Astrocytes are the most abundant cell type in the CNS, providing structural and trophic support to neurons. They have star-shaped morphology, with processes that can be in contact with up to 100,000 neurons (Halassa et al., 2007). Although astrocytes are not capable of producing action potentials, neurotransmitter binding to receptors present on astrocytes can induce Ca2+ waves (Sharma and Vijayaraghavan, 2001; Schipke et al., 2011). In addition to forming close appositions with synapses, astrocytes can influence the chemical environment through secretion of various compounds such as ATP, adenosine and acetylcholine, a process known as gliotransmission. Astrocytes can therefore influence neurotransmission on a synaptic level and could play a role in aggregating neural responses. Collectively, astrocyte involvement with synaptic activity is termed the tripartite synapse (Araque et al., 2014).

Across the CNS, astrocytes display different phenotypes and can adopt different states (Sun and Jakobs, 2012). Activated astrocytes display less branching morphological characteristics, and this is associated with glial fribrillary acid protein (GFAP) upregulation (Pekny and Nilsson, 2005). Activated in injury, astrocytes can be both protective by releasing factors facilitating recovery (Gimsa et al., 2013), and disruptive to neuronal functioning via inhibiting axon growth through formation of glial scars after extended activation (Smith-Thomas et al., 1994; Yuan and He, 2013). Moreover, radial glia located at the subventricular zones also express GFAP, but act as precursor cells capable of differentiating into neurons and astrocytes, which can migrate to several areas including the cortex, displaying ability for adult neurogenesis and cell renewal (Sundholm-Peters et al., 2005). The role of astrocytes is therefore varied and important to normal physiology, but astrocytes can also participate in both neuroimmune signaling and neuroinflammatory disease progression.

In MDD, astrocytes may be involved in the progression of the disease through participating in the reuptake of serotonin. In the presence of TNF-α, serotonin transporters expressed on astrocytes increase reuptake of serotonin in a dose-dependent manner, and this effect is attenuated by administration of SSRIs (Malynn et al., 2013). Interestingly, SSRI are also able to influence astrocytic Ca2+ waves in a similar way to serotonin administration (Schipke et al., 2011). Although unclear whether astrocytes are an integral target for anti-depressant treatment, it appears that astrocytes can play a role in serotonin neurotransmission, and therefore be involved in the pathophysiology of MDD.

Microglia are the resident immunocompetent cells in the CNS, and play an active role in neuroinflammatory actions by release of cytokines, chemokines phagocytosis and removal of debris, directly modulating neuroimmune activity. Microglia are thus the main cell type investigated in neuroimmune signaling events and neuroinflammatory/neurodegenerative conditions. Being implicated in synaptic pruning during CNS development, microglia not only perpetuate damage, but also are integral for normal functioning (Schwartz et al., 2013). Microglia too have different activation states, commonly referred to as M1 and M2, the pro and anti-inflammatory phenotypes, respectively (Olah et al., 2011). When activated to an M1 phenotype, microglia display a more ameboid morphology, and secrete cytokines and chemokines to signal for other immune cells. Microglia can also function as antigen presenting cells through MHC-II expression, therefore possessing the ability to trigger the adaptive immune responses within the CNS (Harms et al., 2013). Expressing pattern recognition receptors such as TLR4, microglia are responsive to DAMPs, MAMPs, and PAMPs. Moreover, TLR4 activation can shift microglia toward a M1 phenotype, inducing pro-inflammatory responses in the CNS (Ajmone-Cat et al., 2013).

Besides functioning as an antigen-presenting cell, microglia can influence neuronal functions through expression of glutamate transporters (Persson et al., 2005), but to a lesser extent as compared to astrocytes (Beschorner et al., 2007). In addition, microglia in the “resting” state actively survey the surrounding area for chemical changes, and can rapidly respond to stimuli (Nimmerjahn et al., 2005). Thus, even during an immunologically dormant state, microglia are able to influence neurotransmission and can rapidly respond to danger signals. Aiding this responsiveness are the toll-like receptors, which can recognize multiple stimuli and modulate the activation states of microglia.

