Biphasic Production of Anti-ApoB100 Autoantibodies in Obese Humans and Mice

Abstract

Obesity is associated with autoimmunity, a phenomenon considered as harmful. Here we show that obese mice and humans produce IgG-type autoantibodies that specifically recognize apolipoprotein B-100 (ApoB100), its native epitope p210, and the synthetic p210 mimotope pB1. By contrast, antibodies against epitopes p45 and p240, which have been associated with atherosclerosis, were not detected in either the humans or mice. In a longitudinal analysis of high fat diet-fed mice, autoantibody production rose with increasing body weight, then decreased and plateaued at morbid obesity. Likewise, in a cross-sectional analysis of sera from 148 human volunteers spanning a wide BMI range and free of comorbidities, the immunoreactivity increased and then decreased with increasing BMI. Thus, the obesity-related ApoB100-specific natural autoantibodies characteristically showed the same epitope recognition, IgG-type, and biphasic serum levels in humans and mice. We previously reported that a pB1-based vaccine induces similar antibodies and can prevent obesity in mice. Therefore, our present results suggest that autoantibodies directed against native ApoB100 may mitigate obesity, and that the vaccination approach may be effective in humans.

Keywords: IgG-type autoantibody; apolipoprotein B-100; body mass index; epitope; high-fat diet induced obesity; mimotope; obese patients.

Figure 1

Characterization of autoantibodies in mouse and human sera. (A) Determination of isotypes. Antibodies were affinity-purified from pooled sera over human ApoB100 and analyzed with paper strip isotyping kits. Blue arrows indicate positive reactions. C; control, G3; IgG3, 2b; IgG2b, 2a; IgG2a, G1; IgG1, G4; IgG4, G2; IgG2, A; IgA, M; IgM, λ; lambda light chain, κ; kappa light chain. (B) Epitope mapping of ApoB100 affinity-purified mouse and human antibodies as well as of mouse monoclonal antibody 22B4 raised against pB1. The mouse sera used for affinity purification were harvested and pooled at the end of the experiments (33 weeks of age) from Chow-fed or HFD-fed mice. The human sera were pooled after the first ELISA screening experiments and also affinity-purified before the dot blot analysis. 0.2 µg of the given antibody preparation were added per well. a, b and c indicate the amounts of the peptides or proteins spotted per well; a, b and c = 5, 2.5 and 1 µg for p45, p210, p240, pB1 and BSA (bovine serum albumin; negative control); a, b and c = 5, 2.5 and 1 ng for immunoglobulins (M/H IgG = mouse or human IgG; positive control), respectively.

Figure 2

Longitudinal analysis of anti-pB1 antibody titers and body weight in Chow-fed and high fat diet (HFD)-fed mice. (A) Body weight curves and superimposition of autoantibody titer. Male C57BL/6 mice were continuously maintained on Chow (n = 11; squares) or switched (black arrow) at 11 weeks of age to a 60% HFD (n = 16; circles). Solid symbols indicate body weights; empty symbols indicate antibody units (absorbance of ×100 diluted serum at 450 nm). Data points are shown for every 3rd week, i.e., 9, 12, 15, 18, 21, 24, 27, 30 and 33 weeks of age. (B) Cluster analysis of the data shown in A. Based on the degree of obesity, its evolution was divided into four stages. Stage I: 9~12 weeks of age (overweight), stage II: 13~18 weeks of age (mildly obese), stage III: 19–25 weeks of age (moderately obese), stage IV: 26–33 weeks of age (severely obese). Error bars in panels A and B indicate means ± s.e.m. * p < 0.0001, ** p < 0.01.

Figure 3

Cross-sectional analysis of human anti-pB1 antibody titer data versus BMI. (A) Scatterplot derived from 107 positive samples (out of 148 human volunteers of South Korean nationality) according to the BMI group. BMI < 23: lean, 23 ≤ BMI < 25: overweight, 25 ≤ BMI < 27: obese, 27 ≤ BMI: severely obese. n = 23, 26, 27 and 31, respectively. Ab (antibody) units indicate the absorbance of ×100 diluted sera at 450 nm. Error bars indicate means ± s.e.m. (B) Quadratic Trend Analysis of data shown in A. Antibody titers refer to serum dilution folds (D.F.) yielding an absorbance read-out of 0.5 (See “Supplementary Materials” for a detailed explanation). The association between autoantibody titers and the BMI group was adjusted for age and sex. The P value for quadratic trend was 0.0494 (p < 0.05). Error bars indicate geometric means ± s.e.m.

Figure 4

Strategy of serum analyses.