Transcript analysis of laser capture microdissected white matter astrocytes and higher phenol sulfotransferase 1A1 expression during autoimmune neuroinflammation

Journal of Neuroinflammation

Arnaud B. Nicot²  David A. Laplaud²  Sophie Brouard¹  Marion Salou²  Alexandra Garcia¹  Flora Guillot² 

[1] CESTI/ITUN, CHU de Nantes, Nantes, France;Université de Nantes, Faculté de Médecine, Nantes, France

关键词: Estrogen metabolism;    Astrogliosis;    Radial glia;    Fibrous astrocyte;    Multiple sclerosis;   

Others  :  1221915
DOI  :  10.1186/s12974-015-0348-y

 received in 2014-10-11, accepted in 2015-06-04,  发布年份 2015

【 摘 要 】

Background

Astrocytes, the most abundant cell population in mammal central nervous system (CNS), contribute to a variety of functions including homeostasis, metabolism, synapse formation, and myelin maintenance. White matter (WM) reactive astrocytes are important players in amplifying autoimmune demyelination and may exhibit different changes in transcriptome profiles and cell function in a disease-context dependent manner. However, their transcriptomic profile has not yet been defined because they are difficult to purify, compared to gray matter astrocytes. Here, we isolated WM astrocytes by laser capture microdissection (LCM) in a murine model of multiple sclerosis to better define their molecular profile focusing on selected genes related to inflammation. Based on previous data indicating anti-inflammatory effects of estrogen only at high nanomolar doses, we also examined mRNA expression for enzymes involved in steroid inactivation.

Methods

Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL6 mice with MOG _35–55immunization. Fluorescence activated cell sorting (FACS) analysis of a portion of individual spinal cords at peak disease was used to assess the composition of immune cell infiltrates. Using custom Taqman low-density-array (TLDA), we analyzed mRNA expression of 40 selected genes from immuno-labeled laser-microdissected WM astrocytes from lumbar spinal cord sections of EAE and control mice. Immunohistochemistry and double immunofluorescence on control and EAE mouse spinal cord sections were used to confirm protein expression in astrocytes.

Results

The spinal cords of EAE mice were infiltrated mostly by effector/memory T CD4+ cells and macrophages. TLDA-based profiling of LCM-astrocytes identified EAE-induced gene expression of cytokines and chemokines as well as inflammatory mediators recently described in gray matter reactive astrocytes in other murine CNS disease models. Strikingly, SULT1A1, but not other members of the sulfotransferase family, was expressed in WM spinal cord astrocytes. Moreover, its expression was further increased in EAE. Immunohistochemistry on spinal cord tissues confirmed preferential expression of this enzyme in WM astrocytic processes but not in gray matter astrocytes.

Conclusions

We described here for the first time the mRNA expression of several genes in WM astrocytes in a mouse model of multiple sclerosis. Besides expected pro-inflammatory chemokines and specific inflammatory mediators increased during EAE, we evidenced relative high astrocytic expression of the cytoplasmic enzyme SULT1A1. As the sulfonation activity of SULT1A1 inactivates estradiol among other phenolic substrates, its high astrocytic expression may account for the relative resistance of this cell population to the anti-neuroinflammatory effects of estradiol. Blocking the activity of this enzyme during neuroinflammation may thus help the injured CNS to maintain the anti-inflammatory activity of endogenous estrogens or limit the dose of estrogen co-regimens for therapeutical purposes.

