The Pennsylvania State University

The Graduate School

The Huck Institutes of Life Sciences

REGULATORY ROLE OF NKT CELLS ON EFFECTS OF

1,25(OH)2D3 IN MICE WITH EAE

A Thesis in

Cell and Developmental Biology

by

Jun Zhao

2012 Jun Zhao

Submitted in Partial Fulfillment of the Requirements

for the Degree of

Master of Science

August 2012

ii

The thesis of Jun Zhao was reviewed and approved* by the following:

Margherita T. Cantorna Professor of Molecular Immunology Thesis Advisor Chair of Intercollege Graduate Degree Program in Immunology and

Infectious Diseases

Na Xiong Associate Professor of Veterinary and Biomedical Sciences

Pamela Hankey Associate Professor of Immunology

Zhi-chun Lai Professor of Biology, Biochemistry and Molecular Biology Chair of Intercollege Graduate Degree Program in Cell and

Developmental Biology

*Signatures are on file in the Graduate School

iii

ABSTRACT

Vitamin D is an immune regulator and has been shown to be involved in the

development and treatment of autoimmune diseases such as multiple sclerosis (MS). The

active form of vitamin D, 1,25(OH)2D3, has been shown to suppress experimental

autoimmune encephalomyelitis (EAE). Invariant Natural Kill T (iNKT) cells have been

proven to be an important suppressor of EAE. The development of iNKT cells was

impaired due to vitamin D and or vitamin D receptor (VDR) deficiency. 1,25(OH)2D3

had less effects in protecting CD1d knockout (KO) mice from EAE than in WT. CD1d

KO mice with 1,25(OH)2D3 supplementation had significantly higher incidence of EAE

and developed significantly higher EAE scores than WT mice with 1,25(OH)2D3

treatment. 1,25(OH)2D3 was also less effective in preventing Jα18 KO mice against EAE

than in WT. Jα18 KO mice on 1,25(OH)2D3 treatment had significantly higher incidence

of EAE and significantly higher cumulative EAE scores than WT mice with 1,25(OH)2D3

treatment. Either 1,25(OH)2D3 treatment or -galactosylceramide (-GalCer)

administration failed to prevent EAE development in Interleukin-4 (IL-4) KO mice.

These data suggests that the protective effects of 1,25(OH)2D3 against EAE might be

regulated by iNKT cells and IL-4 might be a positive regulator in the beneficial effects of

either 1,25(OH)2D3 or -GalCer on EAE inhibition.

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TABLE OF CONTENT

Chapter 1 Introduction ........................................................................................................ 1 

1.1  Vitamin D............................................................................................................ 1 

1.1.1 Metabolism and classical functions of Vitamin D .................................... 1 

1.1.2 Vitamin D in immune regulation ............................................................... 1 

1.1.3 Vitamin D and NKT cells .......................................................................... 2 

1.2 Multiple Sclerosis .................................................................................................. 3 

1.2.1 Background and epidemiology .................................................................. 3 

1.2.2 Vitamin D and multiple sclerosis .............................................................. 4 

1.2.3 EAE and vitamin D ................................................................................... 4 

1.2.4 EAE and NKT cells ................................................................................... 6 

1.3 Conclusion ............................................................................................................. 7 

Chapter 2 Materials and Methods ....................................................................................... 8 

2.1 Mice and diets ........................................................................................................ 8 

2.2 αGalCer stimulation ............................................................................................... 8 

2.3 EAE induction........................................................................................................ 8 

2.4 EAE symptoms ...................................................................................................... 9 

2.5 Statistical analysis .................................................................................................. 9 

Chapter 3 Results .............................................................................................................. 11 

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3.1 1,25(OH)2D3 is ineffective at suppressing EAE in CD1d KO mice .................... 11 

3.2 1,25(OH)2D3 is ineffective at suppressing EAE in Jα18 KO mice ...................... 12 

3.3 Either 1,25(OH)2D3 or α-GalCer fails to prevent EAE in IL-4KO mice ............. 13 

Chapter 4 Discussion ........................................................................................................ 15 

References ......................................................................................................................... 25 

1

Chapter 1 Introduction

1.1 Vitamin D

1.1.1 Metabolism and classical functions of Vitamin D

Vitamin D is a fat soluble vitamin which can be produced in the body or absorbed

through diets or supplements. The precursor of vitamin D, seven-dehydrocholesterol, is

converted into cholecalciferol (vitamin D3) in the skin by UVB light from sunlight

exposure. Vitamin D3 is then converted into 25-hydroxy vitamin D3 [25(OH)D3] in the

liver, which is the major circulating form of vitamin D in human body. 25-hydroxy

vitamin D3 is then hydroxylated by the Cyp27B1 encoded enzyme 1-hydroxylase into

the active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in the kidney

(DeLuca 2004).

