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Decreased Dicer expression is linked to increased expression of co-stimulatory molecule CD80 on B cells in multiple sclerosis Latt Latt Aung and Konstantin E Balashov Mult Scler published online 5 December 2014 DOI: 10.1177/1352458514560923 The online version of this article can be found at: http://msj.sagepub.com/content/early/2014/11/30/1352458514560923
Published by: http://www.sagepublications.com
On behalf of: European Committee for Treatment and Research in Multiple Sclerosis
Americas Committee for Treatment and Research in Multiple Sclerosis
Pan-Asian Committee for Treatment and Research in Multiple Sclerosis
Latin American Committee on Treatment and Research of Multiple Sclerosis
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research-article2014
MSJ0010.1177/1352458514560923Multiple Sclerosis JournalLL Aung and KE Balashov
MULTIPLE SCLEROSIS MSJ JOURNAL
Original Research Paper
Decreased Dicer expression is linked to increased expression of co-stimulatory molecule CD80 on B cells in multiple sclerosis
Multiple Sclerosis Journal 1–8 DOI: 10.1177/ 1352458514560923 © The Author(s), 2014. Reprints and permissions: http://www.sagepub.co.uk/ journalsPermissions.nav
Latt Latt Aung and Konstantin E Balashov
Abstract Background: Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system. B cells have been strongly implicated in disease pathogenesis based on clinical trials with B-cell ablation. There is a growing body of evidence linking microRNAs with regulation of the immune system. Dicer, a key enzyme involved in microRNA biogenesis, is necessary for normal B-cell function. Objective: We aimed to determine whether Dicer expression is impaired in B cells and is linked to increased expression co-stimulatory molecules in patients with MS. Methods: B cells were separated from blood samples of MS patients and healthy subjects. Expression of Dicer and co-stimulatory molecules CD80 and CD86 was tested. The effect of Dicer modulation on CD80 and CD86 expression in B cells was studied. Results: Dicer expression was decreased in B cells but not in monocytes of patients with MS compared with healthy subjects. CD80 and CD86 expression was increased on B cells of MS patients compared with healthy subjects. Inhibition of Dicer expression in B cells by small interfering RNA led to increased expression of CD80. Conclusion: Dicer expression is decreased and is mechanistically linked to increased expression of costimulatory molecule CD80 in B cells of patients with MS. This may contribute to activation of immune responses in MS.
Correspondence to: Konstantin E. Balashov Department of Neurology, Rutger-Robert Wood Johnson Medical School, 125 Paterson Street, Rm 6200, New Brunswick, NJ 08901, USA. konstantin.balashov@ rutgers.edu Latt Latt Aung Konstantin E Balashov Department of Neurology, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ, USA
Keywords: Multiple sclerosis, Dicer, B cells, CD80 Date received: 25 April 2014; revised: 5 October 2014; accepted: 20 October 2014
Introduction The contribution of B cells and their products to the pathogenesis of multiple sclerosis (MS) is well established. B-cell accumulation, clonal expansion and the production of oligoclonal IgG in the cerebrospinal fluid (CSF) of patients with MS have been repeatedly documented.1-3 The elimination of B cells with antiCD20 monoclonal antibodies suppresses disease activity in patients with relapsing–remitting MS (RRMS).4-7 Notably, it is not accompanied by reduction in CSF immunoglobulins.3 In addition, T-cell responses are reduced in MS patients with depleted B cells.8 B cells regulate T-cell response via cytokines8,9 or co-stimulatory molecules,10,11 and can efficiently present myelin antigen to T cells.12 Co-stimulatory
molecules CD80 and CD86 are critical in activating T-cell immune response13,14 and are over-expressed on B cells in patients with MS.15 MicroRNAs (miRNAs) are gene regulatory molecules that influence the output of protein-coding genes in mammals.16,17 Dicer (aka Dicer 1 in humans), a member of the RNase III family of enzymes, is closely involved in miRNA biogenesis.16 Dicer and miRNA expression have a crucial role in immune regulation.18 For example, mice with a conditional deletion of Dicer in B cells have a complete block in B-cell development and an alteration in antibody repertoire.19 Dicer is also involved in establishing B-cell tolerance. The self-reactive autoantibodies are increased in Dicer-deficient mice.20
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Multiple Sclerosis Journal We hypothesized that Dicer expression could be impaired in B cells of patients with MS and linked to increased co-stimulatory function of B cells. Here we report that expression of Dicer is selectively decreased in B cells of patients with MS and is mechanistically linked to increased expression of the co-stimulatory molecule CD80. Materials and methods Patients and healthy controls Control healthy donors and patients diagnosed with clinically definite RRMS21 or clinically isolated syndrome (CIS) with brain magnetic resonance imaging (MRI) suggestive of MS—defined as two or more clinically silent lesions that were at least 3 mm in diameter on MRI scans with at least one lesion which was periventricular or ovoid22—were included in the study, which was approved by an institutional review committee. The patients had not been taking any disease-modifying drugs for at least 3 months prior to their enrollment in the study. Of 17 enrolled MS patients, 14 had experienced at least one clinical