The Pennsylvania State University. The Graduate School. The Huck Institutes of Life Sciences REGULATORY ROLE OF NKT CELLS ON EFFECTS OF

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1 The Pennsylvania State University The Graduate School The Huck Institutes of Life Sciences REGULATORY ROLE OF NKT CELLS ON EFFECTS OF 1,25(OH) 2 D 3 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

2 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

3 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) 2 D 3, 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) 2 D 3 had less effects in protecting CD1d knockout (KO) mice from EAE than in WT. CD1d KO mice with 1,25(OH) 2 D 3 supplementation had significantly higher incidence of EAE and developed significantly higher EAE scores than WT mice with 1,25(OH) 2 D 3 treatment. 1,25(OH) 2 D 3 was also less effective in preventing Jα18 KO mice against EAE than in WT. Jα18 KO mice on 1,25(OH) 2 D 3 treatment had significantly higher incidence of EAE and significantly higher cumulative EAE scores than WT mice with 1,25(OH) 2 D 3 treatment. Either 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 or -GalCer on EAE inhibition.

4 iv TABLE OF CONTENT Chapter 1 Introduction Vitamin D Metabolism and classical functions of Vitamin D Vitamin D in immune regulation Vitamin D and NKT cells Multiple Sclerosis Background and epidemiology Vitamin D and multiple sclerosis EAE and vitamin D EAE and NKT cells Conclusion... 7 Chapter 2 Materials and Methods Mice and diets αgalcer stimulation EAE induction EAE symptoms Statistical analysis... 9 Chapter 3 Results... 11

5 v 3.1 1,25(OH) 2 D 3 is ineffective at suppressing EAE in CD1d KO mice ,25(OH) 2 D 3 is ineffective at suppressing EAE in Jα18 KO mice Either 1,25(OH) 2 D 3 or α-galcer fails to prevent EAE in IL-4KO mice Chapter 4 Discussion References... 25

6 1 Chapter 1 Introduction 1.1 Vitamin D 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 D 3 ) in the skin by UVB light from sunlight exposure. Vitamin D 3 is then converted into 25-hydroxy vitamin D 3 [25(OH)D 3 ] in the liver, which is the major circulating form of vitamin D in human body. 25-hydroxy vitamin D 3 is then hydroxylated by the Cyp27B1 encoded enzyme 1 -hydroxylase into the active form of vitamin D, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] 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) 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) 2 D 3 binds to the VDR and

7 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) 2 D 3 could directly affect T helper1/t helper2 (Th1/Th2) cell differentiation (Boonstra 2001). 1,25(OH) 2 D 3 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) 2 D 3 repressed Th17 and regulatory T cell (Treg) differentiation (Chang, Cha et al. 2010). 1,25(OH) 2 D 3 could also regulate dendritic cell maturation (Piemonti 2000). 1,25(OH) 2 D 3 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) 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

8 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) 2 D 3 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 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).

9 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 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)D 3 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 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

10 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) 2 D 3 could prevent EAE and reversibly block the progression of the disease (Lemire 1991; Cantorna 1996). 1,25(OH) 2 D 3 increased antiencephalitogenic 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) 2 D 3 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) 2 D 3 effects in EAE inhibition. 1,25(OH) 2 D 3 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) 2 D 3 in inhibiting EAE related cytokines. Th17 related-cytokines were also inhibited by 1,25(OH) 2 D 3 in vivo and in vitro in experimental autoimmune uveitis experiments (Tang, Zhou et al. 2009). 1,25(OH) 2 D 3 suppressed IL-17A expression in EAE mouse models through mechanism of transcriptional suppression mediated by VDR (Joshi, Pantalena et al. 2011).

11 6 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) 2 D 3 inhibition of EAE and 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 prevents EAE onset and reverses the progression of symptoms partly through inhibiting Th1 and Th17 cells and inducing Th2 cytokine production 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

12 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) 2 D 3, 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) 2 D 3 is less effective in preventing EAE symptoms in IL-4 KO mice. The goal of this research was to determine whether 1,25(OH) 2 D 3 functions to suppress EAE with the production of IL-4 in inkt cells.

13 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) 2 D 3 supplemented diets were synthetic gel diets that contained 1,25(OH) 2 D 3 (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

14 9 Six to eight week old sex-matched C57BL/6 mice were immunized subcutaneously with 100 µl of 200 µg MOG (myelin oligodendrocyte glycoprotein) peptide (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

15 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.

16 11 Chapter 3 Results 3.1 1,25(OH) 2 D 3 is ineffective at suppressing EAE in CD1d KO mice WT mice on control diet showed EAE symptoms appeared between 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) 2 D 3 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) 2 D 3 diet were significantly lower than that of WT mice on control diet (Fig. 3-1B). Only one WT mouse on 1,25(OH) 2 D 3 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 ; the incidence of the disease was above 91% and the CDI was around 27 (Table 3-1). CD1d KO mice on 1,25(OH) 2 D 3 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) 2 D 3 diet were also significantly lower compared to CD1d KO mice on control diet (Fig. 3-1B). CD1d KO mice on 1,25(OH) 2 D 3 diet had delayed mean day of EAE

17 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) 2 D 3 diet were significantly higher than that of WT mice on 1,25(OH) 2 D 3 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) 2 D 3 diet was also significantly higher than that of WT mice on 1,25(OH) 2 D 3 diet (Fig. 3-1B). The EAE incidence of CD1d KO mice on 1,25(OH) 2 D 3 diet (81%) was also statistically higher than that of WT mice on same diet (8%, Table 3-1) ,25(OH) 2 D 3 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 (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) 2 D 3 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) 2 D 3 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) 2 D 3 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) 2 D 3 diet, the cumulative EAE score of Jα18 KO mice on 1,25(OH) 2 D 3 diet was significantly higher

18 13 than that of WT mice on 1,25(OH) 2 D 3 diet (Fig. 3-2B). Additionally, the EAE incidence of Jα18 KO mice on 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 diet were similar to each other (Fig. 3-3B). 3.3 Either 1,25(OH) 2 D 3 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 (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 ; and the CDI was around (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) 2 D 3 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) 2 D 3 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) 2 D 3 diet (Table 3-3).

