Paricalcitol improves experimental autoimmune encephalomyelitis (EAE) by suppressing inflammation via NF-κB signaling

Biomedicine & Pharmacotherapy 125 (2020) 109528

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Paricalcitol improves experimental autoimmune encephalomyelitis (EAE) by suppressing inflammation via NF-κB signaling

T

Dangfeng Zhanga,1, Lin Qiaob,1, Ting Fuc,* a

Department of Orthopedics, The First Affiliated Hospital, Xi’an Jiaotong University, Xi'an City, Shaanxi Province 710061, China Department of Orthopaedic Surgery, The Third Hospital of Chinese PLA, Baoji City, Shaanxi Province 721004, China c Neural and Spine Rehabilitation Department of TCM Orthopedic Center, Honghui Hospital, Xi'an Jiaotong University, Xi'an City, Shaanxi Province 710054, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Multiple sclerosis (MS) Paricalcitol (Pari) EAE Inflammation NF-κB

Multiple sclerosis (MS) is known as an autoimmune disease in the central nervous system (CNS) characterized by motor deficits, pain, fatigue, cognitive impairment, and sensory and visual dysfunction. MS is considered to be resulted from significant inflammatory response. Paricalcitol (Pari) is a vitamin D2 analogue, which has been indicated to show anti-inflammatory activities in kidney and heart diseases. In the present study, if Pari could ameliorate the experimental autoimmune encephalomyelitis (EAE) was investigated. Here, the C57BL/6 mice were immunized using myelin oligodendrocyte glycoprotein 35–55 (MOG35–55). Subsequently, Pari was intraperitoneally injected into the mice. As for in vitro analysis, RAW264.7 and Jurkat cells were incubated with Pari together with corresponding stimulus. The results indicated that Pari administration reduced the paralytic severity, neuropathology and apoptosis in MOG-treated mice compared to the MOG single group. Pari also exhibited a significantly inhibitory effect on immune cell infiltration, glial cell activation, expression of proinflammatory factors and the activation of nuclear factor κB (NF-κB). The expression of pro-inflammatory regulators and the translocation of NF-κB from cytoplasm into nuclear in RAW264.7 and Jurkat cells under specific stimulation was clearly down-regulated by Pari incubation. Furthermore, we found that suppressing NF-κB with its inhibitor combined with Pari could further reduce the expression of pro-inflammatory factors and associated proteins. These data illustrated that Pari could diminish MOG-triggered EAE, as well as macrophages and T cells activation through blocking NF-κB activation. Collectively, Pari might have therapeutic effects in mouse models with MS.

1. Introduction Multiple sclerosis (MS) is an autoimmune disease, which is characterized by inflammation, demyelination, as well as axonal degeneration in the central nervous system (CNS) [1,2]. The clinical course of MS is significantly heterogeneous, which, however, could be broadly classified into relapsing and progressive forms [3]. Increasing studies have indicated that MS is tightly associated with an intricate interplay between inflammatory effector cells [4]. The inflammatory response is largely due to oligodendrocytes, which are the myelinating cells in CNS and neurons [5]. NF-κB is a family of transcription factors controlling the expression of extensive number of genes associated with cell survival, proliferation, differentiation, death and inflammation [6]. NF-κB has various actions in the nervous system. In contrast to many other tissues, NF-κB is constitutively active at high basal levels in neurons,

indicating its significance in regulating specific physiological functions in the CNS [7,8]. Accordingly, activation of NF-κB is correlated with immune-associated genes, which are up-regulated in the cerebrospinal fluid, serum, brain or spinal cord of patients with MS when compared with the normal individuals [9,10]. Additionally, the NF-κB DNAbinding activation is down-regulated in MS patients treated with interferon-β (IFN-β) [11]. However, presently, only a few therapeutic strategies are available to MS patients [12]. Paricalcitol (Pari) is a vitamin D2 analogue with anti-cancer and anti-inflammatory activities [13]. For example, Pari could inhibit the proliferation of prostate and colorectal cancer cells [14]. Pari reduces peritoneal fibrosis in mice through the modulation of activation of regulatory T cells and reduction in IL-17 production [15]. In addition, Pari shows anti-inflammatory effects on kidney and cardiovascular disease by regulating NF-κB signaling pathway [16,17]. In addition,

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Corresponding author at: Honghui Hospital, Xi'an Jiaotong University, No.76, Nanguo Road, Xi'an City, Shaanxi Province 710054, China. E-mail address: [email protected] (T. Fu). 1 The first two authors contributed equally to this study. https://doi.org/10.1016/j.biopha.2019.109528 Received 18 July 2019; Received in revised form 1 October 2019; Accepted 1 October 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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supplemented with 10 % fetal bovine serum (FBS, Gibco), 25 mM HEPES, 1 mM sodium pyruvate, 1× nonessential amino acids, 55 μM 2mercaptoethanol, 100 units/mL penicillin and 100 μg/mL streptomycin. The obtained cells were then used for further analysis. HT22 cells were derived from HT-4 cells that were immortalized from primary mouse hippocampal neuronal cells. RAW 264.7 cells and Jurkat cells were purchased from Bioleaf (Shanghai, China). All cells were incubated in DMEM (Gibco) and RPMI-1640 (Gibco), respectively, with 10 % fetal bovine serum (FBS) (Gibco) and 100 units/mL penicillin, and incubated in a humidified incubator at 37 °C with 5 % CO2. LPS and CD3/CD28 T cell activator were obtained from Sigma Aldrich (USA) and STEMCELL Technologies (Canada), respectively, to stimulate the cells.