relapse in the year before sample collection. The average annual relapse rate among these 14 active patients was 1.5 relapses/year. Additional exclusion criteria included steroid treatment in the month prior to the blood draw; presence of other disorders that may be associated with abnormal immune response; pregnancy; baseline Expanded Disability Status Scale score greater than 5;23 and clinical signs or history suggesting infection within 2 weeks prior to the blood draw. The age (mean ± SEM) of five healthy subjects (three female/two male) and six patients (four female/two male) tested in gene expression experiments was 36.4 ± 5.2 and 40 ± 4.6, respectively. The age (mean ± SEM) of six healthy subjects (five female/one male) and five patients (four female/one male) tested in Figure 2 was 32.33 ± 4.5 and 35.6 ± 4.2, respectively. The age of 16 healthy subjects (11 female/five male) and 19 patients (14 female/five male) tested in Figure 1 was 35.19 ± 2.59 and 35.42 ± 1.9, respectively. The study was approved by the Rutgers Biomedical and Health Sciences Institutional Review Board (New Brunswick/Piscataway Campus) and all subjects gave written informed consent. Separation of cell subsets Peripheral blood mononuclear cells (PBMC) were separated by Ficoll density gradient (MediaTech
Inc). Separation of B cells and monocytes was done by negative immunomagnetic sorting using a B-cell isolation kit II (Miltenyi Biotec, Cat # 130 091 151) and monocyte isolation kit II (Cat # 130-091-153), respectively, according to the manufacturer’s protocol. More than 95% of separated B cells were CD19-positive. More than 85% of separated monocytes were CD14-positive. Separated cell subsets were immediately resuspended in RNA cell protect reagent solution (Qiagen) for gene expression analysis or frozen at –80°C for Western blot experiments. Cell culture and modulation of Dicer expression with small interfering RNA Ramos B lymphocytes (cat#CRL-1596, ATCC) were cultured with complete medium (RPMI plus 10% FBS and L-glutamine). Cells were transfected with 50 nM of Dicer small interfering RNA (siRNA) (Cat#SI02655492, Qiagen) or Allstars negative control (Cat# SI03650318, Qiagen) using TransIT-TKO transfection reagent (Cat#MIR2154, Mirus Bio) for 72 h according to the manufacturer’s instructions. CD80 and CD86 expressions of siRNA-treated Ramos cells were analyzed by flow cytometry. Similar experiments were done with B cells separated from healthy controls (1×106) transfected with 100 nM of Dicer siRNA (Cat#SI02655492, Qiagen) or Allstars negative control (Cat# SI03650318, Qiagen). Flow cytometry analysis PBMC separated from healthy donors and MS patients were stained with PE-labeled anti-human CD19 mAb (Biolegend), and FITC-labeled antiCD80 mAb, anti-CD86 mAb, anti-CD40 mAb or mouse IgG1 isotype control (Biolegend) followed by a two-color flow cytometry analysis on FC500 flow cytometer (Beckman Coulter). Results were analyzed with CXP software (Beckman Coulter). To analyze the effect of silencing Dicer on CD80 and CD86 expressions, Ramos cells were stained with FITC-labeled anti-CD80 mAb or anti-CD86 mAb (Biolegend). Mean fluorescence intensity (MFI) of CD80 (or CD86) was calculated as a difference between MFI of cell samples stained with anti-CD80 mAb minus MFI of cell samples stained with isotype control Ab. CD80 and CD86 expression change on transfected cells was evaluated by the fold change of MFI, calculated as the ratio of MFI of cells transfected with Dicer siRNA/MFI of cells transfected with negative control siRNA.
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LL Aung and KE Balashov et al.
Figure 1. Dicer protein expression is decreased in B cells of MS patients. B cells were separated from 12 patients with multiple sclerosis (MS) and 11 healthy donors (HD) and tested for Dicer expression by Western blot as described in Material and methods. Dicer protein expression was normalized against endogenous beta-actin protein. Figure 1(a) depicts Western blot from a representative HD and a patient with MS. The mean relative expression of Dicer is shown in Figures 1B. *p = 0.0108.
Gene expression analysis Gene expression analysis was determined by Realtime quantitative polymerase chain reaction (RT-qPCR) as described elsewhere24 using TaqMan Gene Expression Assays (the set of primers and probes) for Dicer 1 and endogenous control HPRT1 purchased from Applied Biosystems, CA. Western blot 1,000,000 cells were lysed by sonication in Nupage LDS Sample buffer (4×) (Invitrogen, Cat #NP0007) with the addition of DTT (50mM) and heated at 70°C for 10 minutes. Cell lysates were loaded on Nupage 4–12% Bis-Tris gels (Invitrogen, Cat #NP0323BOX) and transferred to PVDF membrane (Biorad, Cat #162-0255) by electroblotting. The membranes were blocked in Tris/Tween-20 buffer containing 5% Nonfat dry milk (Biorad, Cat #170-6404) for 1 h at room temperature and then probed with anti-Dicer antibody
(Cat# 3363, Cell Signaling) for overnight at 4°C. Membranes were washed three times with Tris/ Tween-20 buffer (50mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween-20 and probed with anti-rabbit IgG-HRP (Sigma-Aldrich, Cat #A6154) for 1 h at room temperature. The enhanced chemiluminescence agent (Pierce, Cat #32106) was used to detect proteins. The intensities of protein bands were measured with Image J software (NIH). Membranes were stripped with Restore Western blot stripping buffer (Pierce, Cat #21059) and re-probed with mouse monoclonal anti-Beta-actin (Sigma-Aldrich, Cat # A5441) followed by anti-mouse IgG-HRP (Cat# A4416) for protein normalization.
