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Novel role for SLPI in MOG-induced EAE revealed by spinal cord expression analysis.

ABSTRACT: BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte protein (MOG) in female Dark Agouti (DA) rats is a chronic demyelinating animal model of multiple sclerosis (MS). To identify new candidate molecules involved in the evolution or repair of EAE-lesions we used Affymetrix oligonucleotide microarrays to compare the spinal cord transcriptome at the peak of EAE, during remission and at the first relapse with healthy DA rats. METHODS: Untreated DA rats and DA rats immunised with MOG protein were sacrificed at defined time points. Total RNA was isolated from spinal cord tissue and used for hybridization of Affymetrix rat genome arrays RG U34 A-C. Selected expression values were confirmed by RealTime PCR. Adult neural stem cells were incubated with recombinant secretory leukocyte protease inhibitor (SLPI). Proliferation was assessed by BrdU incorporation, cyclin D1 and HES1 expression by RealTime PCR, cell differentiation by immunofluorescence analysis and I kappa B alpha degradation by Western blot. RESULTS: Among approximately 26,000 transcripts studied more than 1,100 were differentially regulated. Focussing on functional themes, we noticed a sustained downregulation of most of the transcripts of the cholesterol biosynthesis pathway. Furthermore, we found new candidate genes possibly contributing to regenerative processes in the spinal cord. Twelve transcripts were solely upregulated in the recovery phase, including genes not previously associated with repair processes. Expression of SLPI was upregulated more than hundredfold during EAE attack. Using immunohistochemistry, SLPI was identified in macrophages, activated microglia, neuronal cells and astrocytes. Incubation of adult neural stem cells (NSC) with recombinant SLPI resulted in an increase of cell proliferation and of differentiation towards oligodendrocytes. These processes were paralleled by an upregulation of the cell-cycle promotor cyclin D1 and a suppression of the cell differentiation regulator HES1. Finally, SLPI prevented the degradation of I kappa B alpha, which may explain the suppression of the cell differentiation inhibitor HES1 suggesting a possible mechanism of oligodendroglial differentiation. CONCLUSION: We identified novel features of gene expression in the CNS during EAE, in particular the suppression of genes of cholesterol biosynthesis and a strong upregulation of SLPI, a gene which is for the first time associated with autoimmune inflammation. The capacity of SLPI to increase proliferation of adult NSC and of oligodendroglial differentiation suggests a novel role for SLPI in the promotion of tissue repair, beyond its known functions in the prevention of tissue damages by protease inhibition damage and modulation of inflammatory reactions.

SUBMITTER: Mueller AM

PROVIDER: S-EPMC2438345 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Novel role for SLPI in MOG-induced EAE revealed by spinal cord expression analysis.

Mueller Andre M AM Pedré Xiomara X Stempfl Thomas T Kleiter Ingo I Couillard-Despres Sebastien S Aigner Ludwig L Giegerich Gerhard G Steinbrecher Andreas A

Journal of neuroinflammation 20080526

<h4>Background</h4>Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte protein (MOG) in female Dark Agouti (DA) rats is a chronic demyelinating animal model of multiple sclerosis (MS). To identify new candidate molecules involved in the evolution or repair of EAE-lesions we used Affymetrix oligonucleotide microarrays to compare the spinal cord transcriptome at the peak of EAE, during remission and at the first relapse with healthy DA rats.<h4>Methods</h4>Untreated D ...[more]