Vitamin D is known for its classical functions of maintaining calcium and phosphate

homeostasis (DeLuca 2004). Vitamin D could regulate absorption and reabsorption of

calcium and phosphate through tissues such as intestines, bones, kidneys, and the

parathyroid gland. Vitamin D could also directly regulate parathyroid hormone

metabolism and itself (Feldman 1999).

1.1.2 Vitamin D in immune regulation

The vitamin D receptor (VDR) is a member of the class II steroid hormone

superfamily of nuclear receptors (Masuyama 1997). 1,25(OH)2D3 binds to the VDR and

2

the formed complex directly binds to DNA to function as a transcriptional regulator in

target cells (Jones 1998). VDR is expressed in almost all tissues and many types of cells

including CD4+ and CD8+ T cells, B cells (Veldman 2000), macrophages (Helming

2005) and monocytes (Provvedini 1983). This suggests that vitamin D can directly

regulate different aspects of the immune system.

1,25(OH)2D3 could directly affect T helper1/T helper2 (Th1/Th2) cell differentiation

(Boonstra 2001). 1,25(OH)2D3 positively regulated anti-encephalitogenic cytokines and

inhibited autoimmune related cytokines in T cells (Cantorna 1998; Mahon, Wittke et al.

2003). In vitro studies have found that through VDR signaling, 1,25 (OH)2D3 repressed

Th17 and regulatory T cell (Treg) differentiation (Chang, Cha et al. 2010). 1,25(OH)2D3

could also regulate dendritic cell maturation (Piemonti 2000). 1,25(OH)2D3 has also been

shown to positively regulate several murine models of autoimmune diseases such as

multiple sclerosis (MS), inflammatory bowel diseases (IBD) (Cantorna 2006),

inflammatory arthritis (Cantorna 1998) and type I diabetes (Zella 2003).

1.1.3 Vitamin D and NKT cells

Natural Killer T (NKT) cells were originally defined as a subset of T cells that

express NK lineage receptors like NK1.1 (Bendelac 1997). NKT cells are divided into

two different subset, including type I and type II NKT cells (Godfrey, MacDonald et al.

2004). Type I NKT cells are also called invariant NKT cells (iNKT cells). The iNKT

cells have an invariant Vα14-Jα18 T cell receptor (TCR) chain (in mice) and can be

specifically activated by a marine sponge derived-glycolipid -galactosylceramide (-

GalCer) presented by CD1d-expressing antigen-presenting cells (Godfrey, MacDonald et

3

al. 2004; Reilly, Wands et al. 2010). iNKT cells possess effector and immunoregulatory

functions. They have cytotoxic effects and secrete large amounts of cytokines very

rapidly, helping effector cells and also regulating Th1/Th2 cell differentiation (Wilson

and Delovitch 2003). Type II NKT cells (non-classical NKT cells) are also CD1d

dependent, but they express diverse TCR chains and are not reactive to -GalCer

(Godfrey, MacDonald et al. 2004). CD1d KO mice are NKT cell deficient and Jα18 KO

mice are iNKT cell deficient, which were used widely to investigate the functions of

NKT cells. The NKT cells serve as important regulators of the immune responses in

different aspects.

The normal development and functions of mouse iNKT cells require vitamin D

receptor (VDR) (Yu and Cantorna 2008). The number of iNKT cells was significantly

reduced in vitamin D deficient mice compared to vitamin D sufficient mice, and the

reduced cell numbers could not be recovered following 1,25(OH)2D3 supplementation in

either neonatal or adult mice (Yu and Cantorna 2011). Vitamin D and the VDR both

regulated the expansion and proliferation of early iNKT cell precursors (Cantorna, Zhao

et al. 2012).

1.2 Multiple Sclerosis

1.2.1 Background and epidemiology

Multiple sclerosis is a chronic autoimmune demyelinating disease affecting the

central nervous system (CNS). The myelin and axons of the brain and spinal cord are

damaged, which leads to demyelination and scarring (Scheinberg 1987; Compston 2008).

