1,25-Dihydroxyvitamin D3 Suppresses TLR8 Expression and TLR8-Mediated Inflammatory Responses in Monocytes In Vitro and Experimental Autoimmune Encephalomyelitis In Vivo

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) suppresses autoimmunity and inflammation; however, the mechanism of its action has not been fully understood. We sought in this study to determine whether the anti-immune/anti-inflammatory action of 1,25(OH)2D3 is in part mediated through an interplay between 1,25(OH)2D3 and toll-like receptor (TLR)7/8 signaling. 1,25(OH)2D3 treatment prior to and/or following experimental autoimmune encephalomyelitis (EAE) induction effectively reduced inflammatory cytokine expression in the spinal cord and ameliorated EAE. These effects were accompanied with a reduction in expression of several TLRs with the most profound effect observed for TLR8. The expression of TLR8 adaptor protein MyD88 was also significantly reduced by 1,25(OH)2D3. To determine the molecular mechanism by which 1,25(OH)2D3 suppresses EAE induction of TLR8 and inflammatory cytokine expression, we evaluated whether 1,25(OH)2D3 can directly inhibit TLR8 signaling and the resulting inflammatory responses in human THP-1 monocytes. 1,25(OH)2D3 treatment not only significantly reduced TLR8 expression but also the expression or activity of MyD88, IRF-4, IRF-7 and NF-kB in monocytes challenged with TLR8 ligands. TLR8 promoter-luciferase reporter assays indicated that 1,25(OH)2D3 decreases TLR8 mRNA level in part via inhibiting TLR8 gene transcription activity. As a result of inhibition on TLR8 signaling cascade at various stages, 1,25(OH)2D3 significantly diminished the TLR8 target gene expression (TNF-α and IL-1β). In summary, our novel findings suggest that TLR8 is a new target of 1,25(OH)2D3 and may mediate the anti-inflammatory action of 1,25(OH)2D3. Our findings also point to a destructive role of TLR8 in EAE and shed lights on pathogenesis of multiple sclerosis.


