The findings of the regression analyses point to a relationship between the proinflammatory cytokine IL-1ß and fatigue only in MS patients suffering from a relapsing remitting disease course. The salivary concentration of IL-1ß was included in all models best predicting fatigue scores in rrMS patients. This finding stands in contrast to two previous studies that did not find a relation between fatigue and blood levels of IL-1ß in MS patients (16, 37). This difference might be due to the fact that Akali and colleagues did not use regression analyses to investigate the relation between fatigue and the IL-1ß concentration. They did not control for factors such as depression, MS type or disease modifying drugs (37). Also Malekzadeh et al. did not consider depressive symptoms in their statistical analysis (16). We did control for an effect of depressive symptoms on the IL-1ß concentration. Furthermore, we included a larger amount of MS patients than the two previous studies and we divided patients into rrMS and spMS patients and checked for a relationship between IL-1ß and fatigue in the separate groups. Our results suggest that especially in the early disease stages of MS, bodily inflammation may be one dominant cause for fatigue. However, the relatively low R2 scores indicate that IL-1ß alone may explain only a limited part of the variance in experienced fatigue.

Other cytokines like TNF-α, IL-6, IL-12, or IL-17 may have an additional impact on subjective fatigue (7).

Overall, fatigue seems to be a multi-factorial symptom resulting from different causes. Besides immunological abnormalities, also structural brain changes may contribute to fatigue. Several studies found a relation between fatigue and gray matter atrophy within specific brain areas such as frontal motor areas and subcortical areas such as the thalamus and basal ganglia (38). Nevertheless, it is noteworthy that in rrMS patients IL-1ß best predicted fatigue scores, whereas in healthy controls the psychological items of the BDI appeared to best predict the variance of the different fatigue scores. Hence, the level of IL-1ß seems to play an important role in fatigue in rrMS patients, whereas—as shown in healthy controls—fatigue might also be related to depressive symptoms as argued in many prior investigations (39–41).

The absence of a significant difference in IL-1ß concentration between rrMS patients and healthy controls raises some questions. One may argue that the peripheral level of IL-1ß cannot explain subjective fatigue, because then rrMS patients should show a higher IL-1ß concentration than healthy controls. The lack of a significant difference in the IL-1ß concentration between rrMS patients and healthy controls may be due to the small group sizes. Furthermore, the majority of rrMS patients received a disease modifying therapy. We found that patients receiving such a therapy had a significantly lower IL-1ß concentration. Hence, the lack of a difference may also be due to fact that most rrMS patients received a disease modifying therapy. Nevertheless, several studies show that chronic neuroinflammation sensitizes the brain to produce an exaggerated response to peripheral inflammation resulting in prolonged sickness behavior and increased cytokine induction within the central nervous system (42, 43). MS is a chronic inflammatory disease of the central nervous system which is characterized by chronic peripheral and central inflammation. Hence, MS patients in general might be sensitized to the effects of peripheral proinflammatory cytokines. Consequently, MS patients might produce an exaggerated response to peripheral inflammation resulting in exaggerated fatigue, whereas peripheral inflammation has no effect on healthy controls. This might explain the finding that even though rrMS and healthy controls present a similar concentration of salivary IL-1ß, rrMS present significantly higher fatigue scores.

According to the regression analyses, age seems to have an influence on the Fatigue Severity Scale Score in healthy controls, rrMS and spMS patients. Age negatively correlated with the fatigue scores in all groups. This unexpected finding contradicts the assumption that increased age is related to higher fatigue scores.

Additional evidence for a relationship between fatigue and bodily inflammation in MS patients comes from recent studies in which we demonstrated the importance of inflammationinduced vagal nerve activity for the generation of fatigue. In a previous study (44), we found that fatigue severity predicts future relapses in MS patients, additionally pointing to a relation between bodily inflammation and fatigue severity in rrMS patients. We also showed that fatigue in MS patients is associated with afferent vagal nerve signaling. A disruption of afferent interoceptive signaling is related to the absence of fatigue in MS patients (45, 46). Furthermore, fatigue correlates with autonomic symptoms (47), especially with those that strongly depend on vagal nerve signaling such as bladder dysfunctions, orthostatic intolerance and pupillomotor dysfunctions.

Given that an elevated systemic concentration of IL-1ß causes fatigue in rrMS patients, anti-inflammatory interventions should have a positive effect on fatigue in this patient population. Recent studies did show that aerobic exercise and resistance training have a combined positive effect on proinflammatory cytokine concentration and on fatigue in mildly impaired MS patients (48–53). Moreover, substances with anti-inflammatory properties such as Alfacalcidol, vitamin A or coenzyme Q10 seem to have the potential to reduce subjective fatigue in MS patients (54–57). Other studies showed that blocking IL-1ß signaling via Anakinra, an IL-1ß receptor antagonist exerts a positive effect on fatigue in patients suffering from inflammatory diseases such as rheumatoid arthritis or Sjögren's syndrome (58, 59).

There is another finding of our study, not directly related to the question of IL-1ß level and fatigue, that should be mentioned. As expected, rrMS and spMS patients differed in age, disease duration and EDSS, but not in their total and cognitive fatigue level. Consequently, the argument that fatigue results from a loss of function and a compensatory effort to solve everyday problems is less likely. Only the motor fatigue score showed a significant difference between the groups. Motor fatigue is strongly associated to muscle strength and motor impairment and might reflect the impact of motor impairment rather than the actual feeling of fatigue (60, 61). If the compensatory effort model of fatigue is correct, then it will concern motor fatigue more than cognitive fatigue.

One limitation of this study is that salivary IL-1ß concentration may not only reflect systemic immune responses but also local immune responses. This might explain the high number of outliers for the salivary IL-1ß concentration scores. To exclude an extremely high IL-1ß concentration due to local inflammation in the mouth, oral examinations need to be included. Nevertheless, gingival crevicular fluid is not only the result of local cytokine production but it is also a fluid of systemic origin indicating systemic inflammation. Moreover, also other factors that were not assessed in this study such as obesity, smoking or stress may also increase bodily inflammation and might have influenced the high concentration of IL-1ß in spMS patients (62–64). Hence, we cannot draw conclusions on causality regarding the relation between fatigue and IL-1ß in MS patients. Future studies on inflammatory markers in MS patients should also check for a potential effect of the above mentioned factors on peripheral inflammation.

# CONCLUSION

We recently developed a model for MS-related fatigue arguing that the feeling of fatigue is caused by systemic inflammation, resulting from inflammation-induced activity changes within interoceptive brain areas (7). The present study points to an association between the salivary concentration of the proinflammatory cytokine IL-1ß and subjective fatigue in rrMS patients. We also found that a disease modifying therapy significantly lowered the IL-1ß concentration. Future studies combining immunological and radiological measures are needed for a better understanding of the relation between subjective fatigue scores, peripheral immune markers and structural and functional changes in the central nervous system.

# AUTHOR CONTRIBUTIONS

KH: elaboration of study design, data collection, saliva analysis, writing of manuscript. CS and LQ: data collection, saliva analysis. H-PS and AK: help on patient recruitment, facilitation of examination room and materials. MH: help on patient recruitment, data collection, facilitation of examination room and materials. PE: writing of manuscript. HH: elaboration of study design, writing of manuscript.

# FUNDING

The study was sponsored by Merck KGaA, Darmstadt, Germany: (MS200136-0058 Fatigue\_\_IST, July 2016–October 2017).

# ACKNOWLEDGMENTS

We would like to thank the Department of Biochemistry of the Carl von Ossietzky University Oldenburg for providing us with the necessary equipment for the salivary analysis. A special thanks goes to Michael Müller for his helpful instructions and the time he spent on familiarizing us with the ELISA analysis.

#### Hanken et al. Fatigue and IL-1ß in Multiple Sclerosis

#### REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Hanken, Sander, Qaiser, Schlake, Kastrup, Haupts, Eling and Hildebrandt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Latitude, Vitamin D, Melatonin, and Gut Microbiota Act in Concert to Initiate Multiple Sclerosis: A New Mechanistic Pathway

Majid Ghareghani 1,2, Russel J. Reiter 3 , Kazem Zibara4 \* and Naser Farhadi 5 \*

*1 CERVO Brain Research Center, Quebec City, QC, Canada, 2 Medicinal Plants Research Center, Yasuj University of Medical Sciences, Yasuj, Iran, 3 Department of Cell Systems and Anatomy, The University of Texas Health Science Center, San Antonio, TX, United States, 4 PRASE, Biology Department, Faculty of Sciences-I, Lebanese University, Beirut, Lebanon, 5 Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran*

#### Edited by:

*Zsolt Illes, University of Southern Denmark Odense, Denmark*

#### Reviewed by:

*Izumi Kawachi, Niigata University, Japan Masaaki Niino, National Hospital Organization, Japan*

#### \*Correspondence:

*Kazem Zibara<EMAIL_ADDRESS>Naser Farhadi<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology*

Received: *30 June 2018* Accepted: *08 October 2018* Published: *30 October 2018*

#### Citation:

*Ghareghani M, Reiter RJ, Zibara K and Farhadi N (2018) Latitude, Vitamin D, Melatonin, and Gut Microbiota Act in Concert to Initiate Multiple Sclerosis: A New Mechanistic Pathway. Front. Immunol. 9:2484. doi: 10.3389/fimmu.2018.02484* Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). While the etiology of MS is still largely unknown, scientists believe that the interaction of several endogenous and exogenous factors may be involved in this disease. Epidemiologists have seen an increased prevalence of MS in countries at high latitudes, where the sunlight is limited and where the populations have vitamin D deficiency and high melatonin levels. Although the functions and synthesis of vitamin D and melatonin are contrary to each other, both are involved in the immune system. While melatonin synthesis is affected by light, vitamin D deficiency may be involved in melatonin secretion. On the other hand, vitamin D deficiency reduces intestinal calcium absorption leading to gut stasis and subsequently increasing gut permeability. The latter allows gut microbiota to transfer more endotoxins such as lipopolysaccharides (LPS) into the blood. LPS stimulates the production of inflammatory cytokines within the CNS, especially the pineal gland. This review summarizes the current findings on the correlation between latitude, sunlight and vitamin D, and details their effects on intestinal calcium absorption, gut microbiota and neuroinflammatory mediators in MS. We also propose a new mechanistic pathway for the initiation of MS.

Keywords: multiple sclerosis, latitude, sunlight, vitamin D, melatonin, gut microbiota

# INTRODUCTION

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) affecting over 2.5 million young adults worldwide; this condition develops when activated immune cells attack the CNS (1). Previous studies using advanced neuroimaging, neuroimmunological, and neuropathological technologies demonstrated that MS is not a single disease but rather a spectrum at disease (2). Symptoms of MS may differ greatly between patients and its progression depends on various factors affecting mainly the nerve processes. The exact mechanism responsible for this destructive disease is still unknown. Several immunological pathways have been suggested to be involved in MS owing to the use of the experimental autoimmune encephalomyelitis (EAE) animal model, the most widely used and best model for clinical MS (3). In the absence of a firm understanding of the mechanisms underlying MS, researchers have suggested a combination of risk factors that are involved in this disease; however, their specific contribution to MS pathogenesis is largely unknown. Among others, these risk factors include age, sex, family history, infections, race, climate, environment, and smoking (4).

Environmental factors, such as exposure to infectious agents, sunlight and vitamin D levels have long been considered as potent risk factors in people under 15 years of age (5). Both epidemiological and immunological data support the idea that some chronic bacterial infections may reside within the CNS and initiate pathological states (6, 7). On the other hand, high prevalence of MS has been reported in areas with short days and long night periods that may last 6–8 months per year (8). Moreover, vitamin D deficiency is commonplace in these regions of limited sunshine durations.

This survey critically reviews the literature in an attempt to clarify whether any connection exists between sunlight, vitamin D, and bacterial infection toward causing MS and suggests a new mechanism by which MS may be triggered.

### LATITUDE, SUNLIGHT, AND VITAMIN D IN MULTIPLE SCLEROSIS

About 85% of the world population lives at latitudes between the 40th parallels North and South, as a result, these individual are routinely exposed to sunlight (8). However, the remainder of the population (15%) lives at higher latitudes in the northern half of the USA, Europe, Canada, and Russia or in the southern hemisphere in New Zealand, Tasmania and Patagonia. The individuals at these high latitudes receive relatively lower amounts of sunshine while they have the highest rate of MS. Indeed, the incidence of MS in these individuals ranges from 110 to 140 cases per 100,000 people, which is two-fold greater than the rate between the 40th parallels which has about 57 to 78 cases per 100,000 (9). The incidence of MS is also higher in colder climates (10).

Inadequate exposure to sunlight has been introduced as the main risk factor for vitamin D deficiency (11). It is well documented that short days and weak sunlight do not trigger vitamin D synthesis in the skin at latitudes above 40 degrees North (10, 12) where the population relies on dietary rather than light-synthetized vitamin D (13). Two forms of vitamin D are present: D2 and D3, ergocalciferol, and cholecalciferol, respectively. Vitamin D2 is produced by some plants in response to UV radiation whereas vitamin D3 is synthesized in the skin of humans and animals via the UV irradiation of 7-dehydrocholesterol to provitamin D3, the most biologically active form (14–16). Importantly, vitamin D is a major regulator of the immune system (17) and various immunological diseases, especially MS (18).