Microglia, through TLR4-dependent signaling (Hines et al., 2013), and production of cytokines (Henry et al., 2009; Dobos et al., 2012), are regarded as mediators of central immune signaling in animal models of sickness behavior. In addition, changes in microglial reactivity states are also associated with the induction of stress-induced depressive like behavior (Pan et al., 2014). In a recent study, minocycline, an antibiotic that has also shown the ability to suppress central immune signaling by acting on microglia, can prevent the development of depressive-like behavior, tested using sucrose preference and social exploration (Kreisel et al., 2014). This reduction of depressive-like behavior was found in conjunction with changes to microglia morphology and proliferation. On the other hand, the study illustrated that simply inhibiting microglia chronically would not be a viable treatment option, since microglial activation states change from an initial proliferative state to later decline from acute to chronic models, and central immune suppression exacerbates depressive-like behavior in a chronic model. Instead, the authors proposed that depression is related to either an over or under activation of microglia, and treatments should strive toward a balance in activation states. Taken together, microglia appear important in central immune signaling and immune to brain communication in MDD, but this relationship is not is not uni-directional, and appears to be time-dependent.

Although the CNS is protected from many peripheral factors by the BBB, peripheral immune cells have been found to infiltrate and drive inflammation within the CNS (Schweingruber et al., 2014; Vogel et al., 2014). Perivascular macrophages and circulating monocytes can cross the BBB into the CNS through the expression of adhesion molecules such as ICAM and VCAM on the endothelium of blood vessels surrounding the CNS (Wong et al., 2007). Additionally, peripheral leukocytes can also roll and adhere to the cerebral vasculature, and cause increased central immune signaling without entering the CNS, thus communicating across the BBB (D'Mello et al., 2013). Glial cells themselves can also release chemokines such as CCL2, which can trigger peripheral immune cell extravasation into CNS tissue (Williams et al., 2013; Shieh et al., 2014). Thus, the central immune system can actively signal for peripheral immune cells to cross the BBB.

Contrary to other models of neuroinflammatory/neurodegenerative disorders Wohleb et al. (2014) have recently shown that peripheral bone marrow-derived cell infiltration is not just evident in models of BBB compromise, but can also occur in sub-inflammatory conditions as well. Interestingly, increased infiltration of peripheral monocytes can influence anxiety-like behavior, once again illustrating the ability for immune signaling to influence higher-order CNS function (Wohleb et al., 2014). This result, however, still needs more investigation, as it is widely held that immune-cell infiltration is a sign of neuroinflammation, and is not evident in sub-maximal levels of central immune signaling.

There is little direct evidence showing peripheral immune cell migration in MDD due to the lack of a representative animal model of MDD. Nevertheless, social defeat and psychological stress can trigger increased trafficking of peripheral cell infiltration (Brevet et al., 2010; Wohleb et al., 2013). Given that MDD is considered a stress disorder (for more information, see Section “what does stress have to do with it?”), this indicates that peripheral cell infiltration could be involved in MDD. However, isolating the exact cause of immune signaling in MDD is challenging, and the extent of peripherally driven immune responses in MDD patients is thus still unknown.

Cytokines are secreted by immune competent cells as a result of innate immune stimulation, including TLR4 activation by various ligands. As previously stated, cytokine administration is sufficient to cause sickness behavior. Cytokines can also upregulate HPA axis signaling on multiple levels through two main ways: (1) through reducing negative feedback on HPA signaling, and (2) by directly stimulating HPA activation. IL-1β, IL-6, and TNF-α are able to reduce the efficacy of GR, thus disinhibiting GR induced negative feedback on HPA activity (Pace et al., 2007; Bogaert et al., 2010). This effect could be due to protein-protein interactions with GR, which enables pro-inflammatory cytokines to influence GR translocation, ligand binding affinity and GR binding to GRE within the nucleus (for review, see Pace and Miller, 2009).

