Nonalcoholic fatty liver disease (NAFLD) has become the most frequent chronic liver disease in children and adolescents in westernized countries (1–4).
NAFLD encompasses a spectrum of diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which is defined by the association of lipid accumulation with evidence of cellular damage, inflammation, and different degree of scarring or fibrosis (5). Although the pathogenesis of NAFLD/NASH, both in children and in adults, remains unclear, a “2-hit hypothesis” is considered most likely (6–8). With this theory, increased fatty liver (steatosis) and hyperinsulinemia may play crucial roles in the pathogenesis of insulin resistance as the first step; then a second event or a sum of multiple hits (multiple-hit hypothesis) lead to the development of NASH. They include abnormalities in lipid metabolism, insulin resistance, oxidative stress, and impaired secretion of cytokines and adipocytokines. However, no conclusive data have been obtained so far (9,10).
Inflammation associated with insulin resistance is characterized by an overproduction of a number of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, leptin, resistin, and conversely, reduced secretion of adiponectin, an anti-inflammatory molecule (9,11,12). All of these factors can promote hepatic inflammation (9,11). Recently, it has been observed that morbidly obese subjects have increased levels of circulating lipopolysaccharide (LPS) and LPS-binding protein, which correlate with both a major liver expression of TNF-α and the presence of NASH (13). Interestingly, LPS is known as an inductor of the expression of fibrinolysis inhibitors (eg, PAI-1). Elevated PAI-1 has been correlated with enhanced LPS-induced liver damage and induction of liver inflammatory response (14,15).
The endotoxin (LPS), derived from Gram-negative gut bacteria, LPS-related sensors, such as Toll-like receptor-4 (TLR4), and plasminogen activator inhibitor-1 (PAI-1) can act in the progression from NAFLD to NASH (16–18). A pivotal role of systemic endotoxemia in the NAFLD pathogenesis has been suggested in both human and animal studies. However, some mechanisms remain obscure. In addition, there are no data about a possible association of endotoxin serum levels and correlated factors (ie, PAI-1, TNF-α, and IL-6) with pediatric NAFLD. A better understanding of the roles of these serum molecules and their pathological changes associated with the development of NAFLD in humans is desirable to unravel the mechanisms leading to NASH but also to ameliorate intervention strategies.
Starting from this background, the aim of our study was to determine whether children with NAFLD/NASH present systemic endotoxemia and whether LPS and other proinflammatory molecules, including TNF-α, IL-6, and PAI-1, may represent serum biomarkers of the severity of liver damage.
PATIENTS AND METHODS
Patients, Anthropometrics, and Clinical Chemistry
The study protocol was approved by the ethics committee of the “Bambino Gesù” Children's Hospital (Rome, Italy), and written informed consent was obtained from all of the subjects included in this study. Forty consecutive patients diagnosed with NAFLD (27 boys and 13 girls), mean age 11.9 ± 2.8 (SD), seen in our center from April 2008 to March 2009 were included in the study.
NAFLD subjects had no medical records of alcohol consumption (15 g/day ethanol); drug-induced hepatotoxicity; evidence of other forms of liver disease, which were excluded based on viral serology, serum iron-binding capacity, ceruloplasmin, anti-nuclear antibodies, anti-mitochondrial and smooth muscle antibodies, and liver histology; history of taking lipid-lowering drugs or drugs affecting lipid metabolism; and clinical indication of impaired nutritional status. Controls (6 boys and 3 girls) were nonobese healthy children. They were available from a previous study (19). Samples were drawn from fasting patients and controls.
Weight and height were measured using standard procedures (20). Body mass index (BMI) was calculated from the formula (weight in kilograms/height in square meters) and data converted into BMI percentile using the UK 1990 reference data for BMI in childhood (21).
Laboratory tests were performed at the first visit. Levels of transaminases (ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamyltransferase) were measured by a routine clinical chemistry laboratory using a MODULAR analyzer (Hitachi/Roche, Milan, Italy). Plasma glucose was measured in triplicate (Beckman, Fullerton, CA); insulin by a specific radioimmunoassay (MYRIA Technogenetics, Milan, Italy). Triglycerides and total cholesterol were measured spectrophotometrically.
