Introduction
Multiple Sclerosis (MS), a chronic autoimmune disease in which myelin sheaths of Central Nervous System (CNS) is targeted by self-immune cells, is a leading cause of neurological disability in young adults [1]. Despite the efforts to determine the causes and aggravating factors of the disease, along with the pivotal role of the immune system, the exact etiology remains unknown [2]. Since women have two- to three-times higher MS incidence compared to men [3], it is plausible that female sex hormones such as estrogen and progesterone affect the progression and development of MS. Furthermore, MS symptoms are significantly ameliorated during the last trimester of pregnancy during which the levels of estrogen and progesterone are at the highest [4]. 
Oral Contraceptives (OCs) containing estrogen and progesterone are known to influence some aspects of autoimmune diseases including MS. Accumulating epidemiological evidence has shown controversial results regarding the role of OCs in the advancement of MS. In addition, studies on experimental models of MS such as Experimental Autoimmune Encephalomyelitis (EAE) have shown that both estrogen and progesterone affect different aspects of the immune system, which might influence the disease in women who suffer from MS. Some studies have also studied the possibility of antagonism between estrogen and progesterone when taken simultaneously. While Mannella and colleagues [5] claimed no antagonism between the two hormones, Yao et al. [6] believed that the effects of estrogen were inhibited by progesterone or synthetic progestins. In this review article, we assessed the effects of estrogen and progesterone on the immune system, as well as the different aspects of immune cells in the pathogenesis of MS.

Estrogen 
Estrogen, the major constituent of OCs, is a steroid hormone mainly synthesized in the ovaries, and to a much lesser extent in the adrenal glands as well as in the testes in males. The biosynthesis of estrogen is a multi-step process initiated from cholesterol and is regulated at each step by hormones, such as Adrenocorticotropic Hormone (ACTH) and Luteinizing Hormone (LH), which controls the first enzymatic steps. In addition, the P-450 enzyme complex aromatase also plays a crucial role in estrogen biosynthesis and is a highly regulated enzyme [7]. Ghayee et al. have shown that estrogen consists of three endogenous biologically different compounds; estradiol, estriol, and estrone [8, 9].
As with all other regulatory substances, estrogen action is mediated by its specific receptors. So far, two distinct intracellular receptors; Estrogen Receptors α (ERα) and β (ERβ) and one membrane-associated Receptor (mER) are known. Nuclear estrogen receptors are known to regulate gene transcription in different target cells while membrane receptors control non-genomic actions such as calcium reflux and some other signaling pathways [10]. 
Gustafsson et al. reported that ERα is mainly localized in the uterus and mammary glands, while ERβ is predominant in the central nervous system, cardiovascular and immune system [11]. In addition, ERα is expressed in different types of immune cells, including CD4+ and CD8+ T cells, B cells, Natural Killer (NK) cells, and macrophages [12, 13]. Recent studies have reported therapeutic effects of estrogen in autoimmune diseases, especially MS. For instance, MS symptoms are ameliorated in the last trimester of pregnancy during which the estrogen levels are the highest and relapse within 3-6 months after delivery with the ebbing of the hormone levels [14]. Experiments on animal MS models (i.e. EAE) have shown that estrogen treatment led to a significant remission of the severity of EAE symptoms [15], even when administered after disease onset [16]. Estrogen is known to have a wide range of effects on the immune system and CNS such as cell differentiation, cytokine production, inhibition of cell migration in CNS, and neuroprotective actions on axons and myelin. We have reviewed these aspects in the sections below.