Introduction
Multiple sclerosis (MS) is a debilitating disease of the central nerve system (CNS) which affects more than 2.5 million people worldwide every year [1]. The pathological hallmarks of MS are demyelination, multifocal inflammation, axonal degeneration and oligodendrocyte loss [1], which have been suggested to be associated with infection [2]. Current therapies use mostly disease-modifying drugs, focusing on blocking and regulating systemic functions and CNS infiltration of immune cells [3], [4]. However, these therapies only attenuate or delay MS symptoms and are not effective in halting the disease progression [3], [4]. Thus, a thorough understanding of the pathogenesis of MS could lead to identification of new drug targets for treatment of MS.
Experimental autoimmune encephalomyelitis (EAE) is a primary T-cell-mediated disorder that has provided important insights into the pathogenesis and treatment of MS. Similar to what occurs in MS, excessive production of inflammatory cytokines by activated microglia and invading inflammatory cells appear to play a critical role [1], [5]. In this regard, treatment of EAE mice with 1,25dihydroxyvitamin D3 (1,25(OH) 2 D 3 ), the active form of vitamin D, was able to suppress inflammatory cytokine production in the inflamed EAE spinal cords and effectively ameliorate EAE [6], [7], [8], [9], [10], [11]. Identification of the molecules which mediate the anti-immune/inflammatory action of 1,25(OH) 2 D 3 may uncover new mechanisms by which this hormone inhibits inflammatory cytokine production.
One pathway that leads to inflammation of CNS under an autoimmune condition could be the overly activation of toll-like receptors (TLRs), which leads to production of inflammatory cytokines via activating NF-kB and IFN regulatory factor (IRF) dependent pathway [12], [13], [14], [15]. Although several in vitro studies demonstrate that 1,25(OH) 2 D 3 was able to reduce TLR2, 4 and 9 expression [16], [17], [18], [19], no information is available on the role of 1,25(OH) 2 D 3 in modulating the action of other important TLRs in vitro or any of TLRs in vivo. TLR7 and 8 belong to the same subfamily and both are located in the intracellular endosomal compartments [20]. These TLRs recognize viral infection in the form of foreign nucleic acids [20]. TLRs can also be activated by endogenous ligands that are released from damaged cells [12], [21]. Since MS has been suggested to be associated with infection [2], TLR7 and 8, which are activated by viral pathogen penetrating the host cells, are of particular relevance to pathogenesis of EAE. Currently information on whether these TLRs, especially the TLR8, play a protective or detrimental role in EAE is scarce. TLR8 is known to be expressed in microglia and neurons [22], [23] and activation of TLR8 by agonists in cultured cortical neurons inhibited neurite outgrowth and induced apoptosis [22]. In vivo, intracerebroventricular inoculation of newborn mice with the TLR7/8 agonist (R837) induced neuroinflammation and production of proinflammatory cytokines and chemokines [24]. In addition, expression of both TLR7 and TLR8 in the spinal cord was increased upon onset of the EAE [25]. In contrast to these studies, which in general suggest a destructive role of TLR8 in CNS, systemic TLR8 gene deletion in mice led to development of autoimmunity and glomerulonephritis [26].This discrepancy further sparked our interest to investigate the role and regulation of this intriguing TLR under an autoimmune condition.
The primary objective of this study was to evaluate if 1,25(OH) 2 D 3 acts to suppress the inflammation in part by regulating the TLR8 signaling. We found that TLR8 was increased by EAE to the largest extent among the several TLRs analyzed. Importantly this up-regulation was abolished by treatment of EAE mice with 1,25(OH) 2 D 3, which ameliorated EAE and reduced expression of key inflammatory cytokines. Our in vitro results are in line with these in vivo data and demonstrate that 1,25(OH) 2 D 3 significantly inhibited TLR8 signaling at multiple stages thereby counteracting the TLR8-induced TNFa and IL-1b expression. These novel findings suggest that TLR8 is a new target of 1,25(OH) 2 D 3 and may mediate the antiinflammatory action of 1,25(OH) 2 D 3 in EAE.

Mice
Female C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were used at ages 8-12 weeks. The investigators adhered to the Animal Welfare Act Regulations and other Federal statutes relating to animals and experiments involving animals and the principles set forth in the current version of the Guide for Care and Use of Laboratory Animals, National Research Council. All experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at the Loma Linda University.

1,25(OH) 2 D 3 Treatment and EAE Induction
For the prevention studies, mice were injected i.p. with 2 mg 1,25(OH) 2 D 3 in peanut oil 24 hours prior to injection of MOG. From day 3 post-MOG injection, mice were injected i.p. with a reduced dose of 1,25(OH) 2 D 3 (100 ng/mouse) every other day till the end of the experiment. For the treatment studies, 200 ng of 1,25(OH) 2 D 3 in peanut oil was injected i.p for every 3 days from day 7 post-MOG injection. In both studies, peanut oil only served as the placebo vehicle control. MOG-immunized mice without 1,25(OH) 2 D 3 administration developed EAE around 10 days post-MOG injection, and disease usually peaked around 3 weeks post-MOG immunization. In our experiments, all the mice were sacrificed at day 14 post-MOG injection. To induce EAE, mice were immunized subcutaneously with 200 mg MOG emulsified in complete Freund's adjuvant (Difco, Lawrence, KS) supplemented with 5 mg/ml Mycobacterium tuberculosis H37Ra (Difco, Lawrence, KS). The mice were injected twice i.p. with 200 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) at the time of immunization and 48 hours later. The mice were then monitored daily for clinical symptoms of disease, and disease severity was scored on a numerical scale from 0-5 as follows: 0, no disease; 1, weak tail or wobbly walk; 2, hind limb paresis; 3, hind limb paralysis; 4, hind and forelimb paralysis; 5, death. Paralyzed mice were given easy access to food and water.

