Immune System Activation and Depression: Roles of Serotonin in the

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Immune System Activation and Depression: Roles of Serotonin in the Central Nervous System and Periphery Matthew James Robson, Meagan A. Quinlan, and Randy D. Blakely ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00412 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 28, 2017

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ACS Chemical Neuroscience

Immune System Activation and Depression: Roles of Serotonin in the Central Nervous System and Periphery Matthew J. Robson1*, Meagan A. Quinlan1,2 and Randy D. Blakely1

1

Department of Biomedical Science, Charles E. Schmidt College of Medicine, Florida

Atlantic University, Jupiter, FL 33458, USA; 2Department of Pharmacology, Vanderbilt University, Nashville, TN 37240-7933, USA

Corresponding Author: Matthew J. Robson, Ph.D. FAU Brain Institute Suite 111, MC-17 5353 Parkside Dr. Charles E. Schmidt College of Medicine Florida Atlantic University Jupiter, FL 33458 TEL: (561)-799-8100 Email: [email protected]

ACS Paragon Plus Environment


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Abstract Serotonin (5-hydroxytryptamine, 5-HT) has long been recognized as a key contributor to the regulation of mood and anxiety and is strongly associated with the etiology of major depressive disorder (MDD). Although more known for its roles within the central nervous system (CNS), 5-HT is recognized to modulate several key aspects of immune system function that may contribute to the development of MDD. Copious amounts of research have outlined a connection between alterations in immune system function, inflammation status and MDD. Supporting this connection, peripheral immune activation results in changes in the function and/or expression of many components of 5-HT signaling that are associated with depressivelike phenotypes. How 5-HT is utilized by the immune system to effect CNS function and ultimately behaviors related to depression is still not well understood. This review summarizes the evidence that immune system alterations related to depression affect CNS 5-HT signaling that can alter MDD-relevant behaviors and that 5-HT regulates immune system signaling within the CNS and periphery. We suggest that targeting the interrelationships between immune and 5-HT signaling may provide more effective treatments for subsets of those suffering from inflammation-associated MDD.

Keywords


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Serotonin, Major Depressive Disorder, Inflammation, Cytokines, Immune System, Serotonin Transporter Introduction Major Depressive Disorder (MDD) affects up to 20% of the world’s population and is currently one of the top ten causes for morbidity and mortality.1-3 Although behavioral and pharmacological treatments have been developed for MDD, it is estimated that up to 75% of people suffering from the disease remain either untreated or treated with medications lacking sufficient efficacy.4 The majority of the current MDD pharmacotherapies target monoaminergic systems, particularly serotonergic and/or noradrenergic neurotransmission, a fact that led to the hypothesis that the disorder derives from disrupted monoaminergic signaling.5 While this hypothesis has driven therapeutic drug development, most see this hypothesis as overly simplistic and only part of a complex etiology that includes most prominently a dysregulated hypothalamic-pituitary

axis

(HPA)6,

impaired

cellular

plasticity

(e.g.

hippocampal

neurogenesis)7, and/or elevated neuroinflammation.8 Recently, a growing body of literature has drawn attention to a connection between immune system activation and neuropsychiatric disorders, including MDD.9-11 This connection is now well-documented across both animal and human studies, with much of the work consistent with an increase in proinflammatory cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α) and inflammatory markers such as C reactive protein (CRP).1216

Although elevations in immune and inflammatory signaling in MDD are evident, whether these

are causal versus a consequence of the disorder has been difficult to disentangle. It is likely that both views are correct, owing to the clinical heterogeneity of MDD and as we will note below, the complex, reciprocal interactions of 5-HT and immune system modulation in both the periphery and central nervous system (CNS).



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It is now well established that the brain is not the immune privileged organ that it was once believed to be, having its own highly complex and regulated immune system closely tied to that of the peripheral immune system.17, 18 There are several ways in which peripheral immune signals enter the brain including cytokine access at the circumventricular organs (which exhibit a weakened or absent blood brain barrier (BBB))19, T cell infiltration20, and active transport of cytokines across the BBB.21-26 Recent work has uncovered a direct link between the CNS and cervical lymph nodes whereby cerebral spinal fluid interacts in a direct nature with the peripheral immune system.26,

27

The intricacies and nuances of how the connections between these

systems are controlled and the full extent of the chemical language they speak is not well understood. Further complicating the efforts of researchers to understand the interplay between the immune system and the CNS are the inherent differences between inflammation and immune system activation in the periphery and neuroinflammatory processes.28, 29 What seems apparent however, is that critical aspects of this language are involved in the etiology of MDD. Herein, we summarize the evidence that serotonin (5-HT) is a prominent component of molecular signaling within the peripheral immune system and the CNS whereby it plays a key role in ultimately how immune signals manifest as psychological and behavioral changes.

