of oligodendroglial differentiation of oligodendroglial progenitor cells in Theiler’s murine encephalomyelitis
Yanyong Sun*†, Arno Kalkuhl‡, Ulrich Deschl‡, Wenhui Sun*†, Karl Rohn§, Wolfgang Baumgärtner*†, Reiner Ulrich*
*Department of Pathology, University of Veterinary Medicine Hannover, Germany,
†Centre for Systems Neuroscience Hannover, Germany, ‡Department of Non-Clinical Drug Safety, Boehringer Ingelheim Pharma, Biberach (Riß), Germany, and
§Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Germany
Manuscript in preparation
Running title: STAT3 in TME
Keywords: Janus kinase; Microarray; Multiple sclerosis; Oligodendrocyte progenitor cell; Signal transducer and activator of transcription;; Theiler’s murine
encephalomyelitis
Correspondence: Dr. med. vet. Reiner Ulrich. Ph.D., Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany. Tel: +49511-953-8670; Fax: +49511-953-8675; E-mail:
Reiner.Ulrich@tiho-hannover.de
Abstract
Aims: Insufficient oligodendroglial differentiation of oligodendroglial progenitor cells (OPCs) is responsible for remyelination failure and astroglial scar formation in Theiler's murine encephalomyelitis (TME). The aim of the present study is to identify molecular key regulators of OPC differentiation in TME, and to proof their mechanism of action in vitro. Methods: TME virus (TMEV) infected SJL/J-mice were evaluated by rotarod analysis, histopathology, immunohistology, and gene expression microarray analysis. The signal transducer and activator of transcription (STAT)3 pathway was activated using meteorin and inhibited using STAT3 inhibitor VII in the glial progenitor cell line BO-1 in vitro. Results: Immunohistology demonstrated a progressively decreasing percentage of myelin basic protein-positive white matter area in TME. In contrast, intralesional nerve/glial antigen 2 (NG2)-positive OPCs as well as glial fibrillary acidic protein (GFAP)-positive astrocytes were increased. Gene Set Enrichment Analysis revealed 26 Gene Ontology terms including Janus kinase (JAK)-STAT cascade to be significantly positively correlated with the density of NG2-positive OPCs. Immunohistology revealed an increased amount of activated, phosphorylated STAT3 (p-STAT3)-expressing cells within the lesions. Most p-STAT3-expressing cells co-expressed GFAP, followed by CD107b and NG2.
Activation or inhibition of STAT3-signaling in BO-1 cells cultured in astrocytic differentiation medium enhanced GFAP- or CNPase-expression, respectively.
Conclusions: Activation of the STAT3 pathway in OPCs is a key regulator shifting their differentiation from an oligodendroglial towards an astrocytic fate, thereby inducing astrogliosis and insufficient remyelination in TME.
Introduction
Multiple sclerosis (MS) is an etiologically unresolved, demyelinating central nervous system (CNS) disease, affecting more than 2.5 million patients worldwide [1]. MS is characterized by an infiltration of inflammatory cells with varying amounts of demyelination, remyelination, astrogliosis and axonal injury [1-3]. Pathomorphological changes in active demyelinating MS lesions suggest different pathomechanisms and a heterogeneous etiology of the disease [4]. Active demyelinating MS plaques can be divided in lesions suggestive of a primary immune-mediated pathogenesis and those possibly due to primary oligodendropathy. Accordingly, 30% of MS cases display an extensive destruction of oligodendrocytes, whereas in the remaining 70%, oligodendrocyte numbers are only reduced in areas of active myelin destruction and reappear in remyelinated areas [5]. A subset of approximately 20% of all MS patients exhibits extensive remyelination, whereas especially those patients with a progressive clinical course display only limited or absent remyelination [6-9]. The demyelinated axons in these chronic lesions are known to be vulnerable to secondary progressive axonal degeneration and atrophy [1,10,11]. Experimental studies show that the proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) is a prerequisite for remyelination and functional recovery, whereas surviving oligodendrocytes are relatively quiescent and do not participate in remyelination [12,13]. Many MS lesions are known to contain OPCs. However, they frequently lack differentiation to myelinating oligodendrocytes [8,14-17]. A similar situation of a progressive demyelination with insufficient remyelination despite the presence of an at least transiently increased number of intralesional OPCs is reported in the Theiler´s murine encephalomyelitis (TME) virus (TMEV)-induced model of demyelination [18].
