Introduction
1,25-Dihydroxyvitamin D
3 (1,25-(OH)
2D
3) is a secosteroid hormone, synthesized through a multistep process, which begins in the skin and is completed in the kidneys. Ultraviolet light photocatalyzes conversion of the precursor, 7-dehydrocholesterol, to vitamin D
3 or cholecalciferol, which has no biological activity until its conversion to the active form, 1,25-(OH
2)D
3 [
1]. The activated vitamin D metabolite has many roles in regulating homeostasis (e.g., calcium homeostasis and maintenance) throughout the body. 1,25-(OH)
2D
3 has effects on the classic target organs (e.g., bones, intestines, and kidneys) and stimulates calcium transport from these organs to the blood. A growing body of evidence has demonstrated that 1,25-(OH)
2D
3 plays an important role in non-classical actions such as regulating immune function [
2]. It is known that 1,25-(OH)
2D
3, as a potent neuromodulator of the immune system, exerts marked effects on neural cells [
3]. 1,25-(OH)
2D
3 was shown to regulate neurotrophic factors in the brain, including nerve growth factors (NGFs) [
4], neurotrophin 3 (NT3) [
5], and glial cell line-derived neurotrophic factor (GDNF) [
6]. Additionally, 1,25-(OH)
2D
3 increases expressions of microtubule-associated protein-2, growth-associated protein-43 [
7], and neurite outgrowth [
8] in cultured neurons, indicating that 1,25-(OH)
2D
3 may also affect neuronal plasticity processes.
Clinical studies suggested that a vitamin D insufficiency is associated with an increased risk of brain insults such as Alzheimer’s disease (AD) [
9], Parkinson’s disease [
10], and ischemic brain injury [
6]. In animal studies, a vitamin D deficiency exacerbated stroke brain injury and dysregulated ischemia-induced inflammation [
11], whereas administration of 1,25-(OH)
2D
3 reduced ischemia-induced brain damage through upregulating GDNF expression [
6]. Pretreatment with 1,25-(OH)
2D
3 attenuated hypokinesia and dopaminergic neurotoxicity induced by 6-OHDA in rats [
12]. Moreover, 1,25-(OH)
2D
3 increased secretion of anti-inflammatory cytokines and reduced secretion of proinflammatory cytokines [
4,
5,
13], suggesting that 1,25-(OH)
2D
3 may be neuroprotective and may regulate neuroinflammation in the brain. However, the underlying mechanisms of vitamin D’s effect on neuroinflammation remain unclear.
Neuroinflammation is a common mechanism and plays a crucial role in the pathogenesis of various nerve diseases. Initiation of a neuroinflammatory response involves a complex interplay of glia. Activated glial cells, mainly astrocytes and microglia, are thus histopathological hallmarks of neurologic diseases. Inflammatory mediators (e.g., nitric oxide (NO), reactive oxygen species (ROS), proinflammatory cytokines, and chemokines) released by activated glia are neurotoxic and can cause neuronal damage [
14]. It is known that lipopolysaccharide (LPS), a gram-negative bacterial cell wall endotoxin, can activate glia through Toll-like receptors, triggering downstream signaling, such as mitogen-activated protein kinases (MAPKs). Three major MAPK subfamilies have been described: p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Activation of MAPK pathways by LPS initiates neuroinflammatory cascades characterized by activation of glia and increasing production of inflammatory mediators including ROS, NO, cytokines, and chemokines [
15‐
17]. Therefore, controlling activated glia can be a therapeutic strategy for neuroinflammation.
Studying the protective roles of antioxidant compounds in inhibiting the inflammatory response in brain diseases is an important vista for further research and clinical applications. Using cortical neuron-glia cultures, we investigated how 1,25-(OH)2D3 affected LPS-induced neuroinflammatory responses, by exploring whether the effects of 1,25-(OH)2D3 are mediated through MAPK pathways.
