Interference with RhoA–ROCK Signaling Mechanism in Autoreactive CD4+ T Cells Enhances the Bioavailability of 1,25-Dihydroxyvitamin D3 in Experimental Autoimmune Encephalomyelitis
2012; Elsevier BV; Volume: 181; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2012.05.028
ISSN1525-2191
AutoresAjaib S. Paintlia, Manjeet K. Paintlia, Bruce W. Hollis, Avtar Singh, Inderjit Singh,
Tópico(s)Psoriasis: Treatment and Pathogenesis
ResumoVitamin D deficiency is a major risk factor for central nervous system (CNS) demyelinating diseases including multiple sclerosis (MS) and its animal model, that of experimental autoimmune encephalomyelitis (EAE). Both vitamin D3 and 1, 25-dihydroxyviatmin-D3 (calcitriol) had beneficial effects in EAE/MS. However, the exact cause of vitamin D deficiency in EAE/MS is not clear. Previously, we documented that lovastatin (LOV) provides protection in EAE animals via inhibition of RhoA–ROCK signaling. Herein, we demonstrate that LOV prevents the lowering of circulating 25-hydroxyvitamin-D3 and 1,25-dihydroxyviatmin-D3 levels including 1,25-dihydroxyviatmin-D3 levels in the peripheral lymphoid organs and CNS of treated EAE animals. These effects of LOV were attributed to enhanced expression of vitamin D synthesizing enzyme (1α-hydroxylase) in kidney and the CNS, with corresponding reduction of vitamin D catabolizing enzyme (24-hydorxylase) expression in the CNS of EAE animals via inhibition of RhoA–ROCK signaling. Ex vivo and in vitro studies established that autoreactive Th1/Th17 cells had higher expression of 24-hydroxylase than Th2/T regulatory cells, that was reverted by LOV or ROCK inhibitor. Interestingly, LOV-mediated regulation of vitamin D metabolism had improved vitamin D3 efficacy to confer protection in EAE animals and that was ascribed to the LOV- and calcitriol-induced immunomodulatory synergy. Together, these data provide evidence that interfering with RhoA–ROCK signaling in autoreactive Th1/Th17 cells can improve vitamin D3 efficacy in clinical trials of MS and related neurodegenerative disorders. Vitamin D deficiency is a major risk factor for central nervous system (CNS) demyelinating diseases including multiple sclerosis (MS) and its animal model, that of experimental autoimmune encephalomyelitis (EAE). Both vitamin D3 and 1, 25-dihydroxyviatmin-D3 (calcitriol) had beneficial effects in EAE/MS. However, the exact cause of vitamin D deficiency in EAE/MS is not clear. Previously, we documented that lovastatin (LOV) provides protection in EAE animals via inhibition of RhoA–ROCK signaling. Herein, we demonstrate that LOV prevents the lowering of circulating 25-hydroxyvitamin-D3 and 1,25-dihydroxyviatmin-D3 levels including 1,25-dihydroxyviatmin-D3 levels in the peripheral lymphoid organs and CNS of treated EAE animals. These effects of LOV were attributed to enhanced expression of vitamin D synthesizing enzyme (1α-hydroxylase) in kidney and the CNS, with corresponding reduction of vitamin D catabolizing enzyme (24-hydorxylase) expression in the CNS of EAE animals via inhibition of RhoA–ROCK signaling. Ex vivo and in vitro studies established that autoreactive Th1/Th17 cells had higher expression of 24-hydroxylase than Th2/T regulatory cells, that was reverted by LOV or ROCK inhibitor. Interestingly, LOV-mediated regulation of vitamin D metabolism had improved vitamin D3 efficacy to confer protection in EAE animals and that was ascribed to the LOV- and calcitriol-induced immunomodulatory synergy. Together, these data provide evidence that interfering with RhoA–ROCK signaling in autoreactive Th1/Th17 cells can