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Estrogen-induced neuroimmunomodulation as facilitator of and barrier to reproductive aging in brain and lymphoid organs
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Estrogen-induced neuroimmunomodulation as facilitator of and barrier to reproductive aging in brain and lymphoid organs ⁎
Srinivasan ThyagaRajan , Lalgi Hima, Uday P. Pratap, Hannah P. Priyanka, Ramasamy Vasantharekha Integrative Medicine Laboratory, Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur 603 203, Tamil Nadu, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Noradrenergic Immunity Neuroprotection Neurodegeneration Inflammation
Reproductive aging in females is marked by alterations in gonadal hormones, estrogen and progesterone, that facilitate cessation of reproductive cycles and onset of female-specific diseases such as autoimmune and neurodegenerative diseases, hormone-dependent cancers, and osteoporosis. Bidirectional communication between the three homeostatic systems, nervous system, endocrine system, and immune system, is essential for the maintenance of health and any dysfunction in the cross-talk promotes the development of diseases and cancer. The pleiotropic effects of estrogen on neural-immune interactions may promote either neuroprotection or inflammatory conditions depending on the site of action, dose and duration of treatment, type of estrogen receptors and its influence on intracellular signaling pathways, etc. Our studies involving treatment of early middle-aged female rats with low and high doses of estrogen and examining the brain areas, thymus, spleen, and lymph nodes revealed that estrogen-induced changes in neural-immune interactions are markedly affected in thymus followed by spleen and lymph nodes while it confers neuroprotection in the brain areas. These alterations are determined by antioxidant enzyme status, growth factors, intracellular signaling pathways involved in cell survival and inflammation, and metabolic enzymes and thus, may regulate the various stages in female reproductive aging. It is imperative that detailed longitudinal studies are carried out to understand the mechanisms of neuroendocrine-immune interactions in reproductive aging to facilitate healthy aging and for the development of better treatment strategies for female-specific diseases.
1. Introduction Aging is characterized by cessation of reproductive cycles in females, alteration in hormonal profiles, and increased incidence of cancers, osteoporosis, neurodegenerative and autoimmune diseases (Meites and Quadri, 1987; Meites, 1988; ThyagaRajan and Priyanka, 2011). The cross-talk between the nervous system, endocrine system, and immune system contributes to the maintenance of homeostasis and normal cellular functions and thus, promote health (Meites, 1988; Ader et al., 2001). It is well established that hypothalamic neuroendocrine system through the release of neurotransmitters and neuropeptides, and releasing hormones regulate hormonal secretion from the pituitary and target endocrine glands to influence various physiological functions including immunity (Meites, 1988; Downs and Wise, 2009). The products of the immune system, cytokines and chemokines, also cross the blood-brain barrier to control neural functions in the brain and thus, regulate physiological processes (Banks, 2015). In addition to the neuroendocrine system, immunity is modulated by the release of
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norepinephrine (NE) and neuropeptides from the sympathetic noradrenergic (NA) and peptidergic nerve fibers, respectively, that innervate the primary (bone marrow and thymus) and secondary (spleen and lymph nodes) lymphoid organs (Bellinger et al., 2008). In general, several studies have reported that age-associated deficiencies in the bidirectional communication between the neuroendocrine system and immune system contribute to the development of diseases and cancer in both rodents and humans (Meites, 1988; Ader et al., 2001; Bellinger et al., 2008; ThyagaRajan and Priyanka, 2011). In contrast to males, aging in females is set apart by distinct changes in the release of hormones, reproductive cyclicity, and increased risks of developing disease pathologies specific to females such as autoimmune diseases, hormone-dependent cancers, and osteoporosis. These crucial changes in hormonal secretion and reproductive cycles occur during middle age in both rodents and women as they transition into acyclicity and menopause, respectively (Randolph et al., 2004; Downs and Wise, 2009; Kermath and Gore, 2012). Among the several hypothalamic, pituitary, and gonadal hormones, estrogen (17β-estradiol; E2) has drawn
Corresponding author. E-mail address: [email protected] (S. ThyagaRajan).
https://doi.org/10.1016/j.jchemneu.2018.02.008 Received 18 August 2017; Received in revised form 22 January 2018; Accepted 20 February 2018 0891-0618/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: ThyagaRajan, S., Journal of Chemical Neuroanatomy (2018), https://doi.org/10.1016/j.jchemneu.2018.02.008
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glutamate and increased activation of GABA(A) receptors in the medial preoptic area may contribute to dampening of LH-surge in middle-aged rats (Neal-Perry et al., 2008). Thus, key changes in population and activity of hypothalamic neurons during middle age may render these neurons less responsive to the gonadal hormones and facilitate further modifications in hormonal secretion and cyclicity during reproductive aging.
primary attention because of its multiple physiological and pathological effects on the cells influenced by its receptor status and function (Burns and Korach, 2012). A number of studies involving in vitro cell lines, in vivo animals, and humans demonstrated that E2 treatment conferred neuroprotection and hence, was a therapeutic option for the treatment of neurodegenerative diseases (Wise et al., 2005; Simpkins et al., 2012). However, treatment of postmenopausal women with estrogen alone or in combination with progesterone provided contradictory results and demonstrated that there was an enhanced risk of stroke and cancer while reducing the incidence of fractures and colorectal cancer in these women (Rossouw et al., 2002; Anderson et al., 2004). Therefore, it is crucial that we understand the physiological role of continued exposure of E2 during reproductive cycles in early life on cellular and molecular mechanisms involving the three homeostatic systems to determine whether it is beneficial or detrimental to women’s health as they transition into various stages of reproductive aging. In this review, we discuss the role of E2 in influencing neural-immune interactions in the few areas of the brain (frontal cortex, striatum, medial basal hypothalamus, and hippocampus)and lymphoid organs (thymus, spleen, and lymph nodes) of early middle-aged rats involving compensatory mechanisms (antioxidant enzymes and growth factors) and signaling molecules and transcription factors (ERK, CREB, Akt, mTOR, and NF-κB) to understand its role in the reproductive aging process (Kale et al., 2014; Pratap et al., 2015; Pratap et al., 2016; Ravichandran et al., 2017).
2.2. Immunosenescence in middle-aged rats Adaptive immune responses markedly decline with advancing age while there is an enhanced pro-inflammatory state due to defects in innate immune responses resulting in the development of diseases and cancer. The cells of the innate immune system have age-related impaired signaling through Toll-like receptor and nucleotide-binding oligomerization domain-like receptors and thus, have compromised phagocytic and chemotactic functions (Shaw et al., 2011). Subsequently, the involvement of NF-κB activation and production of pro-inflammatory cytokines create an inflammatory environment that hastens the pathogenesis of diseases and cancer during the aging process (Franceschi et al., 2000). The decline in adaptive immunity is due to age-associated thymic involution that results in a decrease in naïve T cells paralleling the loss of diversity in T cell receptors repertoire, accumulation of dysfunctional CD8+ memory T cells, and decrease in CD4+/CD8+ T cells. (Alonso-Fernández and De la Fuente, 2011). Also, subsets of T cells belonging to Th1, Th2, Th17, and Treg show significant age-associated functional alterations characterized by an increase in the number of Th2 and Treg cells and changes in intracellular signaling pathways that further compromise the ability of immune system to ward off antigenic challenges or to provide better responses to vaccines (Jagger et al., 2014). Longitudinal studies demonstrating changes in innate and acquired immunity during aging especially, in middle-aged female rats, have not been attempted and thus, it is difficult to understand the modulation by gonadal hormones on immunity. We have provided evidence for a decline in natural killer (NK) cell activity, and Concanavalin (Con A)-induced T cell proliferation and IL-2 production in early middle-aged female rats (ThyagaRajan and Priyanka, 2011). In support of these findings, immune responses such as Con A-induced splenocyte proliferation, IL-2 production, and cytolytic activity declined with advancing age in male F344 rats (Nasrullah and Mazzeo, 1992).