Materials and methods
Chemical reagents and antibodies
1,25-(OH)2D3 (SI-D1530) and LPS (L3129) were purchased from Sigma-Aldrich (St. Louis, MO). The p38 MAPK inhibitor, SB203580, ERK inhibitor, PD98059, JNK inhibitor, SP600125, iNOS, and β-actin were purchased from Calbiochem (San Diego, CA). Antibodies against ERK, p38, JNK, phosphorylated (p)-p38, p-ERK (p-p42/p44), and p-JNK (p-p46/p54) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against microtubule-associated protein-2 (MAP-2) and glial fibrillary acid protein (GFAP) were purchased from Chemicon (Temecula, CA). Antibody against ED1 was purchased from Serotec (Bicester, UK). Antibodies against oligodendrocyte marker 4 (O4), fibronectin 1 (FN1), and rat endothelial cell antigen (RECA-1) were purchased from R&D systems (Minneapolis, MN), Bioworld Technology (MN, USA), and Abcam (Cambridge, MA), respectively.
Primary rat cortical neuron-glia cultures
Primary neuron-glia cultures were prepared from the cerebral cortex of 1-day-old neonatal Sprague–Dawley rats, as previously described [
18‐
31]. All animal procedures were approved by the Institutional Animal Care and Use Committee of Taipei Medical University (Taipei, Taiwan) (permit no.: LAC-101-0249). These procedures were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. After the rats were sacrificed, their brains were quickly removed aseptically, and the blood vessels and meninges were discarded. Cerebral cortices were dissected under sterile conditions and kept on ice in Hank’s solution (without Ca
2+ or Mg
2+). Subsequently, cortical cells were dissociated by trituration using a pipette. Cells were centrifuged (1500 rpm for 5 min) and resuspended in 10 % fetal bovine serum/Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY). To each well of 24-well culture plates was seeded 5 × 10
5 cells in 0.5 ml of culture medium. Cells were incubated at 37 °C and 5 % CO
2 at a humidity of 95 % and used for experiments starting on day 14 of cultivation in vitro. The percentage cell composition was determined by immunostaining, followed by cell counting. The neuron-glia cultures consisted of approximately 35 % neurons, 54 % astrocytes, and 6 % microglia. In addition, cultures also consisted of approximately 4 % of fibroblasts and a small percentage (<1 %) of other cells including oligodendrocytes, and endothelial cells (pictures not shown).
Immunocytochemistry
Cultures were fixed in 4 % paraformaldehyde, as previously described [
18]. Background staining was reduced by blocking nonspecific binding sites with 10 % goat serum for 1 h at room temperature. After washing with phosphate-buffered saline (PBS), endogenous peroxidase activity was quenched by incubation with a 3 % H
2O
2 solution in PBS. Cultures were washed again and then incubated overnight with the appropriate primary antibodies (rabbit anti-MAP-2, 1:500, Chemicon; rabbit anti-GFAP, 1:1000, Chemicon; mouse anti-ED1, 1:500, Serotec; mouse anti-O4, 1:300, R&D system; rabbit-anti fibronectin 1, 1:500, Bioworld Technology; mouse-anti RECA-1, 1:300, Abcam) at 4 °C. Cells were washed and visualized using the avidin-biotin peroxidase complex method (ABC Elite kit; Vector Laboratories, Burlingame, CA). Images were viewed on an inverted Olympus IX 70 microscope (Tokyo City, Japan) equipped with a cooled CCD camera and SPOT advanced software (Diagnostic Instruments, Sterling Heights, MI).
Cell-type identification and counting
Types of cells present in the culture were identified by immunocytochemical staining using cell-specific markers (MAP-2 for neurons, GFAP for astrocytes, ED1 for microglia, FN1 for fibroblasts, O4 for oligodendrocytes, and RACE-1 for endothelial cells). Images of immunostained-positive cells in each well (in five randomly selected fields with a calibrated area) were captured as digital micrographs, and cells were counted by an observer who was blinded to our study and confirmed by the experiment operator.
Measurement of ROS
ROS production was detected using the fluorochrome, 2′,7′-dichlorofluorescin diacetate (DCF-DA). Treated cells were incubated with 30 μM DCF-DA in cultured medium for 20 min at 37 °C. After incubation, the culture medium was removed, and then 500 μl of culture medium was added to each culture well. Plates were read with a fluorometric microplate reader at 485/500 nm [
23]. Production of ROS was calculated as the maximum DCF fluorescence following incubation (20 min). The value of DCF fluorescence is expressed in arbitrary fluorescence units (AU).
