Inflammation and Immunity in Depression: Basic Science and Clinical Applications

disciplines.

Chapter 1

Depression-Associated Cellular Components of the Innate and Adaptive Immune System

Diana Ahmetspahic; Dana Brinker; Judith Alferink    University of Münster, Münster, Germany

Abstract

Major depressive disorder (MDD) is a serious and debilitating mood disorder that affects millions of people each year worldwide. A convergence of evidence has indicated that patients with MDD exhibit altered immune responsivity. The cytokine hypothesis of depression proposed in the 1990s is based on numerous studies showing elevated circulating proinflammatory cytokine levels and impaired cellular immunity in patients with MDD and in animal models of depression. In this section, we highlight aspects of innate and adaptive immunity that have been implicated in the pathophysiology of MDD and depression-associated behavior. We further discuss the significance of key immune cell types that lie at the interface between innate to adaptive immunity in the context of MDD. Finally, we introduce T-helper cell subsets of the adaptive immune responses, while their specific role in depression will be discussed in greater detail in a separate chapter.

Keywords

Depression; Immune response; Innate immunity; Myeloid cells; T-helper cells

The Innate Immune System

The immune system, which defends the organism against potential pathogens and carcinogenesis, involves various tissues, cell types, and soluble components. It is generally divided into two major arms: innate immunity and adaptive immunity (Medzhitov et al., 2011). The innate immune system represents the first line of defense against invading microbial pathogens. In 1989, Charles Janeway Jr. postulated a revolutionary theory to explain what he called the immunologist's dirty little secret (Janeway, 2013). The classical self-nonself paradigm at that time did not explain why a foreign antigen alone was insufficient to elicit an adaptive immune response, and dirty extracts like mycobacteria, mineral oil, or aluminum hydroxide had to be coadministered for induction of an efficient T- and B-cell response. Janeway postulated two key features of innate immunity: the capacity to discriminate between noninfectious self from infectious nonself and to activate the adaptive immune response. He suggested that germline-encoded receptors on innate immune cells may recognize evolutionarily conserved microbial patterns that would thus drive the adaptive response. It is meanwhile well established that within minutes of encountering pathogens, innate immune cells recognize distinct evolutionarily conserved molecular structures called pathogen-associated molecular patterns (PAMPs) (Medzhitov, 2009). In events of trauma or tissue injury, damage-associated molecular-pattern (DAMP) molecules released by dead, damaged, or stressed cells from the host itself can also trigger innate responses. DAMPs encompass a variety of molecule types, including heat-shock proteins, cytoplasmic complex proteins S100A8/S100A9, high-mobility group box 1 DNA-binding proteins in the nucleus, and ATP. These molecules bind various pattern-recognition receptors (PRRs) expressed within the cytosol or on the membranes of innate immune cells, such as toll-like receptors (TLRs), C-type lectin receptors (CLRs), leucine-rich repeat (LRR)-containing (or NOD-like) receptors (NLRs), RIG-I-like receptors (RLRs), and AIM2-like receptors (ALRs). Subsequently, activated PRRs trigger signaling cascades, resulting in the release of cytokines and other factors by myeloid cells that orchestrate the recruitment of leukocytes to inflammatory sites and local effector functions (Iwasaki & Medzhitov, 2015). Myeloid cells that stem from common progenitors, descendants of hematopoietic stem cells in the bone marrow, are a highly heterogeneous population of innate immune cells, including granulocytes (e.g., neutrophils, basophils, eosinophils, and mast cells), monocytes/macrophages, and dendritic cells (DCs) (Akashi, Traver, Miyamoto, & Weissman, 2000) (Fig. 1). Current knowledge about the immune function of specific myeloid cell subsets, namely, neutrophils and monocytes, and their involvement in MDD is summarized in the following section.

Fig. 1 Schematic overview of human hematopoiesis of myeloid and lymphoid lineages. ILC , innate lymphoid cell.

Role of Neutrophils in Innate Immunity and MDD

Neutrophils (aka neutrophil polymorphonuclear granulocytes) account for 35%–80% of circulating leukocytes in mammals and are the ultimate first line of defense against acute infection. Peripherally circulating neutrophils are short-lived, terminally differentiated cells with a half-life of 8–12 h. It is currently discussed that distinct neutrophil subsets that exert inflammatory or antiinflammatory actions may exist under physiological and disease conditions (Kolaczkowska & Kubes, 2013). However, decisive markers for these functionally diverse neutrophil subsets have not been established. Activated neutrophils utilize various cytotoxic mechanisms to restrict pathogen replication, including engulfing invading pathogens by phagocytosis, degrading them by way of releasing granule-derived antimicrobial peptides, and producing reactive oxygen species (ROS). Quite remarkably, activated neutrophils can also trap and kill pathogens by forming so-called neutrophil extracellular traps made up of extracellular weblike fibers of granule antimicrobial proteins and nuclear contents (Kolaczkowska & Kubes, 2013).

Originally, neutrophils were thought to act exclusively as effector cells in innate immunity. However, in recent years, they have been shown to also be capable of modulating macrophage and DC-dependent functions in adaptive immune responses via cell-cell interactions and soluble mediators (Mayadas, Cullere, & Lowell, 2014). Upon making contact with endothelial cells, neutrophils undergo phenotypic changes and upregulate adhesion molecules, chemokine receptors, and proteases, such as neutrophil elastase. When they populate inflammatory sites, they exhibit an extended half-life of up to 1–2 days, a greater capacity to migrate, cytotoxicity, and protease release, and they may also form neutrophil extracellular traps (Kolaczkowska & Kubes, 2013). Interestingly, neutrophils also leave sites of tissue injury and migrate back into the vasculature (de Oliveira, Rosowski, & Huttenlocher, 2016). The contribution of this reverse migration to resolution of inflammation has not yet been resolved.