Cell Culture
Human monocytic THP-1 cells, obtained from American Type Culture Collection (Manassas, VA) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 0.05 mM 2-mercaptoethanol and 100 mg/ml penicillin and streptomycin in 24-well plates in a humidified atmosphere containing 5% CO 2 at 37uC. For the THP-1 cells treatment, the cells were seeded at a density of 1610 6 cells/ml and concentration of 1,25(OH) 2 D 3 routinely used was 100 nM. Thirty min after 1,25(OH) 2 D 3 addition, CL075 or ssRNA was added to the final concentration (1 mg/ml for CL075 and 5 mg/ml for ssRNA). After additional 20 h incubation, cells were harvested for total RNA isolation or FACS analysis.
Quantitative Real-time RT-PCR mRNA levels of the genes of interest in the spinal cord and THP-1 cells were analyzed by real-time RT-PCR. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) and purified with RNeasy mini kit (Qiagen, Valencia, CA). cDNA was prepared using the superscript III cDNA synthesis kit (Invitrogen, Carlsbad, CA), according to instructions. Specific primers were synthesized by Integrated DNA Technologies (San Diego, CA) as shown in Table S1. Real-time PCR was performed with SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA) in 7500 Fast Real Time PCR System (Applied Biosystems, Carlsbad, CA). Each reaction was run in triplicate. The data were normalized to GAPDH or b-actin as a reference and presented as fold change relative to control samples.

Immunohistochemistry
Mice were euthanized and perfused with PBS. The spinal cords were removed and fixed by immersion in 4% paraformaldehyde (PFA) overnight at 4uC, followed by cryopreservation in 30% sucrose in PBS at 4uC until the tissues got submerged. The spinal cords were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) and frozen under liquid nitrogen. Sections (8 mm) were cut on a cryostat, thawedmounted on Superfrost plus slides, air dried, and stored at -20uC for further use. After fixation with 4% PFA and endogenous peroxidase activity blockage, tissue sections were blocked with 10% normal goat serum (Sigma-Aldrich, St. Louis, MO) for 1 h at RT and then incubated with primary antibodies that were diluted (anti-TLR8 monoclonal antibody 1:150 and anti-F4/80 polyclonal antibody 1:200) in PBS containing 2% normal goat serum overnight at 4uC. Samples were subsequently incubated with biotinylated species-specific secondary antibodies (1:300 dilution, Vector Laboratories, Burlingame, CA) for 30 min at RT followed by incubation with HRP (1:300 dilution, Vector Laboratories, Burlingame, CA) for 30 min at RT. Staining was visualized using 3,39-diaminobenzidine (DAB) substrate (Vector Laboratories, Burlingame, CA) and counterstained with haematoxylin (Sigma-Aldrich, St. Louis, MO).

Flow Cytometry
The abundance of TLR8 and TLR7-positive cells in spinal cord was determined using a FACSAria flow cytometer (BD Biosciences, San Jose, CA). Mice were perfused intracardially with PBS to remove blood cells from the organs. Spinal cords were collected and dissociated to single-cell suspension using neural tissue dissociation kits (Miltenyi Biotec, Auburn, CA). Mononuclear cells in the spinal cord digests were isolated by 30%/70% Percoll gradient centrifugation, washed with PBS, fixed in 2% PFA for 1 h at 4uC and then permeablized in a buffer containing 0.1% saponin. Nonspecific antibody binding to Fc receptors was blocked by incubation in 0.1% saponin containing 2% goat serum for 30 min at room temperature. Cells were then incubated with PEconjugated anti-TLR8 antibody, PE-conjugated anti-TLR7 antibody or isotype control for 3 hours at room temperature in dark. Cells were washed twice with permeabilization buffer and once with PBS prior to FACS analysis. Isotype primary conjugated antibodies served as a negative control. Data were analyzed using FACSDiva software (BD Biosciences, San Jose, CA).

