Molecular Mimicry of the Rheumatoid Arthritis-Related Immunodominant T-Cell Epitope within Type II Collagen (CII260-270) by the Bacterial L-Asparaginase

Abstract

The etiology of most autoimmune diseases, including rheumatoid arthritis (RA), remains unclear. Both genetic and environmental factors are believed to be involved in pathogenesis. Molecular mimicry is considered one of the mechanisms for the occurrence of autoimmune diseases. The aim of the study was to determine whether the bacterial peptide L-ASNase67-81, which mimics the immunodominant T-cell epitope CII259-273, can induce T-cell reactivity in blood samples from RA patients and healthy subjects through molecular mimicry. Using bioinformatic molecular modeling methods, we first determined whether the L-ASNase67-81 peptide binds to the HLA-DRB1*04:01 molecule and whether the formed MHCII–peptide complex interacts with the corresponding T-cell receptor. To validate the obtained results, leukocytes isolated from early RA patients and healthy individuals were stimulated in vitro with L-ASNase67-81 and CII259-273 peptides as well as with bacterial L-asparaginase or human type II collagen (huCII). The activated T cells (CD4+CD154+) were analyzed by flow cytometry (FACS), and the levels of cytokines produced (IL-2, IL-17A/F, and IFN-γ) were measured by ELISA. Our in silico analyses showed that the bacterial peptide L-ASNase67-81 binds better to HLA-DRB1*04:01 compared to the immunodominant T-cell epitope CII259-273, mimicking its structure and localization in the binding groove of MHCII. Six contact points were involved in the molecular interaction of the peptide with the TCR. FACS data showed that after in vitro stimulation with the L-ASNase67-81 peptide, the percentage of activated T cells (CD154+CD4+) was significantly increased in both cell cultures isolated from ERA patients and those isolated from healthy individuals, as higher values were observed for the ERA group (9.92 ± 0.23 vs. 4.82 ± 0.22). Furthermore, the ELISA assays revealed that after stimulation with L-ASNase67-81, a significant increase in the production of the cytokines IL-2, IL-17A/F, and IFN-γ was detected in the group of ERA patients. Our data showed that the bacterial L-ASNase67-81 peptide can mimic the immunodominant T-cell epitope CII259-273 and activate HLA-DRB1*04:01-restricted T cells as well as induce cytokine production in cells isolated from ERA patients. These results are the first to demonstrate that a specific bacterial antigen could play a role in the pathogenesis of RA, mimicking the immunodominant T-cell epitope from type II collagen.

Anticitrullinated antibodies recognize rheumatoid arthritis associated T-cell epitopes modified by bacterial L-asparaginase

“…We have already reported that bacterial L-asparaginase can activate the T-cell response in ERA patients through molecular mimicry [24]. As an extension of that study, we report here that this enzyme may also play a role in the B-cell response by modifying immunodominant T-cell epitopes.…”

“…In the present study, the same ERA patients (n = 12) and healthy subjects (n = 11) who participated in a pre-vious study [24] were enrolled again (Table 1). Moreover, we already had frozen sera from these participants.…”

“…To evaluate the T cell reactivity, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with investigated antigens as previously described [24]. After 5 days of stimulation, the concentration of IL-2 and IFN-γ in the supernatants was measured using LEGEND MAX Human IL-2 and Human IFN-γ ELISA kits (BioLegend, San Diego, CA, USA) according to the manufacturer's instructions.…”

“…To assess the humoral response and detect specific antibodies against the studied antigens, enzyme-linked immunosorbent assay (ELISA) tests were performed. Frozen sera from our previous study [24] were used. Corning 96 Well ELISA plates (Merck KGaA, Darmstadt, Germany) were coated overnight at 4°C with 5 µg/ml of the L-ASNase-treated or non-treated synthetic peptides, or 50 μg/ml treated or native huCII, or 10 μg/ml bacterial L-asparaginase.…”

“…The well-known major immunodominant T cell epitope within CII (CII259-273) is a target for other posttranslational modifications but not citrullination. We have previously shown that by mimicking this epitope, the peptide L-ASNase67-81 (part of bacterial L-asparaginase) can activate HLA-DRB1*04:01 restricted CD4 + T cells and induce secretion of interleukin (IL)-2, IL-17A/F and interferon γ (IFN-γ) in patients with early RA (ERA) [24]. L-asparaginase (EC 3.5.1.1 or EC 3.5.1.38) is a homotetrameric enzyme that catalyzes the hydrolysis of asparagine to aspartate (or glutamine to glutamate) and ammonia (Fig.…”