Evaluating sex hormones and cytokine profile in Egyptian females with relapsing-remitting multiple sclerosis

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS), having two pathogenic components, inflammation and neurodegeneration [1]. The female predominance especially during reproductive ages in multiple sclerosis disease suggests that sex hormones interact with the immune system [2].

The exacerbations usually occur immediately premenstrual, when estrogen levels are low [3]. Also, the relapse of disease symptoms lowers during the last 3 months of pregnancy and again increases after delivery [4]. Furthermore, Tomassini and colleagues reported worsening of MS symptoms in about 50% of patients at menopause [5]. Alonso and colleagues reported that subjects receiving contraceptive therapy might be associated with a short-term reduction in the risk of developing MS. These changes are said to be due to immunological and hormonal changes [6]. Sex hormones appear to be involved in regulating the balance of cytokine secretion by T helper 1 and 2 (Th1, Th2), T regulatory (T reg cells), and antigen-presenting cells (APCs). The production of cytokines is the target of sex hormones in the regulation of the clinical manifestation of MS.

The objective of our study is to determine the hormonal profile and serum cytokine in Egyptian female patients with relapsing-remitting MS (RRMS) compared with healthy controls and their associations with disease disability.

Forty patients were included in our study; their age at onset of disease ranged from 16 to 35 years with a mean of 25.38 ± 4.9. The duration of illness ranged from 1 to 12 years with a mean of 4.85 ± 3.11. The number of relapses ranged from 2 to 9 attacks during the duration of illness with a mean of 3.93 ± 1.85. The EDSS ranged from 1 to 5.

Correlative results

Based on Pearson’s correlation coefficient, estrogen level showed significant positive correlation to IL-10 (r = 0.64, p ˂ 0.00001) and IL-4 (r = 0.78, p ˂ 0.00001) and significant negative correlation to TNF-α levels (r = − 0.44, p = 0.004), where higher estrogen levels associated with higher IL-10 and IL-4 and lower levels of TNF-α (Figs. 1, 2, and 3), but no significant correlation was found with EDSS (p ˃ 0.96) as illustrated in Table 2.

	Estrogen levels		Testosterone levels
	R	p value	R	p value
IL-10 level, pg/ml	0.64	˂ 0.00001*	− 0.02	0.9
IL-4 level, pg/ml	0.78	˂ 0.00001*	− 0.1	0.5
TNF-α level, pg/ml	− 0.44	0.004*	0.08	0.6
EDSS	− 0.007	0.96	− 0.25	0.11

IL-10 interleukin 10, IL-4 interleukin 4, TNF tumor necrosis factor alpha, EDSS Expanded Disability Status Scale

*Significant p˂0.05

No statistically significant correlation was found between testosterone, cytokines (IL-10, IL-4, TNF-α), and EDSS (p ˃ 0.05) as illustrated in Table 2.

There was no statistically significant correlation between cytokine profile (IL-10, IL-4, TNF-α) and EDSS (p ˃ 0.05).

Multivariate linear regression analysis using the cytokine profile (IL-10, IL-4, and TNF-α) as the dependent variable revealed estrogen level as the independent variable (p ˂ 0.001, ˂ 0.001, and 0.048, respectively).

Our study confirms the hypothesis that the cooperation between the immune, endocrine, and nervous system could be a possible reason for the influence of sex hormones in MS.

The present study showed that though not reaching statistical significance, estrogen and testosterone levels in the follicular phase were higher in patients compared to control with no significant correlations between sex hormone levels and EDSS score. The results obtained in the present study are in agreement with previous reports.

Zakrzewska-Pniewska and colleagues [7] studied 46 women with a definite diagnosis of MS and 50 healthy women as the control group and determined that 56% of the patients had hormonal disorders, particularly increased levels of estrogen. Furthermore, in a study conducted by Shahdaeizadeh and colleagues [8] on 30 female patients with relapsing-remitting MS (RRMS) and 30 healthy female controls, patients were divided into three groups based on their expanded disability status scale (< 1.5, 1.5–3, and > 3). They reported that the level of estrogen was higher in the follicular phase in all patient groups than in the control group; in addition, it was significantly higher in the patients with EDSS > 3.

