Circulating Autoantibodies Recognizing Immunodominant Epitopes From Human Apolipoprotein B Associate With Cardiometabolic Risk Factors, but Not With Atherosclerotic Disease

Abstract

Rationale: Atherosclerosis is a chronic inflammatory disease of large arteries that involves an autoimmune response with autoreactive T cells and auto-antibodies recognizing Apolipoprotein B (ApoB), the core protein of low-density lipoprotein (LDL). Here, we aimed to establish a clinical association between circulating human ApoB auto-antibodies with atherosclerosis and its clinical risk factors using a novel assay to detect auto-antibodies against a pool of highly immunogenic ApoB-peptides.

Methods and results: To detect polyclonal IgM- and IgG-antibodies recognizing ApoB, we developed a chemiluminescent sandwich ELISA with 30 ApoB peptides selected by an in silico assay for a high binding affinity to MHC-II, which cover more than 80% of known MHC-II variants in a Caucasian population. This pre-selection of immunogenic self-peptides accounted for the high variability of human MHC-II, which is fundamental to allow T cell dependent generation of IgG antibodies. We quantified levels of ApoB-autoantibodies in a clinical cohort of 307 patients that underwent coronary angiography. Plasma anti-ApoB IgG and IgM concentrations showed no differences across healthy individuals (n = 67), patients with coronary artery disease (n = 179), and patients with an acute coronary syndrome (n = 61). However, plasma levels of anti-ApoB IgG, which are considered pro-inflammatory, were significantly increased in patients with obesity (p = 0.044) and arterial hypertension (p < 0.0001). In addition, patients diagnosed with the metabolic syndrome showed significantly elevated Anti-ApoB IgG (p = 0.002). Even when normalized for total plasma IgG, anti-ApoB IgG remained highly upregulated in hypertensive patients (p < 0.0001). We observed no association with triglycerides, total cholesterol, VLDL, or LDL plasma levels. However, total and normalized anti-ApoB IgG levels negatively correlated with HDL. In contrast, total and normalized anti-ApoB IgM, that have been suggested as anti-inflammatory, were significantly lower in diabetic patients (p = 0.012) and in patients with the metabolic syndrome (p = 0.005).

Conclusion: Using a novel ELISA method to detect auto-antibodies against ApoB in humans, we show that anti-ApoB IgG associate with cardiovascular risk factors but not with the clinical appearance of atherosclerosis, suggesting that humoral immune responses against ApoB are shaped by cardiovascular risk factors but not disease status itself. This novel tool will be helpful to develop immune-based risk stratification for clinical atherosclerosis in the future.

Keywords: ApoB; atherosclerosis; auto-antibodies; cardiovascular disease; immunity.

FIGURE 1

Study workflow for the development of a novel ELISA to detect anti-ApoB IgG- and IgM- plasma levels. (1) ApoB-derived peptides with a length of 15 amino acids were screened in silico for their binding affinity to MHC-II variants (18). A pool of 30 peptides that showed a high affinity to MHC-II in direct in vitro affinity measurements were used to coat a microplate at 5 μg/mL. (2) A total of 307 plasma samples from the TRAFIC cohort were analyzed by an in-house chemiluminescent ELISA (3) to detect circulating anti-ApoB IgG and IgM auto-antibodies. The figure was generated with schematics from Biorender.com.

FIGURE 2

Total IgG and IgM plasma levels do not associate with coronary atherosclerosis in coronary angiography. (A) Total IgG and (B) IgM levels were quantified in plasma samples using a Flex Set Cytometric Bead Array (BD Biosciences) and grouped according to the presence of coronary atherosclerosis in coronary angiography. CAD, coronary artery disease; ACS, acute coronary syndrome.

FIGURE 3

Anti-ApoB IgG plasma levels are increased in patients with hypertension and obesity. Anti-ApoB IgG plasma levels were quantified by ELISA and grouped according to (A) patient diagnosis or the presence of cardiometabolic risk factors (B–F). CAD, coronary artery disease; ACS, acute coronary syndrome; DM, diabetes mellitus; HTN, hypertension; MS, metabolic syndrome.

FIGURE 4

Associations of anti-ApoB IgG plasma levels with triglycerides, cholesterol, and apolipoproteins. Anti-ApoB IgG plasma levels were quantified by ELISA and divided into quartiles (Q) of patient plasma levels of (A) triglycerides, (B) total cholesterol, (C) VLDL, (D) LDL, (E) HDL, and (F) ApoB. VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. Data are presented as median ± 95%CI.

FIGURE 5

Associations of anti-ApoB IgM plasma levels with cardiovascular risk factors. Anti-ApoB IgM plasma levels were quantified by ELISA and grouped according to (A) patient diagnosis or (B–F) cardiometabolic risk factors. CAD, coronary artery disease; ACS, acute coronary syndrome; DM, diabetes mellitus; HTN, hypertension; MS, metabolic syndrome.