Estrogen-receptor status determines differential regulation of α1- and α2-adrenoceptor-mediated cell survival, angiogenesis, and intracellular signaling responses in breast cancer cell lines.

Psychosocial stress promotes cancer pathogenesis involving angiogenesis through alterations in neuroendocrine-immune functions that may involve adrenoceptor (AR)-dependent signaling mechanisms in the brain, lymphoid organs, and cancerous cells. Various concentrations of α1- and α2- AR-specific agonists and antagonists were incubated in vitro with estrogen receptor-positive (ER +) MCF-7, and ER (-) MDA MB-231 cells to examine the secretions of VEGF-A, VEGF-C, and nitric oxide (NO), and expression of signaling molecules- p-ERK, p-CREB, and p-Akt on the proliferation of breast cancer cell lines. Cellular proliferation, VEGF-A and NO secretion, expression of p-ERK, p-CREB, and p-Akt were enhanced in MCF-7 cells treated with α1-AR agonist while VEGF-C secretion alone was enhanced in MDA MB-231 cells. Treatment of MCF-7 and MDA MB-231 cells with α2- AR agonist similarly enhanced proliferation and decreased NO production and p-CREB expression while VEGF-C secretion was decreased in MCF-7 cells and p-Akt expression was decreased in MDA MB-231 cells. α1-AR inhibition reversed cellular proliferation and VEGF-A secretion by MCF-7 cells while α2-AR inhibition reversed the proliferation of MCF-7 and MDA MB-231 cells and VEGF-C secretion by MCF-7 cells. Taken together, breast cancer pathogenesis may be influenced by distinct α-AR-mediated signaling mechanisms on angiogenesis and lymphangiogenesis that are dependent on estrogen receptor status.

Alterations in Immune Responses Are Associated with Dysfunctional Intracellular Signaling in Peripheral Blood Mononuclear Cells of Men and Women with Mild Cognitive Impairment and Alzheimer's disease.

Deficits in the neuroendocrine-immune network in the periphery associated with the onset and progression of mild cognitive impairment (MCI) and Alzheimer's disease (AD) have not been extensively studied. The present study correlatively examines the association between cell-mediated immune responses, stress hormones, amyloid precursor protein (APP) expression, peripheral blood mononuclear cells (PBMC), and intracellular signaling molecules in the pathophysiology of MCI and AD compared to adults. Serum APP, lymphocyte proliferation, total cholinesterase (TChE), butyrylcholinesterase (BChE) activities, cytokines (IL-2, IFN-γ, IL-6, and TNF-α), and intracellular signaling molecules (p-ERK, p-CREB, and p-Akt) were measured in the PBMCs of adult, old, MCI, and AD men and women initially and after 3 years in the same population. An age- and disease-associated decline in mini-mental state examination (MMSE) scores and lymphocyte proliferation of MCI and AD men and women were observed. An age- and disease-related increase in serum APP, cortisol levels, and TChE activity were observed in men and women. Enhanced production of Th1 cytokine, IL-2, pro-inflammatory cytokines, and suppressed intracellular transcription factors may promote the inflammatory environment in MCI and AD patients. The expression of CREB and Akt was lower in MCI and AD men, while the expression of p-ERK was higher, and p-CREB was lower in MCI and AD women after 3 years. These results suggest that changes in specific intracellular signaling pathways may influence alterations in cell-mediated immunity to promote disease progression in MCI and AD patients.

17ß-Estradiol Concentration and Direct ß2-Adrenoceptor Inhibition Determine Estrogen-Mediated Reversal of Adrenergic Immunosuppression.

Background: Sympathetic innervation of lymphoid organs, and the presence of 17ß-estradiol (estrogen or E2) and adrenergic receptors (ARs) on lymphocytes, suggests that sympathetic stimulation and hormonal activation may influence immune functions. Purpose: Modeling and simulating these pathways may help to understand the dynamics of neuroendocrine-immune modulation at the cellular and molecular levels. Methods: Dose- and receptor-dependent effects of E2 and AR subtype-specific agonists were established in vitro on lymphocytes from young male Sprague-Dawley rats and were modeled in silico using the MATLAB Simbiology toolbox. Kinetic principles were assigned to define receptor-ligand dynamics, and concentration/time plots were obtained using Ode15s solvers at different time intervals for key regulatory molecules. Comparisons were drawn between in silico and in vitro data for validating the constructed model with sensitivity analysis of key regulatory molecules to assess their individual impacts on the dynamics of the system. Finally, docking studies were conducted with key ligands E2 and norepinephrine (NE) to understand the mechanistic principles underlying their interactions. Results: Adrenergic activation triggered proapoptotic signals, while E2 enhanced survival signals, showing opposing effects as observed in vitro. Treatment of lymphocytes with E2 shows a 10-fold increase in survival signals in a dose-dependent manner. Cyclic adenosine monophosphate (cAMP) activation is crucial for the activation of survival signals through extracellular signal-regulated kinase (p-ERK) and cAMP responsive element binding (p-CREB) protein. Docking studies showed the direct inhibition of ERK by NE and ß2-AR by E2 explaining how estrogen signaling overrides NE-mediated immunosuppression in vitro. Conclusion: The cross-talk between E2 and adrenergic signaling pathways determines lymphocyte functions in a receptor subtype and coactivation-dependent manner in health and disease.

