Contributing factors in multiple sclerosis and the female sex bias.

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ARTICLE IN PRESS

IMLET-5584; No. of Pages 10

Immunology Letters xxx (2014) xxx–xxx

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Contributing factors in multiple sclerosis and the female sex bias Roksana Khalid ∗ University of Guelph, 50 Stone Rd East, Guelph, ON, Canada N1G 2W1

a r t i c l e

i n f o

Article history: Received 27 June 2014 Received in revised form 19 August 2014 Accepted 2 September 2014 Available online xxx Keywords: Multiple Sclerosis T cells Sex bias Endocrine system Immune system Environment Adipokines Adenosine

a b s t r a c t Autoimmune diseases, such as multiple sclerosis (MS), show a higher incidence rate in women compared to men, which may be due to differences in the immune system, sex hormones, or both. Furthermore a disruption in homeostasis within these systems appears to be contributing to the etiology of MS. These systems are also influenced by the environment and metabolic factors necessitating the adoption of a broader viewpoint of the contributing factors in MS in the search for effective therapeutics. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Autoimmune diseases result from a disruption in the normal defense mechanisms of the immune system leading to a failure of self-tolerance. Normally there are various checkpoints along the lymphocyte development pathways to prevent activation against the body’s own tissues, but with an autoimmune disease, these become disrupted through unknown mechanisms leading to damage to a variety of organs and tissues. Recent research has demonstrated that approximately 5% of the population in developed countries suffer from an autoimmune disease and approximately 78% of the patients are women [1,2] leading researchers to believe that the factors contributing to sex differences may be tied to factors contributing to pathogenesis. Multiple sclerosis (MS) is an autoimmune disease in which the interaction between hormones and the immune system plays a role in disease progression but the mechanisms by which this occurs are incompletely understood. In its earliest stages, MS presents as a disruption in self-tolerance of immune cells against the selfantigens of the myelin sheath, triggering the destruction of the myelin layer insulating the axons. This autoimmunity occurs with a state of chronic inflammation that prevents healing while also

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promoting the development of severe lesions that progress MS into a neurodegenerative state. MS presents with a high degree of variability in afflicted individuals partly because the CNS is able to undergo some level of repair. This allows the alleviation of symptoms for some time until another bout of disease causes more damage. The back-and-forth of symptomatic and asymptomatic states is the relapsing-remitting form of MS (RRMS) which is characterized by acute attacks from which recovery can last for months. Another form of MS can be experienced with progressive symptoms that do not completely remit throughout the lifetime of the individual. Progressive MS can be primary, secondary or relapsing and is a gradual but steady progression of disability [3]. The heterogeneous nature of MS makes it a particularly difficult disease to manage. Current standards of medical practice involve the use of immunosuppressive and immune-modulating therapy which generally acts more as a symptom management protocol than a cure. Furthermore treatment routines seem to have only a delaying effect on disease progression while also being challenging to implement since each round of therapeutics has to be directed at a particular phase of the disease [4]. These challenges have sparked an interest in alternative methods of treatment, such as hormonal therapy to alter the female hormonal profile, which could also be indirectly immunomodulatory. Although these changes may be highly beneficial to some MS patients, much is still unknown about the factors contributing to MS which precludes the necessary shift

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Please cite this article in press as: Khalid R. Contributing factors in multiple sclerosis and the female sex bias. Immunol Lett (2014), http://dx.doi.org/10.1016/j.imlet.2014.09.004

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in focus from therapy to prevention of disease. The possible factors contributing to the sex difference in MS are genetic, immune, and hormonal all of which can also be modulated by the environment and metabolic state. The aim of this paper is to report on the current understanding of MS in order to outline areas that could benefit from further research.

chromosome reactivation and haploinsufficiency with disease preponderance in MS. Future research targeting this line of thought may be beneficial in elucidating further roles of the X chromosome in pathogenesis of MS.

3. The immune system in MS 2. Genetics factors: Sex bias in MS

3.1. The influence of sex on the immune system

The contribution of X chromosomes in MS was explored through the experimental autoimmune encephalomyelitis (EAE) animal model commonly used in MS research. The researchers isolated the sex chromosome influence on EAE from the hormonal influence by deleting the testes-determining Sry gene from the Ychromosome of the male mice. This led to the development of a female phenotype in XY− mice. Both XX and XY− mice express a female hormonal profile, therefore the researchers performed ovariectomies on both groups. With the induction of EAE they found that XX mice had a significantly higher susceptibility as well as a more severe disease progression, indicated by a higher degree of inflammation in the CNS. They also assessed castrated XXSry and XY− Sry mice expressing a male hormonal profile and found a similar bias of a female sex chromosome complement predisposing the subjects to the development of EAE. These results offer strong evidence that sex chromosomes have the potential to act independently of sex hormones in conferring disease susceptibility to females. Furthermore, these results were true only for the SJL mouse strain that has been demonstrated as having a female:male difference in EAE thus effectively modeling the sex-bias seen in humans [5]. The “Kast-Stewart hypothesis” implicates disturbances in the X chromosome inactivation process as conferring susceptibility to autoimmune disease [6,7]. X inactivation is the epigenetic silencing of one chromosome in the XX complement which confers a mosaic pattern of expression in each individual female [8]. The process is evolutionarily useful since it is responsible for gene dosage compensation between females and males and it seems to be a random process at the level of the entire organism. A layer of complexity is added to the process with X-inactivation being nonrandom, or skewed, for an area where the same cell types converge for a similar function. In these patches an X-chromosome from one parent shows preferential expression. Skewing in X chromosome inactivation has been observed in haemopoietic stem cells (HSCs) in the blood and bone marrow which give rise to the myeloid and lymphoid lineage of blood cells. The variation in patterns of expression can be constitutive or acquired with age [9] and the pattern has also been found to be heritable [10]. A breakdown in self-tolerance can be explained as a product of skewed inactivation since the differential expression of self-antigens could influence the immune system into action [11]. A study explored differential patterns of X inactivation in patients with MS by comparing 568 female patients with controls but a significant difference in degree of skewing towards one X chromosome’s expression between the two groups was not found. Further comparison between the patients was conducted, grouping them according to the type of MS, RR-MS and progressive MS, and the researchers were able to demonstrate a significant difference in the level of skewing [12]. These findings suggest that the heterogeneity of MS is being expressed at the genetic level and perhaps grouping the various types of MS as different presentations of the same disease may not be accurate. Other hypotheses for increased susceptibility of women to autoimmune diseases are the reactivation hypothesis and haploinsufficiency hypothesis [13] however a literature search relating to these did not reveal studies exploring the association of X

