Citrullinated and homocitrullinated low- density lipoprotein in rheumatoid arthritis

Abstract

Objective: Antibodies to citrullinated and homocitrullinated (also known as carbamylated) proteins, specific for rheumatoid arthritis (RA), are associated with cardiovascular disease (CVD). Immune complexes containing these proteins have been identified in the atherosclerotic plaque of CVD patients. In mice, homocitrullinated low-density lipoprotein (HomoCitLDL) promotes foam cell formation, which is critical in the pathogenesis of atherosclerosis. We aimed to investigate the atherogenic potential of HomoCitLDL and citrullinated low-density lipoprotein (CitLDL) in RA.

Method: Human LDL was homocitrullinated in potassium cyanate and citrullinated by rabbit skeletal muscle peptidyl arginine deiminase-2. The modifications were confirmed by mass spectrometry. Primary human monoctyes from healthy subjects (N = 8) were differentiated to macrophages using macrophage colony-stimulating factor and incubated with modified LDL. Foam cells were visualized using Oil Red O staining. Serum from RA patients (N = 101) and controls (N = 32) was tested for immunoglobulin G antibodies to modified LDL using enzyme-linked immunosorbent assay.

Results: HomoCitLDL and CitLDL strongly induced foam cell production (> 90%) versus unmodified LDL (11%) (p < 0.0001). The characteristics of the RA subjects were: 73% females, median age 60 [interquartile range (IQR) 17] years and disease duration 7.5 (IQR 13) years; 11% had a prior major cardiovascular event, 66% were ever smokers, 32% had hypertension, 33% dyslipidaemia, and 14% diabetes. Antibodies to HomoCitLDL were detected in 18% of RA patients; they were significantly associated with dyslipidaemia [odds ratio (OR) 3.86; 95% confidence interval (CI) 1.22, 12.17] and antibodies to other homocitrullinated antigens (OR 10.61; 95% CI 1.31, 86.11).

Conclusions: HomoCitLDL and CitLDL have atherogenic properties in vitro. Antibody responses to HomoCitLDL, but not CitLDL, were detected in RA patients.

Acknowledgements

Operating funds for the study were received from the Arthritis Society [grant number YIO-14-123] and St Joseph’s Health Care Foundation Research Grant [grant number 060-1819]. EC is supported by an award from the Calder Foundation.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supporting information

Additional supporting information may be found in the online version of this article.

Supplementary figure S1A. Representative mass spectra indicating homocitrullination of human LDL.Supplementary figure S1B. Representative mass spectra indicating citrullination of human LDL.Supplementary figure S2. In vitro homocitrullination and citrullination sites on human ApoB100 as determined by mass spectrometry.Supplementary figure S3. Lipid-treated primary macrophages.Supplementary figure S4. Anti-CitLDL, anti-HomoCitLDL, and anti-LDL antibodies.

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Additional information

Funding

This work was supported by the The Arthritis Society [YIO-14-123]; St. Joseph’s Health Care Foundation [060-1819].