Kurien Biji T, Dave Devavrat, Tsaliki Martha, Quadri Syed M S, Lewis Valerie M, Scofield Robert Hal
Arthritis & Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, OK, USA.
Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma, OK, USA.
Clin Exp Immunol. 2025 Jan 21;219(1). doi: 10.1093/cei/uxae114.
Sjögren's Disease (SjD) subjects have decreased lacrimal/salivary gland function. Studies have proposed that autoantibodies targeting G-protein-coupled muscarinic acetylcholine-type-3-receptor (M3R) are potential clinical markers for SjD. We hypothesized that rabbits/mice immunized with 4-hydroxy-2-nonenal (HNE)-modified/unmodified Ro60 will develop an autoimmunity, specifically a SjD phenotype, thus expressing increased levels of anti-M3R antibodies.
We immunized two rabbits each with 10 mM HNE-modified Ro60/unmodified Ro60 antigen or Ro274-290/Ro413-428/Ro500-517 Ro60 peptides. Two rabbits each were immunized with either M3R second extracellular loop (ECL2) or M3R ECL3 peptide. Finally, five groups of BALB/c mice were immunized as follows-Group-I immunized with Ro60, Groups-II-IV immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively and Group-V-Freund's adjuvant. Serum antibodies to M3R ECL2/ECL3/Ro60/La or Sm were detected by ELISA. Functional assays were also performed.
Immunization with HNE-modified Ro60/unmodified Ro60 antigen or Ro274/Ro 413/Ro500 peptides induced a rapid intermolecular epitope spreading to M3R ECL2/ECL3, especially to M3R ECL3 in HNE-Ro immunized rabbits. These animals did not bind to scrambled M3R peptides. Ro60-immunized rabbit IgG inhibited M3R activity in a functional assay. Rabbits immunized with ECL2/ECL3 developed high reactivity to Ro60 but not against Sm/RNP. We found a differential antibody-induction against M3R ECL2 with Group-3 mice developing significant reactivity.
Our data show induction of increasing anti-M3R antibodies in rabbits immunized with Ro60/HNE-Ro60 or Ro60 peptides and differential induction of these antibodies in mice immunized with Ro60 modified with increasing HNE. These findings suggest that M3R ECL2/ECL3 are involved in SjD autoimmunity progression.
干燥综合征（SjD）患者泪腺/唾液腺功能减退。研究表明，靶向G蛋白偶联毒蕈碱型乙酰胆碱3型受体（M3R）的自身抗体是SjD的潜在临床标志物。我们假设，用4-羟基-2-壬烯醛（HNE）修饰/未修饰的Ro60免疫的兔/小鼠会产生自身免疫，特别是SjD表型，从而使抗M3R抗体水平升高。
我们分别用10 mM HNE修饰的Ro60/未修饰的Ro60抗原或Ro274-290/Ro413-428/Ro500-517 Ro60肽免疫两只兔子。分别用M3R第二细胞外环（ECL2）或M3R ECL3肽免疫两只兔子。最后，将五组BALB/c小鼠按以下方式免疫：第一组用Ro60免疫，第二至四组分别用0.4 mM（低）、2 mM（中）和10 mM（高）HNE修饰的Ro60免疫，第五组用弗氏佐剂免疫。通过酶联免疫吸附测定（ELISA）检测抗M3R ECL2/ECL3/Ro60/La或Sm的血清抗体。还进行了功能测定。
用HNE修饰的Ro60/未修饰的Ro60抗原或Ro274/Ro 413/Ro500肽免疫可诱导分子间表位扩展迅速至M3R ECL2/ECL3，尤其是在HNE-Ro免疫的兔子中扩展至M3R ECL3。这些动物不与混乱的M3R肽结合。在功能测定中，Ro60免疫的兔IgG抑制M3R活性。用ECL2/ECL3免疫的兔子对Ro60有高反应性，但对Sm/RNP无反应。我们发现，第3组小鼠对M3R ECL2的抗体诱导存在差异，产生了显著反应性。
我们的数据表明，用Ro60/HNE-Ro60或Ro60肽免疫的兔子中抗M3R抗体诱导增加，在用HNE增加修饰的Ro60免疫的小鼠中这些抗体的诱导存在差异。这些发现表明，M3R ECL2/ECL3参与SjD自身免疫进展。