Insulin resistance and sensitivity were determined, respectively, by the homeostatic model assessment insulin resistance (HOMA-IR) using the formula IR = (insulin × glucose)/22.5; and by the insulin sensitivity index (ISI) derived from the oral glucose tolerance test (OGTT) using the formula ISI = (10,000/square root of [fasting glucose × fasting insulin] × [mean glucose × mean insulin during OGTT]) (22,23).
Enzyme-linked Immunosorbent Assay (ELISA)
Blood samples were stored at −70°C until analysis. Endotoxin serum concentration was measured using a commercially available limulus amebocyte lysate chromogenic endpoint assay with a concentration range of 0.01 to 10 EU/mL (Hycult Biotechnology, Uden, The Netherlands). Levels of PAI-1, TNF-α, and IL-6 were determined with a commercial ELISA kit (R&D Systems, Wiensbaden, Germany). Levels of endotoxin, PAI-1, TNF-α, and IL-6 were measured in samples of patients (40) with NAFLD and in control healthy subjects (9) matched for age and sex.
Biopsies were performed and processed as described elsewhere (24). Liver tissues were immediately fixed in 10% buffered formalin until later analysis. Steatosis, inflammation, hepatocyte ballooning, and fibrosis were scored using the NAFLD Clinical Research Network criteria. Features of steatosis, lobular inflammation, and hepatocyte ballooning were combined to obtained the NAFLD activity score (NAS) and cases with NAS ≥5 were considered diagnostic of NASH (25).
The Shapiro-Wilk W test was applied for determining whether sample data likely derive from a normal-distributed population. Continuous data were reported as mean ± SD, whereas categorical data were reported as counts and percentages. To compare means between groups, Mann-Whitney U test and analysis of variance were performed. Stepwise linear regression analysis was performed using NAS as the dependent variable and endotoxin, PAI-1, TNF-α, IL-6, and BMI as independent variables. Statistical significance was set to a 2-tailed P < 0.05 value. Analysis was performed using SPSS statistical software version 12.0 (SPSS Inc, Chicago, IL).
Patients' Characteristics and Serum Endotoxin Concentration
Anthropometrics and main laboratory and histological parameters for the 40 patients and the 9 controls are reported in Table 1. Steatosis was present in all of the patients and it was of moderate to severe grade in most patients. Inflammation was of mild severity in most (n = 23, 57.5%) children. 62.5% (n = 12) of cases revealed ballooning of the hepatocytes. Stage 1 fibrosis was present in 29 patients (72.5%). 67.5% of children had NASH, and the average NAS was 4.9 ± 1.6 (SD). No patient had established cirrhosis.
As shown in Figure 1A, levels of endotoxin in serum controls (N = 9) ranged from 0.05 to 0.16 EU/mL (average value 0.10 ± 0.04 EU/mL).
In NAFLD (N = 40), serum concentrations of endotoxin ranged from 0.05 to 3.55 EU/mL (average value 0.85 ± 0.9 EU/mL). Nine (22.5%) patients of 40 had levels of endotoxin in the range of normality and 31 (77.5%) had levels above it.
Serum Levels of TNF-α and IL-6
Next, levels of TNF-α and IL-6 were measured in serum samples of control subjects and patients with NAFLD. As shown in Figure 1B, in patients with NAFLD, TNF-α ranged from 3.08 to 28.98 pg/mL, with an average value of 14.20 ± 5.92 pg/mL versus controls (2.3 ± 0.51 pg/mL). The serum IL-6 concentration in patients with NAFLD ranged from 4.90 to 34.09 pg/mL, and as reported in Figure 1C, the mean (12.92 ± 6.55 pg/mL) was greater than in controls (14.4 ± 0.71 pg/mL).
Analysis of PAI-1 Serum Concentration
We also evaluated the serum concentration of PAI-1 in controls and patients with NAFLD. As shown in Figure 1D, the average serum levels of PAI were 345.95 ± 94.04 ng/mL in NAFLD, ranging from 19.51 to 608.24 ng/mL, which was higher than controls (30.10 ± 3.91 ng/mL).