【 授权许可】

   
2015 Guillot et al.

【 图 表 】

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【 参考文献 】

• [1]Baumann N, Pham-Dinh D: Biology of oligodendrocyte and myelin in the mammalian central nervous system. Physiol Rev 2001, 81:871-927.
• [2]Farina C, Aloisi F, Meinl E: Astrocytes are active players in cerebral innate immunity. Trends Immunol 2007, 28:138-45.
• [3]Brambilla R, Persaud T, Hu X, Karmally S, Shestopalov VI, Dvoriantchikova G, et al.: Transgenic inhibition of astroglial NF-kappa B improves functional outcome in experimental autoimmune encephalomyelitis by suppressing chronic central nervous system inflammation. J Immunol 2009, 182:2628-40.
• [4]Brambilla R, Morton PD, Ashbaugh JJ, Karmally S, Lambertsen KL, Bethea JR: Astrocytes play a key role in EAE pathophysiology by orchestrating in the CNS the inflammatory response of resident and peripheral immune cells and by suppressing remyelination. Glia 2014, 62:452-67.
• [5]Lieury A, Chanal M, Androdias G, Reynolds R, Cavagna S, Giraudon P, et al.: Tissue remodeling in periplaque regions of multiple sclerosis spinal cord lesions. Glia 2014, 62:1645-58.
• [6]Lock C, Hermans G, Pedotti R, Brendolan A, Schadt E, Garren H, et al.: Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med 2002, 8:500-8.
• [7]Nicot A, Ratnakar PV, Ron Y, Chen CC, Elkabes S: Regulation of gene expression in experimental autoimmune encephalomyelitis indicates early neuronal dysfunction. Brain 2003, 126:398-412.
• [8]Steinman L, Zamvil S: Transcriptional analysis of targets in multiple sclerosis. Nat Rev Immunol 2003, 3:483-92.
• [9]Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, et al.: Laser capture microdissection. Science 1996, 274:998-1001.
• [10]Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, et al.: A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 2008, 28:264-78.
• [11]Zamanian JL, Xu L, Foo LC, Nouri N, Zhou L, Giffard RG, et al.: Genomic analysis of reactive astrogliosis. J Neurosci 2012, 32:6391-410.
• [12]Vegeto E, Belcredito S, Ghisletti S, Meda C, Etteri S, Maggi A: The endogenous estrogen status regulates microglia reactivity in animal models of neuroinflammation. Endocrinology 2006, 147:2263-72.
• [13]Giraud SN, Caron CM, Pham-Dinh D, Kitabgi P, Nicot AB: Estradiol inhibits ongoing autoimmune neuroinflammation and NFkappaB-dependent CCL2 expression in reactive astrocytes. Proc Natl Acad Sci U S A 2010, 107:8416-21.
• [14]Raftogianis R, Creveling C, Weinshilboum R, Weisz J. Estrogen metabolism by conjugation. J Natl Cancer Inst Monogr. 2000;113–124.
• [15]Gourdain P, Ballerini C, Nicot AB, Carnaud C: Exacerbation of experimental autoimmune encephalomyelitis in prion protein (PrPc)-null mice: evidence for a critical role of the central nervous system. J Neuroinflammation 2012, 9:25. BioMed Central Full Text
• [16]Lipton HL, Kallio P, Jelachich ML: Simplified quantitative analysis of spinal cord cells from Theiler’s virus-infected mice without the requirement for myelin debris removal. J Immunol Methods 2005, 299:107-15.
• [17]Demarest TG, Murugesan N, Shrestha B, Pachter JS: Rapid expression profiling of brain microvascular endothelial cells by immuno-laser capture microdissection coupled to TaqMan® low density array. J Neurosci Methods 2012, 206:200-4.
• [18]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 2001, 25:402-8.
• [19]Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al.: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3(7):0034.
• [20]Brown AL, Smith DW: Improved RNA preservation for immunolabeling and laser microdissection. RNA 2009, 15:2364-74.
• [21]Okaty BW, Sugino K, Nelson SB: A quantitative comparison of cell-type-specific microarray gene expression profiling methods in the mouse brain. PLoS One 2011., 6Article ID e16493
• [22]Thomsen R, Daugaard TF, Holm IE, Nielsen AL: Alternative mRNA splicing from the glial fibrillary acidic protein (GFAP) gene generates isoforms with distinct subcellular mRNA localization patterns in astrocytes. PLoS One 2013., 8Article ID e72110