ELISA
Serum concentration of IL-17 was measured by an ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol.

Western Immunoblot Analysis
THP-1 cells were collected after treatment with an indicated dose of CL075 and/or 1,25(OH) 2 D 3 , washed twice with ice-cold PBS, and lysed on ice for 20 min in lysis buffer containing 25 mM Tris . HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, phosphatase inhibitors cocktail 2 and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Supernatants were collected after centrifugation at 13,000 rpm for 20 min at 4uC. Protein concentration in the supernatants was measured using a Bio-Rad protein assay kit. Equal amounts of cellular protein were resolved on a 10% SDS-PAGE gel and then transferred to nitrocellulose membrane by electrophoresis. After blocking with 5% dry milk in PBS/Tween 20 (0.1%) for 16 hr at 4uC, membranes were incubated with antibodies specific to NF-kB and IkB followed by horseradish peroxidase-conjugated secondary antibodies incubation for 1 h. After washing with PBS/Tween 20, signals were visualized with autoradiography using ECL system. To control for protein loading, the membranes were reprobed with anti-b-actin antibody.

Transfection and Luciferase Reporter Assay
The cloning of the 3.8 kb 59 regulatory sequence (promoter) of the human TLR8 gene and construction of the TLR8 promoterluciferase report plasmid were described in a previous study [27]. The investigators adhered to the current version of the National Institutes of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules.
THP-1 cells were co-transfected with indicated luciferase reporter plasmids and TK-Renilla plasmid at ratio of 10:1 using X-tremeGENE HP DNA transfection reagent (Roche Applied Science, Indianapolis, IN). After 24 h transfection, cells were stimulated for 20 h with 4 mg/ml CL075 in the absence or presence of 100 nM 1,25(OH) 2 D 3 . The cells were washed once with PBS, lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI).

Statistical Analysis
Data were reported as mean 6 standard error of the mean (SEM). The statistical significance of differences between the group means was assessed using the Student's t test or the Mann-Whitney U test. P,0.05 was considered as statistically significant.

1,25(OH) 2 D 3 Treatment Reduced Inflammatory Cell Infiltration and Inflammatory Cytokine Expression in the EAE Spinal Cord
In order to evaluate the effect of 1,25(OH) 2 D 3 on TLR8 signaling and its potential role in the pathogenesis of EAE, it is important to evaluate if 1,25(OH) 2 D 3 treatment under our experimental conditions is effective in ameliorating EAE. In the prevention model, mice received a single injection of 1,25(OH) 2 D 3 one day prior to and multiple injections of 1,25(OH) 2 D 3 post-MOG injection (Material and Methods). 1,25(OH) 2 D 3 treatment reversed the decrease in body weight of EAE mice ( Figure 1A). At day 14, there was no significant difference in body weight of control mice vs. EAE mice treated with 1,25(OH) 2 D 3. All of the 7 MOG-injected mice developed severe EAE symptoms with an average disease score of 3.5 ( Figure 1B). In contrast, only one of the 7 MOG-injected mice treated 1,25(OH) 2 D 3 developed a moderate EAE symptom ( Figure 1B). The clinical score of an independent experiment in prevention model was recorded daily and presented in the Figure S1A.
Immunostaining of the spinal cord sections for microglia/ macrophage maker F4/80 revealed a severe inflammatory cell infiltration in the EAE mice compared with the control mice ( Figure 2A). The inflammatory cell infiltration, mainly in the white matter, was essentially prevented by 1,25(OH) 2 D 3 ( Figure 2A). mRNA expression of three key inflammatory cytokines (TNF-a, INF-c and IL-17) in spinal cord was significantly increased in mice immunized with MOG ( Figure 2B-D). In addition, EAE mice showed a significant increase in serum concentration of IL 17, the most important disease-driving pro-inflammatory cytokine in this model ( Figure 2E). All of these changes were significantly reduced by 1,25(OH) 2 D 3 treatment ( Figure 2B-E).
In the treatment model, mice were injected, i.p. with 1,25(OH) 2 D 3 for 7 days beginning from day 7 post-MOG injection. We chose to initiate 1,25(OH) 2 D 3 treatment at day 7 as it has been reported that inflammatory cell infiltration and inflammatory cytokine expression in the spinal cord were increased at this disease stage [25], [28]. Moreover, our experiment indicated that expression of TLR8 was significantly increased as early as 5 days post-MOG injection (data not shown). The experiment was terminated at day 14 because most of the MOGimmunized mice developed severe disease symptoms at this time evidenced by paralysis of both hind limbs, whereas the disease progression was essentially halted by the 7-day 1,25(OH) 2 D 3 treatment ( Figure S1B and S2A).
1,25(OH) 2 D 3 treatment for 7 days beginning at day 7 post-MOG immunization significantly attenuated the EAE-induced increase in IL-17 mRNA level in the spinal cord ( Figure S2B) as well as IL17 concentration in the serum ( Figure S2C). Subsequently, modulation of TLR expression by EAE and 1,25(OH) 2 D 3 treatment were evaluated in both models (see section below).

