Citrullinated calreticulin potentiates rheumatoid arthritis shared epitope signaling

Abstract

Objective Citrullinated proteins are immunogenic in rheumatoid arthritis (RA), particularly in patients that carry shared epitope (SE)-coding HLA-DRB1 alleles. The mechanism underlying this association is unknown. We have previously identified SE as a ligand that interacts with cell surface calreticulin (CRT) and activates immune dysregulation. The objective of this study was to determine the effect of CRT citrullination on SE signaling. Methods CRT-SE binding affinity was measured by surface plasmon resonance. The role of individual CRT arginine residues was determined by site-directed mutagenesis. Nitric oxide levels were measured using a fluorochrome-based assay. CRT citrullination in synovial tissues and cell cultures was determined by 2-dimensional gel electrophoresis, immunoblotting and mass spectrometry techniques. Results Synovial tissues and fibroblast-like synoviocytes from RA patients were found to express higher abundance of citrullinated CRT compared to OA samples. Citrullinated CRT showed more robust interaction with the SE ligand, and transduced SE signaling at a 10,000-fold higher potency, compared to non-citrullinated CRT. Site-directed mutation analysis identified Arg205, which is spatially adjacent to the SE binding site in the CRT P-domain, as a dominant inhibitor of SE-CRT interaction and signaling, while a more remote arginine residue, Arg261 was found to enhance these SE functions. Conclusion Citrullinated CRT is over-abundant in the RA synovium, and potentiates SE-activated signaling in vitro. These findings could introduce a new mechanistic model of gene-environment interaction in RA.

Problems of Rheumatoid Arthritis Immunopathology: Evolution of the Disease

“…При РА присутствие антител к ЛТа досто-верно ассоциируется с АЦБ-и РФ-позитивностью. Более того, связь между HLA-SE аллелями и АЦЦП имела место преимущественно у пациентов, в сыворотках которых об-наруживались антитела к ЛТа [154].…”

“…Имеются дан-ные, что наряду с презентацией аутоантигенов аллели SE выполняют функцию, связанную с их ролью как универ-сального лиганда для мембранного кальретикулина (КРТ), представляющего собой рецептор клеток врожден-ной иммунной системы, в первую очередь ДК. Примеча-тельно, что взаимодействие SE и КРТ возрастает при цит-руллинировании КРТ и инициирует передачу «внутрикле-точного сигнала», меняющего фенотип ДК, приводя к по-ляризации иммунного ответа по Th17-типу, снижению об-разования и ослаблению активности Т-регуляторных кле-ток [154,167]. Примечательно, что носительство SE в большей степени ассоциируется с высокими титрами по-лиреактивных АЦБ у пациентов с РА, чем АЦБ-позитив-ных пациентов без РА [48,168].…”

Etiology of Rheumatoid Arthritis: A Historical and Evidence-Based Perspective

“…More recently polymorphisms that govern additional amino acid variations in HLA-DR that are located outside the shared epitope have also been strongly associated with susceptibility to RA [ 8 , 9 ]. The mechanism for MHC predisposition to RA remains to be established-while presentation of a pathogenic autoantigen by RA-associated alleles is an attractive theory, other possibilities exist, for example, unique pro-infl ammatory properties of the shared epitope itself [ 10 ].…”

HLA shared epitope and ACPA: Just a marker or an active player?

“…SE alleles might contribute to the genetic predisposition to RA causing an immune dysregulation or a premature immunosenescence. In fact, SE triggers innate immune signaling via cell surface calreticulin; citrullination of calreticulin enhances the signaling leading to a sustained Th17 expansion [54]. SE alleles are also associated with T cell telomere erosions, observed in lymphocytes and hematopoietic stem cells from RA patients and probably related to immunosenescence [55].…”