Epigenetics in Health and Disease

Chapter 14

Epigenetics in Health and Disease HEALTH, INFLAMMATION, AND DISEASE Health and disease are phases of life and the boundary between them is blurred by the fact that physical dysfunction cannot be noticed or detected and often not be recognized by the subject. One of the modern and widely accepted concepts of health that takes into consideration its physical psychological and social aspects is that “health is the ability to adapt and self-manage in the face of social, physical, and emotional challenges” (Huber et al., 2011). With exception of diseases related to the diet and lifestyle (sedentary life, smoking, drug abuse, etc.), most of the diseases have an external physical causative agent, a pathogen (be it a virus, bacterium, parasite or their toxic products, or other physical agents) that somehow invades and compromises the integrity of the organism, causing an infection in the point of access, to which the organism reacts by mounting the protective response of inflammation. Inflammation is a defensive reaction of the animal organism to infection/ invasion by pathogens (bacteria, viruses, parasites, and their products) and other tissue injuries. Inflammation as a universal expression of pathology was described 20 centuries ago by Roman encyclopedist Celsus (25 BC–AD 50), who characterized it by four cardinal symptoms, rubor (redness), calor (warmth), dolor (pain), tumor (swelling), to which Rudolf Virchow (1785–1865), added the functio laesa (disturbance of function). The ultimate function of inflammation is to restore the disturbed homeostasis in a regulated process of releasing molecular agents and dispatching to the inflammation site of cells that are neurally stimulated to produce proinflammatory chemical signals for killing microbes and preventing their spread beyond the region of infection. Initially this involves cellular and humoral mechanisms of the innate immunity (see “Innate Immunity—The Front Line of Host Defense Against Infection” section). Later the mechanisms of the adaptive/acquired immunity may be involved in the process of the healing of infection. When for any reason, the inflammation process stops prematurely, as well as when it persists longer than necessary, complications may arise that act as causes Epigenetic Principles of Evolution. https://doi.org/10.1016/B978-0-12-814067-3.00014-4 © 2019 Elsevier Inc. All rights reserved.

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of various chronic diseases. In cases of prolonged inflammation, therapeutic activation of the parasympathetic system can inhibit secretion of proinflammatory cytokines by macrophages, thus decreasing inflammation. The process of inflammation is strictly timed. After the macrophages, neutrophils and complement in the inflamed region have killed the pathogens and eliminated the organism’s own apoptotic cells, and macrophages have begun to secrete the epithelium-protecting enzyme, secretory leukocyte protease inhibitor (SLPI), the organism switches from the elimination of the causative agent to the healing stage begins (Nathan, 2002). Inflammatory monocytes are recruited by the inflamed tissue where they change their proinflammatory profile to convert them to antiinflammatory proliferating cells that develop further into macrophages, which, by engulfing organism’s own cell debris, release transforming growth factor-β (TGF-β), thus stimulating repair of the injured tissue (Arnold et al., 2007). The normal course of inflammation that results in restoration of the normal structure of the infected region is known as resolution, with the opposite situation known as nonresolving inflammation. In the latter case the infection persists or reinitiates periodically, leading to the development of a chronic disease. In such cases the inflammatory response itself becomes a bigger factor in the pathogenesis than the microbe or its toxic productions. In the category of such chronic or life-long diseases, resulting from nonresolving inflammation belong tuberculosis, multiple sclerosis, rheumatoid arthritis, asthma, ulcerative colitis, and even forms of cancer whose stroma is infiltrated by macrophages and immature myeloid cells (Fig. 14.1). In other cases, the nonresolving inflammation may cause defective vascularization and inadequate oxygenation of the infected region, so that the tissue repair is flawed, and the normal tissue is replaced by collagen, resulting in fibrosis.

Homeostasis and Stress As open physical systems, living beings are in constant change not only as a result of the thermodynamically determined dynamic equilibrium (Fliessgleichgewicht in von Bertalanffy’s sense) but also because of the predictable (annually changing seasons and diurnal cycles) and unpredictable changes in the environment and the action of adverse environmental agents or stressors. Living organisms need to maintain, within relatively narrow limits, a constant internal environment in order that their cells, tissues, and organs perform normally their complex and delicately balanced vital reactions. Health is a physiological state of an organism and an organism is healthy as long as it can maintain, within narrow limits, its homeostasis, its internal environment (Claude Bernard’s milieu int
erieur), even under conditions of stress. In response to stressors, to resist and neutralize their effects, the healthy organism activates the hypothalamic-pituitary-adrenal axis, the autonomic nervous system, particularly the sympathetic response of the adrenal medulla and the

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NLR Cytokines inflammation

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Nonresolving inflammation Rheumatoid arthritis Atherosclerosis Inflammatory bowel disease Multiple sclerosis Certain cancers

Sickness syndrome Swelling Loss of function Tissue injury Pain Anorexia Fatigue Shock Fever

FIG. 14.1 The innate immune response. Invasion and tissue injury are sensed by pattern recognition receptors that activate innate immune cells. This early response gives rise to a cascade of events, including the release of proinflammatory cytokines and recruitment of leukocytes, aimed to clear pathogens and ultimately restore health. In addition to the local tissue response with swelling and redness, fever, anorexia, and fatigue may develop, indicating that the inflammation is sensed by the central nervous system (CNS). Prolonged immune response may cause excessive tissue damage, nonresolving inflammation and inflammatory disease, and even death. A number of regulatory mechanisms are in place, including suppressor lymphocytes, inhibitory cytokines, and neural reflex circuits. Pattern recognition receptors are indicated as Toll-like receptors (TLRs) and nucleotidebinding oligomerization domain receptors (NLRs). (From Sundman, E., Olofsson, P.S., 2014. Neural control of the immune system. Adv. Physiol. Educ. 38, 135–139.)

sympathetic nerves (McEwen and Wingfield, 2003). The classical stress response is the so-called fight-or-flight behavior (although it often may be preceded by a freezing behavior in many animals, including humans), but humans react with other health-related behaviors including alcohol drinking, smoking, gambling, and so on, and, as a result of the perceived danger, with a feeling of anxiety, all of which aggravate the stress condition and the antistressor potentialities of the organism. The neuroendocrine mediators of the stress response, while restoring the physiological state, in a feedback loop, inhibit their own production as part of the process of the restoration of the steady state (dynamic equilibrium) or homeostasis. This implies return to the normal physicalchemical (e.g., body temperature, glucose, oxygen and electrolyte levels in the body fluids, and osmolarity) and cellular (red blood cells, white blood cells, platelets, etc.) parameters. Initially the activation of the stress neuroendocrine response plays a protective role by increasing production of energy through increased secretion of glucocorticoids, stimulation of immune response, stimulation of adaptive behaviors, such as fight-or-flight response, adaptive social and eating behaviors, and so on. Despite its short-term adaptive effects, the prolonged activation

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of the stress response, i.e., prolonged increased production of catecholamines and glucocorticoids leads to harmful consequences by damaging the immune response, exacerbating the inflammation, increasing cytokine production at the site of inflammation, and so on. McEwen and Wingfield (2003) attempted to supplement the concept of homeostasis with what they call allostasis (from Greek allos/ἄλλος—other and stasis/στᾰ´σῐς—standing) and defined it as a state or process of “achieving stability through change” (McEwen and Wingfield, 2003). Accordingly, the allostatic state is characterized by “altered and sustained activity levels of the primary mediators, e.g., glucocorticosteroids, that integrate physiology and associated behaviors in response to changing environments and challenges such as social interactions, weather, disease, predators, pollution, etc.” and define as allostatic load the resulting state of increased levels of glucocorticoids, catecholamines and cytokines. It is part of the adaptive response to seasonal, diurnal, and other environmental changes when the organism is still able to maintain its energy equilibrium based on external sources, but if the allostatic overload becomes permanent or continues to reappear, it may mark increased risk of pathological condition or development of a disease (McEwen and Wingfield, 2003). How much the concept helps to expand or clarify the Bernard-Cannon concept of homeostasis is not clear (Day, 2005). The most important molecular mediators of allostasis are catecholamines (neurotransmitters norepinephrine and epinephrine) and glucocorticoids released by sympathetic nervous system and the adrenal gland as well as other neuropeptides/neurohormones CRH (corticotropin-releasing hormone), NPY (neuropeptide Y), AVT (arginine vasotocin), β-endorphin, and so on, released in the CNS. In simple terms the difference between the new concept of allostasis and the classical concept of homeostasis was defined as follows: “The evidence is that homeostasis maintains the parameters of life, allostasis is the process that allows the body to adapt through change, and allostatic load and overload are the result of cumulative wear and tear on the brain and body” (McEwen et al., 2012). Over time both repeated acute stress and the chronic stress may lead to disease conditions (Fig. 14.2). All these homeostatic parameters are constantly monitored, controlled, and regulated by the central nervous system, which determines and adaptively adjusts the set points within which the parameters have to be maintained (for an expanded review see Chapter 1). But being in constant interaction with their environment, living beings are also exposed to external (and consequentially also internal) agents that disturb the homeostasis or dynamic equilibrium, to which they respond by activating the innate stress mechanism aiming to restore the disturbed equilibrium. In a clear distinction from all other animals, humans think about the future and make predictions beyond the instinctively driven actions related with immediate future (preying, caching food, etc.). The uncertainty and prediction

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Repeated Stress

Acutely stressful incidents Chronic

Negative health Consequences

Time

FIG. 14.2 Stress leading to negative health outcomes. (McEwen, B., Nasveld, P., Palmer, M., Anderson, R., 2012. Allostatic Load—A Review of Literature. Department of Veterans’ Affairs, Canberra. http://www.dva.gov.au/sites/default/files/files/consultation%20and%20grants/healthstudies/ allostatic/allostatic.pdf)

of the dangers the future might bring, the worrying about our own success, carrier, well-being, and so on, i.e., the psychological stressors are the most common cause of stress condition in humans. To deal with such adverse threats, real but also perceived but nonexistent threats, over the course of their phylogeny, living beings have evolved an innate system of stress response for adapting to, and restoring the homeostasis perturbed by various external or internal stressors. The concept of stress is elusive and there is no universally accepted definition of stress. In the following, the stress condition will be considered as a defensive response for maintaining or restoring the dynamic equilibrium of the organism. The stressor may be real but in humans especially it may be just an illusory or perceived stressor. Under stress condition, the organism set into motion the special neural circuitry (Fig. 14.3) aimed at restoring the perturbed homeostasis. During the last three decades evidence is steadily accumulating on the existence of a relationship between the stress and cancer incidence, progression, and prognosis. As mentioned, while the mild acute, short-term, stress is not harmful and often is beneficial for the organism, the chronic severe stress or repeated acute severe stress may lead to pathological conditions. In the first case the organism reacts with a series of psychological and neurally determined physiological changes. Activation of the stress system during the acute stress leads to increased secretion of glucocorticoids and norepinephrine/epinephrine, which stimulate the immune system by inducing the mobility of immune cells and suppressing secretion of proinflammatory cytokines (Chrousos, 2009). The stress response has a psychological and a physiological component. Perception of stressors, the stress-inducing stimuli, in the CNS stimulates instantly activation of two main stress axes, the hypothalamic-pituitary-adrenal axis (HPA) and the autonomic nervous system with the sympathetic nervous system (SNS) and the parasympathetic nervous system (PNS).

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Hypothalamus

CRH

ds to level of cor

ti s o

l

Pituitary gland

ACTH

am

us

re s

pon

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Cortisol

o yp eh Th

th

al

To immune system FIG. 14.3 A simplified model of the stress response circuitry. (From How to Reduce Your High Cortisol (Stress Hormone) Level Naturally. http://www.top10homeremedies.com/how-to/reduceyour-high-cortisol-stress-hormone-level.html)

The CNS arm of the stress response involves the hypothalamus, pituitary gland, and the adrenal glands. Its activation triggers the cascade:
hypothalamic corticotropin-releasing hormone (CRH) ➔ the pituitary adrenocorticotropic hormone (ACTH) ➔ glucocorticoids/mineralocorticoids. Mediators of the CRH action in stress and reproduction are its receptors CRHR1 and CRH-R2, as well as CRH-binding protein (CRH-BP), a secreted glycoprotein with higher CRH-binding affinity (Ketchesin et al., 2017). The central role of CRH in the stress response is corroborated by the fact that the experimental intracerebral injection of CRH mimics stress condition and displays stress symptoms. Under stress, the CNS activates pathways that stimulate the fight-or-flight behavior, arousal, vigilance, fear, anger, aggression, and so on. Stress response stimulates the immune system by facilitating trafficking of immune cells, but prolonged stress condition can disturb the immune system, both the innate and the acquired immunity, thus predisposing to various infectious diseases and metabolic disorders (Fig. 14.4). The stress-related higher

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Stress Developmental history Nutrition Genetic variation Aging

Stress system CRH AVP

LC NE

Systemic sympathetic adrenomedullary systems

HPA axis Cortisol

NE, E, iCRH, IL-6 Target tissues

GH and/or IGF-I LH, T, E2 TSH, T3

Metabolic syndrome (insulin resistance, visceral obesity, sarcopenia) TG LDL HDL

APR Cytokines

ABP Dyscoagulation

Polycystic ovary syndrome Endothelial dysfunction and/ or inflammation

Sleep apnea

Osteopenia and/or osteoporosis

Atherosclerosis Cardiovascular and neurovascular disease

FIG. 14.4 Chronic stress can lead to development of the metabolic syndrome. Abbreviations: ABP, arterial blood pressure; ACTH, adrenocorticotropic hormone; APR, acute-phase reactants; AVP, arginine vasopressin; CRH, corticotropin-releasing hormone; iCRH, immune CrH; E, epinephrine; E2, estradiol; GH, growth hormone; HPA, hypothalamic-pituitary-adrenal; IGF-I, insulin-like growth factor i; IL-6, interleukin 6; LC, locus ceruleus; LH, luteinizing hormone; NE, norepinephrine; T, testosterone; TG, triglycerides. (From Chrousos, G.P., 2000. The role of stress and the hypothalamic-pituitary-adrenal axis in the pathogenesis of the metabolic syndrome: neuro-endocrine and target tissue-related causes. Int. J. Obesity 24 (Suppl 2), S50–S55.)

levels of plasma cortisol negatively influence the healing of inflammation and infection. Among the pathological conditions caused by the chronic stress are the major depressive disorder, anxiety, and depression. As a result of the hypofunction of the GH (growth hormone) axis, growth is inhibited in children and a as result of increased production of CRH and disturbed HPA axis, obesity, amenorrhea reduced fertility, alcoholism, hypertension, and metabolic syndrome may develop. The major stress neuroendocrine mediators are involved in many diseases. So, e.g., high levels of CRH cause degranulation of mast cells leading to development of asthma, eczema or migraine headaches, fear, hyper- and

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hypotension. On the contrary, low CRH levels may cause other pathologies like postpartum “baby blues” depression as well as seasonal or climacteric depression (Chrousos, 2009). Recently the empirical evidence has directed the attention of investigators toward the role of the medial prefrontal cortex as the master controller of the stress response in mammals, including humans. The acute stress stimulates the excitatory glutamatergic transmission in the medial prefrontal cortex (mPFC) (McKlveen et al., 2015). In synergy with this, during the acute stress act glucocorticoids, which disinhibit the prefrontal glutamatergic neurons of prefrontal cortex by recruiting endocannabinoid signaling, which inhibits γ-aminobutyric acid (GABA) transmission in these neurons (Fig. 14.5). On the contrary, during the repeated and chronic stress glucocorticoids activate the GABA neurotransmission at the expense of glutamate, leading to inhibition of the stress response. The shift between stimulation and inhibition during the acute and chronic stress is context dependent and the general pattern is that glucocorticoids during the acute stress increase the glutamatergic excitatory transmission via the mineralocorticoid receptor (MR) and inhibit the response via the glucocorticoid receptor (GR) during the chronic stress. mPFC GA

BA

SS

PV GA BA

Intermediary structures Glutamat

e

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Neurosecretory cells

BST Hypothalamus Brainstem

+/−

ACh

Preganglionic neurons

FIG. 14.5 Intrinsic circuitry and efferents of the mPFC. The mPFC is under tight regulatory control of local interneuron populations that limit the predominantly glutamatergic output of the mPFC. Somatostatin (SS) and parvalbumin (PV) are two of the primary interneuron subtypes within the mPFC that provide dendritic and perisomatic inhibition, respectively. The mPFC projects to numerous subcortical and hindbrain targets, e.g., the bed nucleus of the stria terminalis (BST), hypothalamic subnuclei, and brainstem that mediate its effects on neuroendocrine and autonomic responses to stress. Other abbreviations as follows: corticotropin-releasing hormone (CRH) and acetylcholine (ACh). (From McKlveen, J.M., Myers, B., Herman, J.P., 2015. The medial prefrontal cortex: coordinator of autonomic, neuroendocrine and behavioural responses to stress. J. Neuroendocrinol. 27, 446–456.)

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The autonomic nervous system also seems to be under partial control of the mPFC, with the pl-(prelimbic) PFC acting as inhibitor and the il-(infralimbic) PFC as a stimulator of the SNS (Hill et al., 2011). According to that model, the mPFC serves as controller of other regions of the brain and energetic systems to generate the adaptive behavioral responses (Fig. 14.6). During the acute stress mPFC increases glutamate synaptic secretion, whereas during the chronic stress it may promote or suppress glutamatergic neurotransmission (McKlveen et al., 2015).

Evolution of Stress Response In the broader meaning of the word, stress is the response of an organism to a potentially injurious external agent by inducing an adaptive counteracting change in its internal environment or in its behavior. In this meaning, stress response is an almost universal response in the animal and the plant worlds. One of the evolutionarily earliest molecular defense mechanisms of animals against stress is expression of Hsps (heat shock proteins). In Daphnia, Hsps expression is an antipredator trait because it tends to maintain homeostasis by protecting and facilitating the binding of messenger molecules and receptors, it contributes to generation of the fight-and-flight. Fishes are a major stressor to the small crustacean Daphnia magna. On detecting the presence in its Autonomic nervous system

HPA axis Medial prefrontal cortex

Behavior

Emotional Mood Fear memory Sociability Coping style

Executive Behavioral flexibility Working memory Decision making Planning

FIG. 14.6 Prefrontal coordination of energetic systems. A schematic representation of the potential role that the medial prefrontal cortex could play in coordinating activity between the autonomic and hypothalamic-pituitary-adrenocortical (HPA) axis to regulate both emotional/reactive and executive functions in response to environmental stimuli. (From McKlveen, J.M., Myers, B., Herman, J.P., 2015. The medial prefrontal cortex: coordinator of autonomic, neuroendocrine and behavioural responses to stress. J. Neuroendocrinol. 27, 446–456.)

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environment of the fish or its kairomones alone, the crustacean reacts not only by increasing levels of the heat-shock protein 60 (Hsp60), but also by instinctive avoidance behaviors, such as the negative phototaxis, by staying and going deeper into the water column where they can’t be visually detected by fish. The investigators suggest that “Hsp60 induction is part of the multi-trait antipredator defence strategies developed by Daphnia clones to cope with one of the major stresses in their life: predation by fish” (Pauwels et al., 2005; Lyu et al., 2015). Maternal age and maternal heat stress lead to individual variation in wing shape in the progeny of the parthenogenetic Drosophila mercatorum (Andersen et al., 2005). Stress might have had a role in the evolution of the adaptive alternative morphologies in animals; stress duration or stress level determines which of the alternative phenotypes develops, thus influencing the evolution of the developmental (phenotypic) plasticity of the trait (Gabriel, 2005). It has been demonstrated that epigenetic marks are involved in the stress response of mammalians to posttraumatic stress. Of the 203 PTSD (posttraumatic stress disorder)-associated CpG sites analyzed in a study, 7% evolved in the LCA (last common ancestor) of humans and rodents, 48%, with Old World monkeys, 73% in the LCA with orangutans, and 93% in the LCA with chimpanzees, demonstrating that the epigenetic regulation of traumatic responses may be shared with nonhuman primates and evolution of this epigenetic regulation is involved in immune response. An important observation of these studies is that the rate of evolution of PTSD-associated CpG sites is a function of CpG evolution (Sipahi et al., 2014) (Fig. 14.7). In a study on CpG sites from more than 14,000 genes among 23 people affected by the PTSD and 77 persons not affected by PTSD, Uddin et al. (2010) observed that PTE induces changes in methylation profiles among some persons and that these changes result in changes in gene expression associated with altered immune function and differences in their immune response. Neurohormone CHR, pituitary ACTH, and neurotransmitters octopamine and norepinephrine are central in the stress response in humans and mammals, but they are found as low in the evolutionary ladder as insects, molluscs, and marine worms (Ottaviani et al., 1998; Nesse and Young, 2000). CHR and its receptors, CHR1 and CHR2 is believed to have evolved in arthropods about 500 million years ago (Cardoso et al., 2014). In molluscs is identified a hormonal cascade similar to the vertebrate CRH ➔ ACTH ➔ glucocorticoids (Ottaviani et al., 1998).

IMMUNITY Immunity is a state of resistance of an organism to invading biotic or abiotic pathogens and their harmful effects that prevents the development of infection and maintains organism’s integrity by counteracing, neutralizing, and clearing

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Mammalia

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FIG. 14.7 Evolutionary history of PTSD-associated CpG dinucleotides. (A) Mammalian phylogenetic tree with species genomes utilized for the inference of molecular evolution here. Divergence dates at internal nodes along human descent, in millions of years ago. (B) The absolute number and cumulative percentage of PTSD-associated CpG sites evolved on branches of human descent, as inferred by parsimony. (C) Branch-specific rates of evolution of PTSD-associated CpG sites through human descent. (From Sipahi, L., Uddin, M., Hou, Z.-C., Aiello, A.E., Koenen, K.C., Galea, S., Wildman, D.E., 2014. Ancient evolutionary origins of epigenetic regulation associated with posttraumatic stress disorder. Front. Hum. Neurosci. 8, 1–10.)

pathogens. Immunity is function of the immune system, which is comprised of two subsystems, the innate (nonspecific) and the acquired (humoral/adaptive) immune systems (Fig. 14.8). The immune system discriminates between “self” and “nonself,” between organism’s own molecular patterns and foreign or even its own injuriously altered molecular patterns. The ability to discriminate self from nonself was the first step in mounting immune responses and a sine qua non prerequisite for the evolution of the innate and acquired immune system. The multicellular life would be unsustainable without it; hence, the ability to distinguish between self and nonself, emerged simultaneously with the advent of the multicellular

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Immune system

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(i) Complement (ii) Cytokines (iii) Chemokines

T cells; NKT cells; NK cells; dendritic cells; macrophages; complement

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Antigenpresenting cells

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CD4+ T cells

B cells (secrete antibodies)

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FIG. 14.8 Overview of the immune system: innate and acquired immunity. An evolutionary bridge between both forms of immunity is observed due to the presence of γδ T cells, NKT cells, NK cells, dendritic cells, macrophages, and complement proteins. The innate immune responses include cells and soluble components that are nonspecific, fast-acting, and first responders in inflammation. In contrast, acquired immunity encompasses immune components that are more specific for targeted antigens and capable of forming immunological memory. (From Pandya, P.H., Murray, M.E., Pollok, K.E., Renbarger, J.L., 2016. The immune system in cancer pathogenesis: potential therapeutic approaches. J. Immunol. Res., Article ID 4273943.)

life. It is also believed that even unicellular organisms can distinguish their own DNA foreign phage DNA, by the specific phage epigenetic mark patterns. From an evolutionary viewpoint, the advent of the innate immune system was the first or the preparatory stage of the acquired immune system. The innate system evolved since the earliest forms of multicellular life, but it is conserved throughout the kingdom Animalia and is operational even in vertebrates.

Innate Immunity—The Front Line of Host Defense Against Infection On entering the organism, pathogens, such as bacteria, viruses, parasites, and their toxic products are identified as nonself by the immune sensors, the so-called PRPs (pattern-recognition proteins), soluble proteins like PRRs (pattern-recognition receptors), which are involved not only in elimination of pathogens but also in the elimination of the organism’s own dead cells (Litvack and Palaniyar, 2010). The most important among PRRs involved in identifying the organism’s own dead cells are pentraxins and scavenger receptors (Jeannin et al., 2008). Cytokines and other products of inflammation and immune response, released at the site of inflammation, may get access to the brain via the brain circumventricular sites lacking a blood-brain barrier and the carrier-mediated transport into the brain. However, the bulk of information on the nature and level of cytokines and other products at the site of inflammation reaches the

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brain via the peripheral nerves of the region where cytokines are released (Kronfol and Remick, 2000). Additional information on the activation of the innate immune system and the beginning of inflammation at the site of the pathogen invasion is provided via the sensory nerves, with receptors for products of inflammation, immune response, and pathogens, such as tumor necrosis factor (TNF), interleukin-1 (IL-1), lipopolysaccharides (LPSs), as well as through the small molecular immune and inflammation products that pass the blood-brain barrier and reach various brain centers (Watkins et al., 1995). This afferent information triggers activation of the neural inflammatory circuit, which is a paradigmatic neural circuit that controls the immune response in the same way other neural circuits regulate the physiology of other organs and organ systems. The information on the accumulation of cytokines and other products of inflammation in the site of infection, via vagus is transmitted for processing in the brain stem nuclei. The output of the processing is released at the site of infection by the efferent vagus nerve, which secretes neurotransmitter acetylcholine in the celiac ganglion. The latter, via the splenic nerve secretes norepinephrine, thus stimulating choline acetylesterase-expressing T cells to secrete acetylcholine, which by binding the α7-acetylcholine receptor subunit inhibits production of cytokines by macrophages and other inflammation products by pertinent cells (Fig. 14.9). This way, the nervous system protects the organism from harmful excessive immune responses (Sundman and Olofsson, 2014; Tracey, 2010). This fact led to the successful use of implantable vagus nerve-stimulating devices for treatment of epilepsy and rheumatoid arthritis (Koopman et al., 2016). Organism’s own immune cells, such as macrophages and lymphocytes, release cytokines Il-1, Il-6, TNFs (tumor necrosis factor), and so on. Cytokines are beneficial because their release by macrophages, dendritic cells, and monocytes serves as signal for arrival of phagocytes and lymphocytes at the site of infection, but some cytokines may also be harmful because they cause nerve inflammation and pain by activating nociceptive sensory neurons. In response to higher levels of cytokines the hypothalamic thermoregulatory center rises the body temperature (Tracey, 2007). Changes in pH, the level of cytokines, and other molecular components at the site of the inflammation are transmitted to the brain via the vagus, splanchnic, and pelvic nerves. The efferent arc of the circuit consists of both sympathetic (adrenergic) and parasympathetic (cholinergic) nerves (Andersson and Tracey, 2012). Based on the afferent information about the exogenous molecules known as pathogen-associated molecular pattern (PAMPs) and endogenous molecules, known as damage-associated molecular pattern (DAMPs), from the site of infection, the brain, via the vagus nerve, sends efferent signals, inhibiting the cytokine secretion by macrophages in the spleen in what is known as inflammatory reflex (Fig. 14.10) and functions just the way other protective reflexes such as coughing, sneezing, immunity, and other reflexes function. The inflammatory reflex in response to exogenous

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Vagus nerve

Neuron

Cholinergic synapse Celiac ganglion

Adrenergic synapse Helper T cell subset

Spleen ACh TNF

Macrophage FIG. 14.9 Neural signal cascade for suppressing inflammatory cytokines. The vagus nerve relays signals to adrenergic neurons in the celiac ganglion that form neuroimmune synapses with the helper T cells. Adrenergic receptors on the T cell trigger production of acetylcholine (ACh), which interacts with cholinergic receptors on macrophages to suppress production of inflammatory cytokines such as TNF. (From Dustin, M.L., 2012. Signaling at neuro/immune synapses. J. Clin. Invest. 122, 1149–1155.)