Evidence of Immune Activation in Major Depressive Disorder The initial hypothesis that inflammatory cytokines released from activated macrophages could elicit symptoms of depression dates back to the work of Smith, in which he postulated a “macrophage theory of depression.”30 Supportive of this theory, subsequent studies revealed the innate immune system of depressed patients is activated.31,

32

Direct activation of the

immune system in the clinic or preclinical models, through infection, lipopolysaccharide (LPS) treatments33-35 or recombinant cytokine therapies, produce elevations in inflammatory cytokines and MDD-like behavioral changes.35, 36, 37, 38 In support of inflammatory responses as mediators


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of behavioral changes, immunotherapies that involve administration of inflammatory cytokines or their inducers (e.g. α-interferon) elicit depressive symptoms in approximately 30-50% of patients.39 Moreover, levels of depressive symptoms and anxiety elicited by LPS treatments are positively correlated with serum IL-1β, IL-6 and TNF-α levels.35, 40 Recent studies have highlighted an overlap between many aspects of the inflammation/immune and monoamine disruption hypotheses of MDD. It has become evident that immune system function is linked to monoaminergic signaling, especially those involving the serotonergic system. In relation to MDD, connections to 5-HT center upon this neurotransmitter’s effects on mood and emotion41,

42

, appetite43, sleep44 and aggression.45,

46

The primary determinant of the cessation of 5-HT signaling is the presynaptic Na+/Cldependent, high affinity 5-HT transporter (SERT, SLC6A4). SERT, a protein comprised of 12 transmembrane domains, was recently crystalized47 and is known to be regulated through various intracellular signaling pathways including protein kinase G (PKG), protein kinase C (PKC) and p38 mitogen activated protein kinase (p38 MAPK) (Figure 1).48-50 Alterations in the structure or expression of SERT have been linked to MDD, anxiety, obsessive-compulsive disorder (OCD) and autism spectrum disorder (ASD).41, 51, 52 For example, a well-characterized SERT polymorphism (5-HTTLPR),53 acts to regulate the expression level of the transporter and in conjunction with early-life and/or current stress is linked to depressive symptomology.54-56 SERT is the direct target of tricyclic antidepressants and selective serotonin reuptake inhibitor (SSRI) therapeutics. It is believed that the primary therapeutic action of SSRIs stems from a tonic elevation of synaptic monoamine levels (primarily 5-HT) in CNS circuits that modulate the key behavioral domains impacted by MDD. Although SSRI’s are widely utilized for the treatment of depression, these therapies have significant limitations in that antidepressant efficacy typically takes weeks to develop and efficacy is partial, with full remission of depressive symptoms in approximately one third of patients.57 This has led to significant efforts to develop



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agents that engage additional determinants of serotonergic signaling, particularly various 5-HT receptors. Examples are vortioxetine and vilazodone, which target 5-HT3, 5-HT7, 5-HT1D receptors and 5-HT1A receptors, respectively.58 Recent studies have provided evidence that vortioxetine does bind to SERT, where it exhibits the classical SSRI activity of blocking 5-HT reuptake.59, 60 However, work from within the Blakely lab with rodent models suggests that the antidepressant efficacy of vortioxetine may not require the blockade of SERT function.61 An alternative strategy to serotonergic polypharmacy is the targeting of regulatory pathways that control SERT, which as we will discuss below, involves inflammatory cytokine signaling pathways.

Evidence of Immune System Modulation of SERT Early in vitro evidence that immune mechanisms could impact SERT expression was provided by Ramamoorthy et al.62 whereby chronic treatment with IL-1β was found to elevate SERT mRNA and protein expression. These effects were found to be dependent on interleukin1 receptor type 1 (IL-1R1) function and arise from the activation of the transcription factor NFκB.63 Other cytokines, such as TNF-α, have also been reported to exhibit similar effects.64, 65 Although these studies have all been conducted in non-neuronal in vitro models, they provide evidence that chronic increases in proinflammatory cytokine levels result in elevated SERT gene expression leading to enhanced 5-HT uptake activity.66 Interestingly, chronic exposure to anti-inflammatory cytokines, such as interleukin-4 (IL-4), has been reported to reduce 5-HT uptake in transformed lymphoblasts in vitro.65 In the latter study, genetic variation in SERT was suggested to moderate the impact of IL-4, suggesting that SERT regulatory mechanisms may represent a convergence point for gene x environment interactions. Evidence also supports a link between the activation of the immune system and the augmentation of CNS SERT activity. The functional status of SERT is highly regulated and