Determination of NO accumulation
Nitrite accumulation was measured using the Griess reaction in culture media. After treatment, culture medium was collected for measurement of nitrite. Briefly, 50 μl of culture medium was mixed with an equal volume of Griess reagent. The absorbance was detected at 540 nm by a microplate reader (Molecular Devices, Menlo Park, CA).
Determination of IL-6 and macrophage inflammatory protein (MIP)-2 release
Levels of IL-6 and MIP-2 secreted into the culture media were measured by enzyme-linked immunosorbent assay (ELISA) kits (BioSource International) according to the manufacturer’s protocol.
Western blotting
Whole cell lysates were prepared from cultured neuronal/glial cells after 24 h of desired treatments. Treated cells were harvested, washed with PBS, and lysed in protein extraction buffer (Mammalian Cell-PE LBTM, Geno Technology) containing protease and phosphatase inhibitors (Complete Mini, Roche Diagnostics, Indianapolis, IN). Cell debris was removed by centrifugation at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected for storage at −80 °C or to perform a Western blot analysis. Equal amounts of protein were separated on 10~15 % sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electroblotted onto polyvinylidene difluoride membranes for 120 min under 100 V (PerkinElmer Life Sciences). Membranes were blocked with 5 % non-fat milk for 1 h, and then incubated overnight at 4 °C with the indicated antibodies, including those for inducible NO synthase (iNOS), p38, phosphorylated (p)-p38, p42/44, p-p42/44, p46/54, and p-p46/54 (1:1000 dilution) followed by an appropriate secondary antibody (at a 1:2 × 104 dilution) for 1 h at room temperature. Signals were visualized using enhanced chemiluminescent detection reagents (PerkinElmer Life Sciences). The membrane was then stripped and reprobed with an antibody specific for β-actin (at a 1:104 dilution) to ensure the accuracy of each loading. The protein signal intensity was quantified by a BioImaging System (Level Biotechnology) and normalized with the corresponding β-actin intensity.
Real-time reverse-transcription polymerase chain reaction (RT-PCR) assay
Total RNA was extracted from treated cells using the TRIzol® reagent (Invitrogen Life Technologies, Paisley, Scotland) according to the manufacturer’s protocol. Three micrograms of total RNA was reverse-transcribed into complementary (c)DNA using the Rever Tra Ace-α First-strand cDNA Synthesis Kit (TOYOBO Life Science, Japan). The resulting cDNA was incubated with the Rotor-Gene SYBR Green kit (QIAGEN Biosystems, CA) and primers for iNOS (sense, 5′-TTCTTTGCTTCTGTGCTAATGC-3′ and antisense, 5′-ATACTGTTCCATGCAGACAACC-3′); IL-6 (sense, 5′-TTCTTGGGACTGATGTTGTTGAC-3′ and antisense, 5′-AATTAAGCCTCCGACTTGTGAAG-3′); MIP-2 (sense, 5′-AAACTGCACCCAGGAAGCC-3′ and antisense, 5′-ACAGTGAGCTGGCCAATGC-3′); and β-actin (sense, 5′-GACCCAGATCATGTTTGAGACCTTC-3′ and antisense, 5′-GGTGACCGTAACACTACCTGAG-3′). For the semiquantitative analysis, we performed 40 amplification cycles (of denaturation at 95 °C for 5 s and annealing at 60 °C for 10 s) on a Rotor-Gene Q PCR Detection System (QIAGEN Biosystems). A melting curve analysis and sequencing data were used to confirm the specificity of the PCR products. Levels of iNOS, IL-6, and MIP-2 messenger (m)RNAs were normalized to those of β-actin and were then expressed as values relative to the control using the comparative threshold cycle (Ct) method.
3-(4,5-Dimethylthianol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) reduction assay
To examine cell viability, a colorimetric MTT reduction assay was performed as previously described [
24]. After a 2-h treatment, the MTT reagent (0.5 mg/ml MTT in PBS) was added to each culture well, and cultured cells were incubated at 37 °C for 40 min. The culture medium was then aspirated from each well, and cells were dissolved by adding DMSO. The absorbance was measured at a test wavelength of 570 nm and a reference wavelength of 630 nm using a microplate reader (Molecular Devices).