4

MS afflicts about 350,000 people in the United States and about 1.1 million people all the

world (Gross and Jager 2011). The etiology of MS still remains unknown, but evidence

shows that environmental and genetic factors involve roles in MS incidence.

Geographical distribution of MS around the world showed that disease prevalence and

severity was dramatically decreased along with increasing sunlight exposure, which was

explained by the increased vitamin D production through the UV sunlight (Gross and

Jager 2011). Vitamin D is a potential environmental factor affecting MS development.

1.2.2 Vitamin D and multiple sclerosis

Vitamin D has been shown to be involved in the development of autoimmune

diseases. We have already proposed that vitamin D status plays an important role at the

environmental level in regulating the development of autoimmunity (Cantorna 2011).

Previous study demonstrated that deficiency of 25(OH)D3 was found in MS patients

compared to healthy population (Sioka 2009). Low levels of circulating vitamin D were

linked to severe symptoms (increased disability) in MS patients in Tasmania, Australia

(van der Mei 2007). Increase in both sun exposure and vitamin D supplements during

childhood and adolescence were proven to be related with decreased MS incidence in

north of the Arctic Circle, and these factors were also linked to MS onset time (Kampman

2007). Thus, vitamin D is an important factor in regulating MS incidence and severity.

1.2.3 EAE and vitamin D

Experimental autoimmune encephalomyelitis (EAE) is a commonly used murine

model for MS. EAE is a Th1 and Th17 CD4+ T cell mediated autoimmune demyelinating

5

disease, targeting the CNS and inducing inflammation and paralysis to the mice.

Preceding evidence has shown that the deficiency of vitamin D caused earlier onset of

EAE symptoms in mice (Cantorna 1996). Recent studies have found that vitamin D

deficiency reduced EAE severity and delayed EAE onset (Fernandes de Abreu, Ibrahim

et al. 2010; DeLuca and Plum 2011; Fernandes de Abreu, Landel et al. 2011). The

vitamin D hormone 1,25(OH)2D3 could prevent EAE and reversibly block the progression

of the disease (Lemire 1991; Cantorna 1996). 1,25(OH)2D3 increased anti-

encephalitogenic cytokine TGF-β1 and IL-4 with EAE, which correlated with the

inhibition of MS like symptoms in mice (Cantorna 1998; Mahon, Wittke et al. 2003).

Serum TGF-β1 levels were increased in MS patients given a vitamin D supplements

(Mahon 2003). In vivo experiments have shown that 1,25(OH)2D3 failed to protect IL-4

KO mice from developing EAE (Cantorna, Humpal-Winter et al. 2000), suggesting that

in vivo up-regulation of IL-4 is an important factor mediating the 1,25(OH)2D3 effects in

EAE inhibition.

1,25(OH)2D3 treatment decreased production of Th1-associated cytokines IFN-γ, IL-

2 and TNF-α in T cells (Rigby 1987; Mahon, Wittke et al. 2003), suggesting a role for

1,25(OH)2D3 in inhibiting EAE related cytokines. Th17 related-cytokines were also

inhibited by 1,25(OH)2D3 in vivo and in vitro in experimental autoimmune uveitis

experiments (Tang, Zhou et al. 2009). 1,25(OH)2D3 suppressed IL-17A expression in

EAE mouse models through mechanism of transcriptional suppression mediated by VDR

(Joshi, Pantalena et al. 2011).

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Recent studies using bone marrow chimeric mice with a disrupted VDR and

conditional targeting experiments have shown that VDR was necessary for 1,25(OH)2D3

inhibition of EAE and 1,25(OH)2D3 acted directly through VDR on CD4+ T cells to

inhibit EAE (Mayne, Spanier et al. 2011). Other researchers have demonstrated that the

development of EAE required mediation of vitamin D and VDR, and 1,25(OH)2D3 may

not play a role in the autoimmune response to initiate EAE (Wang, Marling et al. 2012).

Overall, the data showed that 1,25(OH)2D3 prevents EAE onset and reverses the

progression of symptoms partly through inhibiting Th1 and Th17 cells and inducing Th2

cytokine production.

1.2.4 EAE and NKT cells

NKT cells were also shown to play a regulatory role in EAE. SJL mice which are

highly susceptible to EAE have Vα14 NKT cell dysfunction (Taniguchi, Harada et al.