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includes the control of transporter surface trafficking, phosphorylation states, membrane compartmentalization and 5-HT affinity, each of which can lead to changes in 5-HT clearance rates.48, 67 We and others have previously demonstrated that rapid, acute cytokine treatments, or peripheral immune stimulation, elevates SERT activity using in vitro, ex vivo and/or in vivo preparations.68-71 Evidence suggests that the proinflammatory cytokines, IL-1β and TNF-α increase SERT activity, the former in an IL-1R1-dependent manner.68 These effects are mediated via downstream p38 MAPK and PKG-linked pathways.48 The p38 MAPK pathway plays a major role in immune system signaling and therefore may serve as a distinct link between immune activation and depression in the context of SERT regulation. Previous work has demonstrated a dynamic regulation of SERT by p38 MAPK signaling cascades, affecting the trafficking and activity state of the protein.48,

72

changes in the phosphorylation status of SERT.72,

These effects are believed to arise from 73

The phosphorylation state of SERT is

known to be modulated by 5-HT levels74 and/or the activation of PKC, PKA, PKG1α48, addition to p38 MAPK.76,

77

75

in

Inhibition of p38 MAPK results in a decrease in SERT

phosphorylation, both basally,72 and when phosphorylation is elevated by hypermorphic coding variation.76 The specific residues involved in p38 MAPK-induced phosphorylation of native SERT are currently unknown, though a peptide containing a canonical MAPK site in the transporter C-terminus has been found to be phosphorylated by purified p38 MAPK in vitro.73 Since p38 MAPK is activated by PKG, phosphorylation at SERT residue Thr276, a site previously shown to be phosphorylated dependent on PKG signaling,78, 79 may also be involved in modulating SERT activity. There are four currently known isoforms of p38 MAPK encoded by separate genes (α,β,γ,δ).80 Evidence indicates that the α isoform of p38 MAPK supports the modulation of SERT activity,81, 82 as genetic knockdown (in vitro), elimination (in vivo) and selective pharmacologic inhibition of this specific isoform has been shown to alter the activity state of SERT.82-84 Given



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the evidence accumulating for a specific role of p38α MAPK in SERT regulation, it seems plausible that activation of this isoform by innate immune system activation in MDD subjects could drive pro-depressive changes in SERT activity. Indeed, p38α MAPK activity within serotonergic neurons has been linked to depressive-like behaviors and increases in SERT activity in rodents.82, 83 Bruchas et al. demonstrated that serotonergic neuron expression of p38α MAPK is required for stress-induced social avoidance and depressive-like behavior in the forced swim test. Depressive-like behaviors are associated with the stress-induced activation of p38α MAPK and ultimately results in an increase in SERT activity in mice.82 Similarly, we have recently demonstrated that stimulation of CNS SERT by peripheral immune system activation and resultant depression-like behavior is dependent upon ongoing p38α MAPK activity within 5HT neurons.83 It should be noted that although the general consensus is that p38 MAPK signaling regulates SERT function (effects found in various murine studies), one group has recently reported a failure of acute p38 MAPK signaling to regulate SERT in rats.85 Schwamborn et al. recently published a report whereby increases in SERT activity post-LPS treatment were found to be independent of p38 MAPK activation and independent of changes in total SERT expression.71 Increases in SERT activity were found to occur 24 hr post-LPS administration in contrast to more acute time points previously reported.71 Combined, these findings provide an important,

direct,

and

time-dependent

link

between

a

specific

kinase

involved

in

environmental/immune system signaling and the expression and/or activity state of the most critical determinant of 5-HT signaling with the CNS and periphery. Additional support for a contribution of altered 5-HT signaling in relation to immune system function and MDD has come with the work of Sluzewska et al. whereby increases in 5HT auto-antibodies were found in a small cohort of patients suffering from MDD.86 Confirmatory work in a larger patient cohort by Maes et al. revealed that patients suffering from MDD have higher levels of peripheral 5-HT auto-antibodies as compared to non-depressed controls.87


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These changes were associated with greater levels of melancholia, leading to the hypothesis that levels of anti-5-HT antibodies may be predictive of the severity of depression in certain patient populations.87 Interestingly, changes in anti-5-HT antibody levels were also correlated with increases in plasma TNF-α and IL-1, linking increases in 5-HT auto-antibodies to altered immune system function and cytokine production.87 Although the mechanisms resulting in elevations in 5-HT antibodies is currently unclear, it may potentially stem from excess transporter-mediated accumulation of 5-HT that results in the serotonylation of intracellular proteins88 thereby providing a physical substrate for subsequent antibody production against 5HT.