PMID: 18501024

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Project description:Here we present the data obtained from a label-free quantitative proteomics analysis of soluble spinal cord extract derived from a mouse model of multiple sclerosis (EAE) and sham-induced mice. Samples were prepared offline using the FASP approach and then submitted for nano-LC-MS/MS analysis on an Orbitrap Velos instrument. After statistical evaluation of the data, 431 differentially expressed proteins (KS-test, p < 0.05) out of a total of ~1400 unique proteins were identified in the comparative spinal cord analysis (peptide FDR=0.55%).Database search and protein identification: Tandem mass spectra were extracted from .RAW files and searched using the SEQUEST-PVM v.27 (rev.9) (Eng et al., 1994) database program against a Mouse protein database downloaded as FASTA-formatted sequences from EBI-IPI (database version 3.72) which contains 56957 entries (with priority given to UniProt identifiers) as well as reverse decoy sequences to empirically assess the false identification rate. This search program was executed on a cluster computer to match the MS/MS spectra to the corresponding most highly correlated peptide sequences Mass tolerances for precursor (MS) and product ions (MS/MS) were set to 3 and 0 m/z, respectively. Searches were performed with the enzyme selectivity set to trypsin with one missed cleavage allowed and protein modifications included fixed carbamidomethylation of cysteines (57 Da). Match likelihoods were assigned a statistical confidence score using the STATQUEST probabilistic model (Kislinger et al., 2003) and candidate peptide identifications were filtered using an estimated peptide confidence score of ≥95%. A 10 ppm high accuracy mass filter accounting for isotopic shifts in the spectra was applied post-SEQUEST analysis thus improving the fidelity of protein identifications. Protein quantitation: To estimate relative protein levels, spectral counts were transformed into normalised spectral abundance factors (NSAF) as previously described (Mosley et al., 2009). Briefly, this involves dividing the spectral count (SC) of a protein by its length (Mw) and finally normalises this value to the sum of all SC/Mw.

Project description:BackgroundThe CC chemokine receptors CCR1, CCR2 and CCR5 are critical for the recruitment of mononuclear phagocytes to the central nervous system (CNS) in multiple sclerosis (MS) and other neuroinflammatory diseases. Mononuclear phagocytes are effector cells capable of phagocytosing myelin and damaging axons. In this study, we characterize the regional, temporal and cellular expression of CCR1, CCR2 and CCR5 mRNA in the spinal cord of rats with myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE). While resembling human MS, this animal model allows unique access to CNS-tissue from various time-points of relapsing neuroinflammation and from various lesional stages: early active, late active, and inactive completely demyelinated lesions.MethodsThe expression of CCR1, CCR2 and CCR5 mRNA was studied with in situ hybridization using radio labelled cRNA probes in combination with immunohistochemical staining for phenotypic cell markers. Spinal cord sections from healthy rats and rats with MOG-EAE (acute phase, remission phase, relapse phase) were analysed. In defined lesion stages, the number of cells expressing CCR1, CCR2 and CCR5 mRNA was determined. Data were statistically analysed by the nonparametric Mann-Whitney U test.ResultsIn MOG-EAE rats, extensive up-regulation of CCR1 and CCR5 mRNA, and moderate up-regulation of CCR2 mRNA, was found in the spinal cord during episodes of active inflammation and demyelination. Double staining with phenotypic cell markers identified the chemokine receptor mRNA-expressing cells as macrophages/microglia. Expression of all three receptors was substantially reduced during clinical remission, coinciding with diminished inflammation and demyelination in the spinal cord. Healthy control rats did not show any detectable expression of CCR1, CCR2 or CCR5 mRNA in the spinal cord.ConclusionOur results demonstrate that the acute and chronic-relapsing phases of MOG-EAE are associated with distinct expression of CCR1, CCR2, and CCR5 mRNA by cells of the macrophage/microglia lineage within the CNS lesions. These data support the notion that CCR1, CCR2 and CCR5 mediate recruitment of both infiltrating macrophages and resident microglia to sites of CNS inflammation. Detailed knowledge of expression patterns is crucial for the understanding of therapeutic modulation and the validation of CCR1, CCR2 and CCR5 as feasible targets for therapeutic intervention in MS.

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Novel role for SLPI in MOG-induced EAE revealed by spinal cord expression analysis.

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Novel role for SLPI in MOG-induced EAE revealed by spinal cord expression analysis.

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