Low levels of IgM antibodies recognizing oxidation-specific epitopes are associated with human non-alcoholic fatty liver disease

Abstract

Background: Lipid oxidation of membrane phospholipids is accompanied by the formation of oxidation-specific epitopes (OSE). These epitopes are recognized by specific antibodies and represent danger-associated molecular patterns that are generated during chronic inflammatory processes. In a murine model for hepatic inflammation during non-alcoholic fatty liver disease (NAFLD), increased antibody levels targeting OSE were found to be protective. Here, our aim was to determine an association between OSE-specific antibody titers and NAFLD in humans.

Methods: IgM and IgG levels with specificity for various OSE were assessed in the plasma of patients with NAFLD (n = 71) and healthy controls (n = 68). Antibody titers were comprehensively analyzed in patients with NAFLD after classification by histological evaluation of liver biopsies. Statistical analysis was performed to determine significant correlations and odds ratios. To study the specificity for NAFLD, plasma antibody titers were measured in patients with hepatitis C (n = 40) and inflammatory bowel disease (n = 62).

Results: IgM titers against OSE were lower in patients with NAFLD compared to controls. Further biopsy-based classification of patients with NAFLD did not show any difference in IgM levels. Plasma IgM titers towards the P1 mimotope demonstrated an inverse correlation with markers for obesity, systemic inflammation, and liver damage. In contrast, hepatitis C and increased disease activity during inflammatory bowel disease was not associated with reduced IgM titers.

Conclusions: Our data highlight the importance of immune recognition of OSE by IgM antibodies in the pathophysiology of NAFLD.

Keywords: Adaptive immune response; Fatty liver; IgM; Lipid oxidation; Non-alcoholic steatohepatitis.

Fig. 1

Malondialdehyde (MDA) epitopes in livers of patients with non-alcoholic steatohepatitis (NASH) and relationship between plasma immunoglobulin M (IgM) titers and non-alcoholic fatty liver disease (NAFLD). Immunohistochemical detection of MDA epitopes in liver sections (magnification 20×) of a a negative control and b two patients with NASH – arrows indicate localization of MDA2-positive staining in the liver tissue. Plasma IgM antibody titers against c MDA-low-density lipoprotein (LDL), d malondialdehyde-acetaldehyde (MAA)-LDL, e P1 mimotope, f CuOx-LDL, g PC-BSA, and h total IgM levels in patients with NAFLD and healthy controls. i Ratio of anti-P1 IgM titers to total IgM levels in patients with NAFLD and controls. Data are expressed in log10 relative light units (RLU)/100 ms. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Relationship between plasma P1-immunoglobulin (IgM) levels and non-alcoholic fatty liver disease (NAFLD) upon further characterization of liver biopsies. a IgM titers towards P1 in controls and patients with NAFLD grouped based on their ALT levels. b Plasma IgM antibody titers against the P1 mimotope in patients with NAFLD in which the liver biopsy was evaluated and scored as simple steatosis versus non-alcoholic steatohepatitis (NASH). Anti-P1 IgM titers after scoring liver biopsies for c steatosis, d inflammation, e hepatocyte ballooning, f applying the NAFLD activity score (NAS), and g scoring fibrosis. Data are expressed in relative light units (RLU)/100 ms and presented as means. **p < 0.01

Fig. 3

Single logistic regression model, odds ratio determination, and receiver operating characteristic (ROC) curve analysis for P1-specific IgM levels in non-alcoholic fatty liver disease (NAFLD). a Odds ratios for the predictive power of anti-P1 immunoglobulin (IgM) titers (log2 scale) for fatty liver disease after adjustment for age, gender, body mass index (BMI), and total IgM. b ROC curve analysis for the diagnosis of NAFLD with P1-IgM. c IgM titers towards P1 were divided into quartiles (black: controls; red: NAFLD) and d the odds ratios P1-IgM (log2 scale) for NAFLD were computed across these quartiles after adjusting for age, gender, BMI, and total IgM levels. CI confidence interval

Fig. 4

Correlation between plasma P1-immunoglobulin M (IgM) levels and plasma lipids and systemic markers of inflammation. Shown are the Pearson R correlation between plasma IgM titers towards P1 and a plasma triglycerides (TG), b cholesterol, low-density lipoprotein (LDL), d body mass index (BMI), e waist circumference, f leptin, and the markers for inflammation g monocyte chemoattractant protein 1 (MCP1), h interleukin 6 (IL-6), and i cathepsin D. RLU relative light unit

Fig. 5

Plasma immunoglobulin M (IgM) titers in cohorts consisting of patients with hepatitis C or patients with inflammatory bowel disease (IBD). a Total, b anti-P1 IgM titers, and c P1/total IgM ratio in healthy controls and patients with hepatitis C. Relationship between the activity score during IBD and d total IgM, e P1-specific IgM, and f P1/total IgM ratio. Inactive disease was given score 0, mild to moderate disease activity was given a score of 1–2, and severe activity was given a score of 3. Data are expressed in mg/dl or relative light units (RLU)/100 ms and presented as means and standard deviations. *p < 0.05, ***p < 0.001