• [23]Kigerl KA, Lai W, Rivest S, Hart RP, Satoskar AR, Popovich PG: Toll-like receptor (TLR)-2 and TLR-4 regulate inflammation, gliosis, and myelin sparing after spinal cord injury. J Neurochem 2007, 102:37-50.
• [24]Lee S, Park JY, Lee WH, Kim H, Park HC, Mori K, et al.: Lipocalin-2 is an autocrine mediator of reactive astrocytosis. J Neurosci 2009, 29:234-49.
• [25]Berard JL, Zarruk JG, Arbour N, Prat A, Yong VW, Jacques FH, et al.: Lipocalin 2 is a novel immune mediator of experimental autoimmune encephalomyelitis pathogenesis and is modulated in multiple sclerosis. Glia 2012, 60:1145-59.
• [26]Inglis HR, Greer JM, McCombe PA: Gene expression in the spinal cord in female Lewis rats with experimental autoimmune encephalomyelitis induced with myelin basic protein. PLoS One 2012., 7Article ID e48555
• [27]Marques F, Mesquita SD, Sousa JC, Coppola G, Gao F, Geschwind DH, et al.: Lipocalin 2 is present in the EAE brain and is modulated by natalizumab. Front Cell Neurosci 2012, 6:33.
• [28]Quirie A, Demougeot C, Bertrand N, Mossiat C, Garnier P, Marie C, et al.: Effect of stroke on arginase expression and localization in the rat brain. Eur J Neurosci 2013, 37:1193-202.
• [29]Carmody RJ, Hilliard B, Maguschak K, Chodosh LA, Chen YH: Genomic scale profiling of autoimmune inflammation in the central nervous system: the nervous response to inflammation. J Neuroimmunol 2002, 133:95-107.
• [30]Ahn M, Yang W, Kim H, Jin JK, Moon C, Shin T: Immunohistochemical study of arginase-1 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis. Brain Res 2012, 1453:77-86.
• [31]Renno T, Krakowski M, Piccirillo C, Lin JY, Owens T: TNF-alpha expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental allergic encephalomyelitis. Regulation by Th1 cytokines. J Immunol 1995, 154:944-53.
• [32]Korn T, Magnus T, Jung S: Autoantigen specific T cells inhibit glutamate uptake in astrocytes by decreasing expression of astrocytic glutamate transporter GLAST: a mechanism mediated by tumor necrosis factor-alpha. Faseb J 2005, 19:1878-80.
• [33]Steelman AJ, Li J: Poly(I:C) promotes TNFalpha/TNFR1-dependent oligodendrocyte death in mixed glial cultures. J Neuroinflammation 2011, 8:89. BioMed Central Full Text
• [34]Mandolesi G, Musella A, Gentile A, Grasselli G, Haji N, Sepman H, et al.: Interleukin-1beta alters glutamate transmission at purkinje cell synapses in a mouse model of multiple sclerosis. J Neurosci 2013, 33:12105-21.
• [35]Stone KP, Kastin AJ, Pan W: NFkB is an unexpected major mediator of interleukin-15 signaling in cerebral endothelia. Cell Physiol Biochem 2011, 28:115-24.
• [36]Wu X, Pan W, He Y, Hsuchou H, Kastin AJ: Cerebral interleukin-15 shows upregulation and beneficial effects in experimental autoimmune encephalomyelitis. J Neuroimmunol 2011, 223:65-72.
• [37]Lavisse S, Guillermier M, Herard AS, Petit F, Delahaye M, Van Camp N, et al.: Reactive astrocytes overexpress TSPO and are detected by TSPO positron emission tomography imaging. J Neurosci 2012, 32:10809-18.
• [38]Luchetti S, van Eden CG, Schuurman K, van Strien ME, Swaab DF, Huitinga I: Gender differences in multiple sclerosis: induction of estrogen signaling in male and progesterone signaling in female lesions. J Neuropathol Exp Neurol 2014, 73:123-35.
• [39]Murugaiyan G, Mittal A, Weiner HL: Increased osteopontin expression in dendritic cells amplifies IL-17 production by CD4+ T cells in experimental autoimmune encephalomyelitis and in multiple sclerosis. J Immunol 2008, 181:7480-8.
• [40]Ma N, He Y, Xiao H, Han G, Chen G, Wang Y, et al.: BAFF maintains T-cell survival by inducing OPN expression in B cells. Mol Immunol 2014, 57:129-37.
• [41]Sinclair C, Mirakhur M, Kirk J, Farrell M, McQuaid S: Up-regulation of osteopontin and alphaBeta-crystallin in the normal-appearing white matter of multiple sclerosis: an immunohistochemical study utilizing tissue microarrays. Neuropathol Appl Neurobiol 2005, 31:292-303.