2. Neuroendocrine control of reproductive aging and immunosenescence 2.1. Alterations in hypothalamo-pituitary-gonadal axis hormones in middleaged rats In female rats, regular estrous cycles occur every 4 to 5 days in young (2- to 8-month-old) which is followed by irregular cycles in early middle-aged rats (8- to 9-month-old) characterized by extended days of estrus stage. These stages in young rats are marked by fluctuation in hypothalamic, pituitary, and gonadal hormones and subsequently, the hormonal profiles are altered during middle age characterized by prolonged E2 secretion and delayed ovulation. When the rats are 13 to 14 months of age, they transition into anovulatory stage that is characterized by persistent/constant estrus with intermittent ovulatory activity at irregular intervals. Ultimately, the rats have pseudopregnancy stage followed by persistent anestrus state (Meites and Quadri, 1987; Meites, 1988). These anovulatory stages in old rats are also marked by distinct changes in hormonal secretion from hypothalamus, pituitary, and ovaries. In persistent estrus rats, E2 levels are similar to diestrus day 2 of young rats with lower levels of luteinizing hormone (LH). As the female rats transition to acyclicity, there is a progressive loss of estrogen feedback on LH secretion which favors reproductive aging process (Meites, 1988). Preceding the alterations in E2 release by the ovary, brain-induced changes in releasing hormone secretion via hypothalamic neurotransmitters and neuropeptides and pituitary hormones play a vital role in promoting the transition to reproductive senescence (Downs and Wise, 2009). In middle-aged female rats, there was a characteristic delay and attenuated LH-surge that was associated with changes in cytoarchitecture and a decline in gonadotropin-releasing hormone (GnRH) neuronal activity suggesting that afferent inputs of aminergic and peptidergic neurons to GnRH neurons may have been responsible for alterations in LH-surge (Downs and Wise, 2009; Kermath and Gore, 2012). Accompanying these changes was the impaired functioning of hypothalamic catecholaminergic activity in middle-aged female rodents marked by attenuated release of NE from hypothalamic medial preoptic area and medial basal hypothalamus (Mohankumar et al., 1994; ThyagaRajan et al., 1995). In addition to NE, several other neurotransmitters and neuropeptides such as reduced availability of
3. Sympathetic noradrenergic (NA) innervation influences immune reactivity in the lymphoid organs Numerous studies have demonstrated that noradrenergic and peptidergic nerve fibers are extensively distributed in various compartments of primary (bone marrow and thymus) and secondary (spleen, lymph nodes, and lymphoid tissues) lymphoid organs and that these nerve fibers can modulate immune functions through the release of neurochemicals. Receptor-ligand binding studies have demonstrated the presence of α- and β-adrenergic receptors (AR) on T and B lymphocytes, macrophages, and other immune cells. Pharmacological and surgical manipulation of NA innervation in bone marrow, thymus, spleen, and lymph nodes altered immune responses which further established that norepinephrine (NE) released from autonomic sympathetic NA nerve fibers transduces the message through AR on the lymphocytes and macrophages (Ader et al., 2001; Bellinger et al., 2008; ThyagaRajan and Priyanka, 2011). 3.1. Sympathetic NA innervation in thymus in middle-aged rats In young rodents, sympathetic NA nerve fibers enter the thymus as dense plexus along with large blood vessels either in the capsule into the interlobular septa or continue with the vasculature into the cortex (Bellinger et al., 2008; ThyagaRajan and Priyanka, 2011). Using 2
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fluorescence histochemistry, it has been demonstrated that NA nerve fibers extend into cortical regions of thymus and are in close contact with the thymocytes and yellow cortical autofluorescent cells (CAF cells). An increase in NA innervations was observed in the interlobular septa and cortex with a high density of nerve fibers localized in the corticomedullary junction. At the corticomedullary junction, NA nerves join together with medullary sinuses, which then travel with the vasculature in the septa with some nerve fibers coursing into the adjacent parenchyma. With advancing age, the density of sympathetic NA innervation increased in the thymus of female rats similar to that of male rats (ThyagaRajan and Priyanka, 2011). There was a slight increase in thymic NA innervation in the cortical and paracortical regions associated with an increase in CAF cells in the early middle-aged rats while it was more pronounced in old female rats. A similar increase in thymic NE concentration and NE content was also observed in these female rats. A plausible reason for thymic involution with advancing age may be the result of high levels of gonadal hormones and stress. Estrogeninduced thymic atrophy was characterized by a reduction in cortical area and an increase in medullary area along with alterations in the subsets of thymocytes resulting in altered T cell differentiation and maturation possibly mediated through α- and β-estrogen receptors (ER) along with membrane-associated GPR30 ER (Yao and Hou, 2004; Wang et al., 2008).
travel with the vascular and lymphatic channels to enter the paracortical region that are rich in T lymphocytes. Cortical and paracortical regions containing T lymphocytes had dense NA innervations while it was absent in the nodular regions and germinal centers that are rich in B lymphocytes. There was an age-associated reduction in density of NA nerve fibers in all the compartments of MLN including the paracortical regions of the early middle-aged and old female rats (ThyagaRajan and Priyanka, 2011). Reflecting the age-related loss of NA innervation in the lymph nodes, NE concentration and content also significantly declined in the MLN of middle-aged and old female rats. 4. Estrogen-induced neuroimmunomodulation in brain areas and lymphoid organs in early middle-aged female rats Women’s Health Initiative (WHI) study involving treatment of postmenopausal women with estrogen alone or estrogen and progesterone demonstrated that there was an increased risk of developing cancer and stroke and had adverse effects on cognition following hormone replacement therapy (Rossouw et al., 2002; Anderson et al., 2004; Shumaker et al., 2004). Although it was a multicentric population-based study, the conflicting results were attributed to several factors such as the type and dose of estrogen used, frequency of administration, age of the patients at the start of the study, duration of treatment, etc. These studies raised doubts about the beneficial effects of estrogen such as preventing bone loss, decreasing the risk of coronary heart disease, and neuroprotection in ischemic brain injury and Alzheimer’s disease as reported in the published literature (Wise et al., 2005; Simpkins et al., 2012). Recent review of WHI studies reveal that the benefits of hormone replacement therapy may depend on whether the postmenopausal women were less than 60 years of age, had their last menstrual period less than ten years previous, and their hysterectomy status suggesting that estrogen’s effects depend on the age of the individual warranting longitudinal studies during reproductive aging process (Gurney et al., 2014). We examined whether treatment of early middle-aged rats with estrogen facilitated neuroprotection or is a risk factor for neurodegeneration in the brain and lymphoid organs which may determine the development of diseases at a later stage in reproductive aging. In the following sections, results from a single study involving ovariectomized (OVX) early middle-aged animals (8- to 9-mo-old female Sprague-Dawley rats) treated with two doses of E2 (30-day implantation of 0.6 μg or 300 μg pellets of 17β-estradiol) on various areas of brain [frontal cortex (FC), striatum (STR), medial basal hypothalamus (MBH), and hippocampus (HP)], thymus, spleen, and lymph nodes will be discussed (Kale et al., 2014; Pratap et al., 2015; Pratap et al., 2016; Ravichandran et al., 2017). These results demonstrated that estrogen differentially regulated neural-immune interactions in each of these organ systems and thus, may influence the health status and disease pathogenesis as these rats transition to various stages of reproductive aging.
3.2. Sympathetic NA innervation in spleen in middle-aged rats In young rodents, sympathetic NA nerve fibers travelling along with the splenic artery enter the spleen at the hilar region as dense vascular plexus (Bellinger et al., 2008; ThyagaRajan and Priyanka, 2011). As they course along the arterial branches, NA nerves form subcapsular and trabecular plexuses and finally, enter the lymphoid compartment of the spleen, white pulp, along with the central arteriole. In the white pulp, NA nerve fibers surrounding the central arteriole spread into the adjacent parenchyma and course through the periarteriolar lymphatic sheath (PALS) consisting of T lymphocytes. NA nerve fibers are also localized in the marginal zone that forms the outer region of the white pulp. In contrast to heavy NA innervation in the PALS region, its innervation is scarce among the follicular region of the white pulp that is composed predominantly of B lymphocytes. Both fluorescence histochemistry for NA innervation and immunocytochemistry for tyrosine hydroxylase (TH+)-positive nerve fibers demonstrated the presence of bundles of nerve fibers in the PALS and marginal zone of the white pulp and along the trabeculae forming trabecular plexuses in the red pulp of the spleens of young and early middle-aged female rats (ThyagaRajan and Priyanka, 2011). In contrast to young and early middle-aged female rats, there was a drastic decline in NA nerve fibers in various compartments of old female rats. Splenic NE concentration was reduced in the hilar regions of old female rats but NE content in the end region and whole spleen declined in early middleaged female rats suggesting that the age-related reduction in NA innervation begins at this age. Accompanying these changes in innervation, there was a decline in NK cell activity and IL-2 production by splenocytes in early middle-aged rats while there was a decline in T cell proliferation, IFN-γ and IL-2 production, and NK cell activity in the spleens of old female rats (ThyagaRajan and Priyanka, 2011).