Sex chromosome complement is also associated with differences in immune profiles created by cytokine release from various cell types. Females are more likely to develop a Th1 response except during pregnancy when a Th2 profile prevails. A Th1 response establishes a proinflammatory environment and a Th2 response promotes antibody (Ab) production [14]. This switch leads to a significant inhibition on the responsiveness of the immune system which is also associated with alleviation of MS symptoms. The X chromosome influence on the immune profile is exemplified by the finding that ovariectomized XY− female mice stimulated with autoantigen produce significantly higher levels of Th2 cytokines interleukin (IL) -13 and -5 compared to XX females in the EAE model of MS, with some evidence of higher IL-10 as well [5]. These cytokines have anti-inflammatory roles and contribute to a less severe clinical score of XY− mice compared to XX. IL-13 may also have the ability to limit Th2 responses, further decreasing immune activation by decreasing cytokine secretion and Ab production [15]. IL-13 maps to the X chromosome as do the genes for IL-2R␥ chain and IL-9R (CD129) which promote lymphoid and myeloid cell development [16–19]. Research suggests that the levels of beneficial cytokines could be disrupted with EAE through an unknown mechanism thus reducing the body’s ability to resist its onset. The connection of these cytokines to the sex chromosome complement is incompletely understood, but there is the possibility that the immune profile differences between XY− and XX subjects are the result of the Y chromosome influence on immune factor expression. It has been thought for a while that the most significant contribution of the Y chromosome is the testes-determining factor but recent research has demonstrated that it has influence on immune function as well. Hypothetically speaking, an immune factor produced by genes on the Y chromosome but acting independently of sex holds the potential of becoming an administrable therapeutic. Therefore it is worthwhile to explore sex influences on MS without limiting the research to either sex thus allowing comparison between the two. A capacity for regulation of autoimmunity has also been shown with the regulatory T cell. Naturally occurring CD4+ CD25+ regulatory T cells (nTregs) expressing an X-linked transcription factor, FOXP3, can directly suppress self-reactive T cells. In MS nTregs are upregulated in the CNS with a subsequent decrease in the periphery. As evidenced by the chronicity of MS, this migration is not sufficient in combating pathogenesis, possibly due to impaired functionality of the nTregs [20]. This functional deficit may be a lowered level of expressed FoxP3 protein [21] and upregulation of FoxP3 with stem cell therapy has been shown to ameliorate the symptoms of MS [22]. These findings support the involvement of Tregs and the expression of X-linked transcription factors in disease progression. Other recently implicated immune factors conferring disease risk in a sex-biased manner are human leukocyte antigen (HLA) DR alleles expressing major histocompatibility complex (MHC) II molecules and the strongest genomic association of MS has been with the HLA-DRB1*15 alleles on chromosome 6 [23–25]. MHC haplotypes show a significantly increased inheritance in a motherto-daughter fashion and this finding could explain the development of MS as a product of transgenerational epigenetic effects [26].