We have reviewed the literature related to MS prevalence and vitamin D levels in Sweden, a country that is mostly present above 60◦ North latitude (12), where people experience long nights, especially during the winter. The prevalence of MS in 2011 was 189/100,000 individuals (19). Ultraviolet radiation in Sweden, particularly at northern latitudes, is too low to allow the synthesis of vitamin D during the winter months where the sun is above the horizon (20, 21). Concerning vitamin D levels in the Swedish population, several studies clearly reported vitamin D deficiency (22, 23). These observations are similar to those for New Zealand in the south hemisphere (24). Geographically, the prevalence of MS decreases by moving toward the equator (25), which further implicates sunlight and vitamin D as contributors to this serious disease (26).

#### MELATONIN, VITAMIN D, AND MULTIPLE SCLEROSIS

Melatonin, known as the chemical expression of darkness, is a sunlight dependent molecule released from pineal gland in response to darkness (27). Melatonin levels correlate with neuroimmunological diseases and are inversely related to the severity of MS and its relapse (28–32). These observations prompted researchers to investigate melatonin's effect on MS using experimental autoimmune encephalomyelitis (EAE) animal models. When tested, the severity of this condition was ameliorated using melatonin (33–35). We previously reported, however, that the action of melatonin in EAE rats may be age related (36). At the clinical level, MS patients administered melatonin as a sole treatment for 4 years recovered to 6.0 at the Expanded Disability Status Scale (EDSS), from an initial 8.0 level (32).

While several clinical studies investigated vitamin D-mediated functions in MS, the mechanisms by which vitamin D or melatonin functions relate to MS are not known. Previous studies clearly noted a reduction of vitamin D levels in MS patients, compared to healthy subjects; hence, hypovitaminosis D has been suggested to be a risk factor for MS (37). However, the evidence for a role of vitamin D as a treatment for MS is inconclusive and larger studies are needed (38). As a strategy to ameliorate the severity of MS, low-dose vitamin D supplementation did not show a significant effect on the EDSS score or relapse rate of MS patients (39). Conversely, some studies reported that increased vitamin D levels reduce the incidence and disease course of MS (40–42). A recent study showed an inverse correlation between changes in serum levels of vitamin D and melatonin. Indeed, the night secretion of melatonin was shown to be reduced after 3 months' administration of high dose vitamin D in IFN-β treated MS patients. Moreover, there was a reduction in serum vitamin D levels when melatonin levels rose at night (43).

# VITAMIN D, MELATONIN, AND THE EYE

Both vitamin D and melatonin are individually essential for cellular physiology; their rhythms are contrary to each other.  While vitamin D is present in certain foods, the bulk of it is obtained through exposure to sunlight. Conversely, the pineal gland produces melatonin primarily at night (44, 45), but it is also, like vitamin D, consumed in the diet (46). It is well established that melatonin secretion from the pineal gland peaks near the middle of the dark phase and then declines slowly and gradually (47, 48). It is possible that increasing vitamin D levels during the day may act, in part, as a signal that suppresses melatonin generation (43).

In the mammalian retina, rod and cone photoreceptors, whose photopigments are rhodopsin and photopsin, are responsible for the image-forming vision. Newly identified photoreceptors in the inner retina named "intrinsically photosensitive retinal ganglion cells (ipRGCs)" are responsible for non-image-forming vision such as regulation of circadian rhythms and pupil size (49–52). These retinal ganglion cells (RGCs) are also involved in melatonin regulation where ipRGCs selectively express melanopsin, a novel opsin-like protein and a photopigment whose expression is restricted to <2% of RGCs (53). Melanopsin regeneration is different from that of rhodopsin (54, 55). Melanopsin exists in equilibrium in two stable states under broadband light conditions and exhibits a peak spectral sensitivity in the blue wavelengths at ∼482 nm. It is important to note that ipRGSc are involved in non-visual responses to light, especially blue light (56). The circadian rhythm of pineal melatonin is regulated by signals coming from suprachiasmatic nucleus (SCN) of the hypothalamus (57). ipRGC axons project to the SCN and it is this pathway that ultimately controls pineal melatonin production (58).

It is now known that exposure to blue light activates melanopsin and inhibits the SCN to synthesize and release melatonin (59–61). A recent study indicated that loss of visual axons and RGCs could be associated with vitamin D deficiency, consistent with the neuro-steroid effects of vitamin D in the CNS (62). While RGCs play a critical role in regulating melatonin production/release, the effect of vitamin D deficiency on RGCs could relate vitamin D deficiency with melatonin. This is a subject worthy of investigation. Immunologically, both hormones play a critical role in the blood brain barrier (BBB) integrity (63, 64).

#### VITAMIN D AND INTESTINAL CALCIUM ABSORPTION

Calcium is an abundant element in the human body and exhibits key roles in many physiological processes including blood clotting, hormone secretion, bone mineralization, nerve impulse transmission, and muscle contraction (65). While melatonin influences calcium absorption (66), vitamin D3 is the main hormone controlling intestinal calcium uptake (67). The importance of vitamin D deficiency in impairing calcium absorption from the intestine has been known for decades (68, 69). Several studies have shown, using vitamin D receptor (VDR) knockout mice, that vitamin D directly enhances intestinal calcium absorption (70, 71). In addition, it has been clearly documented that intestinal calcium absorption is reduced in vitamin D deficient animals and patients with low circulating vitamin D levels (72, 73).

Gastrointestinal motility involves a complex tightly coordinated series of contractions and relaxations of gastrointestinal smooth muscles, which are essential to maintain the orderly process of digestion. While most muscle cells use free calcium present in the cytosol for this process, gastrointestinal smooth muscle cells (SMCs) use calcium that has been imported from the extracellular fluid through special channels (74). Intestinal muscle cells need to increase and then reduce the concentration of calcium to initiate the contraction and relaxation of the intestinal muscles, respectively (75). This calcium variation is one of the main regulatory factors that affects intestinal motility. This observation led for simultaneous administration of vitamin D and calcium as a therapeutic strategy to stimulate normal intestinal motility in humans (76). In addition to the critical role of vitamin D3 in intestinal calcium absorption and intestinal motility, it may be involved in maintaining the integrity of the intestinal barrier and protecting it against mucosal injury (77).

# INTESTINAL CALCIUM ABSORPTION, GUT MICROBIOTA, AND MULTIPLE SCLEROSIS

The reduction in intestinal calcium absorption leads to disruptions in intestinal motility and subsequently causes stasis of aboral movement (gut stasis) and gastroparesis in the long term (78). Gut stasis is a potentially deadly condition in which the digestive system slows down or stops completely whereas gastroparesis is a chronic disorder of delayed gastric emptying characterized by food remaining in the stomach for a longer time than normal (79). Gill etal. (80) reported for the first time a role of intestinal aboral movement in MS patients with intractable constipation. Two other studies in MS patients complaining of constipation or fecal incontinence reported that an efficient therapy for MS patients is gut focused behavioral treatment (biofeedback), especially for those with non-progressive limited disability (81, 82). In addition, a similar study in MS patients with constipation symptoms suggested a positive effect of abdominal massage on constipation symptoms and alleviation of MS severity (83).

Abnormalities of slow intestinal movement such as gut stasis, gastroparesis, and constipation seems to cause a rise in intestinal absorption including bacterial toxins. In support of this hypothesis, several previous studies clearly showed that gut stasis leads to elevated gut permeability and bacterial translocation (75, 84–87). This ultimately releases toxic mediators which further increases gut permeability (88–91). Conversely, alterations in the gut microbiota may to be involved in some neurological and autoimmune conditions (92), especially MS (93). For instance, it has been reported that patients in the active or remission phases of relapsing-remitting MS (RRMS) have gut microbial dysbiosis (94). Another similar study in children (≤18 years old within 2 years of MS) showed increased levels of gut gram-negative bacteria that could be associated with neurodegeneration (95). Moreover, gut bacteria can also affect the integrity of BBB, which is critical in MS (96).

#### TABLE 1 | Clinical and review studies.

#### TABLE 2 | Experimental studies on EAE.

FIGURE 1 | Schematic representation correlating various factors such as light, eye, melanopsin, pineal gland, vitamin D, intestinal calcium, and gut microbiota to neuroinflammation and MS. (A) Adequate exposure to sunlight; (1) Long days and adequate exposure to sunlight suppresses the melatonin secretion and (2) leads to activation of melanopsin, generated by RGCs. (3) Activated melanopsin by sunlight sends an inhibitory signal to pineal gland to decreases the melatonin secretion. (Red numbered rectangle). (B) Inadequate exposure to sunlight; (1) Long nights and/or inadequate exposure to sunlight increase the level of melatonin (black arrow), (2) causes melanopsin inactivation and. (3) Promotion in level of inactivated melanopsin by darkness leads to sending a stimulatory signal to pineal gland to cause a further increase in melatonin levels. (4) On the other hand, darkness leads to Vitamin D deficiency. (5) Vitamin D deficiency causes injury to RGCs, (6) reducing melanopsin secretion (dashed black arrow). (7) Vitamin D deficiency also causes disruption in intestinal calcium absorption, which (8) leads to a reduction in smooth muscles of the intestine and subsequently gut stasis. (9) The latter increases gut permeability and LPS translocation toward the CNS. (10) LPS activates CD14/TLR4/MD2 complex which (11) increases the proinflammatory mediators in the brain such as TNF-α. (12) CD14 and TLR4 receptors in the pineal gland respond to LPS by (13) TNF secretion and (14) suppression of melatonin synthesis. (15) Eventually, secreted proinflammatory mediators and activated NF-kB pathway leads to neuroinflammation and possible demyelination at the long term. (Green numbered rectangle).

# GUT MICROBIOTA, CD14, TOLL LIKE RECEPTOR 4 (TLR4), AND MELATONIN

The intestine of animals and humans contain gut microbiota which produce endotoxic compounds including lipopolysaccharides (LPS), a component of gram-negative bacterial outer membrane (97, 98). The rise in LPS levels in gut microbiota increases the blood LPS through gut inflammation (99). LPS is recognized by LPS-binding protein (LBP) in the serum which brings the LPS to the surface of various cells such as macrophages and endothelial cells to form a complex with CD14, a receptor molecule for LPS. CD14 splits LPS aggregates into monomeric molecules and facilitates the transfer of LPS to TLR4/MD2 complex. MD2 is a secreted glycoprotein that functions as an indispensable extracellular adaptor molecule for LPS-signaling events. Activation of TLR4/MD2 complex upon binding to LPS leads to LPS-mediated NFkB activation and production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) (100–107).

The role of the pineal gland/melatonin in response to LPS is controversial. Melatonin was shown to inhibit the LPS-CD14- TLR4 signaling pathway in bovine mammary epithelial cells and decreased LPS-induced expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 (108). In contrast, pineal cells possess both TLR4 and CD14 that bind to LPS and activate NF-kB pathway by increasing the level of TNF, which subsequently suppresses melatonin synthesis (109). Since CD14 is the major cell surface receptor for LPS on monocytes/macrophages (110), the authors established that melatonin increased the secretion of IL-1 in vitro and in vivo (111, 112) and TNF-α and IL-6 in vivo (112, 113). These data support the hypothesis that LPS produced by gut microbiota causes neuroinflammation that in turn induces higher levels of LPS stimulating the pineal gland to activate the NF-kB pathway and to produce TNF-α while suppressing melatonin synthesis.

# UV IRRITATION, VITAMIN D, GASTROPARESIS

Higher latitude has been associated with higher MS incidence and lower UV exposure. In support of the role of latitude in MS susceptibility, a recent study suggested that regional UVB radiation affects MS prevalence which supports the hypothesis that exposure to sunlight can influence MS risk (114). The latter study highlighted the potential role of gender-specific effects of UVB, a suggestion that is also proposed by meta-regression analyses (115) and by incidence studies of MS (116). In fact, an experimental study demonstrated that UVB therapy can suppress EAE; however, its effect does not proceed via the production of vitamin D (117). On the other hand, although vitamin D levels are low in MS patients, evidence that vitamin D prescription can reduce the incidence of MS has not been obtained yet (118–120). Importantly, vitamin D could reduce the severity of disease only when it was accompanied by elevated serum calcium (121).

To investigate the involvement of UV in MS progression, Irving and colleagues demonstrated that EAE incidence was reduced by 74% following UVB radiation (122). Since UVB photons enter the skin to produce vitamin D3 during exposure to sunlight (123), Irving and colleagues showed that UVB therapy in EAE caused an increase in the levels of skin cis-urocanic acid levels, an intermediate in the catabolism of l-histidine. Moreover, they also observed that enhancement of skin cisurocanic acid levels independent of UVB cannot affect the disease onset or progression (122). On the other hand, it has been demonstrated that cis-urocanic acid causes a reduction in the severity of colitis, a chronic inflammatory condition of the gut (124). In addition, colitis was shown to delay gastric emptying and leads to colitis-induced gastroparesis in animal models (124). In accordance, it has been reported that women are more susceptible to gastroparesis than men (125), while the incidence of MS is about 3-fold higher in women than in men. We therefore suggest that gastroparesis could be one of the main factors involved in triggering MS.

The summarized results and highlights about the clinical and experimental studies on MS patients and EAE model have been demonstrated in **Table 1** and **Table 2**, respectively.