recently, as reported, low vitamin D concentration in serum was a cause of MS, and which was independent of established risk factors [18]. Moreover, low concentrations of neonatal vitamin D are associated with an increased risk of MS [19]. Therefore, we supposed that Pri, as a vitamin D analog, might be effective for the treatment or improvement of MS. Nevertheless, no study has been performed to investigate the potential effect of Pari on EAE. Therefore, we supposed that Pari might possess the promising activity to ameliorate inflammation during EAE development. In the present study, we attempted to explore if Pari could suppress neuro-inflammation and progression of MOG35–55-elicited EAE by the modulation of NF-κB signaling via the in vitro and in vivo experiments. 2. Materials and methods

2.3. Clinical calculation 2.1. Animals and treatments Clinical calculation was analyzed according to the clinical signs of neurological deficits showing as followings: grade 0, no abnormality; grade 1, complete tail paralysis; grade 2, complete tail paralysis and hind limb weakness; grade 3, complete hind limb paralysis; grade 4, tetraplegia; grade 5, moribund state or death [22].

8-week old C57BL/6 male mice were purchased from Model Animal Research Center of Xi'an Jiaotong University. They were housed at room temperature (23 ± 1 °C) with a 12-h light-dark cycle and fed food and water ad libitum. All mice were randomly divided into 4 groups: 1) the Control (Ctrl); 2) MOG group; 3) MOG + Pari (0.25 μg/kg); and 4) MOG + Pari (0.5 μg/kg). Mice were immunized with MOG35–55 peptide emulsified with complete Freund’s adjuvant (CFA) with Hooke kits (Hooke Laboratories, USA) following the manufacturer’s protocols. Briefly, 0.1 mL MOG35–55/CFA emulsion was subcutaneously injected into the upper and lower back of mouse (0.2 mL/animal), followed by i.p. injection of 0.1 mL pertussis toxin (PTX, R&D Systems, USA). After 24 h, booster shots of PTX (0.1 mL/animal) were i.p. injected. Paritreated mice were given 0.25 or 0.5 μg/kg on every second day from day 2 to 28 via i.p. injection. Clinical behaviors were monitored. NonPari-treated mice were given saline 50 μL on every second day by i.p. injection (Fig. 1A). The control group of mice (Ctrl) was only treated with 50 μL of saline on every second day by i.p. injection without any other intervention. Paricalcitol (purity 99.95 %) was purchased from MedChemExpress (MCE, USA). MOG (35–55) (mouse) was obtained from Hanhong Chemistry (Shanghai, China). Body weight of mice was recorded each day. After behavioral tests, mice were perfused with phosphate-buffered saline (PBS, pH 7.4) with heparin under inhaled CO2 anesthetization. The mice spinal cords were immediately pulled from the skull. One stored at −80 °C for western blotting and RT-qPCR analysis, and the other were fixed in 4 % paraformaldehyde for seven days at 4 °C and transferred to decalcification solution (14 % EDTA, 2.5 % ammonium hydroxide, pH 7.2) for seven days at RT and transferred to 30 % sucrose solutions at 4 °C. All experiments adhered to the guidelines for the care and use of laboratory animals of Xi'an Jiaotong University.

2.4. Western blotting analysis Protein samples were collected from tissues or cells using Whole Cell Lysis Assay or Nuclear and Cytoplasmic Protein Extraction Kit (KeyGEN BioTECH, China), followed by centrifugation (13,000 g) for 15 min at 4 °C. The obtained supernatants were then subjected to the calculation of protein concentration using a BCA Protein Quantification Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Equal amounts (40 μg) of protein samples were then separated using 10–12 % SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were then blocked for 1 h with TBST containing 0.1 % Tween-20 % and 5 % dry milk at room temperature, followed by incubation with primary antibodies at 4 °C overnight, including: anti-iNOS (1:500, Santa Cruz, USA), anti-COX2 (1:1000, Abcam, USA), anti-p-NF-κB (1:1000, Abcam), anti-NF-κB (1:1000, Abcam), anti-F4/80 (1:500, Santa Cruz), anti-Iba-1 (1:1000, Abcam), anti-F4/80 (1:1000, Abcam), anti-GFAP (1:500, Santa Cruz), anti-p-IκBα (1:1000, Abcam), anti-IκBα (1:1000, Abcam), anti-Lamin B (1:1000, Abcam), anti-APP (1:1000, Abcam), anti-Olig1 (1:1000, Abcam), anti- Olig2 (1:1000, Abcam), anti-Cleaved Caspase-3 (1:1000, Abcam), anti-Cleaved PARP (1:1000, Invitrogen) and anti-GAPDH (1:1000, Abcam). Then, after washing with TBST, all membranes were subjected to incubation with secondary antibodies at room temperature for 1.5 h. Proteins were finally visualized by the enhanced chemiluminescence kit (ECL, Millipore Corporation, USA) and quantified using Image Pro Plus software. The relative expression of target protein was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal standard.