4.1. Neuroprotective and anti-inflammatory effects of estrogen on the brain When early middle-aged OVX rats were treated with E2, it enhanced p-tyrosine hydroxylase (p-TH) expression in the frontal cortex and suppressed NF-κB expression in the frontal cortex and striatum (Pratap et al., 2016). In the hippocampus, low dose E2 treatment enhanced p-TH expression while both the doses suppressed NGF expression. Accompanying these changes in p-TH expression, E2 suppressed total cholinesterase activity in striatum and hippocampus suggesting that it has neuroprotective role on cholinergic neurons in these areas and may have a role in improving movement, and learning and memory functions in old animals. There was an age-related decline in intracellular signaling molecules (p-ERK, p-CREB, and p-Akt) during middle-age, ovariectomy further accentuated it, and estrogen reversed both the ageand OVX-associated decline in these markers depending upon its dose
3.3. Sympathetic NA innervation in mesenteric lymph nodes (MLN) in middle-aged rats In young rodents, NA nerve fibers enter as dense plexuses along with the blood vessels into the lymph nodes distributing themselves into subcapsule or into the medulla through the medullary cords (Bellinger et al., 2008; ThyagaRajan and Priyanka, 2011). NA nerve fibers from the subcapsular plexus course along the vasculature and enter cortical parenchyma. From the medulla, the NA nerve fibers 3
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and differentiation of T cells in thymus and alteration of B lymphopoiesis in bone marrow (Cunningham and Gilkeson, 2011). Perhaps in addition to its effects on acquired immunity, the presence of ERs on dendritic cells and NK cells suggests that they are capable of regulating innate immunity as well, through NF-κB pathway and ultimately, the production of type I IFN or proinflammatory cytokines (Kovats, 2015). Estrogen also influences the maturation and differentiation, and activation and proliferation of lymphoid cells which determines their capacity to produce cytokines and antibodies (Murray, 2001). Development and progression of a particular disease may be the result of a shift in the balance of Th1/Th2 cytokine profile to Th2 type which enhances humoral immunity and decreases cell-mediated immunity (Cutolo et al., 2004). In support of this notion, studies have shown that estrogen treatment of normal rodents and animals with autoimmune diseases augmented Th1 cytokines and proliferation of CD4+ T cells involving ERα-associated mechanism (Maret et al., 2003).
and the brain area involved. In addition, there was an increase in the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx), glutathione-S-transferase (GST)] and nitric oxide (NO) production, and a simultaneous decline in lipid peroxidation which may have facilitated neuroprotection through PI3 K/Akt signaling pathway. Besides suppressing cellular oxidative stress, estrogen enhanced the activities of metabolic enzymes such as hexokinase, pyruvate kinase, and citrate synthase, cytochrome c oxidase, Na+/K+-ATPase in discrete areas of the brain which may have promoted mitochondrial function, energy metabolism and thus, may facilitate neuronal survival in these brain areas. These results demonstrate that both lower and higher doses of estrogen for a short duration were able to confer neuroprotection on catecholaminergic and cholinergic neurons through the suppression inflammatory mediators and enhancement of cellular survival mediators such as antioxidant and metabolic enzymes. Numerous studies have reported diverse effects of E2 from neuroprotection to neurodegeneration based on the type of cells, duration of treatment, and age of the rats used in their studies. When early middleaged rats were treated for 90 days with a similar dose of E2, it enhanced the expression of proinflammatory cytokines, IL-1β and IL-6, and a chemokine, monocyte chemoattractant protein-1(MCP-1), in rostral ventrolateral medulla indicating its role in promoting neuroinflammation during chronic treatment (Subramanian et al., 2015). ERα and ERβ agonists suppressed lipopolysaccharide (LPS)-induced increase in the production of IL-1β, TNF-α, and matrix metalloproteinase-9 (MMP-9) activity in cultured astrocytes but not in microglial cells isolated from 9 to 11 month old middle-aged female rats suggesting that it has differential effects in modulating inflammation depending on the cell type (Lewis et al., 2008). Late middle-aged OVX female rats (13-mo-old) implanted with E2 with a release rate of 2 μg/day for 4 weeks had significantly altered transcriptome of frontal cortex such as dopaminergic and peptidergic neurotransmission, immune surveillance, adenosine and insulin-like growth factor signaling and transport processes and also, suppression of neuroinflammatory-promoting mediators that may possibly explain the beneficial effects of E2 on cognition, reward behavior, and attenuating stress response (Sárvári et al., 2010,2011). Similar to its effects on frontal cortex, E2 promoted the development of protective microglia phenotype, modulated complement expression, and restored semaphorin3A (an axonal growth cone guidance molecule) in hippocampus suggesting that E2-induced neuroprotection may be through the involvement of immune molecules of innate immune system facilitated by genomic regulation by ERβ (Sárvári et al., 2014,2016). In contrast to the above studies demonstrating the neuroprotective functions of estrogen, there are reports of estrogen’s neurodegenerative effects on brain and other organ systems to promote inflammation resulting in diseases and cancer (Straub, 2007). Administration of physiological levels of estrogen has been shown to induce multifocal lesion in the hypothalamic arcuate nucleus characterized by degenerating dendrites, reactive microglial cells and astrocytes, and presence of multi-shaped dense inclusions that are not observed following treatment with androgens or progestins (Brawer et al., 1993). It is plausible that prolonged exposure to estrogen promotes the formation of catecholestrogens and subsequent conversion to o-semiquinone free radicals that are toxic to neurons and thus, induce the development and progression of female-specific diseases and cancer.
4.2.1. Estrogen suppresses neural-immune interactions in thymus Estrogen treatment of early middle-aged female rats for 30 days prevented age- and OVX-related increase in TH expression in the thymus accompanied by differential expression of NGF depending on the dose of estrogen and increased expression of p-ERK, p-CREB, and pAkt that may alter sympathetic NA modulation of thymopoiesis (Ravichandran et al., 2017). Estrogen is known to reduce the population of early thymic progenitor cells and promote the arrest of T cell maturation through ERα and Fas/FasL pathway that may further exacerbate this neural-immune interaction (Yao and Hou, 2004; Zoller and Kersh, 2006). Although there was an increase in the activities of catalase, glutathione peroxidase, and glutathione-S-transferase, estrogen significantly enhanced the lipid peroxidation and production of free radicals suggesting that these pro-oxidants might have promoted degeneration of sympathetic NA innervation in thymus and suppressed T cell maturation and survival by facilitating an inflammatory environment (Roy et al., 2007). 4.2.2. Estrogen modulates neural-immune interactions in spleen Treatment of early middle-aged female rats with estrogen (30-day implantation of 0.6 μg or 300 μg pellets of 17β-estradiol) exerted diverse effects on sympathetic NA neuronal expression and immune responses (Kale et al., 2014). Low dose of estrogen suppressed IFN-γ production while it increased Con A-induced lymphoproliferation, IL-2 production, expression of intracellular signaling molecules (p-ERK, pCREB, and p-Akt), antioxidant enzyme activities (SOD, CAT, and GST), and NO production in the spleen. In contrast, high dose of estrogen treatment suppressed Con A-induced lymphoproliferation but enhanced the expression of p-TH, NGF, intracellular signaling molecules (p-ERK and p-CREB), and activities of antioxidant enzymes (SOD and GST). Accompanying these beneficial effects of estrogen was an increase in the production of lipid peroxidation and protein carbonyl formation by both the doses suggesting that estrogen-induced oxidative stress may compromise immune functions with advancing age. Similar effects were found in the brain areas where chronic treatment with estrogen increased IL-1β, a proinflammatory cytokine, levels in brain noradrenergic nuclei associated with increased nitration of the TH in the hypothalamic medial preoptic area causing a decline in NE synthesis (Kasturi et al., 2013). Supporting evidence for estrogen’s neurodegenerative effects was also demonstrated in myometrium and vagina where estrogen causes diminution of sympathetic neurons associated with suppression of target-derived growth factors and axon-guidance molecules (Mónica Brauer and Smith, 2015). The dose-dependent effects of estrogen on proliferation of splenocytes and cytokine production observed in in vivo studies were similar to that of in vitro studies where splenocytes were incubated with different doses of estrogen. Direct addition of lower doses (nanomolar to picomolar) of estrogen to splenocytes in vitro increased lymphoproliferation and IFN-γ production while higher doses (micromolar) of
4.2. Estrogen-induced sympathetic NA neuronal modulation of immunity in the lymphoid organs Estrogen influences immunity via its receptors on the cells of the immune system and modulation of intracellular signaling cascade in health and diseases. There is a differential expression of estrogen receptors (ER) on lymphocytes with ERα predominantly expressed on CD4+ T cells while ERβ on B cells that may be involved in maturation 4
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vulnerability to diseases and cancer that profoundly affects women’s health. The transition to menopause is one of the most complex and least understood phases of a woman’s life and has been marked by increased risk of development of cardiovascular disease, osteoporosis, type II diabetes, etc. Originally, it was thought that a decrease in estrogen levels during perimenopause might predispose women to these diseases. However, critical evaluation of data suggests that estrogen levels are irregular during perimenopause with a significant increase in its levels during the premenstrual and follicular phases of the menstrual cycle. This is similar to the hormone profile in aging female rats characterized by an increase in estradiol:progesterone ratio during irregular estrous cycles (vom Saal et al., 1994; Randolph et al., 2004). Further, Women’s Health Initiative studies have provided confounding results about the role of estrogen in health because it was reported that prolonged exposure to estrogenic preparations in hormone replacement therapy could increase the risk for breast cancer and cardiovascular diseases (Rossouw et al., 2002; Anderson et al., 2004). Analysis of peripheral blood mononuclear cells (PBMCs) from middle-aged perimenopausal women (mean ± S.D.: 47.1 ± 3.8 yrs.) revealed that the decline in estrogen and progesterone was accompanied by a decrease in T lymphocyte proliferation, IFN-γ production, NGF and p-CREB expression, SOD and catalase activities while there was an increase in lipid peroxidation (Priyanka et al., 2013b). In another study, we found that there was a similar decrease in T cell proliferation, expression of intracellular signaling molecules (p-ERK, pCREB, and p-Akt), and activities of SOD, catalase, and GST accompanied by an increase in TNF-α production in PBMCs of middle-aged perimenopausal (mean ± S.D.; 52.2 ± 3.5 yrs.) women suggesting that compromised cell-mediated immunity is associated with development of inflammatory environment (unpublished data). The results from these studies on middle-aged perimenopausal women are similar to immunosuppression and enhancement of free radical generation in early middle-aged female rats suggesting that it is possible that altered neuroendocrine-immune cross-talk during menopausal transition may favor changes at the cellular level to promote the development of diseases. Recently, we have shown that there is an increase in the cognitive decline in the elderly associated with altered cholesterol metabolism, heme metabolism, calcium homeostasis, and enzyme profiles suggesting that understanding the neuroendocrine-immune interactions is critical to understand aging process and the development of age-associated diseases and cancer (Vasantharekha et al., 2016).