Conversely, Trenova and colleagues [9] found that 60% of patients had below the lower limit of normal serum concentrations of estrogen and/or progesterone in one or both phases of RRMS (relapse and remission). Hormonal levels increased significantly during remission in these patients.

Foroughipour and colleagues [10] found that testosterone levels in MS, especially during the active phase of the disease, are decreased, and this reduction was consistent with the severity of brain lesions; in addition, magnetic resonance imaging (MRI) results confirmed these data [5].

Previous reports have demonstrated that testosterone plays an important role in repairing brain lesions and shows protective effects in patients and animal models of MS [10]. In addition, testosterone and its metabolites such as dehydroepiandrosterone (DHEA) directly or through conversion to estrogen by the aromatase enzyme could affect the estrogen receptor [11]. There is evidence that testosterone and estradiol downregulate reactive gliosis and astrocyte proliferation [12] which are the major problems to axonal regeneration in the mammalian CNS [13].

Our results regarding IL-10 and TNF-α revealed that relapsing-remitting MS patients showed no difference in serum levels of proinflammatory (TNF-α) and anti-inflammatory (IL-10) cytokine levels when compared with controls, and this was consistent with previous studies [14–16]. This can be explained by the fact that increased levels of cytokines are associated with the clinical activity in RRMS and the development of a chronic progressive disease. As regards IL-4, our results came contradictory to the previous studies revealing that IL-4 levels were higher in RRMS patients compared with controls [17, 18].

Our results could not confirm the imbalance between Th1 and Th2 cytokines which is an oversimplified explanation for MS pathogenesis and that other cells may play an important role in pathogenesis such as B cells, T helper 17 (Th17), and T reg. A growing importance has been attributed to Th17 cells, a subset of CD4+ effector T cells, in regulating the immune-mediated inflammatory response, and Th17 cells have been indicated as playing a major role in the pathogenesis of several inflammatory and autoimmune disorders. Th17 cells are mainly characterized by the expression of IL-17 which is a proinflammatory cytokine that activates T cells and other immune cells, producing a variety of cytokines, chemokines, and cell adhesion molecules [19].

In the current study, a statistically significant positive correlation was found between estrogen level and levels of interleukin 10 (IL-10) and interleukin 4 (IL-4); additionally, a significant negative correlation was found between estrogen and tumor necrosis factor alpha (TNF-α) (p = 0.004).

This conforms with Javadian and colleagues [20] who found that estrogen significantly increased IL-10 and IL-4 and significantly decreased the expression of TNF-α mRNA in peripheral blood mononuclear cells (in vivo) and the culture supernatant of cells (in vitro) stimulated with proteolipid protein (PLP) and mitogen phytohemagglutinin (PHA) in RRMS and control group. Moreover, Soldan and colleagues [21] reported that oral estrogen treatment was associated with significant increase in levels of IL-10 and decreased levels of TNF-α in stimulated peripheral blood mononuclear cells isolated during estrogen treatment.

Correlations found between estrogen and cytokine levels (IL-10, IL-4, TNF-α) were attributed to the anti-inflammatory effect of estrogens that mainly inhibit inflammation and demyelination [22] and causes the downregulation of TNF-α production [23] in addition to the fact that estrogen is capable of modulating both pro- and anti-inflammatory activities of CD4+ T cells and consequently has the ability to influence the outcome of CD4+ T cell-mediated immune responsiveness [24]. This outcome can vary depending upon the level of estrogens, cell type, activation state of cells, local environment, and the experimental context, and the estrogen-mediated effects are apparent in all major innate and adaptive immune cells; also, natural varied expression of estrogen receptors in different tissues and cell types balances the overall outcome of estrogen-mediated immune responses [25]. So, estrogen at high concentrations has a stimulatory effect on the anti-inflammatory pathway [26] and shifting cytokine levels towards Th2 immunity [27].

We concluded that sex hormones have an influence on the immune system; besides, estrogen has an anti-inflammatory effect on cytokine milieu, and therefore, it can be tried as a treatment option in multiple sclerosis.