# CONCLUSION

We suggest a new pathway that lead to neuroinflammation and MS by including different factors such as latitude, sunlight, vitamin D, melanopsin, intestinal calcium, pineal gland, gut stasis, gut endotoxins (LPS), and CD14/TLR4 (**Figure 1**). While the prevalence of MS is dramatically higher at latitudes above 40 degrees North and South, populations in these areas receive limited sunlight that may lead to a longer increase in melatonin synthesis and release. Since the functions and synthesis of melatonin and vitamin D are contrary to each other, we believe that therapy using these hormones would not be an effective strategy for the treatment of MS patients with low melatonin levels or vitamin D deficiency. We suggest that a balance should exist between these two hormones. In dark periods, melanopsin in the RGCs is inactivate allowing the pineal gland to synthesize melatonin; however, vitamin D levels decrease dramatically and patients face vitamin D deficiency with long term sunlight deprivation. This low level of vitamin D causes RGC injury which contain melanopsin and also reduces intestinal calcium absorption, essential for intestinal smooth muscle contraction. Vitamin D deficiency and reduction of calcium absorption leads to gut stasis and subsequently increases the gut permeability allowing gut microbiota to transfer more endotoxins such as LPS into the blood. Translocated LPS migrates to the brain and triggers the production of proinflammatory mediators through CD14/TLR4/MD2 complex. CD14 and TLR4 receptors within the pineal gland respond to LPS with induced TNF secretion while melatonin synthesis is suppressed causing neuroinflammation and contributing to the development of MS in the long term. Further experimental and clinical studies are needed to unravel the mechanisms of MS induction.

# AUTHOR CONTRIBUTIONS

MG, RR, KZ, and NF: concept and design of the review, drafting the manuscript and figures. All authors have read, critically revised, and approved the final manuscript before submission. KZ and NF have equal contribution and are co-last and corresponding authors.

# REFERENCES

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increased intestinal permeability. Dig Dis Sci. (1996) 41:2493–8. doi: 10.1007/BF02100148

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Ghareghani, Reiter, Zibara and Farhadi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Sex-Specific Gene-by-Vitamin D Interactions Regulate Susceptibility to Central Nervous System Autoimmunity

*Dimitry N. Krementsov1 \*, Loredana Asarian2 , Qian Fang2 , Mahalia M. McGill 1 and Cory Teuscher 2,3\**

*1Department of Biomedical and Health Sciences, University of Vermont, Burlington, VT, United States, 2Department of Medicine, University of Vermont, Burlington, VT, United States, 3Department of Pathology, University of Vermont, Burlington, VT, United States*

#### *Edited by:*

*Martin S. Weber, Universitätsmedizin Göttingen, Germany*

#### *Reviewed by:*

*Naoto Kawakami, Ludwig-Maximilians-Universität München, Germany Fred Lühder, Georg-August-Universität Göttingen, Germany*

#### *\*Correspondence:*

*Dimitry N. Krementsov<EMAIL_ADDRESS>Cory Teuscher<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology*

*Received: 20 April 2018 Accepted: 29 June 2018 Published: 17 July 2018*

#### *Citation:*

*Krementsov DN, Asarian L, Fang Q, McGill MM and Teuscher C (2018) Sex-Specific Gene-by-Vitamin D Interactions Regulate Susceptibility to Central Nervous System Autoimmunity. Front. Immunol. 9:1622. doi: 10.3389/fimmu.2018.01622*

Vitamin D3 (VitD) insufficiency is postulated to represent a major modifiable risk factor for multiple sclerosis (MS). While low VitD levels strongly correlate with higher MS risk in white populations, this is not the case for other ethnic groups, suggesting the existence of a genetic component. Moreover, VitD supplementation studies in MS so far have not shown a consistent benefit. We sought to determine whether direct manipulation of VitD levels modulates central nervous system autoimmune disease in a sex-by-genotypedependent manner. To this end, we used a dietary model of VitD modulation, together with the autoimmune animal model of MS, experimental autoimmune encephalomyelitis (EAE). To assess the impact of genotype-by-VitD interactions on EAE susceptibility, we utilized a chromosome substitution (consomic) mouse model that incorporates the genetic diversity of wild-derived PWD/PhJ mice. High VitD was protective in EAE in female, but not male C57BL/6J (B6) mice, and had no effect in EAE-resistant PWD/ PhJ (PWD) mice. EAE protection was accompanied by sex- and genotype-specific suppression of proinflammatory transcriptional programs in CD4 T effector cells, but not CD4 regulatory T cells. Decreased expression of proinflammatory genes was observed with high VitD in female CD4 T effector cells, specifically implicating a key role of MHC class II genes, interferon gamma, and Th1 cell-mediated neuroinflammation. In consomic strains, effects of VitD on EAE were also sex- and genotype dependent, whereby high VitD: (1) was protective, (2) had no effect, and (3) unexpectedly had disease-exacerbating effects. Systemic levels of 25(OH)D differed across consomic strains, with higher levels associated with EAE protection only in females. Analysis of expression of key known VitD metabolism genes between B6 and PWD mice revealed that their expression is genetically determined and sex specific and implicated *Cyp27b1* and *Vdr* as candidate genes responsible for differential EAE responses to VitD modulation. Taken together, our results support the observation that the association between VitD status and MS susceptibility is genotype dependent and suggest that the outcome of VitD status in MS is determined by gene-by-sex interactions.

Keywords: vitamin D, multiple sclerosis, CD4 T cells, genetic variation, experimental autoimmune encephalomyelitis (EAE), sex differences, gene–environment interactions, wild-derived inbred strains

# INTRODUCTION

Multiple sclerosis (MS) is a multifactorial autoimmune disease, in which an immune-initiated attack on the central nervous system (CNS) results in demyelination, axonal loss, and eventual neurological dysfunction. Genetics contribute to a significant portion of MS risk, with estimates ranging from 20 to 30% (1). The primary genetic risk factor lies in the major histocompatibility complex class II locus, with up 200 other minor risk loci identified by recent genome-wide association studies (2).

The remainder of MS risk is thought to come from environmental factors or gene-by-environment interactions. A number of different environmental risk factors have been associated with MS susceptibility. The most prominent of these are Epstein–Barr virus (EBV) infection, low sunlight/ultraviolet (UV) radiation exposure, vitamin D3 (VitD) deficiency, and cigarette smoking (3, 4). In addition, over the past 100 years, MS incidence has remained stable in men, but has tripled in women, suggesting the existence of sex/gender-specific risk factors and/or behavioral changes (3).

Vitamin D3 is one of the best-studied MS risk factors. Early epidemiologic studies documented a gradient of increasing MS incidence with increasing distance from the equator, which in later studies was attributed to decreased exposure to sunlight/ UV radiation (3). This protective effect of sunlight in MS has long been thought to be mediated by the immunomodulatory effects of VitD. Photoconversion of 7-dehydrocholesterol to VitD (cholecalciferol) in the skin is catalyzed by UV-B radiation. VitD from the skin and dietary VitD (absorbed in the intestine) enters the circulation and is subsequently converted in the liver to calcidiol [25(OH)D3], and then in the kidney or in target tissues to calcitriol [1,25(OH)2D3], the hormonally active form which can bind and activate the nuclear vitamin D receptor (VDR) in many different target tissues, including bone, kidneys, intestine, and the immune system (5). Calcitriol-mediated activation of VDR in different immune cells is thought to generally result in immunoregulatory transcriptional responses (5). With regard to MS, low systemic VitD levels [typically measured using the most stable VitD metabolite 25(OH)D3 as a surrogate] are associated with increased disease risk (6), relapse rate, and disease progression (7, 8). A number of MS susceptibility genes are predicted to be regulated by VitD (9, 10), but the underlying *in vivo* mechanisms contributing to the etiopathogenesis of MS remain unclear. In addition, recent Mendelian randomization studies have shown that genetic variants that are associated with reduced circulating 25(OH)D3 levels also predict increased risk of MS (11–13). There are ongoing clinical trials to test the benefits of dietary VitD supplementation as a preventative or therapeutic strategy, but to date no clear beneficial effect has been reported (14–17). Importantly, the immunosuppressive effects of UV radiation independent of VitD are also well documented (18, 19). In fact, the results of recent epidemiological studies suggest that VitD and UV radiation can exert independent effects on MS risk (20–22), and data from animal models support this concept (19, 23–25).

Intriguingly, while low VitD levels are strongly associated with MS risk in white populations, a number of studies have shown that this is not the case in blacks and Hispanics (6, 26–30). This is surprising, since these populations typically have darker skin pigmentation and thus lower VitD levels compared with whites living at the same latitude (31), and it demonstrates that the association between VitD and MS risk is modified by unidentified genetic factors. This also suggests that any benefits of VitD supplementation for MS would be genotype dependent.

Effects of VitD have also been explored in animal models of MS. Experimental autoimmune encephalomyelitis (EAE), the principal autoimmune model used to study the pathogenesis of MS, can be induced by immunization with CNS homogenate or specific myelin proteins/peptides, or by transfer of CD4 T cells reactive to these antigens (32). As in MS, autoreactive CD4 T cells enter the CNS to initiate inflammation and pathology, culminating in neurologic disability. Treatment of adult animals with the hormone calcitriol has long been known to suppress EAE in mice (33, 34). More recent mechanistic studies have shown that this suppression requires VDR signaling in T cells and the expression of interferon gamma (IFNγ) (35, 36), thus likely acting directly to inhibit T helper (Th)-1 effector functions. In addition, calcitriol-mediated suppression of EAE is associated with induction of regulatory T cells (Tregs) (37), and hence it has been proposed that VDR signaling may act as a switch between Th1 effector and regulatory CD4 T cells (38). However, the physiologic validity of this approach has been called into question, as treatment with calcitriol can cause hypercalcemia, which itself can suppress EAE (39, 40), although this point remains controversial (38). As a more physiologically relevant approach, dietary supplementation with VitD during adolescence also inhibited EAE in mice (41, 42) and rats (43, 44). Strikingly, this effect was observed only in female, but not male mice, and was dependent on the presence of estrogen (41, 42).

The EAE model is an attractive approach to directly test hypotheses generated from epidemiologic studies on MS risk factors (45). However, one of the common limitations of this approach is that the immense genetic diversity of human populations is not represented among standard inbred laboratory strains of mice (46, 47). We and others have attempted to more closely model human genetic diversity by incorporating into the experimental design wild-derived inbred strains of mice, such as PWD/PhJ (PWD), or chromosome substitution (consomic) strains that carry individual PWD chromosomes on the common B6 background (B6.ChrPWD) (48, 49). We have shown that compared with B6 mice, a classical inbred laboratory strain, wild-derived inbred PWD mice have widely divergent immune cell transcriptomes, which result in differential expression of MS relevant genes and reduced susceptibility to EAE (50). We have also used the B6.ChrPWD consomic model to identify multiple EAE susceptibility loci, many of which were sex specific (51).

In this study, we combined the physiologically relevant dietary approach to manipulate VitD levels (41–43), with the genetic diversity of B6.ChrPWD consomic model, to examine gene-by-sex interactions on the effects of VitD in CNS autoimmune disease. In agreement with previous reports, we show that the effects of VitD supplementation on EAE susceptibility in B6 mice are female specific. This was associated with induction of sex- and genotype-specific transcriptional responses in effector and regulatory CD4 T cells. Strikingly, the EAE response to VitD supplementation varied widely across B6.ChrPWD consomic strains, suggesting that, as in MS, genotype modifies the outcome of VitD status in EAE.

#### RESULTS

### Dietary Manipulation of VitD Levels Modulates Systemic VitD Levels and EAE Severity

To directly manipulate systemic VitD levels in a controlled fashion, we adopted a previously described dietary paradigm initiated during adolescence, which has been shown to modulate EAE in the mouse (41, 42) and rat (43, 44). Briefly, 3-weekold B6 and PWD mice were randomized to either a VitD-low diet or a VitD-high diet, as described in Section "Materials and Methods" (see Figure S1 in Supplementary Material for a diagrammatic overview of the experimental design). Serum samples were collected at 3 and 5 weeks post-dietary intervention, and analyzed for the levels of 25(OH)D, the most abundant and stable metabolite of VitD that is typically used as an indicator of VitD status in clinical studies (6). The dietary regimen induced rapid and sustained changes that appeared to plateau at approximately 3 weeks post-intervention (**Figure 1A**). PWD mice exhibited significantly lower baseline 25(OH)D levels compared with B6 (**Figure 1B**). These differences disappeared on the VitD-low diet (**Figures 1C,D**). Interestingly, on the VitD-high diet, female B6 mice reached higher levels of 25(OH) D compared with B6 males at both time points (**Figures 1E,F**), which were also significantly higher than PWD females at 5 weeks (**Figure 1F**).

Having established the dietary paradigm, we tested whether the robust differences in systemic VitD levels achieved by VitD-high and VitD-low diets impacted clinical progression of EAE. Female and male B6 mice were subjected to the dietary paradigm above, followed by EAE induction at 5 weeks postdietary intervention. Exposure to the VitD-high diet resulted in lower EAE severity compared with VitD-low diet in female B6 mice (**Figure 2A**). By contrast, no significant difference in EAE clinical course on the two diets was observed in male B6 mice (**Figure 2B**). DeLuca and colleagues have observed that suppression of EAE in mice by treatment with the bioactive VitD metabolite, calcitriol, is associated with and dependent on hypercalcemia, questioning the physiological relevance of that approach (39, 40). Hence, we tested whether our dietary paradigm affected systemic Ca2+ levels and found that EAE suppression was not accompanied by significant changes in Ca2+ levels (**Figure 2C**), suggesting that our dietary model influences EAE susceptibility independent of Ca2+.