estrogen had no effect that may be primarily mediated through ERα (Priyanka et al., 2013a). Estrogen and α1- and α2-adrenergic receptor (AR) antagonists had distinct effects on immune responses and intracellular targets suggesting certain types of E2-mediated immune responses may be mediated either through α1- or α2- AR involving distinct intracellular signaling molecules such as p-ERK, p-CREB, and/or pAkt (Priyanka and ThyagaRajan, 2013). β2-ARs are abundant and predominantly distributed on CD4+ T cells and B cells and regulate immune responses depending upon effector cells, method of activation, duration of engagement, and signaling molecule (PKA, PKC, Akt, NF-κB, and other targets) activation. Coincubation of estrogen and β2-AR agonist demonstrated that estrogen modulated β2-AR-induced immune reactivity via ERα involving cell survival (ERK, PKA and PKC) and inflammation-inducing (NF-κB and NO) pathways that may promote differential regulation of Th1/Th2 immunity and inflammation during reproductive aging (Priyanka et al., 2014). 4.2.3. Estrogen modulates neural-immune interactions in lymph nodes Lymph nodes play a crucial role in immunosurveillance where dendritic cells effectively capture antigens for elimination through T and B lymphocytes facilitated by inflammatory and adaptive immune responses. The antigen presentation and priming at cortical areas of lymph nodes also promote the trafficking of naïve T cells to peripheral tissues as activated T cells to prevent the onset of diseases. Treatment of early middle-aged OVX female rats with estrogen as described above induced significant alterations in the neural immune interactions in skin-draining (inguinal and axillary) and gut-draining (mesenteric) lymph nodes. Age-associated decline was observed in proliferation of the lymphocytes, IFN-γ, TNF-α production, and p-Akt/ Total Akt expression in skin-draining lymph node of early middle-aged female rats (Pratap et al., 2015). However, low dose of estrogen treatment in OVX rats enhanced lymphoproliferation, IFN-γ and TNF-α, ROS production, expression of p-NF-κB (p50 and p65), p-mTOR, and pAkt/Total Akt and decreased cytochrome C oxidase activity. Both doses of estrogen increased malondialdehyde (MDA) formation and NO production. These results demonstrated that estrogen promoted the development of inflammatory environment in the skin-draining lymph nodes because of increased IFN-γ and TNF-α production, intracellular signaling markers such p-Akt, p-mTOR, cytochrome c oxidase activity, and NO production aided by an increase in lipid peroxidation by the lymphocytes of early middle-aged female rats. A similar treatment paradigm of estrogen in early middle-aged female rats augmented p-TH and NGF expression in gut-draining (mesenteric) lymph nodes (MLN; Ravichandran et al., 2017). There was a dose-associated increase in the activities of antioxidant enzymes without an increase in free radical generation suggesting that estrogen exerts neuroprotective effects on sympathetic NA activity in MLN. Estrogen treatment also enhanced p-ERK and p-CREB expression that may help in preventing bacterial translocation and intestinal inflammation through the upregulation of T cell proliferation and IL-2 production, and mucosal immunity (Li et al., 2005; Hofmann et al., 2010). Estrogen-induced differences in the neural-immune interactions and free radical generation in these two categories of lymph nodes may have been due to the disparity in the functional capacity of the stromal cells that act as scaffold determining the trafficking of immune cells and immune molecules through the lymph nodes (Pabst et al., 2009). In addition, these stromal cells may independently regulate immune responses through their different homing and adhesion molecules under the influence different cytokines and chemokines in these draining lymph nodes.
6. Conclusions Menopause in women characterized by altered levels of estrogen that may promote development of autoimmune diseases such as rheumatoid arthritis, metabolic diseases such as obesity and diabetes, cardiovascular diseases, and impairment of cognitive functions with advancing age. Alterations in estrogen level during various stages of reproductive cycles may induce remodeling of neural-immune network in the lymphoid organs and brain areas to promote disease development and pathogenesis. The results from our reports demonstrate that estrogen severely affects thymic sympathetic NA activity which may influence thymopoiesis and therefore, compromise cell-mediated immune responses during reproductive aging (Fig. 1). In contrast, estrogen induced both neuroprotection and inflammatory environment in the spleen and lymph nodes while conferring neuroprotection in the brain areas. Such remodeling of neuroendocrine-immune interactions aided by compensatory factors (antioxidant and metabolic enzymes, and growth factors) may promote immunosenescence as they transition to other stages in reproductive aging. In both old female rats and aged women, deficiency in neural-immune interactions characterized by decline in catecholaminergic activity and immunity was observed that may involve discrete changes in intracellular signaling pathways promoting either cell survival or inflammatory environment (ThyagaRajan and Priyanka, 2011; Priyanka et al., 2013b). Further research in the
5. Neuroimmunomodulation in reproductive aging Aging alters all organ systems, particularly, the immune and neuroendocrine (including the hypothalamo-pituitary (HP) adrenal (HPA) and HP gonadal (HPG) axes) systems and thus, increase the 5
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Fig. 1. An overview of the results depicting the probable pathways mediating the neuroprotective and neurodegenerative role of estrogen in various brain areas (Frontal cortex, Striatum, Medial basal hypothalamus, and Hippocampus) and lymphoid organs (thymus, spleen, and lymph nodes). Abbreviations; ER- Estrogen receptor, ERE- Estrogen response element, GnRHGonadotropin releasing hormone, LH- luteinizing hormone, FSH- follicle stimulating hormone, TH-tyrosine hydroxylase, GPCR- G protein-coupled receptor, ERK-Extracellular signal regulated kinase, Akt-Protein kinase B, mTOR- mechanistic target of rapamycin, PI3K- Phosphoinositide 3-kinase, CREB-cAMP response element binding protein, CBP- CREB-binding protein, NF-κB- NF-κB-Nuclear factor kappa-chain-enhancer of activated B cells, NO- nitric oxide, iNOS- inducible nitric oxide synthase, CAT- catalase, GPX- glutathione peroxidase, GSTglutathione-s-transferase, Cyt C- Cytochrome C, ROS- reactive oxygen species, LPO-lipid peroxidation.
following areas might provide a better understanding of the role of estrogen in modulating neural immune interactions during reproductive aging and pave the way for development of better therapeutic targets for healthy aging:
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1. Longitudinal studies during middle and late stages of middle age to understand the extent of fluctuating hormonal profile in promoting the aging process. 2. Examining the role of dose and duration of treatment of both synthetic and bioidentical estrogens. 3. Effects of estrogen on distinct subpopulations of immune cells to understand their diverse effects on health. 4. The area-specific actions of estrogen on the brain areas that may involve type of neurons, receptor status, and genomic and proteomic effects. 5. The type of estrogen receptors (nuclear, cytoplasmic, and membrane-bound) involved in mediating the actions of estrogen and is interactions with adrenergic receptors (alpha and beta) to modulate the intracellular signaling pathways. 6. Modulation of neural-immune interactions by progesterone alone or in combination with estrogen during various stages of reproductive aging Acknowledgments We thank all the students who had worked in our laboratory and contributed to the collection of data. Supported by the Department of Science and Technology (F. NO. SR/SO/HS-46/2007) and Department of Biotechnology (BT/PR9199/Med/30/12/2007), Government of India, New Delhi References Ader, R., Felten, D.L., Cohen, N. (Eds.), 2001. Psychoneuroimmunology, third edition.