TLR8 Expression in the Spinal Cord was Markedly Increased by EAE and this Increase was Significantly Reduced by 1,25(OH) 2 D 3 Treatment
In the prevention model, among the 4 TLRs analyzed, TLR3 and TLR8 showed a significant increase in their steady state mRNA levels in the EAE spinal cords compared to the control spinal cords (10-12 fold) ( Figure 3A). Treatment of EAE mice with 1,25(OH) 2 D 3 only significantly decreased TLR8 mRNA level ( Figure 3A). In the treatment model, the mRNA levels of TLR3, 4, 7 and 8 were all significantly increased by EAE. 1,25(OH) 2 D 3 treatment significantly decreased the EAE-induced expression of all 4 TLRs analyzed ( Figure 3B). Similar to results obtained in the prevention model, TLR8 showed the highest magnitude of induction by EAE and reversal by 1,25(OH) 2 D 3 treatment ( Figure 3B). For this reason, our subsequent analysis was focused on TLR8.
Consistent with real-time PCR analysis of the TLR8 mRNA, we found that the abundance of the spinal cord cells expressing TLR8 protein was significantly increased in the EAE spinal cord and this increase was attenuated by 1,25(OH) 2 D 3 treatment, as determined by immunostaining of the spinal cord sections with TLR8 antibody ( Figure 3C). To further confirm these results, the number of TLR8-positive mononuclear cells in the spinal cord was analyzed by FACS in both prevention and treatment models. The frequency of TLR8 positive cells in the spinal cord of EAE mice was increased by 2.7 fold compared to that of the control spinal cord ( Figure 3D). This increase was reduced moderately in the prevention model and essentially blocked in the treatment model by 1,25(OH) 2 D 3 . In contrast, only a mild increase in abundance of TLR7-postive cells was observed in EAE spinal cord under our experimental conditions ( Figure 3D). Collectively, these data demonstrate that 1,25(OH) 2 D 3 not only reduced EAE-induced TLR8 expression but also the abundance of the spinal cord cells expressing TLR8.
Since MyD88 is an immediate downstream adaptor molecule that interacts with TLRs and its deficiency led to resistance to EAE development [29], [30], we evaluated whether its expression was also affected under EAE. In the prevention model, EAE increased MyD88 mRNA level by ,4 fold and this increase was completely prevented by 1,25(OH) 2 D 3 treatment ( Figure 4A). A similar pattern of regulation was observed for other downstream mediators of TLR8 signaling, including IFN regulatory factor-4binding protein (IBP), IRF-4, and Schlafen-4 ( Figure 4B-D). In the treatment model, 1,25(OH) 2 D 3 suppressed the EAE-induced expression of MyD88 and IRF-7 to the level measured in the control spinal cord ( Figure S2D-E).