Neuroimmunomodulation by estrogen in health and disease.

Systemic homeostasis is maintained by the robust bidirectional regulation of the neuroendocrine-immune network by the active involvement of neural, endocrine and immune mediators. Throughout female reproductive life, gonadal hormones undergo cyclic variations and mediate concomitant modulations of the neuroendocrine-immune network. Dysregulation of the neuroendocrine-immune network occurs during aging as a cumulative effect of declining neural, endocrine and immune functions and loss of compensatory mechanisms including antioxidant enzymes, growth factors and co-factors. This leads to disruption of homeostasis and sets the stage for the development of female-specific age-associated diseases such as autoimmunity, osteoporosis, cardiovascular diseases and hormone-dependent cancers. Ovarian hormones especially estrogen, play a key role in the maintenance of health and homeostasis by modulating the nervous, endocrine and immune functions and thereby altering neuroendocrine-immune homeostasis. Immunologically estrogen's role in the modulation of Th1/Th2 immune functions and contributing to pro-inflammatory conditions and autoimmunity has been widely studied. Centrally, hypothalamic and pituitary hormones influence gonadal hormone secretion in murine models during onset of estrous cycles and are implicated in reproductive aging-associated acyclicity. Loss of estrogen affects neuronal plasticity and the ensuing decline in cognitive functions during reproductive aging in females implicates estrogen in the incidence and progression of neurodegenerative diseases. Peripherally, sympathetic noradrenergic (NA) innervations of lymphoid organs and the presence of both adrenergic (AR) and estrogen receptors (ER) on lymphocytes poise estrogen as a potent neuroimmunomodulator during health and disease. Cyclic variations in estrogen levels throughout reproductive life, perimenopausal surge in estrogen levels followed by its precipitous decline, concomitant with decline in central hypothalamic catecholaminergic activity, peripheral sympathetic NA innervation and associated immunosuppression present an interesting study to explore female-specific age-associated diseases in a new light.

Age-associated decline in neural, endocrine, and immune responses in men and women: Involvement of intracellular signaling pathways.

The aim of this study was to investigate the alterations in the neuroendocrine-immune functions by using human peripheral blood mononuclear cells (hPBMCs) from three age groups (young, middle-aged, and old) of men and women for the analyses of lymphocyte proliferation and cytokine production, expression of cell signaling molecules, nitric oxide (NO) production, and expression of p-tyrosine hydroxylase (TH). Serum was examined for levels of testosterone in men, 17-ß-estradiol in women, and cortisol in both sexes. Lymphoproliferation, expression of p-ERK, p-CREB, p-Akt, and p-TH, and levels of serum sex steroid hormones declined with age in men and women. However, TNF-α production and serum cortisol level increased with age in men and women. mTOR expression was higher in older men while it was lower in older women. IFN-γ and IL-6 production and expression of p-TH and p-mTOR were differentially regulated in men and women. These results suggest that intracellular signaling mediators may be involved in the age-related alterations in the neuroendocrine-immune interactions in men and women.

Sex-Based Differences in the Cytokine Production and Intracellular Signaling Pathways in Patients With Rheumatoid Arthritis.