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Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu
Estrogen-induced neuroimmunomodulation as facilitator of and barrier to reproductive aging in brain and lymphoid organs ⁎
Srinivasan ThyagaRajan , Lalgi Hima, Uday P. Pratap, Hannah P. Priyanka, Ramasamy Vasantharekha Integrative Medicine Laboratory, Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur 603 203, Tamil Nadu, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Noradrenergic Immunity Neuroprotection Neurodegeneration Inflammation
Reproductive aging in females is marked by alterations in gonadal hormones, estrogen and progesterone, that facilitate cessation of reproductive cycles and onset of female-specific diseases such as autoimmune and neurodegenerative diseases, hormone-dependent cancers, and osteoporosis. Bidirectional communication between the three homeostatic systems, nervous system, endocrine system, and immune system, is essential for the maintenance of health and any dysfunction in the cross-talk promotes the development of diseases and cancer. The pleiotropic effects of estrogen on neural-immune interactions may promote either neuroprotection or inflammatory conditions depending on the site of action, dose and duration of treatment, type of estrogen receptors and its influence on intracellular signaling pathways, etc. Our studies involving treatment of early middle-aged female rats with low and high doses of estrogen and examining the brain areas, thymus, spleen, and lymph nodes revealed that estrogen-induced changes in neural-immune interactions are markedly affected in thymus followed by spleen and lymph nodes while it confers neuroprotection in the brain areas. These alterations are determined by antioxidant enzyme status, growth factors, intracellular signaling pathways involved in cell survival and inflammation, and metabolic enzymes and thus, may regulate the various stages in female reproductive aging. It is imperative that detailed longitudinal studies are carried out to understand the mechanisms of neuroendocrine-immune interactions in reproductive aging to facilitate healthy aging and for the development of better treatment strategies for female-specific diseases.
1. Introduction Aging is characterized by cessation of reproductive cycles in females, alteration in hormonal profiles, and increased incidence of cancers, osteoporosis, neurodegenerative and autoimmune diseases (Meites and Quadri, 1987; Meites, 1988; ThyagaRajan and Priyanka, 2011). The cross-talk between the nervous system, endocrine system, and immune system contributes to the maintenance of homeostasis and normal cellular functions and thus, promote health (Meites, 1988; Ader et al., 2001). It is well established that hypothalamic neuroendocrine system through the release of neurotransmitters and neuropeptides, and releasing hormones regulate hormonal secretion from the pituitary and target endocrine glands to influence various physiological functions including immunity (Meites, 1988; Downs and Wise, 2009). The products of the immune system, cytokines and chemokines, also cross the blood-brain barrier to control neural functions in the brain and thus, regulate physiological processes (Banks, 2015). In addition to the neuroendocrine system, immunity is modulated by the release of
⁎
norepinephrine (NE) and neuropeptides from the sympathetic noradrenergic (NA) and peptidergic nerve fibers, respectively, that innervate the primary (bone marrow and thymus) and secondary (spleen and lymph nodes) lymphoid organs (Bellinger et al., 2008). In general, several studies have reported that age-associated deficiencies in the bidirectional communication between the neuroendocrine system and immune system contribute to the development of diseases and cancer in both rodents and humans (Meites, 1988; Ader et al., 2001; Bellinger et al., 2008; ThyagaRajan and Priyanka, 2011). In contrast to males, aging in females is set apart by distinct changes in the release of hormones, reproductive cyclicity, and increased risks of developing disease pathologies specific to females such as autoimmune diseases, hormone-dependent cancers, and osteoporosis. These crucial changes in hormonal secretion and reproductive cycles occur during middle age in both rodents and women as they transition into acyclicity and menopause, respectively (Randolph et al., 2004; Downs and Wise, 2009; Kermath and Gore, 2012). Among the several hypothalamic, pituitary, and gonadal hormones, estrogen (17β-estradiol; E2) has drawn
Corresponding author. E-mail address: [email protected] (S. ThyagaRajan).
https://doi.org/10.1016/j.jchemneu.2018.02.008 Received 18 August 2017; Received in revised form 22 January 2018; Accepted 20 February 2018 0891-0618/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: ThyagaRajan, S., Journal of Chemical Neuroanatomy (2018), https://doi.org/10.1016/j.jchemneu.2018.02.008
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glutamate and increased activation of GABA(A) receptors in the medial preoptic area may contribute to dampening of LH-surge in middle-aged rats (Neal-Perry et al., 2008). Thus, key changes in population and activity of hypothalamic neurons during middle age may render these neurons less responsive to the gonadal hormones and facilitate further modifications in hormonal secretion and cyclicity during reproductive aging.
primary attention because of its multiple physiological and pathological effects on the cells influenced by its receptor status and function (Burns and Korach, 2012). A number of studies involving in vitro cell lines, in vivo animals, and humans demonstrated that E2 treatment conferred neuroprotection and hence, was a therapeutic option for the treatment of neurodegenerative diseases (Wise et al., 2005; Simpkins et al., 2012). However, treatment of postmenopausal women with estrogen alone or in combination with progesterone provided contradictory results and demonstrated that there was an enhanced risk of stroke and cancer while reducing the incidence of fractures and colorectal cancer in these women (Rossouw et al., 2002; Anderson et al., 2004). Therefore, it is crucial that we understand the physiological role of continued exposure of E2 during reproductive cycles in early life on cellular and molecular mechanisms involving the three homeostatic systems to determine whether it is beneficial or detrimental to women’s health as they transition into various stages of reproductive aging. In this review, we discuss the role of E2 in influencing neural-immune interactions in the few areas of the brain (frontal cortex, striatum, medial basal hypothalamus, and hippocampus)and lymphoid organs (thymus, spleen, and lymph nodes) of early middle-aged rats involving compensatory mechanisms (antioxidant enzymes and growth factors) and signaling molecules and transcription factors (ERK, CREB, Akt, mTOR, and NF-κB) to understand its role in the reproductive aging process (Kale et al., 2014; Pratap et al., 2015; Pratap et al., 2016; Ravichandran et al., 2017).
2.2. Immunosenescence in middle-aged rats Adaptive immune responses markedly decline with advancing age while there is an enhanced pro-inflammatory state due to defects in innate immune responses resulting in the development of diseases and cancer. The cells of the innate immune system have age-related impaired signaling through Toll-like receptor and nucleotide-binding oligomerization domain-like receptors and thus, have compromised phagocytic and chemotactic functions (Shaw et al., 2011). Subsequently, the involvement of NF-κB activation and production of pro-inflammatory cytokines create an inflammatory environment that hastens the pathogenesis of diseases and cancer during the aging process (Franceschi et al., 2000). The decline in adaptive immunity is due to age-associated thymic involution that results in a decrease in naïve T cells paralleling the loss of diversity in T cell receptors repertoire, accumulation of dysfunctional CD8+ memory T cells, and decrease in CD4+/CD8+ T cells. (Alonso-Fernández and De la Fuente, 2011). Also, subsets of T cells belonging to Th1, Th2, Th17, and Treg show significant age-associated functional alterations characterized by an increase in the number of Th2 and Treg cells and changes in intracellular signaling pathways that further compromise the ability of immune system to ward off antigenic challenges or to provide better responses to vaccines (Jagger et al., 2014). Longitudinal studies demonstrating changes in innate and acquired immunity during aging especially, in middle-aged female rats, have not been attempted and thus, it is difficult to understand the modulation by gonadal hormones on immunity. We have provided evidence for a decline in natural killer (NK) cell activity, and Concanavalin (Con A)-induced T cell proliferation and IL-2 production in early middle-aged female rats (ThyagaRajan and Priyanka, 2011). In support of these findings, immune responses such as Con A-induced splenocyte proliferation, IL-2 production, and cytolytic activity declined with advancing age in male F344 rats (Nasrullah and Mazzeo, 1992).