• [42]Spence RD, Wisdom AJ, Cao Y, Hill HM, Mongerson CR, Stapornkul B, et al.: Estrogen mediates neuroprotection and anti-inflammatory effects during EAE through ERalpha signaling on astrocytes but not through ERbeta signaling on astrocytes or neurons. J Neurosci 2013, 33:10924-33.
• [43]Baranzini SE, Bernard CC, Oksenberg JR: Modular transcriptional activity characterizes the initiation and progression of autoimmune encephalomyelitis. J Immunol 2005, 174:7412-22.
• [44]Baranzini SE, Elfstrom C, Chang SY, Butunoi C, Murray R, Higuchi R, et al.: Transcriptional analysis of multiple sclerosis brain lesions reveals a complex pattern of cytokine expression. J Immunol 2000, 165:6576-82.
• [45]Simpson JE, Newcombe J, Cuzner ML, Woodroofe MN: Expression of monocyte chemoattractant protein-1 and other beta-chemokines by resident glia and inflammatory cells in multiple sclerosis lesions. J Neuroimmunol 1998, 84:238-49.
• [46]Van Der Voorn P, Tekstra J, Beelen RH, Tensen CP, Van Der Valk P, De Groot CJ: Expression of MCP-1 by reactive astrocytes in demyelinating multiple sclerosis lesions. Am J Pathol 1999, 154:45-51.
• [47]Boven LA, Montagne L, Nottet HS, De Groot CJ: Macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and RANTES mRNA semiquantification and protein expression in active demyelinating multiple sclerosis (MS) lesions. Clin Exp Immunol 2000, 122:257-63.
• [48]Meeuwsen S, Persoon-Deen C, Bsibsi M, Ravid R, van Noort JM: Cytokine, chemokine and growth factor gene profiling of cultured human astrocytes after exposure to proinflammatory stimuli. Glia 2003, 43:243-53.
• [49]Carpentier PA, Begolka WS, Olson JK, Elhofy A, Karpus WJ, Miller SD: Differential activation of astrocytes by innate and adaptive immune stimuli. Glia 2005, 49:360-74.
• [50]Falsig J, Porzgen P, Lund S, Schrattenholz A, Leist M: The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem 2006, 96:893-907.
• [51]Lisak RP, Benjamins JA, Bealmear B, Nedelkoska L, Studzinski D, Retland E, et al.: Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for molecules associated with metabolism, signaling and regulation in central nervous system mixed glial cell cultures. J Neuroinflammation 2009, 6:4. BioMed Central Full Text
• [52]Shrestha B, Ge S, Pachter JS: Resolution of central nervous system astrocytic and endothelial sources of CCL2 gene expression during evolving neuroinflammation. Fluids Barriers CNS 2014, 11:6. BioMed Central Full Text
• [53]Prins M, Dutta R, Baselmans B, Breve JJ, Bol JG, Deckard SA, et al.: Discrepancy in CCL2 and CCR2 expression in white versus grey matter hippocampal lesions of multiple sclerosis patients. Acta Neuropathol Commun 2014, 2:98. BioMed Central Full Text
• [54]McCandless EE, Wang Q, Woerner BM, Harper JM, Klein RS: CXCL12 limits inflammation by localizing mononuclear infiltrates to the perivascular space during experimental autoimmune encephalomyelitis. J Immunol 2006, 177:8053-64.
• [55]Krumbholz M, Theil D, Cepok S, Hemmer B, Kivisakk P, Ransohoff RM, et al.: Chemokines in multiple sclerosis: CXCL12 and CXCL13 up-regulation is differentially linked to CNS immune cell recruitment. Brain 2006, 129:200-11.
• [56]Ludwig A, Schulte A, Schnack C, Hundhausen C, Reiss K, Brodway N, et al.: Enhanced expression and shedding of the transmembrane chemokine CXCL16 by reactive astrocytes and glioma cells. J Neurochem 2005, 93:1293-303.
• [57]Hendrickx DA, Koning N, Schuurman KG, van Strien ME, van Eden CG, Hamann J, et al.: Selective upregulation of scavenger receptors in and around demyelinating areas in multiple sclerosis. J Neuropathol Exp Neurol 2013, 72:106-18.
• [58]Holmoy T, Loken-Amsrud KI, Bakke SJ, Beiske AG, Bjerve KS, Hovdal H, et al.: Inflammation markers in multiple sclerosis: CXCL16 reflects and may also predict disease activity. PLoS One 2013., 8Article ID e75021