2003). Transgenic mice that have enriched CD1d restricted NKT cells failed to develop

EAE and the encephalitogenic autoantigen-specific IFN- production was inhibited in the

spleen. This suggested that the protective role of CD1d restricted NKT cells against EAE

may work through inhibition of autoreactive Th1 cell cytokine responses (Mars 2002).

Activation of NKT cells with -GalCer at the time of EAE induction in C57BL/6 mice

prevented EAE (Jahng 2001). Prior-activation of NKT cells provided protection to

B10.PL mice against EAE development dependent on IL-4 secretion (Jahng 2001). IL-4

KO C57BL/6 mice exhibited increased susceptibility to EAE, and α-GalCer

administration failed to protect IL-4 KO mice against EAE (Singh 2001; Furlan 2003).

NKT cell activation protected mice from EAE in a CD1d-, IL-4- and IL-10-dependent

7

manner and the protection was associated with suppression of Th1 cytokine IFN-

production and increase of Th2 cytokines IL-4 and IL-10 production (Singh 2001; Furlan

2003). The protection from EAE in NKT-enriched mice was associated with NKT cell

infiltrating the CNS and suppression of encephalitogenic Th1 and Th17 cytokine

production responses in the spleen (Mars 2008). WT iNKT cells inhibit both Th1 and

Th17 responses in suppressing EAE while iNKT cells producing either IL-4 or IL-10 but

not IFN- could only inhibit Th1 responses (Oh and Chung 2011). All these data indicate

that NKT cells play a regulatory role in EAE prevention.

1.3 Conclusion

Vitamin D is an immune regulator and has been shown to be involved in the

development and treatment of autoimmune diseases such as MS. The active form of

vitamin D, 1,25(OH)2D3, has been shown to suppress EAE. The iNKT cells have also

been proven to be an important suppressor of EAE. The development of iNKT cells was

impaired due to vitamin D and or vitamin D receptor deficiency. -GalCer administration

prevents EAE development but not in IL-4 KO mice. 1,25(OH)2D3 is less effective in

preventing EAE symptoms in IL-4 KO mice. The goal of this research was to determine

whether 1,25(OH)2D3 functions to suppress EAE with the production of IL-4 in iNKT

cells.

8

Chapter 2 Materials and Methods

2.1 Mice and diets

C57BL/6 mice with the following genotypes: wild type (WT), CD1d KO (which

lack endogenous NKT cells), Jα18 KO (which lack endogenous iNKT cells) and IL-4 KO

were produced in our colony at Pennsylvania State University (University Park, PA).

Breeding pairs of WT and IL-4 KO mice were purchased from Jackson Laboratories (Bar

Harbor, ME) (Cantorna, Humpal-Winter et al. 2000). CD1d KO and Jα18 KO mice were

kind gifts from Vanderbilt University (Nashville, TN). Mice were fed on purified control

gel diets generated in the laboratory of Dr. Cantorna. 1,25(OH)2D3 supplemented diets

were synthetic gel diets that contained 1,25(OH)2D3 (50ng/day/mouse). Mice with

αGalCer treatment were fed on purified control gel diets. For all EAE experiments, the

diets were fed beginning 1 week prior to the EAE induction and continued throughout the

whole experiment (21 days). Mice were fed with freshly made diets which were replaced

freshly 3 times per week. All described experimental procedures were viewed and

approved by the Office of Research Protections, Institutional Animal Care and Use

Committee at the Pennsylvania State University.

2.2 αGalCer stimulation

αGalCer (Axxora, San Diego, CA) was dissolved in PBS containing 0.5% Tween 20.

(Yu and Cantorna 2008). Mice were given 4.4 μg of αGalCer dissolved in PBS or vehicle

through intraperitoneal (i.p.) injection at the time of EAE induction (day 0) (Jahng 2001).

2.3 EAE induction

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Six to eight week old sex-matched C57BL/6 mice were immunized subcutaneously

with 100 µl of 200 µg MOG (myelin oligodendrocyte glycoprotein) peptide 35-55

(AnaSpec, Fremont, CA) emulsified in Freund’s adjuvant (IFA; Difco, Detroit, MI)

supplemented with attenuated Mycobacterium tuberculosis H37RA (Difco, Detroit, MI)

to 4mg/ml. Additionally, on the day of immunization and 2 days after (day 0 and 2),

100µl of 200 ng pertussis toxin (List Biological Laboratories, Campbell, CA) dissolved

in PBS was injected i.p. to the mice (Miller and Karpus 2007).