Increased neutrophil counts and percentages have been observed in peripheral blood of patients with MDD (Darko, Rose, Gillin, Golshan, & Baird, 1988; Irwin, Smith, & Gillin, 1987; Kronfol & House, 1989; Maes, Lambrechts, Suy, Vandervorst, & Bosmans, 1994; McAdams & Leonard, 1993). A meta-analysis demonstrated a relative increase in circulating neutrophils that was paralleled by a decrease in lymphocytes in MDD patients (Zorrilla et al., 2001). Neutrophil-to-lymphocyte ratio (NLR) is an indicator of systemic inflammation whose prognostic value has been demonstrated in cancer, cardiovascular diseases, and inflammatory diseases (Isaac et al., 2016). Nonmedicated patients with MDD have been shown to express a high NLR, compared with healthy individuals, whereas medicated MDD patients (taking selective serotonin reuptake inhibitors (SSRIs)) had normalized physiological NLRs (Demircan, Gozel, Kilinc, Ulu, & Atmaca, 2016). A recent study evaluating NLRs and platelet-lymphocyte ratios (PLRs) of inpatients and outpatients with MDD did not reveal a relationship between severity of depression and NLR, but did demonstrate higher PLRs in patients suffering from MDD with psychotic features than in those suffering from other subtypes of depression (Kayhan, Gunduz, Ersoy, Kandeger, & Annagur, 2017).

Also neutrophil functions such as phagocytosis and ROS production have been investigated in MDD patients. Phagocytosis in immune cells can be assessed by in vitro uptake of fluorescently labeled microorganisms or particles coated with microbial cell-wall components (e.g., the yeast protein zymosan). In depressed individuals, phagocytosis of zymosan particles assessed with chemiluminescence was shown to be impaired in polymorphonuclear cells that include several types of granulocytes among those neutrophils. Reduced polymorphonuclear activity was normalized in response to effective therapy but not in nonresponders (O'Neill & Leonard, 1990). In a later study from the same group, phagocytosis was specifically investigated in monocytes and neutrophils from depressed patients. Neutrophil phagocytosis was reduced during acute phase of disease but returned to control values on recovery. Interestingly, monocyte phagocytosis was increased and returned to control values following remission (McAdams & Leonard, 1993). A subsequent meta-analysis also reported decreased cellular neutrophil function in MDD (Zorrilla et al., 2001). Maes et al. demonstrated that chemotaxis, superoxide release, and phagocytic capacity of circulating neutrophils were similar across diverse subtypes of depression and healthy volunteers (Maes et al., 1992). Also, no difference in neutrophil phagocytosis of Escherichia coli was detected by flow cytometry between hip fracture patients with versus without depressive symptoms (Duggal, Upton, Phillips, & Lord, 2016). Production of polymorphonuclear elastase, an index of neutrophil function, has also been assayed in MDD and found to be enhanced (Deger et al., 1996). Finally, patients with MDD, including elderly patients and patients with comorbidity in heart failure, have been reported to have increased plasma levels of neutrophil gelatinase-associated lipocalin (NGAL), an innate antibacterial factor expressed in activated neutrophils (Naude et al., 2014). Thus, NGAL has emerged as a potential biological link between depression and cardiovascular disease. The relationship between depression and cardiovascular disease will be reviewed in a following section.

Monocytes and MDD

Monocytes are by far the most studied myeloid cell subset in relation to MDD. They are highly plastic regarding their phenotypes and population sizes. Monocytes can act as phagocytes via expression of various receptors, such as Fc, complement, and scavenger receptors (Ginhoux & Jung, 2014; Ziegler-Heitbrock, 2015). Generally, circulating monocytes represent 4%–10% of white blood cells in humans and mice. Monocyte numbers surge within minutes of stress exposure or physical exertion. This phenomenon has been explained by the existence of monocyte reservoirs in areas of slow blood velocity where monocytes adhere weakly to vascular endothelium and can thus be mobilized rapidly in response to catecholamines (Ziegler-Heitbrock, 2015).

Epitopes called cluster of differentiation (CD) molecules are commonly used to identify subsets of cell types, particularly immune system cell types. Based on their surface expression of the lipopolysaccharide (LPS) coreceptor CD14 and the FcγIII receptor CD16, circulating monocytes in humans have been grouped into three monocyte classes: classical CD14+ CD16−, intermediate CD14+ CD16+, and nonclassical CD14lowCD16+ monocytes. Classical monocytes (mouse analog, Ly6Chigh) comprise some 80%–90% of all monocytes, while the other two CD16+ monocyte classes (mouse analog, Ly6Clow) account for the remaining 10%–20% (Ziegler-Heitbrock, 2015). Classical monocytes are recruited to inflammatory sites, where they are highly phagocytic and modulate inflammation via release of ROS and cytokines, including the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)-6 and the antiinflammatory cytokine IL-10. Intermediate CD14+ CD16+ monocytes are associated with active inflammatory conditions and can exert antitumoral actions. They are also phagocytic and produce various proinflammatory molecules, including not only ROS, TNF, IL-1β, IL-6, and IL-12 but also IL-10 (Ziegler-Heitbrock, 2015). Meanwhile, nonclassical monocytes (and their Ly6Clow counterparts in mice), which have a low phagocytic capability, patrol blood vessels and are involved in tissue surveillance and homeostasis (Auffray et al., 2007; Cros et al., 2010).

