Immune Modulation of Interleukin-1α by Noradrenaline and Cortisol in Women with PCOS (Psychoneuroimmunology Aspect)

Background & Objectives: Polycystic ovary syndrome (PCOS) is a common complex condition in women associated with reproductive, metabolic and psychological features. Evidences from studies on women with PCOS and on an experimental rat PCO model suggest that the sympathetic regulatory drive to the ovary may be unbalanced (hyperactivity). The sympathetic nervous system (SAS) the hypothalamic-pituitary-adrenal (HPA) axis (cortisol) the major integrative and components of different immune responses. this to determine cytokines of four cytokines in Serum cytokines: TNFα group: Interleukin 1α (p<0.001) and IL-beta (p=0.017). Discussion: The results of this study indicate that overactivity of sympathetic nervous system (SNS) in women with PCO impairs 1) three cytokines pattern and 2) the increased sympathetic outflow may be related to hormonal and metabolic features of this syndrome. Conclusion: The activation of SNS during an immune response might be aimed to localize the inflammatory response. The results of this study confirm low-grade chronic inflammation in PCOS.


INTRODUCTION
Polycystic ovary syndrome (PCOS), the most common female endocrine disorder, is a complex and heterogenic disease with unknown etiology. PCOS is characterized by reproductive disturbances including chronic anovulation, hyperandrogenism and polycystic ovaries [1]. PCOS, one of the most common causes of female infertility, its prevalence among infertile women is 15-20% [2]. Clinically, PCOS has reproductive, psychological and metabolic features, the latter predisposing to cardiovascular disease (CVD) [3]. Although physical symptoms of PCOS are increasingly recognized by practicing clinicians, little attention has focused on psychological correlates of this frequent endocrine disorder. Emotional disturbances such as depression and chronic stress are associated with certain physiological changes such as increased immune system activity and proinflammatory markers, which increase one's risk for eventual development of chronic disorders such as cardiovascular disease, diabetes, and cancer [4]. Some evidence suggests strong associations between depression and other proinflammatory cytokines such as Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-1β (IL-1β) [5]. Prospective studies employing pharmaceutical treatment illustrate the relationship between mood and inflammation. For example, antidepressants have been shown to inhibit pro-inflammatory cytokine production both In vitro and In vivo [6]. Women with PCOS have chronic low-level inflammation [7] and recent evidence has been focused on a condition of low-grade chronic inflammation as a potential cause of the long-term consequence of the syndrome [8]. Low-grade chronic inflammation seems to play an essential role in insulin resistance and its metabolic consequences [9] and may also play a role in the pathogenesis of PCOS [10]. Normal reproductive functioning in healthy, fertile women exists due to the presence of unique immunologic barriers, and also due to harmonic functioning of immunoregulatory by neuro-immunologic mechanisms. It is believed that cytokines produced by activated lymphocytes, monocytes and macrophages, such as interleukin-1, interleukin-6, gammainterferon and others, affect the processes of fertilization, development and implantation of the fertile egg in the uterus, and this may be a reason for habitual miscarriage and infertility [11]. In female reproductive function neuropeptides, growth factors and cytokines are expressed in reproductive organs and tissues, where they interact with afferent endocrine messages to modulate cell proliferation and differentiation, local hormone secretion and vascular function. These events regulate complex processes such as gonadotropin plasticity, ovulation, implantation and parturition [12]. During female reproductive life, the role of the cytokine cascade and regulation of the expression of cytokine genes in the processes of fertilization is very complex. Studies show Low-grade chronic inflammation is reflected by minor but significant increases in circulating levels of known inflammatory and acute phase proteins such as interleukin (IL)-1, IL-6, tumor necrosis factor-(TNF) and serum Creactive protein (CRP). One of the most prominent mediators of inflammation is the interleukin-1 (IL-1) family that consists of three different cytokines, the proinflammatory cytokines IL1α, IL1β and the physiological antagonist IL1 receptor antagonist (IL-1RA) [13]. IL-1α and IL-1β are produced by monocytes, macrophages, neutrophils and epithelial cells, and affect nearly every cell type [13,14]. Interleukin-1 (IL1) is a multifunctional cytokine and it has highly inflammatory features in reproductive biology and is believed to affect the processes of fertilization and implantation [14]. While human granulosa and cumulus cells synthesize IL1RA, expression of paracrine-acting IL-1 in the ovary could be involved in multiple steps leading to ovulation and, thereby, influence ovarian physiology [15]. The interactions among mood, behavior, and inflammation often manifest in a cluster of symptoms referred to as "sickness behavior" including depressed mood, anhedonia, fatigue, psychomotor retardation, decreased appetite, social withdrawal, sleep disturbances, cognitive dysfunction, and increase sensitivity to pain [16,17]. These symptoms generally are exacerbated when inflammation increases. The relations among these symptoms and inflammation are so robust that even exogenous administration of pro-inflammatory cytokines reliably produces sickness behavior symptoms [18]. Possibly due to chronically elevated inflammatory markers, PCOS women exhibit some sickness behavior symptoms, including fatigue, depressed mood, social withdrawal [19] and sleep disturbances [20]. Based on the immune studies and on the role of inflammatory processes in the pathogenesis of PCOS, we hypothesized that neuroimmune-endocine events are related to overactivity of the sympathetic nervous system that trigger cascade of intra ovary in PCO. There is now sufficient data to conclude that immune modulation by psychosocial stressors and/or interventions can lead to actual health changes.