Microglial Function, Depression and 5-HT Microglia are the resident immune cells of the CNS and for years were believed to only function as the macrophages of the brain, responding to perturbations in the normal, homeostatic function of the CNS. In recent years however, microglia have been recognized as complex cells that modulate the function of neurons through signaling and the pruning of synapses, in addition to their varied roles in injury and neuroinflammatory processes.89 Administration of immune challenges to both humans, as well as rodents (using LPS treatments), results in microglial activation that coincides with the manifestation of depressive symptoms.90,

91

Rodent studies have provided evidence that LPS-mediated depressive-like

effects are dependent upon the activation of microglial cells, as an attenuation of microglial activation

with

minocycline

attenuates

LPS-mediated

depressive-like

symptoms.92,

93

Furthermore, mice that exhibit elevated levels of microglial activation have elevated depressivelike responses to LPS administration.94



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The specific cells that produce cytokines within the CNS, acting to ultimately stimulate SERT have yet to be identified, but are presumably microglial cells, although astrocytes and neurons as sources should not be discounted.95 The activation of microglial cells and the subsequent release of proinflammatory cytokines may act to increase the expression/activity of SERT, thereby decreasing levels of synaptic 5-HT available for neuronal signaling. It is known that inflammation stemming from IL-1β administration (both central and peripheral) results in increased brain tryptophan levels, the metabolism of 5-HT and elevations in 5-HT levels.96, 97 It is possible that increases in extracellular levels of 5-HT elicited by inflammatory stimuli are a compensatory response to increased SERT activity that acts to reduce the availability of synaptic 5-HT. Increases in SERT activity would therefore be viewed as a way for the CNS to remain in homeostatic balance, scaling the amount of 5-HT available at the synapse.70 Interestingly, 5-HT appears to act as a signal to modulate microglia associated responses to inflammatory stimuli. Multiple classes of functional 5-HT receptors have been shown to be expressed by microglia, including 5-HT2, 5-HT5 and 5-HT7 receptors.98-100 5-HT has been shown to modulate microglial responses to injury whereby it enhances the chemotactic responses of microglia, a response attributed to 5-HT2 function.98 In vitro analyses have revealed that the stimulation of 5-HT7 receptors in human microglial cell lines results in an increase in IL-6 expression in these cells, an effect blocked by antagonizing 5-HT7 receptors.100 As mentioned previously, one of the major functions of microglia is to respond to disruptions in homeostatic states through the release of proinflammatory cytokines. Microglia can release cytokines, including IL-1β101, in exosomes by a process that is as of yet is not fully understood.102 5-HT has also been shown to play an important role in the ability of microglia to release exosomes, which is dependent on increases in cytosolic Ca2+ elicited through 5-HT2 and 5-HT4 receptor activation.102 In this manner, 5-HT may ultimately be able to regulate cytokine release and immune processes within the CNS similarly to immune function in the periphery.


