stress-Susceptible and Stress-Resilient Mice

Intriguingly, differences in the microglia transcriptome between the microglia from stress-susceptible and stress-resilient mice (Table 3) were recently identified.^e57 According to the resilience definition used, stress-resilient mice behaved similar to nonstressed controls, while stress-susceptible mice showed decreased social interaction and decreased urine scent marking.^e57 While microglia from stress-susceptible mice had a highly inflammatory transcriptomic profile, certain genes involved in regulating emotional responses were specifically enriched in microglia of stress-resilient mice, thereby giving hints about potential mechanisms that might render these mice resilient against stress. Furthermore, the genes highly enriched in microglia from stress-susceptible mice were increased in the brains of patients with Huntington and Parkinson diseases.^e57 This finding supports the hypothesis that the transcriptome of microglia is similarly altered during chronic stress, as it is altered during some neurodegenerative diseases. Nevertheless, more studies will be needed to further uncover how chronic stress alters microglia profiles in different brain regions and to characterize microglia gene expression profiles of stress-resilient and stress-susceptible animals, ideally also on a single-cell level. In addition, it will also be interesting to investigate the possibility of epigenetic changes occurring in microglia during chronic stress.

It remains to be revealed whether the change in microglia profiles precedes the behavioral response to stress: Do the microglia profiles already differ between stress-susceptible and stress-resilient individuals before the onset of stress, thereby predicting how an individual will respond to chronic stress? Or do differences in other immune system components or differences in other CNS cell types predispose certain individuals to resilience to chronic stress? In turn, these differences could then influence the microglia activation state, leading to the observed differences in microglia signature between susceptible and resilient mice.