PAMPs and endogenous DAMPs, cytokines, and eicosanoids involves the vagus nerve, which releases the neurotransmitter acetylcholine to neurons in the celiac ganglion. In turn, the latter releases the neurotransmitter noradrenaline in the vicinity of spleen T cells, stimulating them to secrete acetylcholine, which, by binding its receptor α7 nAChR on macrophage membrane, suppresses secretion of cytokines, thus alleviating the inflammation (Tracey, 2012). The role of the peripheral nervous system in the inflammatory process comprises the release of neuropeptides at the infection site.

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TLRs

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Tissue injury Cell debris

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Splenic nerve Norepinephrine release

Acetylcholine release

β2AR

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FIG. 14.10 The inflammatory reflex. The current understanding of the inflammatory reflex, an immune-regulatory vagus nerve circuit, is that sensory nerve fibers, e.g., afferent vagus nerve branches, report on localized cytokine levels and inflammation in the periphery. This information is processed in the brain stem, which generates efferent signals that travel through motor fibers in the vagus nerve and activate the adrenergic splenic nerve, which releases norepinephrine in the spleen. Specialized choline acetyltransferase-expressing T cells (ChAT+ T cells) release ACh in response to norepinephrine and inhibit macrophage cytokine production by activating their α7-nicotinic ACh receptors (α7nAChRs). β2AR, β2-adrenergic receptors. (From Sundman, E., Olofsson, P.S., 2014. Neural control of the immune system. Adv. Physiol. Educ. 38, 135–139.)

The brain, via the vagus nerve, modulates secretion of cytokines according to the stage of inflammation, thus preventing harmful to lethal effects of overexpression and secretion of cytokines, which normally tend to suppress inflammation. At a certain stage of inflammation, vagus nerve begins inhibiting cytokines’ production by releasing the neurotransmitter acetylcholine in the inflammation site (Tracey, 2007). Acetylcholine, by binding α7nAChR (nicotinic acetylcholine receptor) of macrophages and other cytokine-producing cells, prevents the signal transduction pathways of proinflammatory agents, cytokines, lipopolysaccharides (LPSs), tumor necrosis factor (TNF), and nuclear HMGB1 (high mobility group box 1). Thus the brain modulates and limits the magnitude of the immune response. Essential for the brain reaction to inflammation is also the adaptive regulation of the body temperature and

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stimulation of the release of antiinflammatory corticosteroids by the adrenal glands. The brain also responds to infection by activating the HPA (hypothalamicpituitary-adrenal) axis. Glucocorticoids secreted by the adrenals have strong antiinflammatory action, but excessive and prolonged production of glucocorticoids may lead to chronic stress, with its negative influence on antibody production and wound healing (Glaser and Kiecolt-Glaser, 2005). Even in one of the smallest and simplest of known nematode worms, Caenorhabditis elegans, the immune response is controlled by the nervous system (Andersson and Tracey, 2012; Styer et al., 2008; Sun et al., 2011). Characteristic for the worm’s innate immunity is production of antimicrobial peptides, mollusc defensin/mycitin-like peptides, neuropeptide-like proteins, caenacins, and so on. (Engelmann and Pujol, 2010), regulated according to “instructions” from the nervous system (Kawli et al., 2010), while the immune response is regulated by specific neurons (Fig. 14.11) (Zhang and Zhang, 2009; Cao and Aballa, 2016; Bogaerts et al., 2010; Sun et al., 2011; Styer et al., 2008). It has been pointed out that “after infection and subsequent inflammation, the aim of the brain is two-fold protective: to contain both the inflammation, as a consequence of the infection, and subsequently to induce antigen specific memory within the immune system to prevent new infections” (Buijs et al., 2008). Summarizing the above, it may be said that the central nervous system in response to the initial secretion of cytokines by APCs (antigen-presenting cells), macrophages and dendritic cells, activates the hypothalamic-pituitary-adrenal axis, via sympathetic nervous system and the parasympathetic nervous system

CEP

CEP

neuron Dopamine

Dopamine Dopamine antagonist

ASG

DOP-4

Dopamine antagonist

neuron

DOP-4 ASG

Intestinal p38/PMK-1

Neural cells

Nonneural cells

p38/PMK-1 pathway Intestine

FIG. 14.11 Proposed model of dopamine regulation of innate immunity. Abbreviations: ASG, one of the 12 chemosensory neurons of the worm; CEP, four mechanoreceptor neurons around the mouth of the worm; DOP-4, D1-like dopamine receptor. (From Cao, X., Aballa, A., 2016. Neural inhibition of dopaminergic signaling enhances immunity in a cell-non-autonomous manner. Curr. Biol. 26, 2329–2334.)

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CNS cytokines

Stressors

Hypothalamus Corticotropinreleasing hormone Pitu tary gland

Adrenocorticotropic hormone

Vagus nerve

Adrenal gland

SNS Cytokines

PNS

Glucocorticoids

Immune cells Spleen

Thymus Immune system

Bone marrow Lymph node

FIG. 14.12 Schematic illustration of connections between the nervous and immune systems. Signaling between the immune system and the central nervous system (CNS) through systemic routes, the vagus nerve, the hypothalamic-pituitary-adrenal (HPA) axis, the sympathetic nervous system (SNS), and the peripheral nervous system (PNS) is shown. (From Sternberg, E.M., 2006. Neural regulation of innate immunity: a coordinated nonspecific host response to pathogens. Nat. Rev. Immunol. 6, 318–328.)

in a systemic attempt to correct the disturbed homeostasis (Sternberg, 2006) (Fig. 14.12). There is some experimental evidence on the involvement of epigenetic factors, such as miR-155 (O’Connell et al., 2007) in enhancing inflammation during innate immunity. Its levels are higher in synovial membrane and synovial

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fluid (SF) macrophages from patients with rheumatoid arthritis (RA) and this is associated with lower expression of miR-155 target genes (Kurowska-Stolarska et al., 2011). It is also observed that the miR-155 level is also increased during the Helicobacter pylori gastric infection and that miR-155 protect bone marrow-derived macrophages from apoptosis induced by DNA damage during H. pylori infection (Koch et al., 2012). Another miRNA, miR-187, is involved in cytokine control during the antiinflammatory processes (Rossato et al., 2012).

Acquired/Humoral Immunity—The Second Line of Defense As indicated by the name, this type of specific immunity against foreign pathogens (bacteria, viruses, parasites, and other biomolecules) is acquired during the life, rather than inherited through the germ line. The vertebrate acquired immune system is function of a group of blood cells, the white blood cells or lymphocytes, among which the central role is played by B cells and T cells, standing, respectively, for Bone marrow cells and Thymus cells, according to the site of the organism where they proliferate and differentiate in the organism. At a certain stage in the individual development, the organism produces a myriad of lymphocytes that randomly produce immunoglobulins, each with specific active (binding) sites, paratope, capable of binding to respective antigenic regions, epitopes. The number of lymphocytes with randomly formed paratopes is that large that, practically for each naturally occurring or artificially prepared antigen there will be an antibody-producing lymphocyte with a paratope matching any antigenic epitope. The ability of the living system to distinguish self from nonself allows it to eliminate early during the individual development all the lymph cells that would produce antibodies against its own species-specific antigens, while preserving the rest of lymphocytes capable of secreting specific antibodies against any existing or imaginable nonself compound. Immunological studies during the last century confirmed the prediction of immunologists Sir Frank McFarlane Burnet (1899–1985) and Niels Kaj Jerne (1911–94) that during embryonic development, lymphocytes capable of producing antibodies against self-antigens somehow are eliminated. We know that almost all the T cells expressing T-cell receptors (TCRs) against self-antigens are deleted in the thymus gland. A fraction of T cells and B cells expressing receptors for self-antigens that escape deletion, via circulation reach the periphery where they may undergo clonal deletion, i.e., removal through apoptosis (Herndon et al., 2005). Acquired or immunity comprises humoral immunity based on production of specific antibodies against specific pathogens/antigens and cell-mediated immunity. Combined action of these subsystems enables the organism to clear pathogens.

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While conserving the ancient innate defense system, vertebrates evolved the incomparably more complex acquired (adaptive) immune system. The system is as plastic as to respond to any particular pathogen molecular pattern by producing a highly specific antibody capable of binding that particular antigen. The first term, acquired, emphasizes the epigenetic nature of the response in the meaning that, in clear distinction from the molecular agents of the innate immunity that are encoded in the DNA, specific antibodies (membrane receptors) are neither inherited nor encoded in the genome but are epigenetically designed and constructed during the individual development. The second term, adaptive, emphasizes the plasticity of the immune response.

Cell-Mediated Immunity in Vertebrates In vertebrates this is part of adaptive immune response and involves T cells, professional phagocytes (macrophages, dendritic cells, monocytes, and neutrophils) and nonprofessional phagocytes, such as epithelial cells, fibroblasts, endothelial cells, and so on. T Cell Lymphocytes On entering the organism, an antigen/pathogen is immediately recognized as a nonself compound at the site of infection, especially by APCs (antigenpresenting cells), macrophages and dendritic cells, based on the possession of PRRs (pattern recognition receptors), TLRs (Toll-like receptors), and so on, which “recognize” membrane motives of widely different microbes, viruses, and bacteria. Upon recognition of the foreignness of the invading microbe, APCs, but some lymphocytes as well, start engulfing and chemically processing the pathogen. The processing implies separation and breaking down of the protein molecule of the antigen into smaller fragments, to form complexes with major histocompatibility (MHC) molecules, a class of molecules that are transported to the APC membrane. Presented in this form, the antigen is recognized by a specific T helper cell containing CD4 glycoprotein in its surface (CD4+ Th cell). Binding of the processed APC receptor to the receptorCD3 complex of the Th serves as a signal for activation of the latter. Remember, earlier in the thymus were eliminated almost all the T cells that happened to express antigens against organism’s own MHCs and MHCs complexed with antigenic peptides. Differentiation of T Cells T cells are functional partners of the B cells in the immune response. They develop from naı¨ve T cells, on antigen stimulation, in the thymus gland where they differentiate into T killer (Tk) cells and T helper (Th) cells. Differentiation of the pro-T cells into naive CD4+ T cells takes place in the thymus gland (Fig. 14.13). Then, in the secondary lymphoid organs CD4+ cells, upon antigenic stimulation by antigen-presenting cells (APCs), undergo chromatin

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Immune response

T cell ontogeny

Microbes CD4+

CD8+

Thymus

IFΝ-γ

Th2

IL-4 IL-13 IL-5

Naïve CD4+

CD4+ CD8+

CD4– CD8–

Th1

Lymph node, then tissues

FIG. 14.13 Developmental sequences of T cell ontogeny and function. Committed pro-T cells (far left, white) enter the thymus expressing neither CD4 nor CD8. After rearrangement of one chain of their antigen receptor, they undergo massive proliferation and express both CD4 and CD8. At this stage, the other chain of the antigen receptor is rearranged. Subsequently, one or the other of the two lineage markers (CD4 or CD8) is down-regulated in a nonmitotic transition. The repression of the Cd4 gene in CD4
CD8
cells is reversible. In contrast, the transition from CD4+ CD8+ to mature CD8+ cell is characterized by heritable silencing of the Cd4 gene. CD4+ (shown) and CD8+ (not shown) thymocytes emigrate to peripheral lymphoid organs. A naive CD4+ T cell (middle, white) proliferates and its progeny undergo further (effector) differentiation in response to pathogens. The mature progeny are categorized, on the basis of their secretion of nonoverlapping patterns of cytokines such as IFN-γ (Th1 cell) or IL-4, IL-13, and IL-5 (Th2 cell). (From Reiner, S.L., 2005. Epigenetic control in the immune response. Hum. Mol. Genet. 14 (Suppl. 1), R41–R46.)

remodeling to differentiate into T helper1 (Th1) expressing IFN-γ, and Th2 (Th2) expressing cytokines Il-4, Il5, Il13, and so on (Ansel et al., 2003; Lee et al., 2006). TH1 cells produce interferon, IL-2, TNF-beta, which activate macrophages and are responsible for cell-mediated immunity, phagocytosis, and elimination of bacteria and viruses. Th2 are involved in humoral immunity, in antibody production, eosinophil activation, and inhibition of several macrophage functions, thus providing phagocyte-independent immune response (Romagnani, 1999). It is demonstrated that acetylation of histones associated with Il4 gene in T cells induces expression of the gene, which is crucial in determining the differentiation fate of the cell (Valapour et al., 2002). And the fact that acetylation of this gene is at the same time necessary for expression of the IFN-γ (interferon gamma) gene indicates that acetylation has a role for Th1/Th2 differentiation (Fields et al., 2002; Lee et al., 2006). Th2 cells that express Il4 and Il13 have promoters of the respective genes demethylated (Lee et al., 2003). Cytokines (Lee et al., 2006) (Fig. 14.13), DNA methylation/demethylation and numerous types of histone modifications are involved in differentiation of Th1- and Th2 cells (Fig. 14.14) from naı¨ve CD4+ T cells.

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Expression Histone-3 and -4 acetylation Histone-3 lysine-4 methylation

β2AR

Naive CD4+ T cell

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Th1

Repression Histone-3 and -4 acetylation Histone-3 lysine-4 methylation Histone-3 lysine-9 and -27 methylation DNA methylation

Th2

FIG. 14.14 Epigenetic factors influence beta2-adrenergic receptor expression by murine CD4+ T cell subsets. Naı¨ve CD4+ T cells express the β2AR protein on the surface and a level of histone acetylation and methylation within the beta2-adrenergic receptor gene promoter. As naı¨ve cells differentiate to a Th1 or Th2 cell that express and repress expression, respectively, the level of histone acetylation and methylation change. The pattern and level of specific histones involved in expression vs. repression is determined by which T cell subset develops. DNA methylation is also involved in beta2-adrenergic receptor repression in TH2 cells. (From Sanders, V.M., 2012. The beta2adrenergic receptor on T and B lymphocytes: do we understand it yet? Brain Behav. Immun. 26, 195–200.)

αβ Lineage HSC

ETP (DN1)

DC, M, NK

CD4 SP Treg

γδ TCRα rearrangement

TCRγδ

CD4 SP

MHC II DN2a cKithi

T lineage commitment

DN2b cKitIo

β DN3a DN3b CD27Io Selection CD27hi

DN4

DP

CD 1d

iNK T

MHC I TCRβ, TCRγδ rearrangement

CD8 SP Death

FIG. 14.15 Overview of T cell development. Expression of CD4 and CD8 separates CD4
CD8
double negative (DN), CD4+ CD8+ double positive (DP) and cells expressing either coreceptor (single positive, SP), whereas the expression of CD44 and CD25 defines four DN subsets: CD44+ CD25
[DN1], CD44+ CD25+ [DN2], CD44
CD25+ [DN3], and CD44
CD25
[DN4]. The earliest precursors, known as ETP (earliest T-lineage progenitors), that enter the thymus from the bone marrow are part of an heterogeneous DN1 subset that includes both subsequent intermediates in the T differentiation pathway and cells belonging to other lineages. The DN2 and DN3 subsets are themselves divided into two stages based on the expression of the receptor cKit and of CD27, respectively. (From Carpenter, A.C., Bosselut, R., 2010. Decision checkpoints in the thymus. Nat. Immunol. 11, 666–673.)

A small fraction of the thymic CD4+ CD8+ DP (double positive) T cells differentiate into another subset of αβ T cells which are known as invariant natural killer T (i NKT) cells because of the presence of an almost invariant TCR α peptide chain (Fig. 14.15). Besides MCH I and II types, they recognize a

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MHC-like molecule, CD1d. They express neither CD4 nor CD8 and possess properties of both T cells and natural killer thymus (NKT) cells. Early, unpolarized transcription and chromatin remodeling of the poised cytokine genes of naive T cells is followed by consolidation and spreading of epigenetic changes and the establishment of self-reinforcing transcription factor networks. Recent studies have begun to elucidate the molecular mechanisms that establish and maintain polarized cytokine gene expression, and thus the cellular identity of differentiated helper T cells. T cells, like B cells, undergo a process of paratope diversification and negative clonal selection leading to the elimination of all the T cells that react with self-epitopes. Specific APCs (e.g., macrophages) that process the pathogen and break its molecules to smaller fragments, expose them on the surface as antigenic epitopes that are recognized and bound by receptors of specific Th cells. This induces activation of the Th cell. The activated Th cell then interacts with a B cell that recognizes the same antigen. Interaction stimulates Th cells to secrete cytokines, which activate the B cell clone to proliferate and develop into mature antibody-producing cells, the circulating plasma cells. The specific circulating antibodies form antibody-antigen complexes, which are eliminated in a process of opsonization (binding of an antibody to a pathogen that facilitates phagocytosis) and phagocytosis, or via the complement cascade. Another function of Th cells is to activate Tk cells, which bind non-self-antigens on the membranes of cells infected by viruses or altered antigens in the cancer cells membranes, leading to elimination of these cells. There is evidence that miRNA 181 is involved in several aspects of the T cell differentiation and maturation. That evidence suggests that miRNA 181 tune T cell sensitivity to antigens at different developmental stages by increasing it in the beginning, when T cells have to recognize low affinity self-antigens, and lowering it at more differentiated T cell stages, and possibly playing role in the development of immune tolerance, T cell differentiation, and T cell autoimmunity (Li et al., 2007). miRNAs are also implied in the Th cell differentiation as an inhibiting factor. So, e.g., overexpression of miR-29 in naı¨ve T cells inhibits Th1 cell differentiation and downregulation of miR-29 gene elevates IFNγ levels (Wang et al., 2017a,b). Generation of T-Cell Receptor (TCR) Diversity The huge diversity of TCR repertoire necessary for T cells to recognize the huge numbers different antigens and self-antigens is generated in the process of the V(D)J recombination by random joining of gene segments, similarly to formation of the repertoire of immunoglobulin molecules. The TCR structure is very similar to that of immunoglobulins in the primary amino acid structure and modes of rearrangement, hence often TCRs are

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considered as members of the immunoglobulin family (Davis and Bjorkman, 1988; Caccia et al., 1985). TCRs represent heterodimers composed of two chains, α (alpha) and β (beta) or γ (gamma) and δ (delta) linked by a disulfide bridge. According to the type of TCRs they express, T cells belong to two subsets. The bulk of the T cells consisting of α (alpha) and β (beta) polypeptide chains represent the αβT cells and a smaller fraction of T cells expressing γ (gamma) and δ (delta) are known as γδT cells. In humans, most of T cells are αβ T cells and only a small fraction, 5%–10% of them, express another T receptor, a heterodimer composed of γ (gamma) and δ (delta), hence the name γδ T cells. Each polypeptide chain of the heterodimer has a variable (V) region and a constant (C) region, which, respectively, are designated AV and AC for the α chain and BV and BC for the β chain. TCRs are located on the surface of the T cells, where they come in contact with processed fragments of antigens, peptides, glycoproteins, lipids, and small metabolites that are presented them by class I/II MHC molecules in antigenpresenting cells (dendritic cells, macrophages, and B cells) (Jorgensen et al., 1992). Human TCR α chain V and C genes (Caccia et al., 1985; Wang et al., 2016) and delta (Isobe et al., 1988) genes are located in chromosome 14, whereas β chain and γ chain genes lie in chromosome 7 (Caccia et al., 1984). The TCR three-dimensional structure resembles that of immunoglobulins and since the antigen molecule is presented to the T cell bound to a MHC molecule, as is to be expected, most of variability of the TCR is concentrated in the center of the antigen-binding or antigen-recognition site in order to complementarily fit the three-dimensional structure of the antigen molecule. The binding site of the TCR with the antigen is known as complementarity-determining region (CDR). However, the diversity of the CDRs of the TCRs is smaller than that of the antibodies because the antigen-binding site, besides the peptide, has also contact with the almost invariable MCH. The TCR diversity results from the somatic recombination of genes similarly to the generation of the antibody diversity (Janeway et al., 2001a,b). The number of diverse TCRs generated by AV and BV domain pairing and by improper joining of gene segments during the process of V(D)J recombination diversity has been estimated to be on the order of 1015 (Davis and Bjorkman, 1988). But this estimate is unattainable under real conditions, when the number of the mouse T lymphocytes is 1  108. The observed diversity of αβ TCRs is 1.85  106, i.e., 10 times lower than the expected value of 2.3–2.7  107 (Casrouge et al., 2000).

The Complement System The complement is an evolutionarily very ancient subsystem of the innate immune system that evolved first in echinoderms (Smith et al., 2001), but is conserved throughout the deuterostomes (chordates, hemichordates, and

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Classical pathway

MB-lectin pathway

Alternative pathway

Antigen:antibody complexes

Lectin binding to pathogen surfaces

Pathogen surfaces

Complement activation

Recruitment of inflammatory cells

Opsonization of pathogens

Killing of pathogens

FIG. 14.16 There are three pathways of complement activation: the classical pathway, which is triggered by antibody or by direct binding of complement component C1q to the pathogen surface; the MB-lectin pathway, which is triggered by mannan-binding lectin, a normal serum constituent that binds some encapsulated bacteria; and the alternative pathway, which is triggered directly on pathogen surfaces. All of these pathways generate a crucial enzymatic activity that, in turn, generates the effector molecules of complement. The three main consequences of complement activation are opsonization of pathogens, the recruitment of inflammatory cells, and direct killing of pathogens. (From Janeway, C.A. Jr, Travers, P., Walport, M., Shlomchik, M., 2001. Immunobiology: The Immune System in Health and Disease, fifth ed. Garland Science, New York, p. 601.)

echinoderms). Complement effector molecules are found even in the simplest of eumetazoans, such as Cnidarians. The system is comprised of about 30 plasma and cell membrane proteins. In vertebrates, the complement system is also integrated in the system of acquired immunity bridging it with the innate system of immunity (Dunkelberger and Song, 2010). It is an essential contributor to clearing infection (Verburg-van Kemenade et al., 2017). Binding of the first protein of the complement cascade, C1q, to an antibody-antigen complex, binding of a specific lectin to viral or bacterial mannose-containing carbohydrates cascade and binding of a compound of the complement system to the pathogen membrane activates the complement cascade in three different pathways, respectively, the classical, lectin, and alternative pathways (Janeway et al., 2001a, b) (Fig. 14.16).

Humoral Immunity Humoral immunity or antibody mediated in vertebrates is part of the adaptive immunity. It is predominantly based on the antibody-secreting activity of lymphocytes known as B cells. Other elements of the humoral immunity in vertebrates are complement, opsonin and a number of other bactericidal biomolecules.

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B Cell Differentiation and Antibody Production B cells and T cells derive from quiescent hematopoietic stem cells (HSCs) in the bone marrow. HSCs in the bone marrow are in a hibernating state and undergo cell division every 1–2 months. The autonomic nervous system via the glial (Schwann) cells directly regulates the HSC homeostasis in bones. The Schwann cells, by ensheathing nerve axons in bones are in contact with a great proportion of HSCs and control their hibernation by regulating TGF-β synthesis (Yamazaki et al., 2011), whereas autonomic nerve denervation decreases the number of Schwann cells (Hanoun et al., 2015). The process of the differentiation of the quiescent HSCs begins with the release by the sympathetic nervous system of norepinephrine, which inhibits production of stromal cell-derived factor (1CXCL12, also known as SDF1) by mesenchymal stem cells (MSCs), enabling thus transition of quiescent HSCs into migrating HSCs (Veiga-Fernandes and Mucida, 2016) (Fig. 14.17). Methylation of DNA in HSC is maintained by DNMT1, which is crucial for their progression to multipotent progenitors and to lineage restricted myeloid progenitors. Histone modifications are also involved in the process (Sharma and Gurudutta, 2016). The development of the HSC into immune B cells proceeds through a number of stages, until they express the transmembrane receptor BCR (B cell receptor). These still immature B cells migrate to spleen to complete differentiation

Sympathetic neurons Schwann cell

Norepinephrine TGFβ RET

MSC

Stem cell

MSC Blood vessel

Neurotrophic factors

CXCR4

MSC

CXCL12 CXCR4

Migrating HSC

RET

Quiescent HSC

FIG. 14.17 Neuroregulators control hematopoietic stem cells. Neuron satellite Schwann cells induce activation of latent TGF-β that ensures hematopoietic stem cell (HSC) quiescence. In turn, neurotrophic factors directly activate HSC via the tyrosine kinase receptor RET, leading to improved HSC survival. Sympathetic nervous fibers produce norepinephrine that downregulates CXCL12 expression in mesenchymal stem cells (MSC). Reduced CXCL12 expression by MSC prompts HSC egress from the niche. (From Veiga-Fernandes, H., Mucida, D., 2016. Neuro-immune interactions at barrier surfaces. Cell 165, 801–811.)