These results suggest that women are more susceptible to the disease because they experience a higher rate of epigenetic modifications, therefore MS could be a by-product of a more actively variable system compared to that of males. The plausibility of this idea is linked to basic gender differences, i.e. women are carriers of children and there is a survival benefit in being able to “adapt” the genetic makeup based on environmental conditions. Although there may be a selective advantage to a higher level of activity, an increase in variation simultaneously increases the potential for error. An epigenetic effect correlated with MS is histone acetylation associated with the oligodendrocyte progenitor cells. A decrease in levels of acetyl-H3 seems to induce differentiation, the brains of MS patients demonstrate a shift towards acetylation with a female bias, and this change in acetylation pattern may be reflective of more widespread changes in the chromatin landscape [27]. Histone acetyltransferase activities are also associated with MHC II expression [28]; contributing to the idea that epigenetic variation of the MHC is a key modulator of MS pathogenesis. Another assessment of HLA-DRB1*1501 expression revealed a significant correlation with disease progression in the spinal cord in a dose-dependent manner [25]. Future research targeting the variability in expression of MHC in a patient population could explain which factors contribute to the epigenetic modifications associated with MS. 3.2. Further exploration of the immune system in MS The alleles of the human MHC, DRB1*1501 (DR2b) and DRB5*0101 (DR2a), are located 85 kb apart which offers the possibility of interaction between the two. Mice expressing a T-cell antigen receptor (TCR) derived from individuals affected with MS also expressing one of the DRB-alleles show different patterns of disease expression with induction of EAE. Mice expressing DR2b were found to spontaneously develop severe EAE while DR2a significantly decreased incidence of disease implying that the latter acts as a modulator of DR2b activity. The mechanism of resistance to disease was described by the researchers as being the result of peripheral deletion of dangerously reactive T cells through activation-induced cell death. They also described the interaction between the two alleles as being the product of epistatic effects and linkage disequilibrium present in the MHC [29]. A more generalized factor contributing to a disruption in selftolerance of MS patients is the process of thymic selection for the generation of the T cell repertoire. This disruption can lead to a circulating population of high-avidity myelin-specific CD4+ T cells which were shown to be on average more than four times higher in MS patients than in controls. This contributed to a skewing towards the proinflammatory phenotype due to the subsequently higher production of interferon gamma (IFN-␥) [30]. The researchers suggested that this T cell subpopulation is able to escape negative selection and act in the periphery due to variability in binding affinities of myelin epitopes. An overlap has been shown to exist in the antigen-presenting function of the HLA class II alleles, especially of the DR and DQ haplotype, which arises from the strong linkage disequilibrium between DRBA*1501, DRB5*0101, and DQB1*0602 [24]. This means that T cells bind somewhat promiscuously to DR and DQ molecules allowing for interaction with different affinities and the possibility of inappropriate activation when there should be silencing. Based on the above described variability with which immune cells bind to each other and are activated, it may be inaccurate to look at one allele in particular when attempting to explain the mechanism of disease development in MS. The possibility of non-MHC risk factors have also been explored in the context of a disrupted immune system homeostasis contributing to the development of MS. A genomewide association study of MS identified a set of single nucleotide polymorphisms

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(SNPs) in genes encoding IL-2R␣ (CD25) and IL-7R␣ (CD127) chains [31]. T cell subpopulations expressing the IL-2R␣ chain can maintain self-tolerance by down-regulating the immune response and elimination of this subpopulation produces a variety of autoimmune diseases in the periphery [32]. The IL7R˛ (CD127) susceptibility locus associated with the SNP, rs6897932, plays a functional role in MS. This gene encodes the IL-7R␣ subunit and the IL-7/IL-7R␣ system is involved in the proliferation and precise regulation of differentiation of thymocytes [33,34]. Research suggests that individuals carrying the risk allele ‘C’ at rs6897932 produce less membrane-bound IL-7R␣ protein compared to carriers of the ‘T’ allele, leading to an increase of the soluble form of IL-7R␣ and the subsequent decrease in regulatory functions [33,35]. The soluble form could also be interacting with thymocytes inappropriately, leading to their activation and propagating disease. It is unclear whether high levels of circulating CD127 are the source of disease onset or its product since changes in the immune profile could be facilitating the expression of the pathogenic allele and this process may also be connected to epigenetic effects. Other research has explored the imbalance in homeostasis of immune cell populations implicating several factors. The involvement of two factors, CD28 and CD45, has been investigated in MS patients. CD28 is a major co-stimulatory receptor responsible for optimal antigen-mediated T-cell activation, proliferation and survival of T cells. CD28− T cells are defective in regulatory functions while having enhanced cytotoxic and suppressive capabilities [36]. CD45 is the protein–tyrosine phosphatase receptor type C (PTPRC), which belongs to the leukocyte common antigen (LCA) family. The LCA is a group of high molecular weight glycoproteins expressed on the surface of all leukocytes and haemopoietic progenitor cells in unique patterns. It consists of multiple isoforms due to alternative splicing of exons leading to the expression of CD45RA, CD45RB, CD45RC and CD45RO [37,38]. A comprehensive analysis of the peripheral blood immunophenotype of 16 MS patients revealed that MS patients have a significant increase in memory (CD45RO+ ) CD4+ T cells with a trend towards a decrease in naïve (CD45RA+ ) T cells in the peripheral blood, as well as a specific decrease in the suppressor precursor subset CD28− CD8+ T cells [39]. The pattern of CD45 expressed in MS seems to be an increase in immune cells with the capacity to react upon activation, which is supportive of the autoimmunity aspect of MS, and this increase appears to be a result of a disruption in homeostasis of active and inactive immune cell populations, but the incident leading to this disruption remains unclear. The role of PTPRC encoding CD45 in MS was investigated in German and North American subjects with a point mutation in the gene. Interestingly it was found that only the German subjects carried an altered CD45 expression phenotype caused by a heterozygous C → G transition in PTPRC that was associated with MS [40] and this mutation was not associated with the development of MS in U.S. patients [41]. These findings were challenged by another study that did find the PTPRC SNP polymorphism in U.S. subjects as well as evidence for its familial transmission [42]. The variability with which cellular systems are affected in MS could be a reflection of the complexity of this disease but it is unlikely that the same disease could have so many different presentations. It may be more accurate to group similar immunologic changes together to specify them as factors of a particular type of MS and to consider each type separately from the rest. Apart from the benefits of better focused research, this change could also lead to improvements in diagnostic tools allowing medical professionals to tailor treatment for each patient’s particular MS profile. Furthermore, based on the research explored so far, MS appears to be a manifestation of homeostatic imbalance but the factors leading to this loss of homeostasis remain a mystery. A focus on the immune system alone cannot explain the etiology of MS and a




