Background/Purpose: Sjögren’s Disease is an autoimmune condition in which patients exhibit decreased salivary/lacrimal gland function and express autoantibodies that target the 60k molecular weight Ro autoantigen. Published works have proposed autoantibodies that target M3R as potential clinical markers for Sjögren’s Disease. Our studies show that 4-hydroxy-2-nonenal (lipid oxidation byproduct) immunized rabbits develop antibodies against G-protein-coupled acetylcholine receptor muscarinic-type-3-receptor (M3R) second (ECL2) and third (ECL3) extracellular loops. We hypothesized that there will be a differential induction of antibodies against ECL2 or ECL3 dependent on the degree of 4-hydroxy-2-nonenal (HNE)-modification of Ro60 in BALB/c mice.

Methods: Five groups of BALB/c mice were used in this study. Group I was immunized with Ro60. Groups II to IV were immunized with Ro60, modified with 0.4 mM (low), 2 mM (medium) and 10 mM (high) HNE respectively. Group V controls received Freund’s adjuvant. Antibodies to M3R ECL2 (amino acids 213-228) and ECL3 (amino acids 514-527) were detected by multiple antigenic peptide ELISA using sera from the immunized mice.

Results: Immunization with unmodified Ro60 induced a rapid and differential intermolecular epitope spreading to the second loop of M3R, but not to the third loop. Bleed 2 sera from Group 1 mice immunized with unmodified Ro60 had an average reactivity of OD₄₀₅ 0.69±0.3 to ECL2 peptide, while Group 2 mice immunized with low HNE-Ro60 showed an average reactivity of OD₄₀₅ 0.58±0.21. The medium HNE-Ro60 immunized mice (Group 3) had the highest reactivity (OD₄₀₅ 1.02±0.39), and the Freund’s control group (Group 5) had the lowest reactivity (OD₄₀₅ 0.01±0.06), while Group 3 mice immunized with high-HNE-Ro60 had a reactivity of OD₄₀₅ 0.25±0.11 to the ECL2 peptide. The anti-ECL2 antibody levels induced in the medium HNE-Ro60 group was significantly higher than the levels induced by immunization with unmodified Ro60 (p< 0.0003). The anti-ECL2 levels correlated well with the induction of anti-Ro60 and anti-La antibodies in the mice immunized with medium HNE-Ro60. A similar trend was observed in Bleed 6 of the five experimental groups, with the medium HNE-Ro60 immunized group having significantly higher anti-ECL2 antibody levels compared to Ro60 immunized group (p< 0.0005). However, there was no significant induction of antibodies to ECL3 in any of the groups for both bleeds.

Conclusion: We found a differential induction of antibodies against the second extracellular loop of M3R, with Group 3 mice immunized with medium HNE-Ro60 developing significantly higher reactivity when compared to all other groups. Such a differential intermolecular epitope spreading may have significant implications for developing a deeper understanding of the progression of autoimmunity in Sjögren’s Disease.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/differential-induction-of-anti-muscarinic-type-3-receptor-antibodies-by-immunization-with-4-hydroxy-2-nonenal-modified-ro60-in-balb-c-mice/