2. Neuroendocrine control of reproductive aging and immunosenescence 2.1. Alterations in hypothalamo-pituitary-gonadal axis hormones in middleaged rats In female rats, regular estrous cycles occur every 4 to 5 days in young (2- to 8-month-old) which is followed by irregular cycles in early middle-aged rats (8- to 9-month-old) characterized by extended days of estrus stage. These stages in young rats are marked by fluctuation in hypothalamic, pituitary, and gonadal hormones and subsequently, the hormonal profiles are altered during middle age characterized by prolonged E2 secretion and delayed ovulation. When the rats are 13 to 14 months of age, they transition into anovulatory stage that is characterized by persistent/constant estrus with intermittent ovulatory activity at irregular intervals. Ultimately, the rats have pseudopregnancy stage followed by persistent anestrus state (Meites and Quadri, 1987; Meites, 1988). These anovulatory stages in old rats are also marked by distinct changes in hormonal secretion from hypothalamus, pituitary, and ovaries. In persistent estrus rats, E2 levels are similar to diestrus day 2 of young rats with lower levels of luteinizing hormone (LH). As the female rats transition to acyclicity, there is a progressive loss of estrogen feedback on LH secretion which favors reproductive aging process (Meites, 1988). Preceding the alterations in E2 release by the ovary, brain-induced changes in releasing hormone secretion via hypothalamic neurotransmitters and neuropeptides and pituitary hormones play a vital role in promoting the transition to reproductive senescence (Downs and Wise, 2009). In middle-aged female rats, there was a characteristic delay and attenuated LH-surge that was associated with changes in cytoarchitecture and a decline in gonadotropin-releasing hormone (GnRH) neuronal activity suggesting that afferent inputs of aminergic and peptidergic neurons to GnRH neurons may have been responsible for alterations in LH-surge (Downs and Wise, 2009; Kermath and Gore, 2012). Accompanying these changes was the impaired functioning of hypothalamic catecholaminergic activity in middle-aged female rodents marked by attenuated release of NE from hypothalamic medial preoptic area and medial basal hypothalamus (Mohankumar et al., 1994; ThyagaRajan et al., 1995). In addition to NE, several other neurotransmitters and neuropeptides such as reduced availability of
3. Sympathetic noradrenergic (NA) innervation influences immune reactivity in the lymphoid organs Numerous studies have demonstrated that noradrenergic and peptidergic nerve fibers are extensively distributed in various compartments of primary (bone marrow and thymus) and secondary (spleen, lymph nodes, and lymphoid tissues) lymphoid organs and that these nerve fibers can modulate immune functions through the release of neurochemicals. Receptor-ligand binding studies have demonstrated the presence of α- and β-adrenergic receptors (AR) on T and B lymphocytes, macrophages, and other immune cells. Pharmacological and surgical manipulation of NA innervation in bone marrow, thymus, spleen, and lymph nodes altered immune responses which further established that norepinephrine (NE) released from autonomic sympathetic NA nerve fibers transduces the message through AR on the lymphocytes and macrophages (Ader et al., 2001; Bellinger et al., 2008; ThyagaRajan and Priyanka, 2011). 3.1. Sympathetic NA innervation in thymus in middle-aged rats In young rodents, sympathetic NA nerve fibers enter the thymus as dense plexus along with large blood vessels either in the capsule into the interlobular septa or continue with the vasculature into the cortex (Bellinger et al., 2008; ThyagaRajan and Priyanka, 2011). Using 2
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fluorescence histochemistry, it has been demonstrated that NA nerve fibers extend into cortical regions of thymus and are in close contact with the thymocytes and yellow cortical autofluorescent cells (CAF cells). An increase in NA innervations was observed in the interlobular septa and cortex with a high density of nerve fibers localized in the corticomedullary junction. At the corticomedullary junction, NA nerves join together with medullary sinuses, which then travel with the vasculature in the septa with some nerve fibers coursing into the adjacent parenchyma. With advancing age, the density of sympathetic NA innervation increased in the thymus of female rats similar to that of male rats (ThyagaRajan and Priyanka, 2011). There was a slight increase in thymic NA innervation in the cortical and paracortical regions associated with an increase in CAF cells in the early middle-aged rats while it was more pronounced in old female rats. A similar increase in thymic NE concentration and NE content was also observed in these female rats. A plausible reason for thymic involution with advancing age may be the result of high levels of gonadal hormones and stress. Estrogeninduced thymic atrophy was characterized by a reduction in cortical area and an increase in medullary area along with alterations in the subsets of thymocytes resulting in altered T cell differentiation and maturation possibly mediated through α- and β-estrogen receptors (ER) along with membrane-associated GPR30 ER (Yao and Hou, 2004; Wang et al., 2008).
travel with the vascular and lymphatic channels to enter the paracortical region that are rich in T lymphocytes. Cortical and paracortical regions containing T lymphocytes had dense NA innervations while it was absent in the nodular regions and germinal centers that are rich in B lymphocytes. There was an age-associated reduction in density of NA nerve fibers in all the compartments of MLN including the paracortical regions of the early middle-aged and old female rats (ThyagaRajan and Priyanka, 2011). Reflecting the age-related loss of NA innervation in the lymph nodes, NE concentration and content also significantly declined in the MLN of middle-aged and old female rats. 4. Estrogen-induced neuroimmunomodulation in brain areas and lymphoid organs in early middle-aged female rats Women’s Health Initiative (WHI) study involving treatment of postmenopausal women with estrogen alone or estrogen and progesterone demonstrated that there was an increased risk of developing cancer and stroke and had adverse effects on cognition following hormone replacement therapy (Rossouw et al., 2002; Anderson et al., 2004; Shumaker et al., 2004). Although it was a multicentric population-based study, the conflicting results were attributed to several factors such as the type and dose of estrogen used, frequency of administration, age of the patients at the start of the study, duration of treatment, etc. These studies raised doubts about the beneficial effects of estrogen such as preventing bone loss, decreasing the risk of coronary heart disease, and neuroprotection in ischemic brain injury and Alzheimer’s disease as reported in the published literature (Wise et al., 2005; Simpkins et al., 2012). Recent review of WHI studies reveal that the benefits of hormone replacement therapy may depend on whether the postmenopausal women were less than 60 years of age, had their last menstrual period less than ten years previous, and their hysterectomy status suggesting that estrogen’s effects depend on the age of the individual warranting longitudinal studies during reproductive aging process (Gurney et al., 2014). We examined whether treatment of early middle-aged rats with estrogen facilitated neuroprotection or is a risk factor for neurodegeneration in the brain and lymphoid organs which may determine the development of diseases at a later stage in reproductive aging. In the following sections, results from a single study involving ovariectomized (OVX) early middle-aged animals (8- to 9-mo-old female Sprague-Dawley rats) treated with two doses of E2 (30-day implantation of 0.6 μg or 300 μg pellets of 17β-estradiol) on various areas of brain [frontal cortex (FC), striatum (STR), medial basal hypothalamus (MBH), and hippocampus (HP)], thymus, spleen, and lymph nodes will be discussed (Kale et al., 2014; Pratap et al., 2015; Pratap et al., 2016; Ravichandran et al., 2017). These results demonstrated that estrogen differentially regulated neural-immune interactions in each of these organ systems and thus, may influence the health status and disease pathogenesis as these rats transition to various stages of reproductive aging.
3.2. Sympathetic NA innervation in spleen in middle-aged rats In young rodents, sympathetic NA nerve fibers travelling along with the splenic artery enter the spleen at the hilar region as dense vascular plexus (Bellinger et al., 2008; ThyagaRajan and Priyanka, 2011). As they course along the arterial branches, NA nerves form subcapsular and trabecular plexuses and finally, enter the lymphoid compartment of the spleen, white pulp, along with the central arteriole. In the white pulp, NA nerve fibers surrounding the central arteriole spread into the adjacent parenchyma and course through the periarteriolar lymphatic sheath (PALS) consisting of T lymphocytes. NA nerve fibers are also localized in the marginal zone that forms the outer region of the white pulp. In contrast to heavy NA innervation in the PALS region, its innervation is scarce among the follicular region of the white pulp that is composed predominantly of B lymphocytes. Both fluorescence histochemistry for NA innervation and immunocytochemistry for tyrosine hydroxylase (TH+)-positive nerve fibers demonstrated the presence of bundles of nerve fibers in the PALS and marginal zone of the white pulp and along the trabeculae forming trabecular plexuses in the red pulp of the spleens of young and early middle-aged female rats (ThyagaRajan and Priyanka, 2011). In contrast to young and early middle-aged female rats, there was a drastic decline in NA nerve fibers in various compartments of old female rats. Splenic NE concentration was reduced in the hilar regions of old female rats but NE content in the end region and whole spleen declined in early middleaged female rats suggesting that the age-related reduction in NA innervation begins at this age. Accompanying these changes in innervation, there was a decline in NK cell activity and IL-2 production by splenocytes in early middle-aged rats while there was a decline in T cell proliferation, IFN-γ and IL-2 production, and NK cell activity in the spleens of old female rats (ThyagaRajan and Priyanka, 2011).
