Verification of B-lymphocyte activating factor’s involvement in the exacerbation of insulin resistance as well as an autoimmune response in patients with nonalcoholic steatohepatitis and patients with HCV-related chronic liver disease

Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent liver disease worldwide, characterized by the accumulation of triglycerides in the liver, and the absence of excessive alcohol consumption [1]. NAFLD covers a spectrum of liver diseases that range from simple steatosis called nonalcoholic fatty liver (NAFL) through nonalcoholic steatohepatitis (NASH), which is associated with hepatic inflammation and fibrosis in addition to simple steatosis [1]. Therefore, NASH is considered to be a risk factor for liver cirrhosis and hepatocellular carcinoma (HCC) [2]. It has been well established that NAFLD is a hepatic manifestation of a metabolic syndrome [3], because insulin resistance is one of the main etiological factors underlying the progression of NAFLD. Thus, the presence of NAFLD is associated with a high risk of developing type 2 diabetes mellitus (DM), dyslipidemia, and hypertension. The incidence of NAFLD will increase rapidly in the future as the frequency of obesity increases worldwide.

Hepatitis C virus (HCV) also induces a spectrum of chronic liver diseases from chronic liver disease to liver cirrhosis, and ultimately to HCC [4]. Persistent HCV infection often evokes numerous types of metabolic abnormalities, including insulin resistance, hepatic steatosis, dyslipidemia, and iron overload [5, 6]. These metabolic abnormalities are greatly involved in the development of liver damage. On the other hand, chronic HCV infection is frequently associated with autoimmune phenomena such as the emergence of non-organ-specific autoantibodies and/or concurrent extrahepatic autoimmune diseases [7].

Oxidative stress, proinflammatory cytokines, and mitochondrial dysfunction seem to be involved in the development of NASH and HCV-related chronic liver disease (CLD-C) [4, 8]. However, it remains unclear whether or not autoimmune responses are initiated during the process of disease progression. Previous studies revealed that 20–40% of patients with NAFLD [9‐15] and approximately 10–50% of patients with CLD-C [16‐20] have non-organ specific autoantibodies, including antinuclear antibodies (ANAs) and/or smooth muscle antibodies (SMAs) in their sera. Some investigators have reported that CLD-C patients seropositive for ANAs had significantly more advanced hepatic fibrosis than those seronegative for ANAs [21], though these findings seems to be controversial in NASH patients [12, 13]. There have been few studies to highlight the relationship between insulin resistance and autoimmune response in patients with NASH so far. Loria et al. [10] found that higher ANA titers were significantly correlated with more severe insulin resistance in patients with NAFLD.

Interestingly, a recent study using diet-induced obese mice revealed that B cells played crucial roles in insulin resistance and glucose intolerance [22]. It is well recognized that B-lymphocyte activating factor (BAFF), a member of the tumor necrosis factor-alpha (TNF-α) superfamily, is essentially involved in the survival and maturation of B cells [23]. BAFF is probably believed to be produced in adipocytes [24] as well as in macrophages, monocytes and dendritic cells, and to affect insulin receptor substrate-1 (IRS-1) in adipocyte directly [25]. Hence, we hypothesized that BAFF might facilitate not only humoral immunity but also insulin resistance in patients with NASH or CLD-C.

Furthermore, obesity has shown a strong correlation with autoimmune diseases, including autoimmune thyroiditis and type 2 DM [26, 27]. Obesity is likely to cause two distinct immunological responses: chronic inflammation through the stimulation of innate immunity and the activation of a humoral response that triggers autoantibody production [28]. We previously reported that autoimmune response was also involved in the process of hepatic steatosis in patients with chronic hepatitis C [29].

The primary purpose of the present study was to investigate the relationships between ANA status and insulin resistance, obesity, or hepatic steatosis in NASH and CLD-C patients.