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broader view encompassing other factors contributing to disease progression and their interaction with the immune system may be a beneficial approach for future research. 4. Hormonal effects in MS A sex bias in MS intuitively points to an effect of sex hormones on disease progression. Paradoxically the effect of hormones in females appears to be protective rather than potentiating MS pathology. The influence of sex hormones in MS is illustrated by the finding that disease course and severity is significantly reduced during pregnancy, especially in the third trimester. Furthermore, it increases during the first three months post-partum before returning to the prepregnancy rate [43]. An effect found in women having undergone pregnancy and those using combined oral contraceptives (COC) was that they developed MS symptoms at a much older age compared to women who had not experienced either childbirth or used COC [44]. Recently there has been some controversy over the effects of contraceptive use in MS however differences in effects found in different studies are attributable to differences in formulations. The effects of these hormones on MS have also been shown in association with the immune system. In one study the reduced relapse rate during the late phase of pregnancy was found to be correlated to the estrogen induced expression of indoleamine 2,3-dioxygenase (IDO) on dendritic cells (DCs) which limits T-cell proliferation and both Th1 and Th2 cytokine production suggesting a novel target for MS therapy, the estrogen–DC–IDO axis [45]. Pregnancy inherently poses a challenge for the maternal immune system because the embryo is immunologically foreign to it, however a close physical association is established that lasts throughout this reproductive phase and it can only come about from a significant modification to the immune system response pattern [46]. Prior to exploring the specificities in cell type and functional modification of the immune system with steroid hormones, it is important to note that changes in ovarian status influence both the morphology and the function of the thymus. Ovariectomized adult female rats show a marked increase in thymus weight compared to sham-operated animals which is associated with an increase in volume and cellularity of both the medulla and cortex. Administration of 17␤-estradiol and progesterone decreased weight of both the lymphoid and nonlymphoid compartments of the thymus [47]. Estrogen treatment can induce thymic atrophy which was shown to occur through apoptosis of thymocytes [48]. In immune cells, functional changes such as the enhancement of Treg function have also been demonstrated with administration of 17␤-estradiol and progesterone [49]. During pregnancy, estradiol is elevated and estriol as well but the majority of recent research has focused on the effects of 17␤estradiol. Research has also explored the effects of other hormones such as progesterone and prolactin which are also upregulated during pregnancy and significantly influence disease progression in MS. 4.1. Estrogen 17␤-Estradiol administration has a profound inhibitory effect on the immune system, strongly down regulating genes coding for the histocompatibility complex, cytokines/receptors, chemokines, adhesion molecules, and signal proteins [50,51]. In support of these findings it has been shown to act prophylactically and therapeutically in MS [52] and some of these effects can be attributed to the upregulation of FoxP3 protein expression similar to levels during pregnancy as determined in an animal model [53]. 17␤-Estradiol has also been shown to increase IL-10 while decreasing TNF-␣ and IFN-␥ release from microglial cells. Its effects are truly wide-spread

since it can also reduce MHC I, CD40, and CD86 surface staining, the basal percentage of cells positive for MHC I and MHC II, CD40, CD152, Fas, and FasL, while increasing cell surface staining of CD80 in microglial cells [54]. There are several pathways through which 17␤-estradiol exerts its effects. Dendritic cells express estrogen receptors (ER) and the activity of 17␤-estradiol on these cells was shown to enhance their CD40 expression. This occurred via the MAPK signaling pathway: the activation of JNK and p38 enhanced minichromosome maintenance protein 6 (MCM6), involved in DNA replication, which subsequently induced CD40 expression [55]. The endogenous metabolite of 17␤-estradiol, 2-methoxyestradiol, also has the capacity to inhibit in vitro lymphocyte activation, cytokine production, and proliferation in a dose-dependent manner through the nuclear factor of activated T-cell pathway (NFAT) [56]. NFAT is a family of transcription factors important in the immune response. One or more members of the family act in most cells of the immune system. These proteins have weak DNA-binding capacity therefore they can cooperate with other nuclear resident transcription factors such as the ras-MAPK [57] to induce changes in transcription. Another cell-type influenced by estrogen-specific modification is the invariant natural killer T (iNKT) cell which contributes to the regulation of the immune system. iNKT cells significantly expand upon stimulation with myelin-derived glycolipids, also inducing robust cytokine secretion, which includes a release of IL-17. This function of iNKT cells was shown to be disrupted in MS [58]. Estradiol administration increases IFN-␥ secretion of iNKT cells in the serum of female mice only. This sex-linked difference was abolished in ER␣ deficient mice [59] therefore iNKT cell activation shows a sex bias that is significant to pathogenesis of MS. Female susceptibility to EAE also appears to be influenced at least in part by IL-13 since female IL-13 knockout (KO) mice experience milder EAE compared to males. The researchers attributed this effect to the opposing roles of estrogen and IL-13 on macrophages with estrogen inhibiting MHC II and IL-13 enhancing MHC II upregulation [60]. It is important to note that this specific evidence for the effects of estrogen on particular immune factors is brief relative to the widespread manner in which this hormone acts on the body. The effects of estrogen are not only dependent on the particular cell type but also its location in the body as different tissues host a microenvironment within which the effect of estrogen action may be modulated in various and sometimes unexpected ways. This is emphasized by the role estrogen plays in inflammation including in that which occurs with MS [61]. The selective activation of estrogen receptors has been targeted with the development of SERMs, selective estrogen receptor modulators, which can act in both men and women without feminizing effects. By focusing on the particular mechanisms through which estrogen induces its effects it is possible to produce a SERM selective enough to be beneficial in MS without the added risks of widespread estrogen receptor activation. The first report of the anti-inflammatory effect with estradiol treatment involved intradermal administration of the hormone. It was shown to minimize edema as well as decrease the number of infiltrating polymorphonuclear cells into the area of foreign antigen injection in the forepaw of the male and female mice of different strains [62]. In machrophages 17␤-estradiol inhibits inflammatory gene expression via the NF-␬B family of transcription factors that are involved in the rapid inflammatory response. Furthermore, they found this activity to be selectively mediated by ER␣ [63]. Inflammation inhibition with selective ER activation can also be mediated by 5-androsten-3␤,17␤-diol (ADIOL) acting on ER␤ of microglia. The researchers administered synthetic ER␤-specific ligands that suppressed transcriptional activation of toll-like receptor (TLR)-4 responsive genes, inhibiting cytokine release from microglia and