Statistical analysis Figures were generated and statistical analysis was performed with a GraphPad Prism 4 package from GraphPad Software, Inc., La Jolla, CA. The results
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Multiple Sclerosis Journal
Figure 2. Expression of CD80 and CD86 on B cells is increased in MS patients. PBMC cells were separated from patients with multiple sclerosis (MS) and healthy donors (HD). CD80 and CD86 expression was analyzed among CD19 positive (gated) B cells. 2(a) and 2(b) depict overlap histograms of CD80 (Figure 2(a)) and CD86 (Figure 2(b)) from a representative healthy donor (black histogram) and a patient with MS (white histogram). The Mean Fluorescent Intensity of CD80 and CD86 on B cells separated from six HDs and five MS patients is shown in Figure 2(c) *p < 0.02
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LL Aung and KE Balashov et al. are presented as Mean ± SEM. The paired or unpaired t-test using two-tailed p-values were utilized as appropriate. Results Decreased Dicer expression in B cells of MS patients As shown in Figure 1, Dicer protein expression was significantly decreased in B cells of patients with MS (0.2616 ± 0.0505 relative units, n = 12) compared with healthy donors (0.4989 ± 0.0695, n = 11), p = 0.0108. In contrast to B cells, Dicer expression was not significantly altered in monocytes of patients with MS (0.2379 ± 0.0833 relative units) compared with healthy controls (0.1998 ± 0.0563), p = 0.7107. We also evaluated Dicer 1 gene expression in five healthy donors and six patients with MS by qRT-PCR. Dicer gene expression in MS patients (0.5462 ± 0.0301) was significantly lower than in healthy donors (0.8433 ± 0.0929), p = 0.0094. Increased expressions of CD80 and CD86 in B cells of MS patients We hypothesized that decreased Dicer expression is linked to altered expression of co-stimulatory molecules on B cells in MS. Co-stimulatory molecules CD80 and CD86 are expressed by B cells and other antigen-presenting cells and promote activation of T cells implicated in MS pathogenesis. As a first step, we studied expression of CD80 and CD86 on B cells in our human subjects. As shown in Figure 2(c), MFI of CD80 on B cells of MS patients (0.3828 ± 0.0324) was significantly increased compared with healthy subjects (0.228 ± 0.0329), p = 0.0091. MFI of CD86 was also increased in MS patients (0.4736 ± 0.0378) compared with healthy subjects (0.2808 ± 0.0467), p = 0.0125. CD40 expression on resting B cells was not changed in MS patients (7.726 ± 0.3407) compared with healthy subjects (7.559 ± 05418). Inhibition of Dicer leads to increased expression of CD80 To evaluate the effect of inhibition of Dicer expression in B cells, we conducted transfection experiments with Dicer-specific or control nonspecific siRNA in vitro. Transfection of Ramos B cells with Dicer-specific siRNA decreased Dicer protein expression, as shown in Figure 3(a). CD80 and CD86 expressions were analyzed in transfected cells by flow cytometry. Inhibition of Dicer expression in cells transfected with Dicer
siRNA led to 1.49 fold increased CD80 expression, p = 0.0167, compared with cell transfected with control siRNA (Figure 3(b)). In contrast, the expression of CD86 was not significantly affected by Dicer modulation. Transfection experiments with Dicer-specific siRNA were also conducted with primary human B cells from healthy subjects. Similar to Ramos B cells, decreased Dicer expression led to increased CD80 expression, p = 0.027 (Figure 3(c)). Discussion CD80 and CD86-positive lymphocytes were increased in peripheral blood of MS patients with active disease compared with healthy donors.15 In contrast to lymphocytes, expression of CD80 or CD86 on monocytes was not increased in MS.15 In line with this, up-regulation of CD80 expression in MS plaques25 and increased concentration of CD80 positive B cells in CSF of MS patients26 were also reported. CD80 and CD86 enhanced the antigenpresenting function of B cells and were required for T-cell activation,14,27,28 and lack of CD80/CD86 co-stimulation could lead to T-cell anergy.28 Notably, inhibition of CD80 activity with antiCD80 antibody could prevent the development of experimental autoimmune encephalitis (EAE), a T-cell-dependent disease and animal model of MS. Interestingly, anti-CD86 antibodies could increase disease severity of EAE.29 CD80 and CD86 expressed on B cells are required for T-celldependent immunoglobulin production.30 Here, we describe increased expression of CD80 and CD86 on B cells of patients with MS. Furthermore, we demonstrated that CD80 expression, but not CD86, was modulated by Dicer. The exact mechanism of decreased Dicer expression in patients with MS is not clear at this time. Dicer was found to be critical in many biological processes which are also important for MS pathogenesis. One may hypothesize that decreased Dicer expression leads to impaired miRNA biogenesis and abnormal expression of protein-coding genes involved in immune response regulation and cell traffic to the central nervous system in patients with MS. For example, inhibition of Dicer in tumor cells was associated with enhanced expression of metallic matrix proteases MMP-2 and MMP-9,31 enzymes implicated in increased permeability of the blood–brain barrier and MS pathogenesis.32 Based on our results, Dicer was selectively decreased in B cells but not in monocytes in patients with MS (Figure 1). It is known that Dicer expression may be cell lineage specific. For example, Dicer expression is increased
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Multiple Sclerosis Journal
Figure 3. CD80 expression is increased upon silencing Dicer. Ramos B cells or B cells from healthy human subjects were transfected with Dicer siRNA or control siRNA. Dicer expression was tested by Western blot. CD80 and CD86 expression was examined by flow cytometry as described in Materials and methods. Figure 3(a) depicts Ramos B cells Western blots from a representative experiment. The average fold change of CD80 and CD86 expression from six different experiments with Ramos B cells is shown in Figure 3(b) (*p = 0.0167). The average fold change of CD80 and CD86 expression from experiments with six different human subjects is shown in Figure 3(c) (*p = 0.027).