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into mature B cells (Almqvist and Ma˚rtensson, 2012) before being released in the blood and lymph. While still in the bone marrow, the immature B cells that happened to express antibodies (BCRs) against antigens of their own cell membranes, undergo a process of programmed cell death (apoptosis), before being cloned. This leads to the development of the B cell immunological tolerance against self or its own specific cell components, while acquiring ability to produce antibodies against substances recognized as nonself. Processes of the proliferation and final differentiation of B cells in the germinal centers begin with epigenetic changes in the progenitors of B cells (Zan and Casali, 2013). After expression of activation-induced cytidine deaminase (AID), B cells undergo somatic hypermutation (SHM), proliferation and differentiation in the dark zone of the germinal center of secondary lymphoid organs, lymph nodes, and spleen (Fig. 14.18). In the germinal center (GC) of the secondary lymphoid organs, lymph nodes and spleen, miR-125b, an important epigenetic marker, inhibits the premature B cell differentiation by repressing expression of genes that promote the differentiation. Experimental overexpression of miR-125b prevents B cell differentiation (Gururajan et al., 2010). Primary stimuli, such as expression of CD40, TLR (toll-like receptor) and BCR, induce B cell activation and proliferation. The activation of B cells induces demethylation of DNA and acetylation of histones H3 at the activation-induced cytidine deaminase (aicda) gene, resulting in its expression and production of the protein activation-induced cytidine deaminase (AID). Involved in the expression of AID also are miRNAs, miR-155 and miR-181b (Teng et al., 2008; de Yebenes et al., 2008). aicda gene in naı¨ve B cells is repressed by hypermethylation of the promoter of the gene (Fujimura et al., 2008) and is activated by demethylation and histone modifications (H3K9ac/ K14ac and H3K4me3) in mature B cells (Daniel et al., 2010). B cell activation by the primary stimuli (expression of CD40, TLR (toll-like receptor)) is followed by the action, in the light zone of the GCs of secondary lymphoid organs (Zan and Casali, 2013) by secondary stimuli represented by cytokines, which induce the class switch DNA recombination (CSR), the process through which B cells switch from production of one type of antibody to another (IgM to IgG, IgA, or IgE). In response to these stimuli, the organism induces demethylation of DNA and acetylation of histones, leading to the decondensation of the chromatin and exposure of the Switch (S) region to the CSR machinery. Epigenetic changes, i.e., new epigenetic marks, are responsible for differentiation of the naı¨ve B cells, as a result of the contact with a specific antigen, into memory B cells, directly or through an intermediary GC stage (Fig. 14.19). Memory B cells survive for long periods of time and on the second exposure to the same antigen proliferate and begin producing antigen-specific antibodies, i.e., transforming into antibody-producing plasma cells (PCs). Naı¨ve B cells, after undergoing CSR and SHM may also follow another pathway to differentiate into GC cells, which, under the action of cytokines Il-21 and the antigen, differentiate into antibody-producing plasma cells. Naive

Antigen-activated GC precursor B cell BCR

Apoptosis

Antigen CD40 FDC Peptide–MHC

Proliferation and SHM

Selection

Plasmablast Differentiation

TCR

Proliferation and SHM

Light zone– dark zone recirculation

CD40L

TFH cell

Memory B cell precursor cell

CSR

Dark zone

Light zone

Germinal center

FIG. 14.18 See legend on next page. (Continued)

FIG. 14.18, CONT’D Dynamics of the GC reaction and selection of high-affinity antibody mutants. Antigen-activated germinal center (GC) precursor B cells form the early GC at day 4, in which they differentiate into blasts that over the following days undergo clonal expansion until the mature GC that is characterized by the dark zone and the light zone forms at day 7. During proliferation, the process of somatic hypermutation (SHM) introduces base-pair changes into the V(D)J region of the rearranged immunoglobulin variable region (IgV) genes (dots); some of these base-pair mutations lead to a change in the amino acid sequence. Dark zone B cells then move to the light zone, where the modified B cell receptor (BCR) of the light zone B cell, with help from immune cells, including T follicular helper cells (TFH cells) and follicular dendritic cells (FDCs), is selected for improved binding to the immunizing antigen. Among the newly generated light zone B cells that express BCR mutants resulting from SHM in the dark zone, higher BCR affinity is directly associated with greater antigen capture and leads to a higher density of peptide-MHC complexes presented on the surface of the B cell. This results in the greatest share of T cell help, which in turn drives positive selection. Therefore newly generated light zone B cells that produce an unfavorable antibody are rendered unable to capture sufficient antigen and undergo apoptosis. Following positive selection, a subset of light zone B cells is instructed to recirculate to the dark zone. Light zone B cells may undergo immunoglobulin class-switch recombination (CSR) before light zone-dark zone recirculation. Back in the dark zone, these cells undergo further proliferation and SHM, thus potentially generating antibody mutants with an improved affinity. Recirculation between the dark zone and the light zone facilitates several iterative rounds of mutation and selection, and within a short time, leads to the generation of high-affinity memory B cells and plasma cells. Antigen-selected light zone B cells eventually differentiate into memory B cell precursor cells and plasmablasts, which are the precursors of plasma cells. CD40L, CD40 ligand; TCR, T cell receptor. (From De Silva, N.S., Klein, U., 2015. Dynamics of B cells in germinal centres. Nat. Rev. Immunol. 15, 137–148.)

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Ag, IL-21

CD40L

Naive IL-4,IL-2

GC

L,I Ag

L

40

D

21

C

Ag, IL-21

Mem

PC

FIG. 14.19 Terminal B cell differentiation. Precursor Naive B cells can differentiate into three possible cell types depending on proper molecular stimuli. Cytokines secreted by T-helper cells play a central role in the determination of B cell fate. IL-2 and IL-4 are required for the transition of Naive to GC cells. Direct contact of B cells with T cells by means of the CD40L receptor promote the differentiation of Naive or GC cells toward the Mem cell type. Antigen (Ag) activation drives terminal differentiation toward the PC cells, a process that is favored by the presence of IL-21. (From M
endez, A., Mendoza, L., 2016. A network model to describe the terminal differentiation of B cells. PLoS Comput. Biol. 12 (1), e1004696.)

B cells, under the influence of the same inducers, can differentiate immediately into antibody-producing PC (M
endez and Mendoza, 2016). Both the memory B cells and antibody-producing plasma cells are determinants of the secondary immune response. Antibodies thus produced exist in the form of circulating molecules or membrane-bound B cell receptors (BCRs). Each of the millions of B cells, expressing specific BCRs bind its respective ligand (antigen), engulfs, processes, and combines its fragments with its membrane MHC (major histocompatibility complex) proteins. When a B cell comes in contact with a matching helper T cell, the latter helps it to finally develop into a mature specific antibody-producing B cell, which proliferates rapidly to produce plasma cells circulating with body fluids (blood plasma and lymph). The process of the exceptional expansion of a single B cell into a huge clone of its descendants is known as clonal selection. The clone secretes into the blood and lymph a vast number of antibodies necessary to specifically bind invading antigens/pathogens and form antibody-antigen complexes that are eliminated via the complement cascade.

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When the invading agents are eliminated, most of cells of the clone die, but a proportion usually survives as memory B cells for different periods of time, from years to lifelong. Upon second contact with the same antigen/pathogen, the organism starts a secondary immune response. The memory cells recognize it promptly and start a new and accelerated process of clonal selection producing specific antibodies necessary for eliminating invaders. The ability of vertebrates to remember any primary response to an antigen/pathogen is known as immunological memory. The immunological memory is essentially not genetically determined; it is not determined by a genetic information, but by the ability of specific B cell populations to survive after the first response. It is not inherited through germ line cells, hence not transmitted through generations, but is epigenetically acquired during the lifetime. Creation of immunological memory is the purpose of all the vaccination procedures that prevent the development and spread of infectious diseases. Based on the ability of the immune system for specific recognition (foreign antigens and allografts), using each-other’s “specific’ molecular elements (neurotransmitters and cytokines), the presence and use of synaptic structures, as well as the ever-growing recognition of the close functional relationship between the nervous system and the immune system, C.J. Bayne would express his insight that “…perhaps it is not so revolutionary to suggest that the adaptive immune system is an evolutionary off-shoot of the vertebrate nervous system!” (Bayne, 2003b).

Evolution of the Acquired Immunity In distinction from the innate immune system that is present throughout the animal kingdom, the acquired/adaptive immunity is a vertebrate privilege. The emergence of the acquired immunity in vertebrates about 500 million years ago was so sudden that the event is often considered the immunological Big Bang. However, it is not so sudden as it was believed before. The innate immune system of invertebrates is based on their ability to recognize self from nonself and react only against nonself. The system involves pattern-recognition receptors (PRRs), nod-like receptors (nLRs), Toll-like receptors (TLRs), and scavenger receptors (SRs), but surprisingly invertebrates also are in possession of VLR-like, RAG1-like, and RAG2-like proteins. So far, no traces of acquired immunity are discovered in invertebrates, whereas in jawed fish the acquired immune system appears in its fully developed form with production of immunoglobulins, specific antibodies, T cell antigen receptors and BCRs (B cell receptors), rags (recombination-activating) genes and RAG enzymes, cell surface MHC (major histocompatibility complex) proteins, which the lower vertebrate group, the jawless fish lack. However, in invertebrates are identified RAG-1-like and RAG-2-like, as well as VLR-like proteins (Fig. 14.20).

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Common deuterostome ancestor: Prototypic Rag1/2-like cluster Primitive Rag1/2 function

V-type Ig-like

Disruption of a primordial V-type Ig domain gene by an RSS-flanked DNA element

Sea urchin Aspects of primitive Rag1/2 function?

V

J

Jawed vertebrates Rag1/2 function in V(D)J recombination

FIG. 14.20 Evolutionary relationship of the early deuterostome Rag1/2-like gene cluster and V(D)J recombination. This model separates the presence of a Rag gene cluster in an ancestral deuterostome species from the appearance of split Ig and TCR genes in jawed vertebrates. The Rag1/2like cluster evolved before the emergence of the last common ancestor of the living deuterostomes (i.e., before the divergence of the lineages that led to the sea urchins and vertebrates). The encoded proteins are likely to have carried out a “primitive” function that was different from its current role in vertebrates. Later, in the lineage leading to the jawed vertebrates, an RSS-flanked DNA element became embedded in an ancestral V-type Ig-like receptor gene and became a substrate for the Rag1/2 complex, which in turn eventually evolved into the V(D)J recombinase required for the reassembly of a functional receptor. In the sea urchin, the Rag1/2 cluster may still carry out the ancestral function or may have evolved to carry out a novel derived function. Color transitions are used for the Rag1/2-like genes to indicate functional divergence. Introns have been left out of this sketch for simplicity but represent key evidence that the Rag cluster in sea urchin consists of functional genes. (From Fugmann, S.D., Messier, C., Novack, L.A., Cameron, R.A., Rast, J.P., 2006. An ancient evolutionary origin of the Rag1/2 gene locus. Proc. Natl. Acad. Sci. USA 103, 3728–3733.)

The jawless fish, lampreys and hagfish, evolved an acquired/adaptive immune system that uses a different immune strategy and different molecular and cell elements. Lacking RAG enzymes, they use a mechanism of rearrangement of leucine-rich repeats (LRRs) by gene conversion for generating variable lymphocyte receptors (VLR), as basis of their immunological diversity. They

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have three cell lineages, variable lymphocyte receptor A (VLRA)-producing cells and VLRB (Cooper and Alder, 1996; Pancer et al., 2004), which serve as functional counterparts of the jawed immunoglobulin-based transmembrane receptors TCR (T-cell receptor) and B-cell receptor (BCR) that have binding sites for specific antigens. A third lymphocyte type in jawless fish is discovered recently (Hirano et al., 2013). The estimated magnitude of the diversity of lower vertebrate LRRs is comparable to that of the mammal IgG antibodies, between 1014 and 1017 (Sirisinha, 2014). The resulting proteins appear as receptor molecules in the lymphocyte cell membranes or are secreted as pentamers of dimers by the VLRB lymphocytes, while the VLRA lymphocytes do not secrete but may be related with the function of macrophages. From this viewpoint jawless fish’s VLRBs and VLRAs are considered as analogs of, respectively, B cells and T cells in the rest of vertebrates (Kaufman, 2010). Both types of acquired immunity observed in jawless fish and the rest of vertebrates evolved sequentially 550 and 500 million years ago. It is believed that evolution of the acquired immunity has been made possible “by the invasion of a putative immunoglobulin-like gene by a transposable element, almost certainly a retrotransposon” (Janeway et al., 2001a,b, p. 601). Transposable elements make up approximately 45% of the human genome (International Human Genome Sequencing Consortium, 2001; Goodier and Kazazian, 2008; Macfarlane et al., 2013), as part of the regulatory DNA, earlier considered to be just “junk DNA.” It has been reported that transposon transcription is epigenetically regulated. So, e.g., transcription of VL30 transposons in mammalian cells is induced by combined action of histone phosphorylation and acetylation (Brunmeir et al., 2011; Evsikov and de Evsikova, 2016). Retrotransposon families known in humans are long interspersed nuclear elements 1 (L1), Arthrobacter luteus (Alu) restriction endonuclease, and SVA, with L1s considered to be the master controllers of retrotransposition (Faulkner, 2011) and the only active and the most important autonomous mobile transposon elements (Doucet et al., 2015). L1s comprise 17% of the human genome and it is estimated to have contributed to addition of 10% of human DNA through mobilization of nonautonomous retrotransposons (e.g., Alu and SVA elements), certain noncoding RNAs, and cellular mRNAs (Macfarlane et al., 2013). A generalized model of retrotransposition mechanism is presented in Fig. 14.21. Regarding the time of the emergence, there is reason to believe that the advent of the acquired immune system coincided with the onset of agnathans (jawless fish) (Shu et al., 1999), i.e., about 555 million years ago. Being both of the acquired type, the immune systems in jawless fish (lampreys and hagfish) and jawed fish (and the rest of vertebrates, including humans) are very different not only because the jawless fish lack such important elements of immune response as B cell receptors (BCRs), T cell receptors (TCR), Major histocompatibility complex (MHC), and RAG1/2 proteins and

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New L1 insertion

An Full-length L1 Second-strand cleavage / cDNA synthesis? 5′ 3′ 5’UTR ORF1

A A AA GC AA TT AA TTT AA

3’UTR

ORF2

3′ 5′

5′

An

A AA

AA

AA

3′-OH

TPRT

Transcription U6 snRNA L1 mRNA An

5′

3′

Nu

Translation of ORF1p and ORF2p

Cy

c leu

top

3′ 5′

TAAAACGT ATTTTTGCA

En target site

s

las m

Nuclear import?

ORF 1p RNP formation

AAAAAAAA

ORF 2p

ORF 1p?

(CCCTCT)n

ALU

VNTR SVA

SINE-R

AAAAAAA

AAAAAAA

Cellular mRNAs

Left monomer AR Right monomer

AAAAAAA

Alu

FIG. 14.21 A LINE-1 retrotransposition cycle. A full-length L1 (chromosome) is transcribed, the L1 messenger RNA (mRNA) is exported to the cytoplasm, and translation of ORF1p and ORF2p leads to ribonucleoprotein (RNP) formation. Components of the L1 RNP are transported to the nucleus, and retrotransposition occurs by target-site primed reverse transcription (TPRT). During TPRT, the L1 endonuclease (EN) nicks genomic DNA, exposing a free 30 -OH that can serve as a primer for reverse transcription of the L1 RNA. TPRT results in the insertion of a new, often 50 -truncated L1 copy at a new genomic location (chromosome) that generally is flanked by target-site duplications. Alu, SINE-R/VNTR/Alu (SVA), and cellular mRNAs may hijack the L1-encoded protein(s) in the cytoplasm to mediate their trans mobilization. U6 small nuclear RNA (snRNA) may be integrated with L1 during TPRT. Question marks denote steps in the retrotransposition pathway of unknown mechanism. (From Beck, C.R., Garcia-Perez, J.L., Badge, R.M., Moran, J.V., 2011. LINE-1 elements in structural variation and disease. Annu. Rev. Genomics Hum. Genet. 12, 187–215.)

use very different effectors (Fig. 14.22), but jawless fish also lack spleen and thymus, two lymphoid organs of crucial importance in mounting immune response in the rest of vertebrates. However, most recently, it was confirmed that marine chordates, amphioxi, are in possession of a ProtoRAG containing both a RAG1-like (BbRAG1L) and a RAG2-like (BbRAG2L) gene, encoding RAG1-like and RAG2-like proteins (Carmona and Schatz, 2017), capable of transposition and resealing on DNA that displays striking mechanistic similarities to vertebrate RAGs. It is considered a relative or “living fossil” of the RAG transposon. It dates back as far as

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VCBP TLR, NLR, and SR Innate immunity based on pattern-recognition receptors

NICIR VLRlike

APAR

RAG1like

RAG1/2like

RAG1 and RAG2 VLR

TCR, BCR, and MHC

Mammals (humans)

Birds (chickens)

Reptiles (snakes)

Amphibians (frogs)

Bony fish (sturgeons)

Bony fish (zebrafish,medaka fish)

Cartilaginous fish (sharks)

Cyclostomes (lampreys)

Cyclostomes (hagfish)

Urochordates (sea squirts)

Cephalochordates (amphioxi)

Hemichordates (acorn worms)

Echinoderms (sea urchins)

Adaptive immunity based on rearranging antigen receptors

222 3R 326 370 476 525

652

2R 1R

794 (Mya)

891 896

FIG. 14.22 The stages in phylogeny of the emergence of immune molecules. Molecules restricted to jawed and jawless vertebrates are in nine right hand side vertical bars and horizontal bars above them. Molecules that emerged at the stage of invertebrates are in four left-hand side vertical bars and the uppermost full-size horizontal bars. Recombination-activating gene (RAG)-like genes are of

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basal chordates, some 550 million years ago. It fails to function with RSS (recombination signal sequences) but this can be explained with the long time of more than half a billion years separating evolution of RAG in lower invertebrates and vertebrates. It is suggested that the RAG transposon “was transmitted vertically through chordate and vertebrate evolution, remaining an (sic!) active in lancelets while being co-opted in jawed vertebrates for the assembly and diversification of antigen receptor loci by V(D)J recombination.” (Huang et al., 2016). Nevertheless, the jawless fish can mount an acquired immune response by producing specific antibodies, just like other vertebrates do. Lampreys and hagfish use for this VLRs (variable lymphocyte receptors), containing multiple leucine-rich repeats. A single VLR locus contains a large bank of diverse LRR (leucine-rich repeat) cassettes. The great diversity of membrane receptors, which is comparable to that of the jawed vertebrates, is generated via somatic DNA rearrangement in lymphocytes and LRR receptors represent diversified versions of a single germ line VLR gene (Pancer et al., 2004). The case of the jawless fish suggests that the organs and molecular effectors used by vertebrates (BCR, TCR, and MHC proteins) may not be indispensable for evolution of a system of acquired immunity in lower animals such as invertebrates. It appears that in this latter group exist many variable molecules such as VLR (variable lymphocyte receptor)-like containing multiple leucine-rich repeats (LRRs) that might perform a similar function (Cerenius and S€ oderh€all, 2013). And biologists still wonder why the adaptive immune system had to “evolve twice in vertebrates whereas invertebrates, representing the vast majority of animals, never developed a similar structure?” (van Niekerk and Engelbrecht, 2015). Although an answer to the question why the acquired immunity didn’t evolve in invertebrates is difficult to provide, the relevant anatomophysiological changes that associated the invertebrate-vertebrate transition, make it tempting the idea that the advent of the hypothalamic-pituitaryinterrenal axis in jawless fish supported the evolution of the acquired immunity in vertebrates.

FIG. 14.22, CONT’D viral or bacterial origin (from the transib transposon family) and are also present in the genomes of sea urchins and amphioxi. Agnathan paired receptors resembling antigen receptors (APAR) and novel immunoreceptor tyrosine-based activation motif-containing immunoglobulin superfamily receptor (NICIR, also known as T cell receptor (TCR)-like) are agnathan immunoglobulin superfamily (IgSF) molecules that are thought to be related to the precursors of TCRs and B cell receptors (BCRs). The divergence time of animals is shown in Mya (million years ago). Abbreviations: 1R, first round of whole-genome duplication (WGD); 2R, second WGD round; 3R, lineage-specific WGD experienced by an ancestor of the majority of ray-finned fish 320 million years ago; MHC, major histocompatibility complex; NLR, Nod-like receptor; SR, scavenger receptor; TLR, Toll-like receptor; VCBP, V-region containing chitin-binding protein; VLR, variable lymphocyte receptor. (From Flajnik, M.F., Kasahara, M., 2010. Origin and evolution of the adaptive immune system: genetic events and selective pressures. Nat. Rev. Genet. 11, 47–59.)

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A model of the evolution of immunoglobulins presented in 2000 describes a hypothetical bacterial transposon consisting of RAG genes, flanked by a single RSS on each side, where a central sequence was flanked by a pair of insertion sequences (IS), each flanked by a pair of RSS. After the transposon was integrated into the germ line, IS elements were excised and integrated elsewhere in the genome (Fugmann et al., 2000). The integration of the transposon resulted in in the splitting of an ancestral V-like gene into two components (Hsu et al., 2006) (Fig. 14.23).

Generation of the Immunoglobulin Diversity The evolution of the jawed vertebrate humoral immunity implies a change in the function of RAG1/2 proteins as a result of the insertion of recombination signal sequence (RSS) “in an ancestral V-type Ig-like receptor gene” (Fugmann et al., RAG transposon Insertion of mobile DNA

V

C TM

V

C TM

V

C TM

Tandem duplication TM lost

Acquisition of D gene

Whole-locus duplication

Germline rearrangement Duplication of D genes, C exons

RS swap

Germline rearrangements

FIG. 14.23 Hypothetical scheme for the evolution of immunoglobulin gene systems. In the ancestral jawed vertebrate, a mobile DNA sequence inserted in an archaic gene, splitting it into V and J segments with recombination signal (RS) sequences at their flanks (black and white triangles representing RS with 12 and 23-bp spacers). We suggested that the ancient gene was part of a family of receptors (TM is transmembrane, black), and the L-chain genes evolved from those that lost the TM. Expansion of the genes could have occurred by tandem duplications of the gene segments, resulting in the translocon organization in tetrapods, or by duplication of the whole locus, which generated the multicluster organization in sharks and skates. The V evolved in structure; D sequences were acquired. The number of C region domains could be increased with tandem gene duplication and divergence. (From Hsu, E., Pulham, N., Rumfelt, L.L., Flajnik, M.F., 2006. The plasticity of immunoglobulin gene systems in evolution. Immunol. Rev. 210, 8–26.)

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2006). These proteins work together to cut DNA at specific sites in a process where RAG1 ubiquitination stimulates DNA cutting and recombination (Singh and Gellert, 2015). The lack of introns in RAG genes suggested that they may be of bacterial or viral origin (Schatz et al., 1989). The evolution of the acquired system of immunity, can be considered as a higher and stage of the evolution of immune response in animals, complementary to the innate immunity. This stage is characterized by a greater involvement of the nervous system and the epigenetic modifications in DNA and histones in production of antibodies specific to each of the invading pathogens, microbes, parasites, nonself molecules and, in pathological cases, antibodies against its own altered molecular components. The cartilaginous fish are the first group to have evolved the ability to produce T cell antigen receptors and specific immunoglobulins. However, the members of the immunoglobulin superfamily (IgSF) are identified as low in the evolutionary ladder as C. elegans, where they are involved in correct axon positioning (Aurelio et al., 1998; Bayne, 2003a,b). Proteins with an immuneglobulin (Ig) domain are found throughout the eumetazoan kingdom and even lower, in sponges. IgSF domains are found even in prokaryote unicellulars such as bacterium Streptococcus agalactiae (Bateman et al., 1996). The empirically demonstrated ability of vertebrates to produce specific antibodies against any possible foreign proteins, in view of the billions of protein/ peptide types produced in the animal and plant world, led biologists to the idea that the variable DNA segments of the VDJ regions of immunoglobulin genes somehow undergo a process of recombination, to generate the huge number of distinct specific immunoglobulins. Production of specific molecules capable of binding any imaginable antigenic molecule in a lock and key fashion is based on their ability to rearrange their V(D)J gene segments (Pancer and Cooper, 2006), thus producing different and specific transmembrane antigenic receptors in their B cells and T cells. Generation of new protein variants (a practically infinite number of specific immunoglobulins), from the same gene, by definition is an epigenetic phenomenon, because the information for producing these specific immunoglobulins is generated during life time; it is neither inborn nor encoded in their DNA. It seems that a determining factor in evolving the acquired system of immunity has been the evolution, from Transib superfamily of transposons, of the enzymes RAG1 and RAG2, which serve as the core of the V(D)J recombination machinery (Kapitonov and Koonin, 2015). The locus accessibility for rearrangement is regulated by an epigenetic mechanism (Levin-Klein and Bergman, 2012) and the direct recruitment of RAG enzyme to discrete rearranging loci (Jaeger et al., 2013). There is a tight correlation between RAG2 plant homology domain (PHD) and H3K4 trimethylation (H3K4me3) that facilitates its association with chromatin in many transcriptionally active regions of the genome. By contrast, RAG1 binding is more specific and restricted to the so-called recombination

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centers containing multiple closely spaced recombination signal sequences (RSSs). The chromatin region of the recombination signal sequence where the RAG1/RAG2 complex binds, is marked by activating histone modifications, which flank V, D, and J segments creating a single-strand nick in the DNA the first base of the RSS and the coding DNA segment. Interaction between RAG2H3K4me3 and RAG-RSS induces recruitment of RAG proteins in this domain, creating the recombination center and leading to double strand DNA cleavage and initiation of the V(D)J recombination (Ji et al., 2010). Somatic hypermutation (SHM), which might have contributed to the generation of vertebrate antibody diversity, is induced in the process of the exposure of the V(D)J region that is made accessible after the DNA demethylation and histone acetylation in the region of immunoglobulin genes (Li et al., 2013). It is believed that activation-induced cytidine deaminase (AID), in conjunction with other transcription factors, induces point mutations in hot spot motifs in the variable region of immunoglobulins. By deaminating cytosines in mutational hotspots, AID produces a mutational change U:G, which is transmitted to the progeny of daughter B cells. The initial, antigen-independent stage takes place in the bone marrow, where differentiation of B cells and the rearrangement of V(D)J DNA directed by the recombinase RAG1-RAG2 and production of clonally unique Ig variable regions that specifically bind antigen occurs. Transition to the antigen-dependent stage of mature B cells takes place in lymphoid organs with expression of membrane immunoglobulin-based antigens and acquisition of immunocompetence and secretion of specific antibodies by plasma cells. According to the “accessibility hypothesis,” the first step in the process of recombination of the variable DNA segments of the V(D)J region is to make the DNA site accessible to the recombination machinery RAG1/RAG2. This occurs as a result of epigenetic changes in histones of the variable region. The RAG1/ RAG2 complex then binds the RSS of two adjacent different coding segments forming a synaptic complex with the help of the DNA-binding high mobility group box (HMGB) proteins (Johnson et al., 2010). Recombination occurs between gene segments of the same chromosome and it involves looping and deletion of DNA between two gene segments at sites determined by RSS. Rearrangement (recombination) starts when RG1/2 complex and HMG proteins recognize RSS flanking sequences to be joined. The RAG1/2 complex cuts one of the DNA strands which reacts with the uncut end of the complementary strand, thus forming a hairpin loop. The hairpin then is cleaved to produce a single stranded DNA, and finally the DNA strands are joined by DNA repair enzymes (Janeway et al., 2001a,b). There is ample evidence (McMurry and Krangel, 2000; Maes et al., 2001; Morshead et al., 2003) that acetylation of histones determines the accessibility of the recombination machinery to the RSS (recombination signal sequences) in DNA. To the contrary, histone methylation is generally related to the repression of gene expression (Johnson et al., 2010).

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Neural Regulation of Humoral Immunity During the last two decades a large body evidence is accumulated not only about the relationship between the immune and nervous system but also about the CNS control of the innate and acquired immunity. The brain exerts its control and regulation of the immune response in two main ways:
globally, by releasing in body fluids immune-stimulating substances, and
locally, via the peripheral nervous system innervating organs and cells of the immune system. The global regulation of the immune response via the HPA axis starts with the activation of the secretory neurons of the hypothalamic paraventricular nucleus (PVN) and magnocellular neurons, which release, respectively, CRH and arginine vasopressin (AVP) in the anterior pituitary, where they stimulate production of ACTH, which induces secretion of glucocorticoids by adrenals. Lymphocytes express membrane receptors for glucocorticoids, enabling the latter to influence the production of antibodies. Not only the glucocorticoids, but all the hormones of the HPA axis are involved in the regulation of the immune response (Webster et al., 2002) and all of them are under neural control. The second way the brain regulates the immune response is by the autonomic arm of the peripheral neural system, involving both the sympathetic and parasympathetic nervous systems secreting, respectively, neurotransmitters norepinephrine and acetylcholine (ACh), whose actions are mediated by their specific receptors expressed by most cells in the body (Straub, 2004; Kin and Sanders, 2006). The autonomic system thus serves as a “messenger from the mind to the body for all organ systems, including the immune system” (Sanders, 2012) (Fig. 14.24). Before the antigen exposure, B cells express only very low levels of the membrane protein CD86, secreted by antigen-presenting cells (APCs). Several hours after the CNS receives sensory information on antigen recognition by immune cells in the site of infection, nerve fibers of the sympathetic nervous system secrete in lymph nodes norepinephrine, which binds to β2 adrenergic receptor (β2 AR) of B cells, activating a transduction pathway that leads to increase in the level of the IgG1 and CD86. Both signaling pathways converge to transcription of the mature immunoglobulin IgG1 (Sanders, 2012) (Fig. 14.25). Neural signals stimulate egress from bone marrow of hematopoietic stem cells (Katayama et al., 2006) and accumulation of the immune cells around nerve fibers of the lymphoid organs (van de Pavert et al., 2009). Mice react against the administration of killed Streptococcus pneumoniae by mobilizing dendritic cells, monocytes, and neutrophils to transport bacteria and their debris to the marginal zone (MZ) of the B cells in the spleen, inducing B cell activation and their differentiation into antibody-producing B cells (Bala´zs et al., 2002). The immune response is detectable by an elevation of the specific antibodies within 48 hours after the exposure.