Lymphocytes 
Bone marrow and thymus are both affected by estrogen due to the expression of estrogen receptors [11] that makes lymphocytes primary mediators of the protective effects of estrogen. Reduction in the number of lymphocytes is one of the several effects of estrogen during pregnancy. In fact, reversible ERα-dependent thymic atrophy is a consequence of estrogen therapy and pregnancy, as determined by the loss of all thymocytes and the CD4+CD8+ population [17]. Furthermore, Singh and colleagues found that estrogen agonists mediated immunosuppression through T-cell apoptosis via activation of the Fas/FasL pathway [17]. Many studies suggest that estrogen and estrogen agonist therapy shifts the T-cell population towards the Th2 phenotype [18] and promotes Treg cells [19]. Studies show that a dominant Th1 population is associated with abortion [20] and autoimmune diseases [21] due to the proinflammatory cytokines like Interferon (IFN)-γ, Tumor Necrosis Factor (TNF)-α, IL-1 and IL6 produced by this cell type. 
In contrast, a Th2 dominant phenotype results in the secretion of anti-inflammatory cytokines such as IL-4, IL-5, and Transforming Growth Factor-β3 (TGF-β3) [22]. Experimental studies on the EAE model show that estrogen mediates the amelioration of symptoms via upregulation of FoxP3 (forkhead box P3) gene, which is related to the development of CD4+ and CD25+ Treg cells. Increased number of Treg cells has been observed both during pregnancy and estrogen therapy which suggest that fetal tolerance is significantly correlated with high estrogen levels during pregnancy [19, 23, 24]. Different stages of B-cell development, such as differentiation, proliferation, and viability, are also negatively affected by estrogen leading to a reduction in the number of B-cell precursors [25]. The effects of estrogen are observed not only in bone marrow but also in the peripheral lymphoid organs.

Macrophages and dendritic cells 
Macrophages and monocytes [13], as well as Dendritic Cells (DCs) [26], are also targeted by estrogen due to the expression and processing of estrogen receptors in these cells. Anti-CD3-stimulated mixture of cytokines released by lymphocytes can reduce the number of macrophages and dendritic cells in estrogen-treated animals by stimulating the expression of inducible Nitric Oxide Synthase (iNOS) and production of nitric oxide in these cells [26]. A shift towards the Th2 dominant T-cell population is responsible for decreased secretion of IL-2 and INF-γ, and increased secretion of IL-10, which suppresses Th1 development. These findings indicate that estrogen reduces the number of macrophages and DCs by altering cytokine production [27]. 
In contrast, Soldan et al. reported that the number of macrophages and monocytes remained intact after oral estriol treatment [28]. Another protective effect of estrogen that has been observed in the EAE animal model is the reduced ability of DCs to present antigen to myelin basic protein-specific T cells [27], which likely ameliorates the MS-like symptoms. The decreased number of dendritic cells is declared to occur with the means of p38 MAPKs due to the expression of CD40, a co-stimulatory molecule playing a significant role in the immune response of effector cells [29]. Decreased expression of TNF-α, IFN-γ, and IL-12 mRNA in DCs following estradiol treatment is another factor in improving MS [27].

Cytokines 
Marked reduction in proinflammatory cytokines and increased anti-inflammatory cytokines have been observed after estrogen therapy [30, 31], mediated via the shift to the Th2 phenotype, which is also observed during pregnancy and is essential for fetal tolerance [28, 32]. Palaszynski and colleagues [19] showed that estriol treatment inhibited the secretion of IFN-γ, TNF-α, IL-2, and IL6. INF-γ is released by different cell types such as CD8+ and CD4+ T cells as well as NK cells and affects cell-mediated immunity, antiviral activity, inflammation, and autoimmune diseases [21]. 
Taken together, these results elucidate the impact of estrogen in shifting cytokine production towards a Th2 dominant phenotype, which suppresses cell-mediated immune response and activates the antibody-mediated immune response instead [33]. This cytokine shift is also responsible for reduced proliferation and development of T-cells, macrophages, DCs and NK cells. On the other hand, as Matejuk et al. declared, considerably suppressed expression of chemokines Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), Macrophage Inflammatory Protein (MIP)-1a, MIP-2, Induced Protein (IP)-10, and Monocyte Chemoattractant Protein (MCP)-1, chemokine receptors C-C Chemokine Receptor (CCR)1, 2, and 5 are truly responsible for decreased inflammatory cell population [34].

