Brain Citrullination Patterns and T Cell Reactivity of Cerebrospinal Fluid-Derived CD4+ T Cells in Multiple Sclerosis

Abstract

Immune responses to citrullinated peptides have been described in autoimmune diseases like rheumatoid arthritis (RA) and multiple sclerosis (MS). We investigated the post-translational modification (PTM), arginine to citrulline, in brain tissue of MS patients and controls (C) by proteomics and subsequently the cellular immune response of cerebrospinal fluid (CSF)-infiltrating T cells to citrullinated and unmodified peptides of myelin basic protein (MBP). Using specifically adapted tissue extraction- and combined data interpretation protocols we could establish a map of citrullinated proteins by identifying more than 80 proteins with two or more citrullinated peptides in human brain tissue. We report many of them for the first time. For the already described citrullinated proteins MBP, GFAP, and vimentin, we could identify additional citrullinated sites. The number of modified proteins in MS white matter was higher than control tissue. Citrullinated peptides are considered neoepitopes that may trigger autoreactivity. We used newly identified epitopes and previously reported immunodominant myelin peptides in their citrullinated and non-citrullinated form to address the recognition of CSF-infiltrating CD4 + T cells from 22 MS patients by measuring proliferation and IFN-γ secretion. We did not detect marked responses to citrullinated peptides, but slightly more strongly to the non-modified version. Based on these data, we conclude that citrullination does not appear to be an important activating factor of a T cell response, but could be the consequence of an immune- or inflammatory response. Our approach allowed us to perform a deep proteome analysis and opens new technical possibilities to analyze complex PTM patterns on minute quantities of rare tissue samples.

Putative Roles for Peptidylarginine Deiminases in COVID-19

“…Detailed exploration into the target proteins of PAD-mediated deimination in COVID-19 will need further assessment, as the subcellular distribution of deiminated proteins is tissue type-dependent [ 108 ]. The citrullinome of chronic diseases has been reported, including rheumatoid arthritis (RA), multiple sclerosis (MS) and Alzheimer’s disease (AD) [ 82 , 109 , 110 ], as well as in a range of taxa throughout the phylogenetic tree, both in plasma and the EV secretome [ 39 , 44 , 45 , 46 , 47 , 48 , 66 , 67 , 111 ]. Interestingly, the enrichment of deiminated proteins for KEGG pathways relating to viral infection has recently been identified in the serum and serum EVs of alligator and cow, both which are animals with unusual anti-viral responses [ 47 , 48 ].…”

GFAP at 50

“…The three α-helical regions of the GFAP central rod domain are indicated by boxes separated by nonhelical linker regions, and the N-terminal head and C-terminal tail domains are indicated by straight lines. Amino acids identified as sites of citrullination are shown in red ( Jin et al., 2013 ; Brenner and Nicholas, 2017 ; Faigle et al., 2019 ), sites of phosphorylation are shown in blue ( Inagaki et al., 1994 ; Battaglia et al., 2019 ), and the single site for lipoxidation is shown in green ( Viedma-Poyatos et al., 2018 ). Evidence also exists for N-glycosylation and O-glycosylation of GFAP, but none of the specific sites for these modifications is known ( Kanninen et al., 2004 ; Korolainen et al., 2005 ).…”

“…Citrullination, the enzymatic conversion of peptidyl-arginine to peptidyl-citrulline through deimination, is another posttranslational modification of potential significance (reviewed in Brenner and Nicholas, 2017 ). Citrullination has been observed at multiple arginine residues in neurologically normal individuals ( Jin et al., 2013 ; Faigle et al., 2019 ); both the number of sites and/or amount increases in several diseases, including multiple sclerosis ( Nicholas et al., 2004 ; Faigle et al., 2019 ), experimental allergic encephalomyelitis ( Nicholas et al., 2005 ), Alzheimer’s disease ( Ishigami et al., 2005 , 2015 ), and hepatic fibrosis ( Kim et al., 2018b ). In vitro studies have suggested that citrullination interferes with GFAP polymerization ( Inagaki et al., 1989 ).…”

The Relationship of the Mechanisms of the Pathogenesis of Multiple Sclerosis and the Expression of Endogenous Retroviruses

“…A larger amount of PADI2 is localized in the periaxonal regions, which could explain why this end of the oligodendrocyte is the first to undergo to apoptosis [ 76 ]. However, it appears that citrullination of MBP has a limited role in activating T lymphocytes, and, therefore, unlikely to play a role in the development of MS [ 77 ]. Therefore, the role of citrullination of MBP in the pathogenesis of MS remains unclear.…”