We have previously shown that PWD mice are almost completely resistant to EAE (50). To test whether this resistance

Figure 1 | Manipulation of dietary vitamin D3 (VitD) results in robust changes in systemic VitD levels that are sex- and genotype dependent. Female and male B6 and PWD mice (*N* = 4 for each sex/strain combination) were assigned to VitD-high and VitD-low diets at 3 weeks of age. Serum samples were collected at the outset (Time = 0), and at 3 and 5 weeks post-treatment. 25(OH)D levels were measured by enzyme-linked immunoassay (see Materials and Methods). An overview of kinetic data is shown in (A), followed by comparisons at individual time points in (B–F), segregated by diet. The significance of the observed differences in (B–F) was assessed by two-way ANOVA, with Holm–Sidak's *post hoc* comparisons: B6 vs. PWD (within sex), and female vs. male (within strain). Symbols indicate a significant difference between the indicated groups, as follows: \**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001; \*\*\*\**p* < 0.0001.

measures two-way ANOVA) are shown. The numbers of animals for each group are provided in Table S1 in Supplementary Material.

could be affected by VitD status, PWD mice were subjected to the dietary regimen as above, followed by EAE induction. VitD had no significant effect on EAE, whereby PWD mice remained highly resistant to EAE induction on either diet (**Figure 2D**; EAE incidence: VitD-high 0/8, VitD-low 2/15; *p* = 0.53). Taken together, our results demonstrate that high VitD provides femalespecific protection from EAE in susceptible B6 mice, in agreement with previous reports (41, 42).

# *In Vivo* Modulation of CD4 T Cell Transcriptome by VitD Is Genotype-, Sex-, and Cell Type Specific

CD4 T cells are thought to initiate the inflammatory cascade in MS. Studies in EAE suggest that these cells are also the most likely target of VitD, either by modification of function of CD4 effector T cells (Teff) or by induction of regulatory CD4 T cells (Tregs) (38). Hence, to understand the molecular mechanisms underlying immune modulation by VitD *in vivo*, we carried out transcriptional profiling of Teff and Tregs isolated from B6 and PWD mice exposed to VitD-low or VitD-high diets. Mice were subjected to the dietary paradigm as described in **Figure 1**, followed by isolation of splenic Teff and Tregs using fluorescence-activated cell sorting (FACS) and transcriptional profiling (see Materials and Methods) at 5 weeks post-dietary intervention (Figure S1 in Supplementary Material). When gene expression data were analyzed for effect of genotype, sex, and VitD, it was found that genotype accounted for the largest effect size, with modest effects of sex and VitD (**Figure 3A**). This is consistent with our published data showing strikingly divergent transcriptomes between B6 and PWD immune cells (50). Consistent with these observations, when both strains and sexes were pooled and analyzed together for effect of VitD, very few differentially expressed (DE) genes were detected in either cell type (**Figures 3B,C**). Considering the sex- and strain-specific effects of VitD on EAE (see **Figure 2**), and the profound effect of genotype, we analyzed the effect of VitD in each of the four sex/strain combinations (B6 females, B6 males, PWD females, and PWD males) separately. Two major findings were noted. First, in Teff cells, a prominent effect of VitD on gene expression was detected in B6 females, which was absent in B6 males or PWD mice of either sex (**Figure 3B**), in concordance with the effect of VitD on EAE outcome (see **Figure 2**). Second, in Tregs, VitD exhibited the strongest effect in PWD females (**Figure 3C**). In both cases, high VitD predominantly induced upregulation of gene expression compared with low VitD (**Figures 3B,C**).

Bioinformatic pathway analyses of DE genes (see Materials and Methods) revealed divergent and sometimes opposing effects of VitD on gene expression, depending on sex and strain

described in Section "Materials and Methods." (A) The proportion of variance in gene expression explained by the indicated experimental variables is shown for Teff and Tregs. (B,C) The number of genes passing the differential expression threshold (|Fold Change| > 2, ANOVA *p* < 0.001) as a function of diet is shown for the indicated groups, in Teff (B) and Tregs (C). Direction of change is depicted as expression level in VitD-high relative to VitD-low (e.g., "up" indicates higher expression in the VitD-high group). (D) Canonical pathway analysis was performed using IPA software (see Materials and Methods). *Z*-scores indicate predicted direction and relative strength of change (VitD-high relative to VitD-low). The top 30 pathways (ranked by *Z*-score) are shown. (E) Enrichment analysis of the indicated differentially expressed (DE) gene sets from Teff within transcripts DE (up or down) in CD4 cells from multiple sclerosis (MS)-CIS patients was performed using IPA (see Materials and Methods). *p*-Values for enrichment are shown as −log(*p*). Dotted line denotes *p* = 0.05.

(**Figure 3D**). Several canonical pathways relevant to EAE/MS pathogenesis (including neuroinflammation, Th1, and a number of other proinflammatory pathways) were downregulated by high VitD in Teff cells from B6 females, yet upregulated in B6 males or PWD males. The gene expression patterns in Teff cells were again concordant with EAE outcomes, whereby downregulation of proinflammatory activity by VitD was associated with EAE suppression in B6 females. Similarly, upstream regulator analysis in B6 female Teff cells predicted significant inhibition of several proinflammatory nodes by high VitD, including TNF, NFκB, CSF2, and IFNγ as the top four nodes. Comparison of DE transcripts within the IFNγ node with two selected MS relevant canonical pathways, "Neuroinflammation Signaling Pathway" (ranked first) and "Th1 Pathway" (ranked 11th) revealed strong functional overlap and several key shared transcripts, including several MHC class II genes, and a central role for IFNγ itself (Figure S2 in Supplementary Material). The former is consistent with the documented regulation of the major MS risk MHC class II allele, HLA-DRB1\*15:01, by VitD. The latter is consistent with the documented critical role of IFNγ in the regulation of EAE by VitD (36). Finally, this analysis implicated *Mapk14* (Figure S2 in Supplementary Material), encoding p38α MAP kinase, a gene that we have previously identified as a central regulator of differential sex-specific genetic effects on EAE in the B6.ChrPWD model (51), a hypothesis that was validated in our targeted analysis of the role of p38α in EAE (52).

To test whether the observed regulation of immune cell transcriptomes by VitD had direct connections to mechanisms of CNS autoimmunity in humans, we compared the level of enrichment of VitD-dependent DE genes in PWD cells within the set of transcripts that were reported to be upregulated or downregulated in CD4 T cells isolated from early onset MS patients (clinically isolated syndrome; MS-CIS) relative to healthy controls (MS-CIS signature genes) (53). VitDdependent DE genes in Teff cells from B6 females exhibited highly significant enrichment (*p* = 1.02e−6) within genes *downregulated* in MS-CIS CD4 cells, while marginal or no enrichment was observed for genes that were *upregulated* in MS-CIS (**Figure 3E**). Marginal to no enrichment was observed in either direction for other strain-sex combinations in Teff cells (**Figure 3E**) or for any of the DE gene sets in Tregs. Together with the observation that the majority of DE genes in B6 female Teff cells are *upregulated* with high VitD (**Figure 3B**), these data suggest that high VitD normalizes the expression of genes that are downregulated in MS-CIS CD4 T cells, which are likely associated with MS pathogenesis. Taken together, our results indicate that high VitD suppresses MS-associated proinflammatory gene expression programs in CD4 T cells in a sex-, cell type-, and genotype-specific manner, in concordance with its protective effects on EAE.

### Sex and Genotype Dictate the Outcome of VitD Modulation in EAE

Since epidemiologic data (6, 26–29) and our findings above suggest the possibility that the outcome of VitD status in MS and EAE may be dependent on genotype, we deliberately introduced the segregation of natural genetic variation into our model. To achieve this, we utilized the B6.ChrPWD consomic model, in which natural genetic variation exhibits a significant impact on EAE susceptibility (51). Twenty B6.ChrPWD consomic strains, encompassing 17 autosomes, X and Y, and a conplastic strain with the PWD mitochondrial genome were included in the study. The mice were exposed to the VitD-high and VitD-low dietary paradigm, followed by EAE induction and evaluation, as in **Figure 2**. Surprisingly, in a combined analysis of all strains and sexes, no significant effect of dietary modulation of VitD on EAE course was detected (**Table 1**; **Figure 4A**). Similarly, when this combined analysis was stratified by sex, no significant effect of VitD in either sex was detected (**Table 1**; **Figure 4A**). Given the known variation in EAE susceptibility across the B6.ChrPWD consomic/conplastic strains (51), and the sex- and strain-specific responses to VitD in EAE and gene expression (see above), each strain was analyzed individually for the effect of VitD on EAE, either combining both sexes or analyzing each sex separately. When analyzing males and females together, five strains showed significant effects of VitD. Chr11.1PWD and Chr19PWD had lower EAE severity on VitDhigh diet compared with VitD-low, as seen in B6 females, but surprisingly, Chr10.2PWD, Chr11.2PWD, and Chr14PWD exhibited the opposite effect (**Figure 4**; Figure S3 in Supplementary Material). Stratification by sex revealed that these effects were primarily driven by a single sex, either female or male, depending on the strain (e.g., **Figure 4**). Stratification by sex also revealed additional significant effects of VitD in females (Chr10.3PWD, Chr15PWD, and mtPWD) and in males (Chr8PWD, Chr12PWD, and ChrYPWD) (**Table 1**). Additional analyses of EAE quantitative trait variables (Table S1 in Supplementary Material) supported the conclusions drawn from disease course analyses (**Table 1**; **Figure 4**; Figure S4 in Supplementary Material).

Table 1 | Effects of vitamin D3 (VitD) on EAE disease course in B6.ChrPWD consomic mice.

*a The significance of the observed differences in the severity of the EAE clinical disease course for each of the B6.ChrPWD consomic strains was analyzed for the effect of VitD diet, as described in Section "Materials and Methods." Data for females and males were analyzed together (pooled) and following stratification by sex. ANOVA p-values for the effect of diet on overall EAE course are shown where significant; ns, not significant; ND, not done. Direction of arrow indicates the direction of change for EAE severity in the VitD-high diet group relative to the VitD-low group (i.e., downward arrow indicates suppression of EAE by VitD-high diet).*

# Systemic 25(OH)D Levels Predict EAE Outcome in Female B6.ChrPWD Consomic Mice

Since circulating VitD levels are genetically controlled in humans (54) and in B6 and PWD mice (**Figure 1**), we determined whether B6.ChrPWD consomic/conplastic strains subjected to our dietary paradigm exhibited any differences in systemic VitD levels. Because the 25(OH)D levels reached on the VitD-high diet were dramatically different from those on the VitD-low diet (see **Figure 1**), the data were analyzed separately for each diet, to assess the effect of strain and sex. Significant differences in 25(OH)D levels were observed between several consomic strains and B6. On the VitD-high diet, a number of strains presented with significantly lower levels of 25(OH)D3 compared with B6, but no strains exhibited higher levels (**Figure 5A**). On the

VitD-low diet, several strains exhibited significantly higher or lower levels of 25(OH)D compared with B6 (**Figure 5B**). Next, we tested whether any of these changes in 25(OH)D levels were correlated with EAE outcomes, e.g., whether a larger change in VitD levels corresponded to a higher degree of disease protection by VitD. EAE cumulative disease score (CDS), as the quantitative trait variable that most accurately reflects the overall severity of the EAE clinical disease course, was used to calculate a normalized difference in CDS between VitD-low and -high diets, for each strain. Similarly, a relative difference in serum 25(OH)D between VitD-low and -high diets was calculated for each strain. The relationship between these two parameters was examined using linear regression. For female consomic mice, a significant positive relationship was observed, suggesting that those strains that had higher serum 25(OH)D responses exhibited protective effects of VitD (e.g., B6, Chr15, and Chr19), while those with low responses had the opposite effects (e.g., Chr10.3), albeit the association was modest (*r*2= 0.29) (**Figure 5C**). Surprisingly, in males, the trend was reversed, although it did not reach significance (**Figure 5D**). Taken together, these results suggest that genotype controls systemic levels of VitD, which in turn may contribute to EAE susceptibility in a sex-specific manner.