OBJECTIVES: This study aims to investigate lymphoproliferation, cytokine production, and intracellular signaling molecules in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals and rheumatoid arthritis (RA) patients to understand the extent of the involvement of these pathways in the pathogenesis of RA. PATIENTS AND METHODS: The study included 65 participants (29 males, 36 females; mean age 51.8±10.3 years; range, 37 to 71 years) who were categorized into four groups as healthy males (n=22, mean age 49.8±10.6 years; range, 39 to 65 years), male RA patients (n=7, mean age 51.8±13.9 years; range, 37 to 68 years), healthy females (n=20, mean age 53.7±8.8 years; range, 42 to 67 years), and female RA patients (n=16, mean age 52.9±10.4 years; range, 40 to 71 years). PBMCs were collected from the participants and analyzed for Concanavalin A (Con A)-induced lymphoproliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cytokine production, and phospho-signal transducer and activator of transcription 3 (p-STAT-3), phospho-extracellular-signal-regulated kinase (p-ERK), phospho-cAMP response element binding (p-CREB), and phospho-protein kinase B expressions using enzyme-linked immunosorbent assay. Short form of the Arthritis Impact Measurement Scales 2 and multidimensional health assessment questionnaire were used to measure the level of disability and the quality of life. RESULTS: In RA patients, production of Con A-induced interleukin (IL)-2 and IL-17 was higher in both sexes while interferon-gamma levels decreased in RA females alone. Expression of p-STAT-3 in PBMCs increased in RA males while it was unaltered in RA females. p-ERK expression was not altered while p-CREB expression was enhanced in RA males and females. Protein-protein interaction analyses demonstrated that these and other key signaling molecules were dysregulated in RA patients. CONCLUSION: Our results suggest that sex-based differences in RA pathogenesis result from differential alterations in signaling pathways to influence the inflammatory process.

Estrogen-induced neuroimmunomodulation as facilitator of and barrier to reproductive aging in brain and lymphoid organs.

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.

Noni (Morinda citrifolia L.) fruit juice delays immunosenescence in the lymphocytes in lymph nodes of old F344 rats.

OBJECTIVE: Aging is associated with the development of diseases because of immunosuppression and altered functioning of the neuroendocrine system. The medicinal properties of Morinda citrifolia L. have been widely exploited for the treatment of age-associated diseases. This study aims to investigate the in vitro and in vivo effects of noni (M. citrifolia) fruit juice (NFJ) on neuro-immunomodulation in the lymph node lymphocytes of F344 rats. METHODS: Lymphocytes isolated from axillary and inguinal lymph nodes of young (3-4 months) and old (18-21 months) rats were treated in vitro with different concentrations (0.0001%, 0.01%, and 1%) of NFJ for a period of 24 h. In the in vivo study, old (16-17 months) male F344 rats were treated with 5 mL/kg body weight of 5%, 10% and 20% of NFJ, twice a day, by oral gavage, and lymph node lymphocytes were isolated after 60 d. Concanavalin A (Con A)-induced lymphocyte proliferation, interleukin-2 (IL-2) and interferon-γ (IFN-γ) production and expression of intracellular markers, such as phospho-extracellular signal-regulated kinase (p-ERK1/2), phospho-cAMP response element-binding protein, phospho-protein kinase B (p-Akt), phospho-tyrosine hydroxylase (p-TH), phospho-nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor-α (p-IκB-α) and phospho-nuclear factor-κB (p-NF-κB p65 and p50) were examined in the lymphocytes of lymph nodes. RESULTS: NFJ increased Con A-induced lymphocyte proliferation, IL-2 and IFN-γ production, and p-ERK1/2 expression both in vitro and in vivo. In in vivo NFJ-treated old rats, lymph node lymphocytes showed increased expression of p-TH and Akt, nitric oxide production and decreased expression of p-NF-κB p65 and p50. CONCLUSION: These results suggest that the immunostimulatory properties of NFJ are facilitated through intracellular signaling pathways involving ERK1/2, Akt and NF-κB.

Noni (Morinda citrifolia L.) fruit juice reverses age-related decline in neural-immune interactions in the spleens of old F344 rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Various parts of the tropical plant, Morinda citrifolia L. (Noni), have been widely used in traditional medicine in South and Southeast Asia for several centuries. The therapeutic effects of the noni are believed to be mediated through several phytochemicals such as anthraquinones, iridoid, fatty acid glycosides, alcohols, etc. AIM OF THE STUDY: The aim of the study is to investigate the effects of Morinda citrifolia fruit juice (noni fruit juice; NFJ) on neural-immune interactions through the involvement of intracellular signaling pathways both in vitro and in vivo in the splenic lymphocytes of young and old male F344 rats. MATERIAL AND METHODS: In the in vitro study, splenocytes from young and old F344 rats were isolated and treated with 0.0001-1% concentrations of NFJ for a period of 24h, while in the in vivo study, old F344 rats were orally administered (5ml/kg body weight) with NFJ (5%, 10% and 20%) twice daily for 60 days. After the treatment period, concanavalin A (Con A)-induced lymphocyte proliferation, cytokines (IL-2, IFN-γ, IL-6, and TNF-α) production, expression of tyrosine hydroxylase (p-TH), nerve growth factor (NGF), m-TOR, IκB-α, p-NF-κB (p50 and p65), p-ERK, p-Akt, p-CREB and lipid peroxidation, protein carbonyl formation, nitric oxide (NO) production were examined in the splenocytes. RESULTS: In vitro NFJ incubation of splenic lymphocytes increased Con A-induced lymphocyte proliferation, IL-2 and IFN-γ production, and expression of p-ERK, p-Akt, and p-CREB in young and old rats. In vivo treatment of old rats with NFJ increased lymphoproliferation, IL-2 and IFN-γ production, the expression of p-TH, NGF, and NO production, and suppressed IL-6 production, lipid peroxidation, protein carbonyl formation, and the expression of IκB-α and p-NF-κB (p50) in the splenocytes. CONCLUSION: Taken together, these results suggest that Morinda citrifolia fruit juice enhanced neural-immune interactions and cell survival pathways while inhibiting inflammatory processes that may be useful in the treatment of age-associated diseases.