2.4 EAE symptoms

Mice were monitored for signs of EAE until 21 days after immunization. Clinical

EAE scores were recorded daily according to the severity of EAE symptoms using the

following scoring system: 1, limp tail; 2, partial hind limb paralysis; 3, complete hind

limb paralysis; 4, complete hind and some fore limb paralysis; 5, moribund or dead

(Cantorna 1996). Mice with EAE scores of 2 or more were considered EAE positive.

Mice with EAE score of 5 were sacrificed due to humane reasons. Day of onset was

calculated by averaging the day of EAE onset for each mouse that got scored equal or

larger than 2. Peak severity scores were the average of the maximum score for each

mouse. Incidence showed the proportion of number of affected mice out of number of all

mice tested in the experiment. Mortality displayed the proportion of mice that died or

were sacrificed in the experiment. Cumulative disease score (CDI) was calculated by

summing all of the EAE scores for the 21 days of the experiment and dividing by the

number of mice in each group (Cantorna 1996; Becklund, Severson et al. 2010).

2.5 Statistical analysis

10

Experimental data were expressed as mean ± SEM. Statistical analyses were

performed using GraphPad Prism software (GraphPad, La Jolla, CA). Clinical scores and

other measurements were compared by the unpaired Student t test and analysis of

variance (ANOVA), incidence ratio was compared by two sample test for binomial

proportions (Wang, Marling et al. 2012). P-value < 0.05 was considered statistically

significant.

11

Chapter 3 Results

3.1 1,25(OH)2D3 is ineffective at suppressing EAE in CD1d KO mice

WT mice on control diet showed EAE symptoms appeared between 12-13 days after

MOG immunization in, with peak EAE scores from around day 15 till day 21(Fig. 3-1A).

In clear contrast, WT mice on 1,25(OH)2D3 diet were protected from developing EAE,

showing significant lower mean EAE scores than mice on control diet throughout the

course of the experiment (Fig. 3-1A). The cumulative EAE scores for WT mice on

1,25(OH)2D3 diet were significantly lower than that of WT mice on control diet (Fig. 3-

1B). Only one WT mouse on 1,25(OH)2D3 diet developed EAE. The incidence of the

disease was significantly lower (8%) than that of WT mice on control diet (91%). The

mean day of EAE onset was also delayed (day 17) compared to WT controls (Table 3-1).

CD1d KO mice on control diet displayed similar course of EAE development as WT

mice on control diet (Fig. 3-1A). For both WT and CD1d KO mice on control diet, the

measures of EAE severity were similar: the mean day of onset was around day 12-13; the

peak severity EAE score was around 3.2-3.3; the incidence of the disease was above 91%

and the CDI was around 27 (Table 3-1). CD1d KO mice on 1,25(OH)2D3 diet had similar

EAE incidence (81%) to that of CD1d KO mice on control diets (95%), but showed

significantly decreased mean EAE scores from day 11 till day 18 compared to CD1d KO

mice on control diet (Fig. 3-1A). The cumulative EAE scores of CD1d KO mice on

1,25(OH)2D3 diet were also significantly lower compared to CD1d KO mice on control

diet (Fig. 3-1B). CD1d KO mice on 1,25(OH)2D3 diet had delayed mean day of EAE

12

onset and significantly lower peak severity scores compared to control treated CD1d KO

mice (Table 3-1).

The mean EAE scores of CD1d KO mice on 1,25(OH)2D3 diet were significantly

higher than that of WT mice on 1,25(OH)2D3 diet from day 16, day 18 to day 21 (Fig. 3-

1A). Additionally, the cumulative EAE score of CD1d KO mice on 1,25(OH)2D3 diet was

also significantly higher than that of WT mice on 1,25(OH)2D3 diet (Fig. 3-1B). The EAE

incidence of CD1d KO mice on 1,25(OH)2D3 diet (81%) was also statistically higher than

that of WT mice on same diet (8%, Table 3-1).

3.2 1,25(OH)2D3 is ineffective at suppressing EAE in Jα18 KO mice

Jα18 KO mice on control diet displayed similar course of EAE development as WT

mice on control diet. EAE symptoms appeared around 13 days after MOG immunization,

with high EAE scores from around day 15-20 (Fig. 3-2A). For both WT and Jα18 KO

mice on control diet, the measures of EAE severity were similar (Table 3-2).