In contrast to TME, a fast and complete remyelination can be observed in most other models of MS, including experimental autoimmune encephalomyelitis (EAE) [19], cuprizone toxicity [20], lysolecithin-induced demyelination in the caudal cerebellar peduncle [21], and murine hepatitis virus (MHV) A-59 strain infection of mice [22], which renders TME to be the most useful model to investigate chronic progressive demyelination with remyelination failure. The principal reasons for a failure of
remyelination can either be a depletion of OPCs, inability of OPCs to proliferate and/or differentiate within the lesion due to a non-permissive environment; axonal loss or the inability of chronically demyelinated axons to be remyelinated [18,23,24].
Cumulative experimental evidence currently suggests that a block of oligodendroglial differentiation seems to represent the key factor in remyelination failure in MS [17,24].
Based on previous own experiments that suggested a shift from oligodendroglial towards astrocytic differentiation of OPCs in TME [18,25], we modified the hypothesis of TMEV-infection directly or indirectly induces a dysregulation of the differentiation of oligodendrocyte progenitor cells (OPCs) resulting in a shift from oligodendrocytic towards an astrocytic fate. Therefore the aims of present study were: 1.) to identify the principal molecular pathways regulating the differentiation of OPCs in TME employing gene expression microarrays, 2.) to verify the cellular source and activation status of the most promising candidate pathway in situ, and 3.) to demonstrate the suggested molecular switch-like mechanism of action in vitro.
Materials and Methods Laboratory animal procedures
Mice were housed in individually ventilated cage systems (Tecniplast, Hohenpeißenberg, Germany), under controlled conditions as previously described [18,26-28].
Five-week-old female SJL/JHanHsd-mice (Harlan Winkelmann, Borchen, Germany) or SJL/JCrl-mice (Charles River Laboratories, Sulzfeld, Germany) were inoculated into the right cerebral hemisphere with 1.6×106 or 4.6x107 plaque forming units per mouse of the BeAn-strain of TMEV or vehicle control (mock-infection), respectively, in two independent experiments, as previously described [18,26,27].
The clinical course was evaluated with a rotarod assay (RotaRod Treadmill, TSE Technical & Scientific Equipment, Bad Homburg, Germany), as previously described [27,29,30]. The rod speed was linearly increased from 5 revolutions per minute (rpm) towards 55 rpm over a time period of 5 min. All living mice were repeatedly assayed at 0 days post infection (dpi, pre-infection) and multiple time points post infection. A
mean score per mouse was calculated from three trials per time point. The attained rpm at drop was analysed for significant differences between TMEV- and mock-infected mice employing independent pair-wise Mann-Whitney U-tests (IBM SPSS Statistics, Version 20, IBM Corporation, Armonk, NY, USA). Statistical significance was designated as p 0.05.
Groups of six mice were necropsied at multiple time points up to 245 dpi as previously described [18,26,30]. The spinal cord was immediately dissected and representative cervical, thoracic and lumbar segments were fixed in 10% formalin, decalcified in ethylenediaminetetraacetic acid solution and embedded in paraffin wax. The remaining parts of the spinal cord were removed from the spinal canal, immediately snap-frozen and stored at -80°C.
All animal experiments were conducted in accordance with local authorities (Regierungspräsidium Hannover, Germany, permission numbers: 33–42502-05/963 and 33-42502-04-07/1292).
Histology and immunohistology
Serial transversal sections of formalin fixed, paraffin embedded cervical, thoracic and lumbar spinal cords were stained with haematoxylin and eosin (HE) or Luxol fast blue-cresyl violet (LFB-CV) and semi-quantitatively assessed as previously described [26,30]. Briefly, each section was evaluated for meningitis and leukomyelitis as follows: 0 = no change, 1 = scattered perivascular infiltrates, 2 = 2 to 3 layers of perivascular inflammatory cells, 3 = more than 3 layers of perivascular inflammatory cells, and the degree of demyelination was evaluated as follows: 0 = no change, 1 =<
25%, 2 = 25–50%, 3 = 50–100% of white matter affected. A mean score per mouse was calculated from all three locations evaluated.