the development of EAE in the mice as well as improving symptoms of already established disease. This mechanism acts in a transrepressing manner, recruiting the repressor C-terminal binding protein to transcriptionally active promoters [64]. This active modulation of immune factors holds therapeutic promise for MS since it appears to be selective enough to not induce secondary effects, although the distribution of ADIOL activity in the brain has yet to be elucidated. The above findings emphasize the point that there is probably no single deregulated pathway of a particular cell type inducing the development of disease in all cases of MS, thus it is unlikely that a single compound can be used therapeutically to cure the disease. Nevertheless estrogen and its derivatives have demonstrated potent immunosuppressant and immunomodulation activity in a widespread manner. They are viable therapeutic candidates in particular contexts [65] and more in-depth evaluation of their efficacy in treating MS is warranted. 4.2. Progesterone As mentioned, progesterone also influences disease progression in MS and this occurs through its immunosuppressive and immunomodulatory properties. By acting on uterine cells progesterone influences the development of an immunologically favorable environment for survival of the fetal-placental semiallograft [66–68]. Progesterone modulation in levels similar to the human placenta and serum induces transient tolerance towards paternal alloantigens through the suppression of Th1 development as well as inhibition of T cell differentiation [67]. Furthermore, these effects seem to be occurring through influence on the immune system’s homeostasis since there is a skewing towards a Th2 type profile during pregnancy [69]. Specific immune changes that occur with progesterone treatment in EAE are a decrease in IL-2 and IL-17 inflammatory cytokines, as well as an upregulation of IL-10, CD19+ cells, CD8+ cells and changes in chemokine receptors in the spinal cord [70]. It also plays a role in differential MHC II immunoreactivity since control rats injected with vehicle show a significantly higher MHC II-related inflammatory response in the CNS compared to rats treated with progesterone [71]. Apart from its influence on the immune system, progesterone has also been found to be neuroprotective in EAE. It modulates oligodendrocyte function and decreases disease severity by inhibiting demyelination, especially in the spinal cord [71–74]. Progesterone and the synthetic derivative Nestorone can promote remyelination of axons by stimulating oligodendrocyte progenitor cell (OPC) recruitment [74] and production of myelin via the activity of insulin growth factor [72]. Treatment with progesterone also has the capacity to enhance axonal density, decreasing damage and the expression of immunoreactive protein GAP43 in the spinal cord of EAE mice [73]. The combined effect of estrogen and progesterone in MS seems to be that the former diminishes responsiveness of the immune system while the latter contributes to the CNS’s capacity for repair. In this scenario, although there may still be some autoimmune damage in the system, the course of disease progression is altered from that of neurodegeneration to neuroprotection. 4.3. Prolactin A discussion around the effects of pregnancy on the immune system would be lacking without the inclusion of prolactin and its influence on disease progression since this hormone is involved in many aspects of fertilization and reproduction. Some initial correlations between prolactin and MS symptoms were that patients of both sexes experienced hyperprolactinemia during the

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relapse stage [75,76] which have been attributed to hypothalamic dysfunction [75]. Hyperprolactinemia in MS was reported in a case study of a 32-year-old male patient with RRMS whose first symptomatic experience occurred during the development of a prolactin-secreting adenoma. Prolactin may have triggered the onset of MS by influencing the inflammatory process [77], and other research has also shown that both men and women with MS are in a hyperprolactinemic state [78]. The investigation into the mechanism of prolactin influencing disease progression has yielded some contradictory results. The immunomodulatory properties of prolactin were explored in the lab through the immune responses of prolactin receptor (PRLR) deficient mice and these mice did not demonstrate any significant immune deficits compared to wildtype controls [79]. Although these findings do not lend support to the influence of prolactin in MS they could be pointing to the possibility of hypothalamic dysfunction in MS [75]. An investigation into the differences in immune and stress response in MS patients vs. controls revealed that there may not be any significant differences in the hypothalamopituitary axis and stress reactivity between the two groups [80] but these inconsistencies in research may be explained by the finding in healthy adults that the prolactin response to acute psychological stress occurs without significant gender differences and the magnitude of response is dependent on estradiol levels [81]. There may not be significant overall hypothalamic dysfunction associated with MS but that does not rule out the possibility of a disruption in a particular hypothalamic pathway which may involve prolactin. Further investigation into changes occurring in the hypothalamus with MS may explain the inconsistency in literature. Prolactin is probably playing some role in MS as a disruption in its levels from the norm has been reported in correlation with MS by several different groups. Future studies may benefit by exploring the interaction of prolactin with other hormones in order to explain how this hormone is influencing pathogenesis in MS. Since pregnancy has been shown to have a profound effect on MS, it may also be beneficial for future studies to investigate differences in pathology with animal models mimicking a pregnant hormonal profile alongside the investigation into the individual roles of these hormones. Such a shift in focus could reveal some key modulatory pathways of hormonal and immune system interaction.