2.2. Cells and incubation Primary adult microglia were prepared from lumbar spinal cords of EAE mice through the modification of a non-enzymatic procedure [20,21]. In brief, mice were lethally anesthetized and perfused intracardially with 50 mL of ice-cold GKN buffer. Spinal cord tissues in each group of mice were mechanically dissociated by passing using a 70-μm cell strainer with a 10 mL syringe plunger, and then strained with a 40 μm filter. Single cell suspensions were then prepared via the centrifugation over a 35 %/70 % discontinuous Percoll gradient (GE Healthcare, USA). The cells were then isolated from the interface, and total cell counts determined. Flow cytometry was used to calculate the cell purification. Cells were pre-blocked using anti-CD16/CD32 (Fc Block, Serotec), and then stained on ice for 30 min with anti-CD11bPerCP-Cy5.5 (BD Biosciences, USA). The data collection was conducted on a LSRII flow cytometer. The flow cytometry analysis was used to determine the microglial cell population to be 95.3 ± 2.0 % pure. Microglia were washed and resuspended in RPMI medium (Gibco, USA)

2.5. Quantitative real-time-PCR Total RNA was isolated with RNA-Solv Reagent (OMEGA, USA). Reverse transcription was performed using Rever Tra Ace (TOYOBO, Japan) and Oligo(dT)18 (TaKaRa, Japan). RT-qPCR was performed with SYBR Premix Ex Taq (TaKaRa) using ABI PRISM 7900 H T detection systems (Applied Biosystems, USA). Reaction procedures were as following: an initial step at 95 °C for 5 min, 40 cycles of 94 °C for 15 s, 60 °C for 34 s. The primers were as the following: mTNF-α Fwd 5′AAG ACG CTC ACA GAA AGG ACA T-3′, Rev 5′-GTG ACG TGA GGT AGG TCC TTC T-3′; mIL-1β Fwd 5′-TGT GGC GAG TGG GAA TAA CTT GA-3′, Rev 5′-CTG GAC GGC GTT ACT TCC TCC G-3′; mIFN-γ Fwd 5′AAA AGC GAA ACT GGA GGC C-3′, Rev 5′-TAG CAT CAC TTG GCG ATG AG-3′; mIL-6 Fwd 5′-GAA GGA ATC GGC CAT GAG ACT-3′, Rev 5′CAC TGA GAA ACG TGC TTG GA-3′; mGAPDH Fwd 5′-TGA CGG TGA 2

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Fig. 1. Effects of Pari on MOG-induced EAE clinical characteristics and histological changes in spinal cord of mice. (A) Description of experimental process design. (B) EAE clinical score (n = 12 samples from 12 mice per group). (C) The change of body weight (n = 12 samples from 12 mice per group). (D) Up, H&E staining of the cervical region of spinal cord (Scale bar was 100 μm.); medium, H&E staining of dorsal horn of the gray matter (Scale bar was 50 μm.); down, luxol fast blue staining (Scale bar was 100 μm.); bottom, IF staining of NeuN in dorsal horn (Scale bar was 50 μm.) (n = 5 samples from 5 mice per group). (E) Histopathologic score from each group. (F) Quantification of NeuN positive cells following IF staining. Western blot analysis of (G) APP, (H) Olig1 and Olig2 in spinal cords of mice. (I) Draining lymph node cells were isolated at the end of study, and were re-stimulated with different doses of MOG35–55 peptide and the cell proliferation was determined by [3H] incorporation (n = 6 samples from 6 mice per group). (J) DTH response in mice was calculated (n = 6 samples from 6 mice per group). (K) Quantitative analysis of infiltrated cells by staining with appropriate fluorescence-conjugated antibodies, followed by flow cytometry (n = 6 samples from 6 mice per group). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 and ***p < 0.001 versus the Ctrl group; +P < 0.05 and ++P < 0.01 versus the MOG group; #P < 0.05; by one-way ANOVA with Bonferroni post hoc analysis or two-tailed Student’s t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

AAC GAG GGA AGG ACC-3′, Rev 5′-ATC ATT GGA GCA TCG GCA TG3′; hGAPDH Fwd 5′-CTA GTG ACA ACG GCC TAG TGA C-3′, Rev 5′-GTG CGA CAT ACA CCC ATG TC-3′.

CCT ACA ACT GC-3′, Rev 5′-CGA CCC ATG CAT GTG ACC T-3′; hTNF-α Fwd 5′-AGC TCA GAC ACA AGA GGA AT-3′, Rev 5′-GGT CGT GGT ACA TGA GTT CGC T-3′; hIL-1β Fwd 5′-TTA TAC TTG CGT GGG AGG AG-3′, Rev 5′-TTA CCA GTG GGC CTC CCT TGG AC-3′; hIFN-γ Fwd 5′-AAC 3

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2.6. ELISA for cytokines

Institute, Shanghai, China) following the manufacturer’s instructions.

The levels of tumor necrosis factor (TNF)-α, γ-interferon (IFN-γ) and interleukin-1β (IL-1β) in spinal cords of mice were calculated by a sandwich enzyme-linked immunosorbent assay (ELISA, R&D Systems, USA) according to the manufacturer’s protocols.

2.11. Delayed-type hypersensitivity (DTH) On day 19 after immunization, mice were injected with MOG35–55 peptide in the right footpad and saline in the left footpad as control. The footpad thickness was calculated 24 and 48 h after the injection using a micrometer (QUICKmini; Mitutoyo, Japan). Specific responses were calculated through subtracting the thickness of the left footpad (saline) from that of the right footpad (MOG).