Measurement of lactate dehydrogenase (LDH) release
Cytotoxicity was detected in culture media using an assay of LDH release, as an index of cell injury [
18,
25]. LDH activity (units/min) was calculated from the slope of the decrease in the optical density at 340 nm over a 3-min period. The level of LDH was expressed as a percentage of values relative to PBS-treated control (Cont.) cultures.
Data analysis
All values are presented as the mean ± standard error of the mean (SEM). Experimental data were assessed using a one-way analysis of variance (ANOVA) followed by the Newman-Keuls’ test using the SigmaStat program (Jandel Scientific, San Rafael, CA). Significance was set at p < 0.05.
Discussion
Glial cells, namely astrocytes and microglia, are the most abundant cells in the brain and play pivotal roles in protecting neurons from harmful insults. Activation of glia following infection or neuronal injury causes neuroinflammation; however, excessive glial activation may exacerbate neuronal damage due to the production of inflammatory molecules. In this study, we used LPS as a stimulant in neuron-glia cultures to initiate a neuroinflammatory response. The excessive production of inflammatory molecules was used as an index of activated glia. In this model, LPS significantly and time-dependently induced the production of inflammatory molecules including NO (reflected by nitrite accumulation), ROS, IL-6, and MIP-2 in cortical neuron-glia cultures. Activation of the p38, ERK, and JNK MAPK pathways is involved in LPS-initiated production of inflammatory molecules. 1,25(OH)2D3 attenuated LPS-induced nitrite accumulation, iNOS expression, and ROS production in concentration-dependent manners. LPS-induced production of IL-6 and MIP-2 was also significantly reduced by 1,25(OH)2D3. Furthermore, LPS-elicited phosphorylation of the p38, ERK, and JNK MAPK pathways was decreased by 1,25(OH)2D3. These results suggest that 1,25(OH)2D3 attenuates LPS-initiated neuroinflammation through inhibiting MAPK activation in neuron-glia cultures.
We previously demonstrated that astrocytes and microglia are important early sources of proinflammatory mediators in cerebrospinal fluid and brain tissues during bacterial (
Klebsiella pneumoniae) infection of the central nervous system (CNS) [
19]. Brain endothelial cells and pericytes which surround endothelial cells not only contribute to maintenance of the barrier function of the blood–brain barrier but also are immunoactive and can respond to LPS [
20‐
22]. It is possible that LPS may stimulate brain immune cells (e.g., astrocyte and microglia) and other brain cells (e.g., pericytes, endothelial cells, and fibroblasts) resulting in a complex cascade of neuroinflammatory responses [
20,
21,
26]. We employed cortical neuron-glia cultures as an
in vitro model for mimicking the
in vivo environment in which neurons and glia can interact. The advantages of our
in vitro model were that (1) cultured cells can bypass any possible physiological feedback interactions
in vivo to allow direct observation of LPS-induced neuroinflammation in specific cell populations and (2) it provides a simpler system to elucidate cellular mechanisms. However, its limitation is that cell cultures are simply an attempt to provide a simulated microenvironment, and cells are not in their normal physiological or original environment. The cortical neuron-glia cultures in this study consisted of approximately 35 % neurons, 54 % astrocytes, 6 % microglia, 4 % fibroblasts, and a small percentage (<1 %) of other cells including oligodendrocytes, and endothelial cells. The low percentage of other cell types may have been due to the fact that we dissected cerebral cortices and carefully removed blood vessel and meninges during our procedures to prepare the cortical neuron-glia cultures. It is possible that the results of this study can be ascribed to complex interactions between the different cell types found in the cultures. However, considering the relatively poor responses of the small percentage of fibroblasts and other cell types (oligodendrocytes and endothelial cells) to LPS, we propose that glial cells (mainly astrocytes and microglia) were the major responder cells in our culture system.