in melanocytes, that occurs via direct transcriptional targeting of Dicer by the melanocyte master transcriptional regulator (MITF).33 In summary, we have found that Dicer expression is selectively decreased in B cells of patients with
relapsing form of MS and is linked to increased expression of co-stimulatory molecule CD80. This may contribute to increased activation of T-cell responses in MS. The cause of decreased Dicer expression in B cells of patients with MS remains to be identified.
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LL Aung and KE Balashov et al. Acknowledgements We thank Joan Moore for proof reading of the article. Conflicts of interest The authors have no commercial conflicts of interests or relationships which will be affected by the article. Dr. Balashov has served as a consultant for Bayer, Novartis and Sandoz. He has been funded by NIH, NMSS, and has received research support from Biogen Idec, TEVA Neuroscience, and Patterson Trust. Funding The study was supported in part by grant number K23NS052553 from the National Institute of Neurological Disorders and Stroke.
References 1. Kuenz B, Lutterotti A, Ehling R, et al. Cerebrospinal fluid B cells correlate with early brain inflammation in multiple sclerosis. PLoS One 2008; 3: e2559. 2. Franciotta D, Salvetti M, Lolli F, et al. B cells and multiple sclerosis. Lancet Neurol 2008; 7: 852–858. 3. Cross AH and Waubant E. MS and the B cell controversy. Biochim Biophys Acta 2011; 1812: 231–238.
multiple T-cell-derived lymphokines/cytokines. Proc Natl Acad Sci U S A 1989; 86: 1333–1337. 11. Sharpe AH and Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol 2002; 2: 116–126. 12. Harp CT, Ireland S, Davis LS, et al. Memory B cells from a subset of treatment-naive relapsing-remitting multiple sclerosis patients elicit CD4(+) T-cell proliferation and IFN-gamma production in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Eur J Immunol 2010; 40: 2942–2956. 13. Good KL, Avery DT and Tangye SG. Resting human memory B cells are intrinsically programmed for enhanced survival and responsiveness to diverse stimuli compared to naive B cells. J Immunol 2009; 182: 890–901. 14. Bar-Or A, Oliveira EM, Anderson DE, et al. Immunological memory: contribution of memory B cells expressing costimulatory molecules in the resting state. J Immunol 2001; 167: 5669–5677. 15. Genc K, Dona DL and Reder AT. Increased CD80(+) B cells in active multiple sclerosis and reversal by interferon beta-1b therapy. J Clin Invest 1997; 99: 2664–2671. 16. Krol J, Loedige I and Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet 2010; 11: 597–610. 17. Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–297.
4. Bar-Or A, Calabresi PA, Arnold D, et al. Rituximab in relapsing-remitting multiple sclerosis: A 72-week, open-label, phase I trial. Ann Neurol 2008; 63: 395–400.
18. O’Connell RM, Rao DS, Chaudhuri AA, et al. Physiological and pathological roles for microRNAs in the immune system. Nat Rev Immunol 2010; 10: 111–122.
5. Hauser SL, Waubant E, Arnold DL, et al. B-cell depletion with rituximab in relapsing–remitting multiple sclerosis. N Engl J Med 2008; 358: 676–688.
19. Koralov SB, Muljo SA, Galler GR, et al. Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage. Cell 2008; 132: 860–874.
6. Kappos L, Li D, Calabresi PA, et al. Ocrelizumab in relapsing–remitting multiple sclerosis: A phase 2, randomised, placebo-controlled, multicentre trial. Lancet 2011; 378: 1779–1787.
20. Belver L, de Yebenes VG and Ramiro AR. MicroRNAs prevent the generation of autoreactive antibodies. Immunity 2010; 33: 713–722.