improve vitamin D3 efficacy in clinical trials of MS and related neurodegenerative disorders. Multiple sclerosis (MS) is an immunologically complex neurodegenerative disease marked by trafficking of autoreactive lymphocytes and mononuclear cells into the central nervous system (CNS) with subsequent demyelination due to loss of oligodendrocytes (OLs) and axonal degeneration.1Al-Omaishi J. Bashir R. Gendelman H.E. The cellular immunology of multiple sclerosis.J Leukoc Biol. 1999; 65: 444-452PubMed Google Scholar, 2Prineas J.W. Barnard R.O. Kwon E.E. Sharer L.R. Cho E.S. Multiple sclerosis: remyelination of nascent lesions.Ann Neurol. 1993; 33: 137-151Crossref PubMed Scopus (494) Google Scholar Increasing evidence suggests that pathogenic CD4+ T helper (Th) cells ie, interferon-γ (IFN-γ)–secreting Th1 and interleukin-17 (IL-17)–secreting Th17 cells play a central role in the inflammatory and demyelinating pathology; whereas IL-4–secreting Th2 and regulatory T (Treg) cells keep the autoimmune response under control.2Prineas J.W. Barnard R.O. Kwon E.E. Sharer L.R. Cho E.S. 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Seasonal variation of serum lipids in an elderly population.Age Ageing. 1993; 22: 273-278Crossref PubMed Scopus (59) Google Scholar indicating that lowering of plasma lipids can increase the bioavailability of vitamin D metabolites in human patients. Consistent with these findings, the elevated circulatory 25-OH-D3 levels were associated with reduced serum lipid profile in heart disease patients treated with lipid-lowering drugs, statins.26Perez-Castrillon J.L. Vega G. Abad L. Sanz A. Chaves J. Hernandez G. Duenas A. Effects of Atorvastatin on vitamin D levels in patients with acute ischemic heart disease.Am J Cardiol. 2007; 99: 903-905Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 27Ertugrul D.T. Yavuz B. Cil H. Ata N. Akin K.O. Kucukazman M. Yalcin A.A. Dal K. Yavuz B.B. Tutal E. STATIN-D Study: comparison of the influences of rosuvastatin and fluvastatin treatment on the levels of 25 hydroxyvitamin D.Cardiovasc Ther. 2010; 2: 146-152Google Scholar Importantly, statins as montherapy and in combination with presently prescribed MS drugs demonstrated significant reduction of gadolinium lesions in the MS brain.28Vollmer T. Key L. Durkalski V. Tyor W. Corboy J. Markovic-Plese S. Preiningerova J. Rizzo M. Singh I. Oral simvastatin treatment in relapsing-remitting multiple sclerosis.Lancet. 2004; 363: 1607-1608Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 29Lanzillo R. Orefice G. Quarantelli M. Rinaldi C. Prinster A. Ventrella G. Spitaleri D. Lus G. Vacca G. Carotenuto B. Salvatore E. Brunetti A. Tedeschi G. Brescia Morra V. Atorvastatin combined to interferon to verify the efficacy (ACTIVE) in relapsing-remitting active multiple sclerosis patients: a longitudinal controlled trial of combination therapy.Mult Scler. 2010; 16: 450-454Crossref PubMed Scopus (63) Google Scholar These effects of statins were ascribed to the activation of autoreactive Th17 cell inhibition and the induction of Th1/Th2 shift in MS patients via lowering of isoprenoids at the cellular level, resulting in inhibition of Rho family small GTPase, RhoA, and its downstream target, Rho kinase (ROCK), as evident from EAE model studies.30Zhang X. Jin J. Peng X. Ramgolam V.S. Markovic-Plese S. Simvastatin inhibits IL-17 secretion by targeting multiple IL-17-regulatory cytokines and by inhibiting the expression of IL-17 transcription factor RORC in CD4+ lymphocytes.J Immunol. 