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The release of these cytokines may then act in a concerted manner to modulate other aspects of 5-HT neurotransmission, such as SERT-mediated 5-HT clearance or exocytotic 5-HT release. Pharmacological blockade of SERT has also been used to study the effects of 5-HT signaling on proinflammatory cytokine release and resulting microglial activation. Treatment with the SSRI, fluoxetine, has been shown to attenuate microglial activation that accompanies ischemia.103 Chronic fluoxetine treatment utilized in conjunction with the chronic unpredictable stress (CUS) rodent model of MDD has been shown to block microglial activation and depressive-like phenotypes elicited by CUS.91, 104, 105 In vitro studies have found similar effects of other SSRI’s, where SSRI treatment resulted in a reduction in microglial activation/cytokine generation.106-108 However, the activation of microglia is a complex process that spans an array of activation states.109 Simplified, microglia exhibit one of two polarized phenotypes; either a proinflammatory M1 phenotype or an anti-inflammatory M2 phenotype.110 The aberrant and unwarranted polarization of microglia to either an M1 or M2 phenotype has been hypothesized to be associated with MDD and other neuropsychiatric disorders.111 A recent study found that both fluoxetine and S-citalopram promote M2 activation in the BV2 cell line and mouse primary microglial cells.112 As to mechanism, Liu and colleagues determined that paroxetine, another SSRI, acts to suppress LPS mediated microglia activation through regulation of JNK1/2 and ERK1/2 activity.113 In contrast to a general trend in the literature indicating a reduction of microglial activation following SSRI treatment, chronic treatment with fluoxetine has been shown to increase OX-42 immunoreactivity, a marker of microglial activation in the substantia nigra of rodents.114 It should be noted that although many of these effects have been attributed to the actions of SERT blockade by various SSRI’s, evidence of SERT expression in microglia is profoundly lacking.98, 99 Further, these in vitro studies have utilized non-physiologically relevant concentrations that may allow for off-target effects.115 Many common SSRI’s interact with other protein targets, including sigma receptors116 and organic cation transporters,115 that could be



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responsible for some or all of the aforementioned in vitro effects. Clearly, more studies are needed to determine the specific role of 5-HT in modulating microglial responses and how these responses effect 5-HT signaling and behaviors that may be associated with MDD.

5-HT and Peripheral Immune System Function Although 5-HT is most well-known for its roles within the CNS, most of the measurable 5-HT present within the body is produced by the gastrointestinal (GI) tract. Initial purification efforts of 5-HT were in fact from the periphery.117, 118 Immune cells such as lymphocytes, mast cells, dendritic cells and monocytes have all been shown to express 5-HT receptors, SERT and enzymes involved in the production and metabolism of 5-HT (Figure 1).70,

119

Additionally,

evidence indicates that 5-HT signaling modulates several functions that are required for normal immune system function such as chemotaxis, leukocyte activation, proliferation, cytokine secretion, and apoptosis.119 T lymphocytes are a subset of immunocytes that express the cellular machinery needed for the production of 5-HT. Tryptophan hydroxylase (TPH) is the enzyme required for the production of 5-HT from its precursor tryptophan. TPH1 is primarily responsible for the peripheral production of 5-HT, whereas TPH2 is relegated to serotonergic neurons present within the CNS.120 Resting, naïve T lymphocytes express very little TPH1; however, following the activation of these cells there is a large increase in the expression of TPH1.121 Moreover, increased production of 5-HT has been detected in activated T lymphocytes,121 consistent with an increase in the activity of TPH1. T cells that have undergone activation also increase their expression of a variety of 5-HT receptors, including 5-HT1B, 5-HT2A and 5-HT7 receptors.121 One could therefore hypothesize the increases in 5-HT production by activated T cells allow for 5-HT to be utilized as an immune signaling molecule in an autocrine or paracrine fashion to other immunocytes that express various 5-HT receptors. It is plausible that the increased expression


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of the cellular machinery required for 5-HT production, coupled with the increased expression of 5-HT receptors in activated T cells, results in a cell autonomous feedback loop whereby 5-HT signaling is required for T cell-mediated immunity. Providing evidence for this hypothesis, LeonPointe and colleagues have shown that impairing 5-HT synthesis in T lymphocytes results in a reduction in T cell proliferation.121 While not as much is currently known about 5-HT function in B cells, these cells have been shown to express 5-HT receptors, including 5-HT3.122-124 Additionally, there is pharmacologic evidence that 5-HT acting at 5-HT1A receptors present on B cells is responsible for mitogen stimulated B cell proliferation.125 B cells appear to also express low levels of SERT in a manner that correlates to activation and proliferation states, however this increase in expression appears to exhibit anti-proliferative, apoptotic effects in B cells.126 SERT expression and corresponding SERT-mediated 5-HT uptake has been found in transformed B cells and Burkitt’s lymphoma cells.127 However, other groups have found negligible SERT-mediated 5-HT clearance in peripheral lymphocyte populations derived from murine, non-human primate and human sources, indicative of a lack of functional, surface SERT expression.128 Further studies and confirmatory genetic analyses are required for a better understanding of precisely how increased 5-HT uptake (possibly through SERT) in B cells modulates the activation states of these cells and how this ultimately effects immune function associated with inflammationassociated MDD. Although the cells of the adaptive immune system noted above express various proteins associated with 5-HT production and signaling, current evidence points more to a role of the innate immune system in driving MDD. One of the primary cell types associated with innate immune system function, monocytes, provide a link between MDD, aberrations in immune system function and 5-HT signaling. Monocytes are a subset of large, immature leukocytes located in the systemic circulation and spleen that ultimately differentiate into macrophages or