7. Naismith RT, Piccio L, Lyons JA, et al. Rituximab add-on therapy for breakthrough relapsing multiple sclerosis: A 52-week phase II trial. Neurology 2010; 74: 1860–1867. 8. Bar-Or A, Fawaz L, Fan B, et al. Abnormal B-cell cytokine responses a trigger of T-cell-mediated disease in MS? Ann Neurol 2010; 67: 452–461. 9. Bouaziz JD, Calbo S, Maho-Vaillant M, et al. IL10 produced by activated human B cells regulates CD4(+) T-cell activation in vitro. Eur J Immunol 2010; 40: 2686–2691. 10. Thompson CB, Lindsten T, Ledbetter JA, et al. CD28 activation pathway regulates the production of
21. Polman CH, Reingold SC, Banwell B, et al. Diagnostic criteria for multiple sclerosis: 2010 Revisions to the McDonald criteria. Ann Neurol 2011; 69: 292–302. 22. Jacobs LD, Beck RW, Simon JH, et al. Intramuscular interferon beta-1a therapy initiated during a first demyelinating event in multiple sclerosis. CHAMPS Study Group. N Engl J Med 2000; 343: 898–904. 23. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: An Expanded Disability Status Scale (EDSS). Neurology 1983; 33: 1444–1452. 24. Balashov KE, Aung LL, Vaknin-Dembinsky A, et al. Interferon-beta inhibits toll-like receptor 9 processing in multiple sclerosis. Ann Neurol 2010; 68: 899–906.
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28. Gimmi CD, Freeman GJ, Gribben JG, et al. Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation. Proc Natl Acad Sci U S A 1993; 90: 6586–6590. 29. Kuchroo VK, Das MP, Brown JA, et al. B7–1 and B7–2 costimulatory molecules activate
differentially the Th1/Th2 developmental pathways: Application to autoimmune disease therapy. Cell 1995; 80: 707–718. 30. Lumsden JM, Williams JA and Hodes RJ. Differential requirements for expression of CD80/86 and CD40 on B cells for T-dependent antibody responses in vivo. J Immunol 2003; 170: 781–787. 31. Han L, Zhang A, Zhou X, et al. Downregulation of Dicer enhances tumor cell proliferation and invasion. Int J Oncol 2010; 37: 299–305. 32. Leppert D, Lindberg RL, Kappos L, et al. Matrix metalloproteinases: Multifunctional effectors of inflammation in multiple sclerosis and bacterial meningitis. Brain Res Brain Res Rev 2001; 36: 249–257. 33. Levy C, Khaled M, Robinson KC, et al. Lineagespecific transcriptional regulation of DICER by MITF in melanocytes. Cell 2010; 141: 994–1005.
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Decreased Dicer expression is linked to increased expression of co-stimulatory molecule CD80 on B cells in multiple sclerosis Latt Latt Aung and Konstantin E Balashov Mult Scler published online 5 December 2014 DOI: 10.1177/1352458514560923 The online version of this article can be found at: http://msj.sagepub.com/content/early/2014/11/30/1352458514560923
Published by: http://www.sagepublications.com
On behalf of: European Committee for Treatment and Research in Multiple Sclerosis
Americas Committee for Treatment and Research in Multiple Sclerosis
Pan-Asian Committee for Treatment and Research in Multiple Sclerosis
Latin American Committee on Treatment and Research of Multiple Sclerosis
Additional services and information for Multiple Sclerosis Journal can be found at: Email Alerts: http://msj.sagepub.com/cgi/alerts Subscriptions: http://msj.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: Downloaded fromhttp://www.sagepub.com/journalsPermissions.nav msj.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 10, 2014
>> OnlineFirst Version of Record - Dec 5, 2014 What is This?
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560923
research-article2014
MSJ0010.1177/1352458514560923Multiple Sclerosis JournalLL Aung and KE Balashov
MULTIPLE SCLEROSIS MSJ JOURNAL
Original Research Paper
Decreased Dicer expression is linked to increased expression of co-stimulatory molecule CD80 on B cells in multiple sclerosis
Multiple Sclerosis Journal 1–8 DOI: 10.1177/ 1352458514560923 © The Author(s), 2014. Reprints and permissions: http://www.sagepub.co.uk/ journalsPermissions.nav
Latt Latt Aung and Konstantin E Balashov
Abstract Background: Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system. B cells have been strongly implicated in disease pathogenesis based on clinical trials with B-cell ablation. There is a growing body of evidence linking microRNAs with regulation of the immune system. Dicer, a key enzyme involved in microRNA biogenesis, is necessary for normal B-cell function. Objective: We aimed to determine whether Dicer expression is impaired in B cells and is linked to increased expression co-stimulatory molecules in patients with MS. Methods: B cells were separated from blood samples of MS patients and healthy subjects. Expression of Dicer and co-stimulatory molecules CD80 and CD86 was tested. The effect of Dicer modulation on CD80 and CD86 expression in B cells was studied. Results: Dicer expression was decreased in B cells but not in monocytes of patients with MS compared with healthy subjects. CD80 and CD86 expression was increased on B cells of MS patients compared with healthy subjects. Inhibition of Dicer expression in B cells by small interfering RNA led to increased expression of CD80. Conclusion: Dicer expression is decreased and is mechanistically linked to increased expression of costimulatory molecule CD80 in B cells of patients with MS. This may contribute to activation of immune responses in MS.