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Brain

Cytokines Cells

Sympathetic nervous system

Immune system

Norepinephrine

FIG. 14.24 The brain-immune communication pathway. Activation of the immune system allows for communication with the brain via the release of cytokines from activated immune cells and/or the trafficking of activated immune cells into the brain. Activation of selective regions in the brain allows for communication with the immune system via activation of the sympathetic nervous system centrally and the release of the neurotransmitter norepinephrine from sympathetic nerve terminals that penetrate lymphoid tissue in the periphery. (From Sanders, V.M., 2012. The beta2-adrenergic receptor on T and B lymphocytes: do we understand it yet? Brain Behav. Immun. 26, 195–200.)

β2AR

CD86

cAMP p-IκB-α B cell

PKA

NF-κB

pCREB OCA-B

Oct-2

3’lgH enhancer binding Mature IgG1 Transcript IgG1 protein FIG. 14.25 The beta2-adrenergic receptor and CD86 signaling pathways converge to induce an increase in the level of IgG1 produced by an activated B cell. The β2AR engagement on an activated B cell activates cAMP/PKA/CREB, while CD86 engagement on an activated B cell activates lyn kinase, CD19, Akt, IkB, and NFkB. The signaling pathway activated by β2AR and CD86 engagement cause an increase in the coactivator protein OCA-B and the transcription factor Oct-2, respectively. OCA-B and Oct-2 interact and translocate to the nucleus and bind to the 30 IgH-enhancer to increase the rate of IgG1 transcription, which increases the level of IgG1 produced by the cell. The increase in IgG1 is not due to an increase in the number of naı¨ve B cell switching to the production of IgG1. (From Sanders, V.M., 2012. The beta2-adrenergic receptor on T and B lymphocytes: do we understand it yet? Brain Behav. Immun. 26, 195–200.)

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The current status of the knowledge about the regulation of the specific antibody production allows us to trace back the causal links from the proximate to the “ultimate” cause of the antibody production. This leads us to the firm conclusion that the information for regulating the processes of innate and acquire immunity, both in health and disease, flows from the central nervous system to the immune system. The regulatory role implies also the continuous monitoring of the immune status by the CNS via the sensory neurons. Both the primary (thymus and bone marrow) and secondary (spleen and lymph nodes) lymphoid organs receive sympathetic nervous fibers. The innervation takes place not only at the organ and tissue levels but even deeper at the level of individual immune cells, lymphocytes, macrophages, and dendritic cells. Although the neural regulation of antibody production by signals coming from the peripheral innervation to the immune organs (lymph nodes, spleen, thymus, and bone marrow) has been known for some time, only these last few years immunologists are shedding some light on the mechanics of the neural regulation at the cell level. The innervation in thymus, spleen, lymph nodes, and gut-associated lymphatic tissue (GALT) is concentrated in T lymphocytes and plasma cell zone, where it branches in parenchyma to extend the nerve ending in close proximity to lymphocytes (Felten et al., 1985). In another study these nerves, which are different from the nerves that innervate blood vessels in lymph nodes, were identified close to lymphocytes, plasmablasts, and mature plasma cells (Novotny and Kliche, 1986; Novotny, 1988). It is suggested that these fibers may affect the antigen presentation of B cells and transmit signals for activation of CD4+/CD8+ T cells (Shi et al., 2017). The neural hardwiring of the lymphoid organs and the cells of the immune system may be a structural manifestation of deeper role of the nervous system in the functions of the immune system cells. The most impressive intimate contact of peripheral nerves with the immunologically relevant cells, lymphocytes, such as macrophages and dendritic cells, T cells and B cells came from the investigation of individual APCs (antigen-presenting cells), dendritic cells, and macrophages, in lymph nodes. It was observed that APCs are closely covered by, and in very narrow contact with, a round or oval neural meshwork originating from a single nerve fiber (Fig. 14.26). The covering neural meshwork suggests it may have a sensory role. The presence of sensory innervation in APCs, in both dendritic cells and macrophages, is in contrast with the lack of innervation in B and T cells. The neural meshwork is found exclusively in the paracortical region of the lymph node where T cells are also found, suggesting that the paracortical sensory innervation may serve for sensing immunological events and act as a guide to finding the appropriate cell in the immune system or provide “the answer to the unsolved question how especially T-cells can find one specific and fitting dendritic cell within billions of cells in a short time frame necessary for an adaptive immune response” (W€ ulfing and G€ unther, 2015).

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FIG. 14.26 Image of the filamentary structure of the mesh around the wAPC (dendritic cell/ € € macrophage) resembling a “glass fishing float” (100 fold). (From Wulfing, C., Gunther, H.S., 2015. Dendritic cells and macrophages neurally hard-wired in the lymph node. Sci. Rep. 5, 16866.)

The idea that B cells and T cells lack sensory innervation is challenged most recently by studies on nonmyelinating Schwann cells (NMSCs), comprising the Schwann cells ensheathing the Remak fibers (nonmyelinating nerve fibers). These cells are involved in immunomodulation (Armati and Mathey, 2013; Ydens et al., 2013) and finally it is observed that NMSC cells and Remak fibers may come in contact with B cells, T helper cells, and T cells and may be involved in the antigen presentation to these cells and in modulating the immune response by producing cytokines (Shi et al., 2017). Sympathetic nerve endings from the superior cervical ganglion come in direct contact with the S100+ immune cells of the cervical lymph nodes, acting as a neuroimmune cross-talker, by providing targeted immune cells instructions via the neurotransmitters norepinephrine, VIP (vasoactive intestinal peptide) and the neuropeptide Y that bind their respective receptors on the immune cell membrane (Huang et al., 2013). Activated B cells, mature in the spleen and migrate along the reticular fibers from the marginal zone to the red pulp as antibody-secreting cells that secrete antibodies into blood (Mina-Osorio et al., 2012). These processes are neurally regulated by the vagus nerve (Fig. 14.27). The electrical stimulation of the vagus inhibits migration of B cells and other immune cells, leading to their accumulation in the marginal zone of the spleen, which is associated with decline in the levels of specific antibodies (Andersson and Tracey, 2012).

Immune Tolerance and the Development of Tolerance to SelfAntigens Immune tolerance is a state in which animals do no respond to a particular antigen, be it foreign or self-antigen. The acquisition of the tolerance against the

Epigenetics in Health and Disease Chapter

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Acetylcholine Norepinephrine

Increased vagus nerve signaling

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Spleen CDIIb+ B cells

Antibody release

Celiac ganglion Vagus nerve

Adrenergic splenic neuron

(A)

Red pulp

Spleen

Marginal zone CDIIb+ B cells

Decreased vagus nerve signaling Celiac ganglion Vagus nerve

(B)

Antibody release

Adrenergic splenic neuron

FIG. 14.27 Neural influence on B cell trafficking and antibody secretion. (A) Stimulation of vagus nerve signals stimulates the adrenergic splenic nerve. This leads to accumulation of CD11+ B cells in the marginal zone and decreased antibody production. (B) In the setting of diminished signaling from the vagus nerve to splenic nerve, antibody-secreting CD11b+ cells traverse the marginal zone and enter the red pulp, where they release antibodies into the circulation. (From Andersson, U., Tracey, K.J., 2012. Neural reflexes in inflammation and immunity. J. Exp. Med. 209, 1057–1068.)

self-antigens during the early development is a necessary condition of preventing the self-destruction of the organism. The acquired tolerance takes place at the level of particular cells known as T cells or T lymphocytes (T stands for thymus, the organ where these cells mature, before entering circulation and reaching the periphery). It is acquired during the embryonic development through a complex process, whose exact mechanism is still unknown.

Development of Central Immune Tolerance Bone marrow hematopoietic stem cells (HSCs) that enter the thymus gland differentiate into thymocytes, progenitors of T lymphocytes, which differentiate first to a double negative (DN) stage expressing neither CD4 nor CD8 transmembrane coreceptors, then as CD4+ CD8+ DP (double positive), expressing

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both coreceptors and, finally, as mature SP (single positive) T cells, expressing either CD4 or CD8. The multipotency inherited from the HSC stage is gradually lost with the T lineage commitment during transition from DN1 to the DN2. After the T lineage commitment, in the DN3 stage occurs the rearrangement of gene segments and separation of the γδ lineage. T cell proliferation leads to formation of millions of T cells expressing specific cell membrane TCRs, capable of binding the most diverse antigens, including self-antigens. Most of the DP (double positive) T cells that show no avidity for MHCps (MCH-bound peptides) die by “neglect” while those that show a high affinity for the MHCps undergo programmed cell death in a process of negative selection that is decisive for developing the immune self-tolerance. Most of the T cells that proved to be reactive to self-antigens are eliminated in the thymus as a primary lymphoid organ. The immune tolerance against selfantigens that develops in the thymus gland is known as central tolerance. By contrast, DP thymocytes that react adequately with the self-MHCps escape the programmed cell death (Carpenter and Bosselut, 2010), i.e., undergo positive selection. The interaction of T cells with MHCp acts as a proliferating and differentiating signal. Their differentiation from DP to SP (single positive) expressing either CD4 or CD8 coreceptor leads to appearance of two different types of cells, CD4 helper cells and CD8 cytotoxic (killer) cells.

Development of Peripheral Tolerance T cells reactive against self-antigens may be found in the periphery, partly because a tiny fraction of them may escape elimination in the thymus, and partly because, after release in circulation, the mature T cell may come in contact with self-antigens that were not produced in thymus or didn’t reach the gland. Dendritic cells (DCs) are activators of T cells and inducers of immune response but under certain circumstances they can act as inducers of peripheral immune tolerance. Evidence shows that DCs in spleen and lymph nodes may induce tolerance in T cell that bear TCRs with high avidity for peptide-loaded major histocompatibility (pMHC) complexes (Mueller, 2010). The switch at a molecular level is determined by inhibition of mTOR (from mammalian Target Of Rapamycin) pathway associated with expression of a number of genes such as Egr2, Cblb, Ctla4Dgkz, and Pdcd1 (Fig. 14.28). At a cell level peripheral immune tolerance is induced by activity of two distinct groups of cells, DCs and lymph node stromal cells (LNSCs). In the first case it is related to a particular category of DCs, which upon binding the T cell fail to activate it and induce a state of T cell anergy. LNSCs such as fibroblastic reticular cells (FRCs), follicular DCs (FDCs), and lymphatic endothelial cells express autoimmune regulator (AIRE), which, by inducing a number of tissuespecific antigens (TSAs), “may play a relevant role in the maintenance of selftolerance” (Gardner et al., 2009; Xing and Hogquist, 2012). When self pMHC complexes increase in the periphery, low avidity autoreactive T cells escape negative selection in the thymus to populate lymph nodes and

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Active DC

Tolerogenic DC

Effector or memory

TSA

mTor Proliferation

Inhibit mTor

TCR Egr2 CbIb Dgkz Ctla4 Pdcd1 Anergy

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T cell

PD-L1

p-MHC

CD28

IL-2R

691

Lymph node stroma

B7 IL-2

14

Egr2 CbIb Dgkz Ctla4 Pdcd1 Anergy T cell

? PD-1

Apoptosis T cell

FIG. 14.28 Peripheral tolerance. T cell anergy is induced by inhibiting mTOR pathways or can be induced by tolerogenic DCs (dendritic cells). The expression of Egr2, Cblb, Ctla4, Dgkz, and Pdcd1 genes is important in T cell anergy. Lymph node stromal cells (LNSCs) express tissue-specific antigens (TSAs) and can mediate the deletion of self-reactive naı¨ve T cells. (From Xing, Y., Hogquist, K.A., 2012. T-cell tolerance: central and peripheral. Cold Spring Harb. Perspect. Biol. 4 (6), pii: a006957.)

spleen. They may respond to self-antigens and the failure of the peripheral tolerance mechanisms marks beginning of autoimmune diseases (Mueller, 2010). Treg (regulatory T) cells also seem to induce immune tolerance. So, e.g., tTreg (thymus derived) cell is believed to be responsible for tolerance to self-antigens, while pTregs (peripheral Tregs)—for tolerance against non-self-antigens, including dietary antigens, allergens and commensal microbiota (Plitas and Rudensky, 2016).

Neural Control of Allergy Stimuli that initially are nonantigenic, after pairing with an antigen, may elicit allergic reactions (Siegel and Kreutzer, 1997). So, e.g., pairing injection of egg albumin with a distinctive environment in guinea pigs led to conditional allergy attacks by exposing animals only to that particular environment. Training rats with neutral stimuli and injection of ovalbumin led to the development of conditional allergic reactions by only exposing them to the audio-visual representations of the neutral stimuli (McQueen et al., 1989). Such facts demonstrate the essential role of the nervous system in development of allergy. Allergic rhinitis is demonstrated to be under neural control in experiments where unilateral administration of the antigen causes nose allergic secretion and congestion bilaterally. The characteristic allergic vasodilatation is determined by activation of muscarinic receptors as proven by the fact that oxitropium bromide, an acetylcholine antagonist, prevents histamine-induced nasal closure in allergic rhinitis patients (Scott and Fryer, 2012).

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In the airway membranes, the inflammation of the mucosa is perceived by sensory receptors and triggers a neural reflexive response with the local sensory nerves releasing at the inflammation site neuropeptides, such as substance P (SP) that modulates the activity of white blood cells and stimulates angiogenesis, calcitonin gene-related peptide (CGRP) with its vasodilatory effects, and the smooth muscle contracting neuropeptide neurokinin A (NKA). This neurogenic inflammatory response can be mimicked by vagus stimulation and may be prevented by blocking the release of neuropeptides by the local sensory neurons (Widdicombe, 2003). Asthma attacks are characterized not only by general inflammation, increased mucus secretion, proliferation of eosinophils, narrowing and constriction of airways, but also by elevated levels of the immunoglobulin IgE and the neurotransmitter serotonin in plasma. The level of neurotransmitter serotonin in plasma correlates with the severity of symptoms and therapeutic reduction of plasma serotonin drastically improved clinical status of patients (Lechin et al., 1998). The afferent input is transmitted for processing in the brain via three types of sensory neurons, slowly adapting receptors (SARs), rapidly adapting receptors (RARs) and C-fiber receptors. The CNS output, via vagus nerve, reaches the inflammatory sites in airways in the form of the parasympathetic neurotransmitter acetylcholine released by parasympathetic postganglionic neurons (Mirotti et al., 2010) (Fig. 14.29).

Chronic Obstructive Pulmonary Disease (COPD) Asthma is always associated with increased activity of the vagal parasympathetic nervous system which controls the airway smooth muscle in the lungs Airway lumen

lnflammation

RAR

Epithelium

C-fiber

Gland

Smooth muscle

ACh

CNS

Ganglion

SAR

FIG. 14.29 The vagal nervous system in the airways. Mechanical forces, inhaled irritants and endogenous inflammatory mediators activate afferent nerve fibers, which send signals to the central nervous system (CNS). This results in the release of acetylcholine (ACh) in the airways. Parasympathetic, postganglionic neurons are located within the airway wall and innervate airway smooth muscle and submucosal glands to induce contraction and mucus secretion, respectively. Abbreviations: RAR, rapidly adapting receptors; SAR, slowly adapting receptors. (From Kistemaker, L.E.M., 2015. Acetylcholine beyond bronchoconstriction A regulator of inflammation and remodeling. PhD Dissert., Univeristy of Groningen, p. 10.)

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CNS + Efferent vagus nerve m2AChR Allergic reaction

Afferent fibers ACh

+ nAChR m2AChR −

Parasympathetic ganglion

m1AChR Postganglionic nerve

ACh α7nAChR m3AChR

m3AChR −

Mucus production

Bronchoconstriction

Inflammatory mediators

FIG. 14.30 The cholinergic pathways in allergic lung. During allergic reactions, the inflammatory mediators released in the tissue activate the sensory afferent fibers, which convey information to the CNS. The CNS sends information back to the inflammatory site by increasing ACh release from efferent vagus nerve. The neurotransmission in the parasympathetic ganglia is mediated by acetylcholine (ACh) via nicotinic (nAChR) or type 1 muscarinic (m1AChR) receptors. The stimulus generated induces ACh release in the pos ganglionic nerve fiber endings. Type 2 muscarinic receptors (m2AChRs) are autoinhibitory, and the dysfunction of this receptor, observed in allergic asthma, induces increased release of ACh. Increased ACh results in augmented mucus secretion via m3AchR expressed in the glandular epithelium, increased airway smooth muscle contraction (bronchoconstriction) via m3AchR expressed in muscle cells, and decreased inflammatory mediators production via a7nAChR receptor expressed on immune cells. (From Mirotti, L., Castro, J., Costa-Pinto, F.A., Russo, M., 2010. Neural pathways in allergic inflammation. J. Allergy 2010, Article ID 491928.)

by releasing acetylcholine which binds its receptors (Fig. 14.30). The proximate cause of asthma bronchoconstriction, hypersensitivity, and increased mucus secretion is an increased release of the neurotransmitter acetylcholine by parasympathetic nerve endings on the smooth muscle cells and cells of submucosal glands of the airways, as a result of the dysfunction of acetylcholine modulating M (muscarinic) autoreceptors in the CNS (Fryer and Wills-Karp, 1991).

Epigenetic Marks in Induction of Allergy T cell activation and differentiation is determined to a certain extent by epigenetic histone modifications. T2 differentiation is triggered by phosphorylation of STAT6 signal transducers and expression of GATA-3 and Th2 cytokines, including Il-4. Th1 differentiation is also triggered by phosphorylation of

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STAT4, expression of the transcription factor T box expressed in T cells (TBET) and cytokine INF-γ is the normal state of CD4 T cells, whereas demethylation of Il-4 promoter leads to allergenic sensitization. Stimulation of CD4+ lymphocytes increases the degree of DNA demethylation (Kwon et al., 2008). Expression patterns of Th2 cell-specific genes are established by histone modifications and DNA methylation. Activation marks of histone hyperacetylation and H3K4 methylation are elevated in TH2 cells compared with Th1 cells. Demethylation of retrotransposon hot spot 1 (RHS1) gene, histone acetylation and H3K4 methylation are observed in Th2 differentiation (Zeng, 2013). Asthmathic individuals show a lower histone deacetylase (HDAC): histone acetylase (HAT) ratio, i.e., a relative decrease of HDAC enzymes, which is corrected by proper treatment (B
egin and Nadeau, 2014).

Autoimmunity and Autoimmune Diseases Autoimmunity is an aberrant response of the vertebrate immune system when the organism losses the immune self-tolerance and produces antibodies against organism’s own compounds. This is what occurs in cases of autoimmune diseases, such as rheumatoid arthritis, Hashimoto thyroiditis, type 1 diabetes mellitus, systemic lupus erythematosus, Sj€ ogren syndrome, Graves disease, vasculitis, and so on. What makes the immune system to behave like a “dog that bites his owner”? The fact that the concordance of developing autoimmune diseases in twins is less than an expected 100% and ranges between 13% and 61% suggests that nongenetic, epigenetic factors must be involved in the onset of the disease. The factors that lead to the development of the autoimmune disease may be production of abnormal antibodies, inability of the immune system to distinguish self-compounds that resemble foreign, nonself compounds, altered selfcompounds as a result of the foreign pathogens, and so on. The sympathetic nervous system (SNS) stimulates the immune response in the initial phases of the inflammation and suppresses the response during the later phases when the infection declines, and the pathogen is cleared. The fact that during a number of autoimmune conditions the SNS continues to be overactive led to the idea that an abnormal function of the SNS may be cause of the autoimmune diseases. Immune self-tolerance is based on maintenance of a balance between Th (Th1, Th2, Th17, etc.) cells. In the process of production of T cells in the thymus glands, a small percentage of autoreactive T cells that have affinity for selfantigens escape the negative selection in the gland and migrate in the periphery. These autoreactive CD4+ Th cells are present in the periphery of not only the autoimmune individuals but in the healthy individuals as well. However, these autoreactive CD4+ cells are harmless and in a latent state because they are kept in check and their activity suppressed by a particular T cell type, known as Treg

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(CD4+ CD25+ regulatory T cells) (Szanya et al., 2002). Both Treg cells and CD4+ cells are found in the peripheral blood of healthy humans. The suppressive action of Treg cells on expansion of the autoreactive CD4+ Th cells prevents the onset of a number autoimmune diseases in experiments (Danke et al., 2004). Treg cells are a subset of T helper cells, produced steadily in the thymus gland, which display a strong autoimmune suppressive action and somehow prevent production of antibodies against self-antigens (Sakaguchi et al., 2007). Treg cell proliferation is positively regulated by miR-21 and negatively by miR-155 (Wang et al., 2017a,b). Treg cells display a specific hypomethylation pattern associated with a specific pattern of gene expression (Kitagawa et al., 2015). Treg’s immunosuppressive action on other Th cells relies on production of the immunosuppressive membrane immunoglobulin CTLA-4 (Wing and Sakaguchi, 2010). Now we know that differentiation and maintenance of the proper balance between various types of T cells is regulated by the SNS (Lorton and Bellinger, 2015). Electrical stimulation induces the vagus nerve to secrete acetylcholine, which binds its own receptor α-7 nicotinic acetylcholine receptor (α7nAChR) on membranes of cytokine-producing monocytes, macrophages, and other cells. In experiments, it reduced tumor necrosis factor α, induced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) (Van Maanen et al., 2009). Evidence from studies in rats suggests that the age-related rise in asthma and autoimmunity is believed to result from the decline in noradrenaline nerve density in lymphoid organs especially in splenic white pulp that leads to an immunosenescence and dysregulation of the sympathetic-to-immune system (Perez et al., 2009). Systemic lupus erythematosus patients have hyperacetylated regions in promoters of a number of genes, such as ITGAL, CD40LG, PRF1, CD70, IFGNR2, MMP14, LCN2, DNA, leading to their overexpression, and generally the balance between the acetylation deacetylation in cases of lupus shifts toward acetylation (Quintero-Ronderos and Montoya-Ortiz, 2012; Huber et al., 2007). Hypomethylation results from decreased levels of Dnmt1 enzyme, as it is suggested by the fact that administration of DNA methylation inhibitors induces DNA hypomethylation (Lu et al., 2002). Downregulation of miR-146 leads to abnormal activation of type I interferon (I-IFN) which is a hallmark of systemic lupus erythematosus (Tang et al., 2009) and administration of demethylated CD4+ T cells alone, in genetically predisposed people breaks the immunological tolerance and activates the disease (Hewagama and Richardson, 2009). The involvement of epigenetic mechanisms in the etiopathogenesis of rheumatoid arthritis (RA) is suggested by lack of genetic mutations in RA synovial fibroblasts. The characteristic cartilage degradation is caused by stimulation of

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secretion of metalloproteinases. During RA, the balance between HAT and HDACs shifts toward HATs, that is acetylation and the level of histone H3 (H3ac) in the IL-6 promoter is much higher in RA synovial fibroblasts than in osteoarthritis (Wada et al., 2014). Unlike systemic lupus, during RA, miR146a is up-regulated in various cell types (Ceribelli et al., 2011; Ceribelli et al., 2012). Besides miR-146, other miRNAs involved in RA pathogenesis are miR-155 and miR-223 (Jerram et al., 2017). Systemic sclerosis is an autoimmune disease of the connective tissue displaying symptoms mainly in skin and other organs and organ systems as a result of increased production of collagen. Among the causes believed to be involved in the etiopathogenesis of the disease are epigenetic modifications, including genome wide DNA hypomethylation as a result of downregulation of methylation enzymes (DNMT1, MBD3, and MBD4), leading to overexpression of CD40L, CD11a, and CD70 (Wang et al., 2017a,b).

Autoimmune Diabetes Autoimmune diabetes in mice initiates at 3–4 weeks of age. The autoimmune response begins with infiltration of T cells into the islets of Langerhans that increases with ageing. In the prediabetic period occurs methylation of Ins1 and Ins2 genes, which are involved in insulin gene expression. In islets also takes place expression of cytokines which induces DNMTs and represses the insulin gene (Rui et al., 2016). A strong interaction is observed between epigenetic and genetic factors in human diabetes mellitus. Epigenetic mechanisms (DNA methylation) in islets of diabetes mellitus patients interact with genes involved in processes of proliferation and apoptosis in pancreatic β cells and may be potential mediators of the genetic association with expression of insulin gene (Olsson et al., 2014). Type 2 diabetes patients displayed in their islets changes in DNA methylation patterns in 254 genes that were not present in type 2 diabetes patients. The dysregulation of these genes may be the cause of mal- and dysfunction of islets in type 2 diabetes (Volkmar et al., 2012). Autoimmune thyroid disease (AITD) includes two distinct diseases Graves’ disease (GD) and Hashimoto’s thyroiditis (HT), and represents one of the frequent autoimmune disorders affecting about 5% of human population. GD is characterized by hyperthyroidism caused by the production of thyroidstimulating immunoglobulins, whereas HT is generally characterized by hypothyroidism as a result of lesioned thyroid gland (Brix and Heged€us, 2012). During Graves’ disease are identified 133 hypomethylated and 132 hypomethylated regions and the DNA methylation pattern in GD patients was different from normal control individuals (Cai et al., 2015). Among the causes of both Graves’ disease and Hashimoto’s is the genetic polymorphism in DNMT genes (Cai et al., 2016).

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EPIGENETICS OF CARCINOGENESIS Despite the earlier reports on the occurrence of tumors in invertebrates, until the beginning of the 20th century, the prevailing opinion was that invertebrates were incapable of developing tumors. Contrary to that initial opinion, tumors occur not only in vertebrates but across the animal kingdom, including lower invertebrates such as annelids, arthropods, molluscs, and ascidians (Scharrer and Lochhead, 1950). However, most of studies on tumorigenesis and carcinogenesis are performed on humans and laboratory mammals because of the effects of cancer to the human health and life. In 2013 cancer was the second leading cause of death after the cardiovascular disease with 14.9 million cases and 8.2 million deaths worldwide (Global Burden of Disease Cancer Collaboration, 2015). The overwhelming majority of cancers is related with the activity of environmental factors (infections, smoking, radiations, pollutants, irritants, external stressors, diet, lifestyle, etc.) Progress made in the last few decades in understanding the pathogenesis of cancer led to the development of new and more efficient treatments of the disease as shown by the impressive increase in the number of cancer survivors, which in 2014, in the US alone, amounted to more than 14 million (De Santis et al., 2014). No matter what the causative agent may be, cancers of all forms display a several common features:
Growth of an abnormal, “foreign” structure,
Increased metabolism, as consequence of the high proliferation rate of cancer cells,
Switch from glycolysis to predominantly oxidative phosphorylation for generation of energy,
A dense network of nerves and blood vessels,

Effects of stress-associated factors on the tumor microenvironment. The responses to stressors involve central nervous system (CNS) perceptions of threat and subsequent activation of the autonomic nervous system (ANS) and the hypothalamic-pituitary-adrenal (HPA) axis. Catecholamines, glucocorticoids, and other stress hormones are subsequently released from the adrenal gland, brain, and sympathetic nerve terminals and can modulate the activity of multiple components of the tumor environment. Effects include the promotion of tumor cell growth, migration and invasive capacity, and stimulation of angiogenesis by inducing production of proangiogenic cytokines. Stress hormones can also activate oncogenic viruses and alter several aspects of immune function, including antibody production, cytokine production profiles and cell trafficking. Collectively, these downstream effects create a permissive environment for tumor initiation, growth, and progression. Abbreviations: CRF, corticotropin-releasing factor; IL-6, interleukin-6; MMP, metalloproteinase; VEGF, vascular endothelial growth factor. (From Antoni, M.H., Lutgendorf, S.K., Cole, S.W., Dhabhar, F.S., Stephton, S.E., McDonald, P.G., Stefanek, M., Sood, A.K., 2006. The influence of bio-behavioural factors on tumour biology: pathways and mechanisms. Nat. Rev. 6, 240–248.)