Alterations in monocyte counts and functionality in patients with MDD have frequently been reported. For example, monocytosis and neutrophilia have been suggested as hallmarks of severe depression (Maes et al., 1992). However, both elevated and reduced and also unchanged monocyte counts and percentages in peripheral blood have been observed in depressed subjects (Darko et al., 1988; Lanquillon, Krieg, Bening-Abu-Shach, & Vedder, 2000; McAdams & Leonard, 1993; Schlatter, Ortuno, & Cervera-Enguix, 2004b; Seidel et al., 1996b). A reduced monocytic phagocytosis of fluorescently labeled particles or bacteria in the peripheral blood of patients with MDD along with lower surface expression of class II human leukocyte antigens (HLAs) and elevated respiratory burst activity was reported (Schlatter et al., 2004b). On the contrary, monocytes from MDD subjects have also been reported to have an increased capacity to engulf zymosan during active depression, but not during symptom remission (McAdams & Leonard, 1993).

Multiple studies, including meta-analyses have indicated that MDD is associated with elevated peripheral levels of cytokines, chemokines, and other inflammatory factors (Dowlati et al., 2010; Eyre, Stuart, & Baune, 2014; Goldsmith, Rapaport, & Miller, 2016; Haapakoski, Mathieu, Ebmeier, Alenius, & Kivimaki, 2015; Howren, Lamkin, & Suls, 2009; Kohler et al., 2017). Proinflammatory cytokines such as TNFα, IL-1β, IL-6, and chemokines CCL2 and CXCL8 (IL-8) are typically produced by activated monocytes (Ziegler-Heitbrock, 2015). Upregulated expression of proinflammatory mediators and chemokines (i.e., IL-1, IL-6, TNF, CCL2, and CCL7) has been demonstrated specifically in purified monocytes from nonmedicated MDD patients (Carvalho et al., 2014). In accordance, a meta-analysis has further demonstrated increased serum levels of CCL2 in depressed subjects (Eyre et al., 2014). Additionally, monocytes and other immune cells from MDD patients have been shown to exhibit an altered responsiveness to LPS stimulation (Humphreys, Schlesinger, Lopez, & Araya, 2006; Krause et al., 2012; Lisi et al., 2013). Stimulated whole blood from depressed patients has yielded high levels of IL-1β and IL-6, but not TNF, compared with control subjects (Schlatter et al., 2004a). In a large cohort study, proinflammatory cytokine production of LPS-stimulated whole blood cells was further associated with symptom severity of depression and anxiety (Vogelzangs, de Jonge, Smit, Bahn, & Penninx, 2016). Conversely, there is also evidence of reduced immune cell responsiveness in MDD, a phenomenon known as the depression-associated exhausted state of innate immunity (Krause et al., 2012). Krause et al. observed reduced interferon (IFN)-γ and IL-10 production in LPS-stimulated whole blood cultures with myeloid cells from MDD patients and found normalization of these differences in response to the antidepressant drug imipramine or the antiinflammatory drug celecoxib (Krause et al., 2012). Additionally, reduced expression of the proinflammatory factors prostaglandin E2 and cyclooxygenase-2 by LPS-stimulated monocytes has been observed in subjects with MDD (Lisi et al., 2013). These controversial findings regarding immune differences in patients with MDD could be due, at least in part, to variability across experimental designs, including the use of differing LPS concentrations. Notably, the ex vivo LPS-induced responsivity of immune cells may be LPS dose-dependent. Additionally, lifestyle and health factors (e.g., smoking, alcohol intake, body mass index, and chronic diseases) may greatly influence the basal cytokine levels and the production capacity of innate immune cells (Vogelzangs et al., 2016). Nonetheless, the majority of studies examining the issue point to depression-associated monocyte alterations.

Innate Lymphoid Cells (ILCs) and Natural Killer (NK) Cells

White blood cells derived from lymphoid tissues are known generally as lymphocytes. Lymphocytes include innate lymphocytes (ILCs), T cells, and B cells (Mjösberg & Spits, 2016). Until recently, the only known ILC was the NK (Vivier et al., 2011). ILCs defined as exhibiting lymphocyte morphology but lacking T-cell or B-cell lineage markers are modulated by cytokine signaling. They are present in mucosae and mucosal-associated lymphoid tissues where they are involved in the initiation and resolution of inflammatory responses and subsequent tissue repair. ILCs play a critical role in graft-versus-host disease, inflammatory bowel disease, asthma, atopic dermatitis, and multiple sclerosis. Based on their expression of transcription factors, cytokines, and cell surface markers, ILCs can be subdivided into three groups. Group 1 ILCs act primarily against intracellular pathogens and produce IFN-γ; this group includes NK cells. Meanwhile, group 2 ILCs act mainly against parasites via production of IL-4 and IL-13. Group 3 is composed of lymphoid tissue inducer cells, which are important for secondary lymphoid tissue formation during embryogenesis, and cells that combat extracellular bacteria via release of IL-17 and IL-22 (Artis & Spits, 2015). Each ILC group has T-cell counterparts with some redundancy in functionality and transcription factor expression (Bando & Colonna, 2016). While the role of ILCs generally in MDD has not yet been studied, numerous studies have examined circulating NK cells in MDD.

NK cells, first described more than 40 years ago, were initially regarded as nonspecific immune cells with the capacity to kill tumor cells and virally infected cells without prior sensitization. However, the discovery of a complex system of activating (e.g., 2B4, NKG2D, NKp46, and NKp44) and inhibiting (e.g., killer-cell Ig-like receptors (KIRs)) NK cell receptors indicated that in fact, they are not truly nonspecific (Vivier et al., 2011) (Fig. 2). In a simplified view, NK cell subsets in human peripheral blood can be distinguished based on the relative expression of CD16 (low-affinity Fc-receptor γ IIIA) and CD56 (neural cell adhesion molecule, NCAM). Some 90% of the circulating NK cells in humans are CD56highCD16low/high NK cells, with the remaining ~ 10% being CD56lowCD16high NK cells that are localized primarily to secondary lymphoid organs (Lünemann, Lünemann, & Münz, 2009). CD56high NK cells are considered to be immune-regulatory cells that produce a wide variety of cytokines (e.g., GM-CSF, IFN-γ, IL-10 or IL-13, and TNF), whereas CD56low NK cells are the major killer population due to their ability to exert cytotoxic effector functions. In the presence of activating signals, NK cells exert cytotoxicity upon cells deemed to be invaders due to their failure to express self-identifying class I HLA molecules. NK cell cytotoxicity is mediated via three distinct pathways: exocytosis of granules with the release of perforin (a pore-forming protein) and granzymes, death receptor-induced apoptosis, and antibody-dependent cell-mediated cytotoxicity (Vivier et al., 2011).

