Bacterial induction of autoantibodies to beta2-glycoprotein-I accounts for the infectious etiology of antiphospholipid syndrome

Abstract

The antiphospholipid syndrome (APS) is characterized by the presence of pathogenic autoantibodies against beta2-glycoprotein-I (beta2GPI). The factors causing production of anti-beta2GPI remain unidentified, but an association with infectious agents has been reported. Recently, we identified a hexapeptide (TLRVYK) that is recognized specifically by a pathogenic anti-beta2GPI mAb. In the present study we evaluated the APS-related pathogenic potential of microbial pathogens carrying sequences related to this hexapeptide. Mice immunized with a panel of microbial preparations were studied for the development of anti-beta2GPI autoantibodies. IgG specific to the TLRVYK peptide were affinity purified from the immunized mice and passively infused intravenously into naive mice at day 0 of pregnancy. APS parameters were evaluated in the infused mice on day 15 of pregnancy. Following immunization, high titers of antipeptide [TLRVYK] anti-beta2GPI Ab's were observed in mice immunized with Haemophilus influenzae, Neisseria gonorrhoeae, or tetanus toxoid. The specificity of binding to the corresponding target molecules was confirmed by competition and immunoblot assays. Naive mice infused with the affinity-purified antipeptide Ab's had significant thrombocytopenia, prolonged activated partial thromboplastin time and elevated percentage of fetal loss, similar to a control group of mice immunized with a pathogenic anti-beta2GPI mAb. Our study establishes a mechanism of molecular mimicry in experimental APS, demonstrating that bacterial peptides homologous with beta2GPI induce pathogenic anti-beta2GPI Ab's along with APS manifestations.

Figure 1

Anti-β2GPI binding properties of mouse sera derived from different bacteria/yeast–immunized mice. Sera derived from mice immunized with H. influenzae (HI), N. gonorrhoeae (NG), tetanus toxoid (TeNT), Streptoccocus pneumoniae (SP), Shigella dysenteriae (SD), C. albicans (CA), or Klebsiella pneumoniae (KP) were assayed at different dilutions for binding to β2GPI by ELISA. Data are presented as OD at 405 nm (mean ± SD of three different experiments).

Figure 2

Affinity determination of mouse affinity-purified Ab’s to the peptide. Using several molar concentrations of NH₄SCN, the percentage of affinity-purified antipeptide Ab’s binding to the peptide was determined by ELISA. The antipeptide Ab’s were derived from mice immunized with H. influenzae (HI), N. gonorrhoeae (NG), tetanus toxoid (TeNT), Streptoccocus pneumoniae (SP), Shigella dysenteriae (SD), or C. albicans (CA) at Ab concentrations of 20 μg/ml. Data are presented as percentage of binding in three separate experiments.

Figure 3

Inhibition of binding of affinity-purified mouse antipeptide Ab to the corresponding synthetic peptide. Antipeptide Ab’s, affinity purified from mice immunized with H. influenzae (HI), N. gonorrhoeae (NG), tetanus toxoid (TeNT), Streptoccocus pneumoniae (SP), Shigella dysenteriae (SD), or C. albicans (CA) competed for binding to the relevant synthetic peptide by the peptide CATLRVYKGG. Scrambled peptide (TGVGKALYCR) was used as a control.

Figure 4

Binding of sera or affinity-purified antipeptide Ab’s to bacterial extracts or to tetanus toxoid. Binding of sera derived from mice immunized with H. influenzae, N. gonorrhoeae, or tetanus toxoid at sera dilution of 1:20, are described in lanes A, C, and E, respectively. Antipeptide Ab’s affinity purified from the same immunized mice are described in lanes B, D, and F, respectively.

Figure 5

Three-dimensional Ribbon diagram of the β2GPI (a) and partial structure of tetanus toxoid (b). The peptide TLRVYK and the relevant mimicry on a tetanus toxoid partial molecule are emphasized in dark gray.