5. Environmental factors There is an inherent risk for MS with a female genetic profile and the exploration of the immune-endocrine interaction reveals some keys areas of disruption contributing to disease, but the triggers disrupting the immune and hormonal balance remain unexplained. One possibility is that environmental factors may trigger changes in the immune and/or hormonal milieu that precipitate MS pathology. The contribution of the environment to disease is exemplified by one study that found a month-of-birth-effect in the risk for MS in people from northern climates where most people with MS were born in May [82]. This suggests that the reduced maternal sunlight exposure over the winter months confers a certain degree of susceptibility to disease in the prenatal fetus. Some skepticism may arise regarding such findings since the vitamin D deprived individual does not experience MS until 2–3 decades after the initial deprivation. Yet there is evidence of modification to certain immune system factors with environmental influence. A study exploring the effect of vitamin D deficiency in utero in mice found a significant reduction in iNKT cells that could not be corrected with vitamin D supplementation in later life [83]. Further exploration into the in utero effect was conducted with the induction of vitamin-D deficiency in female mice 6 weeks prior to conception, it was continued until birth, and the offspring of the




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vitamin-D deficient females were compared to controls. The findings were surprising because the adult offspring of the vitamin-D deficient mice had a significantly milder clinical presentation of EAE compared to controls which was partially explained by the overexpression of the vitamin D receptor (VDR) in the CNS as well as two heat shock proteins and FK506 binding protein 1a which plays a role in immunoregulation [84]. A similar protective effect has yet to be confirmed in humans but these findings do reveal the capacity of maternal imprinting effects on the fetus. Based on the above, it can be hypothesized that early life events may be setting up a system that is vulnerable to disease and these vulnerable offspring could be protected from developing a severe form of the disease with early intervention such as nutritional supplementation. Further research is needed to find out to what extent dietary supplementation can help prevent or fight MS but the potential appears promising. The protective effects of vitamin D have also been explored separate from pregnancy but they still appear to be occurring in a female hormonal profile dependent manner. A comparison of vitamin D supplementation in intact and ovariectomized female mice as well as in intact and castrated males revealed that vitamin D supplementation can significantly reduce EAE symptoms in intact females compared to all other groups. The relationship between ovarian tissue and the vitamin was supported by the reduced number of transcripts encoding the vitamin-D3 -inactivating enzyme in the spinal cord of intact female mice [85]. 17␤-Estradiol administration works synergistically with vitamin D to mediate protection in EAE in ovariectomized females by controlling vitamin D3 metabolism and receptor expression [86]. The exploration of the capacity to which hormonal and nutritional factors may interact to protect from disease is another venue for future research. Depending on the enthusiasm along this line of research, personalized hormonal and nutritional supplementation for people with MS could be developed as a therapeutic option in the near future but it also has the potential to act prophylactically. 6. Adipokines and adenosine Adipokines are signaling molecules produced by adipocytes. The literature contains a number of studies revealing the relevance and importance of adipokines in MS. This section is placed here because the factors discussed within could be the mediators tying together the environment, the endocrine, and the immune system in MS due to their primary function of allocating resources to various systems. Recently adenosine has also been found to play a role in MS and it is included within the same section because of its ability to modulate adipocytes and influence fat cell metabolism thus adding another layer of complexity that is significant enough to warrant further exploration. 6.1. Leptin Leptin, the 16 kD ob protein, is implicated as the satiety signal in energy metabolism [87]. It has been shown to decrease consumption after a certain amount of body fat has accumulated by being released into circulation by adipocytes and binding to its receptors in the hypothalamus [88,89]. There is an upregulation of leptin in obesity however this signaling does not reduce consumption since the chronic activation appears to make the body resistant to its functional activation [88]. Obesity is also correlated with MS based on the observation that, with this condition, the body is also in a state of chronic low-grade inflammation of the adipose tissue [90]. This influences the development of inflammation in the CNS as well due to the constant surge of proinflammatory mediators such as leptin and adipocyte-fatty acid binding protein, each of which has the highest correlation with a different form of MS [91].