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A tendency to expand and metastatize,
Production of neurotrophins,
Common mutations and epigenetic marks. For about one century, cancer has been almost consensually considered to be an abnormal growth of cells that sustained one or a number of mutations in their DNA. This is the kernel of the widely accepted and propounded Somatic Mutation Theory of cancerogenesis, whose origin traces back to the idea of the German biologist Theodor Boveri (1862–1915), the author of the chromosomal theory of inheritance. The tremendous work on the genetics of cancer, especially during the last half a century, produced excessive empirical data, but little new insights into the pathogenesis of cancer. We still have to question whether mutations are the ultimate inducers or mediators or effectors of carcinogenesis. Another well-known hypothesis of carcinogenesis is the Cancer Stem Cell Hypothesis, which posits that long-lived epithelial stem cells, over time, accumulate mutations, differentiate in cancer stem cells, and gain the ability for uncontrolled proliferation. These cancer stem cells represent always a fraction of the cell population of the tumor, which replace the dying cancer cells, thus growing the tumor mass and probably initiating metastases (Sell, 2004; Wu, 2008; Shipitsin and Polyak, 2008). With half a century of continued advances in cancer investigation, with a number of milestone discoveries on the nature of carcinogens, on oncogenes and tumor suppressor genes and their demonstrated role in carcinogenesis, we are still puzzled by its elusive etiopathogenesis and unable to connect all the relevant phenomena in an ordered intelligible narrative. We are still far from a scientifically comprehensive understanding of the biology of cancer.

Etiology of Cancer Causative agents of cancer may be specific external biotic (viruses, other microbes, infections) and abiotic factors (ionizing and nonionizing radiations and various chemicals), internal factors such as spontaneous mutations, epigenetic factors, and, with a high likelihood, psychological factors. Gene mutations, i.e., changes in DNA nucleotide sequences, such as point mutations, deletions, insertions, and translocations, but also complex somatic rearrangements and intrachromosomal tandem duplications, can also lead to tumorigenesis (Stephens et al., 2009). An even greater role in tumorigenesis seem to play various forms of DNA damage that are induced by exogenous and endogenous factors or arise spontaneously because of intrinsic thermodynamic factors. However, the frequency of gene mutations is about three orders of magnitude lower compared to the frequency of the occurrence of epigenetic marks in DNA and histones (Ushijima et al., 2003; Petronis, 2010). These relatively frequent epigenetic changes in cells may accumulate over time to produce significant differences in cell growth

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and differentiation, without relevant changes in DNA sequence identity (Wong et al., 2005). These epigenetic differences can also explain morphological, physiological, and behavioral differences between the monozygotic twins, both when reared together or in different environments (Bouchard et al., 1990). The role of viruses as causative agents of cancer in animals, including humans, is known for a long time. Carcinogenic viruses belong to both classes, DNA viruses and RNA viruses. Among the medically important carcinogenic viruses are the hepatitis B virus, human papilloma viruses and Epstein-Barr virus. Among RNA viruses are human T lymphotrophic virus type 1 and hepatitis C virus. Environmental stress and psychosocial factors are involved in various cancer types. So, e.g., results obtained from a 15-year-long study in Finland show that major stressful events such as divorce/separation, death of a husband, of a close relative or a friend were associated with significant increases in the incidence of cancer (Lillberg et al., 2003). Under experimental conditions of chronic stress, female mice carrying the Bittner oncogenic virus, within a year, increased dramatically the incidence of the mammary tumors to 92%, compared with 7% in the nonstressed control group (Riley, 1975). Social isolation increased 3.3-fold the risk of the mammary tumors in rats (Hermes et al., 2009). In view of the central role of the HPA axis in stress response and the regulation of hormonal secretion of endocrine glands, it is not surprising that hormones of the endocrine glands are also involved in cancerogenesis. The risk of endometrial cancer increases with late menopause and decreases with parity and, generally with the duration of exposure to estrogens unopposed by progestins, suggesting that estrogens stimulate endometrial cell division in endometrium (Key, 1995; Key et al., 2001). According to a model of initiation and progression of tumors presented by Feinberg et al. (2005), the process of tumorigenesis begins with epigenetic changes and genetic changes are later involved in the process (Fig. 14.31). Vogelstein et al. (2013) provided a generalized model of the mutational mechanism of cancer initiation and progression. Accordingly, the first or “gatekeeping” mutation that happens to occur in a gene, provides a selective growth advantage to an epithelial cell, which forms a clone. Mutated gene is a driver gene. Driver genes may carry driver gene mutations and passenger gene mutations. The difference between the two is that driver gene mutations truncate the encoded protein and provide a selective advantage to the cell thus being responsible for tumor initiation whereas passenger gene mutations don’t provide any growth advantage. A second mutation in another gene accelerates the clonal growth. This can be followed by mutations in other genes generating a malignant tumor which invades the underlying membrane and metastasize to other parts and organs of the body. The growth advantage provided by the driver mutation can increase by about 0.4% the difference between cell birth and cell death. Low as it is, over the years, the difference can produce a large tumor mass of billions of cells.

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Differentiated cells

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ONC ONC TSG

TSG

TPG Expanded and/or epigenetically altered progenitor-cell pool

Epigenetic and genetic plasticity

Epigenetic and genetic plasticity

GKM Epigenetic changes Progenitor cells

14

Benign tumor

Primary cancer

Invasion; metastasis; drug resistance

FIG. 14.31 The epigenetic progenitor model of cancer. According to this model, cancer arises in three steps. First is an epigenetic alteration of stem/progenitor cells within a given tissue, which is mediated by aberrant regulation of tumor-progenitor genes (TPG). This alteration can be due to events within the stem cells themselves, the influence of the stromal compartment, or environmental damage or injury. Second is a gatekeeper mutation (GKM), tumor-suppressor gene (TSG) in solid tumors, and rearrangement of oncogene (ONC) in leukemia and lymphoma. Although these GKMs are themselves monoclonal, the expanded or altered progenitor compartment increases the risk of cancer when such a mutation occurs and the frequency of subsequent primary tumors (shown as separately arising tumors). Third is genetic and epigenetic instability, which leads to increased tumor evolution. Note that many of the properties of advanced tumors (invasion, metastasis and drug resistance) are inherent properties of the progenitor cells that give rise to the primary tumor and do not require other mutations (highlighting the importance of epigenetic factors in tumor progression). (From Feinberg, A.P., Ohlsson, R., Henikoff, S., 2005. The epigenetic progenitor origin of human cancer. Nat. Rev. Genet. 7, 21–33.)

The previous estimate indicates that it will take decades for a malignant tumor to develop metastases, which are responsible for the death of vast majority of cancer patients. There is no evidence that metastatic progression of cancer may be related to emergence of new mutations. The number of mutations in self-renewing tissues will be proportional to the age of the patient; it will be higher in aged patients and lower in the young. Obviously, the number of mutations will be lower in non-self-renewal tissues, such as brain cancers or pancreatic ductal adenocarcinomas. In common solid tumors of breast, brain, pancreas, and colon are identified 33–66 genes, most of them consisting in single-base substitutions among which more than 90% are missense changes (Vogelstein et al., 2013). Both changes in miRNA expression patterns and epigenetic regulation of key genes are involved in cancer metastasis (Chatterjee et al., 2017) Cancer cell genome is at the same time globally hypomethylated but hypermethylated in promoters of particular genes, what makes biologists to accept promoter hypermethylation as a mediator of tumorigenesis. It is also observed that during cancer more genes become nonfunctional as a result of epigenetic rather than genetic changes. However, epigenetic and genetic changes may be collaborated to prevent expression of genes during tumorigenesis. There are cases when epigenetic changes precede genetic mutations or predispose for their occurrence and stimulate tumor progression. These cases of epigeneticgenetic collaboration led to the hypothesis of the oncogenic pathway addiction, according to which epigenetic “interactions with genetic changes, could allow

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neoplastic cells to become addicted to various oncogenic driving pathways” (Baylin and Ohm, 2006).

Cancer and DNA Methylation In eukaryotes, DNA exists as a component of chromatin along histones and other chromatin-associated proteins. The basic building block of the eukaryote chromatin is the nucleosome. The nucleosome consists of a histone octamer globular structure, composed of two copies each of the following core histones: H2A, H2B, H3, and H4 with a characteristic N-terminal tail where acetylation and methylation of histones takes place (Luger et al., 1997). Around this core bind and wrap approximately 146 base pairs or 1.7 turns of DNA (Annunziato, 2008). Nucleosomes are linked between them by short DNA segments of 20 base pairs termed “linker DNA.” Epigenetic mechanisms involved in cancer initiation and progress are DNA methylation, histone modification and chromatin remodeling (Feinberg et al., 2016). DNA methylation consists in covalent addition of a methyl group (–CH3) to a cytosine of a cytosine-phosphate-guanine (CpG) dinucleotide in the carbon 5 of the cytosine ring (Albany et al., 2011; Julsing and Peters, 2014). CpG sites are those sites in the DNA sequence where C (cytosine) is followed by G (guanine). Globally the DNA is poorly methylated, but it is punctuated by DNA segments on average 1000 base pairs long sequences with elevated G + C base composition (Deaton and Bird, 2011). Sequences with such high G + C content are known as CpG islands and the human genome contains 29,000 CpG islands (Kim et al., 2009). “CpG islands” are found in promoter and exons regions of about 40% of mammalian genes (Larsen et al., 1992). Only around 1% of human DNA consists of short, C + G rich, compared to a 4% expected. Human chromosomes 21 and 22, comprising about 750 genes, had respectively 5039 and 9023 CpG islands (Takai and Jones, 2002). Invertebrates such as Drosophila and C. elegans have little or no DNA methylation (Deaton and Bird, 2011). In plants, methylation is seen in gene bodies, although nonmethylated regions exist in their extremities. However, it is not clear whether plants have high frequency CpG islands comparable to those of animals (Deaton and Bird, 2011). In vertebrates 70% of gene promoters lie within CpG islands, making this the most common promoter type in the vertebrate genome (Deaton and Bird, 2011). This modification leads to spontaneous deamination of 5-methylcytosine to thymine with resulting modifications in the chromatin structure that inhibit gene transcription (Bird, 1986). DNA methylation is catalyzed by DNA methyltransferases DNMT1, carrying out methylation of the new DNA strand after DNA replication (hence the name maintenance methyltransferase) and DNMT3a and DNMT3b involved in the de novo DNA methylation (Baxter et al., 2014; Rodriguez-Paredes and Esteller, 2011).

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Methylation of CpG islands, especially of those in gene promoters leads to gene repression as a result of methylation, which prevents binding of transcription factors to gene promoters, but it may also be a consequence of the so-called methyl-CpG-binding proteins (MBPs), i.e., the recruitment of one member of a family of proteins that recognize methyl-CpG (Manoharan et al., 2007; Qu et al., 2013). Both hypermethylation and hypomethylation of DNA can stimulate carcinogenesis; the first by preventing expression of the tumor suppressor genes and the second by enabling expression of oncogenes. DNA methylation is reversible, and a DNA cyclical switch methylation/demethylation takes place hourly (Kangaspeska et al., 2008) suggesting an hourly transition expression/ repression of certain genes. A clear correlation between the cancer, especially its metastatic forms, and DNA hypomethylation was observed since early 1990s (Gama-Sosa et al., 1983) and three decades ago biologists observed that in colon adenomas and adenocarcinomas the genomic DNA methylation was reduced by 8%–10% (Feinberg et al., 1988).

Cancer and Histone Modifications Histones are DNA-binding proteins and the chief component of chromatin in cell nucleus. They belong to five types, of which four (H2A, H2B, H3, and H4) serve as the nucleosome core around which DNA strands wind, and H1/ H5, which are known as linker histones that bind to the linker DNA (Bhasin et al., 2006). The processes of gene transcription and DNA replication require unwinding of the DNA strands, which is impeded by the presence of histones in nucleosomes and the convoluted structure of chromatin. To facilitate the unwinding of the DNA and opening of chromatin eumetazoans evolved special mechanisms of modification of the chromatin structure via acetylation, phosphorylation, ubiquitination, and so on, of nucleosome core histones. Histones can be posttranslationally modified at the amino acid residues located on their N- and C-terminal tails (Chervona and Costa, 2012). Histone modifications include acetylation by histone acetyltransferases (HATs) and deacetylation by histone deacetylases (HDACs), histone methylation by histone methyltransferases (HMTs) and demethylation by histone demethylases (HDMs). In histone modification are also involved histone ubiquitination enzymes, histone deubiquitinating enzymes, and so on. The epigenetic state of histones (acetylated, phosphorylated, methylated, or ubiquitinated) and the possible chromatin remodelings, determine whether they will allow or repress the transcription of a gene or group of genes (Fig. 14.32). High levels of histone acetylation are characteristic of the euchromatin, or the loosely packaged and transcriptionally active chromatin, whereas the tightly packed chromatin, the heterochromatin, is transcriptionally inactive.

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Linker DNA (with 20–80 bp)

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H1 H2B H2A H4 H3

Octameric histone core (with 147bp DNA)

H1

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Histone posttranslational modifications

FIG. 14.32 Chromatin structure and histone-modifying enzymes. Chromatin is made of repeating units of nucleosomes, which consist of 147 base pairs of DNA wrapped around a histone octamer consisting of two copies of each of the core histones H2A, H2B, H3, and H4. Linker histone H1 is positioned on top of the nucleosome core particles stabilizing higher order chromatin structure. The histones are subject to a wide variety of posttranslational modifications, primarily on their € N-terminal tails, but also in their globular core region. (From Fullgrabe, J., Hajji, N., Joseph, B., 2010. Cracking the death code: apoptosis-related histone modifications. Cell Death Differ. 17, 1238–1243.)

Acetylation of histones makes genes accessible to transcription factors and is exclusively associated with gene expression. Histone acetylation is function of the enzyme histone acetyltransferases (HATs), which catalyze addition of an acetyl group to a histone. A common modification of histone structure in eumetazoans is acetylation of the histone H4 (Smith et al., 2003). A common posttranslational chromatin modification is induced by acetylation of H4-K16ac (histone 4, lysine 16) (Shogren-Knaak et al., 2006), whereas the loss of acetylation at lysine16 and trimethylation at lysine 20 of histone H4, is a hallmark of human cancer (Fraga et al., 2005). A favorable effect on gene transcription is observed when high levels of acetylation are combined with trimethylation of H3K4 (H3 lysine 4), H3K36 (lysine 36), and H3K79 (lysine 79), while low levels of these modifications generally are associated with inaccessibility and repression of gene expression (Kim et al., 2009). Enhancer of Zeste Homolog 2 (EZH2) is a histone methyltransferase that is increased and represses gene transcription in many breast cancers, playing a role in cancer vascular invasion and metastases and may be used as a marker of aggressive forms of breast cancer (Kleer et al., 2003). In lung cancer, as in other cases of human malignant tumors, a reduction of the level of H4K20me3 takes place in the tumoral tissue and this is associated with an increased invasiveness of cancer cells, hence the level of H4K20me3, may be considered as an indicator of poor prognosis in breast cancer (Van Den Broeck et al., 2008). In prostate cancer the levels of H3K4me1, H3K9me2, H3K9me3, H3Ac, and H4Ac were significantly reduced compared to normal

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prostate tissue and H3Ac and H3K9me2 levels allowed discrimination of the malignant from the normal prostate tissue (Ellinger et al., 2010). A combination of changes in histone modifications of prostate cancer cells proved to be useful in predicting the risk of tumor recurrence in patients with low-grade prostate cancer (Seligson et al., 2005) and oral squamous cell carcinoma (OSCC) (Chen et al., 2013). Global levels of trimethylation of H3K9 (H3K9me3) were positively related with cancer recurrence and poor survival rate of gastric adenocarcinomas (Park et al., 2008), while low levels of H3K4me2, H3K9me2, or H3K18ac were predictors of poor survival in pancreatic cancer (Manuyakorn et al., 2010). While a correlation between the epigenetic changes such as methylation/ demethylation of promoters of oncogenes and tumor suppressor genes, on the one hand, and their silencing/activation, on the other, is demonstrated by vast experimental evidence, the fundamental role of epigenetic modifications and the question on whether they can induce tumorigenesis remains unclear (Baylin and Bestor, 2002; Yu et al., 2014). Only recently, Yu et al. claim to have provided authentic evidence of gene silencing as inducer of tumors and cancerogenesis in mice. They succeeded in increasing the incidence of cancer in mice by hypermethylating an engineered p16Ink4a promoter to induce transcriptional suppression of the gene (Yu et al., 2014). Two epigenetic processes are considered as potential drivers of tumorigenesis, the epigenetic silencing of tumor suppressor genes and activation of oncogenes (Chatterjee et al., 2017).

Tumor-Suppressor Genes Under normal circumstances, tumor suppressor genes (antioncogenes) act as repressors of genes and contribute to programmed cell death in animals. Several tumor suppressor genes are known in mammalians. Their presence represents barriers to molecular pathways leading to carcinogenesis. Silencing of tumor suppressor genes is observed in all forms of human cancers because of hypermethylation of promoter CpG islands (Patel et al., 2017). It is believed that in most of cases, the suppressor genes-induced cancers initiate and progress only when both alleles of the gene are mutated. Among the most important suppressor genes are p53, pRb (encoding retinoblastoma protein), p16 INK4a, p15 INK4b, and BRCA1. Generally, these genes are necessary for the cell cycle to proceed, to induce apoptosis of the damaged cells and to prevent occurrence of malignancies. Mutant p53 proteins, bind to and upregulate chromatin regulatory genes, including three methyltransferase genes, leading to genome-wide histone methylation and suppression of expression of several genes (Zhu et al., 2015). Among these genes may be the one coding for protein p21, which normally binds cyclin-dependent kinase 2 (cdk2). The nonfunctional mutant p53 protein leads to uncontrolled division of cells and cancer initiation and progression. It is also observed that approximately

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10% of cases of unilateral retinoblastoma display aberrant methylation patterns (Greger et al., 1994) and the promoter methylation has a similar effect as mutation of the gene. In the hereditary breast cancer alone are involved ten suppressor genes (Lee and Muller, 2010). DNA repair genes are frequently repressed in cancers due to hypermethylation of CpG islands within their promoters. DNA damage and defective DNA repairs appears to be among the major underlying causes of cancer (Kastan, 2008; Bernstein et al., 2013). If DNA repair is not accurate, DNA damages tend to accumulate, increasing frequency of mutations during DNA replication, but they can also increase DNA methylation (O’Hagan et al., 2008). Methylation of CpG islands preceding suppressor gene promoters results from inactivation of the H3K4me3, leading to gene silencing and tumorigenesis (Lahtz and Pfeifer, 2011; Patel et al., 2017). DNA repair genes are frequently repressed in cancers due to hypermethylation of CpG islands within their promoters (Fig. 14.33).

Oncogenes Oncogenes are potential carcinogenetic factors. Their activation often results from somatic genetic alterations, such as gene mutations, gene arrangements (chromosome inversions and translocations), and gene amplifications (Croce, 2008), which lead to cancer initiation and progression. Since DNA methylation is reversible, demethylation can lead to activation of oncogenes (Chatterjee et al., 2017) from protooncogenes. Unmethylated normal cell promoter Me Me

Me

H3K4

Me

Me Me

H3K4

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CpG island Methylated cancer cell promoter Me Me Me H3K9

Me Me Me H3K9

Gene

CpG island FIG. 14.33 Epigenetic inactivation of a DNA repair gene promoter. Promoters are often embedded within CpG islands. These CpG-rich sequences are usually unmethylated in normal tissues and are associated with the active histone mark H3K4me3. H3K4me3 prevents DNA methylation. During tumorigenesis, the CpG island becomes methylated, is associated with inactive chromatin marks (e.g., H3K9me3), and the gene becomes silenced. (From Lahtz, C., Pfeifer, G.P., 2011. Epigenetic changes of DNA repair genes in cancer. J. Mol. Cell Biol. 3, 51–58.)

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In turn, protooncogenes derive from normal genes and their products belong to different classes of biological inducers such as transcription factors, growth factors, signal transducers, and apoptosis regulators. Transition of the normal genes into the state of protooncogenes results from mutational changes or increased expression, also known as dominant or gain-of-function mutations. An example of point mutations that induce carcinogenesis in humans is the activation of one of the three Ras genes that are found in 70%–90% of pancreatic carcinomas. In this case the mutant Ras protein is present in the active GTP bound form that keeps the cell proliferation going on continuously. A mutation in a typical example of oncogenes is ErbB2, a member of the EGFRs (epidermal growth factor receptors) family, which shows elevated levels in 20%–30% of human breast cancers. Expression/overexpression of the ErbB2 gene in mouse mammary epithelium induces multifocal mammary tumors (Lee and Muller, 2010). It is observed that EGFR-induced human breast cancer tumors don’t express the tumor suppressor gene 14-3-3σ (Hodgson et al., 2005). Activation of oncogenes leads to activation of other transmembrane receptors intensifying the process of cancerization. So, e.g., coexpression of ErbB2 and TGF-β receptor accelerates formation of metastases. What we need at the present time, is a parsimonious unifying principle underlying what quite recently Robert Weinberg called “the increasingly complex, if not chaotic, genetic phenomenology of cancer” (Weinberg, 2014).

The Neurogenic Etiology of Cancer Cases of the involvement of the nervous system in carcinogenesis have been described for quite some time, but until recently, generally were dealt with as counterinstances, or, at best, as unrelevant to cancer pathogenesis and neglected in the process “separation of the chaff from wheat” of the cancer inquiry. All of that evidence went largely unnoticed or were not considered as possible hints on the role of the nervous system in cancer pathogenesis. The first report on the presence of nerves in the tumor mass goes back to 1897. In the following three decades this was corroborated by results of other authors, but due to negative results from others (Martynow, 1930), until the 30th of the last century, the prevailing opinion was that no nerves penetrated the tumor mass (Engel, 1930). However, by the middle of the last century the situation changed, but only in the recent years has the involvement of the nervous system in carcinogenesis emerged as an important topic of cancer research. Four years ago, the biomedical community became aware of the importance of the investigation of the role of the nervous system as a new paradigm in the biology of cancer. The interest was sparked by a report of a Norwegian team, who observed that prostate cancer patients under androgen deprivation therapy (ADT) treatment profited from β-blockers in the form of reduced prostate cancer-specific mortality (Grytli et al., 2013). In prostate cancer the sympathetic innervation controls cancer initiation via β-adrenergic receptors and

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the parasympathetic arm controls the dissemination of cancer cells through activation of the type 1 muscarinic receptor (Chrm1) (Magnon, 2015). The involvement of the nervous system in the pathogenesis of cancer came as an unwelcome complication in the still controversial question of the causal (epigenetic or genetic) basis of cancer.

Role of Inflammation in Cancer Initiation The organism responds to environmental agents invading/attacking the body with inflammation (or sterile inflammation). Inflammation is a defensive reaction of the organism involving the molecular and cell mechanisms of defense at the site of infection. Inflammation may result not only from common pathogens (viruses, bacteria, parasites), their toxic products, and autoimmune conditions, but also from radiation and environmental agents, including diet. Inflammation may also arise as a reaction to the tumor growth and medical procedures for controlling it. The central nervous system reacts to the afferent information from the infection site by releasing a number of neurotransmitter/neuromodulators, which control and regulate the innate immune response (Kronfol and Remick, 2000; Watkins et al., 1995; Sundman and Olofsson, 2014; Tracey, 2010; Tracey, 2007; Andersson and Tracey, 2012; Glaser and Kiecolt-Glaser, 2005; Styer et al., 2008; Sun et al., 2011; Kawli et al., 2010; Zhang and Zhang, 2009; Cao and Aballa, 2016; Bogaerts et al., 2010: Steinberg et al., 2016). The carcinization of the cell is an indirect effect of these external agents and mediator of their carcinogen effect is the inflammation process that takes place at the region of the entrance of the agents in the animal body. Empirical evidence shows that overwhelming majority of cancers originate in an inflamed region of the body. The father of the modern pathology, Rudolph Virchow (1821–1902) may have been the first to notice a causal link between inflammation and carcinogenesis (Trinchieri, 2011) (Fig. 14.34). The causal relationship between the inflammation and the cancer initiation has been repeatedly emphasized and reviewed in the recent years (Mantovani et al., 2008; Freund et al., 2010; Trinchieri, 2011; Coussens et al., 2013; Marelli et al., 2017). From the onset of an infection, mainly via the sensory neurons, the brain receives information on the local inflammation caused by immune cells as first responders to infection. It responds to that information by activating the inflammation circuit (see Fig. 14.10). When the level of cytokines in the inflamed region reaches a higher limit, via neural pathways (the celiac ganglion and splenic nerve) the CNS sends chemical signals in the form of the neurotransmitter acetylcholine, which inhibits the production of cytokines and other products of inflammation from macrophages, thus protecting the organism from the excessive immune response (Sundman and Olofsson, 2014). The peripheral

Dermis

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(A)

(B) Epithelial cell and basement membrane

Endothelial cells and capillary support cells (pericytes, smooth muscle cells)

Platelets and fibrin clot

Neutrophils

Mast cells/eosinophils/basophils

Cytokines/chemokines

Fibroblasts and fibrillar collagens

Malignant epithelial cells

Lymphocytes

Macrophage/monocyte

FIG. 14.34 Wound healing versus invasive tumor growth. (A) Normal tissues have a highly organized and segregated architecture. (B) Invasive carcinomas are less organized. (From Coussens, L.M., Werb, Z., 2002. Inflammation and cancer. Nature 420, 860–866.)