#### Tissue-Specific Differential Expression of VitD Metabolic Pathway Genes in B6 and PWD Mice

Systemic 25(OH)D levels in humans are genetically regulated, and recent GWAS studies have identified common variants in four major genes in the VitD metabolism pathway: *GC*, *DHCR7*, *CYP2R1*, and *CYP24A1*, which together explain a large proportion of the variation in 25(OH)D (54). Subsequent Mendelian randomization studies showed that the genetic control of low 25(OH)D levels by alleles of the four genes is associated with increased susceptibility to MS (11–13). To begin to identify potential candidate genes underlying differential responses across different B6.ChrPWD consomic strains, we examined the level of expression of mouse orthologs of these four key genes (*Gc*, *Dhcr7*, *Cyp2r1*, and *Cyp24a1*) in several relevant tissues (kidney, liver, and spleen) in male and female B6 and PWD mice, using publically available datasets (see Materials and Methods). We included three additional key VitD metabolism genes: *Cyp27a1*, *Cyp27b1*, and *Vdr* (55). A number of these genes were DE across different tissues. In all three tissues, vitamin D-25 hydroxylase, *Cyp27a1*, exhibited twofold to threefold lower expression in PWD compared with B6, with significantly lower expression in females compared with males in both strains in the kidney (**Figures 6A–C**). In the kidney, 1-α-hydroxylase, *Cyp27b1*, exhibited a modest significant increase in expression in PWD compared with B6 (**Figure 6D**), while 1,25(OH)2D3-inactivating 24-hydroxylase, *Cyp24a1*, exhibited a significant increase in expression in B6 (**Figure 6E**). *Vdr* also exhibited higher expression in PWD compared with B6 in the spleen (**Figure 6F**). These results demonstrate genetic control of tissue-specific differential expression of several components of the VitD metabolic pathway which may underlie some of the differences in serum 25(OH)D levels (**Figures 1** and **5**) and EAE responses (**Table 1**; **Figure 4**) in B6.ChrPWD mice. Of these

Figure 6 | Tissue-specific differential expression of vitamin D3 metabolic pathway genes in B6 and PWD mice. Gene expression data were obtained from the Gene Expression Miner database, as described in Section "Materials and Methods." Genes showing a significant differential expression (with a greater than 30% change in expression) as a function of strain or sex are shown in (A–F). The significance of the observed differences was assessed by two-way ANOVA, with Holm– Sidak's *post hoc* comparisons: B6 vs. PWD (overall effect of strain; indicated above the graphs), and female vs. male (within strain; indicated with brackets). The data included 12 C57BL/6J mice (6 females and 6 males), and 11 PWD/PhJ mice (6 females and 5 males). Symbols indicate a significant difference between the indicated groups, as follows: \**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001; \*\*\*\**p* < 0.0001.

genes, two lie within consomic intervals of interest (carried by consomic strains exhibiting significant effects in **Table 1**): *Cyp27b1* (Chr10.3) and *Vdr* (Chr15). As such, they represent candidate genes controlling differential responsiveness to VitD in EAE (see Discussion), and their relevance will be assessed in future studies.

#### DISCUSSION

Genetic and environmental influences on MS risk are well documented. However, gene–environment interactions have been more elusive, with the exception of HLA alleles and their putative interactions with smoking, EBV, and obesity (56). Part of the difficulty in identifying such interactions is the inability to clearly define over time the presence/absence/level of environmental variables/exposures and possible confounding variables (57). The other difficulty inherent to all epidemiologic studies is the inability to separate cause and effect from association/correlation. The association of low VitD with MS risk is an example of such an association, where cause and effect have been difficult to parse out, and therapeutic intervention has not yet provided a clear answer (58). Animal models provide an opportunity to bridge the gap between observation and causation, as putative genetic and environmental risk factors can be precisely controlled (45). As such, they also provide an opportunity to identify gene–environment interactions in a highly controlled experimental setting. In this study, we have applied a welldefined dietary paradigm to intentionally modulate systemic VitD levels, while at the same time introducing controlled genetic variation. This approach revealed that the effects of VitD in a mouse model of MS are regulated by sex and genotype in a cell type-specific fashion.

In a seminal prospective case–control study, Ascherio and colleagues identified an association between low 25(OH) D levels and increased risk of MS in a white U.S. population (6). Interestingly, in the same study, this association was absent in black and Hispanic groups, who in fact had lower 25(OH)D levels compared with whites. This lack of association between 25(OH)D and MS in non-whites was confirmed by several groups (22, 26–29), which has led to the suggestion that VitD-related testing and treatment in MS should be informed by ethnicity (59). In our study, we provide complementary experimental evidence that genotype can influence the outcome of VitD status or supplementation. This may help explain the variability in VitD supplementation trials in MS so far, and our results also suggest that such studies might benefit from a complementary pharmacogenetic approach to identify gene variants associated with positive or negative responses to VitD.

In this regard, there is evidence that common genetic variants in *GC* (encoding the vitamin D binding-protein; DBP) may lead to profound ethnic-specific variations in DBP levels, DBP binding avidity, and bioavailablity of VitD and its metabolites (31). However, a recent study by Barcellos and colleagues found that these variants in *GC* do not account for the lack of association between serum VitD levels and MS in blacks and Hispanics (30). This suggests that other unknown ethnic-specific genetic determinants can regulate VitD metabolism and/or subsequent physiologic responses, such as those underlying MS susceptibility.

In some B6.ChrPWD consomic strains (e.g., male Chr11.2PWD), an unexpected phenotype was observed, whereby higher EAE severity was seen on the VitD-high diet compared with VitDlow diet. However, several previous reports have documented similar unexpected EAE-suppressing effects of VitD insufficiency or deficiency, depending on length and timing of the exposure (60, 61). In addition, mice completely deficient in *Vdr* are unexpectedly resistant to EAE (62), suggesting that VitD signaling is needed to mount a robust T cell response. Thus, we hypothesize that VitD status can serve as a bi-directional rheostat regulating autoimmunity, and this balance can be further modified by genetic background and sex. Interestingly, Chr10.3PWD female mice exhibited lower 25(OH)D responses associated with increased EAE on the VitD-high diet (**Figure 5C**; Figure S3 in Supplementary Material). It is possible that increased kidney *Cyp27b1* expression from the PWD allele in the Chr10.3 locus (**Figure 6D**) drives faster 25(OH)D to 1,25(OH) D metabolism, which could result in different levels or kinetics of VDR activation and divergent EAE outcomes. Conversely, female Chr15PWD mice exhibit the opposite phenotype with regard to VitD status and EAE outcome (**Figure 5C**), while the PWD allele of *Vdr* (located on Chr15) shows higher expression (**Figure 6F**). Since for *Cyp27b1* and *Vdr* the expression differences were not sex specific (**Figures 6D,F**), we postulate that other sex-specific factors (e.g., lower expression of *Cyp27a1* in females; **Figures 6A,B**) interact with the genetically determined differential expression of these VitD pathway genes to give rise to different outcomes in EAE.

In a recent study, Jagodic and colleagues used a dietary paradigm highly similar to ours to examine genomic effects of VitD in the inbred Dark Agouti rat model of EAE (44). The conclusions reached by the authors were largely similar to ours, whereby supplementation of female rats with high levels of VitD induced an anti-inflammatory gene expression program in CD4 Teff cells from immunized mice, in concordance with amelioration of EAE. Comparison of the DE gene set from this study with our DE gene set did not reveal strong gene–gene correlation, suggesting species differences, differences in cell isolation protocols, and/or timing of cell isolation. Nonetheless, many of the differentially regulated pathways showed strong agreement between the two studies (e.g., MAPK signaling, NFκB, etc.), suggesting that a similar immune-regulatory phenotype may be achieved by VitD in both situations.

Despite the strong epidemiological associations, the role of VitD in MS remains complex and unclear. Our studies add another layer to this complexity: the immunologic response to VitD status may differ across individuals due to genetic and sex effects. The sex effects have been shown in mouse models to be likely due to the influence of estrogen (42). Our future studies will be aimed at identifying genetic modifiers of the VitD response in CNS autoimmunity. This information will help inform future VitD supplementation trials, as well as the use of VitD status as a prognostic.

# MATERIALS AND METHODS

#### Animals and Dietary Treatments

C57BL/6J (B6), PWD/PhJ (PWD), and B6.ChrPWD consomic mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA), then bred and housed in a single room within the vivarium at the Larner College of Medicine at the University of Vermont for two to four generations prior to any experimentation. The experimental procedures used in this study were approved by the Animal Care and Use Committee of the University of Vermont.

To ensure their correct identity and to enhance rigor and reproducibility of these studies, B6.ChrPWD consomic mice were subjected to genome-wide SNP genotyping using Dartmouse genotyping services (Dartmouth College, NH, USA). All mice used in this study were of the expected genotypes, with the following exceptions. B6.Chr4PWD mice were excluded from the study, because they were found to be a mix of various genotypes, where much of the Chr4PWD had been replaced with B6 genome. Chr17PWD mice were found to carry a homozygous B6-derived interval between 30 and 45 Mb on Chr17, encompassing *H2*.

At weaning (3 weeks of age), littermate mice were randomly assigned to one of two diets: VitD-low (0 IU VitD/g; 0.87% Ca2+, 0.3% phosphorus, alcohol extracted casein as protein source) or VitD-high [identical composition to VitD-low, supplemented with 5 IU VitD (cholecalciferol)/g]. The composition of the diets was based on the study those described by Spach and Hayes (41) and was prepared by Harlan-Teklad (WI, USA), designated by the following company product numbers: VitD-low, TD.10837; VitD-high, TD.140867.

### Induction and Evaluation of EAE

EAE was induced in B6 and B6.ChrPWD consomic mice using the 2× MOG35-55/CFA protocol, as previously described (63). Mice were injected subcutaneously with 0.1 ml of emulsion containing 0.1 mg of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) peptide (Anaspec Inc., MA, USA) in PBS and 50% complete Freund's adjuvant (CFA; Sigma, USA) on day 0 on the lower flanks (50 µl per flank), followed by an identical injection on upper flanks on day 7. CFA was supplemented with 4 mg/ml *Mycobacterium tuberculosis* H37Ra (Difco, USA). EAE was induced in PWD mice using the following modifications to the protocol above, as previously described (50). Mice were injected subcutaneously with 0.1 ml of emulsion containing 2.5 mg of MSCH in PBS and 50% CFA on day 0 and day 7. On day 0 and day 2, mice also received an i.p. injection of 200 ng pertussis toxin (List Laboratories, USA) as an ancillary adjuvant.

Starting on day 10, mice were scored visually, as follows: 1—partial loss of tail tone, 2—full loss of tail tone, 3—loss of tail tone and weakened hind limbs, 4—hind limb paralysis, 5 hind limb paralysis and incontinence, and 6—quadriplegia or death. EAE scoring was performed by a non-biased observer. EAE quantitative traits were calculated essentially as previously described (64), as follows. The incidence of EAE was recorded as positive for any mouse with clinical signs of EAE (clinical score ≥1) for two or more consecutive days. CDS was calculated as the sum of all daily scores over the course of 30 days. Days affected was calculated as the number of days an animal displayed a clinical score ≥1 for at least two consecutive days. Day of onset was the day a clinical score ≥1 was first observed (not calculated for animals without clinical signs for at least two consecutive days). Severity index was generated by averaging the clinical scores for each animal over the number of days that it exhibited clinical symptoms (unaffected animals were included as 0). Peak score represents the maximum daily score (unaffected animals were included as 0).

# Cell Sorting and RNA Isolation

B6 and PWD mice were subjected to the dietary paradigms, as described in Section "Results." At 5 weeks post-dietary intervention, mice were euthanized, and spleens were collected. Spleens were digested using Spleen Dissociation Medium (STEMCELL Technologies, Inc., Canada). B cells were depleted using the EasySep B cell positive selection kit and EasySep magnet (STEMCELL Technologies, Inc., Canada). The remaining cells were purified by FACS using fluorophore-conjugated antibodies against cell surface markers as follows: CD4 (Tconv) cells (CD19− TCRβ+ CD4+ CD8− CD25−) and Tregs (CD19− TCRβ+ CD4+ CD8− CD25+). Dead cells were excluded using the Far Red Live-Dead staining kit (Thermo Fisher Scientific, USA). Antibodies were purchased from BioLegend, Inc. (San Diego, CA, USA); catalog numbers were as follows: CD19, CD4, CD25, CD8, CD11b, CD11c, and TCRβ; 115534, 100531, 102016, 101206, 117319, and 109222, respectively. RNA was isolated using the Qiagen RNeasy Mini or Micro Kits. RNA quality was assessed using the Agilent Bioanalyzer 2100, and samples were selected for downstream analysis based on RNA integrity number (typically 6–9). RNA quantity was determined using Qubit Fluorometric Quantification (Thermo Fisher Scientific, USA). Four biological replicates (individual mice) for each strain, sex, and diet combination were selected.

#### Transcriptional Profiling

For transcriptional profiling, microarray analysis was performed at the UVM Cancer Center Genomics Facility using the Mouse Affymetrix Clariom D Genechip and the GeneChip™ WT Pico Target Preparation reagent kit (Thermo Fisher Scientific 9026220) as described by the manufactures procedures. Briefly, 5 ng of RNA was used to synthesize cDNA through a First-Strand and Second-Strand reverse transcription reaction followed by conversion to cRNA through an overnight T7 InvitroTranscription reaction. The resulting cRNA was purified and 5.5 μg was converted to sense, single-strand cDNA using UDG (10 U/μL) and APE1 (1,000 U/μL), provided in the GeneChip® WT PLUS Reagent Kit. cDNA was end labeled with biotin using TdT (30 U/μL), and used as input for the hybridization mix for the GeneChip. Mouse Clariom D arrays were incubated in the Affymetrix® GeneChip® Hybridization Oven 645 at 45°C/60 RPM for 16–18 h. Arrays were stained using the Affymetrix® GeneChip® staining reagents and scanned with the 7 G Affymetrix® GeneChip® Scanner 3000.

#### Statistical Analyses of Microarray Data

Raw intensity CEL files were imported into Expression Console software (Affymetrix, USA), and CHP files were generated for gene level analysis. CHP files were imported into Transcriptome Analysis Console (TAC) software v<IP_ADDRESS> (Affymetrix, USA), and gene level expression analysis was performed using the default ANOVA settings (e-Bayesian method), analyzing Tconv and Treg data separately. The following comparison variables were used: strain (B6, PWD), sex (male, female), diet (VitDhigh, VitD-low), as well as a batch variable (cDNA samples were processed and scanned in batches over several different days). To detect DE genes as a function of VitD status, pairwise comparisons were done between VitD-high and VitD-low groups for the following sample groupings: (all mice together, B6 females only, B6 males only, PWD females only, and PWD males only). All raw microarray data have been deposited into the Gene Expression Omnibus database, accession number GSE116457.