It has been hypothesized that leptin may be involved in a variety of physiological processes other than suppressing appetite and promoting lipolysis. Administration of recombinant human leptin alone can correct sterility in ob/ob female mice [92]. These mice do not produce leptin, they are obese, and they are also resistant to EAE [92,93]. The role of leptin in reproductive function is further supported by the finding that administration of leptin to normal prepubertal female mice induces the onset of reproductive maturity significantly earlier than in controls [94]. Administration of leptin to SJL female mice has also been found to exacerbate symptoms of EAE while making previously resistant males susceptible to autoimmunity through the promotion of a Th1 proinflammatory immune profile [95]. The discussion on the influence of leptin on MS will proceed by first exploring its role in pregnancy since the dramatic physiological changes during this time allow some interesting observations and as previously noted there are dramatic changes in MS symptoms during this reproductive phase. Recent research has explored leptin’s influence on the CNS, and subsequently on MS, which allows more specific conclusions about its role in the disease. The investigation into leptin’s role in reproduction revealed that it is involved in an intricate signaling system that alters maternal metabolic pathways in a way to support the metabolic needs of the growing fetus. Measurement of longitudinal changes in leptin levels in maternal serum during pregnancy revealed that there was a 66% increase in leptin concentrations from before pregnancy to late pregnancy and 93% of the total serum increase occurred in early gestation accompanied with an average 3% decrease in body fat mass. Furthermore only 7% of the total increase in leptin concentration was significantly related to an increase in body fat mass which points to the maternal system being in a leptin-resistant state during pregnancy [96]. The changes in leptin levels throughout the various stages of pregnancy may explain the amelioration of MS symptoms during pregnancy and the sudden relapse experienced after birth. Investigation into the changes in serum leptin levels in pregnant women revealed that levels increase significantly between the first 2 trimesters but not between the second and third trimester of pregnancy [97]. Interestingly, it is during the third trimester that the most reduction of MS symptoms is experienced by pregnant patients [43]. Leptin levels also drop significantly immediately after birth which may be related to the removal of the placenta since it is also a significant source of leptin during pregnancy. These changes in leptin levels were not significantly correlated with changes in body mass index and leptin levels were also found to be stimulated by estrogen and hCG but not by progesterone or placental lactogen in incubated adipocyte cultures [97]. Another study revealed that leptin levels in ovariectomized rats are significantly stimulated with administration of prolactin and this effect was diminished with food deprivation pointing to an indirect route of leptin increase in the prolactin–leptin relationship [98]. This indirect route may involve proinflammatory cytokines TNF-␣ and IL-1 that have been shown to increase leptin mRNA expression in mice adipose tissue [98,99] which could also explain the inconsistencies in research relating hyperprolactinemia to MS. The significant reduction of MS symptoms during pregnancy was previously attributed to the high level of circulating steroid hormones as well as switches in cytokine production within the immune system. Based on all of the above data the resistance to disease during pregnancy may also be attributable to the reduced leptin sensitivity in the maternal system. A finding in mice that offers explanation for leptin insensitivity in pregnancy is that there is an upregulation of a soluble leptin binding protein that is coincident with the rise in serum leptin [100]. This increase in soluble leptin receptor alongside serum leptin during pregnancy, as well as the post-partum drop in leptin levels, has been observed in healthy




controls and MS patients. In the same study it was found that the post-partum relapse of MS occurred most frequently in women with the highest relative decrease in serum leptin levels after delivery [101]. This dose-dependent effect of leptin on MS relapse requires further investigation in pregnant and non-pregnant models since the post-partum relapse may have also been influenced by a generalized change in endocrine signaling that occurs after birth or it could be pointing to leptin signaling being sufficient in triggering the onset of MS symptoms. There is an increase in leptin transport into the hippocampus and cervical spinal cord with EAE, especially in the early phase of induction, which occurs alongside an upregulation of the leptin receptor (ObR) in astrocytes of the hippocampus [102,103]. This upregulation is most likely serving a protective role since the absence of leptin signaling in astrocytes promotes EAE [102,104]. A study explored the role of leptin in astrocytes with the astrocytespecific leptin receptor knockout (ALKO) mice and an opposite effect was observed in the CNS from the detrimental effect of peripheral leptin administration. The study included investigation of adenosine which is a major gliotransmitter released from astrocytes and demonstrates the functionality of astrocytes as well as several immune factors. It was found that astrocyte release of adenosine was significantly disrupted in ALKO mice compared to wildtype, indicating the importance of leptin in glial cell signaling. There was also an elevation in infiltrating CD4+ T cells and a lower number of neutrophils in the CNS of ALKO mice leading to worse outcomes with EAE compared to controls [104]. Measurements from obese and nonobese individuals have also revealed that there is a significant reduction in numbers of circulating Treg cells in obesity [105] which may be propagating the development of MS in some individuals.

6.2. Adiponectin Adiponectin is a 30 kD polypeptide highly expressed by adipocytes and inversely related to obesity, insulin resistance, and inflammation [106]. It was also found that it is positively and independently associated with sex hormone binding globulin (SHBG) and IGF binding protein-3 (IGFBP-3) while being inversely correlated with c-peptide [107]. The majority of adiponectin’s contribution to health and disease has been explored with a metabolic perspective; however the influence that this polypeptide may have on MS is evidenced by its association to SHBG and IGFBPs as well as recent research demonstrating that the CNS expresses its receptors [108]. Adiponectin plays a mediating role on endocrine and immune function and can influence the development of EAE. Adiponectin has the capacity to modulate T-cell functions and adiponectin knock-out (ADPKO) mice have enhanced proinflammatory cytokine expression with induction of EAE compared to wildtype controls as well as significantly increased levels of IFN␥, TNF-␣, and IL-6. Furthermore a defect has been found in the numbers of Tregs during EAE in ADPKO mice and treatment with adiponectin ameliorates EAE while increasing the number of Treg cells [109]. It is worthwhile to assess the mechanism of adiponectin action and how it ties in with other EAE ameliorating factors like sex hormones since it could be acting independently or as a supplementary in promoting resilience in MS. Adiponectin levels are greater in women and they increase with age which is also associated with sex-specific fat deposition patterns [110]. It would also be interesting to assess factors involved in energy metabolism alongside environmental factors known to increase risk for MS, such as stress, smoking, etc. to see if there are significant correlations. The data from such studies could potentially lead to the development of specific ways in which patients or at-risk individuals