2.7. Immunofluorescence (IF) staining Mice were deeply anesthetized and intracardially perfused using PBS followed by 4 % paraformaldehyde/PBS (pH 7.4). The dorsal horn (L4-5) samples were removed, post-fixed in the same fixative at 4 °C for 4 h and cryoprotected in 30 % sucrose solution at 4 °C overnight. Transverse lumbar spinal cord sections (30 μm) were cut by a cryostat and mounted on glass slides. Then, the sections were pre-incubated with 5 % BSA 1 h to block nonspecific binding. Next, the sections were incubated with rabbit anti-NeuN (1:150, Santa Cruz, USA), anti-GFAP (1:150, Santa Cruz), or rabbit anti-Iba-1 (1:150, Abcam, USA) at 4 °C overnight. After washing, spinal sections were incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500, Abcam) at 37 °C for 1 h. Then, the spinal sections were washed with PBS, and coverslips were used. When the fluorescent markers were excited, the sections were detected by a fluorescent microscope (ZEISS Axio vert. A1, Germany).

2.12. Cytokine quantification Draining lymph nodes, including axillary, brachial and inguinal, were collected and adjusted to 5 × 106 cells/mL [26,27]. Cells plated in supplemented RPMI medium (Gibco) supplemented with 10 % of fetal calf serum and 2 mM of glutamine were restimulated in vitro with 20 μg/mL of MOG in the absence or presence of Pari (100 nM). Cytokine levels were then evaluated 24 h later by ELISA in culture supernatants using IL-6, IFN-γ, IL-2 and IL-10 (BD Biosciences, USA), and IL-17 and TGF-β (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. 2.13. Histological analysis

2.8. Isolation of infiltrating cells from the CNS

For histopathologicalanalysis, cervical and lumbar part of spinal cord specimens were dehydrated in ascending grades of ethyl alcohol, cleared in xylol, embedded in paraffin wax and sectioned (4 μm thickness). Microscopic sections were subjected to hematoxylin and eosin (H&E) staining, and luxol fast blue to assess the degree of demyelination. As for immunohistochemical (IHC) staining, the frozen sections were incubated with 0.2 % TritonX-100 for membrane permeabilization, blocked with 2 % bovine serum albumin (BSA) for 1 h at room temperature, and then incubated with rabbit anti-CD3 (1:150, Abcam), rabbit anti-F4/80 (1:150, Abcam), rabbit anti-iNOS (1:150, Santa Cruz), mouse anti-COX2 (1:150, Abcam) and rabbit anti-p-NF-κB (1:150, Abcam) in 2 % BSA at 4 °C overnight. Then, the sections were incubated with secondary antibodies (KeyGEN BioTECH) for 1 h, mounted on a glass slide and analyzed. TUNEL staining was used for calculating the apoptosis condition in spinal cord samples (Roche, Germany) or cells (Abcam) according to the manufacturer’s instructions. Finally, the sections were analyzed under a fluorescent microscope (ZEISS Axio vert. A1, Germany).

Mice were euthanized on day 14 post-immunization and perfused with cold PBS via intracardiac route. Spinal cords were removed and pooled for mononuclear cell isolation using Neural Tissue Dissociaton Kits (MACS Miltenyi Biotec, Auburn, CA). The isolated cells were directly analyzed or re-stimulated using 50 ng/ml PMA and 500 ng/ml ionomycin (both from Sigma-Aldrich) in the presence of monensin (GolgiStop, BD Biosciences) for 4 h, and then expression of surface and intracellular markers was calculated by flow cytometry as previously described [23]. 2.9. Flow cytometry analysis Different florescence-conjugated antibodies for flow cytometry were shown as followings: anti-CD45R, anti-CD3, anti-F4/80, anti-Ly-6 G (all from eBioscience, USA). Cells were fixed and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences, USA) and stained with fluorochrome-labelled antibodies. Flow cytometric measurements were conducted on a BD Accuri C6 flow cytometer, and results were analyzed with FlowJo7.6 software (Treestar, Ashland, OR, USA). For isotype control staining, we used APC-conjugated mouse IgG2a, mouse IgG2b, and hamster IgG (all from BioLegend, USA). As for apoptosis, the cells were incubated with annexin V and PI (Beyotime) for 15 min according to the manufacturer’s and analyzed with a FACScan flow cytometer (BD Biosciences).

2.14. Statistical analysis All analysis was conducted using GraphPad PRISM (version 6.0; Graph Pad Software). Data were represented as the means ± SEM. All analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post hoc analysis. Student t-test was used to calculate the diff ;erences between two groups. Statistical significance was defined as P < 0.05.

2.10. T cell proliferation and Cell viability 3. Results Single cell suspension from draining lymph nodes (LN) was prepared as previously described [19,24,25]. Cells (2.5 × 105 cells/well) in 96-well round-bottom cell culture plates were incubated in the presence of 0, 1, 10, or 100 μg/ml MOG35–55 peptide for 72 h. Cultures were pulsed with [3H]-thymidine (1 μCi/well, Perkin Elmer Life Sciences, Boston, USA) in the last 4 h. The cells were then harvested onto glass-fiber mats with a harvester (Perkin Elmer). Cell proliferation was quantified as the amount of [3H]-thymidine incorporated into DNA, which was determined with liquid-scintillation counting in a Micro Beta 2 counter (Perkin Elmer). Data were expressed as mean counts per minute (cpm). As for the calculation of cell viability, after treatments, the cell viability was determined with an MTT assay (Beyotime

3.1. Effects of Pari on MOG-induced EAE clinical characteristics and histological changes in spinal cord of mice As shown in Fig. 1B, the clinical score of EAE mice induced by MOG was up-regulated compared to the Ctrl group, which were alleviated by Pari administration. Additionally, the body weight loss was higher in MOG-treated mice than that of the Ctrl group, while being improved by Pari supplementation (Fig. 1C). H&E staining suggested that MOG treatment led to the significant cell infiltration into the injured area of spinal cords along with reduced histopathologic score and number of neurons in comparison to the Ctrl group, which were rescued by Pari 4