#### Bioinformatic Analyses

Pathway analysis was performed using Ingenuity Pathway Analysis™ (IPA; Qiagen, Inc., USA) software. The gene expression datasets were exported from TAC software and uploaded into IPA. A relaxed cutoff filter of |FC| > 1.5 and ANOVA *p* < 0.05 was used to maximize the number of genes in the analysis (recommended by IPA to enhance the analysis power and accuracy). The IPA Core Analysis function, followed by the Comparison Analysis function was used to compare the effect of VitD across the four strainsex combinations (B6 females, B6 males, PWD females, and PWD male), as follows. The Canonical Pathway function was used to identify the top canonical pathways (*p* < 0.01, *Z*-score > |2|) affected by the DE genes between VitD-high and VitD-low conditions. The sign and magnitude of the *Z*-scores are indicative of the predicted strength and direction of the VitD-high effect. The upstream regulator analysis function was similarly used to predict *Z*-scores and *p*-values for putative upstream regulators.

Enrichment analysis of VitD-regulated genes compared with genes DE in CD4 T cells in MS as performed as follows. The list of transcripts reported to be upregulated in CD4 T cells from MS-CIS subjects vs. controls (53) was imported into IPA. The Core Analysis function was used to determine the significance of enrichment of VitD DE genes within the MS-CIS list.

#### Serum 25(OH)D and Ca2**+** Measurements

Whole blood was collected at the indicated time points, allowed to coagulate for 30 min at room temperature, followed by centrifugation and collection of serum. Sera were stored at −80°C prior to analysis. 25(OH)D levels were determined using a commercially available enzyme-linked immunoassay kit (ImmunoDiagnosticSystems, Inc., MD, USA), which detects 25(OH)D3 and 25(OH)D2. Sera from mice on the high VitD diet were diluted 1:10 with PBS + 1% BSA prior to analysis, to maintain readings within the range of the standard curve. Kit-supplied standards were used with 4-parameter logistic regression to determine concentrations in unknown samples.

Serum Ca2+ measurements were performed at the University of Vermont Medical Center Clinical Chemistry laboratory.

#### Statistical Analyses

Statistical analyses not pertaining to microarray data were carried out using GraphPad Prism software, version 6. Details of the analyses are provided in the figure legends and below. All statistical tests were two-sided, and adjustments for multiple comparisons were made as indicated. All center values represent the mean, and error bars represent the SEM. *p*-Values below 0.05 were considered significant. Sample sizes for animal experiments were chosen based on previous experience with similar analyses. Animals were randomly assigned to different treatment groups (diet), assigning littermates to both groups evenly, whenever possible. For some consomic strains, sample sizes studied were lower due to inadequate breeding performance.

Analyses of EAE clinical scores were performed as follows. Clinical disease time course was analyzed for the effect of diet (VitD) for each strain, using two-way repeated measures ANOVA. The effect of diet was considered significant when a significant strain and/or strain × time interaction term was observed. The latter term is shown in the figures and tables, to indicate overall significance of effect of diet on overall EAE course.

The relationship between serum 25(OH)D levels and EAE severity was determined as follows. First, a weighted difference in EAE severity was calculated, using CDS, as a single quantitative measure of overall disease severity and duration. For each strain, the diet-driven change in CDS was calculated by substracting the mean CDS for the VitD-high diet from the mean CDS for the VitD-low diet. Since the absolute CDS varied significantly across strains independent of VitD, this change was normalized by dividing by the overall mean CDS for that strain:

Normalized change in EAE CDS CDS CDS mean VitD VitD high = − − low − CDSVitD− + ow high . l

Because serum 25(OH)D levels varied by strain, to determine the relative serum 25(OH)D response to diet, a ranked change was calculated for each strain, as follows. 25(OH)D3 levels were ranked for across the consomic strains separately for VitD-high and VitD-low diets. For each strain, the ranked change in 25(OH) D as a function of diet was calculated by substracting the ranked 25(OH)D for the VitD-high diet from the ranked 25(OH)D for the VitD-low diet:

```
Ranked change in 25 OH D
                 ( )
```
ranked 25 OH r low anked 25 = ( ) − DVitD− (OH) . DVitD−high

Subsequently, linear regression was used to determine measure the association between the ranked 25(OH)D3 response and normalized EAE response. *R*2 and the significance for the slope not being equal to zero were calculated as a measure of strength of association.

#### Differential Expression Analyses of VitD-Related Genes in B6 and PWD Mice

Differential expression data were obtained from the Gene Expression Miner database from Jackson Laboratories (http:// cgd.jax.org/gem/strainsurvey26/v1), which contains microarray data from a gene expression survey across 26 inbred strains of mice, in 4 tissues: spleen, liver, and left and right kidneys. The data set included data on 12 C57BL/6J mice (6 females and 6 males), and 11 PWD/PhJ mice (6 females and 5 males). Log2-normalized gene expression data were downloaded for the following genes of interest: *Gc*, *Dhcr7*, *Cyp2r1*, *Cyp24a1*, *Cyp27a1*, *Cyp27b1*, and *Vdr*. *Gc* and *Cyp2r1* did not show appreciable levels of expression in any of the tissues, and thus were not analyzed further. The rest of the gene expression data were analyzed in Graphpad Prism, version 6, as detailed in the figure legends.

# ETHICS STATEMENT

This study was carried out in accordance with the recommendations of AAALAC guidelines. The protocol was approved by the Animal Care and Use Committee of the University of Vermont.

# AUTHOR CONTRIBUTIONS

DK and CT designed experiments. DK, LA, and CT analyzed data and wrote manuscript. DK, QF, and MM performed experiments and analyzed data.

#### REFERENCES

#### FUNDING

This work was supported by the following grants: RG-5170A6/1 from the National Multiple Sclerosis Society (NMSS) to CT, R21 NS095007 from NIH/NINDS to CT, and R01 NS097596 from NIH/NINDS to DK. Additionally, the research performed at the VGN microarray facility was partially supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under grant number P20GM103449, and research performed at the Flow Cytometry and Cell Sorting Facility was partially supported by NIGMS P20GM103496. The manuscript's contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fimmu.2018.01622/ full#supplementary-material.

T cells in vivo in experimental autoimmune encephalomyelitis. *Proc Natl Acad Sci U S A* (2017) 114(9):E1678–87. doi:10.1073/pnas.1615783114

64. Butterfield RJ, Sudweeks JD, Blankenhorn EP, Korngold R, Marini JC, Todd JA, et al. New genetic loci that control susceptibility and symptoms of experimental allergic encephalomyelitis in inbred mice. *J Immunol* (1998) 161(4):1860–7.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer FL and the handling Editor declared their shared university affiliation and declare that they are working in different departments.

*Copyright © 2018 Krementsov, Asarian, Fang, McGill and Teuscher. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Age at Menarche and Risk of Multiple Sclerosis: Current Progress From Epidemiological Investigations

Xia Jiang1,2 \*, Tomas Olsson2 and Lars Alfredsson3

1 Program in Genetic Epidemiology and Statistical Genetics, Harvard T. H. Chan School of Public Health, Boston, MA, United States, 2 Neuroimmunology Unit, Center for Molecular Medicine, Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden, 3 Cardiovascular group, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disorder of the brain and spinal cord in which focal lymphocytic infiltration leads to the damage of myelin and axons. As a multi-factorial complex trait, both genetic background and environmental factors are involved in MS etiology. The disease is more prevalent among women, and an overall female-to-male sex ratio of around 3 is usually reported. The fact that the female preponderance is only apparent among patients with disease onset after age 12 points toward a role of puberty in MS. A key marker of female pubertal development is menarche, however, evidence from previous epidemiological investigations has been sparse and conflicting: although some studies have linked earlier age at menarche (AAM) to an increased risk of MS, others have found no association or an inverse association. Understanding the effect of AAM in MS could increase our knowledge to the disease etiology, as well as deliver meaningful implication to patients' care by aiding clinical diagnosis. Therefore, we reviewed all the currently available epidemiological studies conducted for AAM and risk of MS in adult human populations. We found evidence supporting a possible favorable role of late AAM on MS risk, but this should be further confirmed by well-designed large-scale epidemiological studies and meta-analysis. Future work may be focused on Mendelian randomization analysis incorporating genetic markers to provide additional evidence of a putative causal relationship between AAM and MS. More work should be conducted for non-European populations to increase generalizability, and among the males to complementary with results from females. Future work may also be conducted focusing on hormonal reproductive factors other than menarche, and their effects in MS prognosis, severity, and drug response.

Keywords: puberty, age at menarche, hormone, multiple sclerosis, population-based, epidemiology

# INTRODUCTION

Multiple sclerosis (MS) is primarily an autoimmune inflammatory disorder of the central nervous system (CNS), characterized by the loss of myelin and damage of axons, leading to a variety of neurological deficits (1). The early course of MS is characterized by episodes of neurological dysfunction that usually recover. However, as the disease progresses, pathological changes become

#### Edited by:

Uwe K. Zettl, Universitätsmedizin Rostock, Germany

#### Reviewed by:

Seema Kalra, University Hospitals of North Midlands NHS Trust, United Kingdom Bruno Gran, Nottingham University Hospitals NHS Trust, United Kingdom

> \*Correspondence: Xia Jiang<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology

Received: 03 August 2018 Accepted: 22 October 2018 Published: 13 November 2018

#### Citation:

Jiang X, Olsson T and Alfredsson L (2018) Age at Menarche and Risk of Multiple Sclerosis: Current Progress From Epidemiological Investigations. Front. Immunol. 9:2600. doi: 10.3389/fimmu.2018.02600

**180**

dominated by widespread microglial activation associated with extensive neurodegeneration and accumulated disability. Based on data from WHO, it is estimated that more than two million people (∼2.3–2.5 million) worldwide are living with MS and the disease is one of the most common causes of neurological disability (loss of motor and sensory function) among young adults (2). The incidence of MS varies in different regions of the globe, but usually grows with latitudes, making northern Europe a high-risk zone (3, 4).  While women carry higher disease risk, they usually present less rapid progression to disability (disease severity).

MS is a complex disease with both genetic and environmental factors involved in its etiology. To date, over 200 associated loci have been identified through genome-wide association studies (GWAS) (6), and a low but steadily increasing number of environmental risk factors such as cigarette smoking, EBvirus infection, vitamin D insufficiency (and more) have been consistently observed to be associated with MS risk (7, 8). It has also been long documented that hormonal related factors are crucial in the disease susceptibility and development. Evidence from animal models (experimental allergic encephalomyelitis, EAE) has shown that estrogen, progesterone (at pregnancy range) and testosterone provide anti-inflammatory and neuroprotective effects on both the induction and effectors phases (9). The fact that female preponderance is only apparent among patients with age of disease onset after 12 (female-to-male sex ratio for pediatric MS before 12: 1.2:1) (10) points toward a role of puberty in MS predisposition. A key marker of female pubertal development is menarche, indicating an initiation of a reproductive life. Despite several lines of evidence from epidemiological investigations linking earlier age at menarche (AAM) to an increased risk of MS, other studies have found no association or an inverse association. Most of the previous observational studies have been small, or conducted in selected clinical samples, yielding highly variable estimates. MS is most commonly diagnosed among women who are 20–50 years old and of child-bearing age. It is thus not surprising that patients are usually interested in the topics of pubertal changes, motherhood, and disease.

In this review, we will summarize the results from epidemiological studies conducted so far investigating the relationship between age at menarche and risk of MS in human adults (we collected all the relevant articles through an electronic search from PubMed, with key words "puberty," "pubertal," "menarche," "hormone," and "multiple sclerosis." Only published data were included). We will comment on the advantages and limitations of these studies. We will conclude by presenting challenges, current research gaps and potential future directions for this field. We anticipate that the increased knowledge regarding the association between age at menarche and MS risk as presented by our review will provide insights into the mechanistic developmental processes of MS, as well as facilitate patient care and women's health by aiding clinical consultations.

# AGE AT MENARCHE AND RISK OF MS–RESULTS FROM EARLY RETROSPECTIVE STUDIES

Female menstruation and the risk of MS was first examined by Antonovsky et al. (11) using a population-based case-control study in Israel consisting of 241 patients and 964 controls (of which, 131 female cases and 523 female controls). They found a significantly shorter self-reported menstruated period (the length of menstruation, usually lasts about 3–5 days) among the cases than controls (P = 0.01 for the Chisq-test), but no differences were observed for age at menarche or mean length of menstrual cycle (the length of menstrual cycle, usually 28 days but can be varied from 21 to 35 days). The study had a population-based design where the patients were drawn from a nationwide survey of MS (participation rate: 92%) and controls were randomly selected from the population registry of census, matched on age and sex (case-control ratio 1:4). Study participants answered a questionnaire containing 146 questions covering 11 major areas. The participants were from an admixed population consisting of five regions of birth (Eastern Europe, Central-western Europe, Southern Europe, Asia-Africa, and Israel). The heterogeneous genetic background was not taken into account in the study, which might influence the effect of menstruation (11).