Secondly, cytokines can amplify the feed-forward signaling within the HPA axis. IL-6 has been shown to potentiate CRH activation of ACTH (Mehet et al., 2012), while IL-1β and stress have an additive effect on CRH secretion from the PVN (Chover-Gonzalez et al., 1993). Interestingly, peripheral and central IL-1β is upregulated in response to acute stress within 1 h, and 3 h after stress onset, respectively, and could therefore endogenously prime the HPA response. Moreover, this study also found that the administration of an IL-1β receptor antagonist diminished ACTH responses to restraint stress (Gądek-Michalska et al., 2011), showing that cytokines may partially mediate stress induced HPA activation. Given that an increase in peripheral IL-1β and circulating cortisol is observed in patients with MDD (Maes et al., 1991), this permissive effect on ACTH secretion could be in part driving the dysregulation between the immune and HPA secretion in MDD.

Besides cytokine interactions, COX-2 has also been shown to mediate TLR4 involvement in modulation of HPA activity. COX-2, an enzyme upregulated by TLR4 activation, catalyzes arachidonic acid into prostaglandin E2, which is part of the steroidogenesis pathway. COX-2 is therefore involved in the synthesis of GC at the level of the adrenal, and is shown to mediate LPS induced steroidogenesis within adrenal cells (Vakharia and Hinson, 2004). Furthermore, pharmacologically blocking COX-2 systemically is able to inhibit restraint stress induced GC both in vivo (Mouihate et al., 2010; Ma et al., 2013), and in vitro (Martinez Calejman et al., 2011). The effects of COX-2 extend beyond the adrenal gland, as expression of COX-2 in the PVN is important for sympathetic activation in response to restraint stress (Yamaguchi et al., 2010). In addition, COX-2 inhibition can influence higher order functions, buffering the effects of immobilization stress by reduced anxiety behavior and improve locomotor functioning and learning (Kumari et al., 2007).

Not only can TLR4 activation result in short-term stimulation of the HPA axis, but TLR4 can also influence HPA activity long after the stressor is resolved. For example, a single LPS challenge during early-life is sufficient to hyper-sensitize the CRH and ACTH response to both subsequent LPS and restraint stress when tested in adulthood, without baseline HPA differences when compared to vehicle controls in a rodent model (Mouihate et al., 2010). Early-life TLR4 activation also results in an increase in anxiety behavior during adulthood (Sominsky et al., 2013), thus fundamentally changing the stress response system. This suggests that TLR4 activity during developmentally sensitive periods may shape the HPA system, priming the system toward hyper-reactivity, and may even be changing individual predisposition toward stress-related disorders.

A good way of investigating the developmental consequence of TLR4 is through the use of genetic knockout models. TLR4 knockout mice are observed to have different HPA phenotypes when compared to match wild-type C57Bl/6 counterparts, showing increased adrenal gland volume and correspondingly higher baseline circulating glucocorticoid levels (Zacharowski et al., 2006). The direction of this change is not universally found however, as preliminary findings from our laboratory in Balb/c background TLR4 knockout mice have yielded opposite results. We have found that TLR4 genetic knockout mice have smaller adrenal glands as well as lower circulating GC (unpublished). Additionally, circulating ACTH was also elevated in mice lacking TLR4 when compared to matched controls.

Despite the differences in adrenal size and baseline ACTH levels, we found no difference between wild type and TLR4 knockout mice, in terms of adrenal responsiveness to ACTH administration in the same study (unpublished). Our findings support observations that systemic rather than adrenal MyD88 expression is important in regulating HPA activity (Kanczkowski et al., 2013). Taken together, these results suggest that although the TLR4 pathway can influence HPA activity, the innate immune system has little direct impact on adrenal function, and the observed HPA differences are likely to be driven by mechanisms within the CNS instead.

Given the often-contradictory results on HPA function, the complexity of glucocorticoid signaling is becoming more appreciated. In order to explain the complex actions of glucocorticoids Sapolsky et al. (2000) proposed 4 categories of glucocorticoid action - permissive, inhibitory, excitatory and priming, encapsulating the different receptors activated, timing of response relative to stressors and type of tissue activated. Illustrating this, glucocorticoids are excitatory in terms of heart function but inhibit vascular function, cause the release of glucose aiding energy expenditure, yet also trigger stockpiling of fat in adipose tissue. At the same time, basal glucocorticoid expression can be permissive toward sympathetic activation of the adrenal medulla, thus influencing stress responses even before the HPA activation even occurs. Thus, glucocorticoids have different dose and time response relationships across different tissues, and their actions are dependent on basal or activated HPA states.