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nervous system is also involved in this process by releasing neuropeptides at the site of infection. The failure of the process of healing or the malfunction of the antiinflammatory checkpoint leads to a chronic or repeated form of inflammation (Lawrence and Gilroy, 2007), known as nonresolving inflammation. In such cases, infiltration of the immune cells (macrophages, monocytes, lymphocytes, etc.) leads to degenerative processes and fibrosis. Another characteristic of the nonresolving inflammation is the increased release of free radicals leading to oxidative stress, oxidization of proteins, DNA and cell membrane damages, thus inducing production of carcinogen substances (Marnett, 2000; Khansari et al., 2009), or leading to programmed cell death (Jabs, 1999; Coussens et al., 2013). Empirical evidence indicates that chronic/nonresolving inflammation is often followed by initiation and development of cancer. So, e.g., chronic pancreatitis may lead initially to a form of metaplasia (a change in cell morphology that is reversible) (Pinho et al., 2014), before transforming into cancer. Experimental bouts of nonacute pancreatitis induce pancreatic neoplasias, although the acinar cells are resistant even to the expression of the K-Ras oncogenes (Keim, 2004; Guerra et al., 2011; Hausmann et al., 2014). Adequate empirical evidence of the last decade is showing a dominant role of the nervous system in the initiation and progression of the gastric cancer (Zhao et al., 2014), pancreatic cancer (Saloman et al., 2016), melanoma (Horvathova et al., 2016), opening new venues of thought on the etiology and pathogenesis of cancer. On the Temporal Order of Mutational and Epigenetic Changes in Initiation of Carcinogenesis: Who Precedes Whom? Although most of the researchers believe cancer initiation involves both genetic (mutational) and epigenetic modifications, no conclusive answer can be given to the causally crucial question of the temporal order of their emergence. Genetic and epigenetic aberrations are found in all cancer stem cell lines (Ahmed et al., 2013), but it is a widespread opinion that both may be involved in the initiation and progress of carcinogenesis (Baxter et al., 2014). However, recently an increase is observed in reports suggesting that epigenetics is in the driver’s seat of cancer pathogenesis, i.e., that epigenetic modifications precede occurrence of mutations in cancer cells. It is proposed that “epigenetic abnormalities may prime for changing oncogene senescence to addiction for a single key oncogene involved in lung cancer initiation.” (Vaz et al., 2017), and “deregulation of various epigenetic pathways can contribute to cancer initiation” (Toh et al., 2017). According to Feinberg et al. (2016) “changes in the structure of chromatin are induced very early in the cancer process by epigenetic modulators and even in the non-mutated normal tissues from which tumours arise.” Besides, it is consistently observed that several DNA mismatch repair genes and a number of

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important DNA repair genes are silenced through hypermethylation and the epigenetic defect in mismatch repair mechanisms accelerates the rate of accumulation of mutations in cancer cells (Chatterjee et al., 2017). It is suggested that chromatin modifications make chromatin permissive to “activate oncogene expression or cell fate changes that drive the cancer development” (Flavahan et al., 2017). Administration of the oral carcinogen DBP (dibenzo[def,p]chrysene) in healthy cats induced OSCC (oral squamous cell carcinoma) displaying specific hypomethylation of the gene Fgf3 and suggesting that it is an early symptom of cancerization that may be used for the early diagnosis of the disease (Sun et al., 2017). The idea that “epigenetic errors” induce transformation of the adult stem cells into tumor cells is also supported by experimental fact that administration of inhibitors of the histone deacetylase activity enables reexpression of a number of suppressed genes in colorectal carcinoma cell line (Cameron et al., 1999; Mathews et al., 2009). The previous idea also seems to be supported by ample evidence accumulated during the last three decades or so that cancer suppressor genes in cancer cells are hypermethylated. Tamura (2004) found out that a number of tumor suppressor genes that were unmethylated (hence expressible) in healthy individuals were hypermethylated (suppressed) in patients with gastric cancer. He also observed that incidence of the methylation of tumor suppressor genes increased with age in healthy individuals. Based on results from his investigation he concluded: “Methylation of tumor suppressor and tumor-related genes initially occurs in non-neoplastic gastric epithelia. Though not immediately oncogenic, methylation increases with age and ultimately inactivates gene function to constitute a field-defect (premalignant cells - NRC) where gastric cancer may be prone to develop” (Tamura, 2004). A recent study on hepatocellular carcinoma of 20 tumor suppressor genes showed that the incidence of hypermethylation increased in 6 of 10 tumor suppressor genes (Chen et al., 2015). Methylation of the promoter of the tumor suppressor gene DAL-1, leads to loss of its expression and this is sufficient for the initiation and development of the gastric cancer and the decrease and loss of DAL-1 expression was observed in 90.9% of the primary gastric cancers. Similarly, hypermethylation of the promoter of the tumor suppressor gene p14ARF leads to loss of its expression and initiation of the colorectal cancer (Nyiraneza et al., 2012). Neural Control of Epigenetic Modifications in Cancer The fact that epigenetic marks do not occur randomly, but mostly adaptively, and follow strictly determined temporal and spatial patterns suggests the existence of a programmer. What determines the temporal and spatial patterns of appearance of epigenetic modifications? Let us start with a glimpse on some experimental evidence on the specific placement of epigenetic marks in DNA and histones. Humans with general anxiety disorder (GAD) show increased methylation of glucocorticoid receptor

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(GR) gene promoter exon 1F, along the increased activity of the HPA axis (Wang et al., 2017a,b). A similar increased methylation of the promoter region of the exon 1F of the glucocorticoid receptor gene, NR3CF1 (nuclear receptor subfamily 3, group C, member 1) was observed in samples from suicide victims with a history of childhood abuse, but not in suicide victims that experienced no childhood abuse (McGowan et al., 2009; Zhang et al., 2013), and repetitive restrain stress alters the global hippocampal DNA methylation (Makhathini et al., 2017). In a review of the relevant literature, Turecki and Meaney (2016) found that the overwhelming majority of studies in animals and humans show that the early-life adversity leads to increased methylation of the GR exon variants GR17 in rats and 1F variant in humans (Turecki and Meaney, 2016). Not only stress, but even experimental administration of receptors for neurotransmitters dopamine (DA), muscarinic acetylcholine (mAch), and glulamate (Glu), induce specific modifications in the histone H3 and early gene expression in hippocampus (Crosio et al., 2003). Even the neuronal activity in the primary cortical neuronal cultures recruits the enzyme methyltransferase Asg1L, which methylates the histone H3 of the promoter of the nrxn1α gene, thus repressing the expression of the gene (Zhu et al., 2016). Neuronal activity also increases acetylation of histone H3, inducing specific changes in chromatin structure and ultimately leading to changes in transcription of genes relevant for memory formation (Levenson et al., 2004). The level and patterns of the methylation change with the age of the organism (Hernandez et al., 2011), what seems to support idea of a cell-independent control of the epigenetic modifications. In contrast with gene mutations, epigenetic “mutations” are reversible (Zhang et al., 2013). The dynamic character of the neural induction of methylation processes by neurons may be illustrated by the example of the transient demethylation of the methylated stress response gene growth arrest and DNA-damage-inducible protein 45 beta (Gadd45b) promoter by neuronal activity, which may promote adult neurogenesis (Wu and Sun, 2009; Ma et al., 2009). DNA methylation is performed by DNMT enzymes and it is observed that fear conditioning upregulates DNMT expression in hippocampus (Miller and Sweatt, 2007). Contextual fear conditioning downregulates methylation and increases expression of the BDNF exon IV (Lubin et al., 2008). An already classical impressive example of the neural control of DNA and histone methylation is that observed in high licking and grooming (LG) rat mothers. Their offspring’s high LG behavior is not genetically but epigenetically determined during the first week after birth by decreased expression of the estrogen receptor in the hypothalamic medial preoptic area (MPOA) of the brain as a result of the reduced methylation of Erα gene promoter (Esr1) (Pen˜a et al., 2013). Emphasizing the role of the CNS in directing the methylation processes in the brain that determine stress-related changes in methylation of particular genes and behavior of the offspring, as well as the role of chromatin

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remodeling in transgenerational inheritance, Weaver et al. (2017) pointed out that “the maternal behavior initiates a neural signaling cascade that directs activation of particular transcription factors to recruit and guide chromatin remodelers and DNA methylation enzymes to particular chromatin domains, allowing maternal behavior to affect several behavioral phenotypes in the offspring, including maternal behavior. Herein, both acquired and stable behavioral traits can be propagated across generations through epigenetic modifications to chromatin domains in a brain region- and genome sequence-specific manner.” (Weaver et al., 2017). The only known pathway of the action of stress on somatic structures is the neural pathway and there is adequate evidence of the neural control and regulation of epigenetic modifications in DNA, chromatin and the patterns of expression of miRNAs. Interpreting results of experimental work on the epigenetic modifications, Meaney and Ferguson-Smith expressed their conviction that it is the nervous system that orchestrates and directs the epigenetic modifications in DNA and chromatin: “The ability to mastermind such adaptation to circumstance relies upon the capacity of neurons and glia to dynamically adapt genomic structure and function” (Zhang et al., 2013). Indeed, in 2014 it was argued and supported by adequate empirical evidence that the information for the precise placement of epigenetic marks in DNA and histones originates in the nervous system (Cabej, 2014). Denervation Proves the Role of the Nervous System in Cancer Pathogenesis As early as 1967, it was reported that denervation induces skin cancer (Pawlowski and Weddell, 1967). Almost three decades ago, Romeo et al. (1991) observed that sympathectomy reduced the development of lung tumors implanted in mice skin (Romeo et al., 1991) and in 2001 it was observed that an increased number of neurites grew out of the adjacent nerve/ganglions in direction of prostate cancer cells (Ayala et al., 2001). But, apparently, it was not until the publication of reports on the reduction of the incidence of gastric tumors in rats as a result of denervation (Polli-Lopes et al., 2003) that biologists became aware of the central role of the nervous system in cancer growth. Now, “nerve-cancer cell cross talk” has become a hot topic of cancer research. In a series of experiments on mice with gastric cancer, Zhao et al. (2014) have shown that either surgical or pharmacological (Botox injection) vagotomy not only suppressed gastric tumorigenesis, but decreased markedly the progression of gastric cancer and reduced its incidence (Zhao et al., 2014). It has been experimentally demonstrated that “Denervation therapy was effective in both early preneoplasia and late neoplasia/dysplasia” (Zhao et al., 2014). Vagal denervation (but not sympathectomy) of intestines decreased growth of adenomas in mouse intestines (Liu et al., 2015). The development of prostate cancer implants was inhibited in mice pretreated with

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surgical or pharmacological sympathectomy (Magnon et al., 2013). Ablation of sensory neurons in cases of pancreatic cancers slows the development of the precancerous inflammatory disease and leads to a dramatic prolongation of survival and significant survival in mice with pancreatic cancer (Saloman et al., 2016). Sympathectomy not only delayed initiation of melanomas in mice injected with melanoma cells but it prolonged life of the sympathectomized mice in comparison with unsympathectomized and continued this effect even after the sympathetic nerves started regenerating (Horvathova et al., 2016). Sensory neurons of the skin release substances that inhibit the Hh pathway and the loss of this ability leads to initiation and progress of basal cell carcinogenesis. However, the severance of these neurons prevents both initiation and progress of the cancer (Peterson et al., 2015). Prostate tumor xenografts developed poorly in mice that had been pretreated by chemical or surgical sympathectomy of the prostate gland, or when stromal β2- and β3-adrenergic receptors were genetically deleted. This empirical evidence unequivocally points in direction of an essential role of the nervous system in cancer initiation and progression. Neural Induction of Cancer Metastases Infiltration of nerves in solid tumors is an important hallmark in the process of carcinogenesis. The SNS innervates the tumor mass and under stress conditions secretes norepinephrine. This and the circulating epinephrine, bind their specific β-adrenoreceptors of cancer cells as well as the cells of the cancer microenvironment (endothelial cells, immune cells and fibroblasts). Cancer cells may dissociate from the tumor mass and via the extracellular matrix (ECM) find their way to nerves and blood vessels. The innervation transforms radically the tumor microenvironment by starting a cross-talk between nerves and the cancer stromal cells in a mutually “profitable” way where neuropeptides/neurotransmitters released by local nerves favor tumor growth and facilitate metastasis (Magnon et al., 2013), whereas neurotrophic factors released by tumor cells stimulate a process of increased nerve density in the process of neoneurogenesis (Entschladen et al., 2006), associated with faster progress of the cancer. The cancer neoneurogenesis and angiogenesis not only favors tumor growth, but facilitates and stimulates perineural invasion by cancer cells and their migration to metastasis sites (Ayala et al., 2001) (Fig. 14.35). During chronic stress and other conditions, nerves release norepinephrine, which increases expression of its receptor AdRβ2 in stromal cells, thus downregulating the receptor level. Cancer cells with low levels of expression of the norepinephrine receptor are induced to migrate along the nerves and blood vessels to the metastasis sites (Braadland et al., 2014) (Fig. 14.35). In line with this are findings that β2-adrenoreceptors are key for transmitting neural signals from the tumor microenvironment to stimulate metastatic behavior of invadopodia

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FIG. 14.35 Hypothetical model of how β2-adrenergic signaling may promote progression of prostate cancer and invasive potential of the cells. Cancer cells expressing low levels of ADRB2 will thereby follow the nerves and blood vessels to metastatic sites. (From Braadland, P.R., Ramberg, H., Grytli, H.H., Task
en, K.A., 2014. β-Adrenergic receptor signaling in prostate cancer. Front. Oncol. 4, 375.)

and this explains numerous observations on the causal relationship between the chronic stress and cancer metastasis (Creed et al., 2015). Over the last decade β-blockers (blockers of adrenergic receptors) have been profitably used in the treatment of cancer (Powe et al., 2010; Diaz et al., 2012; Cardwell et al., 2014). Norepinephrine released by the sympathetic nerves (as well as the circulating epinephrine) stimulates expansion of blood vessels within the cancer mass (Cole and Sood, 2012). The increased secretion of neurotransmitters norepinephrine by the sympathetic innervation and the circulating epinephrine during the stress has an immunosuppressive effect, thus facilitating tumor growth (Inbar et al., 2011). Most recently, it is observed that before the start of the metastatic progression of cancer, specific changes occur in the nontumoral tissue around the aggressive prostate cancer in mice (Str€omvall et al., 2017). In bones, sympathetic stimulation as well as the stress-induced activation of the sympathetic system increase release of norepinephrine, which, by binding the AdRβ2 stimulates proliferation of osteoclasts and inhibit osteoblasts in cells of the breast cancer line MDA-231, leading to loss of bone mass (Grano et al., 2000), while the osteoblasts increase expression of the cytokine Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL), which stimulates migration of cancer cells, starting the process of cancer metastasis (Katayama et al., 2006; Campbell et al., 2012). The ability of tumors to initiate and stimulate their own innervation is known as neoneurogenesis (Entschladen et al., 2006; Palm and Entschladen, 2007). The neoneurogenesis changes radically the tumor microenvironment and emerges as one of the determinants of the tumor fate, repression of its growth (in rare cases leading to “spontaneous regression/spontaneous remission” of the tumor/cancer), or its further progress and spread through

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metastases. Adrenergic neurotransmitters released by sympathetic nerves bind β-adrenergic receptors on tumor cell membranes, thus stimulating the proliferation of tumor cells and VEGF (vascular endothelial growth factor), leading to angiogenesis and increase of blood supply in the tumor. Neurotransmitter DA has the opposing effect of inhibiting tumor angiogenesis and tumor growth (Chakroborty et al., 2009).

REFERENCES Ahmed, D., Eide, P.W., Eilertsen, I.A., Danielsen, S.A., Eknæs, M., Hektoen, M., Lind, G.E., Lothe, R.A., 2013. Epigenetic and genetic features of 24 colon cancer cell lines. Oncogenesis. 2(9). Albany, C., Alva, A.S., Aparicio, A.M., Singal, R., Yellapragada, S., Sonpavde, G., Hahn, N.M., 2011. Epigenetics in prostate cancer. Prostate Cancer 2011, 580318. Almqvist, N., Ma˚rtensson, I.-L., 2012. The pre-B cell receptor: selecting for or against autoreactivity. Scand. J. Immunol. 76, 256–262. Andersson, U., Tracey, K.J., 2012. Neural reflexes in inflammation and immunity. J. Exp. Med. 209, 1057–1068. Andersen, D.H., Pertoldi, C., Scali, V., Loeschcke, V., 2005. Heat stress and age induced maternal effects on wing size and shape in parthenogenetic Drosophila mercatorum. J. Evol. Biol. 18, 884–892. Annunziato, A.T., 2008. DNA packaging: nucleosomes and chromatin. Nat. Educ. 1, 26. Ansel, K.M., Lee, D.U., Rao, A., 2003. An epigenetic view of helper T cell differentiation. Nat. Immunol. 4, 616–623. Armati, P.J., Mathey, E.K., 2013. An update on Schwann cell biology—immunomodulation, neural regulation and other surprises. J. Neurol. Sci. 333, 68–72. Arnold, L., Henry, A., Poron, F., Baba-Amer, Y., van Rooijen, N., Plonquet, A., Gherardi, R.K., Chazaud, B., 2007. Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J. Exp. Med. 204, 1057–1069. Aurelio, O., Hall, D.H., Hobert, O., 1998. Immunoglobulin-domain proteins required for maintenance of ventral nerve cord organization. Science 295, 686–690. Ayala, G.E., Wheeler, T.M., Shine, H.D., Schmelz, M., Frolov, A., Chakraborty, S., Rowley, D., 2001. In vitro dorsal root ganglia and human prostate cell line interaction: redefining perineural invasion in prostate cancer. Prostate 49, 213–223. Bala´zs, M., Martin, F., Zhou, T., Kearney, J., 2002. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity 17, 341–352. Bateman, A., Eddy, S.R., Chothya, C., 1996. Members of the immunoglobulin superfamily in bacteria. Protein Sci. 5, 1939–1941. Baxter, E., Windloch, K., Gannon, F., Lee, J.S., 2014. Epigenetic regulation in cancer progression. Cell Biosci. 4, 45. Baylin, S.B., Bestor, T.H., 2002. Altered methylation patterns in cancer cell genomes: cause or consequence? Cancer Cell 1, 299–305. Baylin, S.B., Ohm, J.E., 2006. Epigenetic gene silencing in cancer – a mechanism for early oncogenic pathway addiction? Nat. Rev. Cancer 6, 107–116. Bayne, C.J., 2003a. Co-evolution of innate and adaptive immunity. Integr. Comp. Biol. 43, 291–299. Bayne, C.J., 2003b. Origins and evolutionary relationships between the innate and adaptive arms of immune systems. Integr. Comp. Biol. 43, 293–299.

Epigenetics in Health and Disease Chapter

14

717

B
egin, P., Nadeau, K.C., 2014. Epigenetic regulation of asthma and allergic disease. Allergy, Asthma Clin. Immunol. 2014 (10), 27. Bernstein, C., Prasad, A.R., Nfonsam, V., Bernstein, H., 2013. DNA damage, DNA repair and cancer. In: Chen, C. (Ed.), New Research Directions in DNA Repair. InTech, p. 413. (Chapter 16). Bhasin, M., Reinherz, E.L., Reche, P.A., 2006. Recognition and classification of histones using support vector machine. J. Comput. Biol. 13, 102–112. Bird, A.P., 1986. CpG-rich islands and the function of DNA methylation. Nature 321, 209–213. Bogaerts, A., Beets, I., Schoofs, L., Verleyen, P., 2010. Antimicrobial peptides in Caenorhabditis elegans. Invertebr. Surviv. J. 7, 45–52. Bouchard Jr., T.J., Lykken, D.T., McGue, M., Segal, N.L., Tellegen, A., 1990. Sources of human psychological differences: the Minnesota Study of Twins Reared Apart. Science 250, 223–228. Braadland, P.R., Ramberg, H., Grytli, H.H., Task
en, K.A., 2014. β-Adrenergic receptor signaling in prostate cancer. Front. Oncol. 4, 375. Brix, T.H., Heged€ us, L., 2012. Twin studies as a model for exploring the aetiology of autoimmune thyroid disease. Clin. Endocrinol. 76, 457–464. Brunmeir, R., Lagger, S., Simboeck, E., Sawicka, A., Egger, G., Hagelkruys, A., Zhang, Y., Matthias, P., Miller, W.J., Seiser, C., 2011. Epigenetic regulation of a murine retrotransposon by a dual histone modification mark. PLoS Genet. 7 (1), 10.1371. Buijs, R.M., van der Vliet, J., Garidou, M.-L., Huitinga, I., Escobar, C., 2008. Spleen vagal denervation inhibits the production of antibodies to circulating antigens. PLoS ONE 3 (9), e3152. Cabej, N.R., 2014. On the origin of information in epigenetic structures. Med. Hypotheses 83, 378–386. Caccia, N., Kronenberg, M., Saxe, D., Haars, R., Bruns, G.A.P., Goverman, J., Malissen, M., Willard, H., Yoshikai, Y., Simon, M., Hood, L., Mak, T.W., 1984. The T cell receptor B chain genes are located on chromosome 6 in mice and chromosome 7 in humans. Cell 37, 1091–1099. Caccia, N., Bruns, G.A.P., Kirsch, I.R., Hollis, G.F., Bertness, V., Mak, T.W., 1985. T cell receptor alpha chain genes are located on chromosome 14 at 14q11-14q12 in humans. J. Exp. Med. 161, 1255–1260. Cai, T.-T., Muhali, F.-S., Song, R.-H., Qin, Q., Wang, X., Shi, L.-F., 2015. Genome-wide DNA methylation analysis in Graves’ disease. Genomics 105, 204–210. Cai, T.T., Zhang, J., Wang, X., Song, R.H., Qin, Q., Muhali, F.S., et al., 2016. Gene-gene and genesex epistatic interactions of DNMT1, DNMT3A and DNMT3B in autoimmune thyroid disease. Endocr. J. 63, 643–653. Cameron, E.E., Bachman, K.E., My€oh€anen, S., Herman, J.G., Baylin, S.B., 1999. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet. 21, 103–107. Campbell, J.P., Karolak, M.R., Ma, Y., Perrien, D.S., Masood-Campbell, S.K., Penner, N.L., et al., 2012. Stimulation of host bone marrow stromal cells by sympathetic nerves promotes breast cancer bone metastasis in mice. PLoS Biol. 10(7). Cao, X., Aballa, A., 2016. Neural inhibition of dopaminergic signaling enhances immunity in a cellnon-autonomous manner. Curr. Biol. 26, 2329–2334. Cardoso, J.C.R., F
elix, R.C., Bergqvist, C.A., Larhammar, D., 2014. New insights into the evolution of vertebrate CRH (corticotropin-releasing hormone) and invertebrate DH44 (diuretic hormone 44) receptors in metazoans. Gen. Comp. Endocrinol. 209, 162–170. Cardwell, C.R., Coleman, H.G., Murray, L.J., O’Sullivan, J.M., Powe, D.G., 2014. Beta-blocker usage and prostate cancer survival: a nested case-control study in the UK Clinical Practice Research Datalink cohort. Cancer Epidemiol. 38, 279–285.

718 PART

III Epigenetics of Metazoan Evolution

Carmona, L.M., Schatz, D.G., 2017. New insights into the evolutionary origins of the recombination-activating gene proteins and V(D)J recombination. FEBS J. 284, 1590–1605. Carpenter, A.C., Bosselut, R., 2010. Decision checkpoints in the thymus. Nat. Immunol. 11, 666–673. Casrouge, A., Beaudoing, E., Dalle, S., Pannetier, C., Kanellopoulos, J., Kourilsky, P., 2000. Size estimate of the αβ TCR repertoire of naive mouse splenocytes. J. Immunol. 164, 5782–5787. Cerenius, L., S€ oderh€all, K., 2013. Variable immune molecules in invertebrates. J. Exp. Biol. 216, 4313–4319. Ceribelli, A., Nahid, M.A., Satoh, M., Chan, E.K., 2011. MicroRNAs in rheumatoid arthritis. FEBS Lett. 585, 3667–3674. Ceribelli, A., Satoh, M., Chan, E.K.L., 2012. MicroRNAs and autoimmunity. Curr. Opin. Immunol. 24, 686–691. Chakroborty, D., Sarkar, C., Basu, B., Dasgupta, P.S., Basu, S., 2009. Catecholamines regulate tumor angiogenesis. Cancer Res. 69, 3727–3730. Chatterjee, A., Rodger, E.J., Eccles, M.R., 2017. Epigenetic drivers of tumourigenesis and cancer metastasis. Semin. Cancer Biol. Available: https://www.sciencedirect.com/science/article/pii/ S1044579X17300536#bib0010. In Press (Corrected Proof ). Chen, Y.-W., Kao, S.-Y., Wang, H.-J., Yang, M.-H., 2013. Histone modification patterns correlate with patient outcome in oral squamous cell carcinoma. Cancer 119, 4259–4267. Chen, H., Zhang, T., Sheng, Y., Zhang, C., Peng, Y., Wang, X., Zhan, C., 2015. Methylation profiling of multiple tumor suppressor genes in hepatocellular carcinoma and the epigenetic mechanism of 3OST2 regulation. J. Cancer 6, 740–749. Chervona, Y., Costa, M., 2012. Histone modifications and cancer: biomarkers of prognosis? Am. J. Cancer Res. 2, 589–597. Chrousos, G.P., 2000. The role of stress and the hypothalamic-pituitary-adrenal axis in the pathogenesis of the metabolic syndrome: neuro-endocrine and target tissue-related causes. Int. J. Obesity 24 (Suppl 2), S50–S55. Chrousos, G.P., 2009. Stress and disorders of the stress system. Nat. Rev. Endocrinol. 5, 374–381. Cole, S.W., Sood, A.K., 2012. Molecular pathways: beta-adrenergic signaling in cancer. Clin. Cancer Res. 18, 1201–1206. Cooper, M.D., Alder, M.N., 1996. The evolution of adaptive immune systems. Cell 124, 815–822. Coussens, L.M., Zitvoge, L., Palucka, A.K., 2013. Neutralizing tumor-promoting chronic inflammation: a magic bullet? Science 339, 286–291. Creed, S.J., Le, C.P., Hassan, M., Pon, C.K., Albold, S., Chan, K.T., Berginski, M.E., Huang, Z., Bear, J.E., Lane, J.R., Halls, M.L., Ferrari, D., Nowell, C.J., Sloan, E.K., 2015. β2-Adrenoceptor signaling regulates invadopodia formation to enhance tumor cell invasion. Breast Cancer Res. 17, 145. Croce, C.M., 2008. Oncogenes and cancer. N. Engl. J. Med. 358, 502–511. Crosio, C., Heitz, E., Allis, C.D., Borrelli, E., Sassone-Corsi, P., 2003. Chromatin remodeling and neuronal response: multiple signaling pathways induce specific histone H3 modifications and early gene expression in hippocampal neurons. J. Cell Sci. 116, 4905–4914. Daniel, J.A., Santos, M.A., Wang, Z., Zang, C., Schwab, K.R., Jankovic, M., et al., 2010. PTIP promotes chromatin changes critical for immunoglobulin class switch recombination. Science 329, 917–923. Danke, N.A., Koelle, D.M., Yee, C., Beheray, S., Kwok, W.W., 2004. Autoreactive T cells in healthy individuals. J. Immunol. 172, 5967–5972. Davis, M.M., Bjorkman, P.J., 1988. The T cell antigen receptor genes and T cell recognition. Nature 334, 395–402.