19 14 Comparing the EAE symptoms of IL-4 KO mice on 1,25(OH) 2 D 3 diet with WT mice on 1,25(OH) 2 D 3 diet, the EAE incidence of IL-4 KO mice on 1,25(OH) 2 D 3 diet (100%) was significantly higher than that of WT mice on 1,25(OH) 2 D 3 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).

20 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) 2 D 3 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) 2 D 3 in EAE, suggesting a role for inkt cells in mediating beneficial effects of 1,25(OH) 2 D 3 in mice with EAE. My results show that the supplementation of 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 treatment didn t develop as high EAE scores as CD1d KO controls did. However, the beneficial effects of 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 in EAE prevention. Further, my data demonstrate that 1,25(OH) 2 D 3 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) 2 D 3 in Jα18 KO mice were less than that in WT mice.

21 16 Besides, 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 was a positive regulator for the antiencephalitogenic Th2 cytokine IL-4 (Cantorna 1998; Mahon, Wittke et al. 2003). It has been shown that 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 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) 2 D 3 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) 2 D 3 or α-galcer on EAE inhibition. The protective effects of 1,25(OH) 2 D 3 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

22 17 involved in human MS development and find new therapeutic targets to serve as treatment of the disease.

23 18 Figure 3-1: 1,25(OH) 2 D 3 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) 2 D 3 supplemented diet. *: WT control diet versus WT 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 diet versus CD1d KO 1,25(OH) 2 D 3 diet, day 16: P <0.05, day 18,19,21: P <0.01, day 20: P< (B) The cumulative EAE scores of WT and CD1d KO mice on control or 1,25(OH) 2 D 3 supplemented diet. **: P <0.01, ****: P < The clinical data show the mean ± SEM from three independent experiments, n=

24 19 Figure 3-2: 1,25(OH) 2 D 3 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) 2 D 3 supplemented diet. #: WT control diet versus WT 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 supplemented diet. *: P <0.05, ****: P < The clinical data show the mean ± SEM from three independent experiments, n=

25 20 Figure 3-3: 1,25(OH) 2 D 3 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) 2 D 3 supplemented diet. (B) The cumulative EAE scores of CD1d KO and Jα18 KO mice on control or 1,25(OH) 2 D 3 supplemented diet. The clinical data show the mean ± SEM from three independent experiments, n=

26 21 Figure 3-4: Either 1,25(OH) 2 D 3 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) 2 D 3 supplement or α-galcer treatment. (B) Cumulative EAE scores of IL-4 KO mice on control diet, 1,25(OH) 2 D 3 supplement or α-galcer treatment. *: P <0.05, ****: P < The clinical data show the mean ± SEM from two independent experiments, n= 3-12.

27 22 Table 3-1: Summary table for measures of EAE symptoms of WT and CD1d KO mice on control or 1,25(OH) 2 D 3 diet. ****: WT control diet versus WT 1,25(OH) 2 D 3 diet, incidence, CDI: P <0.0001; CD1d KO control diet versus CD1d KO 1,25(OH) 2 D 3 diet, CDI: P < **: CD1d KO control diet versus CD1d KO 1,25(OH) 2 D 3 diet, day of onset: P<0.01.*: CD1d KO control diet versus CD1d KO 1,25(OH) 2 D 3 diet, peak severity: P < : WT 1,25(OH) 2 D 3 diet versus CD1d KO 1,25(OH) 2 D 3 diet, incidence: P < : WT 1,25(OH) 2 D 3 diet versus CD1d KO 1,25(OH) 2 D 3 diet, CDI: P <0.01. The clinical data demonstrate the mean ± SEM from three independent experiments, n=

28 23 Table 3-2: Summary table for measures of EAE symptoms of WT and Jα18 KO mice on control or 1,25(OH) 2 D 3 diet. ****: WT control diet versus WT 1,25(OH) 2 D 3 diet, incidence, CDI: P <0.0001; **: WT 1,25(OH) 2 D 3 diet versus Jα18 KO 1,25(OH) 2 D 3 diet, incidence: P <0.01; *: Jα18 KO control diet versus Jα18 KO 1,25(OH) 2 D 3 diet, CDI: P <0.05; +: WT 1,25(OH) 2 D 3 diet versus Jα18 KO 1,25(OH) 2 D 3 diet, CDI: P <0.05. The clinical data demonstrate the mean ± SEM from three independent experiments, n=

29 24 Table 3-3: Summary table for measures of EAE symptoms of IL-4 KO mice on control diet, 1,25(OH) 2 D 3 supplement or α-galcer treatment. ****: WT control diet versus WT 1,25(OH) 2 D 3 diet, incidence, CDI: P <0.0001; WT control diet versus WT with α-galcer treatment, incidence: P < *: 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) 2 D 3 diet versus IL-4 KO 1,25(OH) 2 D 3 diet, incidence: P < : WT 1,25(OH) 2 D 3 diet versus IL-4 KO 1,25(OH) 2 D 3 diet, CDI: P < : 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.

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