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Fig. 3C and D suggested a significant elevation of CD3 and F4/80 in spinal cord samples, which was, however, markedly reduced by Pari treatments. MOG treatment could lead to inflammation in central nervous system cells associated with glial cell activation by promoting the expression of inflammatory signals, including iNOS and COX2 [37]. Here, IHC and western blot analysis in Fig. 3C and D indicated that Pari treatment significantly reduced the expression of iNOS and COX2 in spinal cord tissues of MOG-operated mice. Consistently, RAW264.7 cells and Jurkat cells stimulated by LPS or CD3/CD28 showed higher iNOS and COX2 expression levels compared with the Ctrl group, but were reduced by Pari incubation (Fig. 3E).

treatment. Additionally, the demyelination area induced by MOG was significantly improved in Pari-treated mice with MOG by luxol fast blue staining. Meanwhile, IF staining indicated that MOG-decreased the number of NeuN-positive cells was markedly restored by Pari administration (Fig. 1D-F). Then, the expression of amyloid precursor protein (APP), indicating axonal damage [28,29], was found to be reduced in spinal cord tissues of MOG mice. In contrast, the expression levels of Olig1 and Olig2, two critical transcription factors for both oligodendrocyte development and remyelination [30,31], were down-regulated in spinal cord samples of MOG-operated mice. Significantly, Pari treatments could reverse these effects (Fig. 1G and H). Thus, Pari could prevent oligodendrocyte loss and axon damage in mice after MOG treatment. T cells play a critical role in the progression of EAE through their antigen (Ag)-specific effector response. Clonal expansion of Agspecific T cells induced by antigen encounter is a prerequisite for the initiation and development of T-cell regulated immune-pathological processes [32]. We thus used ex vivo re-stimulation of LN cells with immunization antigen MOG35–55 to induce antigen-specific T cell proliferation. We found that the MOG peptide-induced T cell proliferation in a dose-dependent manner and this response of pathological T cells was markedly attenuated in Pari (0.5 mg/kg)-fed animals (Fig. 1I). Similarly, when we used MOG35–55 to induce antigen-specific DTH response as an in vivo indicator of effector function of pathological T cells, we found a smaller DTH response (footpad induration) in mice fed Pari compared to those in the control group (Fig. 1J). Thus, Pari could inhibit autoreactive myelin-specific T cell response in EAE mice. To further explore cellular composition in the CNS inflammatory infiltration, the spinal cords on day 14 post-EAE induction were collected, and the infiltrating cell populations were determined using flow cytometry method. As shown in Fig. 1K, Pari treatment reduced the frequency of infiltrating T cells (CD3), B cells (CD45R), neutrophils (Ly-6 G), and macrophages (F4/80) in the spinal cords.

3.4. Effects of Pari on MOG-induced inflammation in mice with spinal cord injury In this part, we further found that the release of inflammatory cytokines, such as TNF-α, IL-1β and IFN-γ, was highly induced by MOG operation. But these effects were significantly down-regulated by Pari treatments (Fig. 4A). Macrophages are the major cell type that contributes to autoimmune-induced inflammatory demyelination. Thus, the murine macrophage cell line RAW264.7 was used to evaluate the effects of Pari on EAE in vitro, because it acquired an activated dendritic morphology upon LPS induction [38–40]. Jurkat cells have been widely used as in vitro model for EAE to calculate the condition of T cells. To confirm the activation of T cells regulated by Pari, Jurkat cells were therefore used in our study, which was also referred to previous studies [41–43]. Similarly, LPS- and CD3/28-stimulated expression of TNF-α, IL-1β and IFN-γ in RAW264.7 and Jurkat cells, respectively, was apparently decreased by the treatment of Pari using RT-qPCR analysis (Fig. 4B and C). NF-κB signaling plays a critical role in regulating inflammatory response and controlling the glia cell activation, which was then explored [44]. As shown in Fig. 3D, NF-κB phosphorylated expression was induced by MOG operation, which was, however, decreased by Pari treatments. Western blot analysis further indicated that the expression levels of cell p-IκBα, p-NF-κB and nuclear NF-κB in spinal cords of mice were markedly up-regulated by MOG, whereas IκBα expression was down-regulated. However, these findings were evidently reversed by the pre-treatment of Pari (Fig. 4E and F). We also discovered that the expression of cellular p-IκBα, p-NF-κB and nuclear NFκB in LPS- and CD3/28-stimulated RAW264.7 and Jurkat cells, respectively, was significantly increased, but was decreased in cells coincubated with Pari (Fig. 4G). Then, treatment with MOG of isolated cells from the spinal cord of EAE mice showed significantly up-regulated mRNA expression levels of TNF-α, IL-1β, L-6 and IFN-γ, which were however reduced by the Pari incubation, accompanied with markedly decreased expression of p-IκBα and p-NF-κB (Fig. 4H and I). Then, we found that MOG markedly increased pro-inflammatory cytokine production by lymph node cell cultures re-stimulated in vitro with MOG, when compared to the EAE control group, while being alleviated by the treatment of Pari (Fig. 4J). Similarly, a regulatory immune profile was equally present in the lymph node cell cultures from treated EAE mice with MOG treatment, characterized by higher levels of TGF-β, which was also attenuated by Pari incubation (Fig. 4K). Thus, Pari also alleviated peripheral immune response.