METHODS
This study was carried out at February 2012-April 2013. Totally 85 PCO patients (case group) and 86 health women (control group) participated in this study. The diagnosis of PCOS was made according to the joint criteria of the European Society of Human Reproduction and Embryology and the American Society of Reproductive Medicine (ESHRE/ASRM) [21]. Data were collected from clinical and anthropometric variables, including hirsutism score, body mass index (BMI) and a demographic questionnaire inquiring about age, education, occupation, and duration of illness. This study was approved by the Ethics Committee of Tehran University of Medical Sciences. The study objectives were explained to the patients before they entered the study, and an informed consent was obtained from all.

Statistical Analysis
The quantitative variables were presented with mean and standard deviation and were compared between study groups with t test. The qualitative variables were presented with count and percentage and were compared between study groups with chi square test. According to departure from normal distribution, the median was used for presenting inflammatory cytokines, and for comparing of those variables between two study groups Mann-Whitney nonparametric test was used. Multinomial linear regression model was used to deleting the confounding effect. In this analysis, age (year), BMI (kg/m2), occupation, irregular menstrual, hirsutism and painful menstrual were including in the model as confounder.
Suffering from PCO was independent variable and each inflammatory cytokines separately were including in the model as dependent variable. The data were analyzed in IBM SPSS 19 (SPSS Inc, Chicago Ill). P value less than 0.05 considered as significant level.

RESULTS
The mean age of women the control group had an average of 2 years longer than patients in PCO group (P = 0.001). The two groups did not differ in height (P = 0.070), whereas weight in PCOS group was more than control group (P < 0.001) and BMI in PCO group was higher than the control group (P = 0.001). Duration of marriage and infertility also pregnancy and delivery count were not significant difference between two groups (Table 1). Education level of two groups was not statistically difference (P = 0.864). But occupation rate in PCO group was higher than control (P = 0.002). The symptom of PCO was significantly higher in PCO patients ( Table 2).