Interrelationship between Mini-Mental State Examination scores and biochemical parameters in patients with mild cognitive impairment and Alzheimer's disease.

AIM: The aim of the present study was to provide first-hand information about the prevalence of mild cognitive impairment (MCI) and Alzheimer's disease (AD) in Tamil Nadu, a southern state in India, and examine if there exists a relationship between cognitive functions and biochemical parameters in these patients. METHODS: Surveys were collected from adults, older men and women (n = 3126) from different regions of Tamil Nadu, which were followed up after 12 months for 1337 participants. Mini-Mental State Examination (MMSE) scores, lipid profile, and liver function tests were carried out in the elderly, MCI and AD patients. Based on the MMSE scores, the elderly population was classified into old control (28.97 ± 1.49; n = 1868), MCI (19.58 ± 1.17; n = 734) and AD (7.18 ± 1.38; n = 304) groups. Peripheral blood samples were collected after overnight fast from both male and female volunteers (n = 40 per group) who were categorized as young adult control, old control, MCI and AD. RESULTS: AD patients showed lower MMSE scores compared with the young adults, old and MCI groups, and MMSE further decreased at follow-up examination a year later. In the serum of AD patients, high-density lipoprotein, alkaline phosphatase activity and bilirubin levels were lower, whereas low-density lipoprotein, total cholesterol and triglycerides levels were higher. MMSE was positively correlated with high-density lipoprotein, and negatively correlated with other lipid parameters in AD. CONCLUSIONS: Hypercholesterolemia is a risk factor for AD that might result in neurotoxicity and cognitive impairment. Dysfunction of lipoprotein and heme metabolism might also provide additional targets for AD diagnosis. Geriatr Gerontol Int 2017; 17: 1737-1745.

Estrogen-induced neuroprotective and anti-inflammatory effects are dependent on the brain areas of middle-aged female rats.

BACKGROUND: Reproductive aging in females is characterized by fluctuations and precipitous decline in estrogen levels, which may lead to reduction in cognitive function and age-associated neurodegenerative disorders. The nature of estrogen-mediated neuronal plasticity is unknown during reproductive aging. We hypothesize that estrogen treatment of early middle-aged ovariectomized rats may exert specific effects in the brain by modulating signaling pathways regulating metabolic enzymes, inflammatory markers, antioxidant status, cholinergic function and survival signals. PURPOSE: To investigate the mechanisms of estrogen-induced effects on neuroprotection and neuroinflammation through the involvement of intracellular signaling pathways in brain areas of ovariectomized (OVX) middle-aged (MA) female rats. METHODS: Ovariectomized early MA female Sprague-Dawley rats (n=8/group) were implanted with 17ß-estradiol (E2) 30-day release pellets (0.6µg and 300µg). At the end of the treatment period, frontal cortex (FC), striatum (STR), medial basal hypothalamus (MBH), and hippocampus (HP) were isolated and examined for the expression of tyrosine hydroxylase (p-TH), nerve growth factor (NGF), p-NF-κB (p50 and p65)and p-ERK, p-CREB, p-Akt, and activities of cholinesterases and antioxidant enzymes, key regulatory enzymes of metabolic pathways, and nitric oxide production. RESULTS: E2 enhanced p-TH expression in FC and HP, reduced NGF expression in HP, and suppressed p-NF-κB expression in FC and STR. It also increased the expression of molecular markers (p-ERK, p-CREB and p-Akt), and nitric oxide production in various brain areas, while differentially regulating the activities of metabolic enzymes and cholinesterases. CONCLUSION: Estrogen modulates the neural and inflammatory factors, and intracellular markers depending on the brain areas that may influence differential remodeling of neuronal circuitry which can be used to develop therapeutic strategies in cognitive impairment and neurodegenerative disorders in aging.

Phytochemicals in Morinda citrifolia fruit selectively modulate age-associated immunity and antioxidant enzyme activities through ERK pathway in splenic lymphocytes of male F344 rats.