Epigenetics in Health and Disease Chapter

14

719

Day, T.A., 2005. Defining stress as a prelude to mapping its neurocircuitry: no help from allostasis. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 29, 1195–1200. De Santis, C.E., Lin, C.C., Mariotto, A.B., Siegel, R.L., Stein, K.D., Kramer, J.L., et al., 2014. Cancer treatment and survivorship statistics, 2014. CA Cancer J. Clin. 4, 252–271. de Yebenes, V.G., Belver, L., Pisano, D.G., Gonzalez, S., Villasante, A., Croce, C., et al., 2008. miR181b negatively regulates activation-induced cytidine deaminase in B cells. J. Exp. Med. 205, 2199–2206. Deaton, A.M., Bird, A., 2011. CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022. Diaz, E.S., Karlan, B.Y., Li, A.J., 2012. Impact of beta blockers on epithelial ovarian cancer survival. Gynecol. Oncol. 127, 375–378. Doucet, A.J., Wilusz, J.E., Miyoshi, T., Liu, Y., Moran, J.V., 2015. A 3’ poly(A) tract is required for LINE-1 retrotransposition. Mol. Cell 60, 728–741. Dunkelberger, J.R., Song, W.-C., 2010. Complement and its role in innate and adaptive immune responses. Cell Res. 20, 34–50. Ellinger, J., Kahl, P., von der Gathen, J., Rogenhofer, S., Heukamp, L.C., G€utgemann, I., et al., 2010. Global levels of histone modifications predict prostate cancer recurrence. Prostate 70, 61–69. Engel, C.S., 1930. Warum erkranken wirbellose Tiere nicht an Krebs? Z. Krebsforsch. 32, 531–543. Engelmann, I., Pujol, N., 2010. Innate immunity in C. elegans. In: S€oderh€all, K. (Ed.), Invertebrate Immunity. Springer, pp. 105–131. Entschladen, F., Palm, D., Lang, K., Zaenker, K.S., 2006. Neoneurogenesis: tumors may initiate their own innervation by the release of neurotrophic factors in analogy to lymphangiogenesis and neoangiogenesis. Med. Hypotheses 67, 33–35. Evsikov, A.V., de Evsikova, C.M., 2016. Friend or foe: epigenetic regulation of retrotransposons in mammalian oogenesis and early development. Yale J. Biol. Med. 89, 487–497. Faulkner, G.J., 2011. Retrotransposons: mobile and mutagenic from conception to death. Retrotransposons: mobile and mutagenic from conception to death. FEBS Lett. 585, 1589–1594. Feinberg, A.P., Gehrke, C.W., Kuo, K.C., Ehrlich, M., 1988. Reduced genomic 5-methylcytosine content in human colonic neoplasia. Cancer Res. 48, 1159–1161. Feinberg, A.P., Ohlsson, R., Henikoff, S., 2005. The epigenetic progenitor origin of human cancer. Nat. Rev. Genet. 7, 21–33. Feinberg, A.P., Koldobskiy, M.A., G€ond€or, A., 2016. Epigenetic modulators, modifiers and mediators in cancer aetiology and progression. Nat. Rev. Genet. 17, 284–299. Felten, D.L., Felten, S.Y., Carlson, S.L., Olschowka, J.A., Livnat, S., 1985. Noradrenergic and peptidergic innervation of lymphoid tissue. J. Immunol. 135, 755s–765s. Fields, P.E., Kim, S.T., Flavell, R.A., 2002. Cutting edge: changes in histone acetylation atthe IL-4 and IFN-γ loci accompanyTh1/Th2 differentiation. J. Immunol. 169, 647–650. Flavahan, W.A., Gaskell, E., Bernstein, B.E., 2017. Epigenetic plasticity and the hallmarks of cancer. Science 357 (6348), eaal2380. Retrieved on June, 20, 2018. from, https://europepmc.org/ articles/pmc5940341. Fraga, M.F., Ballestar, E., Villar-Garea, A., Boix-Chornet, M., Espada, J., Schotta, G., et al., 2005. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat. Genet. 37, 391–400. Freund, A., Orjalo, A.V., Desprez, P.Y., Campisi, J., 2010. Trends Mol. Med. 16, 238. Fryer, A.D., Wills-Karp, M., 1991. Dysfunction of M2-muscarinic receptors in pulmonary parasympathetic nerves after antigen challenge. J. Appl. Physiol. 71, 2255–2261.

720 PART

III Epigenetics of Metazoan Evolution

Fugmann, S.D., Villey, I.J., Ptaszek, L.M., Schatz, D.G., 2000. Identification of two catalytic residues in RAG1 that define a single active site within the RAG1/RAG2 protein complex. Mol. Cell 5, 97–107. Fugmann, S.D., Messier, C., Novack, L.A., Cameron, R.A., Rast, J.P., 2006. An ancient evolutionary origin of the Rag1/2 gene locus. Proc. Natl. Acad. Sci. U. S. A. 103, 3728–3733. Fujimura, S., Matsui, T., Kuwahara, K., Maeda, K., Sakaguchi, N., 2008. Germinal center B-cellassociated DNA hypomethylation at transcriptional regions of the AID gene. Mol. Immunol. 45, 1712–1719. Gabriel, W., 2005. How stress selects for reversible phenotypic plasticity. J. Evol. Biol. 18, 873–883. Gama-Sosa, M., Slagel, V., Trewyn, R., Oxenhandler, R., Kuo, K., Gehrke, C., Ehrlich, M., 1983. The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res. 11, 6883–6894. Gardner, J.M., Fletcher, A.L., Anderson, M.S., Turley, S.J., 2009. AIRE in the thymus and beyond. Curr. Opin. Immunol. 21, 582–589. Glaser, R., Kiecolt-Glaser, J.K., 2005. Stress-induced immune dysfunction: implications for health. Nat. Rev. Immunol. 5, 243–251. Global Burden of Disease Cancer Collaboration, 2015. The global burden of cancer 2013. JAMA Oncol. 1, 505–527. Goodier, J.L., Kazazian Jr., H.H., 2008. Retrotransposons revisited: the restraint and rehabilitation of parasites. Cell 135, 23–35. Grano, M., Mori, G., Minielli, V., Cantatore, F.P., Colucci, S., Zallone, A.Z., 2000. Breast cancer cell line MDA-231 stimulates osteoclastogenesis and bone resorption in human osteoclasts. Biochem. Biophys. Res. Commun. 270, 1097–1100. Greger, V., Debus, N., Lohmann, D., Hopping, W., Passarge, E., Horsthemke, B., Debus, N., Lohmann, D., Hopping, W., Passarge, E., Horsthemke, B., 1994. Frequency and parental origin of hypermethylated RB1 alleles in retinoblastoma. Hum. Genet. 94, 491–496. Grytli, H.H., Fagerland, M.W., Task
en, K.A., Fossa˚, S.D., Ha˚heim, L.L., 2013. Use of β-blockers is associated with prostate cancer-specific survival in prostate cancer patients on androgen deprivation therapy. Prostate 73, 250–260. Guerra, C., Collado, M., Navas, C., Schuhmacher, A.J., Herna´ndez-Porras, I., Can˜amero, M., Rodriguez-Justo, M., Serrano, M., Barbacid, M., 2011. Pancreatitis-induced inflammation contributes to pancreatic cancer by inhibiting oncogene-induced senescence. Cancer Cell 19, 728–739. Gururajan, M., Haga, C.L., Das, S., Leu, C.-M., Hodson, D., Josson, S., et al., 2010. MicroRNA 125b inhibition of B cell differentiation in germinal centers. Int. Immunol. 22, 583–592. Hanoun, M., Maryanovich, M., Arnal-Estape, A., Frenette, P.S., 2015. Neural regulation of hematopoiesis, inflammation, and cancer. Neuron 86, 360–373. Hausmann, S., Kong, B., Michalski, C., Erkan, M., Friess, H., 2014. The role of inflammation in pancreatic cancer. Adv. Exp. Med. Biol. 816, 129–151. Hermes, G.L., Delgado, B., Tretiakova, M., Cavigelli, S.A., Krausz, T., Conzen, S.D., et al., 2009. Social isolation dysregulates endocrine and behavioral stress while increasing malignant burden of spontaneous mammary tumors. Proc. Natl. Acad. Sci. U. S. A. 106, 22393–22398. Hernandez, D.G., Nalls, M.A., Gibbs, J.R., Arepalli, S., van der Brug, M., Chong, S., Moore, M., Longo, D.L., Cookson, M.R., Traynor, B.J., Singleton, A.B., 2011. Distinct DNA methylation changes highly correlated with chronological age in the human brain. Hum. Mol. Genet. 20, 1164–1172. Herndon, J.M., Stuart, P.M., Ferguson, T.A., 2005. Peripheral deletion of antigen-specific T cells leads to long-term tolerance mediated by CD8+ cytotoxic cells. J. Immunol. 174, 4098–4104.

Epigenetics in Health and Disease Chapter

14

721

Hewagama, A., Richardson, B., 2009. The genetics and epigenetics of autoimmune diseases. J. Autoimmun. 33 (1), 3. Hill, M.N., McLaughlin, R.J., Pan, B., Fitzgerald, M.L., Roberts, C.J., Lee, T.T.-Y., et al., 2011. Recruitment of prefrontal cortical endocannabinoid signaling by glucocorticoids contributes to termination of the stress response. J. Neurosci. 31, 10506–10515. Hirano, M., Guo, P., McCurley, N., Schorpp, M., Das, S., Boehm, T., et al., 2013. Evolutionary implications of a third lymphocyte lineage in lampreys. Nature 501, 435–438. Hodgson, J.G., Malek, T., Bornstein, S., Hariono, S., Ginzinger, D.G., Muller, W.J., et al., 2005. Copy number aberrations in mouse breast tumors reveal loci and genes important in tumorigenic receptor tyrosine kinase signaling. Cancer Res. 65, 9695–9704. Horvathova, L., Tillinger, A., Padova, A., Mravec, B., 2016. Sympathectomized tumor-bearing mice survive longer but develop bigger melanomas. Endocr. Regul. 50, 207–214. Hsu, E., Pulham, N., Rumfelt, L.L., Flajnik, M.F., 2006. The plasticity of immunoglobulin gene systems in evolution. Immunol. Rev. 210, 8–26. Huang, J., Zhu, C., Zhang, P., Zhu, Q., Liu, Y.-M., Zhu, Z., Wang, M.-C., Li, W., Yang, G., Dong, N., Liu, J., Chen, L., Zhang, Y., Yang, R., Deng, L., Fan, J., Wang, X., Liu, J., Ma, B., Fu, Q., Wu, K., 2013. S100+ cells: a new neuro-immune cross-talkers in lymph organs. Sci. Rep. 3, 1114. Huang, S., Tao, X., Yuan, S., Zhang, Y., Li, P., Beilinson, H.A., Zhang, Y., Yu, W., Pontarotti, P., Escriva, H., Le Petillon, Y., Liu, X., Chen, S., Schatz, D.G., Xu, A., 2016. Discovery of an active RAG transposon illuminates the origins of V(D)J recombination. Cell 166, 102–114. Huber, L.C., Brock, M., Hemmatazad, H., Giger, O.T., Moritz, F., Trenkmann, M., et al., 2007. Histone deacetylase/acetylase activity in total synovial tissue derived from rheumatoid arthritis and osteoarthritis patients. Arthritis Rheum. 56, 1087–1093. Huber, M., Knottnerus, J.A., Green, L., van der Horst, H., Jadad, A.R., Kromhout, D., Leonard, B., Lorig, K., Loureiro, M.I., van der Meer, J.W.M., Schnabel, P., Smith, R., van Weel, C., Smid, H., 2011. How should we define health? Br. Med. J. 343, d4163. Inbar, S., Neeman, E., Avraham, R., Benish, M., Rosenne, E., Ben-Eliyahu, S., 2011. Do stress responses promote leukemia progression? An animal study suggesting a role for epinephrine and prostaglandin-E2 through reduced NK activity. PLoS ONE. 6. International Human Genome Sequencing Consortium, 2001. Initial sequencing and analysis of the human genome. Nature 409, 860–921. Isobe, M., Russo, G., Haluska, F.G., Croce, C.M., 1988. Cloning of the gene encoding the deltasubunit of the human T-cell receptor reveals its physical organization within the alpha-subunit locus and its involvement in chromosome translocations in T-cell malignancy. Proc. Natl. Acad. Sci. U. S. A. 85, 3933–3937. Jabs, T., 1999. Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochem. Pharmacol. 57, 231–245. Jaeger, S., Fernandez, B., Ferrier, P., 2013. Epigenetic aspects of lymphocyte antigen receptor gene rearrangement or ‘when stochasticity completes randomness’. Immunology 139, 141–150. Janeway Jr., C.A., Travers, P., Walport, M., Shlomchik, M., 2001a. The complement system and innate immunity. In: Immunobiology: The Immune System in Health and Disease. fifth ed. Garland Science, New York Available from: https://www.ncbi.nlm.nih.gov/books/NBK27100. Janeway Jr., C.A., Travers, P., Walport, M., Shlomchik, M., 2001b. Immunobiology: The Immune System in Health and Disease, fifth ed. Garland Science, New York, p. 601. Jeannin, P., Jaillon, S., Delneste, Y., 2008. Pattern recognition receptors in the immune response against dying cells. Curr. Opin. Immunol. 20, 530–537.

722 PART

III Epigenetics of Metazoan Evolution

Jerram, S.T., Dang, M.N., Leslie, R.D., 2017. The role of epigenetics in type 1 diabetes. Curr. Diab. Rep. 17, 89. Ji, Y., Resch, W., Corbett, E., Yamane, A., Casellas, R., Schatz, D.G., 2010. The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci. Cell 141, 419–431. Johnson, K., Chaumeil, J., Skok, J.A., 2010. Epigenetic regulation of V(D)J recombination. Essays Biochem. 48, 221–243. Jorgensen, J.L., Reay, P.A., Ehrich, E.W., Davis, M.M., 1992. Molecular components of T-cell recognition. Annu. Rev. Immunol. 10, 835–873. Julsing, J.R., Peters, G.J., 2014. Methylation of DNA repair genes and the efficacy of DNA targeted anticancer treatment. Oncol. Discov. 2. https://doi.org/10.7243/2052-6199-2-3. Kangaspeska, S., Stride, B., M
etivier, R., Polycarpou-Schwarz, M., Ibberson, D., Carmouche, R.P., et al., 2008. Transient cyclical methylation of promoter DNA. Nature 452, 112–115. Kapitonov, V.V., Koonin, E.V., 2015. Evolution of the RAG1-RAG2 locus: both proteins came from the same transposon. Biol. Direct 10, 20. Kastan, M.B., 2008. DNA damage responses: mechanisms and roles in human disease: 2007 G.H.A. Clowes Memorial Award Lecture. Mol. Cancer Res. 6, 517–524. Katayama, Y., Battista, M., Kao, W.M., Hidalgo, A., Peired, A.J., Thomas, S.A., Frenette, P.S., 2006. Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell 124, 407–421. Kaufman, J., 2010. Evolution and immunity. Immunology 130, 459–462. Kawli, T., He, F., Tan, M.-W., 2010. It takes nerves to fight infections: insights on neuro-immune interactions from C. elegans. Dis. Model. Mech. 3, 721–731. Keim, V., 2004. Pankreatitis: Von der chronischen Entz€undung zum Karzinom. Dtsch. Med. Wochenschr. 129, S94–S95. Ketchesin, K.D., Stinnett, G.S., Seasholtz, A.F., 2017. Corticotropin-releasing hormone-binding protein and stress: from invertebrates to humans. Stress 20, 449–464. Key, T.J., 1995. Hormones and cancer in humans. Mutat. Res. 333, 59–67. Key, T.J., Verkasalo, P.K., Banks, E., 2001. Epidemiology of breast cancer. Lancet Oncol. 2, 133–140. Khansari, N., Mahmoudi, M., Shakiba, Y., 2009. Chronic inflammation and oxidative stress as a major cause of age-related diseases and cancer. Pat. Inflamm. Allergy Drug Discov. 3, 73–80. Kim, J.K., Samaranayake, M., Pradhan, S., 2009. Epigenetic mechanisms in mammals. Cell. Mol. Life Sci. 66, 596–612. Kin, N.W., Sanders, V.M., 2006. It takes nerve to tell T and B cells what to do. J. Leukoc. Biol. 79, 1–12. Kitagawa, Y., Ohkura, N., Sakagu, S., 2015. Epigenetic control of thymic Treg-cell development. Eur. J. Immunol. 45, 11–16. Kleer, C.G., Cao, Q., Varambally, S., Shen, R., Ota, I., Tomlins, S.A., 2003. EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc. Natl. Acad. Sci. U. S. A. 100, 11606–11611. Koch, M., Mollenkopf, H.-J., Klemm, U., Meyer, T.F., 2012. Induction of microRNA-155 is TLRand type IV secretion system-dependent in macrophages and inhibits DNA-damage induced apoptosis. Proc. Natl. Acad. Sci. U. S. A. 109, E1153–E1162. Koopman, F.A., Chavan, S.S., Miljko, S., Grazio, S., Sokolovic, S., Schuurman, P.R., et al., 2016. Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis. Proc. Natl. Acad. Sci. U. S. A. 113, 8284–8289. Kronfol, Z., Remick, D.G., 2000. Cytokines and the brain: implications for clinical psychiatry. Am. J. Psychiatry 157, 683–694.

Epigenetics in Health and Disease Chapter

14

723

Kurowska-Stolarska, M., Alivernini, S., Ballantine, L.E., Asquith, D.L., Millar, N.L., Gilchrist, D.S., 2011. MicroRNA-155 as a proinflammatory regulator in clinical and experimental arthritis. Proc. Natl. Acad. Sci. U. S. A. 108, 11193–11198. Kwon, N.-H., Kim, J.-S., Lee, J.-Y., Oh, M.-J., Choi, D.-C., 2008. DNA methylation and the expression of IL-4 and IFN-γ promoter genes in patients with bronchial asthma. J. Clin. Immunol. 28, 139–146. Lahtz, C., Pfeifer, G.P., 2011. Epigenetic changes of DNA repair genes in cancer. J. Mol. Cell Biol. 3, 51–58. Larsen, F., Gundersen, G., Lopez, R., Prydz, H., 1992. CpG islands as gene markers in the human genome. Genomics 13, 1095–1107. Lawrence, T., Gilroy, D.W., 2007. Chronic inflammation: a failure of resolution? Int. J. Exp. Pathol. 88, 85–94. Lechin, F., van der Dijs, B., Orozco, B., Jara, H., Rada, I., Lechin, M.E., Lechin, A.E., 1998. Neuropharmacologic treatment of bronchial asthma with the antidepressant tianeptine: a doubleblind, crossover placebo-controlled study. Clin. Pharmacol. Ther. 64, 223–232. Lee, E.Y., Muller, W.J., 2010. Oncogenes and tumor suppressor genes. Cold Spring Harb. Perspect. Biol. 2, a003236. Lee, G.R., Fields, P.E., Griffin, T.J.IV., Flavell, R.A., 2003. Regulation of the Th2 cytokine locus by a locus control region. Immunity 19, 145–153. Lee, G.R., Kim, S.T., Spilianakis, C.G., Fields, P.E., Flavell, R.A., 2006. T helper cell differentiation: regulation by cis elements and epigenetics. Immunity 24, 369–379. Levenson, J.M., O’Riordan, K.J., Brown, K.D., Trinh, M.A., Molfese, D.L., Sweatt, J.D., 2004. Regulation of histone acetylation during memory formation in the hippocampus. J. Biol. Chem. 279, 40545–40559. Levin-Klein, R., Bergman, Y., 2012. Epigenetics of the immune system. In: Meyers, R.A. (Ed.), Epigenetic Regulation and Epigenomics. Current Topics From the Encyclopedia of Molecular Cell Biology and Molecular Medicine. John Wiley & Sons, p. 897. Li, Q.-J., Chau, J., Ebert, P.J.R., Sylvester, G., Min, H., Liu, G., Braich, R., Manoharan, M., Soutschek, J., Skare, P., Klein, L.O., Davis, M.M., Chen, C.-Z., 2007. miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell 129, 147–161. Li, G., Zan, H., Xu, Z., Casali, P., 2013. Epigenetics of the antibody response. Trends Immunol. 34, 460–470. Lillberg, K., Verkasalo, P.K., Kaprio, J., Teppo, L., Helenius, H., Koskenvuo, M., 2003. Stressful life events and risk of breast cancer in 10,808 women: a cohort study. Am. J. Epidemiol. 157, 415–423. Litvack, M.L., Palaniyar, N., 2010. Review: soluble innate immune pattern-recognition proteins for clearing dying cells and cellular components: implications on exacerbating or resolving inflammation. Innate Immun. 16, 191–200. Liu, V., Dietrich, A., Kasparek, M.S., Benhaqi, P., Schneider, M.R., Schemann, M., Seeliger, H., Kreis, M.E., 2015. Extrinsic intestinal denervation modulates tumor development in the small intestine of ApcMin/+ mice. J. Exp. Clin. Cancer Res. 34, 39. Lorton, D., Bellinger, D.L., 2015. Sympathetic nervous system regulation of Th cells in autoimmunity: beyond Th1 and Th2 cell balance. J. Clin. Cell. Immunol. 6, 356. Lu, Q., Ray, D., Gutsch, D., Richardson, B., 2002. Effect of DNA methylation and chromatin structure on ITGAL expression. Blood 99, 4503–4508. Lubin, F.D., Roth, T.L., Sweatt, J.D., 2008. Epigenetic regulation of BDNF gene transcription in the consolidation of fear memory. J. Neurosci. 28, 10576–10586.

724 PART

III Epigenetics of Metazoan Evolution

Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., Richmond, T.J., 1997. Crystal structure of ˚ resolution. Nature 389, 251–260. the nucleosome core particle at 2.8 A Lyu, K., Wang, Q., Li, Z., Chen, R., Zhu, C., Liu, J., Yan, Z., 2015. Age-dependent survival and selected gene expression in Daphnia magna after short-term exposure to low dissolved oxygen. J. Plankton Res. 37, 66–74. Ma, D.K., Jang, M.-H., Guo, J.U., Kitabatake, Y., Chang, M.-L., Pow-anpongkul, N., 2009. Neuronal activity–induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis. 323, 1074–1077. Macfarlane, C.M., Collier, P., Rahbari, R., Beck, C.R., Wagstaff, J.F., Igoe, S., Moran, J.V., Badge, R.M., 2013. Transduction-specific ATLAS (TS-ATLAS) reveals a cohort of highly active L1 retrotransposons in human populations. Hum. Mutat. 34(7). https://doi.org/ 10.1002/humu.22327. Maes, J., O’Neill, L.P., Cavelier, P., Turner, B.M., Rougeon, F., Goodhardt, M., 2001. Chromatin remodeling at the Ig loci prior to V(D)J recombination. J. Immunol. 167, 866–874. Magnon, C., 2015. Role of the autonomic nervous system in tumorigenesis and metastasis. Mol. Cell. Oncol. 2. Magnon, C., Hall, S.J., Lin, J., Xue, X., Gerber, L., Freedland, S.J., Frenette, P.S., 2013. Autonomic nerve development contributes to prostate cancer progression. Science 341, 1236–1361. Makhathini, K.B., Abboussi, O., Stein, D.J., Mabandla, M.V., Daniels, W.M.U., 2017. Repetitive stress leads to impaired cognitive function that is associated with DNA hypomethylation, reduced BDNF and a dysregulated HPA axis. Int. J. Dev. Neurosci. 60, 63–69. Manoharan, M., Ramachandran, K., Soloway, M.S., Singa, R., 2007. Epigenetic targets in the diagnosis and treatment of prostate cancer. Int. Braz. J. Urol. 33, 11–18. Mantovani, A., Allavena, P., Sica, A., Balkwill, F., 2008. Cancer-related inflammation. Nature 454, 436–444. Manuyakorn, A., Paulus, R., Farrell, J., Dawson, N.A., Tze, S., Cheung-Lau, G., et al., 2010. Cellular histone modification patterns predict prognosis and treatment response in resectable pancreatic adenocarcinoma: results from RTOG 9704. J. Clin. Oncol. 28, 1358–1365. Marelli, G., Sica, A., Vannucci, L., Allavena, P., 2017. Inflammation as target in cancer therapy. Curr. Opin. Pharmacol. 35, 57–65. Marnett, L.J., 2000. Oxyradicals and DNA damage. Carcinogenesis 21, 361–370. Martynow, W., 1930. Verhalten der peripheren Nerven zum Plattenepithelkrebs des Menschen. Virchows Arch. Pathol. Anat. 278, 498–517. Mathews, L.A., Crea, F., Farrar, W.L., 2009. Epigenetic gene regulation in stem cells and correlation to cancer. Differentiation 78, 1–17. McEwen, B.S., Wingfield, J.C., 2003. The concept of allostasis in biology and biomedicine. Horm. Behav. 43, 2–15. McEwen, B., Nasveld, P., Palmer, M., Anderson, R., 2012. Allostatic Load—A Review of Literature. Department of Veterans’ Affairs, Canberra. McGowan, P.O., Sasaki, A., D’Alessio, A.C., Dymov, S., Labont
e, B., Szyf, M., Turecki, G., Meaney, M.J., 2009. Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse. Nat. Neurosci. 12, 342–348. McKlveen, J.M., Myers, B., Herman, J.P., 2015. The medial prefrontal cortex: coordinator of autonomic, neuroendocrine and behavioural responses to stress. J. Neuroendocrinol. 27, 446–456. McMurry, M.T., Krangel, M.S., 2000. A role for histone acetylation in the developmental regulation of VDJ recombination. Science 287, 495–498. McQueen, G., Marshall, J., Perdue, M., Siegel, S., Bienenstock, J., 1989. Pavlovian conditioning of rat mucosal mast cells to secrete cell protease II. Science 243, 83–85.

Epigenetics in Health and Disease Chapter

14

725

M
endez, A., Mendoza, L., 2016. A network model to describe the terminal differentiation of B cells. PLoS Comput. Biol. 12(1). Miller, C.A., Sweatt, J.D., 2007. Covalent modification of DNA regulates memory formation. Neuron 52, 857–869. Mina-Osorio, P., Rosas-Ballina, M., Valdes-Ferrer, S.I., Al-Abed, Y., Tracey, K.J., Diamond, B., 2012. Neural signaling in the spleen controls B-cell responses to blood-borne antigen. Mol. Med. 18, 618–627. Mirotti, L., Castro, J., Costa-Pinto, F.A., Russo, M., 2010. Neural pathways in allergic inflammation. J. Allergy. 2010. Morshead, K.B., Ciccone, D.N., Taverna, S.D., Allis, C.D., Oettinger, M.A., 2003. Antigen receptor loci poised for V(D)J rearrangement are broadly associated with BRG1 and flanked by peaks of histone H3 dimethylated at lysine. Proc. Natl. Acad. Sci. U. S. A. 100, 11577–11582. Mueller, D.L., 2010. Mechanisms maintaining peripheral tolerance. Nat. Immunol. 11, 21–27. Nathan, C., 2002. Points of control in inflammation. Nature 420, 846–852. Nesse, R.M., Young, E.A., 2000. Evolutionary origins and functions of the stress response peptide. In: Encyclopedia of Stress. vol. 2. Acaemic Press, San Diego, San Francisco, New York, Boston, London, Sydney, Toronto, pp. 79–84. Novotny, G.E., 1988. Ultrastructural analysis of lymph node innervation in the rat. Acta Anat. (Basel) 133, 57–61. Novotny, G.E., Kliche, K.O., 1986. Innervation of lymph nodes: a combined silver impregnation and electron-microscopic study. Acta Anat. (Basel) 127, 243–248. Nyiraneza, C., Sempoux, C., Detry, R., Dahan, K., 2012. Hypermethylation of the 50 CpG island of the p14ARF flanking exon 1β in human colorectal cancer displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression. Clin. Epigenetics 4 (1), 9. O’Connell, R.M., Taganov, K.D., Boldin, M.P., Cheng, G., Baltimore, D., 2007. MicroRNA-155 is induced during the macrophage inflammatory response. Proc. Natl. Acad. Sci. U. S. A. 104, 1604–1609. O’Hagan, H.M., Mohammad, H.P., Baylin, S.B., 2008. Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island. PLoS Genet. 4(8). Olsson, A.H., Volkov, P., Bacos, K., Dayeh, T., Hall, E., Nilsson, E.A., et al., 2014. Genome-wide associations between genetic and epigenetic variation influence mRNA expression and insulin secretion in human pancreatic islets. PLoS Genet. 10(11). Ottaviani, E., Kletsas, D., Caselgrandi, E., 1998. The CRH-ACTH-biogenic amine axis in invertebrate immunocytes activated by PDGF and TGF-beta. FEBS Lett. 427, 255–258. Palm, D., Entschladen, F., 2007. Neoneurogenesis and the neuro-neoplastic synapse. In: Z€anker, K.S., Entschladen, F. (Eds.), Neuronal Activity in Tumor Tissue. In: Prog. Exp. Tumor Res., vol. 39. Karger, Basel, pp. 91–98. Pancer, Z., Cooper, M.D., 2006. The evolution of adaptive immunity. Annu. Rev. Immunol. 24, 497–518. Pancer, Z., Amemiy, C.T., Ehrhardt, G.R.A., Ceitlin, J., Gartland, G.L., Cooper, M.D., 2004. Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey. Nature 430, 174–180. Park, Y.S., Jin, M.Y., Kim, Y.J., Yook, J.H., Kim, B.S., Jang, S.J., 2008. The global histone modification pattern correlates with cancer recurrence and overall survival in gastric adenocarcinoma. Ann. Surg. Oncol. 15, 1968–1976. Patel, T.N., Roy, S., Ravi, R., 2017. Gastric cancer and related epigenetic alterations. Ecancermedicalscience 11, 714.