1,25(OH) 2 D 3 Inhibited Expression of Inflammatory Cytokines and TLR8 in THP-1 Monocytes Challenged with TLR8 Ligands
To determine the mechanism by which 1,25(OH) 2 D 3 suppresses EAE induction of TLR8 and inflammatory cytokine expression, we tested the possibility that 1,25(OH) 2 D 3 may have exerted these effects by directly acting on inflammatory cells. Human monocytes (THP-1) were used in our studies because the TLR8 ligands/ agonists are better defined in human than mouse species. Moreover, we possess human TLR8 promoter clone for evaluation of TLR8 transcriptional regulation in human cells [27]. Monocytes were challenged with TLR8 agonist/ligand to mimic the in vivo disease state in which self ligands generated from apoptotic/necrotic cells is likely the primary reason for EAEinduced TLR8 activation. Co-treatment with 1,25(OH) 2 D 3 at 100 nM significantly reduced the mRNA level of both TNF-a and IL-1b that were induced by synthetic TLR8 agonist (CL075) ( Figure 5A-B). The expression level of TNF-a but not IL-1b was also significantly inhibited by the natural TLR8 agonist (ssRNA) ( Figure 5A-B). The CL075-induced TNF-a and IL-1b expression was inhibited in a dose-dependent manner by 1,25(OH) 2 D 3 ( Figure 5C-D).
In line with measurements of inflammatory cytokine expression, the steady state mRNA level of TLR8 was dramatically upregulated by stimulation with synthetic or natural human TLR8 agonist, with a much stronger effect observed with the synthetic agonist CL075 ( Figure 6A). The ligand/agonist-induced increase in TLR8 mRNA level was significantly reduced by 1,25(OH) 2 D 3 treatment ( Figure 6A). It should be noted that CL075 had no effect on TLR4 expression and had a much weaker effect on expression of TLR7 compared to that of TLR8 ( Figure S3), suggesting that CL075 is relatively specific in up-regulating TLR8 expression.
To further confirm the inhibitory effect of 1,25(OH) 2 D 3 on TLR8 expression, we analyzed the changes in the amount of intracellular TLR8 in monocytes by FACS analysis. Consistent with a lack of an effect of 1,25(OH) 2 D 3 on basal TLR8 mRNA level, the basal level of TLR8 protein was not altered by 1,25(OH) 2 D 3 treatment ( Figure 6B, peak 2 vs. peak 4). CL075 stimulation increased the TLR8 signal strength ( Figure 6B, peak 5 vs. peak 2). Following 1,25(OH) 2 D 3 treatment, the agonistinduced shift in TLR8 signal returned to its basal level ( Figure 6B, peak 5 vs. peak 3).
Next we determined whether 1,25(OH) 2 D 3 decreases steadystate TLR8 mRNA level in part through inhibiting the TLR8 transcription activity using the recently cloned 59 regulatory sequence of the human TLR8 gene linked to the luciferase reporter [27]. The TLR8 agonist CL075 significantly increased the luciferase reporter activity in the monocytes transfected with the TLR8-luciferase reporter plasmid ( Figure 6C). Treatment with 1,25(OH) 2 D 3 significantly reduced the agonist-induced TLR8 promoter activation ( Figure 6C).