Correspondence to: Konstantin E. Balashov Department of Neurology, Rutger-Robert Wood Johnson Medical School, 125 Paterson Street, Rm 6200, New Brunswick, NJ 08901, USA. konstantin.balashov@ rutgers.edu Latt Latt Aung Konstantin E Balashov Department of Neurology, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ, USA
Keywords: Multiple sclerosis, Dicer, B cells, CD80 Date received: 25 April 2014; revised: 5 October 2014; accepted: 20 October 2014
Introduction The contribution of B cells and their products to the pathogenesis of multiple sclerosis (MS) is well established. B-cell accumulation, clonal expansion and the production of oligoclonal IgG in the cerebrospinal fluid (CSF) of patients with MS have been repeatedly documented.1-3 The elimination of B cells with antiCD20 monoclonal antibodies suppresses disease activity in patients with relapsing–remitting MS (RRMS).4-7 Notably, it is not accompanied by reduction in CSF immunoglobulins.3 In addition, T-cell responses are reduced in MS patients with depleted B cells.8 B cells regulate T-cell response via cytokines8,9 or co-stimulatory molecules,10,11 and can efficiently present myelin antigen to T cells.12 Co-stimulatory
molecules CD80 and CD86 are critical in activating T-cell immune response13,14 and are over-expressed on B cells in patients with MS.15 MicroRNAs (miRNAs) are gene regulatory molecules that influence the output of protein-coding genes in mammals.16,17 Dicer (aka Dicer 1 in humans), a member of the RNase III family of enzymes, is closely involved in miRNA biogenesis.16 Dicer and miRNA expression have a crucial role in immune regulation.18 For example, mice with a conditional deletion of Dicer in B cells have a complete block in B-cell development and an alteration in antibody repertoire.19 Dicer is also involved in establishing B-cell tolerance. The self-reactive autoantibodies are increased in Dicer-deficient mice.20
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Multiple Sclerosis Journal We hypothesized that Dicer expression could be impaired in B cells of patients with MS and linked to increased co-stimulatory function of B cells. Here we report that expression of Dicer is selectively decreased in B cells of patients with MS and is mechanistically linked to increased expression of the co-stimulatory molecule CD80. Materials and methods Patients and healthy controls Control healthy donors and patients diagnosed with clinically definite RRMS21 or clinically isolated syndrome (CIS) with brain magnetic resonance imaging (MRI) suggestive of MS—defined as two or more clinically silent lesions that were at least 3 mm in diameter on MRI scans with at least one lesion which was periventricular or ovoid22—were included in the study, which was approved by an institutional review committee. The patients had not been taking any disease-modifying drugs for at least 3 months prior to their enrollment in the study. Of 17 enrolled MS patients, 14 had experienced at least one clinical relapse in the year before sample collection. The average annual relapse rate among these 14 active patients was 1.5 relapses/year. Additional exclusion criteria included steroid treatment in the month prior to the blood draw; presence of other disorders that may be associated with abnormal immune response; pregnancy; baseline Expanded Disability Status Scale score greater than 5;23 and clinical signs or history suggesting infection within 2 weeks prior to the blood draw. The age (mean ± SEM) of five healthy subjects (three female/two male) and six patients (four female/two male) tested in gene expression experiments was 36.4 ± 5.2 and 40 ± 4.6, respectively. The age (mean ± SEM) of six healthy subjects (five female/one male) and five patients (four female/one male) tested in Figure 2 was 32.33 ± 4.5 and 35.6 ± 4.2, respectively. The age of 16 healthy subjects (11 female/five male) and 19 patients (14 female/five male) tested in Figure 1 was 35.19 ± 2.59 and 35.42 ± 1.9, respectively. The study was approved by the Rutgers Biomedical and Health Sciences Institutional Review Board (New Brunswick/Piscataway Campus) and all subjects gave written informed consent. Separation of cell subsets Peripheral blood mononuclear cells (PBMC) were separated by Ficoll density gradient (MediaTech
Inc). Separation of B cells and monocytes was done by negative immunomagnetic sorting using a B-cell isolation kit II (Miltenyi Biotec, Cat # 130 091 151) and monocyte isolation kit II (Cat # 130-091-153), respectively, according to the manufacturer’s protocol. More than 95% of separated B cells were CD19-positive. More than 85% of separated monocytes were CD14-positive. Separated cell subsets were immediately resuspended in RNA cell protect reagent solution (Qiagen) for gene expression analysis or frozen at –80°C for Western blot experiments. Cell culture and modulation of Dicer expression with small interfering RNA Ramos B lymphocytes (cat#CRL-1596, ATCC) were cultured with complete medium (RPMI plus 10% FBS and L-glutamine). Cells were transfected with 50 nM of Dicer small interfering RNA (siRNA) (Cat#SI02655492, Qiagen) or Allstars negative control (Cat# SI03650318, Qiagen) using TransIT-TKO transfection reagent (Cat#MIR2154, Mirus Bio) for 72 h according to the manufacturer’s instructions. CD80 and CD86 expressions of siRNA-treated Ramos cells were analyzed by flow cytometry. Similar experiments were done with B cells separated from healthy controls (1×106) transfected with 100 nM of Dicer siRNA (Cat#SI02655492, Qiagen) or Allstars negative control (Cat# SI03650318, Qiagen). Flow cytometry analysis PBMC separated from healthy donors and MS patients were stained with PE-labeled anti-human CD19 mAb (Biolegend), and FITC-labeled antiCD80 mAb, anti-CD86 mAb, anti-CD40 mAb or mouse IgG1 isotype control (Biolegend) followed by a two-color flow cytometry analysis on FC500 flow cytometer (Beckman Coulter). Results were analyzed with CXP software (Beckman Coulter). To analyze the effect of silencing Dicer on CD80 and CD86 expressions, Ramos cells were stained with FITC-labeled anti-CD80 mAb or anti-CD86 mAb (Biolegend). Mean fluorescence intensity (MFI) of CD80 (or CD86) was calculated as a difference between MFI of cell samples stained with anti-CD80 mAb minus MFI of cell samples stained with isotype control Ab. CD80 and CD86 expression change on transfected cells was evaluated by the fold change of MFI, calculated as the ratio of MFI of cells transfected with Dicer siRNA/MFI of cells transfected with negative control siRNA.