3.2. Effects of Pari on apoptosis in MOG mice with spinal cord injury TUNEL staining indicated that MOG resulted in cell death in spinal cord samples, which were markedly alleviated by the treatment of Pari (Fig. 2A). Consistently, the protein expression levels of cleaved Caspase3 and PARP triggered by MOG were also significantly inhibited by Pari administration (Fig. 2B). Then, the in vitro studies were performed to explore the effects of Pari on cell death in mouse neuronal HT22 cells. As shown in Fig. 2C, MTT results suggested that LPS resulted in the significant reduction in the cell viability, while being rescued by the treatment of Pari. In contrast, LPS induced up-regulation of apoptosis in HT22 cells was decreased by Pari incubation (Fig. 2D). Also, Pari exposed HT22 cells showed significantly reduced number of TUNEL-positive cells when compared to the LPS group (Fig. 2E and F). Western blot analysis further confirmed the results that Pari treatments markedly reduced the expression of cleaved Caspase-3 and PARP (Fig. 2G). These results demonstrated that Pari could alleviate MOG-induced cell death by the in vivo and in vitro experiments. 3.3. Effects of Pari on glial cell activation and immune cell infiltration induced by MOG in mice with spinal cord injury

3.5. Pari alleviates stimulus-induced inflammatory responses in vitro by regulating NF-κB signaling

To explore the glial cell activation, the expression of GFAP (a marker of astrocytes) [33] and Iba-1 (a marker of microglia) [34] were calculated by IF staining and western blot analysis in the spinal cords of mice. As shown in Fig. 3A and B, MOG operation significantly resulted in the activation of glial cells, as evidenced by the up-regulated expression of GFAP and Iba-1, and these effects were markedly abolished by Pari treatments. To further investigate the infiltrating immune cells that led to inflammation in spinal cord tissues, the expression of CD3 (a hallmark for T cells) [35] and F4/80 (a marker for macrophages) [36] was calculated using IHC and/or western blotting analysis. Results from

In order to further explore the effects of NF-κB signaling on Pariregulated spinal cord injury, the inhibitor of NF-κB, BAY 11-7082, was pre-treated to RAW264.7 and Jurkat cells. As shown in Fig. 5A and B, both Pari and BAY 11-7082 alone treatment could reduce the mRNA levels of TNF-α, IL-1β and IFN-γ in RAW264.7 and Jurkat cells stimulated by LPS and CD3/28, respectively. Significantly, Pari combined with BAY 11-7082 could further decrease TNF-α, IL-1β and IFN-γ mRNA levels compared to Pari or BAY 11-7082 single group in LPS- and 5

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Fig. 2. Effects of Pari on apoptosis in MOG mice with spinal cord injury. (A) TUNEL staining for spinal cord tissue sections, and the number of TUNEL-positive cells (n = 4 samples per group). Scale bar was 50 μm. (B) Western blot analysis for cleaved Caspase-3 and PARP (n = 3 samples per group). (C–G) HT22 cells were treated with LPS (100 ng/ml) with or without Pari (10 and 100 nM) for 24 h. Then, all cells were harvested for the subsequent analysis. (C) MTT analysis was used for cell viability (n = 7 samples per group). (D) Apoptosis in cells was measured using flow cytometry analysis (n = 6 samples per group). (E) TUNEL staining for cells as treated. Scale bar was 50 μm. (F) Quantification for the number of TUNEL-positive cells in vitro (n = 5 samples per group). (G) Western blot analysis for the expression of cleaved Caspase-3 and PARP in cells (n = 5 samples per group). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 and ***p < 0.001 versus the Ctrl group; +P < 0.05, ++P < 0.01 and +++P < 0.001 versus the MOG or LPS group; by one-way ANOVA with Bonferroni post hoc analysis or two-tailed Student’s t-test.

CD3/28-stimulated RAW264.7 and Jurkat cells, respectively. Western blot analysis indicated that the expression of iNOS and COX2 reduced by Pari or BAY 11-7082 was further decreased by the two in

combination in LPS-treated RAW264.7 cells and in CD3/28-incubated Jurkat cells (Fig. 5C and D). Furthermore, the expression of cell p-NF-κB and nuclear NF-κB was down-regulated by Pari and BAY 11-7082 in 6

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Fig. 3. Effects of Pari on glial cell activation and immune cell infiltration induced by MOG in mice with spinal cord injury. (A) IF staining of Iba-1 (up) and GFAP (down) in spinal cords of mice (Scale bar was 100 μm.) (n = 5 samples from 5 mice per group). (B) Western blot analysis of Iba-1 and GFAP in spinal cords of mice (n = 3 western blots). (C) IHC staining of CD3, F4/80, iNOS and COX2 in spinal cords of mice (Scale bar was 100 μm.) (n = 5 samples from 5 mice per group). (D) Western blot analysis of F4/80, iNOS and COX2 in spinal cords of mice (n = 3 western blots). (E) Western blot analysis of iNOS and COX2 in (left) RAW264.7 cells or (right) Jurkat cells treated with LPS (100 ng/ml) or CD3/28 (10 μM) with or without Pari (10 and 100 nM) for 24 h (n = 3 western blots). Data were expressed as mean ± SEM. **P < 0.01 and ***p < 0.001 versus the Ctrl group; ++P < 0.01 and +++P < 0.001 versus the MOG, LPS or CD3/CD28 group; by one-way ANOVA with Bonferroni post hoc analysis or two-tailed Student’s t-test.