Our results support our hypothesis that BAFF is involved in the progression of insulin resistance in NASH or CLD-C patients. Moreover, we confirmed that serum BAFF levels were associated with ANA status in CLD-C patients. BAFF is likely to play a crucial role in HCV-associated B cell proliferation [35]. However, to the best of our knowledge, this is the first report to focus primarily on the relationship between insulin resistance and autoimmune response via BAFF overproduction in CLD-C patients.

Here, we propose a putative mechanism by which BAFF participates in the emergence of ANAs and the enhancement of insulin resistance as follows: overproduction of BAFF in adipocytes leads to the polyclonal activation of B cells and subsequent initiation of ANA production. Simultaneously, excessive BAFF directly affects the down-regulation of IRS-1 and eventually exacerbates insulin resistance.

In the present study, the prevalence of ANAs in patients with NASH or CLD-C was approximately similar to that previously reported [10‐19], and ANA titers in the sera of most NASH or CLD-C patients were as weak as those previously elucidated [10‐14, 16‐18]. The present data demonstrated that CLD-C patients with ANAs had more advanced hepatic fibrosis and steatosis than CLD-C patients without ANAs, while ANA status was not associated with the progression of hepatic fibrosis or steatosis in NASH patients. It has been widely recognized that hepatic fibrosis and steatosis were closely linked to insulin resistance in CLD-C patients [37, 38], and that CLD-C patients with ANAs showed more advanced hepatic fibrosis than those without ANAs [21]. Therefore, greater BAFF production results in more severe insulin resistance, possibly leading to more advanced hepatic fibrosis and steatosis in CLD-C patients with ANAs. However, the elevation of serum BAFF level was somewhat mild in NASH patients with ANA, compared to that in CLD-C patients with ANAs. Mild elevation of BAFF in NASH patients with ANAs was not associated with any significant difference in the degrees of hepatic fibrosis or steatosis between ANA-seropositive and ANA-seronegative NASH patients.

The present study revealed that BMI was associated with neither ANA status nor serum IgG level (data not shown) in NASH or CLD-C patients, suggesting that obesity might not be responsible for the development of autoimmunity in the diseases under consideration. Apoptosis inhibitor of macrophage (AIM), which induces a class-switch of macrophages, and consequently leads to chronic inflammation, appears to participate in obesity-associated autoimmune responses. Taking our results into consideration, AIM is unlikely to be involved in the development of NASH or CLD-C [36].

It was of interest that serum ferritin levels proved to be significantly higher in NASH patients with ANAs than in NASH patients without ANAs. Similarly, CLD-C patients seropositive for ANAs had significantly higher serum ferritin levels than CLD-C patients seronegative for ANAs, indicating that an autoimmune response was involved in iron storage in the liver of these patients. We often experienced CLD-C patients whose serum ferritin levels are transiently elevated during pegylated interferon (PEG-IFN)-based treatment [39]. The elevation of serum ferritin level in patients with CLD-C patients may account for the activation of Kupffer cells by the administration of PEG-IFN [40]. In addition, an elevation of serum ferritin level in patients with systemic lupus erythematosus (SLE) may imply the active stage of the disease [41], suggesting the possibility that ferritin may be a novel serological autoimmune hallmark. Unfortunately, the present study did not show the significant correlations between serum BAFF and ferritin levels in NASH and CLD-C patients.

There were several limitations in the present study. First, our study was limited by a small sample size, although we acquired the valuable results in the present study. A larger-scale cohort study is required to confirm the present results. Second, we were unable to identify the origin to facilitate the synthesis of BAFF, although we speculated that adipocytes are a good candidate. The extent and location of BAFF receptor were not investigated in this study, either. Third, laboratory autoimmune parameters other than ANAs were not determined in the enrolled patients. Therefore, the titers of autoantibodies other than ANAs might be associated with serum BAFF levels in patients with NASH.

We concluded that an increase in BAFF synthesis may evoke exacerbation of insulin resistance as well as an autoimmune response in NASH and CLD-C patients. The exacerbation of insulin resistance by way of BAFF overproduction may result in the progression of hepatic fibrosis and steatosis in ANA-seropositive CLD-C patients.