Glial cells play a role in immune surveillance under normal conditions; however, after a brain injury or exposure to inflammation, glial cells are activated. Under neuroinflammation, glial cells become strongly over-activated through a process that involves phosphorylation of downstream MAPK pathways (e.g., p38, ERK, and JNK) and production of inflammatory molecules [
17]. Consistent with our results that treatment of cultured neurons/glia with LPS significantly induced expression of iNOS and the production of NO, ROS, IL-6, and MIP-2, pharmacological inhibition of MAPK pathways by MAPK inhibitors (SB 203580 for p38, PD98059 for ERK, and SP600125 for JNK MAPK) partially attenuated LPS-induced production of inflammatory molecules (ROS, NO, iNOS, IL-6, and MIP-2). A Western blot analysis also confirmed that treatment of cultured neurons-glia with LPS significantly induced phosphorylation of the p38, ERK, and JNK MAPK pathways, indicating that MAPK activation is involved in LPS-induced neuroinflammation. However, sustained overproduction of these inflammatory molecules may further contribute to neuronal damage. Therefore, inhibition of neuroinflammation has become a promising therapeutic target for neuronal injuries.
A dietary vitamin D
3 (cholecalciferol) deficiency may lead to oxidative stress and elevation of iNOS expression in the brain and further promote cognitive decline in middle-aged and elderly adults [
27]. It was reported that inhibition of iNOS expression can cause improvements in clinical signs observed in rats with experimentally produced allergic encephalomyelitis (EAE) treated with 1,25(OH)
2D
3 [
28]. 1,25(OH)
2D
3 also effectively attenuated inflammatory cytokine expressions in the spinal cord and ameliorated EAE in rats [
29]. In that model, 1,25(OH)
2D
3 treatment significantly reduced Toll-like receptor 8 (TLR8) expression and TLR8 target gene expressions (TNF-α and IL-1β) [
29]. Additionally, 1,25(OH)
2D
3 provides neuroprotection against 1-methyl-4-1,2,3,6-tetrahydropyridine (MPTP)-induced neuronal injury through inhibition of glial activation and proinflammatory cytokine expression [
30]. Those findings suggest that 1,25(OH)
2D
3 has important roles in the brain. In this study, we found that LPS-induced ROS production, nitrite accumulation, iNOS expression, and IL-6 and MIP-2 production were significantly reduced after treatment with 1,25(OH)
2D
3. It is known that 1,25(OH)
2D
3 is one of the important nuclear steroid transcription regulators that can control transcriptions of a large number of genes. 1,25(OH)
2D
3 is known to exert its biological functions by directly influencing cellular processes and also by influencing gene expressions through vitamin D response elements (VDREs). We also found that levels of iNOS, IL-6, and MIP-2 mRNA expressions were also significantly attenuated by 1,25(OH)
2D
3, indicating that 1,25(OH)
2D
3 plays an important role in gene regulation. However, further studies are required to elucidate the role of 1,25(OH)
2D
3 in LPS-initiated gene expression.
The apparent lack of cell damage induced by LPS in our cortical neuron-glia culture may have been due to regional differences in the susceptibility to the concentration (100 ng/ml or 0.1 μg/ml) of LPS in a shorter time (24 h) period. Differential neurotoxicity in mixed neuron-glia cultures from various brain regions after treatment with LPS was demonstrated in a previous study [
31] in which treatment with LPS at an even higher concentration (1 μg/ml) for a longer time (72 h) did not cause neurotoxicity in cortical or hippocampal neuron-glia culture but caused neurotoxicity in cultures derived from the mesencephalon. In another study using mice cortical neuron-glia culture, neurotoxicity was only observed with a longer exposure time (36 h) when cultures were treated with LPS (1 μg/ml) in combination with interferon (IFN)-γ (5 U/ml) [
32]. The concentration of 1,25(OH)
2D
3 we used in this study was similar to that in previous reports [
33,
34]. In order to exclude the possibility that 1,25(OH)
2D
3 attenuated the LPS-induced release of inflammatory molecules because of 1,25(OH)
2D
3 toxicity, our results confirmed that 100 nM 1,25(OH)
2D
3 was not toxic to cultured neuron-glia cells as revealed by the MTT and LDH assays as well as immunocytochemistry. Because the concentration of 1,25(OH)
2D
3 used in our cultures effectively reduced LPS-induced neuroinflammation, we suggest that 1,25(OH)
2D
3 supplementation may be a candidate for prevention and treatment of neuroinflammation.