Immunohistology was performed on serial sections of the formalin fixed, paraffin embedded spinal cords using the avidin-biotin-peroxidase complex (ABC) method
(Vector Laboratories, Burlingame, CA, USA) with
3,3′-diaminobenzidine-tetrahydrochloride (DAB) as chromogen as previously described [18,26,28,30-32]. The used primary antibodies were directed against glial
fibrillary acidic protein (GFAP, polyclonal rabbit anti-cow, diluted 1:1000, Dako Diagnostika, Hamburg, Germany), myelin basic protein (MBP, polyclonal rabbit anti-human, diluted 1:800, Millipore, Schwalbach, Germany), NG2 (polyclonal rabbit anti-rat, diluted 1:200, Millipore, Schwalbach, Germany), signal transducer and activator of transcription 3 (STAT3, polyclonal rabbit anti-human, diluted 1:400, Acris, Herford, Germany), phosphorylated STAT3 (p-STAT3, monoclonal rabbit anti-human, clone EP2147Y, diluted 1:400, Millipore, Schwalbach, Germany), and TMEV (polyclonal rabbit anti-TMEV capsid protein VP1; diluted 1:2000).
For double-labelling immunohistology, we employed a sequential protocol using a rat-on-mouse alkaline phosphatase (AP) polymer kit (Biocare Medical, Concord, CA, USA), mouse-on-mouse AP polymer kit (Biocare Medical, Concord, CA, USA), or anti-rabbit AP polymer kit (Nichirei Biosciences, Tokio, Japan) with nitrotetrazolium blue chloride / 5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt as chromogen (NBT/BCIP) for the first primary antibody, followed by the ABC method (Vector Laboratories, Burlingame, CA, USA) with DAB as chromogen for the second primary antibody. Applied first primary antibodies were directed against CD107b (monoclonal rat anti-mouse, clone M3/84 diluted 1:200, AbD Serotec, Oxford, UK), GFAP (monoclonal mouse anti-pig, clone G-A-5, diluted 1:1000, Sigma-Aldrich, Munich, Germany) or NG2 (polyclonal rabbit anti-rat, diluted 1:200, Millipore, Schwalbach, Germany), and the second primary antibody was directed against p-STAT3 (monoclonal rabbit anti-human, clone EP2147Y diluted 1:400, Millipore, Schwalbach, Germany).
Negative control sections were incubated with equally diluted normal rabbit serum (Sigma-Aldrich, Munich, Germany, R9759), a mouse IgG1 anti-isotype antibody (Millipore, Schwalbach, Germany, CBL600), or a rabbit monoclonal anti-human cytokeratin 20 antibody (clone EPR1622Y, diluted 1:400, Millipore, Schwalbach, Germany) instead of the primary antibodies.
The density of GFAP-, NG2-, STAT3-, p-STAT3- and TMEV-positive cells within the spinal cords was counted as described [18,27,28,30]. The MBP-positive white matter area was measured employing the analysis 3.1 software package (SOFT Imaging
system, Münster, Germany) as described [18,26-28,30].
The semi-quantitative scores and cell counts were evaluated for significant differences between TMEV- and mock-infected mice using pair-wise Mann-Whitney U-tests and between lesioned white matter and normal appearing white matter (NAWM) within the TMEV-infected animals using Wilcoxon signed rank tests (IBM SPSS Statistics, Version 20, IBM Corporation, Armonk, NY, USA). Statistical significance was designated as p 0.05.
Microarray analysis
For microarray analysis RNA was isolated from snap-frozen spinal cord specimens using the RNeasy Mini Kit (Qiagen, Hilden, Germany), amplified and labelled employing the MessageAmp II Biotin Enhanced Kit (Ambion, Austin, TX) and hybridized to GeneChip mouse genome 430 2.0 arrays (Affymetrix, Santa Clara, CA) as previously described [30,33]. Background adjustment and quantile normalization was performed using RMAexpress [34]. MIAME compliant data sets are deposited in the ArrayExpress database (E-MEXP-1717; http://www.ebi.ac.uk/arrayexpress).