CONTEXT/OBJECTIVE: The mechanisms of immunomodulatory effects of Morinda citrifolia (Noni) were examined through intracellular signaling pathways in the splenocytes and their modulation by phytochemicals using bioinformatics tools. MATERIALS AND METHODS: Noni fruit juices without seeds (NSL) and with seeds (NWS) were co-incubated in vitro with splenocytes from young, middle-aged and old F344 male rats and proliferation of lymphocytes, cytokine production, antioxidant enzyme activities and intracellular signaling markers were measured. RESULTS: NSL decreased lymphoproliferation in early middle-aged rats, and IL-2 and IFN-γ production in old rats. In contrast, NWS enhanced lymphoproliferation in young and old rats, IL-2 and IFN-γ production in middle-aged and old rats. The activities of antioxidant enzymes were augmented by NWS and NSL in old rats. NWS reversed age-related increase in lipid peroxidation in all age-groups, while NSL increased lipid peroxidation in old rats. NSL increased p-ERK in old rats and decreased p-CREB in young and middle-aged rats. In contrast, NWS decreased p-ERK in all age groups and increased p-CREB in old rats. Both NSL and NWS increased p-Akt expression in middle-aged and old rats. Both NSL and NWS suppressed p-NF-κB expression in middle-aged and old rats. Docking studies demonstrated that Noni phytochemicals, damnacanthal, myricetin and ursolic acid, are potent inhibitors of ERK with binding sites in the catalytic and phosphorylation sites of the molecule. CONCLUSIONS: These results suggest that Noni fruit juices with or without seeds modulate cell-mediated immunity and antioxidant enzyme activities based on the phytochemicals that may differentially influence cell signaling and therefore, age-associated immunity.

Estrogen upregulates inflammatory signals through NF-κB, IFN-γ, and nitric oxide via Akt/mTOR pathway in the lymph node lymphocytes of middle-aged female rats.

The alterations in the secretion of sex steroids, especially estrogen, in females throughout reproductive life and its decline with age alters the functions of the neuroendocrine-immune network and renders them susceptible to age-related diseases and cancers. This study investigates the mechanisms of estrogen-induced alterations in cell-mediated immune and inflammatory responses in the lymphocytes from lymph nodes (axillary and inguinal) of ovariectomized (OVX) middle-aged female rats. Ovariectomized middle-aged (MA) Sprague-Dawley female rats (n=8) were implanted with 17ß-estradiol (E2) 30-day release pellets (0.6 and 300µg). At the end of the treatment period, lymph nodes (axillary and inguinal) were isolated and examined for serum 17ß-estradiol, lymphoproliferation, cytokine production, expression of p-Akt, p-mTOR, p-IκB-α and p-NF-κB (p50 and p65), extent of lipid peroxidation, nitric oxide (NO) production, cytochrome c oxidase activity and reactive oxygen species (ROS) production. There was an OVX-related decline in serum 17ß-estradiol level, Con A-induced lymphoproliferation, p-Akt and p-mTOR expression, and cytochrome c oxidase (COX) activity. E2 supplementation increased serum 17ß-estradiol level, lymphoproliferation, expression of p-Akt, p-mTOR, p-IκB-α and p-NF-κB (p50 and p65), lipid peroxidation, IFN-γ, TNF-α, ROS and NO production, while it decreased IL-6 production. E2 mediates inflammatory responses by increasing the levels of NO and TNF-α by up regulating IFN-γ and simultaneously promotes aging through the generation of free radicals as reflected by increased lipid peroxidation and ROS production in lymph nodes. These findings may have wide implications to immunity and inflammatory disorders including autoimmune diseases predominantly prevalent in females.

Estrogen modulates ß2-adrenoceptor-induced cell-mediated and inflammatory immune responses through ER-α involving distinct intracellular signaling pathways, antioxidant enzymes, and nitric oxide.