DISCUSSION
Cytokines and other humoral mediators of inflammation are potent activators of the central stress response, constituting the afferent limb of a feedback loop through which the immune/inflammatory system and the CNS communicate [22]. Some of the activating effects of cytokines on the HPA axis may be exerted indirectly by stimulation of the central catecholaminergic pathways.
Conversely, activation of the HPA axis has profound inhibitory effects on the inflammatory/immune response because virtually all the components of the immune response are inhibited by cortisol. Alterations of leukocyte traffic and function, decreases in production of cytokines and mediators of inflammation, and inhibition of the latter's effects on target tissues are among the main immunosuppressive effects of glucocorticoids [23,24].
The mammalian ovary has a rich sympathetic nerve supply with norepinephrine as the major sympathetic neurotransmitter [25]. In human, the ovary has a functional sympathetic innervation coupled to steroid secretion. The value for the concentration of norepinephrine in the ovary was similar to others previously reported and 10 times higher than rat ovarian tissue; this neurotransmitter is coupled to a steroidogenic response [26]. The immune system regulates CNS and interferes with steroidogenesis at the level of the adrenals, testes, and ovaries [27]. This study follows previous studies in the field of overactivity of the sympathetic system in patients with PCO and investigation of sympathoexcitation and cytokinins.
Cytokines and other inflammatory mediators can signal the brain, and the importance of this Neuroimmune-endocrine cross-talk becomes evident in the case of autoimmune and inflammatory diseases. Interleukin-1 (IL-1) has been shown by many investigators to be a potent activator of the HPA axis.
Adrenal steroidogenesis is under the control of the HPA axis. Furthermore, metabolic factors including insulin and obesity-related signals may play a role in the regulation of both enzymes involved in the steroidogenetic pathways, as well as in the regulation of the HPA axis [28]. Smagin et al. [29] in 1996 showed that noradrenergic terminals in the hypothalamus play a role in the IL-l-induced activation of the HPA axis. Hypothalamic noradrenaline (NA) release parallels the increases in plasma corticosterone induced by IL-1.
Noradrenergic innervation of the hypothalamus participates in adrenocortical responses to interleukin-1, and it is possible that noradrenergic terminals in the hypothalamus play a role in the IL-l-induced activation of the HPA axis [30]. There is evidence that the immune-HPA axis circuit is not the only one involving immune components and neuroendocrine mechanisms and that the immune system interacts at several levels with mechanisms integrated by the CNS.
The operation of a complex network of immuneneuroendocrine interactions contributes to immunoregulation and to the homeostatic adjustments necessary during diseases that involve the immune system, cathecolamines and glucocorticoids (stress hormones) are immune madulators and the role of cytokines as modulators of glucocorticoids receptors (GR) has received scarce attention. One of the effects of immune stimulation was elevation of glucocorticoid blood levels [31].
Besedovsky et al. [32] in 1986 originally used conditioned media from immune cells stimulated in vitro, which contain several immune-derived substances including lymphokines and monokines. The first pure lympho-monokine natural or recombinant found to stimulate ACTH and glucocorticoid release by mechanisms integrated at hypothalamic levels was IL-1. Milutinovic's hypothesize in 2011 is that modulation of glucocorticoid receptor (GR) expression and function may underlie possible PCOS-related impairment of feedback inhibition of HPA axis activity and imply that PCOS is associated with increased GR protein concentration and HPA axis sensitivity to dexamethasone [33]. IL-1 has been shown to upregulate GR mRNA expression in hypothalamic CRH secreting cells [34]. Then up-regulation of GR can be the reason of normal rate of cortisol in women with PCO.
Essentially, PCOS is a chronic condition with manifestations that begins most commonly in adolescence with oligomenorrhea / amenorrhea and transition into problems including infertility, metabolic complications and even cancer over time [35].