• [59]Wojkowska DW, Szpakowski P, Ksiazek-Winiarek D, Leszczynski M, Glabinski A: Interactions between neutrophils, Th17 cells, and chemokines during the initiation of experimental model of multiple sclerosis. Mediators Inflamm 2014, 2014:590409.
• [60]Kim JV, Jiang N, Tadokoro CE, Liu L, Ransohoff RM, Lafaille JJ, et al.: Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites. J Immunol Methods 2009, 352:89-100.
• [61]Fukumoto N, Shimaoka T, Fujimura H, Sakoda S, Tanaka M, Kita T, et al.: Critical roles of CXC chemokine ligand 16/scavenger receptor that binds phosphatidylserine and oxidized lipoprotein in the pathogenesis of both acute and adoptive transfer experimental autoimmune encephalomyelitis. J Immunol 2004, 173:1620-7.
• [62]Rosito M, Lauro C, Chece G, Porzia A, Monaco L, Mainiero F, et al.: Trasmembrane chemokines CX3CL1 and CXCL16 drive interplay between neurons, microglia and astrocytes to counteract pMCAO and excitotoxic neuronal death. Front Cell Neurosci 2014, 8:193.
• [63]Martin-Lopez E, Garcia-Marques J, Nunez-Llaves R, Lopez-Mascaraque L: Clonal astrocytic response to cortical injury. PLoS One 2013., 8Article ID e74039
• [64]Slezak M, Goritz C, Niemiec A, Frisen J, Chambon P, Metzger D, et al.: Transgenic mice for conditional gene manipulation in astroglial cells. Glia 2007, 55:1565-76.
• [65]Yang Y, Vidensky S, Jin L, Jie C, Lorenzini I, Frankl M, et al.: Molecular comparison of GLT1+ and ALDH1L1+ astrocytes in vivo in astroglial reporter mice. Glia 2011, 59:200-7.
• [66]Ohgoh M, Hanada T, Smith T, Hashimoto T, Ueno M, Yamanishi Y, et al.: Altered expression of glutamate transporters in experimental autoimmune encephalomyelitis. J Neuroimmunol 2002, 125:170-8.
• [67]Pitt D, Nagelmeier IE, Wilson HC, Raine CS: Glutamate uptake by oligodendrocytes: Implications for excitotoxicity in multiple sclerosis. Neurology 2003, 61:1113-20.
• [68]Doyle JP, Dougherty JD, Heiman M, Schmidt EF, Stevens TR, Ma G, et al.: Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell 2008, 135:749-62.
• [69]Xing L, Goswami M, Trudeau VL: Radial glial cell: critical functions and new perspective as a steroid synthetic cell. Gen Comp Endocrinol 2014, 203C:181-5.
• [70]Noorbakhsh F, Ellestad KK, Maingat F, Warren KG, Han MH, Steinman L, et al.: Impaired neurosteroid synthesis in multiple sclerosis. Brain 2011, 134:2703-21.
• [71]Giraud SN, Seilhean D, Pham-Dinh D, Nicot AB: White matter reactive astrocytes express nuclear estrogen receptor alpha in experimental autoimmune encephalomyelitis and multiple sclerosis. J Neuroimmunol 2010, 248:27.
• [72]Rohn KJ, Cook IT, Leyh TS, Kadlubar SA, Falany CN: Potent inhibition of human sulfotransferase 1A1 by 17alpha-ethinylestradiol: role of 3′-phosphoadenosine 5′-phosphosulfate binding and structural rearrangements in regulating inhibition and activity. Drug Metab Dispos 2012, 40:1588-95.
• [73]Subramanian S, Matejuk A, Zamora A, Vandenbark AA, Offner H: Oral feeding with ethinyl estradiol suppresses and treats experimental autoimmune encephalomyelitis in SJL mice and inhibits the recruitment of inflammatory cells into the central nervous system. J Immunol 2003, 170:1548-55.
• [74]Durand-Dubief F, El-Etr M, Ionescu I, Bracoud L, Cotton F, Merle H, et al.: The POPARTMUS French-Italian multicentric trial of postpartum progestin and estradiol in multiple sclerosis: MRI findings. P063, ACTRIMS-ECTRIMS MSBoston 2014. Multiple Sclerosis J 2014, 20:95.
• [75]Raftogianis RB, Wood TC, Otterness DM, Van Loon JA, Weinshilboum RM: Phenol sulfotransferase pharmacogenetics in humans: association of common SULT1A1 alleles with TS PST phenotype. Biochem Biophys Res Commun 1997, 239:298-304.
• [76]Schulze J, Johansson M, Thorngren JO, Garle M, Rane A, Ekstrom L: SULT2A1 gene copy number variation is associated with urinary excretion rate of steroid sulfates. Front Endocrinol (Lausanne) 2013, 4:88.
• [77]Bradley ME, Benner SA: Phylogenomic approaches to common problems encountered in the analysis of low copy repeats: the sulfotransferase 1A gene family example. BMC Evol Biol 2005, 5:22. BioMed Central Full Text