Recent studies are beginning to classify the priming or sensitizing effect of stress and HPA activity on immune function. Chronic variable and acute social disruptive stress can sensitize HPA and immune response to subsequent LPS challenge, differentially inducing larger neurokine and peripheral cytokine responses (Gibb et al., 2013). Repeated social defeat stress can also prime immune signaling in peripheral monocytes and dendritic cells in response to LPS, coupled with glucocorticoid resistance during the first 48 h after stressor, measured by immune cell expression following GC administration in vitro (Powell et al., 2009). Importantly, this immune priming is also seen in microglial populations 24 h after glucocorticoid treatment (Frank et al., 2011). Moreover, GC administration in vivo has been confirmed to emulate stress induced immune sensitization (Frank et al., 2010; Dey et al., 2014). This sensitizing effect was further blocked by a GR antagonist, indicating that GR signaling is essential (Frank et al., 2012). TLR2 and TLR4 activity could also be integral to glucocorticoid-induced immune priming in microglia, as administration of their respective antagonists prior to tailshock stress can prevent increased sensitivity in hippocampal tissues collected 24 h after the end of the stressor (Weber et al., 2013). Taken together, there appears to be crosstalk between the GR and TLR4 pathways, and both receptors appear to be important in driving immune cell sensitization and increased central immune signaling following stress.

The immune-priming effect of stress is proposed to mediate stress-induced side effects such as allodynia (Loram et al., 2011), and drug abuse (Frank et al., 2011), and could potentially be involved in MDD as well. PBMCs isolated from patients admitted for severe depressive episodes are more responsive to interferon-γ (IFN-γ) stimulation (Schlaak et al., 2012). Along with increased TLR4 mRNA and expression on PBMCs of patients with MDD (Kéri et al., 2014), the increased immune signaling in MDD patients could be indicative of a primed immune system, rather than chronic inflammation.

DAMPs, or alarmins, are released endogenously from stressed, dead and dying cells as a signal for danger. They include HMGB1, various HSP, ATP, and Uric acid. During normal physiological activation, DAMPS have a non-inflammatory function within the cell. Conversely during situations of tissue damage, when released into the extracellular space, DAMPs alert the immune system to the damage in order to promote repair and direct traffic toward the damaged tissue, thus triggering the inflammatory response. This inflammatory response is in part driven by TLR4, since DAMPs including HMGB1 and various HSPs can activate the TLR4 pathway (Hutchinson et al., 2009; Laird et al., 2014). Furthermore, ATP can trigger innate immune signaling by activating a protein complex known as the inflammasome, which induces the maturation and release of cytokines IL-1β and IL-18 via a caspase-1 dependent mechanism (Chen et al., 2013). This effect is known to augment the inflammatory response to LPS, therefore amplifying TLR4 signaling (Ghonime et al., 2014).

DAMPS including HMGB1, uric acid and HSP72 are also released following tail-shock stress (Faraco et al., 2007; Maslanik et al., 2013), and thus are not limited to situations of tissue damage or cell death. DAMPs could therefore mediate the effect of stress on triggering or sensitizing the immune response, and this increased immune signaling may have wider implications for MDD. It is notable, however, that the mechanisms regulating the secretion of DAMPs in response to stress is not well characterized.

High mobility group box 1. HMGB1 functions as a chaperone protein within the cell by binding to proteins and transporting them between the cytoplasm and nucleus. During damage however, HMGB1 can be released into the extracellular space via an inflammasome mediated mechanism (Lu et al., 2012). Neural tissue is capable of releasing HMGB1 in response to glutamatergic excitotoxicity and glial activation as a result of LPS administration in vitro (Faraco et al., 2007). HMGB1 can also activate central immune signaling, as it can trigger TLR4 similar to LPS, via the MD2 and CD14 complex, and requires adaptor protein MyD88 to trigger downstream inflammatory actions (Kim et al., 2013). Moreover, psychological stress itself can induce an increase in HMGB1. For example, thymocytes are responsive to 15 min restraint stress, and release HMGB1 via GR signaling (Billing et al., 2012). There is further evidence showing that HMGB1 and GR can form complexes within the chromatin, increasing the residence time of GR when bound to DNA (Agresti et al., 2005). The functional consequence of this interaction, however, is still unclear. Given that HMGB1 is responsive to stress, interacts with GR, and is able to increase peripheral and central immune signaling, stress-induced immune sensitization through neurokine signaling could therefore be partially mediated by HMGB1.