Jα18 KO mice on 1,25(OH)2D3 diet had similar EAE incidence (81%) to that of Jα18

KO mice on control diets (78%), but showed significantly lower mean EAE scores from

day 14 till day 18 compared to Jα18 KO mice on control diet (Fig. 3-2A). The cumulative

EAE scores of Jα18 KO mice on 1,25(OH)2D3 diet were also significantly lower

compared to Jα18 KO mice on control diet (Fig. 3-2B). But, the mean day of EAE onset

and the peak severity score of Jα18 KO mice on 1,25(OH)2D3 diet was not significantly

different than that of Jα18 KO mice on control diet (Table 3-2). However, when

comparing the EAE symptoms between Jα18 KO and WT mice on 1,25(OH)2D3 diet, the

cumulative EAE score of Jα18 KO mice on 1,25(OH)2D3 diet was significantly higher

13

than that of WT mice on 1,25(OH)2D3 diet (Fig. 3-2B). Additionally, the EAE incidence

of Jα18 KO mice on 1,25(OH)2D3 diet (81%) was also significantly higher than that of

WT mice on same diet (8%, Table 3-2).

CD1d KO mice and Jα18 KO mice on control or 1,25(OH)2D3 diet had similar

course of EAE development (Fig. 3-3A). The cumulative EAE scores of CD1d KO mice

and Jα18 KO mice on control diet did not differ from each other. Also, the cumulative

EAE scores of CD1d KO mice and Jα18 KO mice on 1,25(OH)2D3 diet were similar to

each other (Fig. 3-3B).

3.3 Either 1,25(OH)2D3 or α-GalCer fails to prevent EAE in IL-4KO mice

IL-4 KO mice on control diet displayed a similar course of EAE development as WT

mice on control diet. EAE symptoms appeared around 14 days after MOG immunization,

with high EAE scores from around day 15-21 (Fig. 3-4A). For both WT and IL-4 KO

mice on control diet, the measures of EAE severity were similar: the mean day of EAE

onset was around day 12-14; the peak severity EAE score was around 3.3-3.5; and the

CDI was around 27-28 (Table 3-3). The mortality rate of IL-4 KO mice on control diet

(14%) was higher than that of WT mice on control diet (0%).

IL-4 KO mice on 1,25(OH)2D3 diet showed similar EAE symptoms as IL-4 KO mice

on control diet. EAE symptoms appeared ~12-14 days after MOG immunization, with

high EAE scores from around day 15 to day 21 (Fig. 3-4A). The cumulative EAE scores

of IL-4 KO mice on 1,25(OH)2D3 diet were not significantly different from that of IL-4

KO mice on control diet (Fig. 3-4B). Peak severity score and incidence were also not

significantly different for IL-4 KO mice on control diet and 1,25(OH)2D3 diet (Table 3-3).

14

Comparing the EAE symptoms of IL-4 KO mice on 1,25(OH)2D3 diet with WT mice on

1,25(OH)2D3 diet, the EAE incidence of IL-4 KO mice on 1,25(OH)2D3 diet (100%) was

significantly higher than that of WT mice on 1,25(OH)2D3 diet (8%, Table 3-3).

IL-4 KO mice on control diet with α-GalCer administration also showed similar

EAE symptoms as IL-4 KO mice on control diet (Fig. 3-4A). The mean day of EAE onset

of IL-4 KO mice on control diet with α-GalCer (day 11) was significantly earlier than

that of IL-4 KO mice on control diet without α-GalCer (day 14, Table 3-3). There were

no significant differences in other measures of EAE symptoms including peak severity,

incidence, mortality and CDI between these two groups (Table 3-3, Fig. 3-4B). On the

contrary, WT mice on control diet with α-GalCer administration were protected from

EAE. The EAE incidence of IL-4 KO mice on control diet with α-GalCer administration

(75%) was significantly higher than that of WT mice on the same treatment (0%, Table 3-

3).

15

Chapter 4 Discussion

Vitamin D status plays an important role in regulating the development of MS. It is

also required for the normal development and function of iNKT cells (Yu and Cantorna

2008). The active form of vitamin D, 1,25(OH)2D3 or iNKT cells were both shown to be

positive regulators of EAE (Cantorna 1996; Jahng 2001; Mars 2002). My data shows that

iNKT cells are required for the full beneficial effects of 1,25(OH)2D3 in EAE, suggesting

a role for iNKT cells in mediating beneficial effects of 1,25(OH)2D3 in mice with EAE.