Gene Set Enrichment Analysis (GSEA), Version 2.0.10 was performed employing Pearson´s correlation coefficient as metric to rank the genes within the gene sets derived from the Gene Ontology (GO) biological process category of the Molecular Signatures Database (MSigDB) Version 3.1 according to their correlation to the mean density of NG2-positive cells per spinal cord as observed by immunohistology [30,35,36]. Orthologous official human gene symbols (HUGO), required as gene identifiers by GSEA were retrieved employing MADGene [37]. Gene sets in a size range from 10 to 100 genes with a p ≤ 0.05 and a false discovery rate q ≤ 0.25 were selected and ranked according to the normalized enrichment score [36].
Bottom-up analysis of manually selected candidate genes was done using classical statistics as previously described [11,27,38]. Accordingly, 229 candidate genes comprising ligands, receptors, members, activators, inhibitors and downstream targets of the Janus kinase (JAK)-STAT pathway, were manually extracted from the canonical JAK-STAT pathway of the Kyoto Encyclopedia of Genes and Genomes
(KEGG) database (Entry: map04630) and peer-reviewed published literature (Supplemental Table 1) [39]. The normalized expression values of these genes were evaluated for significant differences between TMEV- and mock-infected mice employing independent pair-wise Mann-Whitney U-tests (IBM SPSS Statistics, Version 20, IBM Corporation, Armonk, NY, USA). Statistical significance was designated as p 0.05.
Cell culture
The glial progenitor cell line BO-1 was maintained in vitro as previously described [25,40]. For the proliferation and differentiation studies, cells were seeded into 96-well microtiter plates (Nunc, Wiesbaden, Germany) at a density of 5,000 cells/well and cultured in B104-conditioned Dulbecco's modified Eagle's medium (DMEM) (proliferation medium), B104-conditioned DMEM containing 5 μM all-trans retinoic acid (oligodendrocytic differentiation medium), or DMEM containing 20% fetal calf serum (astrocytic differentiation medium), respectively for four days. STAT3 was selectively activated or inhibited using 200 ng/ml recombinant mouse meteorin (R&D, Wiesbaden, Germany), or 0.01 μM STAT3 inhibitor VII (Calbiochem, Darmstadt, Germany), respectively [41,42].
Proliferation was assessed by Bromodeoxyuridine (BrdU) uptake using the FLUOS in situ cell proliferation kit (Roche, Mannheim, Germany) according the manufacturer´s instructions.
Cellular differentiation was assessed by immunofluorescence staining as previously described [25,40,43]. The applied primary antibodies were directed against the ganglioside antigen A2B5 (A2B5, monoclonal mouse anti-chicken, diluted 1:2, hybridoma supernatant, provided by J. Trotter, Mainz), 2’, 3’-cyclic nucleotide 3’-phosphodiesterase (CNPase; monoclonal mouse anti-human, clone 11-5B, diluted 1:800, Chemicon Europe, Hofheim/Taunus, Germany), and GFAP (monoclonal mouse anti-pig, clone G-A-5, diluted 1:400, Sigma-Aldrich, Munich, Germany).
Cy3-coupled goat anti-mouse secondary antibodies (1:200, Jackson Immunoresearch, Dianova, Hamburg, Germany) were used for visualization of the
immunoreactions. Nuclei were stained using bisbenzimide Hoechst 33258. Cell cultures were evaluated using an inverted fluorescence microscope equipped with a U-MWBP2 (BP450nm-480nm) / HQ527/30F42-527, U-MNG2 (BP530nm-550nm) / BA590 and U-MNU2 (BP360nm-370nm) / D460/50-F32-000 filter combination for Cy2-, Cy3- and bisbenzimide-fluorescence, respectively (Olympus IX-70, Hamburg, Germany).
Assessed cell counts were evaluated for significant differences using Fisher's exact tests (IBM SPSS Statistics, Version 20, IBM Corporation, Armonk, NY, USA).