4.1. Neuroprotective and anti-inflammatory effects of estrogen on the brain When early middle-aged OVX rats were treated with E2, it enhanced p-tyrosine hydroxylase (p-TH) expression in the frontal cortex and suppressed NF-κB expression in the frontal cortex and striatum (Pratap et al., 2016). In the hippocampus, low dose E2 treatment enhanced p-TH expression while both the doses suppressed NGF expression. Accompanying these changes in p-TH expression, E2 suppressed total cholinesterase activity in striatum and hippocampus suggesting that it has neuroprotective role on cholinergic neurons in these areas and may have a role in improving movement, and learning and memory functions in old animals. There was an age-related decline in intracellular signaling molecules (p-ERK, p-CREB, and p-Akt) during middle-age, ovariectomy further accentuated it, and estrogen reversed both the ageand OVX-associated decline in these markers depending upon its dose
3.3. Sympathetic NA innervation in mesenteric lymph nodes (MLN) in middle-aged rats In young rodents, NA nerve fibers enter as dense plexuses along with the blood vessels into the lymph nodes distributing themselves into subcapsule or into the medulla through the medullary cords (Bellinger et al., 2008; ThyagaRajan and Priyanka, 2011). NA nerve fibers from the subcapsular plexus course along the vasculature and enter cortical parenchyma. From the medulla, the NA nerve fibers 3
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and differentiation of T cells in thymus and alteration of B lymphopoiesis in bone marrow (Cunningham and Gilkeson, 2011). Perhaps in addition to its effects on acquired immunity, the presence of ERs on dendritic cells and NK cells suggests that they are capable of regulating innate immunity as well, through NF-κB pathway and ultimately, the production of type I IFN or proinflammatory cytokines (Kovats, 2015). Estrogen also influences the maturation and differentiation, and activation and proliferation of lymphoid cells which determines their capacity to produce cytokines and antibodies (Murray, 2001). Development and progression of a particular disease may be the result of a shift in the balance of Th1/Th2 cytokine profile to Th2 type which enhances humoral immunity and decreases cell-mediated immunity (Cutolo et al., 2004). In support of this notion, studies have shown that estrogen treatment of normal rodents and animals with autoimmune diseases augmented Th1 cytokines and proliferation of CD4+ T cells involving ERα-associated mechanism (Maret et al., 2003).
and the brain area involved. In addition, there was an increase in the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx), glutathione-S-transferase (GST)] and nitric oxide (NO) production, and a simultaneous decline in lipid peroxidation which may have facilitated neuroprotection through PI3 K/Akt signaling pathway. Besides suppressing cellular oxidative stress, estrogen enhanced the activities of metabolic enzymes such as hexokinase, pyruvate kinase, and citrate synthase, cytochrome c oxidase, Na+/K+-ATPase in discrete areas of the brain which may have promoted mitochondrial function, energy metabolism and thus, may facilitate neuronal survival in these brain areas. These results demonstrate that both lower and higher doses of estrogen for a short duration were able to confer neuroprotection on catecholaminergic and cholinergic neurons through the suppression inflammatory mediators and enhancement of cellular survival mediators such as antioxidant and metabolic enzymes. Numerous studies have reported diverse effects of E2 from neuroprotection to neurodegeneration based on the type of cells, duration of treatment, and age of the rats used in their studies. When early middleaged rats were treated for 90 days with a similar dose of E2, it enhanced the expression of proinflammatory cytokines, IL-1β and IL-6, and a chemokine, monocyte chemoattractant protein-1(MCP-1), in rostral ventrolateral medulla indicating its role in promoting neuroinflammation during chronic treatment (Subramanian et al., 2015). ERα and ERβ agonists suppressed lipopolysaccharide (LPS)-induced increase in the production of IL-1β, TNF-α, and matrix metalloproteinase-9 (MMP-9) activity in cultured astrocytes but not in microglial cells isolated from 9 to 11 month old middle-aged female rats suggesting that it has differential effects in modulating inflammation depending on the cell type (Lewis et al., 2008). Late middle-aged OVX female rats (13-mo-old) implanted with E2 with a release rate of 2 μg/day for 4 weeks had significantly altered transcriptome of frontal cortex such as dopaminergic and peptidergic neurotransmission, immune surveillance, adenosine and insulin-like growth factor signaling and transport processes and also, suppression of neuroinflammatory-promoting mediators that may possibly explain the beneficial effects of E2 on cognition, reward behavior, and attenuating stress response (Sárvári et al., 2010,2011). Similar to its effects on frontal cortex, E2 promoted the development of protective microglia phenotype, modulated complement expression, and restored semaphorin3A (an axonal growth cone guidance molecule) in hippocampus suggesting that E2-induced neuroprotection may be through the involvement of immune molecules of innate immune system facilitated by genomic regulation by ERβ (Sárvári et al., 2014,2016). In contrast to the above studies demonstrating the neuroprotective functions of estrogen, there are reports of estrogen’s neurodegenerative effects on brain and other organ systems to promote inflammation resulting in diseases and cancer (Straub, 2007). Administration of physiological levels of estrogen has been shown to induce multifocal lesion in the hypothalamic arcuate nucleus characterized by degenerating dendrites, reactive microglial cells and astrocytes, and presence of multi-shaped dense inclusions that are not observed following treatment with androgens or progestins (Brawer et al., 1993). It is plausible that prolonged exposure to estrogen promotes the formation of catecholestrogens and subsequent conversion to o-semiquinone free radicals that are toxic to neurons and thus, induce the development and progression of female-specific diseases and cancer.
4.2.1. Estrogen suppresses neural-immune interactions in thymus Estrogen treatment of early middle-aged female rats for 30 days prevented age- and OVX-related increase in TH expression in the thymus accompanied by differential expression of NGF depending on the dose of estrogen and increased expression of p-ERK, p-CREB, and pAkt that may alter sympathetic NA modulation of thymopoiesis (Ravichandran et al., 2017). Estrogen is known to reduce the population of early thymic progenitor cells and promote the arrest of T cell maturation through ERα and Fas/FasL pathway that may further exacerbate this neural-immune interaction (Yao and Hou, 2004; Zoller and Kersh, 2006). Although there was an increase in the activities of catalase, glutathione peroxidase, and glutathione-S-transferase, estrogen significantly enhanced the lipid peroxidation and production of free radicals suggesting that these pro-oxidants might have promoted degeneration of sympathetic NA innervation in thymus and suppressed T cell maturation and survival by facilitating an inflammatory environment (Roy et al., 2007). 4.2.2. Estrogen modulates neural-immune interactions in spleen Treatment of early middle-aged female rats with estrogen (30-day implantation of 0.6 μg or 300 μg pellets of 17β-estradiol) exerted diverse effects on sympathetic NA neuronal expression and immune responses (Kale et al., 2014). Low dose of estrogen suppressed IFN-γ production while it increased Con A-induced lymphoproliferation, IL-2 production, expression of intracellular signaling molecules (p-ERK, pCREB, and p-Akt), antioxidant enzyme activities (SOD, CAT, and GST), and NO production in the spleen. In contrast, high dose of estrogen treatment suppressed Con A-induced lymphoproliferation but enhanced the expression of p-TH, NGF, intracellular signaling molecules (p-ERK and p-CREB), and activities of antioxidant enzymes (SOD and GST). Accompanying these beneficial effects of estrogen was an increase in the production of lipid peroxidation and protein carbonyl formation by both the doses suggesting that estrogen-induced oxidative stress may compromise immune functions with advancing age. Similar effects were found in the brain areas where chronic treatment with estrogen increased IL-1β, a proinflammatory cytokine, levels in brain noradrenergic nuclei associated with increased nitration of the TH in the hypothalamic medial preoptic area causing a decline in NE synthesis (Kasturi et al., 2013). Supporting evidence for estrogen’s neurodegenerative effects was also demonstrated in myometrium and vagina where estrogen causes diminution of sympathetic neurons associated with suppression of target-derived growth factors and axon-guidance molecules (Mónica Brauer and Smith, 2015). The dose-dependent effects of estrogen on proliferation of splenocytes and cytokine production observed in in vivo studies were similar to that of in vitro studies where splenocytes were incubated with different doses of estrogen. Direct addition of lower doses (nanomolar to picomolar) of estrogen to splenocytes in vitro increased lymphoproliferation and IFN-γ production while higher doses (micromolar) of
4.2. Estrogen-induced sympathetic NA neuronal modulation of immunity in the lymphoid organs Estrogen influences immunity via its receptors on the cells of the immune system and modulation of intracellular signaling cascade in health and diseases. There is a differential expression of estrogen receptors (ER) on lymphocytes with ERα predominantly expressed on CD4+ T cells while ERβ on B cells that may be involved in maturation 4
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vulnerability to diseases and cancer that profoundly affects women’s health. The transition to menopause is one of the most complex and least understood phases of a woman’s life and has been marked by increased risk of development of cardiovascular disease, osteoporosis, type II diabetes, etc. Originally, it was thought that a decrease in estrogen levels during perimenopause might predispose women to these diseases. However, critical evaluation of data suggests that estrogen levels are irregular during perimenopause with a significant increase in its levels during the premenstrual and follicular phases of the menstrual cycle. This is similar to the hormone profile in aging female rats characterized by an increase in estradiol:progesterone ratio during irregular estrous cycles (vom Saal et al., 1994; Randolph et al., 2004). Further, Women’s Health Initiative studies have provided confounding results about the role of estrogen in health because it was reported that prolonged exposure to estrogenic preparations in hormone replacement therapy could increase the risk for breast cancer and cardiovascular diseases (Rossouw et al., 2002; Anderson et al., 2004). Analysis of peripheral blood mononuclear cells (PBMCs) from middle-aged perimenopausal women (mean ± S.D.: 47.1 ± 3.8 yrs.) revealed that the decline in estrogen and progesterone was accompanied by a decrease in T lymphocyte proliferation, IFN-γ production, NGF and p-CREB expression, SOD and catalase activities while there was an increase in lipid peroxidation (Priyanka et al., 2013b). In another study, we found that there was a similar decrease in T cell proliferation, expression of intracellular signaling molecules (p-ERK, pCREB, and p-Akt), and activities of SOD, catalase, and GST accompanied by an increase in TNF-α production in PBMCs of middle-aged perimenopausal (mean ± S.D.; 52.2 ± 3.5 yrs.) women suggesting that compromised cell-mediated immunity is associated with development of inflammatory environment (unpublished data). The results from these studies on middle-aged perimenopausal women are similar to immunosuppression and enhancement of free radical generation in early middle-aged female rats suggesting that it is possible that altered neuroendocrine-immune cross-talk during menopausal transition may favor changes at the cellular level to promote the development of diseases. Recently, we have shown that there is an increase in the cognitive decline in the elderly associated with altered cholesterol metabolism, heme metabolism, calcium homeostasis, and enzyme profiles suggesting that understanding the neuroendocrine-immune interactions is critical to understand aging process and the development of age-associated diseases and cancer (Vasantharekha et al., 2016).