Fig. 2 Schematic overview of human NK cell subsets. Exemplary activation and inhibitory NK cell receptors on indicated NK cell subsets are shown. KIR , killer-cell Ig-like receptor; NK , natural killer cell.

NK Cells and Depression

Acute stressors, such as public speaking and stress tests, increase circulating NK cell counts in healthy individuals due, at least in part, to stress-induced catecholamines reducing β2-adrenergic receptor-mediated adhesion of NK cells to endothelial cells, resulting in a rise in circulating NK cells (Benschop, Schedlowski, Wienecke, Jacobs, & Schmidt, 1997; Segerstrom & Miller, 2004). Some research groups have reported evidence of increased circulating NK cells in MDD, while others have reported diminished NK cell numbers in MDD (Evans et al., 1992; Grosse et al., 2016; Ravindran, Griffiths, Merali, & Anisman, 1999; Rothermundt et al., 2001; Seidel et al., 1996a; Zorrilla et al., 2001). However, these incongruent studies differed methodologically in terms of cohorts, interventions, and evaluated outcomes.

A consistently reproduced finding has been reduced NK cell cytolytic activity (NKCA) in MDD, with lesser reduction being associated with a better treatment response (Irwin & Gillin, 1987; Zorrilla et al., 2001). Significant reductions of NKCA were observed in depressed men compared with nondepressed men, but not in women with MDD when compared with nondepressed women (Evans et al., 1992). Reduced NKCA appears to be particularly profound in patients with an early MDD onset age (Frank, Wieseler Frank, Hendricks, Burke, & Johnson, 2002). Variance in NKCA has partly been explained by depressed mood, somatic anxiety, and less diurnal variation (Maes et al., 1994). Sleep disturbance was found to correlate inversely with NKCA in both MDD patients (Cover & Irwin, 1994) and healthy individuals (Irwin, Smith, & Gillin, 1992). Electroconvulsive therapy, a treatment option for therapy-resistant MDD, has been shown to increase NKCA only transiently in medication-resistant MDD patients or MDD patients with psychotic features (Fluitman et al., 2011; Kronfol, Nair, Weinberg, Young, & Aziz, 2002).

Serotonergic mechanisms have been implicated in NK cell activation. Among depressed patients medicated with SSRIs, NKCA levels were augmented more in those patients whose symptoms were responsive to the treatment than in nonresponsive patients (Park, Lee, Jeong, Han, & Jeon, 2015). Moreover, SSRI therapy increased NKCA in depressed outpatients with a reduced NKCA at baseline, and the NKCA of mononuclear cells from MDD patients was increased upon in vitro incubation with SSRIs (Frank, Hendricks, Johnson, Wieseler, & Burke, 1999). Findings showing that enhanced intracellular ROS levels in monocytes correlate with reduced NKCA in MDD suggest that ROS may suppress NKCA (Frank et al., 2001). The reduction of NKCA may be dependent on serotonin, which was reported to clear ROS in the microenvironment consequently preserving NK cell survival and activity (Betten, Dahlgren, Hermodsson, & Hellstrand, 2001). Type 1A serotoninergic receptors on inhibitory monocytes are bound by ROS, thus protecting NK cells from oxidative damage at inflammatory sites (Hellstrand & Hermodsson, 1993). Additionally, reduced levels of the neuronal protein p11, which amplifies serotonin receptor-mediated signaling, have been associated with depression-like behavior (Svenningsson, Kim, Warner-Schmidt, Oh, & Greengard, 2013). Paradoxically, however, Svenningsson and colleagues found that reduction of p11 in NK cells and monocytes within 8 weeks of beginning citalopram antidepressant therapy predicted a positive antidepressant response in MDD patients (Svenningsson et al., 2014).

Various potential underlying mechanisms of reduced NKCA in MDD have been discussed. Clusters of genes involved in NK cell activation have been found to be downregulated selectively in patients with active, as opposed to remitted, MDD (Jansen et al., 2016). Meanwhile, rodent models of stress and depression have demonstrated an adrenergic influence on NK function and distribution (Engler et al., 2004; Tarr et al., 2012). For example, decreased NKCA in a rodent social confrontation model was associated with significantly increased NK-sensitive lung tumor metastasis in a manner that could be mitigated by a β-adrenergic antagonist pretreatment (Stefanski & Ben-Eliyahu, 1996). Additionally, augmented corticotropin-releasing factor (CRF) secretion in depression has been shown to affect NK cell function. Thus, treatment with synthetic CRF reduced NKCA in rats, while pretreatment of these animals by chemical sympathectomy completely abolished CRF-induced plasma catecholamine levels and reduction in splenic NK activity (Irwin, Hauger, Jones, Provencio, & Britton, 1990). Furthermore, stress and depression may impede NK cell migration. Restraint stress in rodents was reported to delay NK cell recruitment to the lungs during influenza infection, and this delay was associated with suppression particularly of chemokines (CCL2 and CCL3) (Hunzeker, Padgett, Sheridan, Dhabhar, & Sheridan, 2004).