Sympathetic noradrenergic neuronal activity in the lymphoid organs regulates immunity through the release and binding of norepinephrine to ß2-adrenergic receptors (AR) on lymphocytes. In women, estrogen modulates immune responses during menstrual cycles, and in aging and age-associated diseases. The intent of the present study is to characterize the extent of immunomodulation by ß2-AR in the presence of estrogen and the involvement of intracellular signaling mechanisms including the role of antioxidant enzymes (AOE) in lymphocytes. In vitro effects of terbutaline, ß2-AR agonist, either alone or in combination with 17ß-estradiol (E2) were examined on splenocyte proliferation, cytokine (IFN-γ, IL-2, and IL-6) production, intracellular signaling molecules (p-ERK, p-CREB, p-Akt, and p-NF-κB) expression, NO production, and AOE activities [superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx)]. The specificity of their actions was investigated using ß-AR antagonist, and inhibitors of signaling targets and inducible nitric oxide synthase (iNOS). Terbutaline suppressed T cell proliferation and IL-6 production and increased AOE activities involving ERK, PKA, PKC, and NF-κB pathways and NO production. E2 alone enhanced T cell proliferation and decreased IL-6 production and NF-κB expression through ER-α. E2 in the presence of terbutaline reversed terbutaline-induced effects on T cell proliferation, IL-6 production, p-ERK and p-CREB expression, AOE activities, NO production, and NF-κB expression. Estrogen through ER-α differentially modulates ß2-AR-induced immune responses involving ERK, PKA, PKC, and NF-κB pathways, and NO that may be responsible for estrogen-induced immunosenescence and development of female-specific diseases.

Estrogen modulates neural-immune interactions through intracellular signaling pathways and antioxidant enzyme activity in the spleen of middle-aged ovariectomized female rats.

Modulation of neural-immune interactions by estrogen in the spleens of ovariectomized (OVX) middle-aged female rats was examined. Con A-induced lymphoproliferation, splenic tyrosine hydroxylase (TH) and nerve growth factor (NGF) expression, levels of p-ERK 1/2, p-CREB, and p-Akt, and activity of superoxide dismutase decreased in OVX rats while estrogen treatment enhanced their expression, levels, and activity. Also, estrogen treatment enhanced Con A-induced IFN-γ production and decreased Con A-induced IL-2 production compared to OVX animals. In contrast, estrogen increased the extent of lipid peroxidation and protein carbonyl formation while OVX induced a decline in protein carbonyl formation. These results suggest that estrogen enhances neural-immune interactions while simultaneously affecting it through generation of free radicals as reflected by increased lipid peroxidation and protein carbonyl formation.

Selective modulation of lymphoproliferation and cytokine production via intracellular signaling targets by α1- and α2-adrenoceptors and estrogen in splenocytes.

UNLABELLED: The mechanistic implications of the presence of sympathetic noradrenergic innervation in lymphoid organs in synaptic association with lymphocytes open to the influence of hormonal fluctuations throughout reproductive age in females has not been investigated yet. OBJECTIVES: The aim of the present study is to investigate the role of alpha-adrenoceptors (α-ARs) and estrogen in modulating immune responses in the spleen through intracellular signaling targets such as ERK 1/2, CREB, Akt, NF-κB. METHODS: Splenocytes from young Sprague-Dawley rats were incubated with α1- and α2- AR specific agonists, phenylephrine and clonidine, without and with 17b-estradiol or specific antagonists prazosin and idazoxan to examine their effects on proliferation, cytokine production, nitric oxide production, and intracellular signaling molecules. RESULTS: α1-AR stimulation inhibited lymphocyte proliferation and IFN-g production and enhanced IL-2, p-ERK and p-CREB expression. Co-stimulation using estrogen enhanced cytokine production and suppressed p-Akt expression. α1-AR blockade reversed agonist-induced IL-2 production alone. α2-AR stimulation inhibited lymphocyte proliferation, p-ERK and p-CREB expression, and increased p-NF-kB and p-Akt expression. Co-stimulation with estrogen increased IL-2 and suppressed p-CREB expression. α2-AR Idazoxan prevented IL-2 production in the absence and presence of estrogen, and reversed clonidine-induced increase in NO production and p-ERK and p-Akt expression in the presence of estrogen. CONCLUSIONS: These results suggest that the cell-mediated immune responses are selectively modulated depending upon the subtypes of α-AR and further, these effects are differentially regulated in the presence of estrogen mediated through selective alteration in the intracellular signaling pathways involving ERK, CREB, Akt, and NF-κB.

Estrogen modulates in vitro T cell responses in a concentration- and receptor-dependent manner: effects on intracellular molecular targets and antioxidant enzymes.