Associations Between Serum Parameters, BMI, and NASH
At the univariate analysis, serum levels of endotoxin (1.18; 0.75–1.60, 95% confidence interval [CI]; P = 0.002), PAI-1 (381; 352.40–410.18, 95% CI; P < 0.001), TNF-α (15.69; 13.89–17.48, 95% CI; P = 0.02), and IL-6 (15.02; 12.30–17.74, 95% CI; P = 0.002), and BMI (93.30; 90.80–95.79, 95% CI; P = 0.028) were the variables that differed significantly in patients with NASH (NAS ≥5) as compared with those without.
We next wondered which parameter would best predict NAS. Stepwise regression analysis, performed with NAS as dependent continuous variable, resulted in 2 models (Tables 2 and 3). In the first model, endotoxin alone explained 47% of the variance in the NAS (P < 0.0001). In the second model, endotoxin and PAI-1 explained together 56% of variance (P < 0.0001). No additional information was gained by modeling for sex, age, waist circumference, AST and ALT levels, insulin resistance, triglycerides, and cholesterol.
Endotoxemia in NAFLD may be a cofactor leading to NAFLD/NASH. This concept is strongly suggested by several studies conducted in both animals and humans (13,26–28). In particular, animal studies demonstrate that LPS in genetically obese rodents with simple fatty liver can induce steatohepatitis, and endotoxin in a nutritional model of NAFLD increases hepatic lipid accumulation and promote development of steatohepatitis (26–28). In human studies, it was reported that obese patients with NAFLD have elevated plasma levels of LPS, which further increase with the severity of liver disease (13).
A possible association between endotoxemia and NASH includes bacterial overgrowth and disruption of intestinal mucosa barrier integrity, which may lead to an increased intestinal permeability, a consequent excessive absorption of endotoxin, an alteration of innate immune response, and liver tissue damage (29). This hypothesis is strongly supported by the high incidence of NASH in obese patients undergoing jejunoileal bypass surgery, a procedure known to be associated with small intestinal bacterial overgrowth (SIBO) and portal endotoxemia (30). In addition, genetically obese mice display enhanced intestinal permeability, which leads to increased portal endotoxemia, which makes hepatic stellate cells more sensitive to bacterial endotoxin (31).
Interestingly, some authors postulate an increased translocation of endotoxin due to disordered intestinal motility as mechanism for NAFLD pathogenesis (16,32). A rich-in-carbohydrate diet may produce ethanol when intestinal stasis favors bacterial overgrowth in the upper parts of the gastrointestinal tract. The increased portal endotoxemia could initiate TLR signaling. Accordingly, the authors found significant correlations between hepatic expression of TLR-4, plasma PAI-1, and endotoxin, even though they are still unable to explain the molecular signaling pathways (32). As occurs in many models of chemical liver inflammation (15), the activation of PAI-1 may be critical for the inflammatory response, which leads to NASH.
Here, we found that the mean value of serum concentration of endotoxin and PAI-1 in pediatric patients with NAFLD was greater, respectively, 8 (P < 0.01) and 11 times (P < 0.0001) than in controls. Furthermore, for the first time our results demonstrate that an increased of gut-derived bacterial products such as endotoxin strongly associates with an increased NAFLD activity score in children, suggesting a potential role of endotoxin as trigger of hepatic necroinflammatory cascades resulting in liver cell injury. Moreover, although the association of PAI-1 and severity of NAFLD histology has been reported in adults by Targher et al (17), our results reinforce the significance of PAI-1 serum levels as a predictor of NASH in children.
Of course, we are aware that clinical significance of the present study is biased by the small number of patients. However, it represents just a preliminary study aimed at verifying our working hypothesis, and novelty in findings must be balanced against difficulties in recruiting a homogeneous sample of such well-characterized children with biopsy-proven NAFLD.
Because the mechanisms involved in the development of NAFLD are not yet fully clarified, therapeutic options are still limited. However, the mechanisms that link serum levels of endotoxin, PAI-1, and pathogenesis of steatohepatitis in children require further investigation and may have significant implications for designing new therapeutic strategies.
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