HMGB1 can also cause an upregulation of Matrix metallopeptidase 9 (MMP9), an enzyme that results in the breakdown of the extracellular matrix (Qiu et al., 2010). Interestingly, increased MMP9 in circulation is associated with mood disorders such as depression and bipolar disorder (Domenici et al., 2010; Rybakowski et al., 2013). Together with evidence that MMP9 can also be upregulated as a consequence of microglial activity (Lively and Schlichter, 2013), MMP9 could be a possible result of stress induced HMGB1 upregulation and immune signaling within the CNS in MDD.

Heat shock proteins. HSPs were first discovered in the drosophila model to be produced in response to hyperthermia, and vary in protein weights ranging up to 110 kDA. HSP regulate the folding and unfolding of other proteins, and are released in response to cellular damage. Signaling danger, extracellular HSP can activate the innate immune system (Colaco et al., 2013). Within the HSP family, HSP70 and HSP90, the 70 and 90 kDa variants, are of most relevance to glucocorticoid and TLR4 signaling. HSP70 and HSP90 can bind TLR4, resulting in release of pro-inflammatory cytokines (Gong et al., 2009; Colaco et al., 2013). Furthermore, HSP90 also serves as a chaperone protein for TLR4, triggering endocytosis in response to ligand binding (Triantafilou and Triantafilou, 2004). Through these mechanisms, HSP90 plays an integral role in TLR4 signaling and in TLR4 related neuropathic pain (Hutchinson et al., 2009). Stress can also induce HSP expression, notably decreasing the ratio between GR and HSP70 and HSP90 expression in the hypothalamus (Simic et al., 2012).

Interestingly, HSP90 is a well-characterized chaperone protein for GR nuclear translocation and permits GC binding to GR (Ricketson et al., 2007). Although important for binding and translocation, Increased HSP90 expression can also impair GR function (Matysiak et al., 2008). This effect increases in chronic stress as compared to acute models of stress, and thus is proposed as one facet of glucocorticoid resistance. Stress induced changes in HSP can therefore either directly activate TLR4, change the trafficking TLR4 and GR, as well alter GR binding capacity. However, to what extent each of these mechanisms is involved, and the magnitude of the change has yet to be investigated in models of depressive-like behavior. Nevertheless, given the involvement in TLR4 and GR signaling, HSP could be an avenue for further research in linking stress and increased immune signaling in MDD.

One way in which TLR4 ligands are upregulated by stress is through intestinal translocation. Recently, the role of gut microbiota in potentiating differences in mood and behavior is gaining traction within the literature (Hsiao et al., 2013). It has been hypothesized that stress can cause a disruption in intestinal tight junctions, which would increase translocation of microbiota into the system, thus inducing inflammatory responses. Indeed, GR appears to be involved in gut HSP70 upregulation and intestinal permeability in response to restraint stress (Ait-Belgnaoui et al., 2012, 2014).

There is some evidence showing that intestinal decontamination using orally administered antibiotics is able to block the tight junction disruption, as well as inflammatory and HPA responses to psychological stress (Gárate et al., 2011). Furthermore, antibiotic treatment is shown to mirror the ability of TLR4 antagonist in blocking stress induced depressive-like behavior (Gárate et al., 2011). This hypothesis is thus showing promise for developing new medication targeting the gut-brain axis in regulating behavior. On the other hand, the exact mechanisms of this gut-brain communication in respect to stress and MDD are unclear, since both ascending pathways and peripheral immune signaling could potentially be involved. In addition, the extent of intestinal translocation in the acute stress response requires more study, since it is not known if this effect is stressor specific.