The Adaptive Immune System and Its Components

The adaptive immune response is mounted by T and B lymphocytes that recognize specific pathogens and enable immune memory to bolster subsequent encounters with recognized pathogens (Medzhitov et al., 2011). Broadly, adaptive immune responses consist of (i) the cellular immune response mounted by T cells and (ii) the humoral response mediated by B cells. B cells recognize native proteins via membrane-bound immunoglobulins, whereas T cells recognize antigenic peptides bound to HLA molecules expressed on antigen-presenting cells (APCs) via their antigen-specific T-cell receptors (TCRs) (Rajewsky, 1996; Zinkernagel & Doherty, 1979). APCs that comprise a heterogeneous group of immune cells, namely, DCs, macrophages, and B cells, exhibit the capacity to process and present antigens. When immature DCs that have been released from bone marrow encounter pathogens in peripheral tissues, they undergo a maturation program and migrate to the lymph nodes where they prime naive T cells (Steinman, 2012). In general, cellular and humoral responses may cooperate in immune defense. Adaptive immune responses are characterized by the capacity for immune memory that provides long-lasting protection and rapid responses to subsequent encounters with pathogens (Farber, Netea, Radbruch, Rajewsky, & Zinkernagel, 2016). Here, we will review T-helper cell subset functions that are potentially relevant for depression. The literature regarding altered cell-mediated adaptive immunity in depression will be reviewed in detail in the following section.

The human body contains about 10¹² T cells (Miles, Douek, & Price, 2011). Committed lymphoid progenitors that arise in the bone marrow and do not yet express TCRs, CD4, or CD8 enter the thymus from the blood stream. The thymus is a multilobed retrosternal organ referred to as a school for T cells owing to its unique role in T-cell differentiation. A complex series of molecular and cellular events involving rearrangement of TCR genes and both negative and positive selection steps result in the production of a broad repertoire of T cells that are restricted by HLAs encoded by the major histocompatibility complex (MHC) gene region but lack self-reactivity. Only a minority of thymocytes that coexpress TCRs and CD4 or CD8 leave the thymus (Kurd & Robey, 2016). The thymus also governs the development of so-called regulatory T cells (Treg) that control potentially autoreactive T cells that evaded negative selection (Sakaguchi, Vignali, Rudensky, Niec, & Waldmann, 2013). In addition to central tolerance mechanisms occurring in the thymus, various mechanisms of peripheral tolerance including deletion, functional inactivation (anergy) of potentially autoreactive T cells, and downregulation of potentially self-reactive TCRs in the periphery may prevent autoimmunity (Arnold, 2002). Most T cells express TCRs composed of a heterodimer of α and β chains associated with CD3 (αβ TCRs), an invariant complex that facilitates signal transduction (Wucherpfennig, Gagnon, Call, Huseby, & Call, 2010). Only a minority of T cells express γδ TCRs, and they, being first-line defense components, populate epithelial tissues commonly exposed to external agents (Chien, Meyer, & Bonneville, 2014).

Recognition of antigens by T cells expressing αβ TCRs depends on the presentation of antigenic peptides bound in the groove of HLA molecules, which are highly polymorphic cell surface molecules encoded by genetic loci in the MHC gene complex region on the short arm of chromosome 6 (Rammensee, Falk, & Rotzschke, 1993). Classical class I HLA molecules, namely, HLA-A, HLA-B, and HLA-C, are expressed by all human cell types except erythrocytes. Meanwhile, class II HLA molecules, namely, HLA-DR, HLA-DQ, and HLA-DP, are expressed constitutively on a subset of antigen-presenting cells, such as DCs, B cells, and macrophages, and can be induced in activated T cells in humans and some other cell types under inflammatory conditions. T cells expressing αβ TCRs consist of two major functionally distinct populations: CD8+ and CD4+ T cells that recognize peptides derived from proteins bound to class I and class II HLA molecules, respectively (Mak, 2007; Masopust, Vezys, Wherry, & Ahmed, 2007).

Historically, class I and class II HLA molecules have been associated with endogenous and exogenous peptide sources, respectively. The original assumption that class I HLAs present intracellular synthesized peptides exclusively was challenged by findings showing that exogenous antigens can also access the cytosolic MHC-I pathway, a phenomenon known as cross presentation (Cruz, Colbert, Merino, Kriegsman, & Rock, 2017). Moreover, self-antigens can also be presented by class II HLAs and be subjected to autophagy (Roche & Furuta, 2015). Activation of T cells expressing αβ TCRs following antigen recognition in the context of a specific cytokine milieu induces a complex signaling cascade that may lead to cell proliferation, cytokine secretion, and differentiation into effector and/or memory cells of specialized phenotypes. Thus, CD8+ and CD4+ T-cell subsets are highly heterogeneous.

Activated CD8+ T cells have the potential to develop into cytotoxic T lymphocytes (CTLs) that play key roles in limiting infection and tumor defense. Upon MHC-I-dependent recognition, CTLs kill target cells with lytic enzymes such as perforin and granzymes. Additionally, CTLs can eliminate infected cells by way of death-inducing ligand-receptor binding (Zhang & Bevan, 2011). Activated CD4+ T cells differentiate into specific T-helper (Th) cell subtypes, principally Th1 and Th2, upon antigen recognition in the context of class II HLAs and an inductive cytokine milieu in the microenvironment (Fig. 3). The presence of IL-12 favors differentiation of CD4+ T cells into Th1 cells, which produce among other cytokines IL-2 and IFN-γ. Meanwhile, the presence of IL-4 promotes the development of Th2 cells that secrete IL-4, IL-10, and IL-13. Th1 and Th2 differentiation depends on distinct transcription factors, most notably T-bet for Th1 cell differentiation and GATA-3 for Th2 development (Zhu & Paul, 2008; Ziegler, 2016). In recent years, considerable effort has been made to understand the diversity and functionality of additional CD4+ Th cell subsets. Based on extensive multiparameter flow cytometry studies, several cell surface markers have been attributed to distinct T-cell functionality, such as CD45RA-mediated T-cell activation, CD27/28-mediated costimulation, and programmed cell death-1-mediated inhibition. Thus, the expression assays of CD45RA, the chemokine receptor CCR7, and the membrane-bound costimulatory molecules CD27 and CD28 have been utilized to characterize naive and antigen-experienced T cells in humans (Mahnke, Brodie, Sallusto, Roederer, & Lugli, 2013). Functionally distinct CD4+ T-cell subsets are also characterized by their chemokine receptor profiles. Accordingly, Th1 cells have been shown to express CCR5 and CXCR3, whereas Th2 cells have been found to express CCR3, CCR4, and CD294 (aka prostaglandin D2 receptor 2). Additional CD4+ Th cell subsets include follicular helper (Tfh), Th9, Th17, and Treg cells. Tfh cells express the chemokine receptor CXCR5 and can migrate to the follicle where they provide B cell help via expression of the CD40 ligand and production of IL-4 and IL-21 (Lefrancois & Marzo, 2006).