1,25(OH)
2D
3 is a neuro-immunomodulator involved in various neurodegenerative and autoimmune diseases [
3]. The neuroprotective effects of 1,25(OH)
2D
3 have been reported in cultured hippocampal neurons against excitotoxic insults through reduced L-type calcium channel expression [
35] and against rotenone-induced neurotoxicity in human neuroblastoma cell line SH-SY5Y cells by enhancing autophagy signaling [
36]. Besides the direct neuroprotective effects on neurons, 1,25(OH)
2D
3 also reduced neurotoxin-induced dopaminergic neuronal loss by inhibiting of microglia activation and proinflammatory cytokine expression [
30]. It is known that 1,25(OH)
2D
3 acts via the vitamin D receptor (VDR), a member of the steroid/thyroid hormone superfamily of transcription factors, and the membrane-associated, rapid-response steroid-binding receptor (MARRS), also known as Erp57/Grp58 [
3]. The VDR is expressed by glia [
4] and neurons [
37] in the brain, and not just those participating in the classic actions of vitamin D. The action of 1,25(OH)
2D
3 mediates its biological effects by binding to the VDR, which then recruits cofactors to form a transcriptional complex that binds to VDREs in the promoter region of target genes to alter transcriptional cascades within cells [
2]. The non-classical action of 1,25(OH)
2D
3 is that it binds to the MARRS receptor, located on the cell surface, initiating non-genomic effects [
3]. A single-nucleotide polymorphism in the VDR gene can influence the affinity of vitamin D to its receptor and thus may be related to neurodegenerative diseases and neuronal damage by altering vitamin D-mediated pathways [
38,
39]. β-amyloid (Aβ) disrupts the vitamin D-VDR pathway and results in iNOS expression in cortical neurons, whereas this effect can be prevented by vitamin D [
40]. Moreover, treatment with vitamin D in that model protected neurons by preventing cytotoxicity and apoptosis and also by upregulating the VDR [
41]. Those results suggest a potential role for vitamin D-VDR-mediated mechanisms in AD. In this study, we were interested in investigating how 1,25(OH)
2D
3 affected LPS-induced neuroinflammatory responses and exploring whether the effects of 1,25(OH)
2D
3 were mediated through MAPK signaling pathways. Involvement of MAPK signaling pathways in the production of inflammatory molecules in neurons and glial cells was previously demonstrated [
42‐
44]. The three major MAPK subfamilies of p38, ERK, and JNK are phosphorylated in neurons and glia following LPS treatment [
45]. MAPKs are related to LPS signaling in glial cells and lead to iNOS expression and the production of NO and proinflammatory cytokines [
45‐
47]. The current results indicate that the phosphorylation of p38, ERK, and JNK MAPKs occurred after LPS treatment. We demonstrated that 1,25(OH)
2D
3 decreased the phosphorylation of p38, ERK, and JNK MAPKs as a result of LPS. This is consistent with a previous report that 1,25(OH)
2D
3 reduced macrophage-induced release of chemokines and cytokines by adipocytes and the chemotaxis of monocytes through inhibiting MAPK signaling pathways [
48]. Similarly, 1,25(OH)
2D
3 diminished LPS-stimulated tumor necrosis factor (TNF)-α and IL-6 release via inhibiting p38 phosphorylation in monocytes/macrophages [
49]. Our results suggest that the 1,25(OH)
2D
3-mediated suppression of the production of neuroinflammatory molecules may occur through inhibition of MAPK pathways. However, further studies are required to better understand the extents of1,25(OH)
2D
3’s anti-inflammatory effects and its involvement in the progression of neuroinflammation.
Authors’ contributions
YNH and JYW conceived and designed the experiments and wrote the paper. YNH and YJH performed the experiments. YNH, CCL, and JYW analyzed the data. YNH, CTC, and JYW contributed reagents/materials/analysis tools. All authors read and approved the final version of the manuscript.