progression [7,8,48]. Paricalcitol (19-nor-1,25-dihydroxyvitamin D2, Pari) is an active, nonhypercalcemic vitamin D analog that exerts antiinflammation, anti-oxidative stress and anti-cancer effects [13,14]. Recently, Pari is suggested to be renoprotective in various experimental nephropathy models, which is closely associated with its anti-inflammatory actions [16,49]. Lipopolysaccharide-induced depressivelike behavior is also alleviated by Pari treatment mainly through inhibiting the hypothalamic microglia activation and neuroinflammation through repressing NF-κB signaling and NLRP3 inflammasome [50]. Moreover, low vitamin D concentration in serum was reported as a cause of MS, and which was independent of established risk factors [51]. In addition, low concentrations of neonatal vitamin D are associated with an increased risk of MS [17,52]. Therefore, we supposed that Pri, as one of a Vitamin D analogs, might be effective for the treatment or improvement of MS. Cell apoptosis is an important factor affecting the progression of MS/EAE. Either excessive neuronal apoptosis or insufficient apoptosis of inflammatory cells may result in recurrence or progression of MS after acute phase, and even leads to severe disability of patients. Excessive activation and inflammatory infiltration of CD4+ T cells could cause CNS demyelination, thus, inducing apoptosis of activated

LPS-treated RAW264.7 cells and in CD3/28-incubated Jurkat cells, and these effects were further elevated by the co-treatments of Pari and BAY 11-7082 (Fig. 5E and F). Thus, the results indicated that Pari decreased LPS- and CD3/28-induced inflammatory factor levels by suppressing the activation of NF-κB. 4. Discussion It is well known that MS is a chronic, autoimmune inflammatory disease in CNS. The brain samples from patients with MS exhibit enhanced injury and cell death in the CNS. MS is considered to result from an intricate interplay between inflammatory effector cells, such as microglia and astrocytes and auto-reactive T cells, which home to the CNS [45,46]. The most widely applied animal model to explore neuroinflammation is EAE, sharing significant characteristics with MS [47]. The NF-κB signaling cascade plays a significant role in the modulation of immune and inflammatory responses, which has been involved in the pathogenesis of autoimmune demyelinating diseases, including MS and EAE, the main animal model of MS. NF-κB is important for peripheral immune cell activation as well as the pathology induction, and also plays a critical role in the resident cells of the CNS during disease 7

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Fig. 4. Effects of Pari on MOG-induced inflammation in mice with spinal cord injury. (A) TNF-α, IL-1β and IFN-γ contents in spinal cord samples were calculated using ELISA (n = 10 samples from 10 mice per group). TNF-α, IL-1β and IFN-γ mRNA levels in (B) RAW264.7 cells and (C) Jurkat cells treated as indicated by RTqPCR analysis (n = 5 samples per group). (D) IHC staining of p-NF-κB in spinal cords of mice (Scale bar was 100 μm.) (n = 5 samples from 5 mice per group). (E,F) Western blot analysis of cell p-IκBα, IκBα, p-NF-κB and nuclear NF-κB in spinal cords of mice (n = 3 western blots). (G) Western blot analysis of cell p-IκBα, IκBα, pNF-κB and nuclear NF-κB in (left) RAW264.7 cells and (right) Jurkat cells treated as indicated (n = 3 western blots). (H) TNF-α, IL-1β, IL-6 and IFN-γ mRNA levels in the isolated microglial cells isolated from the spinal cord tissues of EAE mice treated with or without MOG (20 μg/mL) and/or Pari (100 nM) for 24 h (n = 5 samples per group). (I) Western blot analysis of p-IκBα and p-NF-κB in the isolated microglial cells isolated from the spinal cord tissues of EAE mice treated with or without MOG (20 μg/mL) and/or Pari (100 nM) for 24 h (n = 3 western blots). TNF-α, IL-1β, IL-6, and IFN-γ mRNA levels in the isolated microglial cells from EAE mice treated with MOG (20 μg/mL) and/or Pari for 24 h. ELISA analysis for (J) IL-2, IL-10, IL-17, IL-6, IFN-γ, and (K) TGF-β in regional lymph node cell cultures (5 × 106 cells/mL) stimulated with MOG (20 μg/mL) in the presence or absence of Pari (100 nM) for 24 h (n = 5 samples per group). Data were expressed as mean ± SEM. **P < 0.01 versus the Ctrl group; +P < 0.05 and ++P < 0.01 versus the MOG, LPS or CD3/CD28 group; by one-way ANOVA with Bonferroni post hoc analysis or twotailed Student’s t-test.