The null finding concerning age at menarche was contrasted by two subsequent small case-control studies carried out in 1989 in European ancestry populations. One was conducted by Berr et al. in France which included 63 prevalent cases and 63 controls (46 female case-control pairs). A statistically significant older mean age at menarche was observed among patients than among controls (13.5 vs. 12.7, P < 0.002 for the t-test) (12). The other was conducted by Operskalski et al. in USA which composed of 145 cases and 145 controls (108 female case-control pairs), where an inverse association was observed; the cases were significantly younger at menarche than controls (12.3 vs. 12.7, P = 0.01 for t-test) (13). The conflicting results from these two studies are perhaps not surprising, as both studies were small.

Nevertheless, findings from two recent studies supported again the previous non-significant association between age at menarche and MS as identified by the Antonovsky et al. study. Kurtzke et al. examined 23 MS patients and 127 controls in UK, and reported age at menarche did not differ significantly between cases and controls (13.6 vs. 14.0), although the cases presented a slightly younger AAM. However, this result was based on extremely underpowered data with only 9 female cases and 68 female controls from a selected veteran population (14). Similarly, Gustavsen et al. compared 391 cases and 535 controls in Norway (all female), and found age at menarche did not differ significantly between the two groups (13.07 vs. 12.97), although the cases showed a slightly older AAM (15).

To summarize, these earlier epidemiological studies were mostly underpowered with limited sample size ranging from 9 to 391 cases, yielding highly variable estimates. All studies used selfreported prevalent cases and self-administrated questionnaires for the collection of exposures, introducing a potential recall bias. Moreover, these studies lacked proper design, as most of them did not explicitly studied AAM, but rather collected this exposure complementary to other main exposures of interest. Thus, the numbers of questions in terms of hormonal related factors and AAM are limited and unspecific; and very little information is provided on how the questions were formulated. Finally, none of the aforementioned studies performed a formal statistical analysis with adequate control for confounding factors, but rather performed a simple comparison between the cases and controls using a t-test or a chisq-test, which may yield biased results. More details are presented in **Table 1**. Despite all the limitations and inconclusive findings, these earlier studies provide some preliminary support for a role of AAM in the risk of MS.

## Age at Menarche and Risk of MS–Results From Well-Designed Epidemiological Investigations

During the past decade, several well-designed large-scale epidemiological investigations have been conducted, and consistent results have been reported where earlier age at menarche was found to increase the risk of MS. Ramagopalan et al. identified MS cases and controls through the populationbased longitudinal Canadian Collaborative Project on Genetic Susceptibility to Multiple Sclerosis, for each index case, the spouse was taken as control, and self-reported age at puberty was used. The study collected a total of 4,472 female cases and 658 female controls (the spouse of those male cases or the same-sex partner of female cases), and assessed the effect of age of puberty through logistic regressions controlling for age at birth. The authors found that the average age at menarche for female cases was 12.4 (standard deviation = 1.29) and for female controls was 12.6 (1.33), the difference was small but statistically significant (P = 0.00017). Moreover, each 1-year increased age at menarche was further found to be associated with a decreased risk of MS (odds ratio = 0.90, 95% confidence interval = 0.84–0.95, P = 0.00063) (16).

Similarly, Nielsen et al. followed 77,330 women included in the Danish National Birth Cohort and identified 226 MS cases during an average follow-up period of 11.7 years. Information on menarcheal age was ascertained at the first interview. The authors observed a generally younger age at menarche among the cases than women without MS (13.0 vs. 13.3, P = 0.002). After adjusting for a number of potential confounders such as body mass index, socio-occupational status, age at first pregnancy, parity, smoking and alcohol intake, an 11% reduction in the risk of MS per 1-year increase in age at menarche was found (hazard ratio = 0.89, 95%CI = 0.81–0.98). To eliminate potential bias stemming from using self-reported data, the author further performed a supplementary analysis based on data from a subgroup of girls whom had school health records available (instead of self-reported exposure). In this subgroup a consistent reduction per 1-year increased AAM regarding MS risk was observed (OR = 0.89, 95%CI = 0.70–1.13), indicating that the potential bias due to using self-reported data was minor (17).

These results observed in European populations were further corroborated by two case-control studies conducted in Iran. Rejali et al. recruited 200 incident cases from an MS clinic and 200 sex and residential area matched controls (non-patients from the same clinic), and found a significant younger age at menarche among the cases than controls (12.96 vs. 13.48, P < 0.001). After controlling for the effect of age, marital status, place of residence, family history of MS, other autoimmune disease and viral disease in childhood, a significant relationship between older age at menarche and decreased risk of MS was observed (OR = 0.78, 95%CI = 0.68–0.90, P = 0.001) (18). Likewise,similar findings were reported by Salehi et al. examining 399 MS cases registered at the Iranian Multiple Sclerosis Society and 541 randomly selected controls from the same residential area through standard random digit dialing. The participants were interviewed and after controlling for age, marital status and education, each 1-year increase in the age at menarche was found to reduce MS risk by 10% (OR = 0.90, 95%CI = 0.82– 0.98, P = 0.018) (19). Details of these studies are presented in **Table 2**.

In addition to disease status, two studies have also investigated age at menarche and age at first symptom onset specifically among MS patients. Sloka et al. examined 150 relapsing remitting female MS cases and found that age of first MS symptoms was postponed by 1.16 years as the age of menarche increased per each 1-year, using a linear regression (r 2 = 0.69, P = 0.04). The study, however, wasn't able to account for several important confounders such as socioeconomic status or seasonal variability, and the authors argued that the induction mechanisms linking the two events together, could possibly include sharing of similar induction mechanisms, one event gives rise to another or same genetic susceptibility (20). In a recent study conducted by Bove et al. which included the major genetic risk factor of MS (HLA-DRB1∗ 1501), the risk allele carriers showed an earlier age at onset (as expected), and each 1-year later age at menarche was also associated with later age at onset after adjusting for multiple potential confounders (increased by 0.63 years, P=0.033), consistent with previous findings. However, surprisingly, the MS risk allele carriers were found to have a later age at menarche than non-carriers (P = 0.036) (21). These results suggest the complex dynamics underlying genetics, hormonal factors and disease, as well as the importance of incorporating genetic markers when studying the complex relationship between these factors and MS.

The underlying biology on the effect of puberty on MS risk remains to be elucidated. It may regulate MS risk through multiple pathways. Firstly, experimental autoimmune encephalitis, the animal models of MS, has demonstrated a biphasic dose effect of estrogen on inflammation. At normal ranges (not pregnancy level), estrogen promotes inflammation. Thus, hormonal changes such as rise in estrogen levels after puberty may affect MS onset. In addition, puberty is also known to involve substantial brain maturational changes such as white and gray matter volume increment, and therefore plays a role in neurological modulation. Moreover, puberty appears to be a key period of exposure to some of the wellestablished MS risk factors, such as overweight/high body mass index, vitamin D deficiency, and Epstein-Barr virus infection. Last but not least, it is also likely that metabolic

TABLE 1 | The epidemiological investigations on age at menarche and risk of MS, results from early retrospective studies.

TABLE 2 | Age at menarche and risk of MS, results from well-designed epidemiological investigations.

\*all subjects were females.

factors during puberty such as childhood nutrition, gut microbiome alterations would lead to earlier menarche and altered immunologic modulation thereby contributing to MS risk (22).

#### Challenges and Future Directions

A powerful way to increase our knowledge on the relationship between AAM and MS, and to provide stronger evidence in support for a potentially favored role of late AAM on MS risk, is through a meta-analysis which combines results across different studies. Such analysis is currently unavailable, likely due to the limited number of well-designed and wellpowered epidemiological investigations published so forth on this topic. For example, only five of the previous studies have reported both mean age at menarche and its standard deviations (15–19) and only four epidemiological studies have reported point estimates and 95% confidence intervals, three of which are case-control studies conducted in two distinct populations (16, 18, 19) and one is a cohort study (17). The heterogeneity in design and population ancestry (European or non-European) across those previous studies makes the aggregation of data difficult; and results based on data pooled from these few studies could only be considered as preliminary and suggestive. Meta-analysis or systematic review are warranted when additional well-designed large epidemiological investigations have emerged and accumulated in the future.

In addition, the observational nature of epidemiological studies could only identify association but can hardly make any conclusive causal inference, as the validity of results could be plagued by measurement error, selection bias, confounding, and reverse causality. In the case of AAM and MS, self-reported age at menarche might be inaccurate; selected clinical samples might be unrepresentative to the target population; there are several important confounders need to be taken into account, not all can be collected via conventional questionnaires; and given the long induction period of MS, certain immunological changes might already have taken place several years before the clinical diagnosis or symptom onset, information collected during this period could thus be influenced by the subclinical phase of disease itself.

Mendelian Randomization (MR) fills the gap by incorporating genetic variants (single nucleotide polymorphism, SNPs) as instrumental variables (IV) for assessing a causal effect of a risk factor on an outcome from observational data (23). A typical MR uses genetic variants (SNPs) as proxies for risk factors, rather than self-reported exposure, with the assumption that SNPs are independent of confounders in the population and randomly allocated at conception, mirroring a randomization process (as those in randomized clinical trials). Moreover, SNP allocation always precedes disease onset therefore eliminates reverse causality.

Massive investment in large-scale GWASs over the past years have discovered reliable genetic variants for a wide range of phenotypes including modifiable environmental exposures (e.g., circulating vitamin levels) and complex human behaviors (e.g., nicotine dependence), providing an unprecedented opportunity for genetic epidemiology in particular by utilizing the MR design. The success of MR approach has been demonstrated by numerous relevant works. In MS, using this approach, a causal role of vitamin D insufficiency and obesity has been strengthened (24–27). In the case of AAM, its genetic regulation has been highlighted by a recent GWAS involving 370,000 women which identified 389 independent AAMassociated signals spread over 10 biological pathways (28). A comprehensive understanding of the hypothetical causal roles for AAM in MS have therefore become possible. However, to the best of our knowledge, while MR of reproductive factors has been carried out extensively in sex-steroid-sensitive cancers with a successful identification of causal relationship (28, 29), no MR has been conducted to investigate the hypothetical causal role for pubertal development in MS to date, a sex hormone driven autoimmune disease. This is a potential future direction to be focused on, when data allows for such analysis, e.g., studies have assembled the ideal combination of large numbers of MS cases and controls, highquality questionnaire data and high-throughput genome-wide SNPs.

In addition to investigating a putative causal relationship between AAM and MS, another direction for future research is to focus on non-European populations and males. As most of the current studies have been conducted in European ancestry populations, its generalizability has been restricted. If hormone influences MS onset, it is possible that puberty timing would also affect male MS onset through the emergence of high level sex hormones and its inhibitory effect against autoimmunity. Studying male puberty timing such as age at voice-breaking would increase our knowledge to the mechanistic developmental of MS as well as explain part of the sex disparity. Future work on other hormonal reproductive factors than AAM, and MS outcomes such as disease severity, prognosis and drug response are also warranted.

# CONCLUSION

In this review, we have recapitulated all the published epidemiological studies conducted so far in AAM and MS, detailing their main findings. We have illustrated the advantages and limitations of each study. There are promising evidences in support for a protective effect of late AAM on MS onset. However, the association between AAM and MS remain to be elucidated and confirmed through larger epidemiological investigations and/or meta-analysis. Future work may be conducted to focus on understanding the causal role of AAM in MS by incorporating genetic markers, from which the knowledge gained could answer some of the patients frequently interested questions in topics of hormone and disease. In addition, the broad scope of estrogen, p-pills, as well as the protection against relapses during pregnancy need further investigation. It is also important to conduct studies among non-European populations and male patient subpopulations. We anticipate that our review will inspire to activities increasing our understanding to the biological mechanisms underpinning hormonal factors and autoimmune disease MS, thus deliver meaningful implications to MS etiology.

# AUTHOR CONTRIBUTIONS

XJ reviewed the original studies conducted in the field of age at menarche and multiple sclerosis, and made the tables. XJ, TO, and LA interpreted the data, wrote and revised the manuscript.

# ACKNOWLEDGMENTS

XJ is supported by an International Postdoc Research Grant from The Swedish Research Council.

# REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Jiang, Olsson and Alfredsson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Serum Lipoprotein Profile Is Associated With Protective Effects of Oral Contraceptive Use on Multiple Sclerosis Severity: A Cross-Sectional Study

#### Armando Sena1,2,3 \*, Ana Macedo4,5, Véronique Ferret-Sena1 , Carlos Capela3 and Rui Pedrosa3

1 Centro de Investigação Multidisciplinar Egas Moniz (CiiEM), Instituto Universitário Egas Moniz, Caparica, Portugal, 2 Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisbon, Portugal, 3 Departamento de Neurociências, Serviço de Neurologia, Centro Hospitalar de Lisboa-Central, Lisbon, Portugal, 4 Keypoint, Consultora Científica, Algés, Portugal, 5 Departamento de Ciências Biomédicas e Medicina (DCBM), Universidade do Algarve, Faro, Portugal

#### Edited by:

Zsolt Illes, University of Southern Denmark, Denmark

#### Reviewed by:

Joost Smolders, Canisius Wilhelmina Hospital, Netherlands Maria José Sá, Centro Hospitalar São João, Portugal

> \*Correspondence: Armando Sena<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Neurology

Received: 25 October 2018 Accepted: 17 January 2019 Published: 05 February 2019

#### Citation:

Sena A, Macedo A, Ferret-Sena V, Capela C and Pedrosa R (2019) Serum Lipoprotein Profile Is Associated With Protective Effects of Oral Contraceptive Use on Multiple Sclerosis Severity: A Cross-Sectional Study. Front. Neurol. 10:60. doi: 10.3389/fneur.2019.00060 Background: The mechanisms underlying the influence of sex hormones in multiple sclerosis (MS) are uncertain. Sex steroids interact with cholesterol metabolism and the serum lipid profile has been associated with the severity of the disease. We hypothesized that the putative associations between lipoprotein metabolism and MS could be modulated by sex steroids exposure. The aim of this study was to investigate whether oral contraceptives (OC) use changes the lipoprotein profile associated with disability in patients with multiple sclerosis.