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LL Aung and KE Balashov et al.
Figure 1. Dicer protein expression is decreased in B cells of MS patients. B cells were separated from 12 patients with multiple sclerosis (MS) and 11 healthy donors (HD) and tested for Dicer expression by Western blot as described in Material and methods. Dicer protein expression was normalized against endogenous beta-actin protein. Figure 1(a) depicts Western blot from a representative HD and a patient with MS. The mean relative expression of Dicer is shown in Figures 1B. *p = 0.0108.
Gene expression analysis Gene expression analysis was determined by Realtime quantitative polymerase chain reaction (RT-qPCR) as described elsewhere24 using TaqMan Gene Expression Assays (the set of primers and probes) for Dicer 1 and endogenous control HPRT1 purchased from Applied Biosystems, CA. Western blot 1,000,000 cells were lysed by sonication in Nupage LDS Sample buffer (4×) (Invitrogen, Cat #NP0007) with the addition of DTT (50mM) and heated at 70°C for 10 minutes. Cell lysates were loaded on Nupage 4–12% Bis-Tris gels (Invitrogen, Cat #NP0323BOX) and transferred to PVDF membrane (Biorad, Cat #162-0255) by electroblotting. The membranes were blocked in Tris/Tween-20 buffer containing 5% Nonfat dry milk (Biorad, Cat #170-6404) for 1 h at room temperature and then probed with anti-Dicer antibody
(Cat# 3363, Cell Signaling) for overnight at 4°C. Membranes were washed three times with Tris/ Tween-20 buffer (50mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween-20 and probed with anti-rabbit IgG-HRP (Sigma-Aldrich, Cat #A6154) for 1 h at room temperature. The enhanced chemiluminescence agent (Pierce, Cat #32106) was used to detect proteins. The intensities of protein bands were measured with Image J software (NIH). Membranes were stripped with Restore Western blot stripping buffer (Pierce, Cat #21059) and re-probed with mouse monoclonal anti-Beta-actin (Sigma-Aldrich, Cat # A5441) followed by anti-mouse IgG-HRP (Cat# A4416) for protein normalization.
Statistical analysis Figures were generated and statistical analysis was performed with a GraphPad Prism 4 package from GraphPad Software, Inc., La Jolla, CA. The results
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Multiple Sclerosis Journal
Figure 2. Expression of CD80 and CD86 on B cells is increased in MS patients. PBMC cells were separated from patients with multiple sclerosis (MS) and healthy donors (HD). CD80 and CD86 expression was analyzed among CD19 positive (gated) B cells. 2(a) and 2(b) depict overlap histograms of CD80 (Figure 2(a)) and CD86 (Figure 2(b)) from a representative healthy donor (black histogram) and a patient with MS (white histogram). The Mean Fluorescent Intensity of CD80 and CD86 on B cells separated from six HDs and five MS patients is shown in Figure 2(c) *p < 0.02
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LL Aung and KE Balashov et al. are presented as Mean ± SEM. The paired or unpaired t-test using two-tailed p-values were utilized as appropriate. Results Decreased Dicer expression in B cells of MS patients As shown in Figure 1, Dicer protein expression was significantly decreased in B cells of patients with MS (0.2616 ± 0.0505 relative units, n = 12) compared with healthy donors (0.4989 ± 0.0695, n = 11), p = 0.0108. In contrast to B cells, Dicer expression was not significantly altered in monocytes of patients with MS (0.2379 ± 0.0833 relative units) compared with healthy controls (0.1998 ± 0.0563), p = 0.7107. We also evaluated Dicer 1 gene expression in five healthy donors and six patients with MS by qRT-PCR. Dicer gene expression in MS patients (0.5462 ± 0.0301) was significantly lower than in healthy donors (0.8433 ± 0.0929), p = 0.0094. Increased expressions of CD80 and CD86 in B cells of MS patients We hypothesized that decreased Dicer expression is linked to altered expression of co-stimulatory molecules on B cells in MS. Co-stimulatory molecules CD80 and CD86 are expressed by B cells and other antigen-presenting cells and promote activation of T cells implicated in MS pathogenesis. As a first step, we studied expression of CD80 and CD86 on B cells in our human subjects. As shown in Figure 2(c), MFI of CD80 on B cells of MS patients (0.3828 ± 0.0324) was significantly increased compared with healthy subjects (0.228 ± 0.0329), p = 0.0091. MFI of CD86 was also increased in MS patients (0.4736 ± 0.0378) compared with healthy subjects (0.2808 ± 0.0467), p = 0.0125. CD40 expression on resting B cells was not changed in MS patients (7.726 ± 0.3407) compared with healthy subjects (7.559 ± 05418). Inhibition of Dicer leads to increased expression of CD80 To evaluate the effect of inhibition of Dicer expression in B cells, we conducted transfection experiments with Dicer-specific or control nonspecific siRNA in vitro. Transfection of Ramos B cells with Dicer-specific siRNA decreased Dicer protein expression, as shown in Figure 3(a). CD80 and CD86 expressions were analyzed in transfected cells by flow cytometry. Inhibition of Dicer expression in cells transfected with Dicer