2008; 180: 6988-6996Crossref PubMed Scopus (26) Google Scholar, 31Paintlia A.S. Paintlia M.K. Singh A.K. Singh I. Inhibition of rho family functions by lovastatin promotes myelin repair in ameliorating experimental autoimmune encephalomyelitis.Mol Pharmacol. 2008; 73: 1381-1393Crossref PubMed Scopus (62) Google Scholar, 32Dunn S.E. Youssef S. Goldstein M.J. Prod'homme T. Weber M.S. Zamvil S.S. Steinman L. Isoprenoids determine Th1/Th2 fate in pathogenic T cells, providing a mechanism of modulation of autoimmunity by atorvastatin.J Exp Med. 2006; 203: 401-412Crossref PubMed Scopus (188) Google Scholar RhoA–ROCK signaling controls the variety of cellular processes including cellular signaling, proliferation, and differentiation.33Nobes C.D. Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3700) Google Scholar Considering that statins can increase the circulating levels of 25-OH-D3 in heart disease patients, we proposed to investigate the impact of statin treatment on vitamin D metabolism in EAE animals. To gain more insight into the protective mechanism, we studied the statin-mediated regulation of vitamin D metabolizing enzyme expressions at the cellular level and tested whether statin could improve the efficacy of vitamin D3 in MS clinical trials.Materials and MethodsChemicals and ReagentsUnless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Lovastatin (LOV), Y27643, vitamin D3, and calcitriol were purchased from EMD Chemicals (Philadelphia, PA). Anti-CYP27B1 (recognizes 57 kDa protein band) and anti-CYP24A1 (recognizes 50-kDa protein band) antibodies were purchased from Abcam (Cambridge, MA). Anti–β-actin and, anti-mouse IgG, and anti-rabbit polyclonal IgG secondary antibodies were obtained from Vector Laboratory (Burlingame, CA).AnimalsFemale Lewis rats weighing 250 to 300 g were purchased from Charles River Laboratory (Wilmington, MA) and housed in the animal care facility at the Medical University of South Carolina throughout the experiment and provided with food and water ad libitum. During experimentation, rats were fed commercial rat chow containing 0.33 μg/day vitamin D3 and 1% calcium. For experimental studies using vitamin D3–deficient diet, rats were fed synthetic vitamin D3–deficient diet (Charles River Laboratory) 20 days before MBP priming for EAE induction, and rats showed significant reduction of circulating levels of 25-OH-D3 and 1,25-(OH)2D3 on day 20 (data not shown). All animal experimentations were conducted in accord with accepted standards of humane care, as outlined in the ethical guidelines and approved by the Medical University of South Carolina Animal Ethics Committee.EAE Induction and EvaluationProcedures used for an induction of EAE are as described previously in our publications, with slight modifications.34Paintlia A.S. Paintlia M.K. Khan M. Vollmer T. Singh A.K. Singh I. HMG-CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis.FASEB J. 2005; 19: 1407-1421Crossref PubMed Scopus (88) Google Scholar, 35Paintlia A.S. Paintlia M.K. Singh A.K. Stanislaus R. Gilg A.G. Barbosa E. Singh I. Regulation of gene expression associated with acute experimental autoimmune encephalomyelitis by lovastatin.J Neurosci Res. 2004; 77: 63-81Crossref PubMed Scopus (69) Google Scholar In brief, rats received a subcutaneous injection of 30 μg of guinea pig MBP in 0.1 mL of PBS emulsified with equal volume of CFA supplemented with 2 mg/mL of mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) in the hind limb footpads on days 0 and 7. Immediately and again 24 hours later, rats received pertussis toxin (200 ng, intraperitoneally, ip) in 0.1 mL PBS. Pertussis toxin was administered to rats according to