1,25(OH) 2 D 3 Inhibited Expression of TLR8 Downstream Mediators in THP-1 Monocytes Challenged with TLR8 Ligands
To further determine the mechanism underlying the inhibitory action of 1,25(OH) 2 D 3 on inflammatory response in monocytes, we evaluated whether 1,25(OH) 2 D 3 modulates expression or activity of key TLR8 downstream mediators in addition to TLR8 itself. 1,25(OH) 2 D 3 treatment significantly reduced expression of MyD88 and its interacting proteins IRF-4 and IRF-7 ( Figure 7A-C). Since TLR8 signaling requires activation of NF-kB pathway, we also evaluated whether components of this pathway were modulated by 1,25(OH) 2 D 3 . The total cellular level of NF-kB, evaluated by Western immunoblot analysis, was not affected by various treatments (Figure 7D). The cellular level of IkB, an inhibitor of NF-kB, was reduced by TLR8 agonist treatment ( Figure 7D). Co-treatment with 1,25(OH) 2 D 3 partially reversed the decrease in IkB level resulting from TLR8 activation( Figure  7D). Consistently, treatment with 1,25(OH) 2 D 3 inhibited NF-kB activation as NF-kB-mediated luciferase reporter activity was significantly inhibited by 1,25(OH) 2 D 3 treatment ( Figure 7E). These data suggest that 1,25(OH) 2 D 3 not only suppresses expression of TLR8 itself but also the expression or activity of its downstream mediators either directly or indirectly, leading to inhibition of inflammatory cytokine production.