My results show that the supplementation of 1,25(OH)2D3 in diet reduces the

severity of EAE symptoms but does not decrease the incidence of EAE in CD1d KO

mice. The EAE scores were significantly lower in CD1d KO mice with 1,25(OH)2D3

treatment than in CD1d KO controls. The difference in the EAE clinical measures

between these two treatments is because that the affected mice with 1,25(OH)2D3

treatment didn’t develop as high EAE scores as CD1d KO controls did. However, the

beneficial effects of 1,25(OH)2D3 supplementation on EAE inhibition in CD1d KO mice

were far less than that in WT mice, which suggested that the beneficial effects of

1,25(OH)2D3 in EAE prevention might be mediated by NKT cell regulation. From these

results alone, we are not sure yet whether type I or type II NKT cells or both play the role

to mediate the beneficial effects 1,25(OH)2D3 in EAE prevention.

Further, my data demonstrate that 1,25(OH)2D3 supplementation in diet reduces the

severity of EAE symptoms but does not decrease the incidence of EAE in Jα18 KO mice.

The beneficial effects of 1,25(OH)2D3 in Jα18 KO mice were less than that in WT mice.

16

Besides, 1,25(OH)2D3 has similar effects on the course of EAE development of CD1d KO

mice and Jα18 KO mice and the cumulative EAE scores did not differ from each other.

Since CD1d KO lack both iNKT and type II NKT cells while Jα18 KO mice only lack

iNKT cells, these data suggest that the beneficial effects of 1,25(OH)2D3 in EAE

prevention might be mediated by regulation of iNKT cells but not type II NKT cells.

Previous studies have found that 1,25(OH)2D3 was a positive regulator for the anti-

encephalitogenic Th2 cytokine IL-4 (Cantorna 1998; Mahon, Wittke et al. 2003). It has

been shown that 1,25(OH)2D3 failed to prevent IL-4 KO mice from developing EAE

(Cantorna, Humpal-Winter et al. 2000). α-GalCer administration also failed to protect IL-

4 KO mice on the C57BL/6 background (Falcone 1998; Singh 2001; Furlan 2003). My

results confirm that neither 1,25(OH)2D3 supplementation nor α-GalCer administration

could protect IL-4 KO mice from EAE development. Neither of these treatments had

protective effects against EAE in IL-4 KO mice compared to WT, indicating that in vivo

up-regulation of IL-4 is an important factor in effects of 1,25(OH)2D3 or α-GalCer on

EAE inhibition.

Vitamin D status affects the development of autoimmune diseases like MS. My data

suggest that the preventive effects of 1,25(OH)2D3 in EAE mice might be regulated by

iNKT cells and IL-4 might play an important role in mediating the beneficial effects of

either 1,25(OH)2D3 or α-GalCer on EAE inhibition. The protective effects of

1,25(OH)2D3 against EAE might be regulated by NKT cell derived IL-4. Understanding

the role of NKT cells on the beneficial effects of active vitamin D against EAE might

help us understand more about the protective and pathologic mechanisms of vitamin D

17

involved in human MS development and find new therapeutic targets to serve as

treatment of the disease.

18

Figure 3-1: 1,25(OH)2D3 treatment is ineffective to suppress EAE in CD1d KO mice.

(A) Daily mean EAE scores of WT and CD1d KO mice on control or 1,25(OH)2D3

supplemented diet. *: WT control diet versus WT 1,25(OH)2D3 diet, day 10: P <0.01, day

11,12,14,16-21: P <0.0001, day 13: P <0.05, day 15: P<0.001; #: CD1d KO control diet

versus CD1d KO 1,25(OH)2D3 diet, day 11: P <0.05, day 12,18: P <0.01, day 13:

P<0.001; day14-17: P <0.0001; +: WT 1,25(OH)2D3 diet versus CD1d KO 1,25(OH)2D3

diet, day 16: P <0.05, day 18,19,21: P <0.01, day 20: P<0.001. (B) The cumulative EAE

scores of WT and CD1d KO mice on control or 1,25(OH)2D3 supplemented diet. **: P

<0.01, ****: P <0.0001. The clinical data show the mean ± SEM from three independent

experiments, n= 11-21.

19

Figure 3-2: 1,25(OH)2D3 treatment is ineffective to suppress EAE in Jα18 KO mice.