standardized protocol in our laboratory for induction of EAE. Similarly, control rats received subcutaneous injection of PBS and CFA emulsion in the hind limb footpads on days 0 and 7. Rats were examined for clinical scores by an experimentally blinded investigator daily. Clinical score assessed on a scale of 0 to 5, as follows: 0, no clinical disease; 1.0, piloerection; 2.0, loss in tail tonicity; 3.0, hind-leg paralysis; 4.0, paraplegia, and 5.0, moribund or dead. Several times during the study, rats were weighed. The clinical data of rats with clinical scores >4.0 were not included for statistical analysis. At the conclusion of the study, the rats were euthanized. A blood sample was obtained, and perfusion was done. The kidneys, lymph nodes, and lumbar spinal cords were removed, snap-frozen with liquid nitrogen, and stored at −70°C before RNA, protein, or 1,25-(OH)2D3 extraction. Alternatively, the lumbar spinal cord of each rat was cut into four small pieces and fixed in 4% paraformaldehyde for histopathology. The blood was centrifuged, and decanted plasma was frozen at −70°C before analysis.Drug TreatmentsLOV (4 mg/mL) was suspended in 0.8% ethanol/0.6 N NaOH/PBS, and pH was adjusted to 7.4 with 1N HCl. Mevalonolactone (5 mg/mL) was suspended in PBS; farnesol (5 mg/mL) was suspended in PBS; farnesyl transferase inhibitor–277 (0.5 mg/mL) was suspended in PBS; geranylgeranyl transferase inhibitor–298 was suspended in ethanol and diluted in PBS (0.5 mg/mL); fasudil was dissolved in 100 μl of DMSO and diluted in PBS (1 mg/mL); and 50 μg/mL of calcitriol was suspended in ethanol and diluted to 1 and 2 μg/mL in PBS. Drugs were administered intraperitoneally (ip) at the specified dose, once daily, using a 1-mL insulin syringe. Treatment was started after the onset of EAE disease (11 to 13 days post-immunization [dpi]) and continued until the lessening of paralytic symptoms (25 dpi). Rat developing EAE with no drug treatment received an injection (ip) of vehicle (0.8% ethanol in PBS), once daily. Control rats received an injection (ip) of vehicle or drug, once daily. For vitamin D3 treatment, vitamin D3 (0.25 mg/kg) suspended in saline was administered to rats by gavage every day. Similarly, for combination treatments, LOV and calcitriol were administered once daily in established EAE rats with clinical disease scores ≥3.0 until the lessening of paralytic symptoms (25 dpi).25-OH-D3 and 1,25-(OH)2D3 AnalysisPlasma was separated from blood and the concentrations of 25-OH-D3 and 1,25-(OH)2D3 were determined in duplicate by radioimmunoassay (RIA) developed in the laboratory of Bruce Hollis, as documented earlier.36Hollis B.W. Quantitation of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D by radioimmunoassay using radioiodinated tracers.Methods Enzymol. 1997; 282: 174-186Crossref PubMed Scopus (85) Google Scholar Laboratory personnel were blinded to the samples. The measurements of 1,25-(OH)2D3 in the tissues of spinal cord were performed by extraction methods published earlier.16Spach K.M. Hayes C.E. Vitamin D3 confers protection from autoimmune encephalomyelitis only in female mice.J Immunol. 