Fig. 3 Schematic overview of T-helper subset differentiation. Following antigen presentation by DCs, naive CD4 + T cells differentiate into distinct Th cell subsets in the context of various cytokines. Exemplary differentiation cytokines are shown, and cytokines that are produced by T-cell subsets are shown. PAMPs , pathogen-associated molecular-pattern molecules; DAMPs , damage-associated molecular-pattern molecules; PRR , pattern-recognition receptor; DC , dendritic cell; IL , interleukin; IFN , interferon; Th , T-helper cell; T reg , regulatory T cells; Tfh , follicular T-helper cell.

Numerous studies have investigated the Th1/Th2 dichotomy of CD4+ Th cells in MDD, and some recent studies have focused on the potential role of Th17 cells and Treg cells in depression. Those bodies of work will be reviewed in the following section. In preparation for discussing those findings, key aspects of Th17 cell biology and Treg cell biology are introduced here.

Th17 cells, discovered in 2005, were named for their capacity to produce the prototype cytokine IL-17. They also produce cytokines other than IL-17, such as granulocyte-macrophage colony-stimulating factor, IL-6, and IL-21. Th17 cells have a potent inflammatory influence and have been suggested to be implicated in the pathogenesis of autoimmune and inflammatory disorders, such as multiple sclerosis, rheumatoid arthritis, and allergic skin diseases (Korn, Bettelli, Oukka, & Kuchroo, 2009). Differentiation of Th17 cells is dependent on various transcription factors, such as the signal transducer and activator of transcription 3 (Stat3), retinoic acid receptor-related orphan receptor γ (RORγ), and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB), and Th17 cells are promoted by transforming growth factor (TGF)-β, IL-6, and IL-23 (Crome, Wang, & Levings, 2010). The IL-17 cytokine family consists of six members, namely, IL-17A-F. IL17A, commonly referred to as simply IL-17, and IL-17F share high sequence homology and both mediate proinflammatory responses. While Th17 cells are the major sources of IL-17A and IL-17F in adaptive responses, T cells expressing γδ TCRs, NK cells, and NK-T cells are capable of producing IL-17 (Korn et al., 2009; Mahnke et al., 2013). Autoimmunity studies utilizing mice deficient in various IL17 cytokines or pharmacological blockade of IL17 have produced conflicting data on the role of Th17 cells and IL-17 in the pathogenesis of autoimmunity. While IL-17-producing lymphocytes have been shown to exhibit an enhanced capacity to migrate through the blood-brain barrier and to accumulate in CNS compartments of MS patients, the cytokine IL-17A appears to be largely dispensable for the development of central nervous system autoimmunity in mice (Becher & Segal, 2011).

Acting somewhat in opposition to Th17 cells, CD4+ Treg cells play an important role in maintaining peripheral self-tolerance and controlling autoimmunity. Treg cells were first discovered nearly 50 years ago when they were identified as immune suppressor cells (Gershon & Kondo, 1970). For many years, they were a challenge to isolate due to a lack of appropriate markers. Finally, Sakaguchi et al. demonstrated in 1995 that IL-2 receptor α-chain (CD25) could be used as a surrogate marker for Treg cells (Sakaguchi, Sakaguchi, Asano, Itoh, & Toda, 1995). Subsequently, a variety of functionally and phenotypically diverse Treg cells, including CD25+ and CD25− subsets, have been identified. Notably, two currently recognized major subsets of Treg cells are the (i) thymus-derived (aka naturally occurring) Treg cells that express FOXP3 (a forkhead/winged-helix transcription factor) and the (ii) induced (aka peripheral) Treg cells, commonly referred to as iTreg cells, that are induced in response to pathogens or inflammatory processes (Sakaguchi et al., 2013). Treg cells can exert suppressive effects via cell-cell contact or via soluble immunosuppressive factors, such IL-10 and TGF-β. Treg cells also employ IL-10 and TGF-β to suppress DC maturation and activation. Additionally, CD4+ CD25+ Treg cells utilize cytotoxic T lymphocyte-associated antigen 4 and glucocorticoid-induced tumor necrosis factor receptor to suppress effector T-cell proliferation and IFN-γ production (Vignali, Collison, & Workman, 2008). The biological relevance of Treg cells in autoimmunity has been underscored by findings of a decreased suppressive function of CD4+ CD25+ Treg cells in patients suffering from multiple sclerosis, type I diabetes, psoriasis, and myasthenia gravis, relative to that in healthy controls (Brusko, Putnam, & Bluestone, 2008). Such observations suggest that manipulations of Treg cell function could perhaps be used to treat human disease.