Estrogen is a key hormone in facilitating ovulation and maintenance of pregnancy in young females and subsequent decline in its production contributes to the development of age-associated disorders such as hormone-dependent cancer, osteoporosis, and cardiovascular diseases. The mechanisms through which estrogen promotes female-specific diseases with advancing age are unclear especially, its effects on immune system which is vital for the maintenance of homeostasis and health. Although the diverse effects of estrogen on Th immunity (Th1 vs. Th2) have been characterized in several cell-types and animal models, there is no direct mechanistic study to understand its immunomodulatory actions. The purpose of this study is to investigate whether the in vitro effects of 17ß-estradiol on lymphocytes from the spleen influence cell-mediated immune responses based on its concentration and type of estrogen receptors (ERs) and to assess its mechanism of action at the cellular level. Lymphocytes from the spleens of young Sprague-Dawley rats were isolated and incubated with various concentrations of 17ß-estradiol (10(-6)-10(-14)M) and specific ERα- and ß-agonists (10(-6)M, 10(-8)M and 10(-10)M) without or with concanavalin A (Con A) to measure T lymphocyte proliferation, IFN-γ and IL-2 production, p-ERK 1/2, p-CREB, and p-Akt, activities of antioxidant enzymes[superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)], and nitric oxide (NO) production. The specificity of ER-mediated actions in lymphocytes was examined by coincubation with nonspecific ER antagonists ICI(182,780) or tamoxifen. Lower concentrations of 17ß-estradiol enhanced proliferation of T lymphocytes and IFN-γ production without or with Con A stimulation but had no effect on IL-2 production. ERα and ERß agonists induced an increase in T cell proliferation and IFN-γ production and these effects were inhibited by tamoxifen. ERß agonist alone enhanced IL-2 production by the lymphocytes. Coincubation with 17ß-estradiol and ERα- and ß-agonists augmented p-ERK 1/2, p-CREB, and p-Akt expression in the lymphocytes and tamoxifen reversed the ER agonist-induced effects on these molecular targets. Estrogen increased the activities of SOD, CAT, and GPx in both non-stimulated and Con A-stimulated splenocytes in a concentration-dependent manner. Both ERα- and ß-agonists enhanced CAT and GPx activity while ERα-agonist decreased SOD activity and ERß-agonist increased SOD activity. The effects of ER agonists on the antioxidant enzymes were reversed by ICI(182,780). Coincubation of lower doses of 17ß-estradiol with Con A and both ER agonists enhanced NO production while higher dose of estrogen with Con A and ERα agonist suppressed its production and these effects were reversed by tamoxifen. Taken together, these results suggest that the effects of estrogen on the cell-mediated immune responses are dependent upon its concentrations and mediated through specific estrogen receptors involving intracellular signaling pathways and antioxidant enzymes.

Menstrual cycle and reproductive aging alters immune reactivity, NGF expression, antioxidant enzyme activities, and intracellular signaling pathways in the peripheral blood mononuclear cells of healthy women.

Reproductive senescence in women is a process that begins with regular menstrual cycles and culminates in menopause followed by gradual development of diseases such as autoimmune diseases, osteoporosis, neurodegenerative diseases, and hormone-dependent cancers. The age-associated impairment in the functions of neuroendocrine system and immune system results in menopause which contributes to subsequent development of diseases and cancer. The aim of this study is to characterize the alterations in immune responses, compensatory factors such as nerve growth factor (NGF) and antioxidant enzyme activities, and the molecular mechanisms of actions in the peripheral blood mononuclear cells (PBMCs) of young (follicular and luteal phases), middle-aged, and old healthy women. Peripheral blood mononuclear cells were isolated from young women in follicular and luteal phases of the menstrual cycle (n=20; 22.6±2.9 yrs), middle-aged women (n=19; 47.1±3.8 yrs; perimenopausal) and old (n=16; 63.2±4.7 yrs; post-menopausal) women and analyzed for Concanavalin (Con A)-induced proliferation of lymphocytes and cytokine (IL-2 and IFN-γ) production, expression of NGF, p-NF-κB, p-ERK, p-CREB, and p-Akt, antioxidant enzymes [superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx), glutathione-S-transferase (GST)], extent of lipid peroxidation, and nitric oxide (NO) production. Serum gonadal hormones (17ß-estradiol and progesterone) were also measured. A characteristic age- and menstrual cycle-related change was observed in the serum gonadal hormone secretion (estrogen and progesterone), T lymphocyte proliferation and IFN-γ production. Salient features include the age-related decline observed in target-derived growth factors (lymphocyte NGF expression), signaling molecules (p-ERK/ERK and p-CREB/CREB ratios) and compensatory factors such as the activities of plasma and PBMC antioxidant enzymes (SOD and catalase) and NO production. Further, an age-associated increase in p-NF-κB expression and lipid peroxidation was observed. Also, serum 17ß-estradiol levels were positively correlated with IFN-γ production, SOD activity and NGF expression in the PBMCs. These results suggest that alterations in the levels of gonadal hormones are associated with immunosenescence characterized by decreased IFN-γ production and proliferation of T lymphocytes, decline in NGF expression, SOD and catalase activities, NO production, and signaling mechanisms and thus, may increase the incidence of diseases and cancer in women.

Bacopa monnieri and L-deprenyl differentially enhance the activities of antioxidant enzymes and the expression of tyrosine hydroxylase and nerve growth factor via ERK 1/2 and NF-κB pathways in the spleen of female wistar rats.