(A) Daily mean EAE scores of WT and Jα18 KO mice on control or 1,25(OH)2D3

supplemented diet. #: WT control diet versus WT 1,25(OH)2D3 diet, day 10: P <0.01, day

11,12,14,16-21: P <0.0001, day 13: P <0.05, day 15: P<0.001; *: Jα18 KO control diet

versus Jα18 KO 1,25(OH)2D3 diet, day 14: P <0.001, day 15,18: P <0.05, day 16-17:

P<0.01. (B) The cumulative EAE scores of WT and Jα18 KO mice on control or

1,25(OH)2D3 supplemented diet. *: P <0.05, ****: P <0.0001. The clinical data show the

mean ± SEM from three independent experiments, n= 11-22.

20

Figure 3-3: 1,25(OH)2D3 treatment is ineffective to suppress EAE in either CD1d

KO or Jα18 KO mice. (A) Daily mean EAE scores of CD1d KO and Jα18 KO mice on

control or 1,25(OH)2D3 supplemented diet. (B) The cumulative EAE scores of CD1d KO

and Jα18 KO mice on control or 1,25(OH)2D3 supplemented diet. The clinical data show

the mean ± SEM from three independent experiments, n= 18-22.

21

Figure 3-4: Either 1,25(OH)2D3 or α-GalCer fails to prevent EAE in IL-4 KO mice.

(A) Daily mean EAE scores of IL-4 KO mice on control diet, 1,25(OH)2D3 supplement or

α-GalCer treatment. (B) Cumulative EAE scores of IL-4 KO mice on control diet,

1,25(OH)2D3 supplement or α-GalCer treatment. *: P <0.05, ****: P <0.0001. The

clinical data show the mean ± SEM from two independent experiments, n= 3-12.

22

Table 3-1: Summary table for measures of EAE symptoms of WT and CD1d KO

mice on control or 1,25(OH)2D3 diet. ****: WT control diet versus WT 1,25(OH)2D3 diet,

incidence, CDI: P <0.0001; CD1d KO control diet versus CD1d KO 1,25(OH)2D3 diet,

CDI: P <0.0001. **: CD1d KO control diet versus CD1d KO 1,25(OH)2D3 diet, day of

onset: P<0.01.*: CD1d KO control diet versus CD1d KO 1,25(OH)2D3 diet, peak severity:

P <0.05. ++++: WT 1,25(OH)2D3 diet versus CD1d KO 1,25(OH)2D3 diet, incidence: P

<0.0001. ++: WT 1,25(OH)2D3 diet versus CD1d KO 1,25(OH)2D3 diet, CDI: P <0.01.

The clinical data demonstrate the mean ± SEM from three independent experiments, n=

11-21.

23

Table 3-2: Summary table for measures of EAE symptoms of WT and Jα18 KO

mice on control or 1,25(OH)2D3 diet. ****: WT control diet versus WT 1,25(OH)2D3 diet,

incidence, CDI: P <0.0001; **: WT 1,25(OH)2D3 diet versus Jα18 KO 1,25(OH)2D3 diet,

incidence: P <0.01; *: Jα18 KO control diet versus Jα18 KO 1,25(OH)2D3 diet, CDI: P

<0.05; +: WT 1,25(OH)2D3 diet versus Jα18 KO 1,25(OH)2D3 diet, CDI: P <0.05. The

clinical data demonstrate the mean ± SEM from three independent experiments, n= 11-22.

24

Table 3-3: Summary table for measures of EAE symptoms of IL-4 KO mice on

control diet, 1,25(OH)2D3 supplement or α-GalCer treatment. ****: WT control diet

versus WT 1,25(OH)2D3 diet, incidence, CDI: P <0.0001; WT control diet versus WT

with α-GalCer treatment, incidence: P <0.0001. *: WT control diet versus WT with α-

GalCer treatment, CDI: P <0.05. #: IL-4 KO control diet versus IL-4 KO with α-GalCer

treatment, day of onset: P <0.05. ***: WT 1,25(OH)2D3 diet versus IL-4 KO

1,25(OH)2D3 diet, incidence: P <0.001. ++++: WT 1,25(OH)2D3 diet versus IL-4 KO

1,25(OH)2D3 diet, CDI: P <0.0001. +: WT with α-GalCer versus IL-4 KO with α-GalCer

treatment, incidence, CDI: P <0.05. The clinical data demonstrate the mean ± SEM from

three independent experiments, n= 3-12.

25

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