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could alter their lifestyle to diminish or prevent the experience of MS. 6.3. Adenosine Adenosine receptor (AR) signaling may be directly involved in MS since receptors A1 and A2A can facilitate the entry of intravenously administered macromolecules into the CNS by modulation of the blood brain barrier [111]. There is also an upregulation of A2A AR in lymphocytes isolated from MS patients and the stimulation of these receptors has anti-inflammatory effects [112]. Additionally A1A AR null mice develop severe EAE [113]. It has also been shown that rats treated with an A1 AR agonist have serum leptin levels rise 2- to 10-fold after injection and leptin increases in serum are completely blocked with an A1 antagonist [114]. Thus adipose tissue is directly influenced by adenosine and it can be hypothesized that it also plays a role in MS through processes involving adipokines. Caffeine is a non-selective antagonist of adenosine receptors and chronic caffeine treatments act in a neuroprotective manner by attenuating the EAE disease course possibly through the upregulation of the A1 receptor and TGF-␤ mRNA as well as suppression of IFN-␥ mRNA in rats [115]. Another study explored the effects of A2A AR signaling on immune functioning through the A2A AR− /− hematopoietic cells of chimeric mice. These mice developed severe EAE however the opposite effect was found with a lack of A2A AR expression on non-immune cells. This observation led to the conclusion that A2A AR expression in the CNS can influence EAE development [116]. The assessment of yet another AR was shown to alleviate pathogenesis of EAE. The A2B AR was upregulated in the leukocytes and lymphoid tissues of MS patients and EAE mice and blocking A2B AR was shown to lead to less severe EAE via inhibition of Th17 differentiation and IL-6 production [117]. As with most other factors involved in MS the adenosinergic system shows tissue- and cell-specificity in its disease-causing and disease-ameliorating activity. Although adenosine shows promise as a novel therapeutic based on these studies, because of this specificity in activation, it is necessary to incorporate this system with the others already discussed to assess how adenosine is involved with MS in the bigger picture. 7. Conclusion An under-researched but critical area for consideration in MS is the most fundamental venue for sex differences, the sex chromosomes. Sex chromosomes undergo sex specific regulations and genetic differences can arise through pathways such as nonrandom X chromosome inactivation, cellular mosaicism of females, gene dosage effects, imprinting, sex-determining region of the Y chromosome (Sry) and the expression of genes on non-recombining regions of the Y chromosome [5,13]. All of these mechanisms determining variable genetic profiles could be playing a role in the influence of sex chromosomes on MS. The sex difference in MS is also influenced by sex hormone expression, the interplay of these hormones with the sex chromosomes, and the influences of both on an immune profile which has sparked an interest in the therapeutic potential of hormones in MS. Currently a large European multicenter, placebo-controlled and double blind clinical trial called POPART’MUS is exploring the ability of progestin and estradiol to prevent this post-partum relapse of MS [118]. If the results of the trial are successful this could be the next treatment implemented for women with MS however it would be targeted at specific phases of the disease and might also only be effective for certain types of MS. It is important to widen




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the focus in the pursuit of MS therapy since other immunomodulators such as adipokines and adenosine are also effective in aiding at least some aspects of recovery. Another example of a nontraditional approach to treatment of demyelination was demonstrated with the administration of 9-cis-retinoic acid, an agonist of retinoicX-receptor ␥, to cerebellar slice cultures as well as to aged rats after demyelination which leads to an increase in remyelination of axons [119]. By focusing research on the two main systems implicated in this disease, the endocrine and the immune, it is possible to explain the paradox of a female hormonal profile conferring resistance to disease in the context of a female genetic profile biasing to affliction. But the focus on these systems does not give a satisfactory explanation for the etiology of MS and it is unlikely that any one mechanism is responsible for the disease. Therefore future exploration of this paradox should also include a focus on environmental influences on disease pathogenesis as well as other neurotransmitters and compounds classically regarded as acting only in the periphery. This shift would include the diversity in environmental exposure that comes with individual experience allowing the identification of stressors that can alter the immune reactivity to induce an attack on the self. Incorporating several influential factors into models of the disease may reveal some novel therapeutic venues and this more holistic approach could also reveal important clues into the etiology of MS.

Acknowledgment I would like to thank Dr. Neil MacLusky at the University of Guelph for his support and encouragement in the completion of this project.

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Pregnancy, sex and hormonal factors in multiple sclerosis.

Environmental factors in multiple sclerosis.

The relationship of patients' sex to reproduction in multiple sclerosis.

The Relationship between Psychosocial Factors and Cognition in Multiple Sclerosis.

ALCAM and CD6--multiple sclerosis risk factors.

Sex bias and the validity of believed differences between male and female interscholastic athletic coaches.

Multiple Factors Drive Replicating Strand Composition Bias in Bacterial Genomes.

Growth factors and synaptic plasticity in relapsing-remitting multiple sclerosis.

Effects of multiple sclerosis on female sexuality: a controlled study.

Sex bias in the BMJ: an audit.

Sex versus gender: a touchy subject for multiple sclerosis.

Osteoporosis and multiple sclerosis: risk factors, pathophysiology, and therapeutic interventions.

'Contributing factors to stress'.

The relation between peptide hormones and sex hormone in patients with multiple sclerosis.

Environmental factors and multiple sclerosis severity: a descriptive study.

Risk factors for multiple sclerosis, neuromyelitis optica and transverse myelitis.

Factors Associated with Employment Status in Individuals with Multiple Sclerosis.

Factors affecting bone mineral density in multiple sclerosis patients.

Sex-Based Differences in Multiple Sclerosis (MS): Part II: Rising Incidence of Multiple Sclerosis in Women and the Vulnerability of Men to Progression of this Disease.

The prevalence and risk factors of human papillomavirus in female sex workers.

Sex differences in interpretation bias in adolescents.

Multiple sex chromosomes in the light of female meiotic drive in amniote vertebrates.

Situational factors contributing to the placebos effect.

Female sexual dysfunction in multiple sclerosis: Results of a survey among Dutch urologists and patients.