Summary

In summary, this chapter highlighted depression-associated cellular components of the innate and adaptive immune system. Alterations of key features of myeloid cells, including chemotaxis, phagocytosis, production of nitric oxide, and expression of cytokines, and changes in essential T-cell effector functions could play important roles in the pathological processes underlying MDD. Future investigations that aim to resolve yet unknown immune mechanisms of MDD would benefit from approaches that dissect the temporal and spatial regulation of immune alterations and examine the interrelationship between the innate and acquired arms of the immune system in disease pathophysiology. Targeted research strategies that identify immune factors and cellular components at the interface of the innate and acquired immunity arms that are altered in various disease phases may help to identify predictive immune markers of affective disorders. Resolving the factors underlying MDD-associated alterations in innate and adaptive immunity may further lead to the identification of novel immune-based therapies for depression.

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Chapter 2

Childhood Microbial Experience, Immunoregulation, Inflammation, and Adult Susceptibility to Psychosocial Stressors and Depression

Graham A.W. Rook⁎; Charles L. Raison†; Christopher A. Lowry‡,§,¶,‖    ⁎ Centre for Clinical Microbiology, UCL (University College London), London, United Kingdom

† University of Wisconsin-Madison, Madison, WI, United States

‡ University of Colorado Boulder, Boulder, CO, United States

§ University of Colorado Anschutz Medical Campus, Aurora, CO, United States

¶ Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC), Denver Veterans Affairs Medical Center (VAMC), Denver, CO, United States

‖ Military and Veteran Microbiome Consortium for Research and Education (MVM-CoRE), Denver, CO, United States

Abstract

Microbial exposures early in life modulate our susceptibility to depression via effects on the composition of the microbiota and the development of the immune system. Modern urban lifestyles reduce these exposures resulting in altered microbiota and defective regulation of inflammatory responses. These changes are reflected in the increasing prevalence of chronic inflammatory disorders and of persistently raised biomarkers of inflammation among those living in modern urban societies, compared with those living a hunter-gatherer or subsistence agriculture-based lifestyle. Moreover, the microbiota regulates metabolism, so a distorted microbiota can promote obesity and the associated inflammation. Similarly, the microbiota regulates the size of the inflammatory response to psychosocial stressors. Thus, there are multiple links between childhood exposures to microbes and the later presence of persistent inflammation that contributes to the risk of depression. Here, we evaluate the evidence for the impact of childhood microbial exposures on subsequent vulnerability to stress-related psychiatric disorders.

Keywords

Immunoregulation; Microbiota; Regulatory T cell; Gut-brain axis; Stress; Obesity; Depression

Introduction

This book deals with the role of inflammation and immunity in depression. Our exposures to microorganisms in early life impact on this theme in many ways, and Fig. 1 attempts to provide a simplified summary of the text that follows. Microbes modulate susceptibility to depression via effects on the immune system, local regulation of gut neuroendocrine systems, afferent neuronal signaling, and microbial metabolites that enter the circulation and act on distant targets including the brain. First, microbes drive the development of the immune system by providing the data that the immune system requires before it can function correctly. Second, microbes, not only mostly from mother and other family members but also to a lesser extent from the environment, constitute the symbiotic microbiotas that colonize our body surfaces, notably the gut, airways, skin, and mucosal surfaces of the urinary and reproductive tracts. Vertebrates, which evolved about 500 million years ago, rapidly developed a very complex and diverse gut microbiota that took on crucial roles in the development and subsequent function of essentially all organ systems, including the gut, immune system, and brain (McFall-Ngai et al., 2013). Some of the effects on the brain are mediated by microbial metabolites that are only beginning to be explored. Most evolutionary biologists now think that the adaptive immune system (which invertebrates do not have) evolved to enable the vertebrate immune system to control and regulate this very complex and physiologically essential microbiota (we might almost say it evolved to help farm the microbiota) while simultaneously excluding pathogens, which we can define as organisms that cause damage or disrupt the human-microbiota ecosystem (Pancer & Cooper, 2006). But communication between the microbiota and the immune system goes both ways, and in concert with the data from infections and environmental organisms, the microbiota plays a major role in setting up the regulatory mechanisms that limit and terminate inflammatory processes. Fig. 1 shows inflammation, highlighted in red, associated with chronic inflammatory disorders, such as allergies, autoimmune diseases, and inflammatory bowel diseases (IBDs), and also inflammation manifested as raised C-reactive protein or inflammatory cytokine levels in the absence of any of these diagnosable inflammatory disorders. These are all situations in which failing immunoregulation contributes to inflammation, and all are associated with increased risk of psychiatric problems. Moreover, inflammation can also be caused by poorly regulated metabolism and obesity, in which the microbiota again plays a major role. Finally, Fig. 1 highlights inflammation due to stress. But the inflammatory response to stress and the subsequent behavioral changes are again modulated by the microbiota and attenuated by the regulatory arms of the immune system. In conclusion, childhood microbial exposures, by supplying and modifying the microbiota, modifying the regulation of the immune system, and modifying the regulation of the microbiota by the immune system, have major effects on our susceptibility to some psychiatric disorders.

Fig. 1 Overview of the ways in which exposure to microorganisms and parasites can influence the brain via effects on the immune system and microbiota that modulate inflammation. Immunoregulatory microbial signals include metabolites and are illustrated in Fig. 2. Because the microbiota is critically involved in metabolism, obesity, and responses to psychosocial stressors, the role of these factors in depression is also modulated by microbial exposures. Abbreviations: CRP, C-reactive protein; IBD, inflammatory bowel disease.

In much of what follows, we are forced to discuss the increases in disorders of immunoregulation indicated in Fig. 1 as surrogates for depression, because these are easily diagnosed, common, and intensively studied, though data on depression are used when available, particularly toward the end of the chapter. But it should be remembered that, as indicated in Fig. 1, depression is frequently comorbid with the chronic inflammatory disorders and associated with raised levels of blood biomarkers of inflammation (Hodes, Menard, & Russo, 2016; Maes, 1999; Raison, Lowry, & Rook, 2010). For example, prior hospitalization due to an autoimmune disease has been associated with a 45% increased risk of subsequently developing mood disorder diagnosis (Benros et al., 2013). Migration, urbanization, modern medicine, and high-income lifestyle all lead to the loss of exposure to the organisms with which we coevolved (old friends) and to increases in inflammatory and psychiatric disorders (reviewed and referenced in Rook, Raison, & Lowry, 2014). If we can understand these effects, we may be able to intervene and to counteract the trends toward higher incidences of inflammation and depression.