Statistical significance was designated as p 0.05.
Results
Molecular pathways associated with OPCs in TME
An initial animal experiment was performed with groups of six TMEV- and mock-infected SJL/JHanHsd mice necropsied at 14, 42, 98, and 196 dpi, except a group size of five TMEV-infected mice at 98 dpi. The clinical course and pathohistological changes have been described previously [30].
MBP immunohistochemistry confirmed a progressively increasing amount of demyelination (Figure 1A-C). The morphometrically assessed MBP-positive white matter area reached a statistical significant reduction at 196 dpi in the TMEV- as compared to the mock-infected mice (Figure 2A). An increased amount of intralesional NG2-positive OPCs, mostly exhibiting an enlarged bi- or oligopolar phenotype, was observed within the demyelinating lesions (Figure 1D-F). The density of intralesional NG2-positive cells was significantly increased between 42 and 196 dpi as compared to the NAWM of TMEV- and mock-infected mice, with a peak amount at 42 dpi and a slight decline thereafter (Figure 2B). Furthermore, a progressively increasing amount of GFAP-positive astrocytes, exhibiting a reactive phenotype, was present within the demyelinating lesions (Figure 1G-I). A significantly increased density of intralesional GFAP-positive cells was detected from 42 to 196 dpi as compared to the NAWM of TMEV- and mock-infected mice (Figure 2C). TMEV antigen was detectable in the spinal cords of TMEV-infected animals only (Figure
1J-L). The number of TMEV antigen-positive cells was significantly increased from 14 to 196 dpi in TMEV-infected mice as compared to the mock-infected mice (Figure 2D).
As described in detail previously, a total of 1210 probe sets (1001 genes) were identified to be differentially expressed between TMEV- and mock-infected mice in the spinal cord over the study period at a false discovery rate of 1.0% [30]. In order to focus on transcriptional changes intimately associated with the population of NG2-positive OPCs in the present study, we performed GSEA to identify pathways co-regulated with the density of NG2-positive cells within the spinal cords as revealed by immunohistology. Accordingly, GSEA revealed 26 GO terms significantly positively correlated and no GO terms significantly negatively correlated with the mean density of NG2-positive cells within the spinal cords (Table 1). A leading edge analysis revealed a prominent cluster of 6 GO terms related to tyrosine phosphorylation and the JAK-STAT cascade which are all positively correlated with the density of OPCs (Supplemental Figure 1). Further clusters of GO terms are related to response to other organism, cytokine biosynthesis, and the nuclear factor κβ pathway.
Since GSEA suggested an important association of JAK-STAT signaling with the fate of OPCs in chronic demyelinating TME, a manually curated list of 229 genes involved in JAK-STAT signaling was analysed (Supplemental Table 1). Thereby, 145 of 229 genes involved in JAK-STAT signaling were found to be differentially expressed in TMEV- as compared to mock-infected mice at one or more of the studied time points.
Accordingly, an up-regulation of Jak1, Jak2, Jak3, Stat1, Stat2, Stat3, Stat4, and Stat6 was observed at various time points (Figure 3). Multiple inhibitory molecules including the suppressor of cytokine signaling proteins (Socs)1, Socs2, Socs3 and protein tyrosine phosphatase, non-receptor type 6 (Ptpn6, Synonym SHP-1) were also up-regulated at certain time points, whereas the protein inhibitor of activated STAT2 (Pias2) was temporarily down-regulated. Concerning upstream ligands, interleukin 10 (Il10), Il15, interferon gamma (Ifng), oncostatin M (Osm), bone morphogenic protein 4 (Bmp4), ciliary neurotrophic factor (Cntf) and tumor necrosis factor (Tnf) were up-regulated, whereas Meteorin (Metrn) and platelet-derived growth
factor, D polypeptide (Pdgfd) were down-regulated respectively at various time points.
Furthermore, constantly up-regulated downstream targets included Gfap, and the Proto-oncogene serine/threonine-protein kinase proviral integration site 1 (Pim1).