Neuroprotection
Estrogen has also been shown to be a neuroprotective agent [35] which can promote growth and development of neurons. The developmental effects of estrogen on adult neurons are activated post brain damage. Arvanitis et al. showed the presence of membrane-associated estrogen Receptor (mER) in the oligodendrocyte plasma membrane and within the myelin sheath [36, 37]. Furthermore, estrogen therapy rescued oligodendrocyte cytotoxicity and accelerated oligodendrocyte process formation [38, 39]. Khan and Ansar Ahmed [40]evaluated the effects of estrogen on autoimmune diseases such as MS and Systemic lupus erythematosus and concluded that estrogen had a different effect on autoimmune disease, wherein they hypothesize that estrogen does not affect immune cells.

Progesterone 
Progesterone, which is a component of some OCs, is an immunomodulatory effector during pregnancy along with cortisol, vitamin D, estrogens, Early Pregnancy Factor (EPF), α-Fetoprotein, etc. Progesterone is synthesized in ovaries and placenta in females, and in the testes and adrenal cortex in males. The mitochondrial cytochrome P450scc enzyme is essential for progesterone biosynthesis and helps synthesize pregnenolone from cholesterol. Afterwards, 3b-Hydroxysteroid Dehydrogenase (3b-HSD), which is located in mitochondria and endoplasmic reticulum, converts pregnenolone to progesterone [41]. 
Along with its immunomodulatory and anti-inflammatory roles via its effects on immune cells, progesterone also has neuroprotective effects. These effects are mediated by specific receptors that are present in many immune cells such as lymphocytes, monocytes, macrophages and DCs. Like estrogen, progesterone also has nuclear receptors which allow it to enter the nuclei and bind to specific Progesterone Response Elements (PREs) within the promoter region of target genes to regulate transcription. Furthermore, some membrane Progesterone Receptors (mPRs) have been recognized such as the Gamma-Aminobutyric Acid (GABA) receptor system, which also modulates the chloride ion channels [42, 43]. 
Thomas and Pang [44] have shown mPRs to have seven trans-membrane domains which act through cyclic Adenosine Monophosphate (cAMP). These mPRs, including mPRα, mPRβ, mPRγ, mPRδ, and mPRε, are present on the neurons in the brain [44, 45] while the nuclear receptors are selectively but not ubiquitously distributed within the brain. Studies have also indicated a beneficial impact of progesterone on CNS post injury [46]. The immunomodulatory effects of progesterone include inhibiting DCs and NK cells and steering T-cells towards a Th2 dominant phenotype [47]. The effects of progesterone on the immune and nervous system are explained in more detail in the sections below.

Lymphocytes 
Th1 lymphocytes and their cytokines play major roles in many autoimmune diseases, mainly by causing inflammation. Shifting of the Th1/Th2 balance moves towards the Th2 dominant phenotype diminishes inflammation and reduces cell migration into CNS due to increased levels of anti-inflammatory cytokines. The main effects of progesterone on lymphocytes are suppression of CD4+ T-cell differentiation, modulation of the Th1/Th2 balance and increase in Treg production [48]. These effects are mediated via of the Progesterone-Induced Blocking Factor (PIBF), which shifts the Th1/Th2 balance to Th2 instead of a Th1 predominant population. PIBF targets the phospholipase A2 enzyme and inhibits arachidonic acid release that also influences NK activity and cytokine balance [49]. The Th2 cells activated by the PIBF are also responsible for the production of anti-inflammatory cytokines [50]. Progesterone also triggers the production of asymmetric antibodies by B-cells which block the binding of functional antibodies to their antigens [50, 51].