siRNA led to 1.49 fold increased CD80 expression, p = 0.0167, compared with cell transfected with control siRNA (Figure 3(b)). In contrast, the expression of CD86 was not significantly affected by Dicer modulation. Transfection experiments with Dicer-specific siRNA were also conducted with primary human B cells from healthy subjects. Similar to Ramos B cells, decreased Dicer expression led to increased CD80 expression, p = 0.027 (Figure 3(c)). Discussion CD80 and CD86-positive lymphocytes were increased in peripheral blood of MS patients with active disease compared with healthy donors.15 In contrast to lymphocytes, expression of CD80 or CD86 on monocytes was not increased in MS.15 In line with this, up-regulation of CD80 expression in MS plaques25 and increased concentration of CD80 positive B cells in CSF of MS patients26 were also reported. CD80 and CD86 enhanced the antigenpresenting function of B cells and were required for T-cell activation,14,27,28 and lack of CD80/CD86 co-stimulation could lead to T-cell anergy.28 Notably, inhibition of CD80 activity with antiCD80 antibody could prevent the development of experimental autoimmune encephalitis (EAE), a T-cell-dependent disease and animal model of MS. Interestingly, anti-CD86 antibodies could increase disease severity of EAE.29 CD80 and CD86 expressed on B cells are required for T-celldependent immunoglobulin production.30 Here, we describe increased expression of CD80 and CD86 on B cells of patients with MS. Furthermore, we demonstrated that CD80 expression, but not CD86, was modulated by Dicer. The exact mechanism of decreased Dicer expression in patients with MS is not clear at this time. Dicer was found to be critical in many biological processes which are also important for MS pathogenesis. One may hypothesize that decreased Dicer expression leads to impaired miRNA biogenesis and abnormal expression of protein-coding genes involved in immune response regulation and cell traffic to the central nervous system in patients with MS. For example, inhibition of Dicer in tumor cells was associated with enhanced expression of metallic matrix proteases MMP-2 and MMP-9,31 enzymes implicated in increased permeability of the blood–brain barrier and MS pathogenesis.32 Based on our results, Dicer was selectively decreased in B cells but not in monocytes in patients with MS (Figure 1). It is known that Dicer expression may be cell lineage specific. For example, Dicer expression is increased
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Multiple Sclerosis Journal
Figure 3. CD80 expression is increased upon silencing Dicer. Ramos B cells or B cells from healthy human subjects were transfected with Dicer siRNA or control siRNA. Dicer expression was tested by Western blot. CD80 and CD86 expression was examined by flow cytometry as described in Materials and methods. Figure 3(a) depicts Ramos B cells Western blots from a representative experiment. The average fold change of CD80 and CD86 expression from six different experiments with Ramos B cells is shown in Figure 3(b) (*p = 0.0167). The average fold change of CD80 and CD86 expression from experiments with six different human subjects is shown in Figure 3(c) (*p = 0.027).
in melanocytes, that occurs via direct transcriptional targeting of Dicer by the melanocyte master transcriptional regulator (MITF).33 In summary, we have found that Dicer expression is selectively decreased in B cells of patients with
relapsing form of MS and is linked to increased expression of co-stimulatory molecule CD80. This may contribute to increased activation of T-cell responses in MS. The cause of decreased Dicer expression in B cells of patients with MS remains to be identified.
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LL Aung and KE Balashov et al. Acknowledgements We thank Joan Moore for proof reading of the article. Conflicts of interest The authors have no commercial conflicts of interests or relationships which will be affected by the article. Dr. Balashov has served as a consultant for Bayer, Novartis and Sandoz. He has been funded by NIH, NMSS, and has received research support from Biogen Idec, TEVA Neuroscience, and Patterson Trust. Funding The study was supported in part by grant number K23NS052553 from the National Institute of Neurological Disorders and Stroke.
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