Cellular origin and activation status of the STAT3 pathway in TME
Since STAT3 signaling is known to be an important pathway involved in the control of proliferation and differentiation of neuroglial progenitor cells and astrocytes [44-46], we focussed on STAT3 in our further studies. A second independent animal experiment was performed with groups of six TMEV- and mock-infected SJL/JCrl mice necropsied at 0, 4, 7, 14, 28, 42, 56, 98, 147, 196, and 245 dpi to investigate the cellular origin and activation status of STAT3 with a higher temporal resolution.
Rotarod analysis revealed a slightly increased motor performance at 14 and 28 dpi and a progressively decreasing motor performance from 42 to 245 dpi in TMEV- as compared to mock-infected mice (Figure 4).
Semi-quantitative pathohistological examination of the spinal cords revealed a significantly elevated lymphohistioplasmacytic meningitis and leukomyelitis starting at 4 and 14 dpi, respectively in TMEV- as compared to mock-infected mice (Figure 5A, 6). The extent of these inflammatory changes increased towards a plateau phase from 147 to 245 dpi. A significantly elevated and progressively increasing amount of demyelination was detected from 42 to 245 dpi in TMEV- as compared to mock-infected mice (Figure 5B, 6). The inflammatory as well as the demyelinating changes were mainly found in the thoracic and cervical spinal cord, whereas the lumbal segments displayed only minor changes. None of the mock-infected mice showed demyelinating lesions.
The morphometrically assessed MBP-positive white matter area showed a statistical significant reduction in the TMEV- as compared to the mock-infected mice at 196 and 245 dpi (Figure 7A). Furthermore, a significantly increased density of intralesional GFAP-positive cells was detected from 28 to 245 dpi (Figure 7B). TMEV antigen was detectable in the spinal cords of TMEV-infected animals only, and the number of TMEV antigen-positive cells was significantly increased at 42 dpi and from 147 to 245
dpi in TMEV- as compared to the mock-infected mice (Figure 7C). The density of intralesional STAT3- as well as p-STAT3-positive cells was significantly increased between 14 and 245 dpi (Figure 7D-E). Both, the density of STAT3- and p-STAT3-positive cells displayed a significant, moderate, positive correlation to the density of GFAP-positive cells (Table 2). Furthermore, p-STAT3 exhibited a significant, high, positive correlation to the density of TMEV antigen-positive cells. Most of the STAT3-positive cells exhibited a medium size, bi- or oligopolar spindloid morphology, moderate amount of cytoplasm and central euchromatic oval nuclei resembling astrocytes (Figure 8 A-C). 21% of the STAT3-positive cells in the normal appearing white matter areas of TMEV- and mock-infected mice displayed a nuclear immunoreactivity, whereas an increased percentage of 36% cells with nuclear STAT3 immunoreactivity was observed within the lesions and interpreted as activated STAT3 signaling (Figure 8B-C). This is supported by the exclusively nuclear staining pattern of the phosphorylation-sensitive p-STAT3 antibody (Figure 8D-E). Based on cellular morphology, approximately 78% of the intralesional p-STAT3-positive cells were suggested to be astrocytes, followed by 18% microglia/macrophages and 4% other non-classifiable cells (Figure 7F).
Double-labelling immunohistology showed approximately 40% p-STAT3- and GFAP-positive astrocytes (Figure 8G), 10% p-STAT3- and NG2-positive OPCs (Figure 8H), and 10% p-STAT3- and CD107b-positive microglia/macrophages with Gitter cell morphology (Figure 8I), within the lesioned areas.
Effect of STAT3 activation on cultured BO-1 cells
The basal proliferation rate of BO-1 cells grown in the different media was studied using BrdU labelling (Figure 9A-C). Accordingly, nuclear BrdU-uptake was detectable in 24%, 22% and 5% of the cells cultured in proliferation medium, oligodendrocytic differentiation medium and astrocytic differentiation medium, respectively (Figure 10A). Activation of the STAT3 pathway employing meteorin resulted in no significant alteration of the proliferation rate (Figure 11A-C). In contrast, inhibition of the STAT3 pathway using STAT3 inhibitor VII resulted in severe to complete abrogation of cell