Aging is characterized by development of diseases and cancer due to loss of central and peripheral neuroendocrine-immune responses. Free radicals exert deleterious effects on neural-immune functions in the brain, heart, and lymphoid organs and thus, affecting the health. Bacopa monnieri (brahmi), an Ayurvedic herb, and L-deprenyl, a monoamine oxidase-B inhibitor, have been widely used in the treatment of neurodegenerative diseases. The purpose of this study was to investigate whether brahmi (10 and 40 mg/kg BW) and deprenyl (1 and 2.5 mg/kg BW) treatment of 3-month old female Wistar rats for 10 days can modulate the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)] in the brain and spleen. In addition, the effects of these compounds on the expression of tyrosine hydroxylase (TH), nerve growth factor (NGF), the intracellular signaling markers, p-ERK1/2, p-CREB, and p-NF-kB, and nitric oxide (NO) production were measured in the spleen by Western blot analysis. Both brahmi and deprenyl enhanced CAT activity, and p-TH, NGF, and p-NF-kB expression in the spleen. However, deprenyl alone was found to enhance the p-ERK1/2 and p-CREB expression in the spleen. The activities of SOD, CAT, and GPx in the thymus, mesenteric lymph nodes, heart, and brain areas (frontal cortex, medial basal hypothalamus, striatum, and hippocampus) were differentially altered by brahmi and deprenyl. Brahmi alone enhanced NO production in the spleen. Taken together, these results suggest that both brahmi and deprenyl can protect the central and peripheral neuronal systems through their unique effects on the antioxidant enzyme activities and intracellular signaling pathways.

Diverse age-related effects of Bacopa monnieri and donepezil in vitro on cytokine production, antioxidant enzyme activities, and intracellular targets in splenocytes of F344 male rats.

Aged people are more prone to developing neurodegenerative and infectious diseases, autoimmune disorders, and cancer due to impairment of neuroendocrine-immune functions. Neuronal degeneration and immunosuppression aided by increased generation of reactive oxygen species combined with loss of antioxidant enzyme activities promote the aging process. Bacopa monnieri (brahmi), an Ayurvedic herb, and donepezil, a reversible acetylcholinesterase inhibitor, have been used to reverse cognitive dysfunctions in several neurodegenerative diseases. The aim of this study was to investigate the effects of in vitro incubation of lymphocytes from spleens of young (3-month-old), early middle-aged (8- to 9-month-old), and old (18-month-old) F344 rats with brahmi (0.001%, 0.01%, 0.05%, 0.1%, and 1%) and donepezil (5, 10, 25, 50, and 100 µg/ml) on Concanavalin (Con A)-induced proliferation of T lymphocytes and cytokine production, and the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST)]. In addition, the effects of these compounds on the expression of intracellular signaling pathway markers (ERK, p-ERK, CREB, p-CREB, Akt and p-Akt), nitric oxide (NO) production, and the extent of lipid peroxidation were measured in the splenocytes. Age-related decline in Con A-induced proliferation of T lymphocytes was not reversed by treatment with brahmi and donepezil but donepezil alone further reduced the lymphocyte proliferation in young rats. Lower doses of brahmi treatment reversed the age-related decrease in Con A-induced IL-2 and IFN-γ production by the splenocytes while their production by splenocytes was suppressed by treatment with donepezil in the young and early middle-aged rats. An age-associated decline in the activities of SOD, CAT, GPx, and GST was evident in the lymphocytes of spleen. Brahmi enhanced CAT activity of lymphocytes in all the age groups while donepezil increased SOD activity in old rats. Both brahmi and donepezil increased GPx and GST activities in a dose-dependent manner in the lymphocytes of all age groups. There was an age-related decline in NO production and increase in the extent of lipid peroxidation in the splenocytes. Brahmi and donepezil increased NO production in the lymphocytes of early middle-aged and old rats. Brahmi reversed the age-related increase in lipid peroxidation in the splenocytes of both early-middle-aged and old rats while donepezil suppressed lipid peroxidation only in the splenocytes of old rats. The expressions of p-ERK1/2 and p-CREB in the splenocytes were elevated following treatment with brahmi and donepezil in the early middle-aged and old rats while age-related decline in p-Akt expression was reversed by treatment of lymphocytes with brahmi alone in early-middle-aged and old rats. Taken together, these results suggest that both brahmi and donepezil exert distinct age-related effects on the cell-mediated immune responses through selective modulation of antioxidant enzyme activities and intracellular targets that may influence the therapeutic efficacy of these drugs in neurodegenerative diseases.