726 PART

III Epigenetics of Metazoan Evolution

Pauwels, K., Stoks, R., DeMeester, L., 2005. Coping with predator stress: interclonal differences in induction of heat-shock proteins in the water flea Daphnia magna. 18, 867–872. Pawlowski, A., Weddell, G., 1967. Induction of tumours in denervated skin. Nature 213, 1234–1236. Pen˜a, C.J., Neugut, Y.D., Champagne, F.A., 2013. Developmental timing of the effects of maternal care on gene expression and epigenetic regulation of hormone receptor levels in female rats. Neuroendocrinology 154, 4340–4351. Perez, S.D., Silva, D., Millar, A.B., Molinaro, C.A., Carter, J., Bassett, K., et al., 2009. Sympathetic innervation of the spleen in male Brown Norway rats: a longitudinal aging study. Brain Res. 1302, 106–117. Peterson, S.C., Eberl, M., Vagnozzi, A.N., Belkadi, A., Veniaminova, N.A., Verhaegen, M., Bichakjian, C.K., Ward, N.L., Dlugosz, A.A., Wong, S.Y., 2015. Basal cell carcinoma preferentially arises from stem cells within hair follicle and mechanosensory niches. Cell Stem Cell 16, 400–412. Petronis, A., 2010. Epigenetics as a unifying principle in the aetiology of complex traits and diseases. Nature 465, 721–727. Pinho, A.V., Chantrill, L., Rooman, I., 2014. Chronic pancreatitis: a path to pancreatic cancer. Cancer Lett. 345, 203–209. Plitas, G., Rudensky, A.Y., 2016. Regulatory T cells: differentiation and function. Cancer Immunol. Res. 4, 721–725. Polli-Lopes, A.C., Zucoloto, S., Cunha, F.Q., Figueiredo, L.A.S., Garcia, S.B., 2003. Myenteric denervation reduces the incidence of gastric tumors in rats. Cancer Lett. 190, 45–50. Powe, D.G., Voss, M.J., Zanker, K.S., Habashy, H.O., Green, A.R., Ellis, I.O., Entschladen, F., 2010. Beta-blocker drug therapy reduces secondary cancer formation in breast cancer and improves cancer specific survival. Oncotarget 1, 628–638. Qu, Y., Dang, S., Hou, P., 2013. Gene methylation in gastric cancer. Clin. Chim. Acta 424, 53–65. Quintero-Ronderos, P., Montoya-Ortiz, G., 2012. Epigenetics and autoimmune diseases. Autoimmune Dis. 2012. Riley, V., 1975. Mouse mammary tumors: alteration of incidence as apparent function of stress. Science 189, 465–467. Rodriguez-Paredes, M., Esteller, M., 2011. Cancer epigenetics reaches mainstream oncology. Nat. Med. 17, 330–339. Romagnani, S., 1999. Th1/Th2 cells. Inflamm. Bowel Dis. 5, 285–294. Romeo, H.E., Colombo, L.L., Esquifino, A.I., Rosenstein, R.E., Chuluyan, H.E., Cardinali, D.P., 1991. Slower growth of tumours in sympathetically denervated murine skin. J. Auton. Nerv. Syst. 32, 159–164. Rossato, M., Curtale, G., Tamassia, N., Castellucci, M., Mori, L., Gasperini, S., et al., 2012. IL-10– induced microRNA-187 negatively regulates TNF-α, IL-6, and IL-12p40 production in TLR4stimulated monocytes. Proc. Natl. Acad. Sci. U. S. A. 109 (45), E3101–E3110. Rui, J., Deng, S., Lebastchi, J., Clark, P.L., Usmani-Brown, S., Herold, K.C., 2016. Methylation of insulin DNA in response to proinflammatory cytokines during the progression of autoimmune diabetes in NOD mice. Diabetologia 59, 1021–1029. Sakaguchi, S., Wing, K., Miyara, M., 2007. Regulatory T cells? A brief history and perspective. Eur. J. Immunol. 37, S116–S123. Saloman, J.L., Albers, K.M., Li, D., Hartman, D.J., Crawford, H.C., Muha, E.A., Rhim, A.D., Davis, B.M., 2016. Ablation of sensory neurons in a genetic model of pancreatic ductal adenocarcinoma slows initiation and progression of cancer. Proc. Natl. Acad. Sci. U. S. A. 113, 3078–3083.

Epigenetics in Health and Disease Chapter

14

727

Sanders, V.M., 2012. The beta2-adrenergic receptor on T and B lymphocytes: do we understand it yet? Brain Behav. Immun. 26, 195–200. Scharrer, B., Lochhead, M.S., 1950. Tumors in the invertebrates. Cancer Res. 10, 403–419. Schatz, D.G., Oettinger, M.A., Baltimore, D., 1989. The V(D)J recombination activating gene, RAG-1. Cell 59, 1035–1048. Scott, G.D., Fryer, A.D., 2012. Role of parasympathetic nerves and muscarinic receptors in allergy and asthma. In: Bienenstock, J. (Ed.), Allergy and the Nervous System. Karger Medical and Scientific Publishers, p. 55. Seligson, D.B., Horvath, S., Shi, T., Yu, H., Tze, S., Grunstein, M., Kurdistani, S.K., 2005. Global histone modification patterns predict risk of prostate cancer recurrence. Nature 435, 1262–1266. Sell, S., 2004. Stem cell origin of cancer and differentiation therapy. Crit. Rev. Oncol. Hematol. 51, 1–28. Sharma, S., Gurudutta, G., 2016. Epigenetic regulation of hematopoietic stem cells. Int. J. Stem Cells 9, 36–43. Shi, Z., Greene, W.K., Nicholls, P.K., Hu, D., Tirnitz-Parker, J.E.E., Yuan, Q., Yin, C., Ma, B., 2017. Immunofluorescent characterization of non-myelinating Schwann cells and their interactions with immune cells in mouse mesenteric lymph node. Eur. J. Histochem. 61, 193–203. Shipitsin, M., Polyak, K., 2008. The cancer stem cell hypothesis: in search of definitions, markers, and relevance. Lab. Investig. 88, 459–463. Shogren-Knaak, M., Ishii, H., Sun, J.M., Pazin, M.J., Davie, J.R., Peterson, C.L., 2006. Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science 311, 844–847. Shu, D.-G., Luo, H.-L., Conway Morris, S., Zhang, X.-L., Hu, S.-X., Chen, L., Han, J., Zhu, M., Li, Y., Che, L.-Z., 1999. Lower Cambrian vertebrates from south China. Nature 402, 42–46. Siegel, S., Kreutzer, R., 1997. Pavlovian conditioning and multiple chemical sensitivity. Environ. Health Perspect. 105, 521–526. Singh, S.K., Gellert, M., 2015. Role of RAG1 autoubiquitination in V(D)J recombination. Proc. Natl. Acad. Sci. U. S. A. 112, 8579–8583. Sipahi, L., Uddin, M., Hou, Z.-C., Aiello, A.E., Koenen, K.C., Galea, S., Wildman, D.E., 2014. Ancient evolutionary origins of epigenetic regulation associated with posttraumatic stress disorder. Front. Hum. Neurosci. 8, 1–10. Sirisinha, S., 2014. Evolutionary insights into the origin of innate and adaptive immune systems: different shades of grey. Asian Pac. J. Allergy Immunol. 32, 3–15. Smith, L.C., Clow, L.A., Terwilliger, D.P., 2001. The ancestral complement system in sea urchins. Immunol. Rev. 180, 16–34. Smith, C.M., Gafken, P.R., Zhang, Z., Gottschling, D.E., Smith, J.B., Smith, D.L., 2003. Mass spectrometric quantification of acetylation at specific lysines within the amino-terminal tail of histone H4. Anal. Biochem. 316, 23–33. Steinberg, B.E., Sundman, E., Terrando, N., Eriksson, L.I., Olofsson, P.S., 2016. Neural control of inflammation: implications for perioperative and critical care. Anesthesiology 124, 1174–1189. Stephens, P.J., McBride, D.J., Lin, M.L., Varela, I., Pleasance, E.D., Simpson, J.T., et al., 2009. Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature 462, 1005–1010. Sternberg, E.M., 2006. Neural regulation of innate immunity: a coordinated nonspecific host response to pathogens. Nat. Rev. Immunol. 6, 318–328. Straub, R.H., 2004. Complexity of the bi-directional neuroimmune junction in the spleen. Trends Pharmacol. Sci. 25, 640–646.

728 PART

III Epigenetics of Metazoan Evolution

Str€ omvall, K., Thyssel, E., Bergstrom, S.H., Bergh, A., 2017. Aggressive rat prostate tumors reprogram the benign parts of the prostate and regional lymph nodes prior to metastasis. PLoS ONE 12 (5), e0176679. Styer, K.L., Singh, V., Macosko, E., Steele, S.E., Bargmann, C.I., Aballay, A., 2008. Innate immunity in Caenorhabditis elegans is regulated by neurons expressing NPR-1/GPCR. Science 322, 460–464. Sun, J., Singh, V., Kajino-Sakamoto, R., Aballay, A., 2011. Neuronal GPCR controls innate immunity by regulating non-canonical unfolded protein response genes. Science 332, 729–732. Sun, Y.-W., Chen, K.-M., Imamura Kawasawa, Y., Salzberg, A.C., Cooper, T.K., Caruso, C., Aliaga, C., Zhu, J., Gowda, K., Amin, S., El-Bayoumy, K., 2017. Hypomethylated Fgf3 is a potential biomarker for early detection of oral cancer in mice treated with the tobacco carcinogen dibenzo[def,p]chrysene. PLoS ONE. 12(10). Sundman, E., Olofsson, P.S., 2014. Neural control of the immune system. Adv. Physiol. Educ. 38, 135–139. Szanya, V., Ermann, J., Taylor, C., Holness, C., Fathman, C.G., 2002. The subpopulation of CD4+ CD25+ splenocytes that delays adoptive transfer of diabetes expresses L-selectin and high levels of CCR7. J. Immunol. 169, 2461–2465. Takai, D., Jones, P.A., 2002. Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc. Natl. Acad. Sci. U. S. A. 99, 3740–3745. Tamura, G., 2004. Promoter methylation status of tumor suppressor and tumor-related genes in neoplastic and non-neoplastic gastric epithelia. Histol. Histopathol. 19, 221–228. Tang, Y., Luo, X., Cui, H., Ni, X., Yuan, M., Guo, Y., et al., 2009. MicroRNA-146A contributes to abnormal activation of the type I interferon pathway in human lupus by targeting the key signaling proteins. Arthritis Rheum. 60, 1065–1075. Teng, G., Hakimpour, P., Landgraf, P., Rice, A., Tuschl, T., Casellas, R., Papavasiliou, F.N., 2008. MicroRNA-155 is a negative regulator of activation-induced cytidine deaminase. Immunity 28, 621–629. Toh, T.B., Lim, J.J., Chow, E.K.-H., 2017. Epigenetics in cancer stem cells. Mol. Cancer 16, 29. Tracey, K.J., 2007. Physiology and immunology of the cholinergic antiinflammatory pathway. J. Clin. Invest. 117, 289–296. Tracey, K.J., 2010. Understanding immunity requires more than immunology. Nat. Immunol. 11, 561–564. Tracey, K.J., 2012. Immune cells exploit a neural circuit to enter the CNS. Cell 148, 392–394. Trinchieri, G., 2011. Inflammation in cancer: a therapeutic target? Oncology 25, 418–420Online at cancernetwork. Turecki, G., Meaney, M., 2016. Effects of the social environment and stress on glucocorticoid receptor gene methylation: a systematic review. Biol. Psychiatry 79, 87–96. Uddin, M., Aiello, A.E., Wildman, D.E., Koenen, K.C., Pawelec, G., de los Santos, R., Goldmann, E., Galea, S., 2010. Epigenetic and immune function profiles associated with posttraumatic stress disorder. Proc. Natl. Acad. Sci. U. S. A. 107, 9470–9475. Ushijima, T., Watanabe, N., Okochi, E., Kaneda, A., Sugimura, T., Miyamoto, K., 2003. Fidelity of the methylation pattern and its variation in the genome. Genome Res. 13, 868–874. Valapour, M., Guo, J., Schroeder, J.T., Keen, J., Cianferoni, A., Casolaro, V., Georas, S.N., 2002. Histone deacetylation inhibits IL4gene expression in T cells. J. Allergy Clin. Immunol. 109, 238–245. van de Pavert, S.A., Olivier, B.J., Goverse, G., Vondenhoff, M.F., Greuter, M., Beke, P., et al., 2009. Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation. Nat. Immunol. 10, 1193–1199.

Epigenetics in Health and Disease Chapter

14

729

Van Den Broeck, A., Brambilla, E., Moro-Sibilot, D., Lantuejoul, S., Brambilla, C., Eymin, B., Khochbin, S., Gazzeri, S., 2008. Loss of histone H4K20 trimethylation occurs in preneoplasia and influences prognosis of non-small cell lung cancer. Clin. Cancer Res. 14, 7237–7245. Van Maanen, M.A., Stoof, S.P., Van Der Zanden, E.P., De Jonge, W.J., Janssen, R.A., Fischer, D.F., Vandeghinste, N., Brys, R., Vervoordeldonk, M.J., Tak, P.P., 2009. The α7 nicotinic acetylcholine receptor on fibroblast-like synoviocytes and in synovial tissue from rheumatoid arthritis patients: a possible role for a key neurotransmitter in synovial inflammation. Arthritis Rheum. 60, 1272–1281. van Niekerk, G., Engelbrecht, A.-M., 2015. On the evolutionary origin of the adaptive immune system—the adipocyte hypothesis. Immunol. Lett. 164, 81–87. Vaz, M., Hwang, S.Y., Kagiampakis, I., Phallen, J., Patil, A., O’Hagan, H.M., Murphy, L., Zahnow, C.A., Gabrielson, E., Velculescu, V.E., Easwaran, H.P., Baylin, S.B., 2017. Chronic cigarette smoke-induced epigenomic changes precede sensitization of bronchial epithelial cells to single-step transformation by KRAS mutations. Cancer Cell 32, 360–376. Veiga-Fernandes, H., Mucida, D., 2016. Neuro-immune interactions at barrier surfaces. Cell 165, 801–811. Verburg-van Kemenade, B.M.L., Cohen, N., Chadzinska, M., 2017. Neuroendocrine-immune interaction: evolutionarily conserved mechanisms that maintain allostasis in an ever-changing environment. Dev. Comp. Immunol. 66, 2–23. Vogelstein, B., Papadopoulos, N., Velculescu, V.E., Zhou, S., Diaz Jr., L.A., Kinzler, K.W., 2013. Cancer genome landscapes. Science 339, 1546–1558. Volkmar, M., Dedeurwaerder, S., Cunha, D.A., Ndlovu, M.N., Defrance, M., Deplus, R., et al., 2012. DNA methylation profiling identifies epigenetic dysregulation in pancreatic islets from type 2 diabetic patients. EMBO J. 31, 1331–1619. Wada, T.T., Araki, Y., Sato, K., Aizaki, Y., Yokota, K., Kim, Y.T., et al., 2014. Aberrant histone acetylation contributes to elevated interleukin-6 production in rheumatoid arthritis synovial fibroblasts. Biochem. Biophys. Res. Commun. 444, 682–686. Wang, H., Xu, M., Cui, X., Liu, Y., Zhang, Y., Sui, Y., et al., 2016. Aberrant expression of the candidate tumor suppressor gene DAL-1 due to hypermethylation in gastric cancer. Sci. Rep. 6. Wang, Z., Wang, Z., Lu, Q., 2017a. Epigenetic alterations in cellular immunity: new insights into autoimmune diseases. Cell. Physiol. Biochem. 41, 645–660. Wang, W., Feng, J., Ji, C., Mu, X., Ma, Q., Fan, Y., Chen, C., Gao, C., Ma, X.-C., Zhu, F., 2017b. Increased methylation of glucocorticoid receptor gene promoter 1F in peripheral blood of patients with generalized anxiety disorder. J. Psychiatr. Res. 91, 18–25. Watkins, L.R., Maier, S.F., Goehler, L.E., 1995. Cytokine-to-brain communication: a review & analysis of alternative mechanisms. Life Sci. 57, 1011–1026. Webster, M.J., Knable, M.B., O’Grady, J., Orthmann, J., Weickert, C.S., 2002. Regional specificity of brain glucocorticoid receptor mRNA alterations in subjects with schizophrenia and mood disorders. Mol. Psychiatry 7, 985–994924. Weaver, I.C.G., Korgan, A.C., Lee, K., Wheeler, R.V., Hundert, A.S., Goguen, D., 2017. Stress and the emerging roles of chromatin remodeling in signal integration and stable transmission of reversible phenotypes. Front. Behav. Neurosci. https://doi.org/10.3389/fnbeh.2017.00041. Weinberg, R.A., 2014. Coming full circle-from endless complexity to simplicity and back again. Cell 157, 267–271. Widdicombe, J.G., 2003. Overview of neural pathways in allergy and asthma. Pulm. Pharmacol. Ther. 16, 23–30. Wing, K., Sakaguchi, S., 2010. Regulatory T cells exert checks and balances on self-tolerance and autoimmunity. Nat. Immunol. 11, 7–13.

730 PART

III Epigenetics of Metazoan Evolution

Wong, A.H., Gottesman, I.I., Petronis, A., 2005. Phenotypic differences in genetically identical organisms: the epigenetic perspective. Hum. Mol. Genet. 14, R11–R18. Wu, X.Z., 2008. Origin of cancer stem cells: the role of self-renewal and differentiation. Ann. Surg. Oncol. 15, 407–414. Wu, H., Sun, Y.E., 2009. Reversing DNA methylation: new insights from neuronal activity–induced Gadd45b in adult neurogenesis. Sci. Signal. 2, pe17. W€ ulfing, C., G€ unther, H.S., 2015. Dendritic cells and macrophages neurally hard-wired in the lymph node. Sci. Rep. 5. Xing, Y., Hogquist, K.A., 2012. T-cell tolerance: central and peripheral. Cold Spring Harb. Perspect. Biol. 4 (6). pii: a006957. Yamazaki, S., Ema, H., Karlsson, G., Yamaguchi, T., Miyoshi, H., Shioda, S., Taketo, M.M., Karlsson, S., Iwama, A., Nakauchi, H., 2011. Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche. Cell 147, 1146–1158. Ydens, E., Lornet, G., Smits, V., Goethals, S., Timmerman, V., Janssens, S., 2013. The neuroinflammatory role of Schwann cells in disease. Neurobiol. Dis. 55, 95–103. Yu, D.H., Waterland, R.A., Zhang, P., Schady, D., Chen, M.H., Guan, Y., et al., 2014. Targeted p16Ink4a epimutation causes tumorigenesis and reduces survival in mice. J. Clin. Invest. 124, 3708–3712. Zan, H., Casali, P., 2013. Regulation of Aicda expression and AID activity. Autoimmunity 46, 81–99. Zeng, W.-P., 2013. ‘All things considered’: transcriptional regulation of T helper type 2 cell differentiation from precursor to effector activation. Immunology 140, 31–38. Zhang, X., Zhang, Y., 2009. Neural-immune communication in Caenorhabditis elegans. Cell Host Microbe 5, 425–429. Zhang, T.Y., Labont
e, B., Wen, X.L., Turecki, G., Meaney, M.J., 2013. Epigenetic mechanisms for the early environmental regulation of hippocampal glucocorticoid receptor gene expression in rodents and humans. Neuropsychopharmacology 38, 111–123. Zhao, C.-M., Hayakawa, Y., Kodama, Y., Muthupalani, S., Westphalen, C.B., Andersen, G.T., et al., 2014. Denervation suppresses gastric tumorigenesis. Sci. Transl. Med. 6 (250), 250ra115. Zhu, J., Sammons, M.A., Donahue, G., Dou, Z., Vedadi, M., Getlik, M., 2015. Gain-of-function p53 mutants co-opt chromatin pathways to drive cancer growth. Nature 525, 206–211. Zhu, T., Liang, C., Li, D., Tian, M., Liu, S., Gao, G., Guan, J.-S., 2016. Histone methyltransferase Ash1L mediates activity-dependent repression of neurexin-1α. Sci. Rep. 6, 26597.

FURTHER READING Barker, D.J., Bull, A.R., Osmond, C., Simmonds, S.J., 1990. Fetal and placental size and risk of hypertension in adult life. BMJ 301, 259–262. Beck, C.R., Garcia-Perez, J.L., Badge, R.M., Moran, J.V., 2011. LINE-1 elements in structural variation and disease. Annu. Rev. Genomics Hum. Genet. 12, 187–215. Boilly, B., Faulkner, S., Jobling, P., Hondermarck, H., 2017. Nerve dependence: from regeneration to cancer. Cancer Cell 31, 342–354. Cooper, M., 2010. 99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: evolution of adaptive immunity in vertebrates. Clin. Exp. Immunol. 160, 1365–2249. De Silva, N.S., Klein, U., 2015. Dynamics of B cells in germinal centres. Nat. Rev. Immunol. 15, 137–148. Dustin, M.L., 2012. Signaling at neuro/immune synapses. J. Clin. Invest. 122, 1149–1155.

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Estampador, A.C., Franks, P.W., 2014. Genetic and epigenetic catalysts in early-life programming of adult cardiometabolic disorders. Diabetes Metab. Syndr. Obes. 7, 575–586. Flajnik, M.F., Kasahara, M., 2010. Origin and evolution of the adaptive immune system: genetic events and selective pressures. Nat. Rev. Genet. 11, 47–59. F€ ullgrabe, J., Hajji, N., Joseph, B., 2010. Cracking the death code: apoptosis-related histone modifications. Cell Death Differ. 17, 1238–1243. Gimenez, J.L.G., Carbonell, N.E., Mateo, C.R., Lo´pez, E.G., Palacios, L., Chova, L.P., Berenguer, E., Garzo, C.G., Pallardo´, F.V., Blanquer, J., 2016. Epigenetics as the driving force in long-term immunosuppression. J. Clin. Epigenet. 2, 2. Heijmans, B.T., Tobi, E.W., Stein, A.D., Putter, H., Blauw, G.J., Susser, E.S., et al., 2008. Persistent epigenetic differences associated with prenatal exposure to famine in humans. Proc. Natl. Acad. Sci. U. S. A. 105, 17046–17049. Kagawa, N., Mugiya, Y., 2002. Brain Hsp70 mRNA expression is linked with plasma cortisol levels in goldfish (Carassius auratus) exposed to a potential predator. Zool. Sci. 19, 735–740. Kanwal, R., Gupta, S., 2012. Epigenetic modifications in cancer. Clin. Genet. 81, 303–311. Kistemaker, L.E.M., 2015. Acetylcholine beyond bronchoconstriction A regulator of inflammation and remodeling. PhD Dissert. Univeristy of Groningen, p. 10. Mukouyama, Y.-S., Gerber, H.-P., Ferrara, N., Gu, C., Anderson, D.J., 2005. Peripheral nervederived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback. Development 132, 941–952. Nestler, E.J., 2016. Transgenerational epigenetic contributions to stress responses: fact or fiction? PLoS Biol. 14(3). Ng, S.-F., Lin, R.C.Y., Laybutt, D.R., Barres, R., Owens, J.A., Morris, M.J., 2010. Chronic high-fat diet in fathers programs β-cell dysfunction in female rat offspring. Nature 467, 963–967. Palm, N.W., Rosenstein, R.K., Medzhitov, R., 2012. Allergic host defenses. Nature 484, 465–472. Pandya, P.H., Murray, M.E., Pollok, K.E., Renbarger, J.L., 2016. The immune system in cancer pathogenesis: potential therapeutic approaches. J Immunol Res. Qi, Q., Liu, Y., Cheng, Y., Glanville, J., Zhang, D., Lee, J.-Y., Olshen, R.A., Weyand, C.M., Boyd, S.D., Goronzy, J.J., 2014. Diversity and clonal selection in the human T-cell repertoire. Proc. Natl. Acad. Sci. U. S. A. 111, 13139–13144. Ravelli, A.C., van der Meulen, J.H., Michels, R.P.J., Osmond, C., Barker, D.J.P., Hales, C.A., Bleker, O., 1998. Glucose tolerance in adults after prenatal exposure to famine. Lancet 351, 173–177. Reiner, S.L., 2005. Epigenetic control in the immune response. Hum. Mol. Genet. 14 (Suppl. 1), R41–R46. Stirzaker, C., Millar, D.S., Paul, C.L., Warnecke, P.M., Harrison, J., Vincent, P.C., Frommer, M., Clark, S.J., Millar, D.S., Paul, C.L., Warnecke, P.M., Harrison, J., Vincent, P.C., Frommer, M., Clark, S.J., 1997. Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors. Cancer Res. 57 (11), 2229–2237. Swenne, I., Crace, C.J., Milner, D.G., 1987. Persistent impairment of insulin secretory response to glucose in adult rats after limited period of protein-calorie malnutrition early in life. Diabetes 36, 454–458. Torpy, D.J., Papanicolaou, D.A., Lotsikas, A.J., Wilder, R.L., Chrousos, G.P., Pillemer, S.R., 2000. Responses of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis to interleukin-6. A pilot study in fibromyalgia. Arthritis Rheum. 43, 872–880. Tykocinski, L.O., Hajkova, P., Chang, H.D., Stamm, T., Sozeri, O., Lohning, M., et al., 2005. A critical control element for interleukin-4 memory expres-sion in T helper lymphocytes. J. Biol. Chem. 280, 28177–28185.