The role of citrullination of central nervous system proteins in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system (CNS) characterised by focal lesions of inflammation and demyelination, with subsequent axonal damage. Although the cause is not yet known, it is thought to be autoimmune in origin. Antibodies are implicated in the disease process, as these are detectable in the cerebrospinal fluid (CSF) seen as oligoclonal bands (OCBs) following isoelectric focussing. However, the target antigen of these antibodies has not yet been fully characterised. Post-translational modification of myelin basic protein (MBP) and other CNS-specific proteins, including citrullination of arginine residues, results in conformational changes in the protein. This leads to increased degradation by proteases and exposure of new epitopes, which may then cause the production of autoantibodies targeting the myelin sheath. The process of citrullination is carried out by a family of enzymes known as peptidylarginine deiminases (PADs) with PAD2 and PAD4 expressed in the brain. Excess citrullination occurs in MS, with MBP isolated from MS white matter being more highly citrullinated than in healthy control white matter. This thesis aimed to investigate the in vitro expression of PAD2 and PAD4 by cells of the CNS to gain a better understanding of how these enzymes might be regulated and their possible role in the pathogenesis of MS. Cells were treated with pro-inflammatory cytokines in vitro, to mimic in vivo inflammatory conditions, and following this, PAD2 and PAD4 mRNA expression was determined using real-time PCR. PAD2 mRNA was significantly decreased in the presence of pro-inflammatory cytokines IL-1beta, TNF-alpha and IFN-gamma in three preparations of primary human astrocytes, a human foetal microglial cell line (CHME3) and a brain endothelial cell line (hCMEC/D3). Furthermore, treatment of CHME3 cells with antiinflammatory cytokines IL-4, IL-10 and TGF-? also resulted in the down-regulation of PAD2 mRNA. PAD4 was undetectable. Thus pro- and anti-inflammatory cytokines down-regulate expression of PAD2 mRNA in vitro, which indicates that increased citrullination of CNS proteins in MS is not due to the inflammatory milieu in the CNS, suggesting additional mechanisms must be involved in the up-regulation of PAD2 and PAD4 enzymes reported in MS tissue.Subsequently, in vivo studies were carried out investigating the distribution and expression of PAD2 and PAD4 enzymes, and citrullinated proteins, in post-mortem control, MS normal appearing white matter (NAWM) and MS lesional brain tissue by immunohistochemistry. Higher levels of citrullinated proteins were observed in MS lesions compared to control and NAWM brain tissue, and were associated with areas of myelin thinning with on-going demyelination, and was co-localised to GFAP-positive astrocytes. PAD2 expression was difficult to detect. Western blotting of proteins extracted from these MS brains showed citrullination of multiple proteins, two of which were identified as glial fibrillary acidic protein (GFAP) and MBP, the latter also confirmed by mass spectrometry. This agrees with previous studies implicating citrullination in the pathogenesis of MS. Lastly, an enzyme-linked immunosorbant assay (ELISA) method was developed to detect anti-citrullinated protein antibodies (ACPAs) against citrullinated MBP peptides in the CSF and serum of patients newly diagnosed with MS, other neurological diseases (ONDs) and controls, to determine whether higher levels of antibodies were observed in individuals with MS. No significant increases in the levels of ACPAs were found in the CSF and serum of individuals with MS compared to ONDs and controls. Although not significant, increased reactivity of MS patient CSF immunoglobulins towards citrullinated MBP peptides was observed. Furthermore, an association was found between reactivity towards citrullinated MBP peptides and patients testing positive for OCBs. Evidence presented in this thesis demonstrates an important role of citrullination in the pathogenesis of MS. The results of this thesis indicate that further investigations into the role of citrullination in MS, particularly with regards to assessment of further citrullinated peptides as antigenic targets in ELISA assays, are warranted.