Microbial Exposures and Human Evolution

Before analyzing these issues in greater depth, we need to identify the groups of organisms with which humans coevolved, and that might have become physiological necessities. The notion that modern life might deprive us of essential exposures initially emerged as the hygiene hypothesis, following the observation that allergies are less frequent in children brought up with older siblings (Strachan, 1989). It was suggested that older siblings provided increased exposure to childhood infections that might somehow protect from allergic disorders. This was a valuable insight, but it implied a crucial role for the common infections of childhood and for hygiene, whereas neither implication is correct. The childhood infections are mostly crowd infections such as measles that either kill the host or induce solid sterilizing immunity, so they could not survive in isolated Paleolithic hunter-gatherer groups (Wolfe, Dunavan, & Diamond, 2007). They did not coevolve a necessary immunoregulatory role, and they do not protect from chronic inflammatory disorders and often actually trigger them (Bremner et al., 2008; Yoo, Tcheurekdjian, Lynch, Cabana, & Boushey, 2007). They are recent arrivals in human communities, endemic only since populations have increased (Wertheim & Kosakovsky Pond, 2011). Meanwhile, implicating hygiene was a reasonable guess, but as will be demonstrated later, hygiene is a minor factor in the contemporary reduction in microbial exposures. Therefore, we prefer terms such as the biodiversity or old friends hypothesis (Rook, 2010; von Hertzen, Hanski, & Haahtela, 2011), which place emphasis on our evolutionary heritage and are leading to the identification of relevant organisms and mechanisms.

Microbiota

Humans evolved in small hunter-gatherer groups. As outlined in the introduction, humans, like all vertebrates, were colonized internally and externally by a vast range of symbiotic species including viruses, archaea, bacteria, fungi, protozoa, and even multicellular mites found in hair follicles and sebaceous glands. These diverse organisms constitute the microbiotas of epithelial linings, including the skin, genitourinary system, airways, oropharynx, and gut. At least 50%, perhaps more, of the cells that make up our bodies are microbial (Sender, Fuchs, & Milo, 2016), and they contribute far more genes, DNA, and metabolic pathways than are encoded in our human genomes (O'Hara & Shanahan, 2006). Studies of human metabolomics reveal that much of our metabolism is in fact microbial (Wikoff et al., 2009).

Spores

The issue of spores has been neglected. Spores are remarkably resistant and can remain viable for thousands, possibly millions of years (reviewed in Nicholson, 2002). They are relevant in two contexts. First, about 1/3 of the organisms in the gut microbiota are spore-forming, and spores are readily demonstrable in human feces (Hong et al., 2009a). Human feces average up to 10⁴ spores/g, while soil contains approximately 10⁶ spores/g (Hong et al., 2009b). Wherever humans have lived, the natural environment is inevitably seeded with human gut-adapted bacterial strains. A recent study revealed that the spore-forming strains within the human microbiota are more diverse than nonspore-forming bacteria and show a higher species turnover or a greater shift in relative abundance over the course of a year (Browne et al., 2016). Therefore, it is possible that when a gut organism becomes extinct as a result of dietary inadequacy or antibiotic misuse (Cox et al., 2014; Sonnenburg et al., 2016), it can be reinstalled via spores from the environment.

Other spore-forming organisms from the environment might also be important despite not being definite components of the human microbiota. Spores of Bacillus subtilis can germinate in the small bowels of mice and rabbits (Casula & Cutting, 2002; Tam et al., 2006) and also humans (Hong, To, et al., 2009b). Moreover, after germination, they replicate in the small bowel and then resporulate as they enter the colon. This might be very relevant to the old friends mechanism, particularly to the clear importance of exposure to animals, agricultural land, and green spaces. After germinating in the small bowel, these organisms will provide data to the immune system in the ileum where dendritic cells sample gut contents and where recently ingested organisms can constitute a significant proportion of the microbes present (Schulz & Pabst, 2013).

Environmental Microorganisms

In addition to spores of gut-adapted organisms discussed above (Browne et al., 2016; Mulder et al., 2009), our ancestors were also exposed to many other microorganisms from the natural environment, many of which would have had significant immunologic impact, even when not able to establish themselves within the microbiotas. Large epidemiological studies demonstrate that living close to the natural rural or coastal environment, often denoted green space or blue space," respectively, reduces overall mortality, cardiovascular disease, and depressive symptoms and increases subjective feelings of well-being (Maas, Verheij, Groenewegen, de Vries, & Spreeuwenberg, 2006; Mitchell & Popham, 2008; Wheeler, White, Stahl-Timmins, & Depledge, 2012). The beneficial effects of exposure to green and blue space are particularly prominent in urban individuals of low socioeconomic status who tend to be most severely deprived of green space (Dadvand et al., 2012; Maas et al., 2006; Mitchell & Popham, 2008; Wheeler et al., 2012). It used to be assumed that these effects are explained by psychological mechanisms, but this view is untenable and supported only by experiments that lack relevant controls (Rook, 2013). While there undoubtedly are health benefits attributable to relaxation induced by exposure to the delights of nature and also benefits from accompanying exercise and sunlight, there is solid evidence for biological effects on the immune system mediated by exposures to environmental microorganisms.

Also, supporting the importance of natural environments in immunoregulation are studies demonstrating that exposure of pregnant mothers or infants to the farming