estrogen had no effect that may be primarily mediated through ERα (Priyanka et al., 2013a). Estrogen and α1- and α2-adrenergic receptor (AR) antagonists had distinct effects on immune responses and intracellular targets suggesting certain types of E2-mediated immune responses may be mediated either through α1- or α2- AR involving distinct intracellular signaling molecules such as p-ERK, p-CREB, and/or pAkt (Priyanka and ThyagaRajan, 2013). β2-ARs are abundant and predominantly distributed on CD4+ T cells and B cells and regulate immune responses depending upon effector cells, method of activation, duration of engagement, and signaling molecule (PKA, PKC, Akt, NF-κB, and other targets) activation. Coincubation of estrogen and β2-AR agonist demonstrated that estrogen modulated β2-AR-induced immune reactivity via ERα involving cell survival (ERK, PKA and PKC) and inflammation-inducing (NF-κB and NO) pathways that may promote differential regulation of Th1/Th2 immunity and inflammation during reproductive aging (Priyanka et al., 2014). 4.2.3. Estrogen modulates neural-immune interactions in lymph nodes Lymph nodes play a crucial role in immunosurveillance where dendritic cells effectively capture antigens for elimination through T and B lymphocytes facilitated by inflammatory and adaptive immune responses. The antigen presentation and priming at cortical areas of lymph nodes also promote the trafficking of naïve T cells to peripheral tissues as activated T cells to prevent the onset of diseases. Treatment of early middle-aged OVX female rats with estrogen as described above induced significant alterations in the neural immune interactions in skin-draining (inguinal and axillary) and gut-draining (mesenteric) lymph nodes. Age-associated decline was observed in proliferation of the lymphocytes, IFN-γ, TNF-α production, and p-Akt/ Total Akt expression in skin-draining lymph node of early middle-aged female rats (Pratap et al., 2015). However, low dose of estrogen treatment in OVX rats enhanced lymphoproliferation, IFN-γ and TNF-α, ROS production, expression of p-NF-κB (p50 and p65), p-mTOR, and pAkt/Total Akt and decreased cytochrome C oxidase activity. Both doses of estrogen increased malondialdehyde (MDA) formation and NO production. These results demonstrated that estrogen promoted the development of inflammatory environment in the skin-draining lymph nodes because of increased IFN-γ and TNF-α production, intracellular signaling markers such p-Akt, p-mTOR, cytochrome c oxidase activity, and NO production aided by an increase in lipid peroxidation by the lymphocytes of early middle-aged female rats. A similar treatment paradigm of estrogen in early middle-aged female rats augmented p-TH and NGF expression in gut-draining (mesenteric) lymph nodes (MLN; Ravichandran et al., 2017). There was a dose-associated increase in the activities of antioxidant enzymes without an increase in free radical generation suggesting that estrogen exerts neuroprotective effects on sympathetic NA activity in MLN. Estrogen treatment also enhanced p-ERK and p-CREB expression that may help in preventing bacterial translocation and intestinal inflammation through the upregulation of T cell proliferation and IL-2 production, and mucosal immunity (Li et al., 2005; Hofmann et al., 2010). Estrogen-induced differences in the neural-immune interactions and free radical generation in these two categories of lymph nodes may have been due to the disparity in the functional capacity of the stromal cells that act as scaffold determining the trafficking of immune cells and immune molecules through the lymph nodes (Pabst et al., 2009). In addition, these stromal cells may independently regulate immune responses through their different homing and adhesion molecules under the influence different cytokines and chemokines in these draining lymph nodes.
6. Conclusions Menopause in women characterized by altered levels of estrogen that may promote development of autoimmune diseases such as rheumatoid arthritis, metabolic diseases such as obesity and diabetes, cardiovascular diseases, and impairment of cognitive functions with advancing age. Alterations in estrogen level during various stages of reproductive cycles may induce remodeling of neural-immune network in the lymphoid organs and brain areas to promote disease development and pathogenesis. The results from our reports demonstrate that estrogen severely affects thymic sympathetic NA activity which may influence thymopoiesis and therefore, compromise cell-mediated immune responses during reproductive aging (Fig. 1). In contrast, estrogen induced both neuroprotection and inflammatory environment in the spleen and lymph nodes while conferring neuroprotection in the brain areas. Such remodeling of neuroendocrine-immune interactions aided by compensatory factors (antioxidant and metabolic enzymes, and growth factors) may promote immunosenescence as they transition to other stages in reproductive aging. In both old female rats and aged women, deficiency in neural-immune interactions characterized by decline in catecholaminergic activity and immunity was observed that may involve discrete changes in intracellular signaling pathways promoting either cell survival or inflammatory environment (ThyagaRajan and Priyanka, 2011; Priyanka et al., 2013b). Further research in the
5. Neuroimmunomodulation in reproductive aging Aging alters all organ systems, particularly, the immune and neuroendocrine (including the hypothalamo-pituitary (HP) adrenal (HPA) and HP gonadal (HPG) axes) systems and thus, increase the 5
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Fig. 1. An overview of the results depicting the probable pathways mediating the neuroprotective and neurodegenerative role of estrogen in various brain areas (Frontal cortex, Striatum, Medial basal hypothalamus, and Hippocampus) and lymphoid organs (thymus, spleen, and lymph nodes). Abbreviations; ER- Estrogen receptor, ERE- Estrogen response element, GnRHGonadotropin releasing hormone, LH- luteinizing hormone, FSH- follicle stimulating hormone, TH-tyrosine hydroxylase, GPCR- G protein-coupled receptor, ERK-Extracellular signal regulated kinase, Akt-Protein kinase B, mTOR- mechanistic target of rapamycin, PI3K- Phosphoinositide 3-kinase, CREB-cAMP response element binding protein, CBP- CREB-binding protein, NF-κB- NF-κB-Nuclear factor kappa-chain-enhancer of activated B cells, NO- nitric oxide, iNOS- inducible nitric oxide synthase, CAT- catalase, GPX- glutathione peroxidase, GSTglutathione-s-transferase, Cyt C- Cytochrome C, ROS- reactive oxygen species, LPO-lipid peroxidation.
following areas might provide a better understanding of the role of estrogen in modulating neural immune interactions during reproductive aging and pave the way for development of better therapeutic targets for healthy aging:
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