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dendritic cells at the site of injury.129 5-HT has been shown to enhance LPS-induced release of IL-6 and IL-1β from monocytes upon activation.130 These effects were found to be dependent upon 5-HT4 and 5-HT7 receptor signaling.119,

130, 131

Similar effects have been seen in mature

monocyte-derived dendritic cells (DCs) whereby 5-HT acting through 5-HT4 and 5-HT7 receptors results in an enhancement of IL-1β and IL-8 release.132 Providing a direct link between 5-HT system function, MDD and monocytes, the 5-HT/5-HIAA ratio in DCs is lower in those suffering from MDD than in their non-depressed counterparts.133 Monocytes appear to be the primary immunocyte that exhibits alterations in SERT expression in the context of MDD,134 and further the total number of monocytes expressing SERT has been shown to be reduced in those suffering from MDD.133 Plausibly, the elevations of proinflammatory cytokines present in certain populations of patients suffering from MDD may act systemically to alter the regulation of 5-HT in peripheral immune cells. The disruption of 5-HT signaling and metabolism in the periphery may be mimicked within the CNS, which could affect behavior and mood. Additionally, crosstalk between the periphery and CNS may directly alter 5-HT signaling within the CNS. Monocytes and/or macrophages carrying these messages may be able to traverse the BBB135 whereby they may alter the immune status of the CNS through the activation of glial cells or alter cytokine levels.136 Dendritic cells have the ability to activate T lymphocytes and act as liaisons between the innate and adaptive arms of the immune system. In this manner, DCs may be vitally important for the link from the immune system to MDD. Notably, DCs have been shown to express various 5-HT receptors including 5-HT1B, 5-HT1E, 5-HT2B, 5-HT3, 5-HT4 and 5-HT7.132 Stimulation of 5HT4 and 5-HT7 receptors in mature DCs has been shown to result in an enhancement of IL-1β and IL-8 release.132 It is plausible that the activation of T lymphocytes by DCs is modulated by orchestrated 5-HT signaling through 5-HT receptors which act to alter cytokine release and ultimately the response of the adaptive immune system. Seemingly small changes in this


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function could be present in populations of patients suffering from MDD and may result in sustained low grade inflammation/cytokine increases over time. Further, chronic changes in T cell responsiveness and the corresponding alteration of cytokine release may act to modulate the activity states of CNS targets such as SERT. It is plausible that this sustained, low grade chronic inflammation could act to alter the expression of SERT through transcriptional means;66 however, this has yet to be experimentally confirmed. Although specific cells of the immune system appear to express small quantities of the molecular machinery required for 5-HT production, the vast majority (99%) of 5-HT located in the systemic circulation is sequestered in platelets.137 Since platelets lack the enzymes needed to produce 5-HT, platelet 5-HT levels derive from sequestration through SERT as they pass through the bloodstream of the GI tract. 5-HT not sequestered by SERT is rapidly degraded, as revealed by the very low plasma levels of 5-HT in the blood of SERT KO mice.138 Platelets are also therefore the single largest store of 5-HT readily available to modulate the function of immune cells. It is plausible that the 5-HT taken up and then released by platelets acts as an immune modulator in certain instances by interacting with the 5-HT receptors located on immunocytes as outlined above.139 Hypothetically, SSRI treatment, by blocking platelet SERT and therefore reducing platelet 5-HT stores for subsequent release, will prevent platelet modulation of immune responses by not allowing 5-HT to interact with 5-HT receptors present on these immunocytes. The blockade of platelet SERT function and its effects on immune system function, inflammation and/or MDD-associated inflammation is unknown, but certainly warrants future study.

Serotonin, the GI Tract Immune System and Major Depressive Disorder



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The large-scale production of 5-HT by GI enterochromaffin cells and the obvious connection between 5-HT and neuropsychiatric disorders begs the questions as to whether 5HT production, release and uptake with the confines of the GI tract affects neurologic function and mood. This connection between the GI tract and neuropsychological processes has been dubbed, “the gut-brain axis.”140 Both 5-HT and SERT also play an important role in diseases associated with GI-specific inflammation and immune dysfunction. Inhibition of peripheral 5-HT production decreases cytokine responses to treatment with trinitrobenzene sulfonic acid (TNBS) (an activator of the innate immune system),141 suggesting that 5-HT release plays a role in perpetuating GI inflammation.