It has become increasingly clear that PCOS is an important metabolic disorder, as women with PCO may have significant insulin resistance, glucose intolerance, obesity, and dyslipidemia [36]. Therefore, it is not surprising that the risk of serious metabolic disorders such as type2 diabetes, metabolic syndrome, and subsequent cardiovascular disease [37] are significantly increased in women with PCOS. This is suggestive of possible involvement of sympathoexcitation in the pathogenesis of this condition [38]. The higher sympathetic tone is confirmed within PCOS, chronic inflammatory and/or psycho-emotional distress provoke a series of neuroimmunoendocrine interactions such as increased tissue and plasma levels of proinflammatory cytokines and neurotrophins, vegetodystonia, misbalance of neurotransmitters, hormones and immunity markers, activation of the HPA axis, insulin resistance, and atherosclerosis [38].
Our works in 2011 confirmed overactivity in sympathetic nervous system in rat modeling of PCOS [39,40] and in women with PCO [38].
Research over the past decade shows proinflammatory cytokine interleukin-1 (IL-1) produced during psychological and immunological stress plays a significant role in the neuroendocrine and stress responses [41].
Specifically, production of brain IL-1 is an important link in stress-induced activation of the HPA axis and secretion of glucocorticoids. Furthermore, IL-1 signaling and the resultant glucocorticoid secretion mediate the development of depressive symptoms associated with exposure to acute and chronic stressors, at least partly via suppression of hippocampal neurogenesis [42].
Kolbus et al. [14] in 2007 have shown that a common polymorphism of the interleukin-1alpha but not interleukin-1beta gene is associated with the presence of PCOS and with clinical parameters of women affected by this condition. IL-1β stimulates basal progesterone secretion by human granulosa and theca cells [43] and small and large follicles [44] In vitro and also IL-1β induce and increase levels of progesterone and PGF2-α in equine granulosa and cumulus cells which demonstrated that IL-1 is involved in equine oocyte In vitro maturation [45]. When stimulated by gonadotropins, IL-1β inhibits both LH/hCG and FSH stimulated progesterone and estradiol secretion by follicular theca and granulosa cells, affecting cAMP production, suggesting a follicle-stage dependent regulatory role of IL-1 on ovarian follicles [46].
Atsumi et al. [47] in 2014 have redefined inflammation as local activation of the inflammation amplifier, which causes an accumulation of various immune cells followed by deregulation of local homeostasis. The inflammation amplifier is activated by the stimulation of cytokines, such as TNF-α, IL-17, and noradrenaline, resulting in the subsequent expression of various target genes for chemokines.
Then, the significant decreasing of IL17 can be because of the regulatory role of noradrenaline. In this study, increasing of IL1α and β can obtain from a special relationship with noradrenaline and cortisol in women with PCOS. These results can indicate that 1) the increase in IL-1β (p <0.01) could be due to lack of ovulation (anovulation) in women with PCOS. One of the genetic factors that have been associated with PCOS is IL-1β, particularly synergy with psychogenic and neurogenic stressors are described. It has proactive effects on mood states. 2) Study of Kolbus et al. [14] in 2007 showed that a common polymorphism of the interleukin-1alpha gene is associated with the presence of PCOS. Increasing of IL1 α could be due to sympathoexitation in these patients [48].
Although crosstalk between these two systems is clear, the role of NE in mediating inflammatory responses is less clear, with evidence of both pro-and anti-inflammatory effects. 3) Regulation of IL-17 by adrenal hormones caused a significant reduction of this cytokine in the serum of patients [49]. 3) PCO is a chronic condition and recent studies show that PCO as chronic low-grade subclinical inflammation has been increasingly recognized as an interposer in the endocrine, metabolic and reproductive disturbances [50]. 4) In addition, this study shows that an abnormal lymphocyte subset distribution was observed in PCOS, possibly associated with an impaired cytokine pattern that can play a major role in the immunopathogenesis of PCO. IL1 extremely is high in PCO patients which; confirm the high activity of HPA axis and sympathetic overactivity. Then noradrenaline exerts anti-inflammatory and neuroprotective effects In vitro and In vivo and can regulates IL17 activity in PCO. 5) Immune-modulatory role of cortisol and noradrenaline in HPA axis and reproductive health of women. 6) SNS may thus offer a new therapeutic target in PCO and can be a novel biological target for the treatment of it [38].

CONCLUSION
This study shows that an abnormal lymphocyte subset distribution was observed in PCOS, possibly associated with an impaired cytokine pattern that can play a major role in the immunepathogenesis of PCOS.