DCs and NK cells
Progesterone can modulate CD83+ DC differentiation by altering cytokine secretion. It diminishes pro-inflammatory cytokine secretion by the DCs, thereby, reducing inflammation and decreasing cell migration to CNS. Specifically, progesterone administration suppresses IL-23 secretion by the monocyte-derived DCs, and stimulates them to secrete the anti-inflammatory IL-10, IL-27, IL-8 and IL-13, which in turn block IL-12 production and stimulate the Th2 or antibody-mediated immune responses [31, 52, 53]. NK cell activity is also directly or indirectly inhibited by progesterone [47]. During pregnancy, the placenta produces high amounts of progesterone that diminishes NK-cell activity and IFN-γ production via PIBF and Major Histocompatibility Complex (MHC) I, G (HLA-G) [54, 55]. NK bustle is also significantly decreased due to PIBF which alters cytokine balance by influencing T-cell phenotype. Lower IL-12 production and secretion is correlated with PIBF production, resulting in lower NK activity [56].

Cytokines 
As already mentioned, the balance between pro-inflammatory and anti-inflammatory cytokines play a crucial role in immune responses, and can be targeted to relieve the symptoms of autoimmune diseases. Progesterone induces PIBF production in lymphocytes [54, 57], which stimulates the production of Th2 dominant cytokines and promotes antibody-mediated immune response [58]. Hudi and colleagues [59] showed that lower serum concentration of PIBF and IL-10 and a higher level of proinflammatory cytokines is associated with higher risk of preterm labor. Furthermore, progesterone shifts the cytokine production in monocyte-derived DCs towards the anti-inflammatory spectrum. The inhibitory effects of progesterone on NK-cell activity and INF-γ secretion are likely due to increased IL-10 production. Munoz-Cruz et al. [58] have shown that lipopolysaccharide induced nitric oxide biosynthesis and secretion of pro-inflammatory cytokines by macrophages are inhibited by progesterone. Taken together, these findings show a highly significant function of progesterone in altering cytokine balance, which suppresses inflammation and cell migration into CNS in MS patients, and results in less myelin destruction. 

Neuroprotection and Myelination 
One study showed that progesterone decreased the lesion volume of cerebral brain ischemia or Traumatic Brain Injury (TBI), and also prevented the death of facial motor neurons after nerve transection [60]. It is hypothesized that the neuroprotective effects of progesterone in mammalian brain tissue is mediated either directly via the neurons or indirectly via the glial cells. The presence of membrane progesterone receptors or the GABA receptor can also trigger some neuroprotective signaling pathways, such as upregulation of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein [61]. 
Progesterone is known to influence the function of glial cells that are important mediators of neuronal survival. Labombarda and colleagues have reported decreased proliferation of astrocytes and microglia by progesterone [62]. Myelination is a major defense action against MS and is controlled by various mediators. Progesterone has also been shown to activate NG2+ and O4+ Oligodendrocyte Precursor Cells (OPCs) which are the fundamental cells in myelination and myelin repair [63, 64]. Furthermore, Ghoumari et al. reported increased levels of the Myelin Basic Protein (MBP) and the mature oligodendrocyte marker 2’,3’-Cyclic-Nucleotide 3’-Phosphodiesterase (CNPase) in the slice cultures of cerebellum (mixed glial cells and organotypic) in the presence of progesterone, indicating that accelerated myelination is mediated by progesterone [65]. 
Consistent with these reports, Hughes [66] demonstrated that low levels of progesterone would enhance IFN-α pathways in SLE which further aggravate the symptoms. Taken together, progesterone can suppress MS and rheumatoid arthritis by inhibiting the Th1 and Th17 pathways and inducing an anti-inflammatory effect. The effects of estrogen and progesterone on immune cells were summarized in Table 1.
 

 

Conclusion
Estrogen and progesterone are able to shift the immune response towards an antibody-mediated one, inhibit pro-inflammatory cytokine production, prohibit cell migration into CNS, suppress different pro-inflam­matory immune cells, stabilize neuronal environment and inhibit neural death. As MS is primarily an inflamma­tory disease in which NK cells, cytotoxic T cells, DCs and macrophages play pivotal roles, therefor estrogen and progesterone can play role in MS. This concept can be considered as a potential therapeutic opportunity.

Ethical Considerations
Funding
This research did not receive any specific grant from funding agencies in public, commercial or not-for-profit sectors

Conflict of interest
The authors declared no conflict of interest.




References