2005; 175: 4119-4126PubMed Google Scholar Briefly, tissues were extracted with chloroform-methanol-4% KCl in water (1:2:0.8 v/v) mixture to recover the vitamin D metabolites. The extracts were then vacuum dried, suspended in ethanol, diluted in sample buffer, and analyzed in duplicate for 1,25-(OH)2D3 using rat 1,25-(OH)2D3 ELISA kit (NovaTeinBio, Cambridge, MA). A spike recovery control was performed with each 1,25-(OH)2D3 extraction by adding 100 pg of 1,25-(OH)2D3 to a crushed spinal cord of rat fed a vitamin D3-depleted diet for 20 days.Stimulation of MBP-Primed Spleen Cells and Characterization of CD4+ T CellsThe spleens of MBP-primed rats were collected after the onset of EAE with clinical score 97% purity. Total RNA was purified from collected CD4+ T cells and used for real-time PCR analysis.In Vitro T Helper Cell DifferentiationNaive CD4+CD25− T cells were purified from the spleen single cell suspensions using magnetic cell sorting (Miltenyi Biotec) with 98% to 99% of purity. Purified CD4+CD25− T cells were stimulated with antibodies to CD3 (5 μg/mL) and CD28 (2 μg/mL) under Th17 cell differentiation conditions (rmIL-6, 20 ng/mL; R&D systems; rhTGF-β1, 3 ng/mL; anti-IL-4, 10 μg/mL; and anti–IFN-γ, 10 μg/mL; BD Biosciences), Th1 cell differentiation conditions (rmIL-12, 10 ng/mL and IL-4, 10 μg/mL; BD Biosciences), Th2 cell differentiation conditions (rmIL-4, 20 ng/mL; rmIL-2, 10 ng/mL, R&D Systems; and anti-IFN-γ, 10 μg/mL), or Treg cell differentiation conditions (rhTGF-β1, 5 ng/mL and rmILs-2, 40 ng/mL; R&D Systems) for 48 hours. This was followed by treatment with LOV (5 μmol/L) for another 24 hours to assess the expression of CYP24A1 using quantitative real-time PCR.HistopathologyFixed lumbar spinal cords sections of 5 μm were stained with Luxol fast blue or H&E (Sigma-Aldrich) and examined using light microscopy (Olympus BX-60) and images were acquired with an Olympus digital camera (Optronics; Goleta, CA). The region of spinal cord depicting >10 nuclei was considered as a lesion. The meninges, and the gray and white matter of the sections were scored in a blinded fashion based on the presence or the absence of infiltrating cells in each of the regions of the spinal cord. The histopathology score was recorded as the number of lesions in the spinal cord of each group rats that showed a readily identifiable inflammatory cell infiltrate. Similarly, luxol fast blue–stained spinal cord sections were analyzed for degree of demyelination and scored. The demyelination degree was scored as grade 0, no disease; grade 1, foci of demyelination/axonal loss that was superficial and involved 25% of the lateral columns; and grade 3, diffuse and widespread demyelination and axonal loss.Real-Time PCR and Transcript AnalysisFor quantitative assessments of relative mRNA levels, total RNA was prepared using TRIzol LS reagent (Invitrogen, Carlsbad, CA), following the manufacturer's instructions. RNA was reverse transcribed using an MMLV-RT reverse transcription kit with random hexamer primers (Bio-Rad, Hercules CA). Relative levels of transcripts of various genes described in Table 1 were determined by real-time PCR (iCycler IQ, Bio-Rad) using a SYBR green PCR mix (Bio-Rad). Rat-specific primers for each gene were designed using Primer Quest software and oligonucleotides were purchased from Integrated DNA Technologies Inc. (Coralville, IA). Thermal cycling conditions were as follows: activation of iTaq DNA polymerase at 95°C for 10 minutes, followed by 40 cycles of amplification at 95°C for 30 seconds and 60°C for 1 minute. The performance of each real-time PCR run was determined by examining the melting curve. The detection of threshold was set above the mean baseline fluorescence determined by the first 20 cycles. Amplification reaction in which the fluorescence increased above the threshold was defined as positive. Values obtained for each gene of interest were normalized to β-actin or 18S rRNA transcripts as an internal control.Table 1List of Primers Used for Quantitative Real-Time PCR AnalysisGene