Along these lines, the SSRI fluoxetine was able to reverse

intestinal inflammation in IL-1 deficient mice, a model of inflammatory bowel disease.142 Mice lacking TPH1 expression exhibit reduced levels of 5-HT within the GI tract and have been shown to exhibit blunted inflammatory responses to dextran sulfate sodium (DSS)-mediated GI inflammation, a common model of colitis. Specifically, DCs and CD4+ T cell-mediated production of IL-12 and IL-17 in response to DSS-induced colitis, respectively, was found to be blunted in the TPH1 null mice as opposed to TPH1 expressing counterparts.143 Combined, these studies provide evidence that 5-HT production and release within the GI tract is associated with the ability of the local immune system to respond to inflammatory insults. This link between disruption in normal 5-HT signaling and GI inflammation leads to the possibility that the immune system present within the GI tract plays a role in depressive-like phenotypes via the gut-brain axis.144 Although not causal in nature, studies in rodents have provided evidence that components of diet (in this case Mg2+ content) alter depressive-like behaviors and the composition of the microbiota.145 Interestingly, manipulation of the diet in these studies resulted in the increase expression of IL-6 in the hippocampus,145 a 5-HT neuron projection region associated with MDD.


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Studies aimed at determining the role of the GI tract and microbiota colonies contained within the GI tract in 5-HT production and signaling are beginning to emerge. Microbiota changes in rodent models has been associated with depressive- and anxiety-like behavioral readouts largely associated with 5-HT signaling. Recently, Yano and colleagues published notable findings about how the microbiome regulates 5-HT synthesis within the GI tract.146 Germ free mice exhibit a reduction in serum levels of 5-HT, an effect linked to a reduction in TPH1 expression and a concomitant increase in SERT expression. These effects were reversed by colonization with specific-pathogen free microbiota, as were alterations in GI tract function. Importantly, alterations in 5-HT production in germ free mice extended to platelet function, effects that were also rescued through the normalization of gut microbiota.146 As addressed above, these alterations in the ability of platelets to sequester 5-HT and deficits in function may extend to immune processes that involve 5-HT acting to modulate immune system functionality and corresponding changes in CNS-mediated depressive-like behaviors.

SSRI Treatment and Immune System Function Increasing evidence indicates that SSRIs also have the ability to modulate immune system responsiveness to inflammation and infection.147-149 Importantly, studies have shown that immune system-induced changes in mood are amenable to reversal by classical depression treatments, including SSRIs.150, 151 MDD patients after SSRI treatment exhibit lower levels of the proinflammatory cytokine, IL-6,152 consistent with a reduction in depression symptoms. In contrast, sertraline (an SSRI) treatment has been shown to increase proinflammatory cytokine levels (IL-2, IL-12 and TNF-α) and decrease anti-inflammatory cytokines (IL-4 and TGF-β1) in depressed patients to normal levels.153 In rodents, Roumestan et al. found that post-LPS administration, SSRI treatment with fluoxetine reduces the number of macrophages, lymphocytes, neutrophils and eosinophils. Desipramine, a TCA that has a higher affinity for NET



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than SERT, however only decreased the number of macrophages and lymphocytes.154 Interestingly, both fluoxetine and desipramine inhibit the activity of the transcription factor NF-κB and activator protein 1 (AP-1) which have both been shown to control the expression of proinflammatory cytokine TNF-α.154 Studies have begun to elucidate the specific cellular compartments in which SSRI treatment exerts anti-inflammatory effects that confer specificity in the ability of these medications to alter immune status. Depressed patients treated with escitalopram exhibit increased levels of the natural inhibitor of IL-1, IL-1RA, and IL-2, that is proposed to contribute to a shift toward T helper 2 (TH2) response, an effect that acts to quell stimulation of the immune system.155 Another recent study has linked polybasic hemagglutinin (PHA)- and LPS-stimulated peripheral blood monocyte cells (PBMCs) treated with SSRI’s to a decrease in expression of CD4, CCR5 and CXCR4, suggesting that SSRI’s may be useful in reducing inflammation.156 The general consensus, bolstered by the studies noted above, is that SSRIs promote a decrease in proinflammatory cytokines and/or increase in anti-inflammatory cytokine levels.157-159 The specific sites of action by which these SSRIs produce these changes in immune signaling though are still not well characterized. Clearly, much more work is needed to understand the full complement of cytokines altered by antidepressant therapies. Although there is substantial evidence to suggest that the immunomodulatory effect of SSRI’s are dependent upon their actions on serotonergic pathways, some studies have shown that antidepressants may act through novel, independent mechanisms to modulate immune function, which have still yet to be revealed.160, 161 Several SSRI’s exhibit effects through targets other than SERT, including high affinity interactions with sigma receptors.116, 162 One model that may be particularly useful testing the SERT dependence of SSRI action on immune system function is the SERT Met172 mouse. This mouse expresses a single amino acid substitution in SERT from an isoleucine to methionine that disrupts high-affinity binding of many common