CD4+ T cells may facilitate the treatment of MS [53]. In the study, we found that EAE mice induced by MOG showed significantly higher expression of cleaved Caspase-3 and PARP, which are well known proapoptotic factors [54]. However, these effects were markedly alleviated by Pari treatments. Pari has been suggested to attenuate LPS-induced inflammation and apoptosis in proximal tubular cells [55]. Indoxyl sulfate-induced apoptosis in renal cells could also be inhibited by Pari treatment [56]. Similar in vitro results were detected in our study that LPS-induced apoptosis in mouse neuronal HT22 cells was also blunted

by Pari incubation. Thus, the effects of Pari on the suppression of EAE were partly attributed to its repression of apoptosis. In this study, we also found that Pari treatment alleviated histopathologic scores and improved the body weight loss in mice with EAE induced by MOG. Additionally, activation of glial cells, including astrocytes and microglial cells, was markedly inhibited by Pari treatment in spinal cord samples of MOG-operated mice, as evidenced by the reduced expression of GFAP and Iba-1. These suppressive effects of Pari on glial cell activation were in line with previous study [50]. 8

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Fig. 5. Pari alleviates stimulus-induced inflammatory responses in vitro by regulating NF-κB signaling. (A) RAW264.7 cells were pre-treated with NF-κB inhibitor BAY 11-7082 (BAY, 1 μM) for 2 h, followed by LPS (100 ng/ml) and Pari (10 and 100 nM) treatments for another 24 h (n = 5 samples per group). (B) Jurkat cells were pre-treated with BAY (1 μM) for 2 h, followed by CD3/28 (10 μM) and Pari (10 and 100 nM) incubation for another 24 h. Then, RT-qPCR analysis was used to measure TNF-α, IL-1β and IFN-γ mRNA expression levels in cells (n = 5 samples per group). Western blot analysis of iNOS and COX2 in (C) RAW264.7 cells and (D) Jurkat cells treated as shown (n = 3 western blots). Western blot analysis of cell p-NF-κB and nuclear NF-κB in (E) RAW264.7 cells and (F) Jurkat cells treated as indicated (n = 3 western blots). Data were expressed as mean ± SEM. *P < 0.05 and **P < 0.01; by one-way ANOVA with Bonferroni post hoc analysis or two-tailed Student’s t-test.

and cytokine production [62]. NF-κB also induces the production of inflammatory mediators by dendritic cells, enhances antigen processing and presentation in macrophages, and causes production of pro-inflammatory cytokines, such as TNF-α, IL-1β, IFN-γ, iNOS and COX2, and neurotoxic mediators in microglia and astrocytes [63–65]. It is clear that excessive expression of NF-κB could promote a pro-inflammatory milieu in which autoimmune diseases could develop [66]. As reported, the promoted expression of TNF-α, IL-1β, iNOS and COX2 has been suggested to be involved in the progression of EAE [47,67]. Previous study reported that suppressing TNF-α expression ameliorated EAE development by the results of rodent animals [68]. More experiments demonstrated that IL-1β was a pivotal inflammatory cytokine implicated in EAE onset and progression [69]. Furthermore, IFN-γ induces the neuro-inflammatory disorders, which is largely by the actions exhibited in CNS [70]. Clinical studies indicated that TNF-α, IFN-γ and IL-1β expression was up-regulated in the peripheral blood mononuclear cells from patients with MS [71,72]. Many studies have found that NFκB is activated in the brain tissue of patients with MS [73]. Microarray analysis of MS brain tissue has also identified up-regulation of NF-κB itself as well as genes related to NF-κB [61]. Another study showed that in active MS lesions, there was up-regulation of nuclear NF-κB in a large proportion of oligodendrocytes located at the edge of active lesions and in microglia throughout plaques but not in healthy white matter or silent MS plaques [74]. Thus, suppressing NF-κB activation could be

Furthermore, infiltration of immune cells, including macrophages and T cells, in spinal cord tissues of MOG-treated mice was clearly ameliorated, which was evidenced by the reduced expression of F4/80 and CD3, respectively. What’s more, we found that the release and expression of inflammatory factors, including TNF-α, IL-1β, IFN-γ, iNOS and COX2, by MOG were markedly reduced in spinal cord samples from Pari-treated mice. Similar anti-inflammatory effects of Pari were detected in Raw264.7 cells and Jurkat cells after specific stimulus, which was largely through the inhibition of NF-κB activation. These anti-inflammatory effects of Pari on EAE were consistent with previous study [50]. NF-κB signaling pathways are involved in cell immune responses, apoptosis and infections. In MS, NF-κB pathways are changed, leading to increased levels of NF-κB activation in cells. This may indicate a key role for NF-κB in MS pathogenesis [57]. NF-κB signaling is complex, with many elements involved in its activation and regulation [58]. Interestingly, current MS treatments are found to be directly or indirectly linked to NF-κB pathways and act to adjust the innate and adaptive immune system in patients. MS is associated with constitutive activation of NF-κB which results in excessive expression of the effector molecules whose transcription relies on the NF-κB pathway [59,60]. NF-κB acts on many immune cells, producing effects that increase inflammation, as seen in MS [61]. NF-κB is crucial to the development, proliferation and survival of B and T lymphocytes and is involved in processes such as antibody class switching, CD4+ T cell differentiation 9

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effective for developing therapeutic strategies. In the present study, the expression levels of TNF-α, IL-1β and IFN-γ were reduced in Paritreated mice, LPS-stimulated RAW264.7 cells as well as CD3/CD28incubated Jurkat cells, which were further reduced by the combination with NF-κB inhibitor of BAY 11-7082. Therefore, the findings here suggested that the suppressive role of Pari in NF-κB activation played remarkable effects on EAE pathogenesis prtly through reducing the expression of inflammatory factors. Accordingly, Vitamin D has different analogs. Thus, whether other analogs or regulators of Vitamin D also exhibit anti-inflammatory response during the progression of EAE, further studies are still warranted in future. In summary, our results indicated that Pari treatment markedly alleviated the progression of EAE induced by MOG through reducing the cell infiltration, glial cell activation and inflammatory response, which was largely dependent on the blockage of NF-κB signaling. Thus, Pari might be a promising therapeutic agent to prevent the progression of MS.

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