namePrimer sequenceβ-actinFP: 5′-AGCTGTGCTATGTTGCCCTAGACT-3′; RP: 5′-ACCGCTCATTGCCGATAGTGATGA-3′18SrRNAFP: 5′-CCAGAGCGAAAGCATTTGCCAAGA-3′; RP: 5′-TCGGCATCGTTTATGGTCGGAACT-3′CYP24A1FP: 5′-TTAGACCCGAACGCTGGCTTGAAA-3′; RP: 5′-AGCAAAGAGCCAAGTGGAGCTGTA-3′CYP27B1FP: 5′-GGATCAGATGTTTGCCTTTGCCCA-3′; RP: 5′-AGGAAGTGGGTTAAGTGATGCCCA-3′IL-4FP: 5′-GGTATCCACGGATGTAACGACAGC-3′; RP: 5′-CCGTGGTGTTCCTTGTTGCCGTAA −3′IFN-γFP: 5′-TTTGAGGTCAACAACCCACAGGTC-3′; RP: 5′-TTTCCGCTTCCTGAGGCTGGATT-3′IL-6FP: 5′-CAGAAGGAGTGGCTAAGGAC-3′; RP: 5′-CACTAGGTTTGCCGAGTAGA-3′IL-23FP: 5′-ATCACCACTGGGAGACTCAACAGA-3′; RP: 5′-TGCGAAGGATCTTGGAACGGAGAA-3′IL-17AFP: 5′-TCTGAGCCAGCCAAGAAGAAGTGT-3′; RP: 5′-TTCCAACCCAAACATAGGCAC-3′FoxP3FP: 5′-AGAGTTTCTCAAGCACTGCCAAGC-3′; RP: 5′-TGCATAGCTCCCAGCTTCTCCTTT-3′RORγtFP: 5′-AGGCCATTCAGTATGTGGTGGAGT-3′; RP: 5′-TGTGTGGTTGTTGGCATTGTAGGC-3′FP, forward primer; RP, reverse primer. Open table in a new tab Western BlottingTissues were homogenized in ice-cold lysis buffer (50 mm Tris-HCl, pH 7.4, containing 50 mmol/L NaCl, 1 mmol/L EDTA, 0.5 mmol/L EGTA, 10% glycerol, and protease inhibitor mixture), using a soincator with three to four pulses for 30 seconds each. Samples were centrifuged at 12,000 × g for 10 minutes at 4°C, and protein concentration was determined in the supernatant using Bradford reagent (Bio-Rad). SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed as described previously.34Paintlia A.S. Paintlia M.K. Khan M. Vollmer T. Singh A.K. Singh I. HMG-CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis.FASEB J. 2005; 19: 1407-1421Crossref PubMed Scopus (88) Google Scholar Autoradiograph of immunoblots was generated using enhanced chemiluminescence detection kits (Amersham Biosciences, Arlington Heights, IL). Densitometeric analysis of autoradiograph was performed using ImageJ software from the NIH (available at http://rsb.info.nih.gov/ij).Quantification of CytokinesSlices of the spinal cords (20 to 50 mg) of rats were soincated in 0.5 mL of PBS and centrifuged at 12,000 × g for 10 minutes at 4°C. The protein concentration in each sample supernatant was determined using Bradford reagent (Bio-Rad). To quantify the levels of various cytokines, ELISA kits (eBiosciences Inc., San Diego, CA) were used as per instructions in the product manuals. Recombinant protein of cytokines was used as standard to measure their concentration in test samples. Levels of cytokines in each sample in triplicate were measured using an ELISA reader (UV-VIS spectrophotometer), and data were normalized with protein concentration in each sample.Statistical AnalysisFor multiple comparisons, one-way multiple range analysis of variance followed by Bonferroni post-test was used and P values were determined for each experiment of three to four separate experiments using GraphPad Prism 3.0 software (GraphPad Software, San Diego, CA). Two-way analysis of variance plus Bonferroni post-test was used to compare two groups over multiple time points. Student's t-test was used to compare two groups of matched samples. P values <0.05 were considered significant.ResultsEffect of LOV Treatment on Plasma Levels of 25-OH-D3 and 1,25-(OH)2D3 in EAE Rats in Relation to Clinical SymptomsThe experiments reported here used the acute EAE model to investigate whether LOV-mediated signaling mechanism(s) enhances the systemic levels of vitamin D metabolites to influence MS risk. Earlier, we and others documented that statin-mediated inhibition of Rho family GTPases in immune cells participates in the attenuation of EAE pathogenesis.31Paintlia A
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