Discussion
Preclinical studies have shown that 1,25(OH) 2 D 3 treatment effectively ameliorated EAE in mice [6], [7], [8], [9], [10], [11]. However, the mechanism by which this hormone provides the therapeutic effects has not been fully understood. Our findings in this study from both in vivo and in vitro experiments suggest that TLR8 is potentially a new target of 1,25(OH) 2 D 3 and may, in part, mediate the anti-inflammatory action of 1,25(OH) 2 D 3 .
The murine TLR8 was once suggested to be biologically inactive based on the finding that viral ssRNA, the natural ligand for human TLR7/8, failed to activate mouse TLR8 [31]. The lack of mouse TLR8 activation by human TLR7/8 agonist was postulated to be due to the structural difference in the ligand binding domain of TLR8 between these two species [32], [33]. This prediction was consistent with the finding that a combination of TLR7/8 agonist and polyT oligodeoxynucleotides, was able to enhance activation of NF-kB in the human embryonic kidney 293T (HEK293T) cells overexpressing the  mouse TLR8 [32], [33]. Now there is compelling evidence, which indicates that mouse TLR8 is indeed expressed and biologically functional in mouse tissues, especially in the central nerve system [22], [24]. Thus, a mouse EAE model is useful in evaluating the role and regulation of TLR8 in EAE.
To our knowledge, this is the first study which demonstrates that 1,25(OH) 2 D 3 treatment effectively suppressed TLR8 expression in an autoimmune condition. This suppressive effect is likely to be mediated by both of the following mechanisms. First, the decrease in TLR8 mRNA level in the spinal cord is at least in part due to the decrease in the number of spinal cord cells expressing TLR8 ( Figure 3C-D). This conclusion is consistent with the data showing that 1,25(OH) 2 D 3 treatment remarkably reduced the abundance of infiltrating macrophages (Figure 2A) which are known to abundantly express TLR8 [22], [23]. Second, 1,25(OH) 2 D 3 may directly act on the infiltrating inflammatory cells and/or resident microglia cells to inhibit their TLR8 expression and our in vitro data support this contention. The homologous up-regulation of the TLR8 expression by their synthetic or natural ligand was strongly inhibited by 1,25(OH) 2 D 3 ( Figure 6A-B). This inhibition could occur as early as 12 hours post-1,25(OH) 2 D 3 treatment (data not shown). The 1,25(OH) 2 D 3 inhibition of the TLR8 expression in monocytes is biologically significant as it links to an inhibition on both NF-kB and IRF-7 pathways and reduction in the expression of inflammatory cytokines TNF-a and IL-1b. Hence, our data suggest that 1,25(OH) 2 D 3 suppression of TLR8 activation in EAE mice may have resulted from a direct inhibition on TLR8 signaling pathway within the infiltrating monocytes and/or activated microglia cells in the spinal cord lesion.  Figure 5A-B legend. Cells were collected and subjected to RNA and protein extraction. mRNA levels of MyD88 (A), IRF-4 (B) and IRF-7 (C) were determined by real-time RT-PCR. Data represent means 6 SEM (n = 4) and are representative of three independent experiments. (D) Western blotting for NF-kB and IkB-I protein detection was performed on total cell lysates from THP-1cells stimulated with CL075 (1 mg/ml) in the presence of vehicle or 1,25(OH) 2 D 3 (100 nM) for 20 h. Representative results were obtained from two independent experiments. (E) The THP-1 cells were co-transfected with NF-kB promoter luciferase and TK-Renilla plasmid (Materials and Methods). After 24 h, the cells were pretreated with 1,25(OH) 2 D 3 (100 nM) for 30 min and then stimulated with CL075 (4 mg/ml) for 20 h. Luciferase activity was measured and presented as the ratio of luciferase activity to TK-Renilla activity. The data shown are means 6 SEM (n = 4) and are representative of three independent experiments. * P,0.05; ** P,0.01; *** P,0.001. doi:10.1371/journal.pone.0058808.g007 To begin to understand the mechanism by which 1,25(OH) 2 D 3 suppresses the ligand-induced TLR8 expression in monocytes, we evaluated whether this regulation could occur at the transcriptional level. Consistent with a recent report [27], treatment of human monocytes with TLR8 agonist significantly increased the transcriptional activity of the 3.8 kb distal human TLR8 promoter. The agonist-induced TLR8 promoter activation is significantly inhibited by 1,25(OH) 2 D 3 treatment. These data provide evidence that 1,25(OH) 2 D 3 can acutely regulate TLR8 expression at the transcriptional level and support our premise that 1,25(OH) 2 D 3 down-regulation of the TLR8 expression in the EAE spinal cord is in part mediated through a direct action of 1,25(OH) 2 D 3 on inflammatory cells. 1,25(OH) 2 D 3 exerts its biological functions through vitamin D receptor (VDR). Analysis of the 3.8 kb proximal TLR8 promoter sequence did not reveal presence of a consensus VDR response element. Thus, future studies are needed to determine whether VDR could interact with transcription factors regulating TLR8 transcription, such as C/ EBPd [27], leading to a decrease in TLR8 gene transcription.
In summary, this study provides strong evidence that 1,25(OH) 2 D 3 regulates TLR8 expression and signaling in EAE leading to inhibition of the TLR8-mediated inflammatory responses. While our novel findings suggest that TLR8 activation may play an important role in initiation/propagation of EAE and potentially mediate the anti-inflammatory action of 1,25(OH) 2 D 3 in CNS, this premise needs to be confirmed in future by more definitive studies. Such studies may include use of TLR8 knockout mice to determine if susceptibility to EAE could be reduced in the absence of TLR8 in knockout mice. Also, the TLR8 knockout mouse model could be employed to define if TLR8 plays an critical role in 1,25(OH) 2 D 3 suppression of EAE. If this is the case, one would expect 1,25(OH) 2 D 3 to be ineffective to ameliorate EAE in TLR8 knockout mice. Then therapy aimed at reducing TLR8 expression or activation could be developed to treat inflammatory neurological diseases including MS. Regarding the systemic 1,25(OH) 2 D 3 therapy of multiple sclerosis, it is currently avoided to use in patients because the doses needed to achieve therapeutic efficacy is accompanied with hypercalcemia, which is an unacceptable side effect [34]. Future clinical application of this therapy will rely on development of 1,25(OH) 2 D 3 analogues which retain immunosuppressing activity but will not cause hypercalcemia. More practically, targeting this therapy specifically to diseased organs may ameliorate the disease without causing systemic hypercalcemia. Figure S1 The disease courses in both prevention and treatment models. Mice immunization and treatments were