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SSRI’s to SERT while not disrupting the transporter recognition of 5-HT.163, 164 The utilization of these mice may provide a platform to determine the role of SERT in the immunomodulatory actions of SSRI’s and how these actions alter immune system status and ultimately behaviors relevant to depression.

Conclusion Several lines of evidence now point to immune system alterations associated with inflammatory processes being associated with MDD. The precise molecular mechanisms behind how these changes manifest as the behavioral and mood alterations in MDD are currently unclear. Nevertheless, it is clear that immune system activation and inflammation result in changes in serotonergic signaling in the CNS and periphery. Future work should be focused on causally linking changes in immune system status to the etiology of MDD and how disruptions in normal, physiologic serotonergic signaling play an important role in these effects. Immune system activation within the context of MDD may involve more than alterations in serotonergic signaling, as in recent years, novel, rapid acting antidepressants that act through glutamatergic targets have been found.165,

166

Future studies aimed at elucidating how 5-HT, the immune

system and the CNS interact to effect inflammation, mood and ultimately behavior will provide answer to many of these questions and may point to opportunities for novel therapeutics aimed at treating those suffering from MDD, particularly those who’s disorder is characterized by heightened inflammation and/or adaptive immune system function.

Acknowledgments



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Support for this work was generously provided by NIH MH078028, MH094527 and the Simons Foundation (RDB). Additional support was provided by the PhRMA Foundation and the Brain & Behavior Research Foundation (MJR).

Author Contributions MJR, MAQ and RDB co-wrote the current manuscript. MAQ and MR designed and produced figures contained within the manuscript.

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Table of Contents Figure Immune System Activation and Depression: Roles of Serotonin in the Central Nervous System and Periphery

Matthew J. Robson, Meagan A. Quinlan and Randy D. Blakely


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Figure 1: Serotonin (5-HT) controls various CNS, immune system and gastrointestinal functions. Several critical aspects of 5-HT signaling are present within these three physiologic systems, including enzymes needed for the production of 5-HT, SERT and 5-HT receptors. SERT is present within 5-HT neurons emanating from the raphe nucleus where is regulates serotonergic signaling within the CNS and is present on the surface of enterochromaffin cells where it controls 5-HT signaling and ultimately GI function. Further, SERT is present on cells within the systemic circulation including platelets where it regulates extracellular levels of 5-HT. 5-HT receptors within the CNS, GI tract and immune system act to relay 5-HT signaling throughout these various physiologic systems. 5-HT receptors of various subclasses have been discovered on monocytes, T lymphocytes, B lymphocytes and dendritic cells where they are believed to be involved in immune functionality. Expression levels of 5-HT receptors and enzymes required for 5-HT synthesis are altered in immunocytes in MDD.


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Figure 1: Serotonin (5-HT) controls various CNS, immune system and gastrointestinal functions. Several critical aspects of 5-HT signaling are present within these three physiologic systems, including enzymes needed for the production of 5-HT, SERT and 5-HT receptors. SERT is present within 5-HT neurons emanating from the raphe nucleus where is regulates serotonergic signaling within the CNS and is present on the surface of enterochromaffin cells where it controls 5-HT signaling and ultimately GI function. Further, SERT is present on cells within the systemic circulation including platelets where it regulates extracellular levels of 5-HT. 5-HT receptors within the CNS, GI tract and immune system act to relay 5-HT signaling throughout these various physiologic systems. 5-HT receptors of various subclasses have been discovered on monocytes, T lymphocytes, B lymphocytes and dendritic cells where they are believed to be involved in immune functionality. Expression levels of 5-HT receptors and enzymes required for 5-HT synthesis are altered in immunocytes in MDD. 215x279mm (300 x 300 DPI)



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Immune System Activation and Depression: Roles of Serotonin in the

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