Methods: Clinical data was collected from 133 relapsing-remitting multiple sclerosis (RRMS) women with a mean of 6.5 years of disease duration and prior to the start of disease-modifying therapies. Patients who were using OC after disease onset (DO) (OC+, n = 57) were compared to those who never used OC or discontinued its intake before DO (OC–, n = 76). In both cohorts of subjects, the associations between the apolipoprotein E (ApoE) polymorphism, and plasma lipid levels, and the annualized relapse rate (RR), the Expanded Disability Status Score (EDSS), and the Multiple Sclerosis Severity Score (MSSS) were evaluated using a hierarchic multiple regression analysis after adjustment for confounders.

Results: Low density lipoprotein (LDL) levels were associated with higher EDSS (p = 0.010) and MSSS (p = 0.024) in the whole studied cohort. In E3/E3 phenotype carriers (73.7%), EDSS and MSSS were lower in OC+ in comparison with OC– subgroup of patients (p < 0.01). LDL and total cholesterol were associated with EDSS (p = 0.005 and p = 0.043, respectively), and LDL and the triglyceride/high density lipoprotein ratio with MSSS (p = 0.011 and p = 0.048, respectively) in OC+ patients. In OC– subgroup of patients, ApoE levels were associated with EDSS (p = 0.012) and MSSS (p = 0.031). No significant interactions between the lipid variables or OC use and RR were observed.

**186**

Conclusions: Serum lipid profile is associated with protective effects of OC use on disability of RRMS patients. Lipoprotein metabolism may be involved in the modulatory effects of sex steroids on the severity of the disease.

Keywords: multiple sclerosis, lipoproteins, cholesterol, apolipoprotein E, oral contraceptives, sex steroids

#### INTRODUCTION

Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease in which onset and course may be modulated by gender and sex hormones (1).  Recent studies have implicated serum cholesterol metabolism and lipoprotein profile in the pathophysiology and severity of the disease (2). Nevertheless, results are mixed, and whether these relations are causal or secondary to the disease process, treatment regimens or other confounding factors remain uncertain. Complex interactions between cholesterol metabolism and sex steroids are well-known (3) and may modulate the clinical activity of experimental autoimmune encephalomyelitis (EAE), the animal model for MS (4, 5). Therefore, we hypothesized that the putative associations between lipoprotein metabolism and the severity of MS could be modulated by sex hormone exposure.

Oral contraceptives (OC) use has provided an opportunity to assess the influence of sex hormones on the risk and course of MS. Recent data suggested a less severe clinical course (6, 7) and decreased inflammatory brain lesions (8) in relapsingremitting (RRMS) women taking OC. Protective effects of estriol, the estrogen unique to parity (9), and of hormone therapy in postmenopausal MS women were also reported (10). In healthy women, OC use induces variable alterations in serum lipids and apolipoproteins levels modulated by ApoE polymorphism (11, 12). Although ApoE polymorphism is not generally considered to affect the risk of MS, its association with the neurodegenerative process and severity of the disease is still controversial (4, 5). ApoE is implicated in the immune dysfunction and clinical activity of EAE (5) and recent studies have suggested that EAE disease severity is differently modulated by cholesterol and ApoE metabolism in female and male mice (4, 13). Oestrogens may regulate the expression of ApoE gene (2) and estrogen treatment has protective effects in EAE (14). Oestrogens-ApoE interactions are suggested to be involved in other neurological conditions with a sex bias and abnormal cholesterol metabolism, such as Alzheimer disease (15). Based on these data, the aim of the present study was to investigate whether OC intake in RRMS patients influence the associations between the serum lipoprotein profile and the clinical severity of the disease.

#### MATERIALS AND METHODS

#### Study Population

The studied population include 133 women with the diagnosis of RRMS according to the revised McDonald criteria (16) followed at the MS outpatient clinic of a University Hospital in Lisbon (Portugal). Most women enrolled in this study belong to a population of Caucasian origin included in a previous work published by our group (7). The present study includes all patients followed in our clinic since 1995 diagnosed with RRMS with at least 2 years of disease duration and whose lipid data were available prior to the start of disease-modifying therapies. No woman was taking lipid-lowering agents. Disease onset (DO) was defined as the age of appearance of the first symptoms suggestive of MS. The annualized relapse rate (RR) and the Expanded Disability Status Scale (EDSS) and Multiple Sclerosis Severity (MSSS) values were determined at a stable phase of the disease. MSSS scores were obtained from Figure 3 of the paper of Roxburg et al. (17). The MSSS is based on EDSS scores adjusted for disease duration and it is a method to compare disability progression in groups of patients (7, 17). Women in menopause or history of gynecological surgical interventions or a delivery the last 6 months were excluded. Clinical indexed information included the body mass index (BMI) (Kg/m2 ), age of first menstruation (menarche) and history of childbirths (parity), smoking habit, and OC intake. Patients labeled smokers reported to smoke regularly at least five cigarettes per day since DO. Women were classified as OC non-users if they never used OC or discontinued its intake for at least 1 year before DO (OC–); and OC users if they maintained pill intake after DO for at least a continuous period of 1 year (OC+). We were unable to take the composition of the prescribed pill into account because this information was lacking for some patients and many changed the brand of the drug. However, all women who remembered the formulation of OC used took formulations of 20 or 30 µg of ethinyl estradiol combined with progestin. No woman reported to use progestin-only formulations. This study was approved by the Ethics Committee of the Centro Hospitalar, Lisboa Central (Lisbon, Portugal). All patients gave written informed consent, including for publication of results, in accordance with the Declaration of Helsinki.

#### Biochemical Analysis

Blood samples were collected in fasting conditions shortly after clinical data collection and neurological examination. Plasma or serum samples were stored at −80◦C and biochemical measurements performed in blind conditions regarding subject participants. At sampling, patients were in a remission phase of RRMS and none of them had initiated disease-modifying therapies, suffered from a relapse or were treated with steroids for at least 1 month. Serum triglycerides (TG), total cholesterol (TC), high density lipoprotein (HDL)-cholesterol, apolipoprotein A-1 (ApoA1), and apolipoprotein B (ApoB) were determined with enzymatic methods and lipoprotein (a) [Lp(a)] by turbidimetric immunoassay by using a Hitachi 911 autoanalyzer and commercial kits (Roche Diagnostic, Mannheim, Germany). Non-HDL cholesterol levels were calculated by subtracting HDL from TC. Low density lipoprotein (LDL)-cholesterol was determined by using the Friedewald equation (18) and oxidized LDL (oxLDL) by Enzyme-Linked Immunosorbent Assay (Mercodia, eBioscience). Apolipoprotein E (ApoE) protein levels were determined by electroimmunodiffusion (Sebia, Emery, France) and ApoE polymorphism examined by using an isoelectric focusing (IEF) method as described previously (19). Briefly, 15 µl of delipidated plasma samples was run on agarose with sorbitol, urea, ampholine (pH 5–7) and pharmalyte (pH 4–6.5) (Amersham Pharmacia Biotech, Little Chalfont, UK). After IEF, the proteins were transferred to nitrocellulose membranes (Immobilon, pore size 0.2µm; Millipore Corporate Headquarters, Billerica, USA). The membranes were incubated with polyclonal-goat anti-human ApoE antibody (Daichi Pure Chemicals, Tokyo, Japan) and IgG peroxidase-conjugate antigoat antibody (Sigma-Aldrich Biotechnology, St Louis, USA). The ApoE isoforms were visualized in a solution containing 3,3' diaminobenzidine tetrahydrochloride reagent (Sigma). For common ApoE polymorphism, protein phenotyping is in good agreement with DNA-based genotyping (20).

#### Statistical Analysis

Patient demographic and clinical characteristics were described using mean, median, standard deviation, and interquartile range for continuous variables. In the text, standard deviation is presented as mean (standard deviation). For categorical variables absolute and relative frequencies were calculated. The relation between severity parameters of clinical disease activity (RR, MSSS, and EDSS) and ApoE phenotypes was evaluated using Kruskal-Wallis ranking test. Comparisons of disease severity between OC+ and OC– subgroups of patients were performed using a Mann Whitney test. The associations between disease severity parameters such as MSSS, EDSS, and RR and the lipid profile were evaluated using a two tailed Spearman Correlation analysis. The correlation analysis was performed for the total subset of patients carrying the E3/E3 phenotype and also split for those in OC+ and OC– subgroups. A hierarchical multiple linear regression was used assuming the EDSS and MSSS as dependent variables and characterization variables such as age, disease onset, oral contraception, disease duration and parity (block 1), and lipid profile parameters that had significant correlation with EDSS and MSSS in the correlation analysis (block 2), as independent variables. A enter model was used for the variables in the block 1 and a stepwise model was used for variables in block 2.A significance level of 0.05 was considered in all analysis.

#### RESULTS

The main demographic and clinical characteristics of the studied population are summarized in **Table 1**. Twenty-nine patients (21.8%) were classified as overweight (25 ≥ BMI < 30) or obese (BMI ≥ 30). In the OC+ subgroup (n = 57), the mean duration of OC use was 10 years (6.6) and all but nine women started intake before DO. In the OC– subgroup, fifty patients were never prescribed with OC and 26 discontinued the intake before DO. OC+ patients were younger and had the onset of the disease at an earlier age than OC– subgroup of patients. Significant associations were found between EDSS and MSSS and age (p < 0.001 and p = 0.013), DO (p = 0.004 and p < 0.001), disease duration (p = 0.004 and p < 0.001), OC use (p = 0.001 and p = 0.002), and parity after DO (p < 0.001 and p = 0.006). RR was only associated with disease duration (p = 0.006). Menarche age, duration of OC intake, BMI, and smoker habit were not associated with RR or disability scores (data not shown). Concerning the lipid data, HDL and Apo A1 levels were higher in OC+ patients. In a hierarchic multiple regression analysis adjusted for age, DO, disease duration, and OC use, LDL was the only lipid variable associated with EDSS and MSSS (β = 0.008, 95% CI (0.002 to 0.015) p = 0.010 and β = 0.013, 95% CI (0.002 to 0.025) p = 0.024, respectively). ApoE phenotypes found in the studied cohort were E3/E3 (n = 98, 73.7%), E4/E3 (n = 20, 15%), E2/E3 (n = 12, 9%). Analysis of ApoE polymorphism was missing from one patient and two additional patients carried the E4/E2 phenotype. No homozygotes for the E4 and E2 alleles were detected. The observed frequencies of ApoE alleles were comparable with those reported for the general populations in Portugal and other countries in South Europe (21).

No associations between the three common ApoE phenotypes and EDSS, MSSS, or RR were found. However, in the E3/E3 subset of subjects, EDSS, and MSSS values were lower in OC+ in comparison to OC– subgroup of patients (p < 0.01) (**Figure 1**). These results remain significant after hierarchical multiple linear regression analysis adjusting for demographic features among the ApoE genetic groups. RR was not significantly changed by OC intake (p = 0.457). In consequence, serum lipid and apolipoprotein levels were investigated in this subset of patients according to OC use. Overall, there was no statistical difference in the lipid profile with the exception of higher ApoA1 and lower ApoE levels in OC+ in comparison to OC– patients [171.3 mg/dl (40.6) vs. 151.5 mg/dl, (27.5); p < 0.01 and 67.3 mg/dl (29.6) vs. 80.1 mg/dl (28.7); p < 0.05, respectively]. Correlation between lipoprotein levels and disability scores were analyzed in E3/E3 subset of patients stratified according to OC use. In OC+ subgroup of patients, LDL was associated with EDSS (p = 0.018) and ApoB was associated with MSSS (p = 0.043). In contrast, in the OC– subgroup of patients, ApoE was associated with MSSS values (p = 0.008); TC and non-HDL were associated with EDSS (p = 0.025 and p = 0.035, respectively); and TC (p = 0.035), LDL (p = 0.028), non-HDL (p = 0.005), and ApoB (p = 0.008) with RR (**Table 2**).

A hierarchic multiple regression analysis in E3/E3 subjects split for OC+ and OC– patients and adjusted for age, DO and disease duration was then performed (**Table 3**). EDSS was related to LDL [β = 0.026, 95% CI (0.009 to 0.044); p = 0.005] and TC [β = −0.018, 95% CI (−0.034 to −0.001); p = 0.043]; model R square = 0.328 (p = 0.015) in OC+ population; and to ApoE [β = 0.015, 95% CI (0.004 to 0.028); p = 0.012]; model R square = 0.312 (p = 0.001) in the OC– subgroup of patients. In a similar model, MSSS was related to LDL, [β = 0.022, 95% CI (0.002 to 0.033); p = 0.011] and to TG/HDL ratio, [β = 0.389, 95% CI (0.088 to 0.800); p = 0.048];

#### TABLE 1 | Patient demographic and clinical characteristics.

The continues variables are expressed as mean ± SD; \$ median and IQR [].